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1. A fixing device for co-operating with a tool having a tool body in the form of a circular disk, with a central opening, which can be mounted on a machine tool or with which a machine tool is directly equipped, comprising at least one radially outwardly facing projection of a locking means, which can be actuated without a tool and with which the fixing device can be releasably connected to the tool, wherein in the locking position the at least one radially outwardly facing projection of the fixing device engages over a radially inwardly facing locking surface of the tool and the tool body is axially secured and held non-rotatably, characterized in that the at least one radially outwardly facing projection (5) of the fixing device (1, 32) and a support (7, 35) for the tool (23, 38) are arranged axially displaceably relative to each other, wherein the axial displacement travel is blockable by a centrifugal force device (14, 17; 41, 43). 2. A fixing device as set forth in claim 1 characterized in that the at least one radially outwardly facing projection (5) and the support (7, 35) for the tool (23, 38) are arranged axially rotatably relative to each other. 3. A fixing device as set forth in claim 1 characterized in that the locking means is in the form of a bayonet locking means by which the tool (23, 38) and the fixing device (1, 32) can be clamped fast in the locking position. 4. A fixing device as set forth in claim 1 characterized in that the centrifugal force device (14, 17; 41, 43) is operatively connected to at least one radially displaceable tongue (16) which can be introduced into the axial displacement travel. 5. A fixing device as set forth in claim 4 characterized in that the centrifugal force device (14, 17; 41, 43) has an element (14, 41) which is engaged, to provide a return force, by a spring means, in particular a spring (17) or an elastic O-ring (43) which prevents radial expansion or displacement, wherein upon rotation of the fixing device (1, 32) a centrifugal force can be produced which is in opposite relationship to the force means, and the element is operatively connected to the tongue (16). 6. A fixing device as set forth in claim 4 characterized in that the tongue (16) is designed to face radially outwardly and is extended in the axial direction and upon the production of a sufficiently high centrifugal force it closes an axial gap, in particular an axial gap (15) between axially relatively displaceable parts (5, 7) of the fixing device (1, 32). 7. A fixing device as set forth in claim 4 characterized in that the element is in the form of a semicircular pair of rings (14) with the at least one radially outwardly facing and axially extended tongue (16), which can be fixed by a spring (17) to form a ring, wherein the two half-rings move radially away from each other under the effect of centrifugal force and with the tongue close an axial gap (15) between the relatively displaceable parts (5, 7) of the fixing device (1). 8. A fixing device as set forth in claim 1 characterized in that radial displacement of the tongue (16) causes non-parallel surfaces (40, 42) of axially relatively displaceable parts (5, 35) to bear against each other, whereby an axially acting force can be produced to provide a clamping force for the tool. 9. A fixing device as set forth in claim 1 characterized in that there is provided an additional arresting device by which the tool (23, 38) can be secured non-rotatably with respect to the fixing device (2, 32) in the locking position. 10. A fixing device as set forth in claim 1 characterized by at least one arresting nose (18, 19) which extends axially and in the direction of the machine tool on a radially outwardly facing projection (5) of the locking means and which co-operates with an associated segment (22) on the tool, providing the locking surface (22) for affording a positively locking engagement between the projection (5) and the segment in the locking position. 11. A fixing device as set forth in claim 10 characterized by two peripherally spaced such arresting noses (18, 19) for providing a pocket on the projection (5), in which pocket the associated segment (22a) of the tool can be brought into a condition of positively locking engagement in the locking position. 12. A fixing device as set forth in claim 10 characterized in that one of the arresting noses (18) is adapted to transmit the drive force and the other arresting nose (19) is adapted to transmit the inertia forces produced upon braking of the tool, wherein to provide a rotary abutment for the tool the former is of greater axial extent than the other arresting nose. 13. A fixing device as set forth in claim 1 characterized in that the at least one radially outwardly facing projection (5) of the locking means, in the locking position, is adapted to provide positively locking engagement with a segment (22a) of the tool, which affords an associated locking surface (22), wherein formed on the segment of the tool is a radially inwardly open pocket into which the projection (5) can be introduced to provide the positively locking engagement. 14. A fixing device as set forth in claim 13 characterized in that at least one respective axial surface on the at least one projection (5) in the locking position transmits the drive force and the inertia forces produced upon braking of the tool. 15. A fixing device as set forth in claim 1 characterized by a female screwthread with which the fixing device can be screwed onto a screwthreaded portion of the machine tool. 16. A fixing device as set forth in claim 1 characterized in that the female screwthread is arranged on a screwthreaded sleeve which is disposed in a central opening of the fixing device. 17. A fixing device as set forth in claim 16 characterized in that the screwthreaded sleeve is provided at an end with an annular flange which engages over the edge of the central opening at the side of the fixing device, that is remote from the screwing direction. 18. A fixing device as set forth in claim 1 characterized by a central opening (13) for passing therethrough an axial screw with which the fixing device (1, 32) can be mounted to the machine tool. 19. A clamping system comprising a fixing device, in particular a fixing device as set forth in claim 1 for co-operating with a tool having a tool body in the form of a circular disk, with a central opening, and the associated tool, wherein the fixing device has at least one radially outwardly facing projection of a locking means which is actuable without a tool and with which the fixing device can be releasably connected to the tool, and wherein in the locking position the at least one radially outwardly facing projection of the fixing device engages over a radially inwardly facing locking surface of the tool and the tool body is axially secured and held non-rotatably, and the fixing device and the associated tool are so adapted that in the locking position there is a positively locking engagement between the fixing device and the associated tool, characterized in that upon a rotary movement of the fixing device (1, 32) the positively locking engagement is fixed by a centrifugal force device (14, 17; 41, 43). 20. A clamping system as set forth in claim 19 characterized in that the radially inwardly facing locking surfaces of the tool are formed by the tool body itself. 21. A clamping system as set forth in claim 19 characterized in that radially inwardly facing locking surfaces (22) of the tool are formed by a carrier device (20, 24, 31) which is provided in the opening of the tool body and is fixed to the tool body. 22. A clamping system as set forth in claim 19 characterized in that the carrier device (24) engages over the edge of the opening at least at one side of the tool body (28). 23. A clamping system as set forth in claim 19 characterized in that two arresting noses (25, 26) extend axially and in a direction away from the machine tool from a radially inwardly facing locking surface (22) of a segment (22a) of the tool, wherein the arresting noses are peripherally spaced to provide on the locking surface a pocket in which the associated projection (5) of the fixing device can be brought into positively locking engagement with the segment of the locking surface in the locking position. 24. A clamping system as set forth in claim 23 characterized in that one of the arresting noses (25) is adapted to transmit the drive force and the other arresting nose (26) is adapted to transmit the inertia forces produced upon deceleration of the tool, wherein to provide a rotary abutment for the tool the former is of a greater axial extent than the other arresting nose. 25. A clamping system as set forth in claim 19 characterized in that after the positively locking engagement has been made between the at least one axially outwardly facing projection (5) of the fixing device (1, 32) and the segment on the tool which provides the associated locking surface (22) the centrifugal force device is oriented axially for blocking an axial displacement travel between the projection (5) and the support (7, 35). 26. A clamping system as set forth in claim 25 characterized in that when the machine tool is switched on the centrifugal force device provides a means which urges the support (35) in the direction of the projection (5) of the fixing device to provide axial, force-implemented clamping of the tool. 27. A tool for use with a fixing device as set forth in claim 1 or in a clamping system as set forth in claim 19. 28. A method of tool-free clamping a tool (23, 38) having a tool body (21, 28) in the form of a circular disk and having a central opening, on a fixing device (1, 32) for a machine tool, in particular a fixing device as set forth in claim 1, including the steps: axially orienting and bringing together the tool (23, 38) and the fixing device in an open position of a locking means between the fixing device and the tool, by the application of a force directed axially towards the machine tool; producing an overlap position by relative rotation of radially outwardly facing projections (5) on the fixing device in relation to radially inwardly facing locking surfaces (22) of the tool so that projections (5) of the fixing device engage over radially inwardly facing locking surfaces (22) of the tool; forming a positively locking connection between the fixing device and the tool by removal of the axially directed force; and automatically securing the positively locking engagement by means of a centrifugal force device by operation of the machine tool. 29. A method as set forth in claim 28 characterized in that the tool is caused to bear against a support (7, 35) against a spring force and said support is rotated with the tool relative to the projections (5) into the overlap position. |
Measuring head for an ultrasonic flowmeter |
In a measuring head for an ultrasonic flowmeter, ultrasonic coupling element 40 and measuring head housing 10 are cast together and form, along with the casting material 50, a contact surface E, which allows an effective transfer of heat to the measuring head 2. In order to compensate manufacturing tolerances, the distance a between the measuring head housing 10 and the ultrasonic coupling element 40 is at least 1 mm in the area of the contact surface E. |
1. Measuring head for an ultrasonic flowmeter having a partially open measuring head housing (10), in which an ultrasonic coupling element (40) with piezoelement (20) and connection unit (30) is fixed, characterized in that the ultrasonic coupling element (40) and measuring head housing (10) are cast together and form with the cast material (50) a contact surface E, wherein the separation a between measuring head housing (10) and ultrasonic coupling element (40) in the area of the contact surface E amounts to at least 1 mm. 2. Measuring head as claimed in claim 1, characterized in that the casting material (50) has a thermal conductivity of at least 1 W/mK. 3. Measuring head as claimed in claim 1, characterized in that the contact surface E is flat. 4. Measuring head as claimed in claim 1, characterized in that the measuring head housing (10) is not completely filled with casting material (50). 5. Measuring head as claimed in claim 1, characterized in that anchoring elements (80) are provided on the measuring head housing (10). 6. Measuring head as claimed in claim 5, characterized in that a groove is provided as the anchoring element (80). 7. Measuring head as claimed in claim 5, characterized in that a lug is provided as the anchoring element (80). |
Pressure sensor |
A pressure sensor having a measuring cell (1) and sensor electronics (13) connected with the measuring cell (1), wherein the sensor electronics (13) and its electrical connections are protected from external influences, wherein on front side of the measuring cell (1) a pressure sensitive element is arranged, on which a pressure (p) acts during operation, and wherein the sensor electronics is arranged on a rear side of the measuring cell (1). The sensor has connected with the measuring cell (1) a cap (19, 27), which encloses the sensor electronics (13), and at least one bushing (25), through which the connection lines (17) connected to the sensor electronics (13) pass. |
1. Pressure sensor having a measuring cell (1), a pressure sensitive element arranged on a front side of the measuring cell (1), on which element a pressure (p) to be measured acts during operation, a sensor electronics (13) arranged at a rear side of the measuring cell (1), and a cap (19, 27) connected with the measuring cell (1), which encloses the sensor electronics (13), and which exhibits at least one bushing (25), through which pass connection lines (17) connected to the sensor electronics (13). 2. Pressure sensor as claimed in claim 1, wherein a bushing (26) for a tube (15) is provided in the cap (19, 27), and wherein a reference pressure (pR), to which the pressure to be measured is to be referenced, is brought to the measuring cell (1) through the tube (15) during operation. 3. Pressure sensor as claimed in claim 1, wherein the cap (19) is secured with its open end on an outer edge of the rear side of the measuring cell (1). 4. Pressure sensor as claimed in claim 1, wherein the cap (27) is inverted over the measuring cell (1) and connected at its open end with a closed surface portion of a lateral surface leading from the front side to the rear side of the measuring cell (1). 5. Pressure sensor as claimed in claim 1, wherein the measuring cell (1) comprises ceramic, the cap (19, 27) comprises metal, and the measuring cell (1) and the cap (19, 27) are hermetically sealed together by means of a seam. 6. Pressure sensor as claimed in claim 5, wherein the measuring cell (1) comprises aluminum oxide, the cap (19, 27) comprises a nickel-iron-cobalt alloy, and the seam comprises an active braze, or hard solder. 7. Pressure sensor as claimed in claim 1, wherein the measuring cell (1) comprises ceramic, the cap (27) comprises ceramic, and the measuring cell (1) and the cap (27) are hermetically sealed together by means of a seal of active braze, or hard solder. 8. Pressure sensor as claimed in claim 1, wherein the sensor electronics (13) is arranged directly on the rear side of the pressure measuring cell (1). 9. Pressure sensor as claimed in claim 1, wherein the sensor electronics (13) is mounted spaced from the rear side of the pressure measuring cell (1). |
Liquid foods and process for producing the same |
The invention is to provide a process for producing a liquid food, characterized by subjecting at least one of coffee, a milk composition, a food material (liquid) containing a component(s) having an antioxidation ability (radical-scavenging activity) and mixed liquids containing one(s) of them to an electrolysis treatment and/or a current passage treatment. Since the invention can prevent quality deterioration of coffee or milk composition over a long period of time without using additives and also improve safety, it is appropriate for producing canned coffee, milk-containing coffee or milk-containing tea drink to be sold in vending machines or can warmers. Further, since the invention can improve the antioxidation ability of foods, the resulting foods can suppress the in-vivo increase of active oxygen and free radicals, it greatly contributes to health. |
1. A process for producing a liquid food, characterized by subjecting a main material for the liquid food and/or a mixed liquid containing the main material for the desired liquid food to at least one of an electrolysis treatment and a current passage treatment. 2. The process as claimed in claim 1, characterized in that a main material prepared previously by subjecting to an electrolysis treatment and/or a current passage treatment is used as the main material. 3. The process as claimed in claim 1 or 2, characterized in that in the electrolysis treatment, the main material and/or the mixed liquid treated is collected from the cathode side (reduction side). 4. The process as claimed in any one of claims 1 to 3, which process is conducted in a deoxidated condition. 5. The process as claimed in any one of claims 1 to 4, characterized in that at least one selected from coffee, a milk composition and a food material containing at least one component having an antioxidation ability (radical-trapping ability) is used as the main material. 6. The process as claimed in claim 5, characterized in that at least one selected from an extracted liquid, an extract, a concentrated liquid, a dilute liquid and a component fraction liquid of coffee and a solution of instant coffee is used as the coffee. 7. The process as claimed in claim 5, characterized in that at least one selected from raw milk (cow's milk), processed milk, skim milk powder, whole milk powder, skim milk, concentrated milk, a liquid containing at least one thereof, a liquid obtained by dissolving at least one thereof, each concentrated liquid of those liquids and each dilute liquid of these liquids is used as the milk composition, and an emulsifying agent or a stabilizer is further used as required. 8. The process as claimed in claim 5, characterized in that the component having the antioxidation ability is at least one selected from vitamins, flavonoids and polyphenols. 9. A liquid food which is produced by the process as claimed in any of claims 1 to 8. 10. The liquid food as claimed in claim 9, characterized in that the liquid food is at least one selected from coffee drinks, milk-containing drinks, tea drinks, fruit juice drinks, soups and sports drinks. 11. The liquid food as claimed in claim 10, characterized in that the milk-containing drink is at least one selected from milk-containing coffees, milk-containing tea drinks, milk-containing fruit juices, milk shake, cocoa and acidic milk drinks. 12. The liquid food as claimed in any of claims 9 to 11, characterized in that the drink is at least one of drinks with flavor and quality retained, drinks with a raw milk feel improved of the milk composition, drinks with a rich taste enhanced and drinks with an antioxidation property improved. 13. The liquid food as claimed in claim 12, characterized in that the retention of flavor and quality is at least one of prevention of decrease in flavor and quality at a high temperature over a long period of time, suppression of decrease in chlorogenic acid, suppression of formation of organic acids and prevention of scales of milk components. |
<SOH> FIELD OF WHICH THE INVENTION BELONGS <EOH>The present invention relates to various liquid foods including drinks and processes for producing the same. More specifically, the invention is characterized in that materials of liquid foods are subjected to electrolysis treatment and/or current passage treatment. The resulting liquid foods retain taste and quality over a long period of time, and further liquid foods having an antioxidation ability (radical-scavenging activity) are also improved in antioxidation ability and available as foods good for health. |
Image processing apparatus, image processing method, and image processing program |
Using an image signal SDin of a received image, a scene change detection unit 21 detects scene change of the received images. A movement detection unit 22 detects a movement in each of the received images. A display position determination unit 231 determines a display position of each of the received images based on the movement in each of the received images detected in a period of continuous scene. A wide field angle image generation unit superimposes the received images in said period of said continuous scene, a display position of which has been determined, to generate an image signal SB of the wide field angle image. A received image slide unit 233 generates image signal SDg wherein the received images are slid to the display positions. An image superimposition unit 234 superimposes the received images whose display positions have been slid on the wide field angle image, to generate an image signal SDp of a wide field angle display image in which the display position of each of the received images is slid in response to the movement in each of the received images. |
