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<SOH> SUMMARY OF THE INVENTION <EOH>The foregoing object is achieved by providing a putter comprising a shaft and a club head having a striking surface, wherein the striking surface is arranged on an elevation provided on the club head. Guide brackets are provided on the club head and form a corridor in a direction away from the striking surface. The guide brackets form an opening for the ball passage with their ends. This opening can be changed by the guide brackets using a unit-spacing arrangement and can be coordinated during practice with the respective level of play. It is also conceivable for these guide brackets to be plugged onto a commercially available putter, for example onto the rear of the latter, by means of a corresponding retaining device. In the case of a successfully executed stroke, the ball passes through the opening and the ball corridor, formed by the guide brackets, and, following impact against the centrally hit striking surface, leaves the ball corridor again through the opening, in the opposite direction, in extension of the swing line. If the ball, in contrast, does not precisely hit the center of the striking surface, i.e. if the ball strikes against one of the longitudinal edge of the striking surface or against a deflecting surface adjacent to the longitudinal edges of the striking surface, it is deflected by the impulse component occurring transversely to the swing line and gets caught in the ball corridor, as a result of which the failure of the stroke is immediately apparent. A striking plate assigned to the striking side preferably has a striking surface which is provided on an elevation comprising four deflecting surfaces and of which the width is kept narrow in comparison with the club-head width, which is important for the centrality with which the ball is hit insofar as the ball here is hit at the center of gravity of the putter. The vertically oriented width of the striking surface here is preferably approximately 5 mm, although it may also be adapted to other requirements. For professionals, this striking surface may be round; for normal players, a rectangular shape is preferred since there is more hitting surface available with the longer rectangle sides arranged horizontally. If the user is then more proficient, it is possible for the rectangular striking surface to be positioned vertically and for the hitting surface thus to be reduced in size. A round or relatively small hitting surface teaches the hitting moment and allows even better teaching of the horizontal and vertical sweetspot. It is possible for the striking plate to be assigned not just to a commercially available club head of a putter or to a special club head of a practice putter, but also, for example, to a corresponding attachment on a commercially available putter or practice putter. A further striking plate of the same design is preferably located on the rear side of the club. It differs from the striking plate on the striking side merely by way of the striking surface being approximately double the width. The two striking plates can be exchanged for one another, with the result that striking surfaces of different sizes are available. Since the opening for the ball passage can be coordinated both as desired and with the size of the striking surface, the putter can be set individually to any level of play for practicing a straight putting movement and for the centrality with which the ball is hit. Moreover, a fair number of proficient players use the striking plate with ball corridor for stroke practice, but thereafter use the second striking plate on the rear side of the club head. A further hitting-moment factor is constituted by the vertical striking angle at which, with correct stroke execution together with the rest of the hitting-moment factors, the ball is hit precisely at the vertex of the curve of the swing. Rotating the striking plate through 90° results in the width of the striking surface being located transversely to the putter and thus deliberately provides the ball with less striking surface in the vertical direction, with the result that this ideal hitting point (sweetspot) is only hit when followthrough takes place with the putter at an ideal height in relation to the practice green. If the ball is hit beneath or above the vertex, i.e. if it strikes against one of the longitudinal edges of the striking surface or against one of the adjacent deflecting surfaces thereof, the ball “jumps” or behaves in a manner similar to when the width of the striking surface is positioned vertically, which always gives an immediate indication of the failure of the stroke. The task of orienting the striking-surface position in relation to the aiming or putting line at the hitting moment is assisted in a sustained manner by two direction markings which are arranged in alignment, at different height, on the top side of the club head, directly behind the shaft. Since the direction markings are located at different heights, they can only be brought together with the aiming or putting line on a common direction line if the player's eyes are vertically above the direction markings when the player is in the conventional putting posture, which is advantageous in any case for a straight