An electrophoretic display apparatus includes a first substrate and a second substrate; an electrophoretic layer that is allocated between the first substrate and the second substrate; and a first electrode and a second electrode that are each formed in an island shape, for each pixel, at the electrophoretic layer side of the first substrate, and are mutually independently driven. The first and second electrodes form a comb-teeth shaped electrode in a plan view, which include a plurality of branch portions and a trunk portion combining the plurality of branch portions, and each of first ones of the branch portions, which are located at respective edge portions of a pixel area, has a width smaller than a width of each of second ones of the branch portions, which are branch portions other than the first branch portions.

1. Technical Field
The present invention relates to an electrophoretic display apparatus and an electronics device.
2. Related Art
With respect to electrophoretic display apparatuses each having a function of displaying color images, heretofore, an electrophoretic display element enveloping therein three kinds of particles, i.e., white color ones, black color ones and another color ones, has been proposed. In such an electrophoretic display element, individual particles are sorted into positively charged particles, negatively charged particles and non-charged particles. At the side of an active matrix substrate for driving the display elements, each pixel is provided with two pixel electrodes. Further, on an opposite substrate, a common electrode that is common to all pixels is provided.
Once both of the two pixel electrodes on the active matrix substrate and the common electrode are supplied with a positive electric potential and a negative electric potential, negatively charged particles and positively charged particles are attracted to the active matrix substrate side and the common electrode side, respectively. Therefore, users view a color tone created by the positively charged particles.
Further, once the two pixel electrodes on the active matrix substrate are supplied with a positive electric potential and a negative electric potential, respectively, and the common electrode is supplied with an intermediate electric potential therebetween (for example, a ground electric potential), both of the positively charged particles and the negatively charged particles are attracted to the active-matrix substrate side owing to electric fields arising from the two pixel electrodes. Therefore, users view a color tone created by the none-charged particles (refer to JP-A-2009-9092 and JP-A-2009-98382).
Here, in order to obtain the color tone created by the non-charged particles, it is necessary to cause electric fields to arise uniformly throughout the whole area of each pixel so that all of the positively charged particles and the negatively charged particles can be attracted to the active matrix substrate side. In order to realize this condition, a configuration resulting from combining and allocating comb-teeth shaped pixel electrodes is used in lots of cases.
However, supplying respective adjacent pixel electrodes with mutually different electric potentials results in occurrence of leakage current between the pixel electrodes via an electrophoretic layer. This leakage current flows between the two pixel electrodes, and thus, an amount of leakage current becomes larger in proportion to the length of a boundary between the two pixel electrodes. Therefore, in the case of the comb-teeth shaped pixel electrodes, a large amount of leakage current flows. Furthermore, the occurrence of leakage current leads to an increase of power consumption of an electrophoretic display panel.
Moreover, owing to such leakage current, there is a possibility of causing an electrochemical reaction between the electrophoretic layer and each of the pixel electrodes. Namely, there is a possibility of detracting reliability of the pixel electrodes. That is, there is a high possibility of occurrence of ionic migrations and/or corrode. If a precious metal, such as gold or platinum, is used as a material of the pixel electrodes, the reliability is enhanced; however, the use of such a material leads to an increase of cost and a growth of complexity of manufacturing processes thereof. Therefore, it has been difficult to enhance the reliability, and concurrently therewith, suppress increase of the cost. Consequently, in order to reduce an amount of such leakage current, a method for reducing the length of a boundary between the mutually adjacent pixel electrodes by enlarging a width of each of branch portions of the comb-teeth shaped pixel electrodes and/or enlarging each distance between the mutually adjacent two pixel electrodes can be conceived.
However, depending on the width of each of the pixel electrodes and the distance between the mutually adjacent two pixel electrodes, a problem in that there occur areas that are not subjected to any electric fields occurs, and as a result, particles existing at the common pixel side cannot be attracted. This phenomenon remarkably occurs in edge portions of each pixel area. Namely, particles distributed immediately above any one of branch portions located at the middle side of each pixel area are attracted by actions of electric fields arising from either of branch portions of a different pixel electrode, which are located at respective both sides of the branch portion located at the middle side thereof; however, each of branch portions located at respective peripheral edge portions of the pixel area is subjected to actions of electric fields arising from only one branch portion of a different pixel electrode, which is located at one side of and adjacent to the branch portion located at the peripheral edge portion thereof. Therefore, as a result, such a phenomenon causes a problem in that particles located at peripheral edge portions of the pixel area cannot be attracted.
Unintentional particles remaining at the common electrode side results in occurrence of unevenness of display.

Electrophoretic display apparatus and electronics device


An electrical energy generation device includes an energy conversion device thermally coupled to an electrical component and configured to convert thermal energy produced by the electrical component to mechanical motion. An electricity generation device is coupled to the energy conversion device and is configured to convert the mechanical motion to electrical energy. At least a portion of the electrical energy energizes the electrical component.

As electronic components become more powerful, the amount of thermal energy these components generate greatly increases. Accordingly, various devices (e.g. heat sinks and cooling fans) are utilized to dissipate such thermal energy. Unfortunately, while such devices may dissipate such thermal energy, this energy is wasted and simply exhausted into the surrounding environment. This problem is further complicated when numerous computing devices (e.g. servers) are placed into a common environment (e.g. a server room), wherein a considerable amount of thermal energy is generated and more complex thermal management systems (e.g. refrigeration systems, air circulation and exhaust systems) are utilized to thermally manage such a common environment.

System and method for generating electricity from component waste heat


An exerciser is provided for application of an anatomically applied force to the jaw for pivoting of the jaw at its tempero-mandibular joint or to provide a motion to the jaw, or resistance to motion of the jaw which is beneficial in facial orthopedics. A structure is provided having curvilinear guide tracks which guide movement of a carriage according to the character of movement that is desired. The carriage is movable to thereby allow corresponding movement of a mandibular jaw member supported by the carriage relative to a maxillary jaw member supported by the exerciser body. Through suitable linkages, motion may be imparted to the carriage or resistance exerted against its motion by manual action, by a spring or by a motor. The linkage may provide for adjustment of mechanical advantage or range of motion through selection of slots or holes formed in parts of the linkage structure.

Exercise of jaw muscles is an important part of treatment and rehabilitation for many jaw injuries and disorders. For example, the common practice of wiring jaws closed as a splinting approach to fractures and bone surgical procedures leads to a shortening of the muscles that close the jaw and a weakening of the muscles that open it. As a result, patients may find it impossible to open their mouths after the wiring has been removed. Physicians and surgeons resort to expedients which include prying with tongue depressors and the use of screw operated wedge devices to stretch the muscles and thereby accomplish opening of the mouth. Once the jaws are opened, it is necessary for patients to undertake exercises to stretch the muscles in order to regain full freedom of motion. Though to date jaw exercise devices have considered only opening and closing movement of the jaw, it may be desirable to impart lateral as well as pivotal movement to the mandible during its normal opening and closing movement as therapeutic movement of the temporo-mandibular joint as well as the muscle system for mandibular control. Passive elastic devices have been suggested for accomplishing jaw exercising. Also, expensive spring operated instruments have been developed for this purpose.
A typical defect of most commercially available jaw exercising devices is that they either cause pain and injury through the exertion of excessive force or they may apply pressure to the molar region in a vertical manner thus displacing the condyles downward and disrupting the temporo-mandibular joint. None of these devices moves the lower jaw in a fashion which takes into account the structural features of the jaw hinge established by the temporo-mandibular joint, namely that the pivotal area of the jaw hinge is above and to the rear of the mouth opening by virtue of the curvature of the jaw at its upper rear portion and that the temporo-mandibular joint does not establish a single precise, pivot point about which the mandible rotates. Rather, the temporo-mandibular joint forms a movable pivot which causes compound movement of the mandible throughout its rotation.
It is a principal feature, therefore, to provide a novel jaw exercising mechanism which accomplishes movement of the jaw by applying pressure to the teeth along the natural anatomical lines taking account of the natural angles of motion of the mandible.
It is also a feature of this invention to provide a novel passive jaw exercising device that avoids injury by allowing the patient to continuously meter the force that is applied to accomplish opening movement of the jaw.
It is an even further feature of this invention to provide a novel passive jaw exercising device that adjusts for both jaw size and for range of motion and provides for graded application of force for both opening and closing motions.
It is another feature of this invention to provide a novel passive jaw exercising mechanism that utilizes a hand grip designed to take advantage of the natural strength and endurance of the patient's first and second fingers and the patient's hand in general to provide both opening force and closing resistance to the jaw and thereby accomplishes exercising of the muscles that open and close the jaw.
It is another feature of this invention to provide a novel therapeutic jaw exerciser that is capable of inducing lateral movement as well as pivotal movement to the mandible or any specifically designed movement that may be desired for appropriate therapy for the patient.
It is a further feature of this invention to provide a novel passive jaw exercising mechanism that can be made inexpensively as a plastic molding for use by a single patient thus insuring against cross contamination between patients and providing for infection control.
It is also a feature of this invention to provide a novel passive jaw exerciser that is adaptable for use by a wide range of patient's having different size jaws through the use of different sized jaw members and/or jaw member extensions.
It is an even further feature of this invention to provide a novel passive jaw exerciser that is capable of providing a motion that is beneficial in facial orthopedics through employment of a motion controlling track of a particular configuration.


A target ratio module selectively determines a target ratio of transmission input shaft speed to transmission output shaft speed independently of an accelerator pedal position and as a function of a vehicle speed and a driver axle torque request. A first pulley valve control module controls opening of a first valve based on the target ratio. The first valve controls transmission fluid flow to a first pulley actuator. The first pulley actuator is coupled to the transmission input shaft of a continuously variable transmission (CVT) and expands and contracts based on transmission fluid pressure. A second pulley valve control module controls opening of a second valve based on the target ratio. The second valve controls transmission fluid flow to a second pulley actuator. The second pulley actuator is coupled to the transmission output shaft of the CVT and expands and contracts based on transmission fluid pressure.

The background description provided herein is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present disclosure.
An internal combustion engine combusts an air and fuel mixture within cylinders to drive pistons, which produces drive torque. Air flow into the engine may be regulated via a throttle. More specifically, the throttle adjusts throttle area, which increases or decreases air flow into the engine. As the throttle area increases, the air flow into the engine increases. A fuel control system adjusts the rate that fuel is injected to provide a target air/fuel mixture to the cylinders and/or to achieve a target torque output. Increasing the amount of air and fuel provided to the cylinders increases the torque output of the engine.
Some transmission control systems control a plurality of solenoids to shift gears of an automatic transmission. In one example, the transmission control system actuates (e.g., opens) one or more solenoids to shift the transmission to a particular gear. The transmission control system actuates a solenoid by adjusting the pressure in a line that supplies hydraulic fluid to the solenoid. In another example, the transmission control system increases the hydraulic pressure supplied to a solenoid to actuate the solenoid.
Manual transmissions require a driver to actuate a clutch pedal and select a gear ratio using a gear selection lever. The number of gear ratios available to the driver to select in a manual transmission may be five to 7 although a greater or lesser number of gear ratios may be possible. A continuously variable transmission is an automatic transmission that can adjust its gear ratio continuously to a large number of gear ratios between maximum and minimum values.