1. An image processing apparatus according to the present invention comprises: scene change detection means for detecting scene change of received images; movement detection means for detecting a movement in each of said received images; and display image generation means for determining a period of each continuous scene based on the scene change detected by said scene change detection means and superimposing said received images of the period of said continuous scene based on the movement in each of said images detected by said movement detection means in the period of said continuous scene, and generating a display image which has a wider field angle than said received images and in which a position of each of said received images is slid in response to said movement in each of said images. 2. The image processing apparatus according to claim 1, wherein said scene change detection means includes: correlation value calculation means for calculating a correlation value between mutually adjacent frames in said received images; and determination means for determining whether scene change has occurred based on said correlation value. 3. The image processing apparatus according to claim 2, wherein said correlation value is a correlation coefficient. 4. The image processing apparatus according to claim 2, wherein said correlation value calculation means comprises: difference average value calculation means for calculating an average value of difference values between mutually adjacent frames as a difference average value; pixel average value calculation means for calculating an average value of pixel values of one frame as a pixel average value; and normalization means for normalizing said difference average value using said pixel average value to output said difference average value thus normalized as said correlation value. 5. The image processing apparatus according to claim 1, wherein said display image generation means comprises: display position determination means for determining a display position of each of said received images based on the movement in each of the images detected by said movement detection means in said period of said continuous scene; wide field angle image generation means for superimposing the received images in said period of said continuous scene, display positions of said images having been determined by said display position determination means, and for generating a wide field angle image having a wider field angle than said received images; received image slide means for sliding said received images to the display position determined by said display position determination means; and image superimposition means for superimposing the received images whose display position has been slid by said received image slide means on said wide field angle image, to generate said display image. 6. The image processing apparatus according to claim 5, wherein said wide field angle image generation means sequentially superimposes the received images in reverse order of time in said period of said continuous scene, display positions of said received images having been determined by said display position determination means, to generate said wide field angle image. 7. The image processing apparatus according to claim 5, wherein said wide field angle image generation means slides a display position of said wide field angle image based on the movement in each of the images detected by said movement detection means in said period of said continuous scene, to prevent a portion of said received images in said display image from going out of an image display region in which said display image is displayed; and wherein said received image slide means corrects the display position determined by said display position determination means with said wide field angle image sliding in said wide field angle image generation means. 8. The image processing apparatus according to claim 5, wherein said wide field angle image generation means slides said display position thereof based on the movement in each of the images detected by said movement detection means in said period of said continuous scene, in accordance with sizes of said wide field angle image and said image display region; and wherein said received image slide means corrects the display position determined by said display position determination means with said wide field angle image sliding in said wide field angle image generation means. 9. The image processing apparatus according to claim 1, further comprising image division means for dividing said display image, wherein, when an image display region for displaying said display image is comprised of multiple display regions, said image division means divides said display image corresponding to said multiple display regions. 10. An image processing apparatus for changing a display position of an image in response to a movement in a received image, said apparatus comprising: movement detection means for detecting said movement in the received image; display position determination means for determining a display position of said received image based on information of multiple frames on the movement in the image detected by said movement detection means; and received image slide means for sliding said received image to said display position determined by said display position determination means. 11. An image processing method comprising the steps of: detecting scene change of received images and a movement in each of said received images; and determining a period of each continuous scene based on the scene change thus detected and superimposing said received images in said period of said continuous scene based on the movement in each of the images detected in said period of said continuous scene, to generate a display image which has a wider field angle than said received images and in which a position of each of said received images is slid in response to said movement in each of said images. 12. The image processing method according to claim 11, further comprising the steps of: calculating a correlation value between mutually adjacent frames in said received images; and determining whether scene change has occurred based on said correlation value. 13. The image processing method according to claim 12, wherein said correlation value is a correlation coefficient. 14. The image processing method according to claim 12, further comprising the steps of: calculating an average value of difference values between mutually adjacent frames, as a difference average value; calculating an average value of pixel values of one frame as a pixel average value; and normalizing said difference average value using said pixel average value, to use said normalized difference average value as said correlation value. 15. The image processing method according to claim 11, further comprising the steps of: determining a display position of each of said received images based on the movement in each of the images detected in said period of said continuous scene; superimposing the received images in said period of said continuous scene, display positions of said images having been determined, and generating a wide field angle image having a larger wide field angle than said received images; sliding said received images to said display position; and superimposing said received images whose display position has been slid on said wide field angle image to thereby generate said display image. 16. The image processing method according to claim 15, wherein received images in said period of said continuous scene whose display positions have been determined are superimposed in reverse order of time to generate said wide field angle image. 17. The image processing method according to claim 15, further comprising the steps of: sliding a display position of said wide field angle image based on the movement in each of the images detected in said period of said continuous scene, to prevent a portion of said received images in said display image from going out of an image display region in which said display image is displayed; and correcting a display position with respect to said received image in said period of said continuous scene with said wide field angle image sliding. 18. The image processing method according to claim 11, further comprising the steps of: when an image display region for displaying said display image is comprised of multiple display regions, dividing said display image corresponding to said multiple display regions. 19. An image processing method for changing a display position of an image in response to a movement in a received image, said method comprising the steps of: detecting said movement in said received image; determining a display position of said received image based on information of multiple frames on said movement in the image thus detected; and sliding said received image to said determined display position. 20. An image processing program for causing a computer to execute: a procedure for detecting scene change of received images; a procedure for detecting a movement in each of said received images; and a procedure for determining a period of each continuous scene based on the scene change thus detected and superimposing said received images in said period of said continuous scene based on the movement in each of the images detected in said period of said continuous scene, to generate a display image which has a wider field angle than said received images and in which a position of each of said received images is slid in response to said movement in each of said images. |
<SOH> BACKGROUND ART <EOH>Conventionally, in an image display system for displaying an image of a TV broadcasting program, a movie, etc., the image has been displayed using one fixed image frame such as a display screen of a TV set, a movie screen, etc. Image contents of a broadcast program, a movie, etc. have also been made on the assumption that the image is to be displayed using such a fixed image frame. Furthermore, recently, in order to enhance realistic sensations in image display, a multi-screen display system and such displays as a curved-surface display, a wide field angle display, and a head-mount display have been made practicable. However, even when such a multi-screen display system and the like are utilized, a contents-production has been realized on the assumption that related images are displayed in such a manner as to match the fixed screen frame using a image display region formed in the system as one fixed image frame. In the image contents thus made so as to match the fixed screen frame, if they contain an image that has shot by panning of a camera, conventionally the image thus shot has been displayed in the fixed screen frame and also the camera panning has been expressed using a movement of a background in this image thus shot. That is, by watching the movement of the background of the image displayed in the fixed screen frame, a viewer recognizes a subject in the frame as it moves in a direction opposite to that of the movement of the background. The viewer also feels as if he or she turns around in response to the motion of the subject although he or she does not move actually. This is because an image that has been shot in a wide space is forcedly projected to a two-dimensional plane in the fixed screen frame. Since the viewer feels as if he or she has moved owing to the movement of the background although he or she does not move actually, this feeling is accompanied by unnaturalness due to a mismatch with a real space, thus making it impossible to display an image having realistic sensations. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is an illustration for showing a configuration of an image display system; FIG. 2 is a block diagram for showing a configuration of an image processing apparatus 20 - 1 ; FIG. 3 is a block diagram for showing a configuration of a scene change detection unit; FIG. 4 is a diagram for showing a relationship between a frame position and a normalized value; FIG. 5 is an illustration for showing how to skip some pixels; FIG. 6 is a block diagram for showing another configuration of the scene change detection unit; FIG. 7 is a diagram for showing a relationship between a frame position and a correlation coefficient; FIG. 8 is a block diagram for showing a configuration of a movement detection unit; FIG. 9 is a block diagram for showing a configuration of a wide field angle image generation unit; FIG. 10 are illustrations each explaining how to determine a continuous scene; FIGS. 11A and 11B are illustrations each explaining operations of position determination unit; FIG. 12 are illustrations each explaining how to generate a wide field angle image; FIGS. 13A and 13B are diagrams each explaining a display position correction method; FIGS. 14A-14G are diagrams each explaining another display position correction method; FIGS. 15A-15C are diagrams each explaining how to generate a display image; FIG. 16 is a block diagram for showing a configuration in a case where a computer is used; FIG. 17 is a flowchart for showing image processing operations; FIG. 18 is a flowchart for showing scene change detection operations; FIG. 19 is a flowchart for showing wide field angle image generation operations; FIG. 20 is a flowchart for showing display image generation operations; FIGS. 21A and 21B are illustrations each explaining image display operations in a second embodiment; FIG. 22 is a block diagram for showing a configuration of an image processing apparatus 20 - 2 a; FIG. 23 is a block diagram for showing a configuration of an image processing apparatus 20 - 2 b; FIGS. 24A and 24B are illustrations each explaining image display operations; FIG. 25 is a block diagram for showing a configuration of an image processing apparatus 20 - 2 c; FIG. 26 is a block diagram for showing a configuration of a display position control unit 25 c and a display control unit 27 c; FIGS. 27A-27C are illustrations each explaining image signal generation operations; FIGS. 28A and 28B are illustrations each explaining operations of the image processing apparatus; FIG. 29 is a block diagram for showing a configuration of an image processing apparatus 20 - 2 d; FIG. 30 is a block diagram for showing a configuration of a display position control unit 25 d; FIGS. 31A and 31B are diagrams each explaining an accumulated movement value; FIG. 32 is an illustration explaining slidable width calculation operations; FIGS. 33A-33D are illustrations each explaining initial position setting operations; FIGS. 34A-34C are illustrations each explaining slide correction operations; FIG. 35 is a flowchart for showing an overall configuration of an image processing program in the second embodiment; FIG. 36 is a flowchart for showing operations in the case of real time processing; FIG. 37 is a flowchart for showing operations in the case of off-line processing; FIG. 38 is a flowchart for showing display operations in the case of off-line processing; FIG. 39 is a flowchart for showing operations in the case of varying a display position in response to a movement in an image in real time processing; FIG. 40 is a flowchart for showing operations in the case of making it possible to change a display position in response to a movement in an image in off-line processing; FIG. 41 is a flowchart for showing display operations in off-line processing; FIG. 42 is a block diagram for showing a configuration of an image processing apparatus 20 - 3 and an information acquisition apparatus 30 ; FIG. 43 is a block diagram for showing another configuration of the information acquisition apparatus 30 ; FIGS. 44A and 44B are illustrations for showing a configuration of an image memory; FIGS. 45A and 45B are illustrations for showing a relationship between a shooting direction and an image presenting direction; FIGS. 46A and 46B are illustrations for showing display position calculation operations; FIGS. 47A and 47B are illustrations for showing operations of an image display system; FIG. 48 is a block diagram for showing a configuration in the case of real-time image display; FIGS. 49A and 49B are illustrations for showing a configuration in the case of off-line image display; FIGS. 50A and 50B are illustrations for showing another configuration in the case of off-line image display; and FIG. 51 is a flowchart for showing image processing operations in a third embodiment. detailed-description description="Detailed Description" end="lead"? |
Biological control decidous tress with new strains of chonodrostereum purpureum |
The present invention relates to the biological control of weedy deciduous trees. More particularly, the invention relates to novel purified cultures of Chondrostereum purpureum fungus, their use in compositions and methods for biologically controlling deciduous weed trees in forestry management. |
1. A purified culture of Chondrostereum purpureum fungus deposited under deposit number 090502-01 at the International Depository Authority of Canada (IDAC) on May 9, 2002. 2. A purified culture of Chondrostereum purpureum fungus deposited under deposit number 090502-02 at the International Depository Authority of Canada (IDAC) on May 9, 2002. 3. A composition for biologically controlling weedy deciduous trees, comprising an effective amount of at least one fungus as defined in claim 1 or 2, and an environmentally acceptable carrier. 4. The composition of claim 3, wherein the fungus is present in the form of mycelia. 5. The composition of claim 3, comprising from about 105 to 107 CFU of said fungus per milliliter of the composition. 6. A composition for inhibiting sprouting and regrowth of freshly cut weedy deciduous trees, the composition comprising: an effective amount of at least one fungus as defined in claims 1 or 2; and an environmentally acceptable carrier. 7. The composition of claim 6, wherein the carrier comprises a nutritive element suitable for sustaining growth of said fungus. 8. The composition of claim 6, wherein the carrier comprises a biodegradable inert agent for increasing adherence of the fungus on the tree. 9. A method for biologically controlling weedy deciduous trees, said method comprising the step of colonizing said trees with a composition comprising an effective amount of at least one fungus as defined in claim 1 or 2. 10. The method of claims 9, wherein the effective amount consists of about 105 to 107 CFU of the fungus per milliliter of the composition. 11. A method for biologically controlling weedy deciduous trees, said method comprising the steps of: a) cutting a stem of a weed tree to provide a stump; and b) applying on said stump, a composition comprising an effective amount of at least one fungus as defined in claim 1 or 2. 12. A method for inhibiting sprouting and regrowth of freshly cut stems of weedy deciduous trees which comprises the step of applying to a cut stem a composition comprising an effective amount of at least one fungus as defined in claim 1 or 2 13-14. (canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>It is often necessary to control the undesirable vegetation or weedy deciduous trees to provide an optimal growth of forestry plantations or to ensure access for maintenance of equipment installed or used in forested areas. Control methods currently used in the field are mainly mechanical cutting and chemical herbicide application. However, mechanical cutting of deciduous trees stimulates strong regrowth of sprouts, resulting in the need for more frequent cutting and a continual increase in stem density and maintenance cost. Furthermore, environmental pressures and government regulations clearly require a significant reduction in chemical pesticide use, and in some country, the use of chemicals is already banished. The biological control of the vegetation is an effective alternative to methods used presently in the field since it promotes a compatible return pattern of the vegetal cover to: ensure a higher degree of productivity of forest plantations permitting the growth of valuable species while limiting undesirable non value-added species on the same site; ensure that equipment installed in forest are functioning appropriately; reduce the return cycle of the mechanical cut which give the reverse effect of the initial objective because of the strong sprouting and regrowth; optimally manage the maintenance costs; and eliminate the use of toxic chemical pesticides harmful to the environment and the population. The naturally occurring fungus Chondrostereum purpureum (sometimes referred to hereinafter as C. purpureum ) is a known agent for biologically controlling deciduous weedy trees. The fungus plays a beneficial role in wood decay and recycling processes. When applied to stems freshly cut, it colonizes the stump and inhibits sprouting and regrowth. Because the fungus can be applied selectively, its use for control of the target trees or species of trees does not threaten the use of trees of the same species for commercial or other purposes. However, the commercial production and application of C. purpureum as a biological control has been hindered by the lack of effective strains of C. purpureum. Because of the problems associated with the use of chemical herbicides or mechanical cuttings, safer and more effective methods for biologically controlling weed trees are clearly needed. To date, no isolate of C. purpureum used as a biological control agent has been commercially successful for the biological control of weed trees. Therefore, there exists a need for new isolates of C. purpureum that has the effectiveness required for commercial use as a biological control agent of deciduous weedy trees. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides novel purified cultures of Chondrostereum purpureum fungus which have been deposited under deposit numbers 090502-01 and 090502-02 at the International Depository Authority of Canada (IDAC) on May 9, 2002. More particularly, the present invention is concerned with the use of these strains of C. purpureum for the biological control of weedy deciduous trees. According to an aspect, the present invention provides a composition for biologically controlling weedy deciduous trees. According to another aspect, the present invention provides a composition for inhibiting sprouting and regrowth of freshly cut weedy deciduous trees. The compositions of the invention comprise an effective amount of at least one fungus as defined above, and an environmentally acceptable carrier. According to a further aspect, the present invention provides a method for biologically controlling weedy deciduous trees which comprises the step of colonizing the trees with an effective amount of at least one fungus as defined above or with a composition as defined above. According to another aspect, the present invention provides a method for inhibiting sprouting and regrowth of freshly cut stems of weedy deciduous trees which comprises the step of applying to a stump an effective amount of at least one strain of fungus as defined above or with a composition as defined above in order to obtain a colonization of the stump by the fungus. Yet, according to another aspect, the present invention provides a method for biologically controlling weedy deciduous trees, said method comprising the steps of: a) cutting a stem of a weed tree to provide a stump; and b) applying on said stump, an effective amount of at least one fungus as defined above or a composition as defined above. An advantage of the present invention is that the use of the new strains of C. purpureum as a biological control agent does not involve any of the hazards commonly associated with conventional chemical herbicides. Furthermore, it has been found to be particularly advantageous to use C. purpureum IDAC 090502-01 and/or IDAC 090502-02 due to their effectiveness to inhibit sprouting and regrowth in cut stumps of deciduous tree species in vegetation management situations. |
Non-falsifiable information carrier material, information carrier produced therefrom and test device therefor |
In order to protect against falsification, an information carrier is doped with a photochromic substance. The location of the points (2), wherein the photochromic substance is embedded, is stored in readable form. For authentication purposes, the location of said points (2) is read out optically and compared with the stored information (FIG. 1), whereby a suitable initialization device is used. |
1. Counterfeitproof information carrier material, which comprises a substrate and at least one photochromic substance, which can be converted by light radiation from a first state to at least one second state, which can be spectroscopically distinguished from the first state, wherein the photochromic substance is embedded in the substrate, and the substrate is sufficiently transparent to the visible wavelengths that serve to convert the photochromic substance from the first to the second state. 2. Counterfeitproof information carrier material in accordance with claim 1, wherein at least one second state can be converted back to the first state by light radiation, and the substrate is sufficiently transparent to these wavelengths. 3. Counterfeitproof information carrier material in accordance with claim 1, wherein the photochromic material is a bistable material. 4. Counterfeitproof information carrier material in accordance with claim 1, wherein the photochromic substance is a chromoprotein. 5. Counterfeitproof information carrier material in accordance with claim 1, wherein the photochromic substance in the information carrier material is localized on particles. 6. Counterfeitproof information carrier material in accordance with claim 1, wherein the substrate is a paper. 7. Information carrier produced from a counterfeitproof information carrier material in accordance with claim 1, wherein the substance that has been converted to the second state is localized in at least one point (2) of the information carrier (1) 8. Information carrier in accordance with claim 7, wherein position information representing the local position of the point (2) in the information carrier is recorded in readable form on the information carrier. 9. Apparatus for authenticating an information carrier in accordance with claim 7, wherein a device (5), which emits a scanning light beam (6) with a wavelength suitable for distinguishing the second state, for detecting the local positions of the points (2) on the information carrier that have the second state, a device (5) that analyzes the position information corresponding to these points (2), and optionally, a device (5) for reading position information recorded on the information carrier (1) and a device (5) for comparing the detected and the recorded position information. 10. Apparatus for writing an information carrier in accordance with claim 7, wherein a device that emits a writing light beam for converting the photochromic substance from a first to a second state and, optionally, a device that emits an erasing light beam for converting a second state to a first state. 11. Method for writing an information carrier produced from an information carrier material in accordance with claim 1 with binary-coded information, wherein the two binary values “0” and “1” are recorded by the two states of the photochromic substance in a predetermined grid pattern. |