putting movement. Optical problems mean that the putter is still difficult to align correctly in relation to the aiming line, this often also being the cause of frequent errors during putting. A remedy to this source of error is often to be found by drawing a line on the ball by means of a template, aligning the ball with the hole with reference to this marking and positioning the striking surface of the putter at right angles to the ball marking. This method is further improved by an aiming clip. The aiming gap of the aiming clip, which is positioned in a removable manner on the club head, is easily brought into alignment, above the ball, with the marking on the latter as the putter sits on the practice green. Since the aiming gap is aligned with the longitudinal axis of the club head, the putter is inevitably brought into correct alignment, which also results in the optimum ball position between the guide brackets. Improved aiming may also be achieved using a rubber cable which is tensioned between the club head and a ground anchor behind the hole. When the ball is struck, the cable then has to extend precisely over the ball. It is possible for the cable simply to be fitted into the aiming clip or to be fitted by means of a clamping device on the shaft or on the shaft extension of this practice putter or any desired commercially available putter. In an exemplary embodiment according to the invention, a releasable mount is provided for securing the rubber cable on the shaft. This releasable mount essentially comprises a strip and a tensioning lever. The strip is changeable in length and/or flexible. In the use position, the strip wraps around the shaft of the putter, it being possible for the tensioning lever to be tilted in relation to the strip so that it is supported firmly against the shaft. The strip is thus tensioned and secured on the shaft itself. For better guidance of the tensioning lever in relation to the shaft, an end surface of the tensioning lever which strikes against the shaft is designed as a notched surface. In the tensioning position, the tensioning lever is located approximately horizontally, with the result that it can serve for securing the rubber cable. A retaining needle can preferably be inserted into the tensioning lever for this purpose, the rubber cable being secured on said retaining needle. In order to take account of a different sweetspot of the putter, the tensioning lever has a plurality of bores, with the result that the retaining needle can be adjusted precisely over the sweetspot. The significant advantage of such an aiming means is that the player's eye can remain on the ball rather than having to follow the putter. The rubber cable has to be located precisely over the point at which the ball is hit. The rubber cord itself is highly elastic, with the result that it does not counteract swinging of the putter by any opposing force. Even once the ball has been struck, the player can track whether the striking surface is still running perpendicularly to the cord. As a result, he/she controls the stroke not just before the ball is hit but also thereafter. It is also conceivable for a rubber cable to be tensioned between two ground anchors, one behind the hole and the other behind the ball. The aiming clip may then be replaced by a vertically positioned clip with a hollow in which the cable runs when the ball is struck. Simply by releasing the two striking plates and screwing them tight, it is possible to change the shaft-inclination angle therebetween, in accordance with requirements of the practicing golfer, by virtue of a shaft-retaining plate with the shaft being rotated. The putter can be used equally well for right-handed players and left-handed players by the striking plate which is mounted on the striking side being unscrewed, the adjacent shaft-retaining plate, together with the shaft, being drawn off from the threaded pin and being plugged onto the threaded pin again, in a state in which it has been rotated through 180°, and positioned in a force-fitting manner by means of the striking plate being screwed on again. Different shaft lengths can readily be adapted to different heights and arm lengths by virtue of the shaft, including the shaft-retaining plate, being exchanged. By means of a damping plate, which can be used in materials of different weights, the firmness of stroke of the practice putter can easily be adapted to that of the usual putter. The present invention can, of course, be used on the practice green to play the ball to any desired hole, which seems indispensable for comprehensive training success. At the same time, this variable putter can be used to teach the feel for stroke control and to practice reading of the green.

Methods of recycling and/or upgrading olefin (co)polymers
The present invention relates to a method of recycling and/or upgrading an olefin (co)polymer, olefin (co)polymer scrap and/or mixtures thereof including adding effective amounts of a vinyl silane and a free radical initiator to graft the vinyl silane to the olefin (co)polymer. The present invention also relates to olefin (co)polymers, olefin (co)polymer scrap and/or mixtures thereof whenever recycled and/or upgraded by the method defined above and articles which are composed wholly or partly of them.