Transmission ratio control systems and methods


A collapsible multilayer container includes a cover and a plurality of housings in which the housings are pivotally connected one by one so as to be collapsible for spacial savings and expandable for charming outlooks. The cover includes a pair of handles pivotally connected thereto for carrying purpose. And, the cover and the housings are securely locked together by a locking device.

A conventional container for storing articles such as chain, ring, and needle and thread generally comprises a cover and a housing in which the cover is secured to the housing in an openable manner.
But, storing articles may be the only one function such container can have. And, the size of the container is huge and inconvenient for carrying when lots of articles are stored. Or, only a packet size container is possible for easy transport but impractical.


Apparatus for syndicated data stream content provisioning, the apparatus comprising: an interaction tracker, configured to track at least one interaction of a user with at least one content object of a respective one of a group consisting of at least one syndicated data stream received on a computer device, the interaction being implicitly indicative of a preference of the user, a ranking function calculator, in communication with the interaction tracker, configured to calculate a ranking function based on the tracked interaction, and a content ranker, in communication with the ranking function calculator, configured to rank a plurality of content objects of the syndicated data streams of the group, in an order based on the calculated ranking function.

The present invention relates to content provisioning, and more particularly, but not exclusively, to a system and method for syndicated data stream content provisioning.
As the volume of data accessible via computer systems continues to increase, the need for automated tools for assisting the user to home in valuable for him information within the data also increases. Queries to search engines are routinely employed to find relevant information on the many web pages or enterprise data.
A substantial portion of consumption of digitized content gradually shifts from query-driven search in the web or enterprise digital libraries to passively following the information continuously made available though various syndicated data streams of content. The syndicated data streams include, but are not limited, to RSS (Really Simple Syndication) feeds, streams of user messages on social network platforms (say, Twitter™), Atom syndication format feeds, e-mail messages from a mail server, web site content delivered using a dedicated API (such as Facebook™ connect), etc., as known in the art.
Typically, a user subscribes to one or more syndicated data streams, such as an RSS news feed, a web feed, or a stream of user messages in a social network platform, as known in the art. Optionally, the data stream is syndicated by a content provider (say by a news network, such as CNN™), by a Web Blogger, etc. The syndicated data stream is used by the content provider, to provide users who subscribe to the syndicated data stream with a frequently updated, structured list of content objects.
A content object is a data object with a usually short textual description which may be presented to the user. The textual description may include a content item itself (say a user message published on a social network platform), a button which presents a content item to the user upon clicking on the button, a hyperlink which links the user to a page of a web site, a link which initiates downloading of the content item, etc., as known in the art.
In a typical current scenario of syndicated data stream provisioning, a content provider publishes a feed link on the provider's web site. End-users can subscribe to the feed, using an aggregator program (also called a feed reader or a news reader). The aggregator program runs on the end-users' machines. The subscription process is usually as simple as dragging the link from the web browser to the aggregator. When instructed, the aggregator asks all the servers in its feed list if they have new content. If so, the aggregator either makes a note of the new content or downloads it. Aggregators can be scheduled to check for new content periodically.
The motivation for the shift from query-driven search in the web or enterprise digital libraries to passively following the information continuously made available though various syndicated data streams is manifold. In particular, users don't want to miss information the users consider valuable and yet the interests of the users are typically wide and dynamic, and thus it is not easy to express the user's interests via standard queries.
Subscription-based information consumption from syndicated data streams, such as RSS feeds, or a user specific Twitter™ feeds of messages from Twitter™ followers of a user, provides the user with a potential of timely access to information valuable for him. However, the subscription-based information further creates an overload of data provided to the user from one or more data streams.
It is unavoidable that most pieces of information (i.e. content objects, say video clips, news reports, articles, Twitter™ short messages ("Twits"), etc.) distributed via even a single syndicated data stream (say a single RSS feed) are optimized for a specific single user.
Even if all the streams the user subscribed to are fully personal, the multitude of such streams may still create the effect of an information overload. Consequently, the user has to separate signal from noise, and browse through multitude of content objects, in an attempt to locate the content objects that are more valuable for the user. As this process is time consuming, for many users getting to the valuable content becomes virtually impossible.
Several current systems target information overload on the user and aim at reducing the burden put on the user.
For example, the Google™ Reader system allows the user to browse through content objects not only in the chronological order of creation, but also in an order reflecting the rate at which various streams the user subscribed to are updated.
FeedHub™′, an mSpoke™ powered system, exploits content categorization, together with absolute, explicit feedback of a user on content objects (of the form "I like this item"), to establish a relevance ranking, which is a relative numerical estimate of the statistical likelihood that a certain content object is of interest to the user. The efficiency of this method heavily relies on the user's willingness to continuously provide explicit feedback on content objects provided to him, as well as on credibility and semantic consistency of the explicit feedback.
U.S. Pat. No. 7,089,237, to Turnbull et al., filed on December Jan. 26, 2001, discloses a search and recommendation system, which employs the preferences and profiles of individual users and groups within a community of users.
U.S. Pat. No. 7,345,232, to Toivonen, filed on Nov. 6, 2003, describes a play list selection module, which creates a play list of media pieces stored in databases accessible by the selection module. The play list includes media pieces that are adapted to a specific user together with randomly selected media pieces, in a ratio determined by a surprise factor provided by the specific user. The play list may begin with a set of media pieces that are known to be enjoyed by the specific user, say media pieces previously consumed by the specific user, and ends with the randomly selected media pieces.
U.S. patent application Ser. No. 10/727,444, to Karnawat, filed on Dec. 3, 2003, describes a personalized internet search system based on a context-based user feedback gathered regarding searches performed on a search mechanism.
U.S. patent application Ser. No. 10/861,154, to Michelitsch et al., filed on Jun. 4, 2004, describes a content recommendation device with user feedback The device described by Michelitsch includes a selection engine that selects content objects from a content object pool according to a user profile.
U.S. Pat. No. 7,089,194, to Berstis et al., filed on Jun. 17, 1999, describes a method and apparatus for adaptively targeting advertisements to a specific client computer from a server within a distributed data processing system. As a user of the client browses the internet, the material that is downloaded to the client is scanned to generate a list of keywords. The keywords are used to select an advertisement from a database, the selected advertisement is inserted into the material, and the material and selected advertisement are presented together to the user.

Syndicated data stream content provisioning


A drawer type heating apparatus includes a heating case forming a heating space, and an openable door adapted to be moved in a drawing manner relative to the heating case for opening and closing an opening portion, and includes a radio-wave transmission suppression portion which is faced to inner wall surfaces of the opening portion of the heating case, and placed such that there is a predetermined gap between the radio-wave transmission suppression portion and the entire periphery of the inner wall surfaces of the opening portion in the heating case, in a state where the openable door is closed, the radio-wave transmission suppression portion including a first radio-wave-propagation-direction suppression-area constituted by a surface with a plurality of steps, and a second radio-wave-propagation-direction suppression-area having suppression protruding portions which are periodically formed and placed in such a way as to face the first radio-wave-propagation-direction suppression-area with a predetermined interval.

As conventional drawer type heating apparatuses, there have been suggested various heating apparatuses relating to pull-out mechanisms, placement of heating means, and the like (refer to Patent Literature 1, for example).
Ordinary microwave heating apparatuses employ hinge couplings, as mechanisms for opening and closing openable doors and such openable doors are of turning types. In microwave heating apparatuses having such turning-type openable doors, there have been suggested structures which provide, in an openable door, a rectangular plate-shaped choke portion (a radio-wave transmission suppression portion) facing the inner wall surfaces of a heating chamber, in order to suppress leakages of microwaves supplied to the heating chamber for housing objects to be heated, through the openable door (refer to Patent Literature 2, for example).
Further, there have been suggested methods of determining states of thawing of objects to be heated within a heating chamber, by detecting reflected electric power returning from the heating chamber while varying the frequency of microwaves supplied to the heating chamber (refer to Patent Literature 3, for example).    PLT 1: Unexamined Japanese Patent Publication No. 2005-164091    PLT 2: Unexamined Japanese Patent Publication No. 2005-090942    PLT 3: Unexamined Japanese Patent Publication No. 2006-086004

PANASONIC INTELLECTUAL PROPERTY MANAGEMENT CO., LTD.


The invention relates to a protein which presents identical or different catalytically active domains of glycosyltransferases and has a processive action. In particular, the same protein is successively active in at least two successive process steps.