Method of mass spectrometry |
A method of analyzing the cleavage of a polymer wherein the polymer is differentially labeled with an isotope and the cleaved fragments of the polymer are analysed by mass spectrometry is disclosed. The method is particularly useful for analyzing the cleavage of proteins and more particularly for determining substrate specificity for enzymes such as proteases. |
1. A method of analyzing cleavage of a polymer, the method comprising: (a) providing a sample of said polymer, a portion of said polymer molecules having been labeled at a position on one side of a potential cleavage site with a first isotopic label and a portion of said polymer molecules having been labeled at a position on the opposite side of the potential cleavage site with a second isotopic label; (b) incubating said sample under conditions suitable for cleavage at said potential cleavage site, and (c) analyzing the mass(es) of any cleaved fragments by mass spectrometry and thereby determining whether and/or where cleavage has taken place. 2. A method according to claim 1 wherein the mass change caused by the presence of the first isotopic label compared with the mass in the presence of the normal element(s) at that position in different to the mass change caused by the presence of the second isotopic label compared with the mass in the presence of the normal element(s) at that position. 3. A method according to claim 1 wherein (a) 50% of the polymer molecules in said sample comprise said first isotopic label, and/or (b) 50% of the polymer molecules in said sample comprise said second isotopic label. 4. A method according to claim 3 wherein said sample comprises a portion of polymer molecules which comprise both said first isotopic label and said second isotopic label, a portion of polymer molecules which comprise said first isotopic label but not said second isotopic label, a portion of polymer molecules which comprise said second isotopic label but not said first isotopic label and a portion of said polymer labels which comprise neither said first nor second isotopic labels. 5. A method according to claim 1 wherein the first and second isotopic labels comprise different chemical isotopes. 6. A method according to claim 1 wherein the first and second isotopic labels comprise the same chemical isotope and wherein each of said first and second isotopic labels comprise a different number of atoms of said chemical isotope. 7. A method according to claim 1 wherein said isotopic labels comprise 2H, 13C, 18O, 15N and/or 81Br. 8. A method according to claim 1 wherein said polymer is a linear polymer. 9. A method according to claim 1 wherein said polymer comprises a peptide or protein. 10. A method according to claim 9 wherein said first and second isotopic labels are located in the terminal amino acids of the peptide or protein. 11. A method according to claim 1 wherein said polymer further comprises a moiety which allows for separation of the molecule attached to the moiety from other material. 12. A method according to claim 1 wherein the sample or library is incubated with an enzyme under conditions suitable for cleavage of a substrate. 13. A method according to claim 12 wherein said enzyme is a protease. 14. A method according to claim 1 wherein the mass(es) of the cleaved fragments are used to calculate the location of the cleavage site. 15. A method according to claim 1, the method further comprising the step of quantifying the amount of the polymer or cleaved fragment(s) present after cleavage. 16. A method according to claim 1 wherein said polymer further comprises a detectable non-isotopic label on one side of the potential cleavage site and an inhibitor for said label on the opposite side of the potential cleavage site, the method comprising the additional step of detecting said detectable non-isotopic label, wherein the detection of said label indicates that said polymer has been cleaved. 17. A method according to claim 16 wherein said detectable label is a fluorescent group and said inhibitor is a quencher for said fluorescent group. 18. A method according to claim 1 wherein said polymer is attached to a solid support. 19. A method according to claim 18 wherein the polymer is attached via a releasable linker molecule. 20. A method according to claim 18 wherein said solid support is a resin bead. 21. A method according to claim 1 further comprising determining the structure or sequence of one or both of the cleaved fragments. 22. A method according to claim 21 wherein the cleaved fragment(s) are further cleaved and the masses of these further fragments determined using mass spectrometry. 23. A kit for screening a library of polymers for cleavage, comprising two or more polymers a portion of said polymers having been labeled at a position on one side of a potential cleavage site with a first isotopic label and a portion of said polymers having been labeled at a position on the opposite side of the potential cleavage site with a second isotopic label. 24. A kit according to claim 23 wherein said polymers are supplied in solid form or in solutions. 25. A kit according to claim 23 wherein said polymers are supplied on a solid support and attached via a releasable linker molecule. 26. A kit according to claim 25 further comprising a linker cleaving reagent. 27. A kit for the preparation of a polymer as described in claim 1, comprising a set of chemical monomers which comprises monomers which have been isotopically labeled and monomers which have no isotopic label, in a form suitable for polymer synthesis. 28. A kit according to claim 27 further comprising means for adding an isotopic label to an unlabelled monomer. 29. A kit according to claim 27 wherein said set of monomers comprises: (a) a number of isotopically labeled amino acids and a number of amino acids which have no isotopic label; or (b) a number of isotopically labeled nucleotides and a number of nucleotides which have no isotopic label. 30. A method of screening a library of polymers for cleavage, the method comprising: (a) providing a library comprising two or more polymers, a portion of said polymers having been labeled at a position on one side of a potential cleavage site and a portion of said polymers having been labeled at a position on the opposite side of the potential cleavage site with a second isotopic label, (b) incubating said library of polymers under conditions suitable for cleavage, and (c) analyzing the mass(es) of any cleaved fragments by mass spectrometry and thereby determining whether and/or where cleavage has taken place. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Chemical labels are widely used in chemical analysis. Among the types of molecules used are radioactive atoms, fluorescent reagents, luminescent reagents, metal-containing compounds, electron absorbing substances and light absorbing compounds. Such chemical labels may be covalently attached to the target to enable the substance to be detected. However, chemical moeities present on the target surface may interfere with the detection of the label. Such labels have been used in methods to identify the substrates of enzymes and their cleavage sites. However, such labels may have further limitations since the labels themselves can have an influence on the cleavage kinetics. There is therefore a need for a method of labelling potential substrates for such cleavage analysis which involves minimal perturbation of the structure of the substrate, presenting the substrate molecule almost indistinguishable from its unlabelled state. In recent years, the approach of mass spectrometry has been used in the identification of proteins. Proteins are usually identified by the best match between the measured and calculated m/z (mass to charge ratio) values of the peptides. This approach however, encounters a number of problems such as low sample availablility, the presence of background or artifact ions and postsynthetic modification of proteins. Ultrahigh accuracy instruments may be required to determine absolute m/z values. Recently, a more accurate approach has been reported wherein stable isotopic labels are incorporated into proteins. Uniformly 15 N-labelled proteins have been generated for improved accuracy of mass measurements by mass spectrometry (Jensen et al Anal Chem 71: 2076-2084 (1999)). Specific amino acids have been labelled with stable isotopes for incorporation into proteins so that these proteins may be distinguished from other molecules in a mass spectrum (Chen et al Anal Chem 72: 1134-1143 (2000)). 18 O labels have been used in the identification of C terminal peptides of proteins (Kosaka et al Anal Chem 72:1179-1185 (2000)). |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a method of analysing cleavage of a polymer, the method comprising: (a) providing a sample of said polymer, a portion of said polymer molecules having been labelled at a position on one side of a potential cleavage site with a first isotopic label and a portion of said polymer molecules having been labelled at a position on the opposite side of the potential cleavage site with a second isotopic label, (b) incubating said sample under conditions suitable for cleavage at said potential cleavage site, and (c) analysing the mass(es) of any cleaved fragments by mass spectrometry and thereby determining whether and/or where cleavage has taken place. Preferably, the mass change caused by the presence of the first isotopic label compared with the mass in the presence of the normal element(s) at that position is different to the mass change caused by the presence of the second isotopic label compared with the mass in the presence of the normal element(s) at that position, The present invention further provides a method of screening a library of polymers for cleavage, the method comprising: (a) providing a library comprising two or more polymers labelled as described in claim 1 , (b) incubating said library of polymers under conditions suitable for cleavage, and (c) analysing the mass(es) of any cleaved fragments by mass spectrometry and thereby determining whether and/or where cleavage has taken place. The sample may be incubated with an enzyme under conditions suitable for the cleavage of a substrate. In a further aspect of the invention, the method further comprises the step of quantifying the amount of the polymer or cleaved fragment(s) present after cleavage. In another aspect of the invention, said polymer further comprises a detectable non-isotopic label on one side of the potential cleavage site and an inhibitor for said label on the opposite side of the potential cleavage site, and the method comprises the additional step of detecting said detectable non-isotopic label, wherein the detection of said label indicates that said polymer has been cleaved. The methods of the present invention may further comprise determining the structure or sequence of said polymer or of one or both of the cleaved fragments, optionally by further cleaving these fragments and determining the masses of these further fragments using mass spectrometry. The present invention further comprises a kit for screening a library of polymers for cleavage, comprising two or more differentially isotopically labelled polymers as described above. The invention also comprises a kit for the preparation of such a polymer, comprising a set of chemical monomers which comprises monomers which have been isotopically labelled and monomers which have no isotopic label, in a form suitable for polymer synthesis. |
Pole |
The invention relates to a walking or ski pole used in Nordic walking or skiing. To a prior art pole comprising a body part (1), a grip part (2), and a tip part (3), is connected a power sensor (6), a user interface (7), and means (9) for transmitting the data from the power sensor to the user interface for the purpose of measuring the physiological effect of the pole-assisted exercice and for controlling the exercise performance. |
1. A pole for pole-assisted exercise, the said pole being comprised of a body part (1), a grip part (2), and a tip part (3), the pole further comprising at least one power sensor (6), a user interface (7) including a display (8) and means (9, 9a, 9b) for processing the data coming from the power sensor (6) and transmitting it to the display (8), characterised in that the power sensor (6) is located in connection with the grip part (2) or the tip part (3) in such a way that it is located between the said part (2, 3) and the body part (1). 2. A pole as claimed in claim 1, characterised in that the user interface (7) comprises wired data transmission means (9, 9a) between the power sensor (6) and the data processing means (9b). 3. A pole as claimed in claim 2, characterised in that the user interface (7) comprises means (9b, 10, 11, 12) for entering data in the memory, and that the data entered includes limit values set for the exercise performance. 4. A pole as claimed in claim 3, characterised in that the user interface (7) comprises one or more warning lights (13, 14), a sound signalling device (15), or a combination thereof for informing the user of deviations in the exercise performance with respect to the set limit values. 5. A pole as claimed in claim 1, characterised in that as the driving power of the system is used an accumulator/battery, which is arranged to be charged by the energy obtained from the exercise performance. 6. A pole as claimed in claim 1, characterised in that after the exercise, the information on the exercise can be unloaded from the memory onto a display (8), a PC or combination thereof. 7. A pole as claimed in claim 1, characterised in that the information to be entered into the memory by the data entry means (10, 11, 12) includes the length of the stride, and the information to be entered in the memory from the power sensor (6) includes the number of thrusts. 8. A pole as claimed in claim 1, characterised in that at least one of the grip part (2) or tip part (3) are detachable with respect to the body part (1) and re-attachable by means of a quick-release coupling (19, 20, 21). |
Incandescent body |
An improved incandescent mantle formed of a woven fabric tube and having at least one open end with a fireproof attaching material provided for attaching at least one of the ends of the tube to a burner. At least one additional fireproof material portion is provided between the burner and mantle to reinforce and protect the mounted mantle from jarring forces. |
1. An incandescent mantle comprising a woven fabric tube having at least one open end, with a fireproof attaching material provided for attaching the ends of the tube to a burner or to a shaped part for a burner of an incandescent lamp, wherein at the at least one open end of the mantle and at least on the side facing the burner or the shaped part for the burner at least one additional fireproof material, preferably connected with the mantle, is provided between the burner and mantle, and the mantle has increased mechanical stability with respect to the burner as a result. 2. The incandescent mantle according to claim 1, wherein the additional fireproof material is in a crossing arrangement in at least one location when the incandescent mantle is in the attached state. 3. The incandescent mantle according to claim 2, wherein the additional fireproof material is in the form of a preferably bead-like solid. 4. The incandescent mantle according to claim 2 or 3, wherein the additional fireproof material is in the form of a flexible body, preferably as a thread. 5. The incandescent mantle according to claim 2, 3 or 4, wherein the at least one thread in the attached state is passed around the circumference of the fabric tube in at least one location, forming a loop. 6. The incandescent mantle according to one or more of the preceding claims, wherein the thread is routed so that it winds around the attaching material in at least one place, preferably winding around the inside. 7. The incandescent mantle according to one or more of the preceding claims, wherein the thread is routed so that it winds around the end of the fabric tube. 8. The incandescent mantle according to one or more of the preceding claims, wherein the attaching material is routed so that it does not penetrate the fabric tube. 9. The incandescent mantle according to one or more of the preceding claims, wherein the fireproof material is a woven fabric that preferably is attached to the incandescent mantle by means of the attaching material. 10. The incandescent mantle according to one or more of the preceding claims, wherein the end of the fabric tube is turned inside out. 11. The incandescent mantle according to one or more of the preceding claims, wherein the end of the fabric tube is dyed. 12. The incandescent mantle according to one or more of the preceding claims, wherein the end of the fabric tube is chemically reinforced at least in the area of the attaching material. 13. The incandescent mantle according to one or more of the preceding claims, wherein the string and/or the attaching material, if it is in the form of a thread, is introduced into the fabric tube during the knitting, transversely to the direction of knitting. 14. A burner of an incandescent lamp, having a rotationally symmetrical surface for attaching at least one end of an incandescent mantle, wherein the surface has a three-dimensional macroscopic structure. 15. The burner according to claim 14, wherein the structure is in the form of waves, whose crests are oriented parallel to one axis of the rotationally symmetrical surface. 16. The burner according to claim 14, wherein the structure is in the form of nubs. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Such incandescent mantles comprise a circular knitted base material of generally viscose mixed with metallic salts. The mantle is usually attached with a tie string, either directly onto a burner or else first on a prefabricated shaped part for the burner (the shaped part with the attached mantle is placed on the burner by the end user), and is then burned off and brought to luminescence in the gas flame. When this is done, the base material burns away completely, and all that remains is the oxide skeleton of the metallic salts. Even slight mechanical stresses are sufficient to destroy it. If this oxide skeleton is subjected to jolts or vibrations, as for example during transporting or careless handling of the lamp, the mantle is usually destroyed at its weakest point, namely at the burner. To increase the mechanical stability of the mantle, the following methods are known in the prior art. One consists in reinforcing the fabric at the point of contact with the burner. To strengthen the oxide skeleton at its point of contact with the burner, the fabric is turned inside out in the vicinity of the burner and chemically reinforced in this area, so that it is doubled. The fabric and the oxide skeleton that remain after combustion now comprise two layers, which are chemically reinforced. This two-layer oxide skeleton increases the time the mantle remains intact on the burner when subjected to jolts and impacts. Another method consists in the choice of the tie string with which the mantle is tied to the burner. To attach the mantle to the burner, a thread is sewn into the fabric. In the usual attachment method, during production a thread is threaded into the attachment opening or the two attachment openings of the mantle with a greater or lesser number of stitches; the end user then pulls the mantle onto a ceramic ring or an elongated one-piece or two-piece burner and draws the ends of the thread tight and ties them with a knot. For other types of burners, the mantle is drawn in and pre-knotted already during the production process, the diameter of the opening that thus remains being specified to a precision of tenths of a millimeter. The attaching material influences the durability of the mantle. The tie string is the most widely used method of attaching the mantle worldwide. However, the selection of the tie string is subject to a variety of criteria. It must be resistant to high temperatures, should retain textile properties even at high temperature and become neither hard nor brittle, and the knot must hold tightly. In addition to use of a tie string, use of a metal clip instead of a tie string has also been proposed. |