1. A method of recycling and/or upgrading olefin (co)polymer scrap and/or mixtures of olefin (co)polymer scrap and an olefin (co)polymer, said method comprising adding effective amounts of a vinyl silane and a free radical initiator to graft the vinyl silane to the olefin (co)polymer. 2. A method of upgrading olefin (co)polymer scrap and/or mixtures of olefin (co)polymer scrap and an olefin (co)polymer which fails the ESCR test as defined in ASTM No. D1693B, said method comprising adding effective amounts of a vinyl silane and a free radical initiator to graft the vinyl silane to the olefin (co)polymer. 3. A method according to claim 1, in which the olefin (co)polymer is an ethylene (co)polymer. 4. A method according to claim 3, in which the ethylene (co)polymer is polyethylene, ethylene-propylene copolymer, ethylene-propylene-diene terpolymer (EPDM), ethylene vinyl acetate copolymer (EVA) or copolymers of ethylene-alkyl acrylates. 5. A method according to claim 4, in which the polyethylene is high density polyethylene (HDPE), medium density polyethylene (MDPE), low density polyethylene (LDPE), very low density polyethylene (VLDPE), ultra low density polyethylene (ULDPE) or linear low density polyethylene (LLDPE). 6. A method according to claim 4, in which the copolymers of ethylene-alkyl acrylates are ethylene-ethyl acrylate (EEA), ethylene-butyl acrylate (EBA) and their terpolymers with maleic anhydride or mixtures thereof. 7. A method according to claim 1, in which the olefin (co)polymers are metallocene catalyst (co)polymers. 8. A method according to claim 1, in which the (co)polymers have a specific gravity (S.G.) of above about 0.936. 9. A method according to claim 8, in which the (co)polymer is HDPE. 10. A method according to claim 9, in which the HDPE has a S.G. above about 0.942. 11. A method according to claim 10, in which the HDPE has a S.G. above about 0.945. 12. A method according to claim 10, in which the HDPE has a S.G. about 0.95 to about 0.96. 13. A method according to claim 1, in which the polymer is a homopolymer. 14. A method according to claim 1, in which the (co)polymer is collected, sorted, washed, granulated, pelletised, ground and/or filtered prior to the grafting step. 15. A method according to claim 14, in which the (co)polymer is dried prior to the grafting step. 16. A method according to claim 15, in which the (co)polymer is dried to moisture levels less than about 500 ppm. 17. A method according to claim 16, in which the (co)polymer is dried to moisture levels less than about 200 ppm. 18. A method according to claim 1, in which the vinyl silane is a vinyl alkoxy silane. 19. A method according to claim 18, in which the vinyl alkoxy silane is vinyl-tris-methoxy-silane (VTMOS), vinyl-tris-methcxy-ethoxy-silane, vinyl-tris-ethoxy-silane, vinyl-methyl-dimethoxy-silane or gama-methacryl-oxypropyl-tris methoxy-silane. 20. A method according to claim 19 in which the amount of vinyl silane and free radical initiator is about 0.5 to about 2.4% by weight of the (co)polymer. 21. A method according to claim 20, in which the amount of vinyl silane and free radical initiator is about 0.8% to about 2% by weight of the (co)polymer. 22. A method according to claim 18, in which the free radical initiator is a peroxide. 23. A method according to claim 22, in which the peroxide is dialkyl peroxide or diaryl peroxide. 24. A method according to claim 23, in which the dialkyl peroxide or diaryl peroxide is dicumyl peroxide (DCP, Dicup), di-tertiary-butyl peroxide (DTEP), di-tertiary-butyl-cumyl peroxide (DTBCP), di (tert-butylperoxy-isopropyl) benzene (Luperox F) or 2,5-dimethyl-2,5-di (tert butylperoxy) hexane (Luperox 101). 25. A method according to claim 23, in which the amount of the free radical initiator is about 0.05% to about 0.3% by weight of the (co)polymer. 26. A method according to claim 25, in which the amount of the free radical initiator is about 0.08% to about 0.2% by weight of the (co)polymer. 27. A method according to claim 26, in which the amount of the free radical initiator is about 0.10% to about 0.16% by weight of the (co)polymer. 28. A method according to claim 1, in which the vinyl silane and free radical initiator are pre-mixed and added to the (co)polymer in their mixed form. 29. A method according to claim 1, in which the ratio of free radical initiator to vinyl silane is about 1:10 to about 1:15. 