The invention relates to the use of processive UDP-sugar: 1,2-diacylglycerol-3-xcex2-, UDP-sugar: 3-xcex2-1,3xe2x80x2-phospho-sn-glycerol-1xe2x80x2,2xe2x80x2-diacyl-sn-glycerol)- and UDP-sugar: 3-[O-xcex2-D-glucopyrartosyl]-sn-glycerol-1,3 xe2x80x2-phospho-1xe2x80x2,2xe2x80x2-diacyl-sn-glycerol-D-sugar transferases and similar proteins as well as the corresponding coding nucleic acids for the manipulation of the contents and/or the structure of glycosyldiacylglycerols and/or the synthetic secondary products thereof, as well as other substrates which are glycosylated by these enzymes, in transgenic cells and/or organisms.
Glycosyldiacylglycerols were produced enzymatically by means of a sugar transferase (glycosyl transferase). For this purpose, the gene coding for a UDP-sugar transferase was isolated from genomic DNA of Bacillus subtilis and Staphylococcus aureus, and cloned into, and expressed in, E. coli. The activity of the enzymes was confirmed by means of specific in vitro-enzyme assays. The products were also detected and identified in lipid extracts of transgenic E. coli cells. The products are various novel glycolipids having different number of glucose residues (maximum of 4) linked via a xcex2(1xe2x86x926)glycosidic bond, and utilizing diacylgylcerol (DAG) or phosphatidylglycerol (PG) as the primary acceptor.
In addition, these novel glycolipids comprise two differently structured novel phosphoglycolipids (PL1 and PL2) with a different number of glucose residues (maximum of two) which in the case of (PL2) are also linked via a xcex2(1xe2x86x926)glycosidic bond and utilize phosphatidylglycerol as the acceptor (i.e. both diastereomers, i.e. with respect to the configuration of the non-acylated glycerol residue). The glycosyl residues may further be acylated in position 6xe2x80x2xe2x80x3 of the terminal glucose:
1) MGlcD: 3-[O-xcex2-D-glucopyranosyl]-1,2-diacylglycerol (Staphylococcus aureus ypfP)
2) DGlcD: 3-[O-xcex2-D-glucopyranosyl-(1xe2x86x926)-O-xcex2-D-glucopyranosyl]-1,2-diacylglycerol
3) TGlcD: 3-[O-xcex2-D-glucopyranosyl-(1xe2x86x926)-O-xcex2-D-glucopyranosyl-(1xe2x86x926)-O-xcex2-D-glucopyranosyl]-1,2-diacylglycerol
4) TeGlcD: 3-[O-xcex2-D-glucopyranosyl-(1xe2x86x926)-O-xcex2-D-glucopyranosyl-(1xe2x86x926)-O-xcex2-D-glucopyranosyl-(1xe2x86x926)-O-xcex2-D-glucopyranosyl]-1,2-diacylglycerol
5) Phospholipid 1:3-[O-xcex2-D-glucopyranosyl]-sn-glycerol-1,3xe2x80x2-phospho-1xe2x80x2,2xe2x80x2-diacyl-sn-glycerol)
6) Phospholipid 2:{3-[O-(6xe2x80x2xe2x80x3-O-acyl)-xcex2-D-glucopyranosyl-(1xe2x80x2xe2x80x36xe2x80x2)-O-xcex2-D-glucopyranosyl]-2-acyl-sn-glycerol-1,3xe2x80x2-phospho-1xe2x80x2,2xe2x80x2-diacyl-sn-glycerol}
Note: The numbering of the glycerol residues I (GroI) and II (GroII) corresponds herein to the numbering 1-3 and 1xe2x80x2-3xe2x80x2, respectively, i.e. GroI is xe2x80x9cleft-handxe2x80x9d and GroII is xe2x80x9cright-handxe2x80x9d in accordance with FIG. 13.
Surprisingly, the enzymes act in a processive manner, i.e. all detected novel glycolipids are formed by successive addition of UDP-glucose to the respective preceding product of the enzymes. Further, alkyl-xcex2-D-glucosides, ceramides (both enzymes), sterols and sterol glucosides (only the enzyme of S. aureus) are used as acceptors for a further glucosylation reaction.
Glyceroglycolipids represent a group of membrane components which are very hetero-geneous with respect to their structure. They are found in bacteria (Kates, 1990), plants and in very low amounts also in animals. Many structures especially of bacterial glycolipids have already been described many years ago (Kates, 1999), however, none of the genes synthesizing these glycolipids have been cloned, so that these substances can be obtained from the corresponding organisms only in analytical amounts. Only at the beginning of 1997 was the first publication issued, wherein the cloning and expression of a plant galactose: 1,2-diacylglycerol galactosyl transferase is described (Shimojina et al., 1997). However, this enzyme is no xe2x80x9cprocessivexe2x80x9d glycosyl transferase.
Database searches in the xe2x80x9cU.S. Patent Databasexe2x80x9d revealed that two further patents relating to glycosyl transferases exist: U.S. Pat. No. 5,545,554: Glycosyl transferases for biosynthesis of oligosaccharides, and genes and encoding them, and U.S. Pat. No. 5,641,668: Proteins having glycosyl transferase activity. It appears that the first-mentioned patent only relates to glycosyl transferases which synthesize oligosaccharide, so that this patent is not relevant with respect to the enzyme, viz a lipid glycosyl transferase, described herein. The second-mentioned patent relates to glycosyl transferases in general, in view of which the processive enzyme described in this specification is novel.
Glycosyl diacylglycerols are naturally occurring compounds found in plants, animals and bacteria. However, an inexpensive, large-scale production of these compounds was not possible so far, since corresponding genes were not yet cloned. Glycosyl diacylglycerols can be used in a variety of applications, depending on the number of sugar residues and the structure of the fatty acids.
When esterified with usual C18 unsaturated fatty acids, diglucosyl diacylglycerols have emulsifier properties which are useful in food industrial applications (in mayonnaise, margarine, ice cream, confectionery etc.).
In the presence of highly unsaturated fatty acids, glycolipids may be introduced into polymers, which then obtain new characteristics and surfaces. Finally, glycosyl diacylgylcerols may obtain detergent characteristics, when the fatty acid chain length is drastically shortened. This would already now be possible in transgenic rape seed with predominant lauric acid. Such detergents could be produced in large amounts in an inexpensive manner, and such detergents would be biologically degradable.
The phospholipids which are glucosylated by the enzyme of S. aureus receive new physico-chemical characteristics due to the charge of the phosphate residue between the two glycerol residues on the one hand, and on the other hand due to acylation of the sugar residue(s). Thus, by use of the described processive sugar transferases, not only neutral lipids, but also charged glycolipids can be specifically produced and varied. Thus, a further class of charged glycophospholipids are developed via the sugar transferases.
In the production of plant oils from oil seeds, a lecithin fraction is obtained, wherein phospholipids and glycolipids are accumulated. By over-expressing the genes disclosed in this specification in these plants, a variety of glycolipids (glucosyl diacylglycerols, steryl glucoside, glucocerebroside and other lipids described herein) could be concentrated, with a favourable effect on the baking properties of bakery products, to which the lecithin fraction is added.
In addition, the phospholipids glucosylated by the S. aureus enzyme receive further physicochemical properties due to the charge of the phosphate residue.
This invention, therefore, relates to a process for the production of glycolipids in transgenic cells and/or organisms, comprising the following steps:
transfer of a nucleic acid molecule that codes for a protein having the biological activity of a processive diacylglycerol glycosyltransferase to the cells or organism,
expression of the protein having a biological activity of a processive diacylglycerol glycosyltransferase under suitable regulatory sequences in the cells or the organism, and
if desired, recovery of the glycolipids synthesized by the biological activity of a processive diacylglycerol glycosyltransferase from the cells or the organism.
In a preferred embodiment of the invention, the nucleic acid molecule codes for a protein having the biological activity of a processive diacylglycerol glycosyltransferase from Bacillus subtilis or Staphylococcus aureus.
The transgenic cells may be any cells that are useful for the production of the new glycolipids, preferably the cells are plant, yeast or bacteria cells. The transformed organism is preferably a plant, yeast or bacterium.
As mentioned above and as will be clear from the following description, the glycolipids produces by the process of the invention are preferably glycosyl diacylglycerols and/or phosphoglycolipids. More preferably, the glycolipids are monoglycosyldiacylglycerol, diglycosyldiacylglycerol, triglycosyl diacylglycerol, tetraglycosyldiacylglycerol, glycosyl ceramide, diglycosyl ceramide, steryl glycoside, steryl diglycoside, glycosyl phosphatidylglycerol, and/or diglycosyl phosphatidylglycerol. Most preferably, the glycolipids are monoglucosyldiacylglycerol, diglucosyldiacylglycerol, triglucosyldiacylglycerol, tetraglucosyldiacylglycerol, glucosyl ceramide, diglucosyl ceramide, steryl glucoside, steryl diglucoside, glucosyl phosphatidylglycerol, and/or diglucosylphosphatidylglycerol.
The invention is also directed to the use of a nucleic acid molecule coding for a protein having the biological activity of a processive diacylglycerol glycosyltnsferase or of a proteins having the biological activity of a processive diacylglycerol glycosyltransferase for processive glycosylation, in particular for production of glycolipids. Processive glycosylation, in particular the production of glycolipids, may take place in vivo or in vitro.
Further, the invention is directed to tetraglucosyldiacylglycerol, synthesized and described herein for the first time. The same applies to glucosylphosphatidylglycerol and diglucosylphosphatidylglycerol.
The invention is further directed to the use of the glycolipids produced by processive glycosylation according to the invention in the food industry, as an emulsifier or as a detergent.
A processive glycosyl transferase, as described herein, catalyzes the successive transfer of one or more hexose residues to an acceptor molecule. In particular the enzyme catalyzes at least one of the following reactions:
a) addition of hexose xcex2(1xe2x86x926) to diacylglycerol,
b) addition of hexose xcex2(1xe2x86x926) to a MHexD,
c) addition of hexose xcex2(1xe2x86x926) to a DHexD,
d) addition of hexose xcex2(1xe2x86x926) to a THexD,
e) addition of hexose xcex2(1xe2x86x926) to a TeHexD,
f) addition of hexose xcex2 to a ceramide,
g) addition of hexose xcex2(1xe2x86x926) to a monohexosyl ceramide,
h) addition of hexose xcex2 to a sterol,
i) addition of hexose xcex2(1xe2x86x926) to a steryl glucoside,
j) addition of hexose in xcex2-glycosidic linkage to the primary hydroxyl group of phosphatidylglycerol,
k) addition of hexose xcex2(1xe2x86x926) to the first hexose of phosphatidylglycerol-xcex2-D- glucoside.
In paricular the glycosyl transferase catalyzes the successive transfer of one or more hexose residues to at least one acceptor molecule for synthesis of glycolipids, in particular phosphoglycolipids, in particular catalyzing one of the following reactions:
a) addition of Glc xcex2(1xe2x86x926) to a diacylglycerol,
b) addition of Glc xcex2(1xe2x86x926) to a MGlcD,
c) addition of Glc xcex2(1xe2x86x926) to a DGlcD,
d) addition of Glc xcex2(1xe2x86x926) to a TGlcD,
e) addition of Glc xcex2(1xe2x86x926) to a TeGlcD,
f) addition of Glc xcex2 to a ceramide,
g) addition of Glc xcex2(1xe2x86x926) to a monoglucosyl ceramide,
h) addition of Glc xcex2 to a sterol,