<SOH> SUMMARY OF THE INVENTION <EOH>This object is fulfilled with a mantle comprising a fabric tube having at least one open end, a fireproof attaching material being provided for attaching the ends of the tube to a burner or to a shaped part for a burner of an incandescent lamp, by providing at least one additional fireproof material, preferably connected to the mantle, between the burner and the mantle at the at least one open end of the mantle and at least on the side facing the burner or the shaped part for the burner, so that the mantle exhibits increased mechanical stability on the burner as a result. In addition, the object is fulfilled in a burner of an incandescent lamp having a rotationally symmetrical surface for attaching at least one end of a mantle, by the fact that the surface has a three-dimensional macroscopic structure. To test the mechanical stability of the mantle, the burned-off mantle together with the burner is attached to a jolting machine and shaken until the mantle is damaged. The shaking time is the measure of the mechanical resistance to jolts and impacts. The mantles produced in accordance with the present invention have increased stability and service life, by a factor of more than three. The mantle was also produced in accordance with the present invention with a round-shaped metal clip (U.S. Pat. No. 5,116,220) instead of the tie string. The produced mantle yielded more than 20 times the stability of the mantle equipped only with the metal clip. When subjected to jolts, impacts and vibrations, the mantle in accordance with the present invention wears out in an entirely different manner than conventional mantles. The conventional mantle is destroyed by the fact that the oxide skeleton becomes detached successively from the tie string, by jolting and other forces, until it is no longer held by the latter and falls off of the burner. If the mantle produced in accordance with the present invention is subjected to such jolts, impacts and vibrations, the oxide skeleton no longer becomes detached from the tie string, and is held on the burner by the tie string for a significantly longer period. The disclosed mantle herein is destroyed beneath the attachment, namely in the mantle itself, so that the stability of this mantle depends only on the character of the oxide skeleton of which the mantle is made and on the shape of the burner. Thus, when the mantle is subjected to jolts, impacts and vibrations, the additional fireproof material connected to the mantle thereby serves as a protective medium to separate the oxide skeleton from the attaching material that functions as a tying medium, to protect it from these forces. The present invention is based on the separation of these two functions. The protective medium can also assume the function of a spacer. A similar effect is also shown by the macroscopic structure of the burner surface. Thus, in addition to the tying medium, an additional medium is incorporated into the mantle in the area of the burner. This protective medium may be incorporated for example as a thread, as follows: a. it is incorporated into the fabric of the mantle with stitches (similar to the way in which the tie string is stitched in.) b. it is incorporated loop-like around and through the fabric of the mantle in the area of the opening. These may be multiple loops that are incorporated continuously, but may also be individual, separate loops placed at various locations around the opening, each of which may be knotted separately. c. it may be incorporated by being suspended only on the tying medium on the inner side of the mantle in the area of the burner, by means of a great variety of stitches (such as buttonhole stitching.) d. it is attached on the inner side of the mantle in the area of the burner (for example by cementing or clipping.) e. it is incorporated into the fabric of the mantle on the knitting machine, at least in the area of the burner, in the process of knitting the base material (usually viscose) of which the mantle is made. The thread may be, for example, a thread made up of a minimum proportion of high-temperature material and/or a fabric comprising a minimum proportion of high-temperature material, or a braided thread made up of a minimum proportion of high-temperature material or knitted thread made up of a minimum proportion of high-temperature material. One advantageous embodiment of the present invention provides that the additional fireproof material is in a crossing arrangement in at least one location when the incandescent mantle is in the attached state. This achieves the advantageous result that the attaching material is kept at a distance at least at some points, namely at the crossing points. Another advantageous and preferred embodiment of the present invention provides that the additional fireproof material is in the form of a preferably bead-like solid. The beads are strung onto the tie string on the inside of the mantle when the tie string is threaded in, as a means to limit contact with the burner so that at least in partial areas the mantle is no longer in contact with the burner. It is then held exclusively by the tie string. The mechanical explanation of the increased stability is that in static terms the hold is no longer fixed but a rotationally movable hold, so that no bending moments can be introduced into the fabric by the holding point. But shaped parts that are made up of metal, ceramic, porcelain or other high temperature materials are also conceivable as solid bodies. These shaped parts may have any form and design characteristics desired. These shaped parts are attached to the mantle in the area of the burner in such a way that they fulfill a protective function for the mantle as described above. In an alternative preferred embodiment of the present invention, the fireproof material of the protective body is structured in the form of a flexible body, preferably as a thread. In the production process such a thread may be threaded or knitted into the fabric of the mantle especially advantageously. When the tie string is pulled in to attach the mantle to the burner, the thread forms loops, so that these areas act as a protective medium. A combination of tying medium and protective medium is also conceivable, connected to each other in such a way that the tying medium may be tightened and knotted more or less independently of the protective medium. One example of such a combination is a fabric in which a warp thread is distinguished by being used as a tie string. If such a fabric is stitched into the opening of the mantle like the classic tie string, and if the warp thread which is distinguished as the tie string is drawn tight, the rest of the fabric remains in the opening and bunches up in such a way that it lies between the mantle and the burner. Another example is a tie string which is combined with a protective thread (for example by enmeshing it) and is stitched in the area of the opening of the mantle like the classic tie string in such a way that the tie string may be tightened and knotted so that the protective thread bunches together in the opening and lies between the mantle and the burner. At the same time, an advantageous refinement of the present invention provides for the thread to be passed loop-like through the fabric tube around its circumference. The at least one loop or the loops do not necessarily need to be present when the mantle is not attached, but may be formed only during attachment of the mantle to the burner. The loops also do not have to be closed, but may also take the form, for example, of meander lines. The formation of loops may be influenced in an advantageously reproducible way by routing the thread so that it encircles the attaching material, for example the tie string, in at least one place. A further increase of stability is achievable by routing the thread so that it encircles the end of the fabric tube. That results in the end of the fabric being reinforced at the same time by the loops that are formed. It is also possible to work in the tie string itself around or along the opening of the mantle, after it has been sewn in around the mantle by the usual method, so that it becomes a protective medium in the second pass. An advantageous refinement of the present invention provides for the tie string to be routed so that it does not penetrate the fabric tube. The result is that the tie string no longer holds the mantle directly, but indirectly via the protective thread. Thus the thread is first sewn, for example, into the mantle, and then the tie string is drawn through the loops of the thread that serves as a protective thread. That makes the holding of the mantle even more flexible. The mantle is held entirely free of the binding forces of the tie string. To further increase the strength, provision may be made for the end of the fabric tube to be turned inside out. It is preferably turned inward, and the thread may be passed through one or both layers. For production, to make it easier to recognize the areas that are structured in accordance with the present invention, these may be dyed in the initially continuously knitted tube in such a way that the end of the fabric tube is dyed after it has been separated. A further advantageous increase in service life is obtained if the end of the fabric tube is chemically reinforced, at least in the area of the protective medium. For the production process, a design is especially advantageous in which the thread and/or the tie string are incorporated into the fabric tube transversely to the direction of knitting. The mantles need to be modified only slightly in order to achieve the advantages of the present invention, if the tie string penetrates the fabric tube at a distance from its end, and is attached to the fabric tube in such a way that it forms loops when it is tightened. With this design it is possible to advantageously forego additional spacers or threads. The tie string itself functions as the spacer through the loops that are formed. For example, the tie string itself, after it has been stitched around the mantle in the usual way, is worked in again around or along the opening of the mantle, thus becoming a protective medium in the second pass. Or alternatively, the tie string is not stitched in around the mantle by the usual method. The tie string is stitched in around the opening of the mantle so that in the area of the mantle that faces the burner, the string lies in between. It thus becomes simultaneously tying medium and protective medium. An advantageous design of the burner provides for the surface to have a three-dimensional macroscopic structure, with the structure preferably being in the form of waves whose crests are oriented parallel to one axis of the rotationally symmetrical surface. This design is especially advantageous in terms of the production technique. Alternatively, the structure may be in the form of nubs. |
Device for image detecting objects, people or similar in the area surrounding a vehicle |
A device for detecting objects, persons or the like, including, for example, in the surroundings of a vehicle, has a stereo-sensitive image recording unit which includes at least two image sensors and an evaluation unit to evaluate the signals, supplied by the image sensors, using which, in a simple manner, a three-dimensional geometry of the surroundings of a vehicle is variably recordable at a desired operating range and at a desired viewing angle, and in which the optical axes of the image sensors are changeably alignable relative to each other and/or relative to vehicle. |
1-10. (Canceled). 11. A device for detecting at least one of an object and a person, comprising: a stereo-sensitive image recording unit including at least two image sensors; and an evaluation unit to evaluate signals supplied by the at least two image sensors, wherein optical axes of the at least two image sensors are aligned in a changed manner relative to each other, so that an overlapping region of recording regions of the at least two image sensors is one of enlarged and reduced, respectively, corresponding to a change of the optical axes. 12. The device of claim 11, wherein the device is operable to detect the at least one of the object and the person in a surrounding of a vehicle. 13. The device of claim 11, wherein the optical axes are aligned in a changed manner relative to the vehicle. 14. The device of claim 13, wherein the optical axes are aligned in a changed manner with respect to at least one of a vehicle longitudinal axis, a vehicle transverse axis, and a vehicle vertical axis. 15. The device of claim 11, further comprising: a drive device that is assigned to each of the at least two image sensors. 16. The device of claim 11, further comprising: a carrier to arrange the at least two image sensors on; and a drive device assigned to the carrier. 17. The device of claim 11, wherein the optical axes are alignable in a horizontal plane. 18. The device of claim 11, wherein the optical axes are alignable on cone envelope surfaces. 19. The device of claim 11, wherein the image recording unit is arranged at a roof region of a vehicle. 20. The device of claim 11, wherein the image sensors include CMOS cameras. 21. The device of claim 15, wherein the drive device includes one of a stepper motor and a motor that is continuously adjustable. 22. The device of claim 11, wherein the evaluation unit generates control signals for a drive device as a function of supplied image signals of the at least two image sensors. |
<SOH> BACKGROUND INFORMATION <EOH>According to the document of IEEE Intelligent Vehicles Symposium, October 2000, “Real-Time Stereo Vision for Urban Traffic Scene Understanding”, U. Franke, one may record the surroundings of a vehicle using an image recording unit, which includes a stereo camera for taking three-dimensional images. By evaluating the scene taken, the position of, for example, additional vehicles, persons or objects in the surroundings of the vehicle may be ascertained. In the light of this information, the longitudinal and/or lateral guidance of the vehicle may be influenced by a controller, so that the driver may be supported in his vehicle guidance, and driving comfort and traffic safety may be improved. For example, in the case of a threatening collision, an automatic evasive maneuver or braking maneuver may avoid the collision or at least reduce the collision energy. According to MIT Press document, 1995, “Three-Dimensional Computer Vision: A Geometric Viewpoint”, O. Faugeras, a stereo camera system may ascertain a three-dimensional geometry of the surroundings projected onto the image pair by triangulation, i.e., by determining the intersection of the light beams falling into the left and right camera, respectively. Thereby it may be established that the position of the camera heads, that is, the alignment of their optical axes, of a stereo camera considerably influences the detecting properties, and many objects make stereo-sensing more difficult on account of their position or texture. The choice of the optical focal length of a rigid stereo set-up may require a compromise between operating range and viewing angle. In vehicle applications, however, high requirements may be set for both variables. Thus, in the case of travel speeds of ca. 200 km/h, an operating range of more than 100 m may be required, for example, according to the so-called “half tachometer rule”. At the same time, a reliable recording of traffic signs may require large viewing angles, such as, for example, 70°. These requirements may not be met using other stereo cameras. As a remedy, IEEE Intelligent Vehicles Symposium, October 2000, “EMS Vision: Gaze Control in Autonomous Vehicles”, M. Pellkofer and E. D. Dickmanns, discusses that one may mount individual mono-cameras having different focal lengths on a rotatable platform to widen the recordable viewing angle. In this connection, the camera platform may be aligned with a distant object or with an object that is present laterally to the travel direction, such as a traffic sign. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to a device, in which a three-dimensional geometry of a vehicle environment may be recorded both at great operating reach and at a wide viewing angle. Because the image recording unit may include at least two image sensors, the optical axes of the image sensors being variably aligned relative to one another and/or relative to the vehicle, the geometry of the image recording unit may be adjusted to the currently required operating range and the viewing angle, respectively. A desired operating range may be provided via the choice of a long focal length, despite a momentarily lower viewing angle, since an exemplary device according to the present invention may permit a reenlargement of the viewing angle at any time. Furthermore, a variation of a stereo region of the image recording unit may be achieved by the optical axes of the image sensors being able to be aligned variably relative to one another. Depending on the alignment of the optical axes relative to one another, the overlapping region of the recording regions of the individual image sensors may be enlarged or reduced, respectively. In this respect, exemplary embodiments may also be provided, according to which at least one of the image sensors is aligned in the direction of travel, while another one of the image sensors is aligned transversely to the travel direction. Thus, an intermittently monocular image evaluation of each of the signals supplied by the image sensors may be achieved, if, in an appropriate situation, this seems to be required or sufficient. Because the optical axes are, in addition, alignable variably relative to the vehicle, the overlapping region of the recording regions of the image sensors may also be swivelled relative to the vehicle, and there in that position also be varied in its size. Thus, for example, during a parking procedure, a stereo measuring of a sighted parking space may be made, which may be, for example, at the side of the vehicle. Furthermore, it may be provided that, because of the ability variably to set the optical axes, bodies or the like in the surroundings, that are stereo-sensable only with difficulty because of their shape and/or their texture, may be recorded and evaluated. |
Birth stimulator |
The invention concerns an active and an interactive birth simulator for duplicating human pregnancy and the birth process, whereby the birth simulator has the following features: A womb torso 1, a child model 2, which is arranged in the womb torso 1, whereby natural shape, size and positional ratios are preferably maintained, and the child model 2 is connected to a controllable drive 5 via a coupling device 7, in order to move the child model 2 in the womb torso or to expel it from the womb torso 1 through the birth canal and a programmable control device is provided for controlling the drive 5. |
1. A birth simulator with the following features: a womb torso of flexible material, a child model of flexible material, which is arranged in the womb torso, whereby the natural shape and size ratios and haptics are preferably maintained, is hereby characterized in that the child model is connected to a controllable drive via a coupling device, in order to move the child model in the womb torso or to expel it from the womb torso through the birth canal, and a programmable control device is provided for controlling the drive, whereby the control is produced in such a way that natural movements of the child in the womb are duplicated. 2. The birth simulator according to claim 1, further characterized in that several coupling devices and several controllable drives are provided for the movement of the child model. 3. The birth simulator according to claim 1, further characterized in that a sensor arrangement is provided for detecting forces and movements, which are introduced by a person in training with his/her hands or with medical instruments on the womb torso or on the child model, wherein the sensors of the sensor arrangement are arranged on the coupling device and/or on the drive elements and a programmable control device having a computer is provided for controlling the drive elements, wherein the control device is designed such that the measured signals processed by the sensor arrangement are introduced into the computer, in which a simulation program is stored, which, in cooperation with the control device, causes that the child model is moved by action of external force by means of the drive elements so that the child model carries out adequate reaction movements, which correspond to the natural movement behavior of a child in the womb during the examination concerned or during the birth segment involved. 4. The birth simulator according to claim 1, further characterized in that an optic display device connected by signal technology to the control device is provided, which shows the movements of the child in real time. 5. The birth simulator according to claim 4, further characterized in that the optic display device indicates hints and additional information. 6. The birth simulator according to claim 1, further characterized in that an acoustic generator connected by signal technology to the control device is provided for the generation of typical noises and articulations, which can occur during real examinations or during natural birth. 7. The birth simulator according to claim 6, further characterized in that the acoustic generators are integrated in the womb torso. 8. A child model, preferably for a birth simulator according to one of the preceding claims, further characterized in that distance and/or force and/or pressure sensors, which are connected to the control device via signal technology, are arranged on the child model in the neck region and/or in the region of the skull, which consists of deformable segments. |
Aptamers and antiaptamers |
The present invention relates to: An aptamer comprising a circular oligonucleotide defining one to four target binding regions; An aptamer comprising an oligonucleotide defining two, three or four thrombin binding quadruplex regions separated by at least partially duplex regions, wherein the quadruplex regions comprise a GGTMGGXGGTTGG sequence wherein M represents A or T and X represents a sequence of two to five nucleotides and/or nucleotide analogues; An aptamer represented by formula (I): 5′D1, wQxD1D2yQzD2,3′—the variables are as defined in the specification; and Aptamers selected from specific sequences. |
1. An aptamer comprising a circular oligonucleotide defining one to four target binding regions. 2. The aptamer of claim 1 which defines two, three or four target binding regions wherein said binding regions are separated by at least partially duplex regions. 3. The aptamer of claim 1 which defines one or more protein, cellular, cell component or material binding region. 4. The aptamer of claim 3 wherein the protein binding region is a thrombin binding region 5. The aptamer of claim 3 wherein the cellular binding region is an L-selectin binding domain. 6. The aptamer of claim 1 consisting of nucleotides. 7. The aptamer of claim 1 consisting of RNA. 8. The aptamer of claim 1 consisting of DNA. 9. An aptamer comprising an oligonucleotide defining two, three or four thrombin binding quadruplex regions separated by at least partially duplex regions, wherein the quadruplex regions comprise a GGTWGGXGGTTGG (SEQ ID NO:3) sequence wherein M represents A or T and X represents a sequence of two to five nucleotides and/or nucleotide analogues. 10. The aptamer of claim 9 ligated at its termini to form a circular oligonucleotide. 11. The aptamer of claim 10 wherein the termini have been chemically ligated. 12. The aptamer of claim 10 wherein the termini have been enzymatically ligated. 13. The aptamer of claim 9 consisting of nucleotides. 14. The aptamer of claim 9 consisting of RNA. 15. The aptamer of claim 9 consisting of DNA. 16. The aptamer of claim 15 wherein X represents a sequence selected from TGT, GCA and TGA. 17. An aptamer represented by formula I: 5N D1NwQxD1D2yQzD2N 3N Formula I wherein Q represents a sequence GGTWGGXGGTTGG (SEQ ID NO:3) where M represents A or T and X represents a sequence of two to five nucleotides and/or nucleotide analogues; w, x, y and z are the same or different and represent a sequence of zero to ten nucleotides and/or nucleotide analogues; D1 and D2 are the same or different and each represent a sequence of zero to twenty-five nucleotides and/or nucleotide analogues with the proviso that D1 and D2 together comprise at least two nucleotides or nucleotide analogues; D1N and D2N are the same or different and each represent a sequence of zero to fifty nucleotides and/or nucleotide analogues, wherein at least two consecutive nucleotides or nucleotide analogues of D1N and/or D2N are complimentary to at least two consecutive nucleotides or nucleotide analogues of D1 and/or D2, so as to allow duplex formation between complimentary nucleotides or nucleotide analogues. 18. The aptamer of claim 17 wherein the 5N terminus is phosphorylated. 19. The aptamer of claim 17 wherein w, x, y and z are the same or different and each represent zero, one or two nucleotides and/or nucleotide analogues. 20. The aptamer of claim 17 wherein D1 and D2 in total represent two to twenty nucleotides and/or nucleotide analogues. 21. The aptamer of claim 20 wherein D1 and D2 in total represent four to twelve nucleotides and/or nucleotide analogues. 22. The aptamer of claim 17 wherein D1N and D2N in total represent two to twenty nucleotides and/or nucleotide analogues. 23. The aptamer of claim 22 wherein D1N and D2N in total represent four to twelve nucleotides and/or nucleotide analogues. 24. The aptamer of claim 17 ligated at its termini to form a circular sequence of nucleotides and/or nucleotide analogues. 25. The aptamer of claim 24 wherein the termini have been chemically ligated. 26. The aptamer of claim 24 wherein the termini have been enzymatically ligated. 27. The aptamer of claim 17 consisting of nucleotides. 28. The aptamer of claim 17 consisting of RNA. 29. The aptamer of claim 17 consisting of DNA. 30. The aptamer of claim 17 wherein X represents a sequence selected from TGT, GCA and TGA. 31. The aptamer of claim 17 wherein D1 and D1N are selected from the following respective pairs: CAG and CTG; CAGC and GCTG; CATGC and GCATG; CATCGC and GCGATG. 32. The aptamer of claim 17 wherein D2 and D2N are selected from the following respective pairs: CAC and GTG; GCAC and GTGC; GCTAC and GTAGC; GACTAC and GTAGTC. 