30. A method according to claim 1, in which the grafting step is performed at a melt temperature of from about 180° C. to about 230° C. 31. A method according to claim 30, in which the grafting step is performed at a melt temperature of from about 190° C. to about 220° C. 32. A method according to claim 31, in which the grafting step is performed at a melt temperature of from about 200° C. to about 220° C. 33. A method according to claim 1, in which one or more additives and/or fillers known in the art of polymer processing are added either before, during or after grafting of the vinyl silane. 34. A method according to claim 33, in which the additives are antioxidants, processing and/or thermal stabilisers, metal deactivators and/or copper inhibitors, UV stabilisers, blowing agents, catalysts, pigments, fillers and/or flame retardants. 35. A method according to 34, in which the amount of additive is up to about 10% by weight based on the (co)polymer and the amount of filler is up to about 50% by weight of the (co)polymer. 36. A method according to claim 1, in which either before, during or after the grafting step, other (co)polymers, their scrap and/or mixtures thereof are added to enhance the properties of the recycled and/or upgraded (co) polymer. 37. A method according to claim 36 in which the other (co)polymer is polyolefin, polypropylene, nitrile butadiene rubber, chlorinated polyethylene or chloro-sulfonated polyethylene. 38. A method according to claim 37, in which the polyolefin is ethylene vinyl acetate copolymer (EVA), ethylene ethyl acrylate copolymer (EEA), ethylene butyl acrylate copolymer (EBA), ethylene propylene rubber (EPR), ethylene-propylene copolymer (EPM), ethylene propylene-diene terpolymer (EPDM), ethylene-butylene copolymer (EBM), ethylene butylene-diene terpolymer (EBDM), very low density polyethylene (VLDPE) ultra linear low density polyethylene (ULDPE) linear low density polyethylene (LLDPE), low density polyethylene (LDPE) or medium density polyethylene (MDPE). 39. A method according to claim 38, in which the other (co)polymer is added in an amount of from about 5% to about 50% by weight of the (co)polymer. 40. A method according to claim 1, in which the recycled and/or upgraded (co)polymer is formed. 41. A method according to claim 40, in which the forming is conducted by injection moulding, blow moulding, compression moulding, extrusion calendering or conversion processes. 42. A method according to claim 41, in which the (co)polymer is cross-linked. 43. A method according to claim 42, in which the (co)polymer is cross-linked using a cross-linking catalyst. 44. A method according to claim 43, in which the cross-linking catalyst is added before, during or after the grafting step. 45. A method according to claim 42, in which the grafted and formed (co)polymer cross-links naturally in the presence of humidity without a catalyst. 46. A method according to claim 42, in which the grafted and formed (co)polymer is cross-linked in the presence of water, water vapour or steam at temperatures from about ambient up to about 115° C. 47. Olefin (co)polymer scrap and/or mixtures of olefin (co)polymer scrap and an olefin (co)polymer recycled and/or upgraded by the method defined in claim 1. 48. An article comprising at least a portion of the olefin (co)polymer scrap and/or mixtures of olefin (co)polymer scrap and an olefin (co)polymer according to claim 47. 49. An article according to claim 48, wherein the article comprises a bottle, container, box, tube, pipe, cable, profile, sheet, film or pre-form. 50. A method according to claim 20, in which the amount of vinyl silane and free radical initiator is about 0.9% to about 1.4% by weight of the (co)polymer. 51. A method according to claim 36, in which the other (co)polymer is added in an amount of from 5 to about 95% by weight of the (co)polymer. 52. A method according to claim 1, wherein the olefin (co)polymer scrap and/or mixtures of olefin (co)polymer scrap and an olefin (co)polymer fails the ESCR test as defined in ASTM No. D 1693B. 53. A method of modifying olefin polymer scrap and/or mixtures of olefin polymer scrap and an olefin polymer, said method comprising: providing olefin polymer scrap material; providing an effective amount of a vinyl silane and a free radical initiator; and reacting the components to graft the vinyl silane to the olefin polymer to form a modified olefin polymer having vinyl silane grafted thereto.