i) addition of Glc xcex2(1xe2x86x926) to a steryl glucoside,
j) addition of Glc in xcex2-(1xe2x86x926)-glycosidic linkage to the primary hydroxyl group of phosphatidylglycerol,
k) addition of Glc xcex2(1xe2x86x926) to the first Glc of phosphatidylglycerol-xcex2-D-glucoside.
The invention also relates to the DNA sequences coding for an protein having enzyme activity of a processive glycosyl transferase from Bacillus subtilis and/or Staphylococcus aureus. Further the invention is directed to DNA sequences coding for a protein which shows at least 50%, preferably at least 70%, more preferably at least 90%, and most preferably at least 95% identity with the deduced protein of ypfP (Clustal X). More particular, the DNA sequence codes for a protein having more than 5 amino acids within the amino acid sequence EHQPDIII (SEQ ID NO. 5) which are identical with the amino acid sequence of the proteins from B. subtilis and/or S. aureus, preferably having more than 6 amino acids within the amino acid sequence QVVVVCGKN (SEQ ID NO. 6) or the amino acid sequence DCMITKPG (SEQ ID NO. 7) which are identical with the amino acid sequence of the proteins from B. subtilis and/or S. aureus. More preferably, the DNA sequence codes for a protein the amino acid sequence of which comprises the amino acid sequence MITKPGGITxTE (SEQ ID NO. 8) (wherein x is any amino acid), or the amino acid sequence VKxTGIPI (SEQ ID NO. 9) (wherein x is any AA) or the amino acid sequence of which comprises more than 5 amino acids within the sequence ZPDIIIxxxP (SEQ ID NO. 10) (wherein Z represents the amino acid Q or K and x is any amino acid) which are identical to the sequence found in Bacillus subtilis and/or Staphylococcus aureus.
The invention also relates to the use of a processive glycosyltransferase for biosynthetic production of glycolipids having the following structure:
a) xcex2-D-Glcp-(1xe2x86x926)-xcex2-D-Glcp-(1xe2x86x926)-xcex2-D-Glcp-(1xe2x86x926)-xcex2-D-Glcp-(1xe2x86x926)-Gro,
b) 3-[O-xcex2-D-Glucopyranosyl]-phosphatidylglycerol (PL1), or
c) {3-[O-(6xe2x80x2xe2x80x3-O-acyl)-xcex2-D-glucopyranosyl-(1xe2x80x2xe2x80x3xe2x86x926xe2x80x3)-O-xcex2-D-glucopyranosyl]-2-acyl-phosphatidylglycerol} (PL2).
Finally, the invention is directed to secondary products which are produced by biosynthetically and gentechnically engineered microorganisms and/or plants using a processive glycosyl transferase by further conversion of the products produced by the action of the processive sugar transferases, in particular by addition of a fatty acid to the position 6xe2x80x2xe2x80x3 of the terminal hexose in {3-[O-xcex2-D-glucopyranosyl-(1xe2x80x2xe2x80x3xe2x86x926xe2x80x3)-O-xcex2-D-glucopyranosyl]-2-acyl-phosphatidylglycerol}.
PL1, PL2 and their respective diastereomeric counterparts are illustrated in FIG. 13. PL1xe2x80x2 is the diastereomeric counterpart of PL1 and PL2xe2x80x2 is the diastereomeric counterpart of PL2.
1. Isolation and Cloning of ypfP
The ypfP gene was isolated from B. subtilis, the gene being described in the SubtiList Database as an open reading frame of unknown function (accession number P54166). The other gene that was isolated and cloned was a sequence from Staphylococcus aureus (accession number Y14370) described as an open reading frame of unknown function.
For DNA isolation, restriction analysis and ligation, standard techniques were used (Sambrook et al., 1989). Genomic DNA from Bacillus subtilis 019 was isolated according to Cutting et al., 1990. Genomic DNA of S. aureus was provided by Prof. Dr. Witte, (Robert Koch-Institute, Postfach 650280, 13302 Berlin). Restriction endonucleases and DNA-modifying enzymes were purchased from New England Biolabs and Boehringer Mannheim, and used as recommended by the suppliers.
E. coli XL1 Blue (MRFxe2x80x2) (Stragene), E. coli BL21 (DE3) (Novagen) and Bacillus subtilis 019 were grown at 37xc2x0 C. in a Luria Broth (LB) (Sambrook et al., 1989). For plasmid-bearing E. coli strains, the antibiotics ampicillin (100 xcexcg ml) and kanamycin (30 xcexcg ml) were included in the medium. The vectors pUC18 (Yanish-Perron et al., 1985) and pET24c(+) and pET24d(+) (Novagen) were used as cloning vectors. The ypfP genes were isolated from genomic DNA of B. subtilis and S. aureus by PCR. For this purpose the specific primers PJ1 (5xe2x80x2-CCGAGCTCCCATATGAATACCAATAAAAGAG 3xe2x80x2) (SEQ ID NO. 11) and PJ2 (5xe2x80x2 TCCGGATCCTTACGATAGCACTTTGGC 3xe2x80x2) (SEQ ID NO. 12) for B. subtilis ypfP and the primers PJ10 5xe2x80x2 TTCCATGGTTACTCAAAATAAAAAGATATTG 3xe2x80x2 (SEQ ID NO. 13) and PJ11 5xe2x80x2 TTTGGATCCTTATTTAACGAAGAATCTTGCATATAA 3xe2x80x2 (SEQ ID NO. 14) for the S. aureus gene (say) were used, the underlined part of which annealed to the 5xe2x80x2 and 3xe2x80x2 end of the ypfP/say genes. The following amplification program was used: 10 min at 94xc2x0 C.; 30 cycles of 0.5 min at 55xc2x0 C. and 60xc2x0 C. for S. aureus ypfP, respectively, 2 min at 72xc2x0 C., 1 min at 94xc2x0 C.; one cycle of 10 min at 74xc2x0 C., Pwo-polymerase (Boehringer) was used for the amplification of the 1170 bp product of the genomic DNA of B. subtilis, Pfu-polymerase (Stratagene) was used for the amplification of the 1190 bp product from S. aureus genomic DNA. The amplified genes were cloned into SmaI-linearized pUC18 vector, resulting in pypfP3 and psay1. For construction of the expression vectors pEypfP 24 and pEsay24, the ypfP fragments were released by BamHI and NdeI and NcoI digestion, respectively, from pypfP3 and psay1, and inserted into BamHi-, NdeI- and NcoI-linearized pET24c(+) and pET24d(+), respectively. E. coli XL1 Blue (MRFxe2x80x2) was transformed with pypfP3 and psay1 and E. coli BL21 (DE3) was transformed with pEypfP24 and pEsay24. Correct in-frame cloning was confirmed by sequencing. One strand of the DNA of pypfP3 and psay1 was sequenced using the dideoxy method (automatic sequencer 373A and 377, Applied Biosystems). For computer analysis of the sequences, Clone manager for Windows 4.1 (Scientific and Educational Software) was used. Database searches were performed using the BLAST algorithm (Altschul et al., 1990). Sequence alignments were performed using Clustal X (Higgins and Sharp, 1988).
2. Expression of the ypfP/say-genes
For expression of the genes, ypfP was cloned into pET24c(+) and pET24d(+), respectively, and E.coli BL21 (DE3) was transformed with the resulting constructs pEypfP24 and pEsay24. Pre-cultures of E. coli BL21 (DE3), E. coli BL21 (DE3) pEypfP24 and E. coli BL21 (DE3) pEsay24 were grown overnight at 37xc2x0 C., and expression cultures were started at an optical density (O.D.)580 of 0.05. Induction was performed by adding 0.4 mM IPTG at an optical density of 0.8 and further incubation for 2 h at 37xc2x0 C. All subsequent steps were carried out at 4xc2x0 C. Cells were collected by centrifugation (15 min, 5000xc3x97g). The cell pellet was re-suspended in a buffer 1 (50 mM Tris-HCl, pH 8.0; 20% (v/v) glycerol) (4% of the volume of the expression culture). The cells were frozen and sonicated after thawing (3xc3x9740 s; Braun, Labsonic 2000). Inclusion bodies were collected by centrifugation (15 min, 4000xc3x97g) and the supernatant was then divided into the membrane fraction and the soluble supernatant (B. subtilis ypfP) by ultrasonification (1 h, 147000xc3x97g).
The inclusion bodies, the membrane fraction and the soluble supernatant were separated in an SDS-PAGE. The SDS-PAGE was carried out as described by Laemmli, 1970, and the gels were stained with Coomassie brilliant blue R250 (Serva).
By SDS-PAGE analysis, over-expression of a protein having an apparent molecular mass of 44 kDa could be identified in the membrane fraction and the inclusion body fraction. The molecular weight corresponds to the calculated mass of 43.6 kDa or 44.7 kDa, respectively, for YpfP. This protein was not present in the soluble fraction and in untransforned E. coli.
3. Lipid Extraction and Analysis
Expression cultures of E. coli BL21 (DE3) pEypjP24 and pfsay 24 and cultures of the late logarithmic growth stage of Bacillus subtilis 019 were harvested by centrifugation (15 min, 5000xc3x97g), and the sedimented cells boiled for 10 min in water. Lipid extraction was performed as described by Linscheid et al., 1997. For separation of individual lipids by preparative chromatography, the lipids were subjected to thin-layer chromatography in the following solvent systems: (1) chloroformn/methanol/H2O (70:30:4. v/v/v) for separation of MGlcD, DGlcD, TGlcD and TeGlcD from phospholipids; (2) diethyl ether/petroleum ether (2:1, v/v) for separation of acetylated DGlcD from non-acetylated DGlcD; (3) diethyl ether/petroleum for separation of acetylated DGlcD from non-acetylated DGlcD; (3) diethyl ether/petroleum ether (4:1, v/v) for separation of acetylated TGlcD from non-acetylated TGlcD; (4) chloroform/acetone (9:1, v/v) for separation of acetylated TeGlcD from non-acetylated TeGlcD.
Separation of the two acetylated phosphoglucolipids PL1 and PL2 was performed in the solvent chloroform/methanol (80:20, v/v). Then both acetylated lipids were extractedfrom the silica gel and re-suspended in chloroform. The lipids were methylated by addition of diazomethane and subsequently separated in the solvent toluene/methanol (9:1, v/v).
Acetylation of the glycolipids was performed as described by Tulloch et al., 1973. Synthesis of the fatty acid methyl esters from DGlcD with sodium methylate was performed according to Roughan and Beevers, 1981. Release of the fatty acid from the sn 1 position of DGlcD was achieved by incubation with Rhizopus Lipase (Boehringer) according to suppliers"" protocol. Incubation with Cerebrosidase (provided by Prof. Dr. Sandhoff, University Bonn) was performed as described by Vaccaro et al., 1993.
The lipid extracts of E. coli BL21 (DE3) pEypfP24 and pEsay24 showed various new glycolipids, which could not be detected in the wild-type (FIG. 1). These glycolipids reacted with a sugar-specific spray reagent, but they were ninhydrin and phosphate negative (the native PL1and PL2 were phosphate positive). One of the glycolipids co-migrated with a diglucosyl diacylglycerol (DGlcD) standard of B. cereus. The different glycolipids were purified and acetylated. The glycolipid band with the polarity of DGlcD also co-migrated after acetylation with the acetylated DGlcD standard of B. cereus.
4. Analysis of the New Glycolipids by MS and NMR
Mass spectrometric (MS) and nuclear magnetic resonance spectroscopic (NMR) analysis of the new glycolipids was exclusively performed with the per-O-acetylated derivatives (1,2,3,4) and the phosphomethyl esters (PL1, PL2) of the glycolipids, respectively.
4.1. Mass Spectrometric Analysis (CI-MS and MALDI-MS)
4.4.1. EI-MS and CI-MS (DIP-mode)
Mass spectrometric analysis of the neutral glycolipids was carried out with a Hewlett Packard mass spectrometer (Model 5989) using the direct insert probe (DIP) mode. The sample was evaporated from 80xc2x0 C. to 325xc2x0 C. at a rate of 30xc2x0 C./min.
While all per-O-acetylated di-(2), tri-(3) and tetrahexosyl-(4) diacylgycerolipids could be analyzed by MS analysis in the DIP mode directly, the two phospholipids PL1 and PL2 could not be analyzed by this technique. Due to the high polarity and the complexity of the molecule, the phospholipids were, therefore, dephosphorylated with hydrogen fluoride (48% HF, 4xc2x0 C., 20 h) prior to MS analysis, the dephosphorylated fragment was per-O-acetylated and only after this treatment analyzed by mass spectrometry (DIP mode). Electron impact spectra (EI-MS) were recorded at 70 eV and chemical ionization spectra (CI MS) were obtained using ammonia (0.5 torr).