33. Aptamers selected from those with the following sequences: DH6-1 5′ p CTG GGT TGG TGA GGT (SEQ ID NO: 4) TGG TCA GCA CGG TTG GTG AGG TTG GTG TG 3′ DH8-1 5′ p GCT GTG GTT GGT GAG (SEQ ID NO: 5) GTT GGC AGC GCA CTG GTT GGT GAG GTT GGG TGC 3′ DH10-1 5′ p GCA TGT GGT TGG TGA (SEQ ID NO: 6) GGT TGG CAT GCG CTA CTG GTT GGT GAG GTT GGG TAG C 3′ DH12-1 5′ p GCG ATG TGG TTG GTG (SEQ ID NO: 7) AGG TTG GCA TCG CGA CTA CTG GTT GGT GAG GTT GGG TAG TC 3′ TS1-1 5′ p GCT GTG GTT GGT GAG (SEQ ID NO: 8) GTT GGC AGC AGC CAA GGT AAC CAG TAC AAG GTG CTA AAC GTA ATG GCT TCG GCT 3′ TS2-1 5′ p GCT GTG GTT GGT GAG (SEQ ID NO: 17) GTT GGC AGC AGC TGG CGG TAC GGG CCG TGC ACC CAC TTA CCT GGG AAG TGA GCT 3′ TS3-1 5′ p GCT GTG GTT GGT GAG (SEQ ID NO: 18) GTT GGC AGC AGC CAT TCA CCA TGG CCC CTT CCT ACG TAT GTT CTG CGG GTG GCT 3′ DH8-Br 5′ GCT GTG GTT GGB GAG (SEQ ID NO: 12) GBB GGC AGC GCA CBG GBB - GGB GAG GBB GGG BGC 3′ where B=5-bromo-2′-deoxyuridine, 5-iodo-2′-deoxyuridine or other photoactive nucleotide analogue 34. An antidote aptamer comprising at least ten nucleotides and/or nucleotide analogues complimentary to a sequence of at least ten nucleotides and/or nucleotide analogues from an aptamer according to claim 17. 35. An antisense oligonucleotide to an aptamer of claim 17. 36. An antidote aptamer according to claim 34 ligated at its termini to form a circular oligonucleotide. 37. The antidote aptamer or the antisense oligonucleotide of claim 36 wherein the termini have been chemically ligated. 38. The antidote aptamer or the antisense oligonucleotide of claim 37 wherein the termini have been enzymatically ligated. 39. An aptamer according to claim 34 having the following sequence: ADH8-1 5′ pGCA CCC AAC CTC ACC AAC (SEQ ID NO: 19) CAG TGC GCT GCC AAC CTC ACC AAG CAC AGC 3′. 40. A method of treatment of thrombosis in a patient requiring such treatment which comprises administering to said patient an effective amount of an aptamer according to claim 1. 41. A method of preventing or reducing coagulation of blood or blood derived products which comprises contacting the blood or blood derived product with an effective amount of an aptamer according to claim 1. 42. Use of a compound according to claim 1 in preparation of a medicament for the treatment of thrombosis. 43. A method for capturing leukocytes from a physiological fluid comprising contacting the physiological fluid with an effective amount of an aptamer according to claim 1. 44. A composition comprising an aptamer according to claim 1 or its antisense antidote together with one or more pharmaceutically acceptable carriers or excipients. 45. A composition according to claim 41 in oral dosage form. 46. A method for counteracting the effect of an aptamer according to claim 1 comprising contacting the aptamer with a counteracting effective amount of an antidote aptamer thereof. 47. An antisense oligonucleotide according to claim 35 ligated at its termini to form a circular oligonucleotide. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The processes of blood clotting, tissue repair and clot dissolution are referred to generally as haemostasis, which requires the coordinated action of platelets, clotting factors, endothelial cells and smooth muscle cells within blood vessels (Wu, 1984). Thrombin is an essential component of the haemostatic processes and is responsible for activation of platelets to adhere to exposed subendothelial structures, conversion of soluble fibrinogen into insoluble fibrin and activation of factor XIIIa, which in turn causes crosslinking of fibrin molecules to form a hard clot. Apart from its haemostatic functions, thrombin is recognised as having a number of other activities, for example as a mitogen (Carney et al, 1985). It is also thought to exert a chemotactic effect on monocytes (Bar Shavit and Wilner, 1986). In light of these functions thrombin has been implicated as a pro-metastatic agent (Nierodzik et al, 1992) as well as a factor involved in neurodegenerative disease (Tapparelli et al, 1993). Therefore, apart from the obvious roles of thrombin inhibitors in prevention or reduction of thrombosis and blood or blood product coagulation, thrombin inhibitors have the potential to be used in the treatment of a wide range of disorders including inflammation, cancer and neural disease. Present anticoagulant and antithrombotic therapies rely upon the use of heparin and coumarin derivatives that indirectly and incompletely inhibit the coagulation system. The coumarins are the only class of currently available thrombin inhibitors to possess significant oral activity, which makes them acceptable to patients and useful in long term treatments. However, as a result of their mode of action which involves inhibition of hepatic synthesis of vitamin K-dependent coagulation proteins (Tapparelli et al, 1993), the coumarins are associated with a number of disadvantages. In particular the coumarins exhibit pharmacological interactions with food and other drugs, require several days for a full thrombin inhibitory effect to manifest and several days for resynthesis of coagulation factors to normalise on cessation of treatment. Coumarin therapy is also characterised by variability between patients, which necessitates close monitoring. The most important drugs presently used in the prevention and treatment of thromboembolic diseases are the heparins, which are administered in surgery and to patients suffering from stroke, acute myocardial infarction, respiratory failure and during immobilisation of patients when extracorporeal circulation or renal dialysis is required (Stubbs and Bode, 1995). Unlike the coumarins, which take days to manifest their effects, heparin compounds have an immediate effect on blood coagulation. However, they are also associated with a wide range of biological effects due to their binding of a variety of cells including platelets, endothelial cells, red blood corpuscles and lymphocytes (Stubbs and Bode, 1995) as well as an interaction with more than fifty enzymes (Jaques, 1980). Heparin administration can be associated with side effects including heparin-associated thrombocytopenia and osteoporosis. Although there have been advances with fractionated, more orally bioavailable heparins, conventional unfractionated heparins are characterised by low oral bioavailability which means they must be parenterally administered, such that they are restricted to short term usage. A major, further limitation relating to the heparins is their ineffectiveness in treatment of arterial thrombosis (Topoi et al, 1989). Although a number of anticoagulant agents have been trialled in treatment of thromboembolic diseases, none so far has supplanted heparin. There is therefore a pressing need to develop anticoagulant agents, that preferably are effective in the treatment of arterial thrombosis, are orally administrable and exhibit long lasting activity in vivo, with minimal side-effects. Some consideration has been given to the development of nucleic acid aptamers as antithrombotic agents. Aptamers are nucleic acids capable of three dimensional recognition that bind specific proteins or other molecules. Many known thrombin binding aptamers are composed of oligodeoxynucleotides containing the consensus sequence d(GGTTGGXGGTTGG), (<400>1), where G and T nucleotides are invariant and X is any two to five nucleotides. The 15-mer d(GGTTGGTGTGGTTGG), (<400>2), also known as GS-522 has been the subject of a number of structural and functional studies. These known thrombin-binding aptamers are characterised by a central core of two guanine quartets (Guschlbauer et al, 1990) formed from eight conserved guanine residues. These two G-quartets are linked by two TT loops at one end and a TGT loop at the other end of a quadruplex, as shown in FIG. 1A . Thrombin binding aptamers of this type have been identified as binding to thrombin exosite II (Padmanabhan et al, 1993). Although GS-522 and molecules like it have been shown to be effective in inhibiting clot- and matrix-bound thrombin (Li et al, 1994), the antithrombotic aptamers known to date have been characterised by low oral bioavailability and short in vivo half life. In an attempt to overcome problems with known thrombin binding aptamers such as those disclosed in WO 92/14842 a number of approaches have been attempted. For example, U.S. Pat. No. 5,399,676 proposes DNA-binding oligonucleotides stabilised against exonuclease degradation by virtue of combining tandem sequences of inverted polarity via a linker molecule. U.S. Pat. No. 5,668,265 discloses a bi-directional nucleic acid ligand that may be used as a diagnostic or therapeutic and which combines at least two oligonucleotides of opposite sequence polarity via a linker molecule. The publication of Macaya et al (1995) described quadruplex-duplex aptamers stabilized by either disulfide or triethylene glycol (TEG) linkages between the terminal nucleotides. Although molecules of these types demonstrate improved stability to exonuclease degradation, they are limited in their therapeutic and diagnostic utility. It is with the difficulties associated with prior art antithrombotic agents in mind that the compounds according to the present invention have been conceived. By virtue of their molecular recognition properties, these compounds may also be employed in diagnostic applications. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides an aptamer comprising a circular oligonucleotide defining one to four target binding regions. In a preferred form of the invention, the aptamer defines two, three or four target binding regions. Preferably, the aptamer defines one or more protein, cellular, cell component or material binding regions. A preferred cellular binding region is an L-selectin binding domain. A preferred protein binding region is a thrombin binding region. Accordingly, in one embodiment of the present invention there is provided an aptamer comprising a circular oligonucleotide defining one to four thrombin binding regions. Preferably, the aptamer defines two, three or four thrombin binding regions wherein said regions are separated by at least partially duplex regions. Preferably, the thrombin binding regions are quadruplex structures. According to another embodiment of the invention there is provided an aptamer defining two, three or four thrombin binding quadruplex regions separated by at least partially duplex regions, wherein the quadruplex regions comprise a GGTMGGXGGTTGG, sequence (<400>3) wherein M represents A or T and X represents a sequence of two to five nucleotides and/or nucleotide analogues. Preferably, the aptamer is ligated at its termini to form a circular oligonucleotide. Preferably, the termini have been enzymatically ligated, or alternatively chemically ligated. Preferably, X represents a sequence selected from TGT, GCA and TGA. According to another embodiment of the invention there is provided an aptamer represented by formula I: in-line-formulae description="In-line Formulae" end="lead"? 5′ D 1 ′wQxD 1 D 2 yQzD 2 ′ 3′ Formula I in-line-formulae description="In-line Formulae" end="tail"? wherein Q represents a sequence GGTMGGXGGTTGG where M represents A or T and X represents a sequence of two to five nucleotides and/or nucleotide analogues; w, x, y and z are the same or different and represent a sequence of zero to ten nucleotides and/or nucleotide analogues; D 1 and D 2 are the same or different and each represent a sequence of zero to twenty-five nucleotides and/or nucleotide analogues, with the proviso that D 1 and D 2 together comprise at least two nucleotides or nucleotide analogues; D 1 ′ and D 2 ′ are the same or different and each represent a sequence of zero to fifty nucleotides and/or nucleotide analogues, wherein at least two consecutive nucleotides or nucleotide analogues of D 1 ′ and/or D 2 ′ are complimentary to at least two consecutive nucleotides or nucleotide analogues of D 1 and/or D 2 , so as to allow duplex formation between complimentary nucleotides or nucleotide analogues. Preferably, the 5′ terminus is phosphorylated. Preferably, w, x, y and z are the same or different and each represent zero, one or two nucleotides and/or nucleotide analogues. Preferably, D 1 and D 2 in total represent two to twenty nucleotides and/or nucleotide analogues. Particularly preferably, D 1 and D 2 in total represent four to twelve nucleotides and/or nucleotide analogues. Preferably, D 1 ′ and D 2 ′ in total represent two to twenty nucleotides and/or nucleotide analogues. Particularly preferably, D 1 ′ and D 2 ′ in total represent four to twelve nucleotides and/or nucleotide analogues. Preferably, the aptamer is ligated at its termini to form a circular oligonucleotide. Preferably, the termini have been enzymatically ligated or chemically ligated. In a preferred embodiment of the invention the aptamer consists of nucleotides. Preferably, the aptamer consists of RNA and more preferably the aptamer consists of DNA. Preferably, X represents a sequence selected from TGT, GCA and TGA. Preferably, D 1 and D 1 ′ are selected from the following respective pairs: CAG and CTG; CAGC and GCTG; CATGC and GCATG; CATCGC and GCGATG. Preferably, D 2 and D 2 ′ are selected from the following respective pairs: CAC and GTG; GCAC and GTGC; GCTAC and GTAGC; GACTAC and GTAGTC. According to another embodiment of the invention there are provided aptamers selected from those comprising the following sequences: DH6-1 5′ p CTG GGT TGG TGA GGT TGG TCA GCA CGG TTG GTG AGG TTG GTG TG 3′ (<400>4) DH8-1 5′ p GCT GTG GTT GGT GAG GTT GGC AGC GCA CTG GTT GGT GAG GTT GGG TGC 3′ (<400>5) DH10-1 5′ p GCA TGT GGT TGG TGA GGT TGG CAT GCG CTA CTG GTT GGT GAG GTT GGG TAG C 3′ (<400>6) DH12-1 5′ p GCG ATG TGG TTG GTG AGG TTG GCA TCG CGA CTA CTG GTT GGT GAG GTT GGG TAG TC 3′ (<400>7) TS1-1 5′ p GCT GTG GTT GGT GAG GTT GGC AGC AGC CAA GGT AAC CAG TAC AAG GTG CTA AAC GTA ATG GCT TCG GCT 3′ (<400>8) TT4-1 5′ GAG TCC GTG GTA GGG CAG GTT GGG GTG ACT CGC TGT GGT TGG TGA GGT TGG CAG C 3′ (<400>9) TT4-2 5′ GAG TCC GTG GTA GGG CAG GTT GGG GTG ACT CGC TGT GGT TGG TGA GGT TGG ACA GC 3′ (<400>10) TT4-3 5′ GAG TCC GTG GTA GGG CAG GTT GGG GTG ACT CGC TGC GGT TGG TGA GGT TGG GCA GC 3′ (<400>11) DH8-Br1 5′ GCT GTG GTT GGB GAG GBB GGC AGC GCA CBG GBB GGB GAG GBB GGG BGC 3′ (<400>12) where B=5-bromo-2′-deoxyuridine, 5-iodo-2′-deoxyuridine or other photoactive nucleotide analogue. According to another embodiment of the invention there is provided an antidote aptamer comprising at least ten nucleotides and/or nucleotide analogues complimentary to a sequence of at least ten nucleotides and/or nucleotide analogues from an aptamer as referred to above. In one embodiment, the antidote aptamer comprises the following sequence: ADH8-1 5′ pGCA CCC AAC CTC ACC AAC CAG TGC GCT GCC AAC CTC ACC AAG CAC AGC 3′ (<400>19) In another embodiment there is provided an antisense oligonucleotide of an aptamer according to the invention. In another embodiment there is provided a method of treatment of thrombosis in a patient requiring such treatment which comprises administering to said patient an effective amount of an aptamer according to the invention. In another embodiment there is provided a method of preventing or reducing coagulation of blood or blood derived products which comprises contacting the blood or blood derived product with an effective amount of an aptamer according to the invention. In another embodiment there is provided use of a compound according to the invention in preparation of a medicament for the treatment of thrombosis. In a further embodiment, there is provided a method for capturing leukocytes from a physiological fluid comprising contacting the physiological fluid with an effective amount of an aptamer of the invention. The invention also provides a composition comprising an aptamer of the invention together with one or more pharmaceutically acceptable carriers or excipients. |
Optical semiconductor housing with transparent chip and method for making same |
Optical semiconductor package and its method of fabrication, which package comprises a semiconductor component (6), a rear face of which is attached to a front face of a mounting and electrical connection support (2) and a front face of which comprises an optical sensor (9), means (11) for electrically connecting the semiconductor component to the support, a transparent chip (12) placed in front of the semiconductor component, which lies at least in front of the optical sensor, and encapsulation means (21) comprising an encapsulation material which envelopes, in front of the support, the periphery of the semiconductor component and of the chip, without covering at least the central part of the front face of this chip. |
1-5. (canceled) 6. An optical semiconductor package comprising: a mounting and electrical connection support; a semiconductor component, a rear face of which is attached to a front face of the support and a front face of which comprises an optical sensor; means for electrically connecting the semiconductor component to the support; a transparent chip placed in front of the semiconductor component, which lies at least in front of the optical sensor; and encapsulation means comprising an encapsulation material which envelopes, in front of the support, the periphery of the semiconductor component and of the chip, without covering at least the central part of the front face of the transparent chip. 7. The optical semiconductor package according to claim 6, wherein an annular spacer is inserted between the front face of the semiconductor component and the rear face of the transparent chip, around the optical sensor. 8. The optical semiconductor package according to claim 6, further comprising a sealing ring lying in the peripheral interface region between the semiconductor component and the chip. 9. The optical semiconductor package according to claim 6, wherein the transparent chip is attached to the front face of the optical semiconductor component by means of a layer of transparent adhesive. 10. The optical semiconductor package according to claim 9, further comprising a sealing ring lying in the peripheral interface region between the semiconductor component and the chip. 11. The optical semiconductor package according to claim 9, wherein an annular spacer is inserted between the front face of the semiconductor component and the rear face of the transparent chip, around the optical sensor. 12. The optical semiconductor package according to claim 11, further comprising a sealing ring lying in the peripheral interface region between the semiconductor component and the chip. 13. A method of fabricating an optical semiconductor package, the method comprising the steps of: attaching and electrically connecting, to a front face of a mounting and electrical connection support, a rear face of a semiconductor component, a front face of which comprises an optical sensor; attaching to the front face of the semiconductor component a transparent chip lying in front of the optical sensor and sealing the interface region between the optical sensor and the rear face of the transparent chip; placing the support fitted with the semiconductor component and the chip in a cavity of an injection mold, in a position such that at least the central part of the front face of the chip located facing the optical sensor is covered; and injecting an encapsulation material into the cavity so as to encapsulate the periphery of the semiconductor component and of the chip in front of the support. 14. The method according to claim 13, comprising the step of: attaching the rear face of the transparent chip to the front face of the semiconductor component by means of an annular spacer lying around the optical sensor. 15. The method according to claim 13, comprising the steps of: before the molding operation, keeping the transparent chip at some distance from the semiconductor component by virtue of an annular spacer; and forming a sealing ring lying in the peripheral interface region between the semiconductor component and the chip. 16. The method according to claim 13, comprising the step of: attaching the rear face of the transparent chip to the front face of the semiconductor component by means of a layer of transparent adhesive. 17. The method according to claim 16, comprising the steps of: before the molding operation, keeping the transparent chip at some distance from the semiconductor component by virtue of an annular spacer; and forming a sealing ring lying in the peripheral interface region between the semiconductor component and the chip. 18. The method according to claim 16, comprising the step of: attaching the rear face of the transparent chip to the front face of the semiconductor component by means of an annular spacer lying around the optical sensor. 19. The method according to claim 18, comprising the steps of: before the molding operation, keeping the transparent chip at some distance from the semiconductor component by virtue of an annular spacer; and forming a sealing ring lying in the peripheral interface region between the semiconductor component and the chip. 20. An optical semiconductor device comprising: an electronic circuit; and an optical semiconductor packaging arrangement comprising: a mounting and electrical connection support; a semiconductor component, a rear face of which is attached to a front face of the support and a front face of which comprises an optical sensor electrically coupled to the electronic circuit; means for electrically connecting the semiconductor component to the support; a transparent chip placed in front of the semiconductor component, which lies at least in front of the optical sensor; and encapsulation means comprising an encapsulation material which envelopes, in front of the support, the periphery of the semiconductor component and of the chip, without covering at least the central part of the front face of the transparent chip. 21. The optical semiconductor device of claim 20, wherein an annular spacer is inserted between the front face of the semiconductor component and the rear face of the transparent chip, around the optical sensor. 22. The optical semiconductor device of claim 20, further comprising a sealing ring lying in the peripheral interface region between the semiconductor component and the chip. 23. The optical semiconductor device of claim 20, wherein the transparent chip is attached to the front face of the optical semiconductor component by means of a layer of transparent adhesive. 24. The optical semiconductor device of claim 23, further comprising a sealing ring lying in the peripheral interface region between the semiconductor component and the chip. 25. The optical semiconductor device of claim 23, wherein an annular spacer is inserted between the front face of the semiconductor component and the rear face of the transparent chip, around the optical sensor. 26. The optical semiconductor device of claim 25, further comprising a sealing ring lying in the peripheral interface region between the semiconductor component and the chip. |
Fuel injector with variable control chamber pressurization |
A fuel injector for an internal combustion engine, with a control chamber contained in an injector housing and acted on with highly pressurized fuel via a permanently acting inlet throttle in an inlet from a high-pressure accumulator. A nozzle needle/tappet device protrudes into this control chamber and is actuated in a movement directed (10) by the pressure decrease or pressure increase in the control chamber a pressure relief in the control chamber being produced by means of a multi-way valve activated by an actuator. The valve chamber of the multi-way valve and the control chamber in the injector housing are connected to each other by means of two conduits, one of which is closed when the multi-way valve is in a second switched position and is open when the multi-way valve is in a middle position. |