Microorganisms and cells for diagnosis and therapy of tumors
Described are diagnostic and pharmaceutical compositions comprising a microorganism or cell containing a DNA sequence encoding a detectable protein or a protein capable of inducing a detectable signal, e.g. a luminescent or fluorescent protein, and, in a particular embodiment, furthermore (a) DNA sequence(s) encoding (a) protein(s) suitable for tumor therapy and/or elimination of metastatic tumors, e.g. a cytotoxic or cytostatic protein.
1. A method for tumor imaging, monitoring therapeutic tumor treatment or therapeutically treating a tumor, the method comprising: preparing a composition comprising a microorganism or cell containing a DNA sequence encoding a detectable protein or a protein capable of inducing a detectable signal; and administering said composition to a subject in need thereof, wherein said microorganism or cell is capable of accumulating in the tumor and is recognized by the immune system of the patient. 2. The method according to claim 1, wherein said microorganism or cell furthermore contains (a) DNA sequence(s) encoding (a) protein(s) suitable for tumor therapy and/or elimination of metastatic tumors. 3. The method according to claim 1 or 2, wherein said protein capable of inducing a detectable signal is a luminescent and/or fluorescent protein. 4. The method according to claim 2, wherein said protein is a cell receptor capable of binding a ligand. 5. The method according to claim 4, wherein said composition further comprises a ligand which is a radionuclide-labelled ligand. 6. The method according to claim 4, wherein said composition further comprises a ligand which is a paramagnetic- or superparamagnetic-metal-labelled ligand. 7. The method according to claim 4, wherein said composition further comprises a ligand which is coupled to a toxin. 8. The method according to claim 4, wherein said composition further comprises a ligand which is coupled to a therapeutic protein. 9. The method according to claim 8, wherein said ligand is an antibody. 10. The method according to claim 1, wherein said protein capable of inducing a detectable signal is a protein inducing a signal detectable by magnetic resonance imaging (MRI) or capable of binding a contrasting agent, chromophore or a ligand required for visualization of tissues. 11. The method according to, wherein said microorganism is a bacterium or a virus. 12. The method according to claim 11, wherein said bacterium is a magnetic bacterium. 13. The method according to claim 11, wherein said virus is Vaccinia virus. 14. The method according to claim 11, wherein said bacterium is attenuated Vibrio cholerae. 15. The method according to claim 1, wherein said cell is a mammalian cell. 16. The method according to claim 3, wherein said luminescent or fluorescent protein is luciferase, RFP or GFP. 17. The method according to claim 16, wherein said microorganism or cell additionally contains a gene encoding a substrate for a luciferase. 18. The method according to anyone of claims 1 to 17, wherein tumor-imaging or monitoring is carried out by MRI. 19. The method according to claim 1 wherein said composition further comprises (a) an antimicrobial compound fused to a protein suitable for tumor therapy and/or elimination of metastatic tumors and/or (b) an antimicrobial compound fused to a detectable protein or a protein capable of inducing a detectable signal. 20. The method according to claim 1, wherein said protein suitable for therapeutically treating a tumor is endostatin or recombinant chimeric toxin PE37/transforming growth factor alpha (TGF-alpha). 21. The method according to claim 1, wherein said protein suitable for therapeutically treating a tumor is a protein capable of converting a nontoxic prodrug into a toxic drug which is cytotoxic to a tumor. 22. The method according to claim 1, wherein said DNA sequence(s) is (are) on a BAC, MAC, cyber cell or cyber virus. 23. The method according to claim 1, wherein said tumor is a bladder tumor, breast tumor, prostate tumor, glioma tumor, liver tumor, skin tumor, colon tumor, lung tumor, ovarial carcinoma or pancreatic carcinoma.