In the DIP MS analysis all per-O-acetylated di-(2), tri-(3) and tetrahexosyl-(4) diacylgycerolipids in the EI mode showed characteristic fragments for terminal mono-hexosyl (m/z=331) and di-hexosyl (m/z=619) residues and differed from each other in the evaporation rate maximum (9.5 min, 2, 10.6 min, 3 and 12.0 min, 4). The disaccharid 2 showed in the CI MS a pseudomolecular ion [M+NH4]+ at m/z=1202, wherein hexadecanoyl (16:0) and hexadecenoyl (16:1) could be identified as the fatty acid residues. In addition, a second ion [M+NH4]+ at m/z=1230 was observed, which could be identified as disaccharide with 16:0 and 18:1 (or 18:0 and 16:1) as fatty acids. The amounts of these differently acetylated diglycosyl lipids were present in a relative proportion of 2:3.
The trisaccharide 3 showed the expected pseudomolecular ion [M+NH4]+ at m/z=1490 and 1516 with the same heterogeneity in its acylation pattern, however in a slightly different proportion (2:1).
The tetrasaccharide 4 showed an evaporation profile with an increased maximum in the evaporation time (12.0 min) in comparison to 2 and 3. Pseudomolecular ions in the CI MS could not be produced with this compound. The presence of the tetrasaccharide 4 could, therefore, only be indirectly deduced under these conditions from the characteristic fragments of the non-reducing glycosyl residue (m/z=331 and 619, respectively).
In the DIP MS (CI mode) both PL1 and PL2 showed a characteristic biphasic evaporation profile, wherein the first maximum (xcx9c6 min) from the dephosphorylated and re-acetylated partial structures and the second maximum (xcx9c11 min) from the diagnostic pyrolysis fragments of the intact, not cleaved by HF hydrolysis phospholipids could be assigned. From this result it had to be noted that the applied reaction time (6-20 h) for removal of the phosphate residue with aqueous (48%) HF was not sufficient, since both phospholipids were cleaved only partially into their dephosphorylated partial fragments. In spite of this limitation, the fatty acid distribution pattern for both of the glycerol residues GroI and GroII could be unambiguously determined.
For PL1, the first peak revealed two pseudomolecular ions [M+NH4]+ at f/z=626 and 654, which could be assigned to the molecule ions and, thus, to the mono-acetylated glycerol residue GroII with 16:0 and 16:1 (M=608) and a 16:0 and 18:1 (or 18:0 and 16:1) (M=636), respectively. Thus, this peak contained the expected product of the HF treatment and re-acetylation. In contrast, an analogous reaction product deduced from GroI (with Glc as the substituent) was not observed.
The second peak (xcx9c11.0 min) revealed three fragments, which in contrast to the first peak represented only pyrolysis products of PL1, produced during MS analysis, but no intact derivatives. The first ion (m/z=447) was assigned to a fragment deduced from GroI, substituted with a peracetylated Glc and with an acetyl residue. The two other fragments (m/z=549 and 577) originate from GroII. They carried a diacylglycerol, wherein each a palmitic acid (16:0) and a palmitoleic acid (16:1) were esterified (m/z=549), and a second fragment (m/z=577) with each a palmitic acid (16:0) and an oleic acid (18:1) (alternatively 16:1 and 18:0). Already from this fragmentation pattern it could be concluded that PL1 is an xe2x80x9casymmetrically acylatedxe2x80x9d phospholipid, because GroII carries both fatty acids, and GroI in the native phospholipid with a free hydroxyl group and a glucose appears to be relatively hydrophilic.
The dephosphorylated and per-O-acetylated PL2 also showed a biphasic evaporation profile in the DIP MS analysis. The first peak had an evaporation maximum (xcx9c6 min) and a fragmentation pattern identical with PL1, indicating that PL2, is identical with PL1 with respect to the fatty acid substitution pattern in GroII. In contrast, the second peak (11.0 min) revealed four fragmentations. The first couple (m/z=550 and 577) was identified as a pyrolysis fragment deduced from GroII with 16:0 and 18:1 (16:1 and 18:0, respectively) and was therefore analogous to PL1. The second couple (m/z=1202.9 and 1231.1) could be identified as [M+NH4]+ ion of an intact derivative, produced from PL2 after de-phosphorylation and re-acetylation. This PL2 derivative is a diglucosyl diacylglycerol being further esterified with two fatty acids 16:0 and 16:1 (M=1185) and 16:0 and 18:1 (M=1203) respectively, as well as an acetyl residue (on the initial phosphate position). Although it was possible to assign the fatty acid substitution pattern to the two glycerol residue GroI and GroII by these analyses, the exact substitution pattern of the fatty acids could only be determined by NMR analysis (see below). Due to thermal instability of the molecules, the DIP MS analyses of the two intact phospholipid derivatives PL1 and PL2 could not be completely analyzed and were therefore further examined by means of MALDI-TOF-MS analysis.
4.1.2. MALDI-TOF-MS
MALDI-TOF-MS analyses were carried out on a Bruker Reflex II spectrometer in the reflector mode at an acceleration potential of 20 kV by means of the xe2x80x9cdelayed ion extractionxe2x80x9d in the positive mode. The per-O-acetylated and phosphomethylated samples of PL1 and PL2 were re-suspended in chloroform (10 xcexcml) and 2 xcexcl solution thereof were mixed with 2 xcexcl of a matrix solution (0.5 M 2,4,6-trihydroxyacetophenone; Aldrich, Steinheim). An aliquot of this mixture (0.5 xcexcl) was applied to a metal support, dried with warm air and immediately thereafter placed into the spectrometer. Calibration of the spectra was performed using an internal standard (Angiotensin). All mass data apply exclusively to the monoisotopic mass of the molecules.
In each case, two derivatives of both phospholipids were analyzed by MALDI-TOF-MS: the free phosphoric acid derivatives and the phosphomethyl esters (PL1 and PL2, see FIG. 2). The free phosphoric acid derivative of PL1 showed prior to esterification of the phosphate residue (diazomethane) in the positive reflector mode a pseudomolecular ion [Mxe2x88x92H+Na]+ at m/z=1116,48, corresponding to the calculated formula C54H95O20P (M=1094,56). The non-esterified phosphoric acid derivative of PL2 showed under the same conditions a pseudomolecular ion [Mxe2x88x92H+Na]+ at m/z=1796,43, corresponding to the formula C94H167O28P (M=1775,06) and thus carrying in addition a hexose and a fatty acid (16:0 and/or 18:1) in comparison to PL1.
Note: The mass of the MALDI-TOF-MS analyses given herein in all cases only relate to the smallest monoisotopic pseudomolecule ion or mass fragment. That means, in all mass data only the smallest fatty acid (16:0) was considered (see FIG. 2). All pseudomolecular ions presented herein showed per fatty acid always a heterogeneity resulting from the exchange of 16:0, 16:1, 18:0 and 18:1, this heterogeneity influencing all mass spectra (DIP and MALDI), but is not considered in the mass data provided herein.
PL1 showed after treatment with diazomethane a pseudomolecular ion [Mxe2x88x92H+Na]30  at m/z=1130,69 which corresponds to the formula C55H97O20P (M=1108,57) and thus contains only one additional methyl group (xe2x96xa1m/z=14) in comparison to the free acid. PL2 showed under the same conditions a pseudomolecular ion [Mxe2x88x92H+Na]30  at m/z=1811,42, which corresponds to the formula C95Hl69O28P (M=1789,07) and also contains only one additional methyl group (xe2x96xa1m/z=14) in comparison with the free acid. Thus, not only the preceding MS analyses (DIP MS) were confirmed, but it was also unambiguously demonstrated that both PL1 and PL2 represent phosphodiesters, which are likely to be substituted with two glycerol residues. In both cases only one methyl group was introduced by a diazomethane treatment and transformation into the corresponding methyl ester.
4.2. Proton Nuclear Magnetic Spectroscopic Analysis (1H-NMR)
The per-O-acetylated and purified samples (2-4, 30-200 xcexcg) were dissolved in 100 xcexcl CDCl3 (99.96% Cambridge Isotope Laboratories, Andover, Mass., USA), and transferred into special capillary NMR microtubes (2.5 mm OD, Wilmad, Buena, N.J., U.S.A.). The proton spectra (1H-NMR) were recorded on a 600 MHz Spectrometer (Bruker Avance DRX 600), equipped with a special microprobe head (PH TXI 600SB). The samples were measured at 300K with reference to internal trimethlysilane (TMS, xcex4H=0.000 ppm). One- and two-dimensional homonuclear spectra (1H, 1H COSY, ROESY, and relayed COSY) were performed using standard Bruker software (XWINNMR, Version 1.3).
The one-dimensional (1D) 1H-NMR spectra (600 MHz, microprobe head) of the di-(2,≈200 xcexcg), tri-(3≈200 xcexcg), and tetrahexosyl diacylglycerolipids (4≈50 xcexcg) of compounds 2, 3, and 4 are shown in FIGS. 3a-c and the results are set forth in Table 1 (Annex).
Assignment of the signals was carried out by 1D and two-dimensional (2D) proton nuclear magnetic resonance spectrometry (1H, 1H COSY, relayed 1H, 1H COSY, ROESY) in comparison with the structurally related xcex2-gentiobiose octaacetate (1) which served as a reference substance for unambiguous assignment and which is therefore also included in Table I. The xcex2-anomeric configuration of all hexoses in the substance results from the coupling constants J1,2 being between 7.6 and 8 Hz for all glucoses. The other coupling constants of the pyranosidic ring protons H-2, H-3, and H-4 and H-5 (J2,3, J3,4 J4,5) were all larger than 9.5 Hz, indicating glucopyranose. The chemical shift of the methylene protons (H-6a and H-6b) as well as their coupling constants (J6a,6b) in the terminal Glc residue (A) were found to be identical in all oligosaccharides (4.062xc2x10.005 ppm for H-6a and 4.205xc2x10.005 ppm for H-6b) as compared with those of H-6a,6b in residue A of the xcex2-gentiobiose octaacetate, thus allowing the assignment of the spin systems of all terminal Glc residues A in the oligosaccbarides 2, 3, and 4 on the one hand, but also in PL1 and PL2.
The xcex2(1xe2x86x926)glycosidic bond could be determined by means of the shift towards a higher field of the (overlapping) signals of H-6a and H-6b in residues B, C and D (3.855xc2x10.05 ppm) since these signals clearly differed from the non-substituted methylene signals of the terminal H-6a,6b (A). This fact clearly indicates that all Glc residues of the compounds 2, 3, and 4 are identically, i.e. xcex2(1xe2x86x926) glycosidically interlinked. This observation could be confirmed by means of a two-dimensional spectrum (1H, 1H COSY, FIG. 4, bottom) and a nuclear Overhauser spectrum (rotating-frame NOE spectroscopy, ROESY, FIG. 4, above) of trisaccharide 3. A ROESY spectrum showed (indicated) cross-peaks of the anomeric H-1 protons H-1A, H-1B, and H-1c which could be observed between H-1A/H-6a,6bB, H-1B/H-6a,6bC, and H-1C/H-3a,3bGro (FIG. 5), allowing an unambiguous assignment of the three spin systems to each of the glucosyl residues A, B and C.