1-10. (canceled) 11. A fuel injector for injecting fuel into the combustion chamber of an internal combustion engine, the injector comprising an injector housing (2) containing a control chamber (5) acted on with highly pressurized fuel via a permanently acting inlet throttle (4) by means of an inlet (3) from a high-pressure accumulator; a nozzle needle/tappet device (8) protruding into the control chamber (5), the needle/tappet device (8) being actuatable in a movement direction (10) by means of a pressure decrease or pressure increase in the control chamber (5), can be actuated in a movement direction (10), and a multi-way valve (13) actuatable by an actuator to relieve pressure in the control chamber (5), at least one conduit (25, 28) connecting a valve chamber (20) of the multi-way valve (13) and the control chamber (5) in the injector housing (2) to each other to permit a flow both in the inlet direction and in the outlet direction in relation to the control chamber (5), and one conduit (28) of said at least one conduit (25, 28) being closed by the multi-way valve (13) being placed in a second valve seat (17) and being open when a valve body (14) of the multi-way valve (13) is in a position (19) between a first valve seat (15) and the second valve seat (17). 12. The fuel injector according to claim 11, further comprising throttle elements (29, 30) are incorporated into at least one of the conduits (25, 28). 13. The fuel injector according to claim 11, wherein the at least one conduit (25, 28) permits a flow in the inlet direction (26) toward the control chamber (5) and a flow in the outlet direction (27) from the control chamber (5) to the valve chamber (20). 14. The fuel injector according to claim 12, wherein the at least one conduit (25, 28) permits a flow in the inlet direction (26) toward the control chamber (5) and a flow in the outlet direction (27) from the control chamber (5) to the valve chamber (20), and wherein the throttle elements (29, 30) in the at least one conduit (25, 28) act as inlet throttles in the inlet direction (26) and act as an outlet throttle element (29) or as parallel-connected outlet throttles (29, 30) in the outlet direction (27), depending on the switched position of the multi-way valve (13). 15. The fuel injector according to claim 11, further comprising an additional inlet (11) from the high-pressure accumulator feeding into the valve chamber (20) perpendicular to the operational direction of the valve body (14). 16. The fuel injector according to claim 11, further comprising an additional inlet (11) from the high-pressure accumulator feeding into one of the conduits (25, 28), above the respective throttle element (29, 30). 17. The fuel injector according to claim 16, wherein the additional inlet (11) feeds into the one of the two conduits (25, 28) that feeds into the valve chamber (20) next to the valve body (14) of the multi-way valve (13). 18. The fuel injector according to claim 11, further comprising an inlet conduit (53) branching off from the inlet (3) of the high-pressure accumulator and connected to the one of the conduits (25, 28) that can be opened or closed by the valve body (14). 19. The fuel injector according to claim 18, wherein the inlet conduit (53) feeds into an annular chamber (50) of the conduit (28), which annular chamber (50) is disposed spaced a distance (52) apart from the throttle element (30) contained in this conduit (28). 20. The fuel injector according to claim 19, wherein the space (52) between the opening of the inlet conduit (53) and the additional throttle element (30) is embodied with a length that places the fuel flow diverted from the control chamber (5) in contact with the wall of the section (51) of the second conduit (28). |
<SOH> TECHNICAL FIELD <EOH>In modern air-compressing internal combustion engines, increasing use is being made of accumulator injection systems, which supply highly pressurized fuel to the individual fuel injectors associated with the cylinders of the engine. Through the use of a high-pressure accumulator (common rail), it is possible to damp pressure pulsations in the fuel so that the fuel pressure in the individual injection openings of the fuel injectors oriented toward the combustion chamber can be kept virtually constant. In order to control the nozzle needle movement, control chambers are integrated into the housing of the fuel injectors and when the pressure in these control chambers is relieved, a nozzle needle or a tappet for indirect nozzle needle actuation, can be actuated in order to open or close injection openings. In general, a volume of highly pressurized fuel from the high-pressure source can act on the control chamber via an inlet throttle. |
Pael receptor, cells and animal expressing pael receptor and method of screning remedy for parkinson's disease |
An object of the present invention is to provide a substrate of Parkin, a system for elucidating the cause of Parkinson's disease using the substrate and a method of screening for a remedy for Parkinson's disease. An animal cell having a DNA encoding a human Pael receptor or a DNA having a variant of the DNA incorporated therein; a non-human mammal having a DNA carrying the aforementioned DNA incorporated therein; a probe comprising the whole or a part of DNA encoding the human Pael receptor; and an antibody specifically recognizing Pael receptor. |
1. A human Pael receptor, for screening for a remedy for Parkinson's disease. 2. The human Pael receptor of claim 1, wherein Parkinson's disease is autosomal recessive hereditary Parkinson's disease. 3. An animal cell for screening for a remedy for Parkinson's disease, having a foreign DNA encoding a human Pael receptor or a DNA containing a variant of the DNA incorporated therein. 4. The animal cell of claim 3, wherein the foreign DNA encoding a human Pael receptor is represented by SEQ ID NO: 1. 5. The animal cell of claim 3 or 4, further having the Parkin gene or a variant of the Parkin gene. 6. The animal cell of claim 5, further having a gene encoding at least one protein selected from the group consisting of endothelin receptor type A, endothelin receptor type B, UBC6 and UBC7. 7. The animal cell of any one of claims 3 to 6, wherein the cell is an HEK293T cell or SH-SY5Y cell. 8. The animal cell of any one of claims 3 to 7, wherein Parkinson's disease is autosomal recessive hereditary Parkinson's disease. 9. A non-human mammal, having a foreign DNA encoding a human Pael receptor, or a DNA containing a variant of the DNA incorporated therein. 10. The non-human mammal of claim 9, wherein the foreign DNA encoding a human Pael receptor is represented by SEQ ID NO: 1. 11. The non-human mammal of claim 9 or 10, further having the Parkin gene or a variant of the Parkin gene. 12. The non-human mammal of claim II, further having a gene encoding at least one protein selected from the group consisting of endothelin receptor type A, endothelin receptor type B, UBC6 and UBC7. 13. The animal of any one of claims 9 to 12 above, wherein the non-human mammal is a rodent. 14. The animal of claim 13, wherein the rodent is a mouse. 15. The non-human mammal of any one of claims 9 to 14, which is a mammal obtained by ontogenesis of a totipotent cell having DNA encoding a human Pael receptor or its variant introduced therein, or the progeny of such mammal, and which carries the transgene in the somatic cell chromosome. 16. A cell isolated from the animal of any one of claims 9 to 15. 17. A method of screening for a remedy for Parkinson's disease, which comprises causing the human Pael receptor of claim 1 or 2 to come into contact with a candidate remedy for Parkinson's disease, and using the Pael receptor quantity in animal cells as an indicator. 18. A method of screening for a remedy for Parkinson's disease, which comprises causing the animal cells of any one of claims 3 to 8 and claim 16 to come into contact with a candidate remedy for Parkinson's disease, and using the Pael receptor quantity in the animal cells as an indicator. 19. A method of screening for a remedy for Parkinson's disease, which comprises administering a candidate remedy for Parkinson's disease to the animal of any one of claims 9 to 15, and using the Pael receptor quantity in the cell of the animal as an indicator. 20. The method of any one of claims 17 to 19, wherein Parkinson's disease is autosomal recessive hereditary Parkinson's disease. 21. A probe, consisting of the whole or a part of a DNA encoding a human Pael receptor for detecting a risk factor for the onset of Parkinson's disease. 22. The probe of claim 21, wherein Parkinson's disease is autosomal recessive hereditary Parkinson's disease. 23. A method of detecting a risk factor for the onset of Parkinson's disease, using the probe of claim 21 or 22. 24. The method of claim 23, wherein Parkinson's disease is autosomal recessive hereditary Parkinson's disease. 25. An antibody recognizing the Pael receptor, which reacts with the Pael receptor but does not react with ETBR-LP-2 which is a Pael receptor homolog. 26. The antibody of claim 25, which further does not react with endothelin receptor type A or endothelin receptor type B, which is a Pael receptor homolog. 27. The antibody of claim 25 or 26, which is a monoclonal antibody. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's Disease (prevalence rate: 50 to 60 or more people per 100,000 people). Parkinson's disease begins in old age, and is clinically a movement disorder mainly characterized by muscle rigidity, tremors, immobility and the like. The pathological feature of Parkinson's disease is a selective loss of dopamine neurons in the substantia nigra pars compacta of the midbrain. The cause of Parkinson's disease is completely unknown. However, gene mutations have been recently found in cases of familial Parkinson's disease, so that the molecular mechanism is now being elucidated. Two rare mutations (A53T and A30P) in α-synuclein gene cause autosomal dominant hereditary familial Parkinson's disease (Kruger, R. et al., (1998) Nat Genet 18, 106-108; Polymeropoulos, M. H. et al., (1997) Science 276, 2045-2047). α-synuclein is a presynaptic protein and is a main component of the Lewy body. Lewy bodies are highly ubiquitinated intracellular aggregates, which are pathological features of all the sporadic and a part of familial Parkinson's diseases (Trojanowski, J. Q. et al., (1998) Cell Death Differ 5, 832-837). Lewy bodies are often found in degenerative neurons including dopamine neurons in the substantia nigra, and now are strongly suspected to be involved in the mechanism of the onset of Parkinson's disease (Goldberg, M. S. et al., (2000) Nat Cell Biol 2, E115-119; Spillantini, M. G. et al., (1998) Proc Natl Acad Sci USA 95, 6469-6473). Autosomal recessive hereditary juvenile Parkinson's disease (AR-JP, hereinafter referred to as AR-JP in this specification) is the most frequent hereditary Parkinson's disease and is caused by mutations in the Parkin gene. Patients with AR-JP are characterized by Parkinson's-disease-like symptoms resulting from the loss of dopamine neurons without Lewy body formation (Mizuno, Y. et al., (1998) J Neurochem 71, 893-902). The Parkin gene is 1.5 megabase and is one of the largest human genes. The Parkin gene consists of 12 exons, and encodes a 465-amino acid protein with a molecular weight of 52 kDa (Kitada, T. et al., (1998) Nature 392, 605-608; Shimura, H. et al., (1999) Ann Neurol 45, 668-672). The amino terminal 76 amino acids of Parkin share 62% homology with ubiquitin. There are two RING fingers at the carboxy terminus of Parkin, and a motif resembling RING fingers, which is referred to as “in between RING fingers,” is present between the two RING fingers (Morett, E. et al., (1999) Trends Biochem Sci 24, 229-231). Recent studies have revealed that many RING finger proteins have ubiquitin ligase (E3) activity (Jackson, P. K. et al., (2000) Trends Cell Biol 10, 429-439; Joazeiro, C. A. et al., (2000) Cell 102, 549-552). Protein to be degraded by proteasome is modified by covalent binding of ubiquitin. Ubiquitination reaction is performed by 3 types of enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3) (Hershko, A. et al., (1998) Annu Rev Biochem 67, 425-479; Hochstrasser, M. (1996) Annu Rev Genet 30, 405-439). E3 is responsible for promoting ubiquitination by specifically recognizing and binding to the substrate protein. High specificity to recognize a protein to be ubiquitinated is determined by E3, a considerable number of types of which are expected to be present. Multiple groups including our group have revealed that Parkin is E3 and a variant Parkin observed in AR-JP patients lacks E3 activity (Imai, Y. et al., (2000) J Biol Chem 275, 35661-35664; Shimura, H. et al., (2000) Nat Genet 25, 302-305; Zhang, Y. et al., (2000) Proc Natl Acad Sci USA 97, 13354-13359). |
<SOH> SUMMARY OF THE INVENTION <EOH>The onset of AR-JP is caused by dysfunction of Parkin which is E3. Thus it is anticipated that when ubiquitination mediated by Parkin does not occur, the substrate protein remains undegraded and abnormally accumulates intracellularly, so as to induce death in dopamine neurons of the substantia nigra. However, no substrate of Parkin has been discovered so far. Therefore, identification of the substrate proteins of Parkin may be a key to elucidate the pathological mechanism of AR-JP, and to develop a therapy to treat it. Furthermore, dopamine neurons may be specifically degenerated even in the case of sporadic Parkinson's disease under a mechanism shared in common with AR-JP. Thus, it is expected that the identification of the substrates of Parkin can contribute to the elucidation of the causes and the development of therapies for all types of Parkinson's disease. Hence, an object of the present invention is to provide a substrate of Parkin, a system for elucidating the cause of Parkinson's disease using the substrate and a method of screening for a remedy for Parkinson's disease. As a result of intensive studies to achieve the above objective, we have completed the present invention related to the Pael receptor by discovering that a protein thought to be a G protein-conjugated membrane protein and named the Pael (Parkin-associated endothelin receptor-like) receptor is the substrate of Parkin, and also by discovering that Pael receptor accumulation is likely to induce dopamine neuronal death in AR-JP. The present invention is as summarized by the following matters. (1) A human Pael receptor, for screening for a remedy for Parkinson's disease. (2) The human Pael receptor of (I) above, wherein Parkinson's disease is autosomal recessive hereditary Parkinson's disease. (3) An animal cell for screening for a remedy for Parkinson's disease, having a foreign DNA encoding a human Pael receptor or a DNA containing a variant of the DNA incorporated therein. (4) The animal cell of (3) above, wherein the foreign DNA encoding a human Pael receptor is represented by SEQ ID NO: 1. (5) The animal cell of (3) or (4) above, further having the Parkin gene or a variant of the Parkin gene. (6) The animal cell of (5) above further having a gene encoding at least one protein selected from the group consisting of endothelin receptor type A, endothelin receptor type B, UBC6 and UBC7. (7) The animal cell of any one of (3) to (6) above, wherein the cell is an HEK293T cell or SH-SY5Y cell. (8) The animal cell of any one of (3) to (7) above, wherein Parkinson's disease is autosomal recessive hereditary Parkinson's disease. (9) A non-human mammal, having a foreign DNA encoding a human Pael receptor, or a DNA containing a variant of the DNA incorporated therein. (10) The non-human mammal of (9) above, wherein the foreign DNA encoding a human Pael receptor is represented by SEQ ID NO: 1. (11) The non-human mammal of (9) or (10) above, further having the Parkin gene or a variant of the Parkin gene. (12) The non-human mammal of (11) above, further having a gene encoding at least one protein selected from the group consisting of endothelin receptor type A, endothelin receptor type B, UBC6 and UBC7. (13) The animal of any one of (9) to (12) above, wherein the non-human mammal is a rodent. (14) The animal of (13) above, wherein the rodent is a mouse. (15) The non-human mammal of any one of (9) to (14) above, which is a mammal obtained by ontogenesis of a totipotent cell having DNA encoding a human Pael receptor or its variant introduced therein, or the progeny of such mammal, and which carries the above transgene in the somatic cell chromosome. (16) A cell isolated from the animal of any one of (9) to (15) above. (17) A method of screening for a remedy for Parkinson's disease, which comprises causing the human Pael receptor of (1) or (2) above to come into contact with a candidate remedy for Parkinson's disease, and using the Pael receptor quantity in animal cells as an indicator. (18) A method of screening for a remedy for Parkinson's disease, which comprises causing the animal cells of any one of (3) to (8) and (16) above to come into contact with a candidate remedy for Parkinson's disease, and using the Pael receptor quantity in the animal cells as an indicator. (19) A method of screening for a remedy for Parkinson's disease, which comprises administering a candidate remedy for Parkinson's disease to the animal of any one of (9) to (15) above, and using the Pael receptor quantity in the cell of the animal as an indicator. (20) The method of any one of (17) to (19) above, wherein Parkinson's disease is autosomal recessive hereditary Parkinson's disease. (21) A probe, consisting of the whole or a part of a DNA encoding a human Pael receptor for detecting a risk factor for the onset of Parkinson's disease. (22) The probe of (21) above, wherein Parkinson's disease is autosomal recessive hereditary Parkinson's disease. (23) A method of detecting a risk factor for the onset of Parkinson's disease, using the probe of (21) or (22) above. (24) The method of (23) above, wherein Parkinson's disease is autosomal recessive hereditary Parkinson's disease. (25) An antibody recognizing the Pael receptor, which reacts with the Pael receptor but does not react with ETBR-LP-2 which is a Pael receptor homolog. (26) The antibody of (25) above, which further does not react with endothelin receptor type A or endothelin receptor type B, which is a Pael receptor homolog. (27) The antibody of (25) or (26) above, which is a monoclonal antibody. The present invention is described in detail as follows. 1. Obtainment of DNA Encoding the Pael Receptor DNA encoding the Pael receptor can be obtained according to a method described in a publication well known by a person skilled in the art, such as J. Sambrook, E. F. Fritsch & T. Maniatis (1989), Molecular Cloning, a laboratory manual, second edition, Cold Spring Harbor Laboratory Press, or Ed Harlow and David Lanc (1988) Antibodies, a laboratory manual, Cold Spring Harbor Laboratory Press. For example, the DNA can be obtained as the substrate of Parkin by constructing cDNA library from RNA extracted from the brain of a human, and then screening the library according to the yeast two-hybrid method using Parkin as bait. Here the term “Parkin” means the expression product of the Parkin gene involved in hereditary Parkinson's disease. The DNA can also be obtained by screening the library using an oligonucleotide probe synthesized based on the DNA sequence encoding the Pael receptor. Examples of a DNA encoding the Pael receptor include a DNA containing the nucleotide sequence represented by SEQ ID NO: 1, and a DNA encoding the amino acid sequence represented by SEQ ID NO: 2. A variant of the DNA encoding the Pael receptor can be prepared by inducing mutations in the cDNA of a wild type Pael receptor by a known method. Examples of a variant of the DNA encoding Pael receptor include a DNA which hybridizes under stringent conditions to a DNA containing the nucleotide sequence represented by SEQ ID NO: 1 and encodes a protein that can be the substrate of Parkin, or a protein which contains an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 2 by deletion, substitution or addition of one or a plurality of amino acids and can be the substrate of Parkin. Here the term “stringent conditions” mean conditions wherein a so-called specific hybrid is formed, but non-specific hybrids are not formed. Under an example of such conditions, DNAs having high homology, specifically, DNAs having homology of 60% or more, or preferably 80% or more hybridize to each other, whereby nucleic acids sharing low homology do not hybridize to each other. More specifically, the stringent conditions comprise a sodium concentration of 150 to 900 mM or preferably 600 to 900 mM, and a temperature of 60 to 68° C. or preferably 65° C. As an amino acid sequence that is derived from the amino acid sequence represented by SEQ ID NO: 2 by deletion, substitution or addition of one or a plurality of amino acids, for example, an amino acid sequence may be derived from the amino acid sequence of SEQ ID NO: 2 by the deletion of 1 to 10, preferably 1 to 5, and more preferably 1 or 2 amino acids, or by the substitution of 1 to 10, preferably 1 to 5, and more preferably 1 or 2 amino acids with other amino acids. Moreover, 1 to 10, preferably 1 to 5, and more preferably 1 or 2 amino acids may be added to the amino acid sequence represented by SEQ ID NO: 2. An amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 2 by deletion, substitution or addition of one or a plurality of amino acids includes, for example, at least 60% or more, preferably 80% or more, and further preferably 95% or more homology with the amino acid sequence of SEQ ID NO: 2, when calculated by BLAST. Once the nucleotide sequence of a gene is determined, a DNA encoding the Pael receptor or a variant thereof can then be obtained by chemical synthesis, PCR using the cloned cDNA as a template, or hybridization using a DNA fragment having the nucleotide sequence as a probe. Thus by the use of the obtained DNA, a protein that contains an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 2 by deletion, substitution or addition of one or a plurality of amino acids and can be the substrate of Parkin can be obtained. Furthermore, the DNA encoding a human Pael receptor of the present invention includes a DNA containing not only the ORF portion of the Pael gene but also the 5′ untranslated region and the 3′ untranslated region. Here, the term “substrate of Parkin” means a protein that is Parkin-dependently ubiquitinated and degraded by in the presence of ATP, E1, E2 and ubiquitin. For example, whether or not a protein is the substrate of Parkin can be confirmed by mixing the protein in vitro with ATP, E1, E2 and ubiquitin, and then confirming if it is ubiquitinated and degraded. Furthermore, gene diagnosis can be performed using the whole or a part of the DNA encoding the Pael receptor, so that whether or not the gene carries a risk factor for the onset of Parkinson's disease can be diagnosed. At this time, the length of a DNA sequence to be used ranges from 12 nucleotides to 16 nucleotides or more, and is preferably 20 nucleotides or more. Moreover, polynucleotides having these sequences and nucleotides that are capable of hybridizing under the above stringent conditions to the polynucleotides can also be used for diagnosis. 