<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 : External imaging of GFP expression in subcutaneous C6 glioma tumors in nude mice C6 glioma cells (5×10 5 ) were implanted subcutaneously into the right lateral thigh. At designated days after tumor cell implantation, the animals were infected intravenously with 1×10 8 pfu of rVV-ruc-gfp virus particles. GFP expression was monitored under a fluorescence stereomicroscope. Bright field (top), fluorescence (middle), and bright field, fluorescence overlay (bottom) images of subcutaneous glioma tumor are shown. GFP signal can be observed in tumors as small as 22 m 3 in size (B-B″), or as old as 18 days (about 2500 mm 3 in size) (A-A″). In older tumors, GFP expression was seen in “patch”-like patterns (indicated by arrows in A′). Marker gene expression in the tumor of the same animal can be monitored continuously 4 (C-C″), 7 (D-D″), and 14 (E-E″) days after intravenous viral injection. (Bars=5 mm.) FIG. 2 : Visualization of tumor angiogenesis C6 glioma cells (5×10 5 ) were implanted subcutaneously into the right lateral thigh of nude mice. Ten days after tumor cell implantation, the animals were infected intravenously with 1×10 8 pfu of rVV-ruc-gfp. GFP expression was monitored 7 days post-viral injection. Vascularization at the surface of the subcutaneous C6 glioma tumor is shown against the bright green fluorescent background in the tumor following Vaccinia-mediated gene expressions. Bright field (A), fluorescence (B), and bright field, fluorescence overlay (C) images of subcutaneous glioma tumor are illustrated. (Bars=5 mm.) FIG. 3 : Expression of GFP in subcutaneous glioma tumor of the same animal Five days after the subcutaneous implantation of 5×10 5 C6 glioma cells into the right lateral thigh, 10 8 of rWV-ruc-gfp virus particles were injected intravenously. Five days after viral injection, the animal was anesthetized and sacrificed for analysis of GFP expression under fluorescence microscope. The tumor was visualized externally (A-K), with the overlying skin reflected (B-B″), in cross section (C-C″), and in the amputated leg (D-D″). Bright field (A), fluorescence (B), and bright field, fluorescence overlay (C) images of subcutaneous glioma tumor are illustrated. The strongest GFP expressions are seen as patches located along the outer surface of the tumor on the right (double arrows in C-C″). Sharp difference of GFP expression in tumor tissue and in the normal muscle tissue (arrows in D-D″) is clearly visible. Asterisks mark the reflected skin (B-B″ and D-D″). (Bars=5 mm.) FIG. 4 : Bright field (A) and fluorescence (B) images of tumor cells expressing GFP Frozen sections (30 μm thick) of the glioma tumor tissues were prepared from a nude mouse that has been intravenously injected with 1×10 8 of rVV-ruc-gfp virus particles. (Bars=50 Wm.) FIG. 5 : Low light image of the anesthetized nude mouse to indicate the location of Renilla luciferase-triggered light emission in the presence of intravenously injected substrate coelenterazine (5 μg ethanol solution) FIG. 6 : Monitoring tumor-specific viral infection based on GFP gene expression in a variety of tumor models including subcutaneous PC-3 human prostate tumor (A-A″) and MCF-7 human breast tumor (B-B″) in nude mice, intracranial C6 rat glioma tumor (C-C″, arrows indicate the location of the tumor) in Lewis rats, and MB-49 mouse bladder tumor (D-D″) in C57 mice. Animals were monitored 7 days after intravenous injections of 1×10 9 of rVV-ruc-gfp virus particles. Bright field (top), fluorescence (middle), and bright field, fluorescence overlay (bottom) images of the tumor are illustrated. (Bars=5 mm.) FIG. 7 Monitoring Vaccinia-mediated GFP expression in a breast tumor model Nude mouse carrying breast tumor was injected intravenously with 1×10 8 of rVV-ruc-gfp virus particles. Both the primary tumor (A-A″, B-B″, and C-C″) and the metastasized tumor (D-D″, E-E″, and F-F″) were visualized externally (A-A″ and D-D″), with overlying skin removed (B-B″ and E-E″), and when they were split open (C-C″ and F-F′) in a set of bright field, fluorescence (′) and bright field, fluorescence overlay (″) images. GFP expression in lung metastases in the same animal was also visualized (G-G″). (Bars=5 mm (A-A″ to F-F″), and Bars 1 mm (G-G″) FIG. 8 : Visualization of the clearance of light emitting bacteria from nude mice based on the detection of light emission under the low light imager Nude mice were intravenously injected with 10 7 cells of attenuated S. typhimurium (A, B) and V. cholera (C, D). Both strains were transformed with pLITE201 carrying the lux operon. Photon collection was done 20 min (A, C) and 2 days (B, D) after bacterial injections. FIG. 9 : Homing of glioma gumors by attenuated bacteria Nude mice with a C6 glioma tumor in the right hind leg were intravenously injected with 10 7 