In addition to the glycosyl residues in all 1H-NMR spectra, signals of the glycerol moiety (H-1a, 1bGro, H-2Gro, and H-3a,3bGro) could also be identified (FIG. 3, Table 1). The fatty acids showed the expected methylene (xe2x80x94CH2xe2x80x94, 1.185 ppm) and methyl protons (xe2x80x94CH3, 0.812 ppm). Finally, signals from olefinic protons (xe2x80x94CHxe2x95x90CHxe2x80x94, ≈5.27 ppm) could also be found in all glycolipids 2-4, PL1 and PL2, which from the MS spectra could be assigned to the unsaturated fatty acids 16:1 and/or 18:1.
In accordance with the non-phosphorylated compounds 1-4, NMR analyses were carried out only with the per-O-acetylated mono methyl ester derivatives Samples (0.1-0.2 mg) were dissolved in 500 xcexcl CDCl3 (99.96% Cambridge Isotope Laboratories, Andover, Mass., U.S.A.) and measured in 5 mm NMR tubes (Ultra Precision NMR sample tubes., Isocom, Landshut) at 300 K. Proton and Phosphorous-31 spectra (1H- and 31P NMR) were recorded with a 600 MHz spectrometer (Bruker Avance DRX 600) equipped with an inverse probe head (5 mm TXI 13C), and the carbon 13 (13C NMR) spectra were recorded with 360 MHz Bruker AM spectrometer (5 mm dual probe head) at 90,6 MHz. The chemical shift was measured with reference to internal tetramethylsilane (TMS, xcex4H=0.000 ppm) and chloroform (CHCl3, xcex4C=77.00 ppm), respectively. 31P NMR spectra were recorded at 242.9 MHz and calibrated with reference to an external standard (85% H3PO4=0.0 ppm) One (1D) and two-dimensional (2D) homonuclear spectra (1H, 1H COSY, NOESY, and relayed COSY) and heteronuclear spectra [1H, 13C and 1H, 31P HMQC (heteronuclear multiple quantum coherence) as well as 1H, 13C HMBC (hetero multiple bond correlation)] were recorded with a standard Bruker software (XWINNMR, Version 1.3).
In the 1H-NMR spectrum (FIG. 6 and Table 2), PL1 showed, in accordance with MGlcD: {3-[O-xcex2-D-glucopyranosyl]-1,2diacylglycerol }, characteristic signals corresponding to a xcex2-glycosidically bonded Glc residue. Surprisingly, the anomeric proton H-1 was cleaved into a couple of signals (H-1 and H-1xe2x80x2) with a similar intensity (H-1, 4.461 ppm, J1,2 7.9 Hz; H-1xe2x80x2, 4.457 ppm, J1,2 7.9 Hz. While the other protons (H-2, H-3, H-4, H-5, H-6a,b) showed identical chemical shift and coupling constants as compared with MGlcD, the protons of H-3aGro I and H-3bGro I (3.62 and 3.88 ppm) on the one hand and H-1aGro II and H-1bGro II (4.11 and 4.31 ppm) on the other hand were split: fine resolution of the two other methylene proton signals of the glycerol residues I and II (H-1a,bGro I and H-3a,bGro II; xcx9c4.08-4.14 ppm) could, however, not be observed. Further, we observed two singular methine protons for H-2Gro I (ddd, 5.082 ppm, 5.3 Hz) and H-2Gro II (ddd, 5.168 ppm, 5.3 Hz) as to be expected for a diglyceride.
Further characteristic signals were of the methyl group of the phosphate ester, which also exhibited a characteristic split doublet (POCH3, 3.828 and 3.810 ppm) with a characteristic JH,P coupling of 11.2 Hz. A phosphate monomethylester, PL1 could be identified as a phosphodiester via the integral of the signal of the phosphomethyl group (3H). This confirmed the results of MS analyses. Finally, 5 OAc signals could be detected (2.026, 2.018, 1.989, 1.954, 1.934 ppm; all s), which led to the conclusion that, besides the four OAc groups of the terminal Glc residue, presumably a fifth OAc group was bound to one of the two glycerol residues. The accurate fatty acid distribution pattern could be partially determined using an HMBC experiment. A fatty acid in position sn-1 of glycerol residue II could be assigned via the connectivities of the xcex1-methyl protons of the fatty acids (xe2x80x94Oxe2x80x94COCH2xe2x80x94). However, due to the small amount of substance, the substitution of the second fatty acid could not be determined in the HMBC experiment and could only be investigated based on MS analyses.
In 31P NMR (FIG. 7 and Table 3), the phosphate signal of PL1 is split (0.514 and 0.444 ppm) and appears as a singlet in the decoupled spectrum. The 1H, 31P-HMQC experiment (i) showed the expected connectivity with the phosphomethyl ester group (3.828 and 3.810 ppm), and (ii) revealed two methylene protons of glycerol residue I (H-1a,bGro I and H-3a,bGro II)(xcx9c4.08-4.14 ppm) to which the phosphomethyl group is bound. Hence, the phosphate substitution could be determined. Thus, based on this experiment, the connection of glycerol residues I and II via a phosphate diester could be proven, which could already be assumed due the presence of phosphomonomethyl ester in NMR analysis and due to characteristic fragments in MS analysis.
The splitting of the signals of H-1Glc, H-3Gro I and H-1a,bGro II is especially notable. This anomaly in 1H NMR can be explained by the presence of a pair of diastereomers of PL1. By introducing a methyl group, the prochiral phosphate (Rxe2x80x94Oxe2x80x94PO(OH)xe2x80x94ORxe2x80x2) in the middle of the molecule becomes chiral (Rxe2x80x94Oxe2x80x94PO(OMe)xe2x80x94ORxe2x80x2) which results in two diastereomeric phospholipids PL1 and PL1xe2x80x2. A corresponding chirality of the phosphorous atom was already observed and described for other phospholipids in 1H-, 13C and 31P-NMR spectra (Bruzik et al., 1983).
Phospholipid 2 (PL2) showed the same characteristic splitting of two anomeric protons in 1H-NMR. In this case, the anomeric protons are of the two glucose residues GlcA and GlcB (H-1A and H-1xe2x80x2A; 4.647 and 4.635 ppm, J1,2 7.7 Hz) and H-1B and H-1xe2x80x2B (4.533 and 4.524 ppm, J1,2 7.9 Hz). These splittings arc characteristic for diastereomeric pairs analogous to PL1. Thus, a structural relationship of both phospholipids and, as a consequence, a correlation in the biosynthesis of these phospholipids can be assumed. The 1H-NMR spectrum of PL2 showed high similarity with the one of DGlcD (2) which made the interpretation of the NMR spectra and thus the structural analysis easier. By comparison of both 1H-NMR spectra, the substitution of the fourth acyl residue (16:0 or 18:1) in position C-6A of terminal glucose could be determined. (The third acyl residue is connected with the C2 group of GroI).
Further characteristic signals were of the phosphomethyl ester, which also exhibited a split doublet revealing the chirality of the phosphate residue, and thus the diastereomeric nature of the molecule (3.835 and 3.818 ppm, JP,H 11.2 Hz). In 31P-NMR, the phosphate signal of PL2 was also split (0.414 and 0.275 ppm) analogous to PL1, which is characteristic for the diastereomeric pair PL2 and PL2xe2x80x2 (Bruzik et al., 1983).
In conclusion, our MS (DIP and MALDI) and 1H-, 13C- and 31 P-NMR analyes unequivodcally identified three neutral and two inogenic glyolipids, which could be identified as di-, tri-, and tetrasaccharide-diacylglycerols 2, 3, and 4 with the following structure in the glycosyl moiety (FIG. 8):
xcex2-D-Glcp-(1xe2x86x926)-xcex2-D-Glcp-(1xe2x86x926)-Gro (2)(DGlcD),
xcex2-D-Glcp-(1xe2x86x926)-xcex2-D-Glcp-(1xe2x86x926)-xcex2-D-Glcp-(1xe2x86x926)-Gro (3)(TGlcD), and
xcex2-D-Glcp-(1xe2x86x926)-xcex2-D-Glcp-(1xe2x86x926)-xcex2-D-Glcp-(1xe2x86x926)-xcex2-D-Glcp-(1xe2x86x926)-Gro (4)(TeGlcD),
as well as the phospholipid 1:3-[O-xcex2-D-glucopyranosyl]-sn-glycerol-13xe2x80x2-phospho-1xe2x80x2,2xe2x80x2-diacyl-sn-glycerol (PL1) and the phospholipid 2: {3-[O-(6xe2x80x2xe2x80x3-O-acyl)-xcex2-D-glucopyranosyl-(1xe2x80x2xe2x80x3xe2x86x926xe2x80x3)-O-xcex2-D-glucopyranosyl]-2-acyl-sn-glycerol-1,3 xe2x80x2-phospho-1xe2x80x22xe2x80x2-diacyl-sn-glycerol} (PL2) (FIG. 9).
5. Enzyme Assay
Standard enzyme assays for determination of the activity of the processive glycosyl transferase were performed in a final volume of 100 xcexcl, containing buffer 1, 20 xcexcl E. coli BL21 (DE3) pEypfP24 and pEsay24 membrane fraction (20-40 xcexcg of protein) and 250 000 dpm UDP-[14C]-glucose (specific activity 10.8 GBq/mmol; 3.85 xcexcM final concentration). The reaction was carried out for 1 h at 30xc2x0 C. and stopped by the addition of chloroform/methanol (2:1; 2 ml). The organic mixture was washed with 0.7 ml of NaCl solution (0.45% (w/v)) and the resultant subphase recovered. An aliquot of the subphase was subjected to scintillation counting, and after removal of the solvent by evaporation with argon, the remaining part was used for separation by thin-layer chromatography.
Detergents such as octyl-xcex2-D-glucopyranoside (Sigma), decyl-xcex2-D-glucopyranoside (Sigma), SDS, Chaps (Signa), Tween 20, dodecyl-xcex2-D-maltoside (Sigma) and sodium cholate (Sigma) were added in concentrations according to twice their critical micellar concentration (this applies only to the proccssive glycosyl transferase from B. subtilis ypfP).
Ceramide was added as fluorescent D-erythro-C6-NBD-ceramide (Matreya, INC.), cholesterol was added as [4-14C ]cholesterol and steryl glucoside was added as 14C-labelled steryl glycoside (FIGS. 10/11). Radioactive products on thin-layer chromatography plates were detected by radio scanning (BAS-1000 Bio Imaging Analyzer, Fuji) (see FIG. 10).
Assays with UDP-[14C]glucose showed the highest incorporation of radioactivity with membrane fractions compared with the soluble and inclusion body fractions of E. coli BL21 (DE3) pEypfP24 and pEsay24. Therefore, all subsequent in vitro standard assays were carried out with membrane fractions and UDP-[14C]glucose. The [14C]-labeled lipophilic products counted for 70-80% of the label offered in the assay. Separation by TLC was used to identify lipophilic radioactive products, using a monogalactosyl diacylglycerol (MGD), DGlcD and TGlcD as non-radioactive standards. The highest proportion of radioactivity was found in DGlcD, whereas labeling of MGlcD and TGlcD (TeGlcD only for B. subtilis) was low (FIG. 11). Assays with membrane fractions of the untransformed E. coli did not show incorporation of radioactivity into lipophilic products. To increase the DAG concentration in the enzyme assay, the effects of several detergents on the enzymatic activity were tested. With the exception of lyso-PC (Sigma) and alkyl-xcex2-D-glucopyranosides, the addition of all above-mentioned detergents resulted in complete inhibition of enzymatic activity. [14C]-MGlcD and [14C]-DGlcD from assays with transformed E. coli were isolated and subjected to various chemical and enzymatic treatments to identify their structure.
The DAG moiety in [14C]DGlcD was confirmed by treatment with Rhizopus lipase. This lipase specifically releases the fatty acid from the sn 1-position of the DAG-containing lipid. As expected, the resulting radioactive product co-migrated with a lyso-DGlcD that had been prepared from non-radioactive DGlcD by the same treatment. Incubation of [14C]DGlcD with sodium methylate resulted in the release of a free fatty acid methyl ester and [14C] glucosyl diacylglycerol, the same products were produced when using non-radioactive DGlcD of known structure. Characterization of the linkage between the first glucose and the DAG was carried out by incubation of the labeled MGlcD with cerebrosidase. This enzyme is specific for the xcex2-glucosidic linkage, but is relatively unspecific for the hydrophobic part of its substrate (Vandeijagt et al., 1994). The incubation of [14C]glucose-labeled MGlcD with cerebrosidase resulted in the release of labeled glucose and unlabeled DAG. The success of the hydrolysis was measured by scintillation counting of the aqueous and organic phase after phase partitioning. 90% of the label was found in the aqueous phase as compared with 15% in ski the control experiment, in which 85% of the radioactivity was recovered as [14C]MGlcD in the organic phase. These results support the assumption of a xcex2-glucosidic linkage between the first glucose and DAG in MGlcD.