2. Obtainment of Recombinant Pael Receptor-Expressing Cell and Pael Receptor DNA encoding the Pael receptor of the present invention represented by SEQ ID NO: 1 and the variant thereof are inserted to an expression vector, and then the vector is introduced into an appropriate host cell, so as to be able to obtain Pael receptor-expressing cells. Any vector such as a plasmid, phage, or virus can be used as long as it is replicable in host cells. Examples of a vector include Escherichia coli plasmids such as pBR322, pBR325, pUC118, pUC119, pKC30 and pCFM536, Bacillus subtilis plasmids such as pUB110, yeast plasmids such as pG-1, YEp13 and YCp50, and phage DNA such as λgt110 and XZAPII. Examples of a vector for mammalian cells include virus DNA such as baculovirus, vaccinia virus and adenovirus, and SV40 and the derivative thereof. The vector contains the replication initiation point, a selection marker and a promoter, and may also contain an enhancer, a transcription termination sequence (terminator), a ribosome binding site, or a polyadenylation signal and the like, if necessary. Any promoter may be used, as long as it is effectively expressed in host cells. Examples of a promoter include an SRα promoter, an SV40 promoter, an LTR promoter, a CMV promoter and an HSV-TK promoter. In addition, when a protein is expressed on the membrane surface, a nucleic acid encoding a known signal peptide may be added to the N-terminus. Examples of a host cell include bacterial cells such as Escherichia coli, Streptomyces or Bacillus subtilis , Fungi cells such as strains of the genus Aspergillus , yeast cells such as baker's yeast or methanol-assimilating yeast, insect cells such as those of Drosophila S2 or Spodoptera Sf9, and mammalian cells such as HEK293T, SH-SY5Y, CHO, COS, BHK, 3T3 or C127. Transformation can be performed by a known method such as calcium chloride, calcium phosphate, DEAE-dextran-mediated transfection or electroporation. Pael receptors can be isolated from the thus obtained Pael receptor-expressing cells. Furthermore, animal cells expressing Pael receptors can be used in screening for an agent that can be used for treating Parkinson's disease such as a substance that suppresses Pael receptor accumulation, a substance that promotes Pael receptor ubiquitination or degradation, or a substance that promotes Pael receptor discharge outside the body. Furthermore, not only can screening for an agent that can be used for treating Parkinson's disease be performed, but also a cell line that can be used for elucidating the cause of Parkinson's disease can be obtained by the introduction and co-expression of a gene encoding the Pael receptor with the Parkin gene (Kitada, T. et al., (1998) Nature 392, 605-608; Shimura, H, et al., (1999) Ann Neurol 45, 668-672), a variant of the Parkin gene, a gene encoding Pael-receptor-associated receptor such as an endothelin receptor type A or type B (Arai, H et al. (1990) Nature 348, 730-732; Sakurai, T et al. (1990) Nature 348, 732-735), and/or an endoplasmic reticulum-associated E2 gene (Katsanis N, Fisher EM (1998) Genomics 51, 128-31; Lester D, et al. (2000) Biochem Biophys Res Commun 269, 474-80). Here, a variant of the Parkin gene means a gene whose expression product lacks the biological action of Parkin such as E3 activity or the like. Examples of a variant of the Parkin gene include a Parkin gene isolated from an AR-JP patient (Imai, Y. et al., (2000) J Biol Chem 275, 35661-35664; Shimura, H. et al., (2000) Nat Genet 25, 302-305; Zhang, Y. et al., (2000) Proc Natl Acad Sci USA 97, 13354-13359), and Parkin-N, Q311X or ARING losing the C-terminal amino acid sequence of Parkin ( FIG. 2 ). 3. Obtainment of Anti-Pael Receptor Antibody Anti-Pael receptor antibodies can be prepared by inoculating animals such as mice, guinea pigs, rabbits or goats with Pael receptors as antigens subcutaneously, intramuscularly, intraperitoneally or intravenously several times according to a method well known by a person skilled in the art for sufficient immunization, collecting blood from the animals, and performing serum separation. At this time, an appropriate adjuvant can also be used. A monoclonal antibody can also be prepared by a known method. For example, monoclonal antibodies can be prepared by fusing splenocytes of a mouse immunized with the Pael receptor with mouse myeloma cells to produce hybridomas, administering intraperitoneally the culture supernatant of the hybridomas or the hybridomas to mice, and then preparing from the mouse ascites. The Pael receptor to be used as an antigen for immunization may be a natural protein extracted from brain tissue, a recombinant protein, or a chemically-synthesized protein. Furthermore, the Pael receptor may also be a protein having the entire amino acid sequence, or a peptide fragment having a partial structure of the protein and a fusion protein with another protein. A peptide fragment that can be used herein may be a fragment obtained by degrading the protein with an appropriate protease, or may be a product expressed by incorporating the whole or a part of the nucleotide sequence represented by SEQ ID NO: 1 into an expression vector. A polypeptide fragment that is chemically bound with an appropriate carrier protein may also be used. The reactivity of the thus obtained antibody can be measured by a method well known by a person skilled in the art such as enzyme immunoassay (EIA), radioimmunoassay (RIA) or Western blotting. At this time, by the use of a homolog of the Pael receptor such as LP-2 as an antigen to be used for the assay of reactivity in addition to the Pael receptor, a specific antibody that recognizes the Pael receptor but does not recognize the homolog of the Pael receptor such as LP-2 can be obtained. 4. Obtainment of Pael Receptor-Expressing Model Animal A promoter sequence or an enhancer sequence to regulate expression is ligated to a gene to be introduced into a Pael receptor-expressing model animal. These sequences are not specifically limited, and appropriate combinations of sequences that are generally employed can be used. To specifically express a transgene in the brain, β-actin promoter or the like is preferably used. Transgenic animals can be generated by, for example, introducing the above transgene into totipotent cells of a mammal according to the method of Pro. Natl. Acad. Sci. USA 77:7380-7384, 1980 or another method, allowing the cells to develop into individual animals, and then selecting individual animals carrying the transgene incorporated in the genome of the somatic cells. A preferred mammal is a mouse, of which many inbred lines have been produced, and for which techniques for culturing the fertilized eggs, for external fertilization and the like have been well established. However, technically, any animal species can be a target. Examples of a totipotent cell, into which a gene is introduced, include, in the case of a mouse, culture cells such as ES cells having pluripotency, in addition to fertilized eggs and early embryos. As a method of introducing a gene into a culture cell, for example, a known electrostatic pulse method, the liposome method or the calcium phosphate method can be used. In terms of generation efficiency regarding individual transgenic animals and transmission efficiency regarding the transmission of a transgene to the next generation, a physical method (microinjection) of injecting a DNA solution into a fertilized egg is preferred. A totipotent cell into which a gene has been injected is transplanted in the fallopian tubes of a pseudo parent, and then the cell is allowed to develop into an individual animal, so that a Pael receptor-expressing animal can be obtained. DNA is extracted from the somatic cell, and then the presence of the transgene can be confirmed by Southern blot analysis, PCR assay or the like. By the use of animals confirmed to carry the transgene as Founders for breeding, animals wherein Pael receptor gene or a variant thereof has been stably incorporated into a part of the chromosome can be efficiently generated. Furthermore, similar to the above Pael receptor-expressing cells, the Parkin gene, a variant of the Parkin gene isolated from an AR-JP patient, and/or a gene encoding a Pael-receptor-associated receptor such as the endothelin receptor may be incorporated simultaneously. The thus generated transgenic animal can be an optimum model animal for elucidating the cause of Parkinson's disease and screening for an agent that can be used for treating Parkinson's disease. Furthermore, transgenic animals provided by the present invention carry transgenes in all of their somatic cells, so that cells isolated from the individual animal also express Pael receptors. Hence, the culture system of these cells can also be utilized for elucidating the cause of Parkinson's disease and screening for an agent that can be used for treating Parkinson's disease, similarly to the case of the above individual animals. 5. Screening for Agent that can be Used for Treating Parkinson's Disease The above Pael receptor, Pael receptor-expressing cells, transgenic animals and the cells obtained from the transgenic animal can be used for screening for an agent that can be used for treating Parkinson's disease. Examples of an agent that can be used for treating Parkinson's disease include a substance that suppresses Pael receptor accumulation, a substance that promotes Pael receptor ubiquitination or degradation, and a substance that promotes Pael receptor discharge outside the body. Applicability of a candidate substance as an agent can be determined by adding the candidate substance as an agent that can be used for treating Parkinson's disease to Pael receptor-expressing culture cells, or administering it to Pael receptor-expressing animals, and then measuring the quantity or the ubiquitination degree of the Pael receptor within the cell or within the animal body. Thus, screening for a remedy can be performed. |
Oncology drug innovation |
The present invention describes methods for identification of molecules expressed at a different level on the cell surface of cancer cells compared to non-malignant cells and methods of identification of cancer specific promoters to be used singly or in combination for delivery and expression of therapeutic genes for treatment of cancer. The invention furthermore describes targeting complexes targeted to cell surface molecules identified by the methods of the invention. In embodiments of the invention said targeting complexes comprise the promoters identified by the methods of the invention. In addition the invention describes methods of identifying binding partners for the cell surface molecules and the binding partners per se. Methods of treatment using the targeting complexes and uses of the targeting complexes for the preparation of a medicament arc also disclosed by the invention. Furthermore, the invention describes uses of the cell surface molecules or fragments thereof for preparation of vaccines. |
1. A method for identifying a plurality of cell surface molecules, which are expressed at a different level in malignant cells compared with normal cells, comprising the steps of: i) providing at least 3 malignant cell lines selected from the group consisting of CPH 54 A, CPH 54 B, GLC 2, GLC 3, GLC 14, GLC 16, GLC 19, GLC 26, GLC 28, DMS 53, DMS 79, DMS 92, DMS 114, DMS 153, DMS 273, DMS 406, DMS 456, NCI H69, NCI N417, MAR H24, MAR 86 MI, SHP-77, NCI-H2171, NCI-H2195, NCI-H2196, NCI-H2198, NCI-H2227, NCI-H2286, NCI-H2330, NCI-H735, NCI-H1339, NCI-H1963, NCI-H2107, NCI-H2108, NCI-H1304, NCI-H1341, NCI-H1417, NCI-H1436, NCI-H1522, NCI-H1618, NCI-H1672, NCI-H1694, NCI-H1836, NCI-H1870, NCI-H1876, NCI-H1882, NCI-H1926, NCI-H1930, NCI-H1994, NCI-H2029, NCI-H2059, NCI-H2066, NCI-H2081, NCI-H2141, NCI-H211, NCI-H220, NCI-H250, NCI-H524, NCI-H592, NCI-H711, NCI-H719, NCI-H740, NCI-H748, NCI-H774, NCI-H841, NCI-H847, NCI-H865, NCI-H1048, NCI-H1059, NCI-H1092, NCI-H1105, NCI-H1184, NCI-H1238, NCI-H1284, NCI-H1688, NCI-H187, NCI-H378, NCI-H526, NCI-H660, NCI-H889, NCI-H60, NCI-H196, NCI-H446, NCI-H209, NCI-H146, NCI-H82, NCI-H460, NCI-H345, NCI-H510A, NCI-128, NCI-446, SW 1271; and ii) providing at least 3 total RNA samples derived from normal tissue selected from the group consisting of liver, heart, kidney, lung, adrenal gland, colon, pancreas, small intestine, spleen, skeletal muscle, trachea, prostate, placenta, salivary gland, testes, leucocytes, brain, adipose tissue, bladder, breast, cervix, esophagus, larynx, ovary, rectum, skin, spinal cord, stomach, thymus, thyroid and uterus; and iii) comparing the expression of mRNA in the cell lines according to i) and tissue samples according to ii); and iv) identifying nucleic acid sequences, wherein a) there is a difference between the amount of mRNA expressed in one or more cell lines according to i) and the amount of mRNA expressed in one or more tissues according to ii); and/or b) there is essentially no difference in the amount of mRNA expressed in at least two cell lines according to i); and/or c) there is essentially no difference in the amount of mRNA expressed in at least two tissue samples according to ii); and v) selecting among the nucleic acid sequences according to iv), nucleic acid sequences encoding for potential cell surface molecules. 2. (Canceled) 3. The method according to claim 1, wherein step ii) involves tissue samples derived from lung, liver, heart, and kidney. 4.-12. (Canceled) 13. The method according to claim 1, wherein nucleic acid sequences encoding for potential cell surface molecules according to step v) are selected according to information available in commonly accessible databases selected from the group consisting of PubMed (NCBI), Nucleotide (NCBI), Protein (NCBI), Structure (NCBI), OMIM (NCBI) and LocusLink (NCBI). 14.-16. (Canceled) 17. A method of identifying first nucleic acid sequences, which are capable of directing expression of second nucleic acid sequences operably linked thereto, wherein the level of said expression is different in malignant cells compared with normal cells comprising the steps of: i) providing at least 3 malignant cell lines selected from the group consisting of CPH 54 A, CPH 54 B, GLC 2, GLC 3, GLC 14, GLC 16, GLC 19, GLC 26, GLC 28, DMS 53, DMS 79, DMS 92, DMS 114, DMS 153, DMS 273, DMS 406, DMS 456, NCI H69, NCI N417, MAR H24, MAR 86 MI, SHP-77, NCI-H2171, NCI-H2195, NCI-H2196, NCI-H2198, NCI-H2227, NCI-H2286, NCI-H2330, NCI-H735, NCI-H1339, NCI-H1963, NCI-H2107, NCI-H2108, NCI-H1304, NCI-H1341, NCI-H1417, NCI-H1436, NCI-H1522, NCI-H1618, NCI-H1672, NCI-H1694, NCI-H1836, NCI-H1870, NCI-H1876, NCI-H1882, NCI-H1926, NCI-H1930, NCI-H1994, NCI-H2029, NCI-H2059, NCI-H2066, NCI-H2081, NCI-H2141, NCI-H211, NCI-H220, NCI-H250, NCI-H524, NCI-H592, NCI-H711, NCI-H719, NCI-H740, NCI-H748, NCI-H774, NCI-H841, NCI-H847, NCI-H865, NCI-H1048, NCI-H1059, NCI-H1092, NCI-H1105, NCI-H1184, NCI-H1238, NCI-H1284, NCI-H1688, NCI-H187, NCI-H378, NCI-H526, NCI-H660, NCI-H889, NCI-H60, NCI-H196, NCI-H446, NCI-H209, NCI-H146, NCI-H82, NCI-H460, NCI-H345, NCI-H510A, NCI-128, NCI-446 and SW 1271, and ii) providing at least 3 RNA samples derived from normal tissue samples derived from the group consisting of liver, heart, kidney, lung, adrenal gland, colon, pancreas, small intestine, spleen, skeletal muscle, trachea, prostate, placenta, salivary gland, testes, leucocytes, brain, adipose tissue, bladder, breast, cervix, esophagus, larynx, ovary, rectum, skin, spinal cord, stomach, thymus, thyroid and uterus; and iii) comparing the expression of mRNA in the cell lines according to i) and tissue samples according to ii); and iv) identifying second nucleic acid sequences, wherein a) there is a difference between the amount of mRNA expressed in one or more cell lines according to i) and the amount of mRNA expressed in one or more tissues according to ii); and/or b) there is essentially no difference in the amount of mRNA expressed in at least two cell lines according to i); and/or c) there is essentially no difference in the amount of mRNA expressed in at least two tissue samples according to ii); and v) identifying first nucleic acid sequences operably linked to the second nucleotide sequences identified in step iv) 18. (Canceled) 19. The method according to claim 17, wherein step ii) involves tissue samples derived from the group consisting of lung, liver, heart and kidney. 20.-30. (Canceled) 31. The method according to claim 17, wherein any first nucleic acid sequence operably linked to a second nucleic acid sequence comprises up to up to 5000 base pairs upstream of the translation start codon of said second nucleic acid sequence on the chromosome. 32. (Canceled) 33. The method according to claim claim 17, wherein any first nucleic acid sequence operably linked to a second nucleic acid sequence comprise intron sequences found downstream of the translation start codon of said second nucleic acid sequence on the chromosome. 34. The method according to claim 17, wherein any first nucleic acid sequence operably linked to a second nucleic acid sequence comprise an enhancer sequence located more than 10,000 base pairs upstream or downstream from the translation start codon of said second nucleic acid sequence on the chromosome. 35.-36. (Canceled) 37. A targeting complex comprising: vi) a binding partner capable of binding a cell surface molecule identified by the method according to claim 1, wherein said cell surface molecule is selected from the group consisting of GRIA2, GRM8, ITGAV, ITGAE, NCAM1, NPTXR, LRP8 and CHRNA5; and vii) a bioreactive species 38.-41. (Canceled) 42. The targeting complex according to claim 37, wherein the binding partner is selected from the group consisting of L-glutamate, kainate, 5-(bromomethyl)-4-isoxazolepropionic acid, analogues of glutamate, substituted quinoxaline 2,3 diones, GYKI52466, 5-I-Willardine, 5-F-Willardine, agonist and antagonist ligands to the AMPA ((RS)-αx-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, NBQX, CNQX, DNQX, GYKI 52466, 6-Chlorokynurenic acid, JSTX, L-APA, L-SOP, ACPT, (R,S)-PPG, CPPG, MAP4, (S)-3,4-DCPG, vitronectin, cytactin, fibronectin, fibrinogen, laminin, MMP-2, osteopontin, prothrombin, thrombospondin, von Willebrandts Factor, recombinant fragments of L1CAM, salmosin, E-cadherin and peptides thereof, including the peptide: NRDKETKV, NCAM1 domain Ig I+II, NCAM1 domain IgIII and peptides thereof, peptides C3: ASKKPKRNIKA, D3: AKKERQRKDTU, D4: ARALNWGAKP, monoclonal antibody 123C3, NPTX1, NPTX2, taipoxin, TCBP49, Oxynor, ApoE2, ApoE3, ApoE4, peptides from ApoE (E141;-155; LRKLRKRLLRDADDL and its tandem E(141;-155)2; LRKLRKRLLRDADDLLRKLRKRLL RDADDL) reelin, nicotine, acetylcholine, α-bungarotoxin, carbachol and specific antibodies against any of said surface molecules. 43. The targeting complex according to claim 37, wherein said cell surface molecule is capable of internalising the targeting complex. 44. The targeting complex according to claim 37, wherein said bioreactive species comprises a nucleic acid. 45. The targeting complex according to claim 44, wherein the nucleic acid comprises a second nucleic acid operably linked to a first nucleic acid sequence comprising an expression signal. 46. A targeting complex comprising i) a binding partner capable of binding a cell surface molecule identified by the method according to claim 1, wherein said cell surface molecule is capable of internalising the targeting complex; and ii) a bioreactive species comprising a nucleic acid sequence comprising a second nucleic acid operably linked to a first nucleic acid sequence comprising an expression signal, wherein said first nucleic acid sequence has been identified by the method according to claim 17. 47. The targeting complex according to claim 46, wherein the cell surface molecule comprises or essentially consists of a cell surface molecule selected from the group consisting of NCAM1, NPTXR, LRP8 and CHRNA5. 48. The targeting complex according to claim 46, wherein the cell surface molecule comprises or essentially consists of NCAM1. 49. The targeting complex according to claim 46, wherein the cell surface molecule comprises or essentially consists of NPTXR. 50.-51. (Canceled) 52. The targeting complex according to claim 46, wherein the binding partner is selected from the group consisting of NCAM1 domain Ig I+II, NCAM1 domain IgIII and peptides thereof, peptides C3: ASKKPKRNIKA, D3: AKKERQRKDTU, D4: ARALNWGAKP, monoclonal antibody 123C3, NPTX1, NPTX2, taipoxin, TCBP49, Oxynor, ApoE2, ApoE3, ApoE4, peptides from ApoE (E141;-155; LRKLRKRLLRDADDL and its tandem E(141;-155)2; LRKLRKRLLRDADDL-LRKLRKRLL RDADDL) reelin, nicotine, acetylcholine, α-bungarotoxin, carbachol and specific antibodies to said surface molecules. 53. The targeting complex according to claim 46, wherein said first nucleic acid sequence comprises an expression signal which direct a higher level of expression of said second nucleic acid sequence in malignant cells compared with non-malignant cells. 54. (Canceled) 55. The targeting complex according to claim 46, wherein said first nucleic acid sequence is selected from the group consisting of pro221, pro210, pro71, pro41, pro30, pro2, pro209, pro14, pro4, pro8, pro246, pro16, pro27, pro5, pro49, pro19, pro140, pro139, pro207, pro81, pro273 and pro362. 56. The targeting complex according to claim 46, wherein said first nucleic acid sequence comprises fragments of nucleotide sequences selected from the group consisting of pro221, pro210, pro71, pro41, pro30, pro2, pro209, pro14, pro4, pro8, pro246, pro16, pro27, pro5, pro49, pro19, pro140, pro139, pro207, pro81, pro273 and pro362. 57.-60. (Canceled) 61. The targeting complex according to claim 46, wherein said first nucleic acid sequence further comprises nucleic acid sequences not natively associated therewith. 62. (Canceled) 63. The targeting complex according to claim 46, wherein said second nucleic acid sequence encodes a therapeutic protein. 64.-69. (Canceled) 70. The targeting complex according to claim 37, wherein said bioreactive species is a toxin. 71. (Canceled) 72. The targeting complex according to claim 37, wherein said bioreactive species is an inducer of apoptosis. 73. (Canceled) 74. The targeting complex according to claim 37, wherein said bioreactive species comprises a radioisotope. 75. The targeting complex according to claim 37, wherein said bioreactive species comprises a cytostatica. 76. The targeting complex according to claim 37, wherein said bioreactive species comprises or essentially consists of a polypeptide. 77.-85. (Canceled) 86. The targeting complex according to claim 63, which comprises more than one first nucleotide sequence encoding a therapeutic protein or more than one therapeutic protein. 87.-89. (Canceled) 90. The targeting complex according to claim 37, wherein the complex further comprises a endosomal lytic agent selected from the group consisting of polyethylenimine (PEI), a replication defective virus and a viral protein capside. 91.-93. (Canceled) 94. The targeting complex according to claim 37, wherein the binding partner associates with the bioreactive species via a nucleic acid binding agent covalently attached to said binding partner. 95. The targeting complex according to claim 94, wherein the nucleic acid binding agent is selected from the group consisting of poly-L-lysine (PLL), spermine, spermidine and histone proteins. 96. (Canceled) 97. The targeting complex according to claim 37, wherein the binding partner associates with the bioreactive species indirectly via a pair of specific interacting components wherein one component is covalently attached to the bioreactive species and the second component is covalently attached to the binding partner. 98. (Canceled) 99. A method of using a cell surface molecule identified according to claim 1 as a drug target, wherein said drug target is capable of binding a binding partner and internalising said binding partner into cells expressing said cell surface molecule. 100. The method according to claim 99, wherein the cell surface molecule is selected from the group consisting of NCAM1, NPTXR, LRP8, CHRNA5, GRIA2, GRM8, ITGAV, ITGAE, TNFRSF12, L1CAM, GPR49 and TMEFF1. 101.-129. (Canceled) 130. A complex comprising a cell surface molecule identified according to claim 1 and a targeting complex comprising i) a binding partner capable of binding said cell surface molecule, wherein said cell surface molecule is selected from the group consisting of GRIA2, GRM8, ITGAV, ITGAE, NCAM1, NPTXR, LRP8 and CHRNA5; and ii) a bioreactive species. 