attenuated S. typhimurium (A-D) and with V. cholera (E-H) both transformed with pLITE201 plasmid DNA encoding the lux operon. Photon collection was carried out for one minute under the low light imager. Mice injected with S. typhimurium exhibited luminescence immediately through the whole animal (A). In contrast, luminescence in the mice injected with V. cholera was visible in the liver area (E). Two days after bacterial injection, both groups of mice demonstrated luminescence only in the tumor region (B, F). The light emission in the tumors infected with S. typhimurium slowly diminished four (C) and six (D) days after bacterial injection. Tumors infected with V. cholera showed enormously increased light emission four (G) and six (H) days after injection suggesting continued replication of the bacteria in the tumor tissues. FIG. 10 : Homing in of bacteria to breast tumors Nude mice with breast tumors in the right breast pad were intravenously injected with 10 7 attenuated V. cholera (A-D) or with 10 7 B. coli (E-F) transformed with pLITE201 plasmid DNA encoding the lux operon. Photon collection was carried out for one minute under the low light imager. Twenty minutes after (bacterial delivery, luminescent V. cholera were observed in the liver (A). Forty-eight hours after injection, light emission was noted in the primary breast tumor in the right breast area and a metastatic tumor (arrow) in the left breast area, and in the incision wound (B). At five days, the light emission was visible only in the tumor regions, and not at the wound (C). Eight days after bacterial injection, the luminescent activity was abolished from the smaller tumor region but remained strong in the primary breast tumor (D). Homing in of E. coli to breast tumors in nude mice was also observed two days after intravenous bacterial injection (E: side view, F: ventral view). FIG. 11 : Homing in of bacteria to bladder tumors in C57 mice C57 mice were intravenously injected with 10 7 attenuated V. cholera transformed with pLITE201 encoding the lux operon. Nine days after bacterial delivery, luminescence was noted in the bladder region of the whole animal (A). The animal was sacrificed and an abdominal incision was made to expose the bladder. The light emission was limited to the bladder region (B). With the removal of the bladder (C) from the mouse, the entire source of light emission was removed (D) as demonstrated by the overlay of the low light photon emission image over the photographic image of the excised bladder. FIG. 12 : Homing in of bacteria to brain glioma tumors in Lewis rats Lewis rats were intravenously injected with 10 8 cells of attenuated V. cholera transformed with pLITE201 encoding the lux operon. Twenty-four hours after bacterial injection, faint luminescence was noted in the head region of the whole animal during visualization under the low light imager. The animals were sacrificed and their brain removed. Photon collection was carried out for one minute from rats with (A) and without (B) brain tumors. Strong luminescence was confirmed in regions of the brain of the rats with the brain tumor (marked with arrows in A). Luminescence was completely absent in the control brain tissues (B). FIG. 13 : Transformed human fibrosarcoma cells home in on subcutaneous glioma tumors in nude mice Nude mice with subcutaneous glioma tumors were injected intravenously with 5×10 5 human fibrosarcoma cells, which were permanently transformed with retrovirus derived from pLEIN. Seven days post-injection, the animals were anesthetized and monitored under a fluorescent stereomicroscope. Fluorescent cells were noted only in the tumor region of the whole mice through the skin (A1-3). Upon exposure of the tumor tissues by reflection of the overlying skin (B1-3), and in cross sections of the tumors (C1-3), fluorescent patches were visible in distinct regions. Close examination of the organs of the mice showed the presence of small clusters of fluorescent cells in the lungs of the animals, demonstrating the affinity of the fibrosarcoma cells for the lungs in addition to the tumorous tissues (D1-3). (Bars=5 mm (A1-C3), =1 mm (D1-D3)). FIG. 14 : Homing of attenuated Listeria monocytogene into subcutaneous prostate tumors Nude mice with subcutaneous human PC3 prostate tumor in the right hind leg were intravenously injected with 10 7 attenuated L. monocytogenes transformed with psod-gfp plasmid DNA carrying the gfp cDNA, GFP fluorescence was observed under a fluorescence stereo microscope. Twenty-seven hours after bacterial injection, GFP signal was detected only in the tumor region. The tumor is shown in a set of visible light (a), fluorescent (b), and visible and fluorescent light overlay (C) images. (Bars=5 mm.) detailed-description description="Detailed Description" end="lead"? The present invention is explained by the examples.