6. Characterization of Glycosyltransferase Activity
The formation of three different radioactive products in the in vitro enzyme assay raises the question whether all of these products are produced by a single enzyme coded by ypfP genes. To answer this question, three of the possible sugar acceptors were incubated separately in labeled form with unlabeled UDP-glucose in the presence of the membrane fraction. The sugar acceptors were isolated from previous assays. Assays with radioactive [14C]DAG [14C]MGlcD and [14C]DGlcD were performed by sonification of the radioactive substrates in 0.5 mN lyso-phosphatidylcholine (for [14C]DAG) or in ethanol before adding the membrane fraction, buffer I and UDP-glucose (3.6 mM final concentration). The maximum ethanol concentration in assays was 5% (v/v). After conversion of the substrates, the lipophilic products were separated by TLC and detected by radio scanning (FIG. 12). [14C]DAG was converted to [14C]DGlcD and [14C]TGlcD, [14C]MGlcD to [14C]DGlcD and [14C]TGlcD an [14C]DGlcD to [14C]TGlcD. Conversion of radioactive labeled DGlcD to TGlcD did not occur any more with the S. aureus enzyme. Control experiments using the same substrates and untransformed E. coli membrane fractions did not result in any of the mentioned products. The results suggest processivity of the enzyme, whereby the starting reaction can be described as a UDP-glucose: 1,2-diacylglycerol-3-xcex2-D-glycosyltransferase reaction. In subsequent reaction steps, however, the glucose acceptors vary and represent the products of previous additions of xcex2-glucosyl residues.
To exclude a reaction mechanism based on the transfer of glycosyl residues from glycosides to various acceptors, as observed for glycosidases, the enzyme assay was carried out in the presence of a radioactively labeled MGlcD, but in the absence of UDP-glucose. No conversion of a radioactively labeled MGlcD could be observed. Incubation of YpfP with the glucosidase inhibitor deoxynoijrimycin (Alexis Deutschland GmbH) and substance 3 (provided by Dr. Y. Ichikawa) was perforrned as described by Ichikawa and Igaiashi, 1995. These compounds interfere with the transfer of glucose in reactions catalyzed by glucosylhydrolases, but not with the transfer of a sugarnucleotide-dcpendent glucosyltransferases. None of the inhibitors was able to inhibit the enzyme reaction. Both approaches suggest a transfer of glucose by a sugar nucleotide-dependent reaction. On the other hand, ricinoleic acid and oleic acid were able to inhibit the enzyme, inhibition varying with the concentration in the assay. Additions between 25 and 50 xcexcg in 100 xcexcl assay volume resulted in inhibition of DGlcD and TGlcD formation, the second and third step of the enzyme reaction. In these experiments, MGlcD accumulated in the assay, whereas MGlcD accumulated in normal assays in a low amount. Concentrations above 50 xcexcg in the assay led to a complete inhibition of the enzyme. Hydrolysis experiments with sodium methylate excluded the possibility that ricinoleic acid (=12-D-hydroxy-oleic acid) was glucosylated.
7. Substrate Specificity
Substrate specificity was characterized regarding the sugar donor and the sugar acceptor. Apart from UDP-[14C]glucose, UDP-[14C]galactose was also tested, but galactose was not incorporated into lipophilic products. Experiments concerning the sugar acceptor showed that besides DAG, MGlcD and DGlcD also alkyl-xcex2-D-glucopyranosides can serve as acceptor (this applies only for the B. subtilis enzyme). This resulted in products, which tentatively have been identified as alkyl diglucosides. However, the only evidence available so far are the Rf-values of the resulting products and their stability towards alkaline hydrolysis. Neither alkyl-xcex1-D-glucopyranoside nor alkyl-xcex2-D-glucopyranoside could serve as acceptor. The S. aureus enzyme could convert sterol, as well as steryl glucoside (FIG. 10). This data shows that the YpfP enzymes are less specific concerning the sugar acceptor, but have a higher specificity for the sugar donor UDP-glucose.
The recombinant DNA molecules according to the invention can be produced by standard techniques, as, for example, described in the Laboratory Manual by Sambrook et al., vide supra. Also, production of transgenic cells and organisms can be performed using conventional transformation methods, well-known in the art. This applies to microorganisms and yeast, as well as to plants. For introducing DNA into a plant host cell several techniques are available and the person skilled in the art can easily choose a suitable transformation procedure. These techniques comprise the transformation of plant cells with T-DNA using Aerobacterium tumefaciens or Agrobacterium rhizozenes as transformation means, fusion of protoplasts, direct gene transfer of isolated DNA into protoplasts, microinjection or electroporation of DNA, introducing DNA via biolistic methods and other procedures. In an alternative embodiment of the invention the nucleic acid molecules of the invention can be introduced into plant cells via viral infection. These techniques are all described in the literature, as are suitable binary vectors and expression vectors.
For constructing the recombinant nucleic acid molecules according to the invention, the skilled person can use any DNA sequence that codes for a protein having the biological activity of a processive diacylglycerol glycosyltransferase, including DNA sequences which hybridize with the DNA sequences disclosed herein. In the context of the invention, the term xe2x80x9chybridizationxe2x80x9d means a hybridization under convential conditions, preferably under stringent conditions, as e.g. described in Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Nucleic acid molecules that hybridize with the molecules of the invention may isolated e.g. from genomic or cDNA libraries. Identification and isolation of such nucleic molecules can be carried out using the nucleic acid molecules of the invention or fragments or reverse complements thereof, e.g. by hybridization under standard conditions (Sambrook et al., supra). For instance, nucleic acid molecules which display exactly or essentially the nucleic acid sequences of the invention or portions thereof can be used as hybridization probes. Also synthetic fragments, which are synthesized using common synthesis processes and which correspond basically to one of the DNA sequences or nucleic acid molecules of the invention can be used as hybridization probes. When genes are identified and isolated, which hybridize to the DNA sequences of the invention, it is necessary to determine their sequence and the sequence and characteristics of the proteins encoded by them. The man skilled in the art has a variety of biochemical, biotechnological and genetic engineering methods for the characterization of the nucleic acid molecules and the proteins at his disposal. The molecules that hybridize to the nucleic acid molecules of the invention comprise also fragments and (degenerated or allelic) derivatives of the nucleic acid molecules described herein. The terms xe2x80x9cderivativesxe2x80x9d means in this context that the sequences of such molecules differ from the molecules of the invention (as herein described) in one or more positions and show a high degree of homology to the sequences provides by the invention. Homology means an identity in sequence of at least 40%, in particular of at least 60%,preferably of more than 80% and more preferably of more than 90%. The deviations from the sequences of the invention may be generated by deletion, addition, substitution, insertion or recombination.
Nucleotide Sequence
B. subtilis ypfp (SEQ ID NO. 1)
ttgaatacca ataaaagagt attaattttg actgcaaatt acggaaatgg acatgtgcag gtagccaaaa cactttatga acaatgtgta cggctcggct ttcagcatgt aacagtttct aatttgtacc aagagtcaaa tccgattgtt tcagaggtaa ctcaatacct ttatttaaaa agcttctcaa tcgggaaaca gttttatcgt ttgtmatt acggagttga caaaatctat aataaacgta aattcaatat ttactttaaa atgggtaata aaagattggg cgaacttgtc gatgaacatc agcccgatat tattattaat acatttccga tgatcgtcgt gccggaatac agacgccgaa ctggaagagt cattcctacc ttcaacgtta tgactgatt ttgtcttcat aaaatttggg ttcacgaaaa cgtggataaa tattatgtgg cgacagatta cgtgaaggaa aaactgctgg agatcggcac tcatccaagc aatgaaaa tcacaggaat tccaatcagg ccgcaatttg aagaatccat gcctgttggc ccgatatata aaaagtacaa tctttcacca aacaaaaaag tgcttctgat catggcaggt gctcacggtg tattaaagaa cgtaaaagag ctgtgcgaaa accttgtcaa ggatgaccaa gtgcaagtag ttgtcgtgtg cgggaaaat acggctttaa aagaatcttt gagtgcgctt gaagcggaaa atggtgacaa attaaaagtt ctgggctatg tggagcgcat tgatgagcta tttcggatca cagattgcat gattaccaag cccggcggca ttactttgac agaagccaca gccattggag tgcctgtcat tctgtacaaa cccgtgcctg gccaggaaaa agaaaatgca aacuctttg aagaccgcgg agctgccatc gttgtgaacc gtcatgaaga gattctcgag tcagtcactt cccttcttgc agatgaagat accttgcatc gcatgaagaa aaacattaag gaccttcatt tagcaaactc ctctgaagtg attttagagg atatcctgaa ggaatcagaa atgatgaccg ccaaacaaaa agccaaagtg ctatcgtaa
S. aureus ypfP (SEQ ID NO. 3)
Atggttactca aaataaaaag atattgatta ttactggctc attcggtaac ggtcatatgcaagttacaca gagtatcgtt aatcaactta atgatatgaa tctagaccat ttaagcgtcattgagcacga tttatttatg gaagctcatc caattttgac ttctatttgt aaaaaatggt atatcaatag ctttaaatat tttagaaata tgtacaaagg gttttattac agccgcccag ataaactaga caaatgtttt tacaaatact atggacttaa taagttaatt aatttattgataaaagaaaa gccagattta atattattaa cgtttcctac accagttatg tcggtactaa ctgagcaatt taacattaat attccagttg ctacagtgat gacagactat cgcttacata aaaactggat tacgccgtat tcaacaagat attatgtggc aacaaaagaa acgaaacaag acttcataga cgtaggtatt gatccttcaa cagttaaagt gacaggtatt cctatgata acaaatttga aacgcctatt aatcaaaagc agtggttaat agacaacaac ttagatccag ataagcaaac tattttaatg tcagctggtg catttggtgt atctaaaggt tttgacacga tgattactga tatattagcg aaaagtgcaa atgcacaagt agttatgatt tgtggtaaga gcaaagagct aaagcgttct ttaacagcta agtaaatt aacgagaatg tatttgattc taggttatac caaacacatg aatgaatgga tggcatcaag tcaacttatg attacgaaac ctggtggtat cacaataact gaaggtttcg cccgttgtat tccaatgatt ttcctaaatc ctgcacctgg tcaagagctt gaaaatgcct tttactttga agaaaggt tttggtaaaa cgctgatac tccag
Amino Acid Sequence
B.subtilis YpfP (SEQ ID NO. 2) MNTNKRVLIL TANYGNGHVQ VAKTLYEQCV RLGFQHVTVS NLYQESNPIV SEVTQYLYLK SFSIGKQFYR LFYYGVDKIY NKRKFNIYFK MGNKRLGELV DEHQPDIIIN TFPMIVVPEY RRRTGRVIPT FNVMTDFCLH KIWVHENVDK YYVATDYVK EKLLEIGTHPS NVKITGIPIR PQFEESMPVG PIYKKYNLSP NKKVLLIMAG AHGVLKNVKE LCENLVKDDQ VQVVVVCGKN TALKESLSAL EAENGDKLKV LGYVERIDEL FRITDCMITK PGGITLTEAT AIGVPVILYK PVPGQEKENA NFFEDRGAAI VVNRHEEILE SVTSLLADED TLHRMKKNIK DLHLANSSEV ILEDILKESE MMTAKQKAKV LS
S. aureus YpfP (SEQ ID NO. 4) MVTQNKKILI ITGSFGNGHM QVTQSIVNQL NDMNLDHLSV IEHDLFMEAH PILTSICKKW YINSFKYFRN MYKGFYYSRP DKLDKCFYKY YGLNKLINLL IKEKPDLILL TFPTPVMSVL TEQFNINIPV ATVMTDYRLH KNWITPYSTR YYVATKETKQ DFIDVGIDPS TVKVTGIPID NKFETPINQK QWLIDNNLDP DKQTILMSAG AFGVSKGFDT MITDILAKSA NAQVVMICGK SKELKRSLTA KFKLTRMYLI LGYTKHMNEW MASSQLMITK PGGITITEGF ARCIPMJFLN PAPGQELENA FYFEEKGFGK IADTPEEAIK IVASLTNGNE QLTNMISTME QDKIKYATQT ICRDLLDLIG HSSQPQEIYG KVPLYARFFV K