131. The complex according to claim 130, wherein the cell surface molecule is selected from the group consisting of NCAM1, NPTXR, LRP8, CHRNA5, GRIA2, GRM8, ITGAV, ITGAE, TNFRSF12, L1CAM, GPR49, TMEFF. 132. (Canceled) 133. A pharmaceutical composition comprising of the targeting complex according to claim 37 together with a pharmaceutically acceptable carrier. 134. A method of treatment of a premalignant and/or malignant conditions in an individual in need thereof, comprising administering to said individual a pharmaceutically effective amount of the targeting complex according to claim 37. 135.-137. (Canceled) 138. The method according to claim 134, wherein said condition is a cancer selected from the group consisting of melanoma, brain tumour, neuroblastoma, breast cancer, lung cancer, prostate cancer, cervix cancer, uterine cancer, ovarian cancer, leukaemia, colon cancer, rectum cancer and bladder cancer. 139.-145. (Canceled) 146. The method according to claim 134, wherein said method further comprises one or more second treatments. 147.-159. (Canceled) 160. A method of using a pharmaceutically effective amount of a cell surface molecule identified according to claim 1, or a nucleic acid encoding said cell surface molecule, for the preparation of a vaccine. 161. (Canceled) 162. The method according to claim 160, wherein the cell surface molecule is selected from the group consisting of NCAM1, NPTXR, LRP8, CHRNA5, GRIA2, GRM8, ITGAV, ITGAE, TNFRSF12, L1CAM, GPR49 and TMEFF1. 163.-166. (Canceled) 167. The use method according to claim 160, wherein said vaccine is suitable for ameliorating and/or curative and/or prophylactic treatment of a premalignant and/or malignant conditions. 168. The targeting complex according to claim 45, wherein said second nucleic acid sequence encodes a therapeutic protein. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Approximately half of all patients with cancer have disseminated disease at the time of diagnosis. Existing cancer therapies are able to cure only 5-7% of these patients. Consequently, there is a great need for more effective drugs, which can be administered systemically alone or in combination with existing treatments. Methods utilising gene therapy to deliver efficient and specific treatment of cancer cells is therefore a promising strategy. However, strategies applied to this date have only had limited success and the development of suitable delivery systems need further development. Delivery Vectors The choice of the delivery vector for gene therapy is a major issue. Many vector systems have been tested for their suitability for gene transfer, including viral vectors such as retrovirus, adenovirus, adeno-associated virus, lentivirus and non viral vectors such as complexing with liposomes, cationic lipids or polycations. However, all of these vectors have specific advantages and limitations. Retrovirus requires mitotic division for transduction, but mediate long term expression, as they integrate in the genome. Adenovirus will transduce both dividing and non-dividing cells, but only transiently as they remain episomal. Adenovirus, however, are highly immunogenic and retrovirus are rapidly inactivated by the human complement system. Lentivirus does not induce immune response, but involve specific safety concerns; as it is a member of the immunodeficiency virus. More than 75% of all protocols so far have used viral vectors despite these are difficult and expensive to produce, there is a limited insert size of the therapeutic gene and there are many safety considerations to be made. Therefore, the majority of the protocols used for adenoviral vectors have administered the therapeutic gene by local delivery (injection into the tumour) to increase the local titer of the virus and avoid immunogenic response, but even the highest titer system has not yet been sufficient to cure local tumours. A major disadvantage of viral vector systems is that their uptake is unspecific and not targeted to the cancer cells. However, as adenovirus still is the preferred vector due to its efficiency of delivery, ways of reducing the immune response and target the virus to specific cells are under development. On the other hand, liposomes and polycation complexes, which are less immunogenic, easier to produce and do not need the safety considerations of viral vectors have much lower transfection efficiency than viral transduction and also lack the cell specificity. However, polycations have the ability to compact and neutralise the charge of the delivered DNA and PEI complexes appear relatively stable in the blood system (Goula et al., 1998; reviewed in Mountain, 2000). To assure high specificity and to limit undesired side effects of the treatment, it is of importance to design a vector or vehicle, which targets and delivers the therapeutic gene in question to the cancer cells efficiently and with high specificity. However, as described below, this involves assembly of a multi component vector. Receptor Targeting. Functional receptors or other cell surface molecules, which can internalise by ligand or antibody binding on the cancer cell surfaces, can be used to target the gene delivery to the cells. Receptor targeted gene delivery by means of DNA conjugated to a ligand of the receptor offers a promising approach. The major advantages of targeted gene delivery are that receptor targeting can be performed without virus, thus eliminating many of the obstacles present in current strategies of gene therapy, Successful deliverance of genes to cancer cells using receptor targeting has been reported to a variety of different surface receptors including receptors for epidermal growth factor (Cristano and Roth, 1996, Frederiksen et al., 2000), folate (Gottschalk et al., 1994), transferrin (Wagneret al., 1990). High expression of a specific receptor is not always a pre-requisite for efficient receptor mediated uptake, as has been demonstrated for the epidermal growth factor receptor (Frederiksen et al., 2000). However, many of the receptors expressed by cancer cells are also expressed by normal cells to some extent, meaning that normal cells will often be targeted as well. This issue emphasises the need for further requirements for specificity for the expression or nature of the therapeutic gene. Molecular Conjugates For targeted gene therapy it is essential that the ligand to be internalised and DNA expressing the therapeutic gene are physically associated for receptor mediated uptake. Several methods have been used for preparing non-viral, synthetic vectors of targeted DNA molecular conjugates by associating cationic polymers, such as poly-L-lysine (Frederiksen et al., 2000) or polyethylenimine (PEI) (Kircheis et al., 1997) (polyplexes) with the ligand and DNA. Successful gene targeting has been reported for a number of molecular conjugates. The ligand has either been covalently linked to the polycation, or biotinylated ligand and polylysine were complexed via streptavidine to form condensed conjugates with DNA, which are internalised by the receptor of the ligand. One of the advantages of these system over virus mediated transfer is the lack of size limitation of the DNA. PEI complexes, in addition, appear to be able to pass the capillary barrier in lung, making this compound one agent for molecular conjugates. Endosomal Release of Molecular Conjugate. After endocytosis of the DNA/ligand conjugate by the receptor, the normal pathway would lead to degradation and loss of DNA. It has therefore proven essential to include an endosomolytic agent in the molecular conjugate. Adenovirus, replication deficient adenovirus and the viral capside have all proven to be very efficient for endosomal lysis, when included in the molecular conjugate. However, all the reservations of unspecific uptake, safety and immunogenic response applying to use of using adenovirus as vectors also apply for this system. Inclusion of other fusogenic peptides containing amino acid sequences from e.g. influenza virus, toxins or synthetic peptides in the molecular conjugate have been tested for cytoplasmic release. These have the advantage of less immunogenicity and lower cost, but have been shown to be less effective in endosomal lysis than adenovirus. However, if the molecular conjugate is formed using the polycationic PEI, inclusion of endosomolytic agents are not necessary, as PEI has an intrinsic endosome-buffering capacity resulting in endosomal swelling and rupture. Cancer Specific Promoters An increase in the specificity of the targeting of a therapeutic gene to cancer cells can be obtained if a tumour specific promoter controlling the expression can be used (reviewed in Nettelbeck et al., 2000). Promoters for genes, whose expression is specific for the malignant phenotype, but show no tissue specificity such as telomerase have been used. Also, promoters regulating oncofetal antigens, which are not normally expressed in the adult, have been found to be active in tumor cells, such as carcinoembryonic antigen (CEA). However, the activity of these promoters (compared to strong, constitutive active viral promoters) have often proven not to mediate sufficient expression of the therapeutic gene, wherefore the tumour specific genes have been used for activation of another, stronger promoter controlling the therapeutic gene. Another disadvantage of oncofetal promoters is that these promoters will only be active in a subset of tumour types, depending on the tissue origin of the tumour. Alternatively, synthetic promoters have been designed taking advantage of the fact that many oncogenes which are overexpressed in cancer cells are transcription factors, which can mediate high transcriptional activity from their respective DNA recognition sequences. Therapeutic Genes The product of a therapeutic gene must be able to effectively induce cell death. Gene therapy strategies for cancer treatment have used many different approaches. These include immunogene therapy such as cytokine stimulation of immune system (enhancing the immune response against tumour cells), selective prodrug activation, suicide genes, restoration of tumor suppressor genes and inhibition of activated oncogenes (reviewed in Frederiksen et al., 1999; Gunji et al., 2000). Indeed, most of the present therapeutic protocols in clinical trials against cancer involve immunotherapy. However, as the molecular phenotype of many types of cancer regarding aberrant expression or mutations of oncogenes and tumour suppressor genes, these are obvious candidates to target. Therapeutic gene products reducing expression or activity of oncogenes, such as antisense RNA or neutralising antibody fragments, have been tried and shown to inhibit proliferation. However, oncogene inactivation does not necessarily kill the cells and is therefore probably not applicable for short term treatment. One of the at present promising strategies is to reintroduce tumour suppressor genes, as most cancer cells exhibit loss of function of one or more of these genes. Of particular interest is the tumour suppressor gene TP53 encoding p53, which is a transcription factor, which activates genes known to be involved in cell cycle arrest and induction of apoptosis. Reintroduction of wild type p53 has been shown to markedly reduce tumour cell growth or induce apoptosis of cancer cells in both in vitro and in vivo systems (Roth et al., 1996; Nielsen and Maneval, 1998). However, gene products rendering cells sensitive to otherwise harmless drugs has also been extensively used for gene therapy trials. In particular, the herpes simplex virus thymidine kinase (HSV-tk) in combination with the nucleoside analogue drug gangcyclovir has been used. However, the conversion of the drug to a toxic nucleo side analogue by the enzyme only will kill cells, which are dividing. However, the toxic products are transmitted to surrounding cells by the so-called “by-stander” effect, making the approach potential for systems with low targeting efficiency. |
<SOH> SUMMARY OF THE INVENTION <EOH>Accordingly, it is a first objective of the present invention to provide methods for identifying a plurality of cell surface molecules, which are expressed at a different level in malignant cells compared with normal cells, comprising the steps of: i) Providing at least 3 malignant cell lines selected from the group consisting of cell lines mentioned in table 1 ii) Providing at least 3 total RNA samples derived from normal tissue selected from the group consisting of liver, heart, kidney, lung, adrenal gland, colon, pancreas, small intestine, spleen, skeletal muscle, trachea, prostate, placenta, salivary gland, testes, leucocytes, leucocytes, brain, adipose tissue, bladder, breast, cervix, esophagus, larynx, ovary, rectum, skin, spinal cord, stomach, thymus, thyroid and uterus. iii) Comparing the expression of mRNA in the cell lines according to step i) and tissue samples according to step ii) iv) Identifying nucleic acid sequences, wherein a) there is a difference between the amount of mRNA expressed in one or more cell lines according to i) and the amount of mRNA expressed in one or more tissues according to ii); and/or b) there is essentially no difference in the amount of mRNA expressed in at least two cell lines according to i); and/or c) there is essentially no difference in the amount of mRNA expressed in at least two tissue samples according to ii); and v) Selecting among the nucleic acid sequences according to iv), nucleic acid sequences encoding for potential cell surface molecules. It is a second objective of the present invention to provide methods of identifying first nucleic acid sequences, which are capable of directing expression of second nucleic acid sequences operably linked thereto, wherein the level of said expression is different in malignant cells compared with normal cells comprising the steps of: i) Providing at least 3 malignant cell lines selected from the group consisting of cell lines mentioned in table 1 ii) Providing at least 3 RNA samples derived from normal tissue samples derived from the group consisting of liver, heart, kidney, lung, adrenal gland, colon, pancreas, small intestine, spleen, skeletal muscle, trachea, prostate, placenta, salivary gland, testes, leucocytes, brain, adipose tissue, bladder, breast, cervix, esophagus, larynx, ovary, rectum, skin, spinal cord, stomach, thymus, thyroid and uterus. iii) Comparing the expression of mRNA in the cell lines according to i) and tissue samples according to ii) iv) Identifying second nucleic acid sequences, wherein a) there is a difference between the amount of mRNA expressed in one or more cell lines according to i) and the amount of mRNA expressed in one or more tissues according to ii); and/or b) there is essentially no difference in the amount of mRNA expressed in at least two cell lines according to i); and/or c) there is essentially no difference in the amount of mRNA expressed in at least two tissue samples according to ii) v) Identifying first nucleic acid sequences operably linked to the second nucleotide sequences identified in step iv) It is a third objective of the present invention to provide uses of a pharmaceutically effective amount of the cell surface molecules identified according to the present invention for the preparation of a vaccine. Furthermore, the present invention provides uses of a pharmaceutically effective amount of a nucleic acid sequence encoding a cell surface molecule identified according to the methods of the present invention for the preparation of a vaccine. The present invention also provides uses of a pharmaceutically effective amount of a cell surface molecule and/or a nucleic acid sequence encoding such a cell surface molecule for the preparation of a vaccine, wherein said cell surface molecule preferably comprises or essentially consists of or for example is a cell surface molecule mentioned in table 2. It is a fourth objective of the present invention to provide uses of a cell surface molecule identified according to the methods described by the present invention as a drug target, wherein said drug target is capable of binding a binding partner and internalising said binding partner into cells expressing said cell surface molecule. Furthermore, the present invention provides uses of a cell surface molecule which preferably comprises or essentially consists of or for example is a cell surface molecule mentioned in table 2 as drug target, wherein said drug target is capable of binding a binding partner and internalising said binding partner into cells expressing said cell surface molecule. It is a fifth objective of the present invention to provide methods of identifying and/or preparing specific binding partners comprising the steps of i) Providing a cell surface molecule identified by the methods described by the present invention ii) Identifying and/or preparing binding partners capable of associating with said cell surface molecules It is furthermore an objective of the present invention to provide methods of identifying and/or preparing specific binding partners comprising the steps of. i) Providing a cell surface molecule which preferably comprises or essentially consists of or for example is Transferrin receptor; such as type II membrane protein cone: for example is HP10481; such as type II membrane protein clone: such as HP10390; for example is PG40; such as TRC8; for example is TR2-11; such as OA3 antigenic surface determinant; for example is integrin alpha 6, For example GPIIb; such as vitronectin receptor alpha subunit; for example is integrin alpha-7; such as integrin alpha E precursor; for example is integrin alpha 6B; such as integrin alpha 5 subunit; for example is integrin beta-5 subunit; such as integrin alpha-3 chain; for example is RYK; such as amyloid precursor protein-binding protein 1; for example is putative transmembrane GTPase; such as membrane cofactor protein; FOR EXAMPLE GLVR1; for example is Mr 110,000 antigen; for example is syndecan-1; such as putative seven transmembrane domain protein; for example is LCA-homolog/LAR protein; such as M6 antigen; for example is Me491/CD63 antigen; such as multispanning membrane protein; for example is DDR; such as autocrine motility factor receptor; for example is insulin receptor precursor; such as IGF1R, for example is insulin-like growth factor II receptor; such as SAS; for example is TAPA-1; such as MICB; for example is MHC class II HLA-DR7-associated glycoprotein beta-chain; such as HLA-DP; for example is bone small proteoglycan I biglycan; such as CAR; for example is MEA11; such as interferon-gamma receptor alpha chain; for example is Polymeric immunoglobulin receptor; such as metabotropic glutamate receptor type 4; for example is metabotropic glutamate receptor 8; such as CLPTM1; for example is MAGE-4b; such as MAGE5a; for example is MAGE-3; such as MAGE-1; for example is MAGE6; such as MAGE-9; for example is MAGE11; such as CD24; for example is CD59; such as CD44; for example is low density lipoprotein receptor; such as very low density lipoprotein receptor, for example is N-CAM; such as lamin B receptor homolog TM7SF2; for example is putative T1/ST2 receptor binding protein precursor; such as NTR2 receptor; for example is RAGE-4; such as HLA-G1; for example is MOAT-C; such as alpha 2 delta calcium channel subunit isoform I; for example is LFA-3; such as L1-CAM; for example is AVPR2; such as C1 p115 C1; for example is TE2; such as RbP; for example is HCF1; such as IRAK; for example is CD151; such as surface antigen; for example is MAG; such as GPR19; for example is pcta-1; such as PRAME; for example is vasopressin activated calcium mobilizing receptor-like protein; such as serotonin receptor 5-HT4B; for example is serotonin 1 D receptor (5-HT1D˜); such as CD9; for example is LDL receptor member LR3; such as DR6; for example is tumor necrosis factor receptor; such as HG38; for example is urokinase-type plasminogen receptor; such as FGF receptor; for example is nerve growth factor receptor; such as cystine/glutamate transporter; for example is CB1 cannabinoid receptor (CNR1); such as PSG; for example is PSG13′; such as CPE-receptor; for example, is CRH2R; such as OCI5; for example is TRAIL receptor 2; such as HNMP-1; for example is kidney alpha-2-adrenergic receptor; such as erythropoietin receptor; for example is chondroitin sulphate proteoglycan versican V1; for example is mGluR1beta; such as CD97; for example is L6; such as NY-ESO-1; for example is T-cell receptor alpha delta; such as ror1; for example is ror2; such as SSTR2; for example is VESPR; such as IgG Fc receptor; for example is glutamate receptor subunit GluRC; such as HEK2; for example is PVR; such as CEA; for example is CC-chemokine-binding receptor JAB61; such as HER2; for example is HER3; such as hypothetical protein FL22357 similar to Epidermal growth factor receptor-related protein; for example is putative endothelin receptor type B-like protein; such as GLVR2; for example is P2X4 purinoreceptor; such as FPRL1; for example is Atrial natriuretic peptide clearance receptor; for example is gastrin/CCK-B receptor; such as Neuromedin B receptor; for example is GFRA3; such as GRPR; for example is CDH1; such as CDH2; for example is TGFBR1; such as TGFBR2; for example is TGFBR3; such as precursor of epidermal growth factor receptor. ii) Identifying and/or preparing binding partners capable of associating with said cell surface molecules. A further objective of the present invention is to provide isolated and/or purified specific binding partners capable of associating with cell surface molecules, which are expressed at a different level in malignant cells compared with normal cells, identified by the methods provided by the present invention. The present invention also provides isolated and/or purified specific binding partners capable of associating with a cell surface molecule which preferably comprises or essentially consists of or for example is a cell surface molecule mentioned in table 2. It is also an objective of the present invention to provide methods of identifying novel drug targets, comprising the steps of i) Providing a binding partner as described in the present invention, ii) Identifying potential drug targets capable of associating with said binding partner It is yet another objective of the present invention to provide drug targets identified by the methods described by the present invention. Furthermore, it is an objective of the present invention to provide targeting complexes comprising: i) A binding partner as described by the present invention; and ii) A bioreactive species wherein the targeting complex is capable of binding a cell surface molecule identified according to the methods described by the present invention and capable of being internalised into cells bearing said cell surface molecule. The present invention also provides uses of binding partners as describes by the invention for the preparation of targeting complexes according to the invention. It is yet a further objective of the present invention to provide pharmaceutical compositions comprising of the targeting complexes described by the present invention together with a pharmaceutically acceptable carrier. It is even a further objective of the present invention to provide methods of treatment of a premalignant and/or malignant conditions in an individual in need thereof, comprising administering to said individual a pharmaceutically effective amount of the targeting complexes described by the present invention. Furthermore, it is an objective of the present invention to provide uses of the targeting complex described by the present invention for the preparation of a medicament for the treatment of a premalignant and/or malignant conditions in an individual in need thereof. |
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