Gesellschaft Fxc3xcr Erwerb und Verwertung von Schutzrechten-GVS mbH


Examples may include a non-volatile memory having a memory including a first table of device physical addresses and a second table of physical device addresses; a control register to receive a clone command to clone a second memory region of the memory as a copy of a first memory region of the memory, the first and second memory regions being referenced by different device physical addresses; and address translation logic, upon receipt of the clone command, create a first entry in the first table for each page of the first memory region and create a second entry in the first table for each page of the second memory region, each first table entry for the first memory region and each first table entry for the second memory region pointing to a same entry in the second table.

In some situations, data is copied multiple times within a memory. One approach to making copies of data within a memory is to use repeated operating system (OS) commands to write the same data over and over, to new locations in memory each time. This is inefficient because it involves many read/write operations, the entire copied area of memory has to be read and then written to a new memory area, and the reading and writing is executed in blocks. Further, this requires heavy use of the memory media, and this may impair the endurance (e.g., lifespan) of the memory since some memory technologies provide for limited numbers of write operations before possible memory failures.
Another approach is to use a file system that implements a copy-on-write (CoW) paradigm. Copy-on-write, sometimes referred to as implicit sharing or shadowing, is a resource-management technique used in computer programming to efficiently implement a "duplicate" or "copy" operation on modifiable resources. If a resource is duplicated but not modified, it is not necessary to create a new resource; the resource can be shared between the copy and the original. Modifications must still create a copy, hence the technique: the copy operation is deferred to the first write. By sharing resources in this way, it is possible to significantly reduce the resource consumption of unmodified copies, while adding a small overhead to resource-modifying operations. However, using a file system which implements a CoW paradigm has disadvantages. This approach limits a user of a computing system to a particular file system. It requires additional metadata to be stored on the memory media, it's efficiency depends strongly on a proper hardware configuration and is not transparent to the user.

Non-volatile memory cloning with hardware copy-on-write support


A beverage extraction device includes: a cylinder, having a bottomed cylindrical shape, having a lower-surface opening which is closed with a bottom portion; a mesh member, having a disk shape, having a plurality of through holes, moving close to and away from the bottom portion to extract beverage from beverage raw material and hot water, fed through an upper-surface opening of the cylinder, so that the extracted beverage is discharged through an extraction passage connected to the bottom portion; an ultrasonic vibrator provided on the bottom portion; and a control unit, when a cleaning instruction is given, driving the ultrasonic vibrator in a state where the hot water is stored in the cylinder to apply ultrasonic waves to the hot water, and causing the mesh member to move close to and away from the bottom portion.

The present disclosure relates to a beverage extraction device.
In the related art, as a beverage extraction device applied to a beverage server and a cup-type automatic beverage vending machine, for example, a beverage extraction device including a cylinder and a mesh member has been known.
The cylinder is an extraction container having a bottomed cylindrical shape the lower-surface opening of which is closed with a bottom portion. The mesh member has a disk shape in which a plurality of through holes are formed, and can be vertically moved close to and away from the bottom portion with its side surface being in contact with an inner surface of the cylinder.
In such a beverage extraction device, when beverage raw material and hot water have been fed therein through an upper-surface opening of the cylinder, the mesh member is vertically moved to extract a beverage, and the extracted beverage is discharged through an extraction passage connected to the bottom portion (see Japanese Patent No. 5044558, for example).
In the beverage extraction device described above, the mesh member is movably provided inside the cylinder, and the side surface of the mesh member is in contact with the inner surface of the cylinder. This configuration makes it difficult to remove the mesh member from the cylinder. Thus, when the beverage extraction device is cleaned, cleaning of the mesh member may be insufficient.


An illuminator includes an illuminator frame having a cut-out portion in which a light source unit is removably positioned. The light source unit includes a cold-lamp light source supported in a plate-like member of the unit's frame. The plate-like member has extended portions which are also plate-like for fitting into guide slits formed in a light source unit supporting member of the illuminator. The supporting member has a retainer which helps hold the plate-like extensions of the plate-like member of the light source unit. The illuminator includes a power supply linked with a connector positioned so that when the light support unit is inserted into the illuminator in its proper position, a connector provided on the light source unit will connect the light source to the power supply. Thus, the light source unit can be removed from or inserted into the illuminator in a single step process.

The present invention relates to an illuminator, particularly to a portable illuminator for use in conjunction with a photographic camera or a video camera.
In the medical field, a picture of a surgical operation is taken by a photographic camera or a video camera for research. A conventional illuminator is used to project light of appropriate luminance, form and color temperature to take the picture. It is desirable to make the illuminator so compact, light and portable that it is easy to position so as to illuminate the affected part of the patient undergoing the operation. The light source of the illuminator is a cold light source which does not make the patient feel uncomfortable due to hot rays of light or does not dry the illuminated area. For example, a metal vapor lamp hermetically containing a rare-earth metal halide is used as the cold light source. The light source of the illuminator is built in the body thereof.
When the lamp filament of the above-mentioned conventional illuminator ceases to function, or the luminance, form or color temperature of the light from the illuminator is inappropriate for the affected part of the patient, the illuminator needs to be disassembled to replace the light source built therein. Thus, replacement of the light source of the illuminator is troublesome.

Illuminator having an easily replaceable light source unit


A thin and light weight eyeglass case that utilizes a protective member between an inner lining and an outer skin to prevent possible damage to the eyeglasses. A flap to which a strap is joined is also provided for securing the frame and temple ear pieces to the case. The eyeglass case provides maximum optical protection while assuming minimal space with an accompanying sleeked-down shape.

The present invention relates to an eyeglass case and, in particular, to a case which provides maximum protection in a compact storage member.
It is well recognized that present-day eyeglass cases do not satisfy a need for a receptacle that is slim and sleek in design but which nevertheless carries a variety of different frames safely and securely. Eyeglass cases that afford maximum protection are a necessity in today's marketplace in view of the high cost associated with prescription lenses including the frames. This is particularly true with respect to prescription glasses that are polychromic in nature.
The present invention has effectively solved the limitations of the prior art by providing a contoured shape eyeglass case that is compact in outline but includes a hard shell on the front side of the case for supporting and protecting the lenses.
This invention also includes an adjustable strap which enables the temples of the frame to be folded and securely held in place outside the rear flexible panel of the case.


In a position indicating device, especially for an industrial sewing machine, with a slotted wheel (3) and a first track (6) of slots (4) having, e.g., 512 slots (4), to permit a null-point synchronization over a relatively small range of rotational angles, and the establishment of definite angular positions, a second track (8) with slots (7) of various widths is provided. The first track (6) of slots (4) is associated with two light barrier arrangements comprising emitters (11a, 11b), receivers (12a, 12b), and masks (13a, 13b) while the second track (8) is associated with one light barrier arrangement. Evaluating systems are connected to the receivers (12) so that, on the basis of the number of slots (4) of the first track (6), the size of the slots (7) passing in each case and corresponding approximately to a multiple of the slot width may be determined through evaluating systems (14, 28) connected to the output, and in this manner to define the rotary angle position.

The invention is directed to a position signaling device, especially for industrial sewing machines, having a rotatably mounted slotted wheel which can be coupled with a drive shaft of the machine and which has a plurality of equally wide and equidistantly disposed slots and lands forming a first circular track. The device also has fixed slotted masks disposed in back of the slotted wheel in the axial direction, offset from one another circumferentially by a defined angle, and disposed coaxially with the slotted wheel, comprising slots and lands corresponding to the division of the slotted wheel. Each fixed mask has a light emitter or a light receiver disposed axially parallel thereto behind each slotted mask, and having each a light receiver or light emitter, respectively, disposed axially parallel on the other side of the slotted wheel, an electronic evaluating system for determining the rotatory speed and the angle of rotation according to the number of counted light pulses caused by the slots, and for determining the direction of rotation according to the phase shift of the signals on the basis of the angular offset of the slotted masks.
A position indicating device of this kind is disclosed in U.S. Pat. No. 3,723,748, which is hereby incorporated by reference.
For many applications of position indicating devices, e.g., in machine tools, it is necessary that the absolute angular position of certain angular positions of the drive shaft be detected, since these positions correspond to corresponding positions of the tool. In sewing machines, for example, there are two common stop positions, which are designated as position 1 and position 2 and correspond to the needle-raised position and the needle lowered position, respectively. These positions are defined in reference to a null point of the position indicating device. After the machine is turned on, a null-point synchronization must be performed. Conventional position signaling devices must for this purpose cover at least a range of angles of 180.degree. and 360.degree., respectively, in order to permit the null point to be known with certainty. For the establishment of the definite angular positions referred to above, e.g., position 1 and position 2, in addition to the slotted wheel for measuring the rotatory speed, additional wheels are conventionally needed coaxially with the slotted wheel and at a distance from the latter, e.g., a second wheel for establishing stop position 1 and a third wheel to establish stop position 2.

Device for determining the angular position of the drive shaft in a sewing machine

Frankl & Kirchner GmbH & Co KG Fabrik fur Elektromotoren u. elektrische Apparate