Use of biologically active hiv-1 tat, fragments or derivatives thereof, to target and/or to activate antigen-presenting cells, and/or to deliver cargo molecules for preventive or therapeutic vaccination and/or to treat other diseases
The present invention concerns a method for prophylactic and/or therapeutic vaccination and/or treatment and/or diagnosis of HIV/AIDS, other infectious diseases, inflammatory and angiogenic diseases and tumours which utilizes a biologically active HIV-1 Tat protein, fragments or derivates thereof, as a module with one or more of the following features: antigen, adjuvant and targeting-delivery system to specific antigen-presenting cells including dendritic cells, endothelial cells and macrophages. In particular, it is claimed that Tat can be used only in its biologically active form as an antigen combined with one or more other antigens, to prime or to boost protective immune responses against itself as well as other antigens and/or to selectively deliver these antigen(s) as well as active compounds to dendritic cells, endothelial cells and macrophages, due to its capability of targeting these A PC and of activating their maturation and functions and of increasing Th-1 type immune responses as an adjuvant. Therefore, due to these characteristics and to the distribution of these cells in the body (during physiological and pathological disorders), biologically active Tat, fragments or derivates thereof containing the RGD region, can be used for preventive, therapeutic and/or diagnostic purposes for HIV/AIDS, other infectious diseases, inflammatory and angiogenic diseases and tumors.
1. Use of isolated native, substantially monomeric, and biologically active HIV-1 Tat, fragments or derivatives thereof, to selectively target antigen presenting cells expressing α5β1 and/or αvβ3 integrins, including dendritic cells, endothelial cells and macrophages, to deliver cargo molecules across their cellular and/or nuclear membrane and to induce their maturation and/or their antigen presenting functions. 2-55. (Canceled) 56. Method to selectively target antigen presenting cells expressing α5β1 and/or αvβ3 integrins, including dendritic cells, endothelial cells and macrophages, to deliver cargo molecules across their cellular and/or nuclear membrane and to induce their maturation and/or their antigen presenting functions said method comprising the step of putting in contact the cells with an effective amount of isolated native, substantially monomeric, and biologically active HIV-1 Tat or tat DNA, fragments and derivatives thereof. 57. Method according to claim 56 wherein isolated native, substantially monomeric, and biologically active HIV-1 Tat, fragments or derivatives thereof, will selectively target antigen presenting cells expressing α5 β1 and αvβ3 integrins, including dendritic cells, endothelial cells and macrophages, for the uptake of both Tat, fragments, derivatives thereof selectively bound to these cells, and cargo molecules bound to Tat, fragments, derivatives thereof. 58. Method according to claim 56 wherein isolated native, substantially monomeric, and biologically active HIV-1 Tat, fragments or derivatives thereof, will selectively target, bind and enter antigen presenting cells expressing the α5β1 and αvβ3 integrins, including dendritic cells, endothelial cells and macrophages, to induce their maturation and/or their antigen presenting functions. 59. Method according to claim 57 to selectively target antigen presenting cells expressing α5β1 and αvβ3 integrins, or other integrins, including, but not limited to, dendritic cells, endothelial cells and macrophages capable of taking up Tat via the integrin-mediated pathway and/or other uptake pathways conferring a selective uptake, in order to deliver antigens and/or therapeutic compounds. 60. Method according to claim 59 wherein Tat delivers antigens in the form of peptides, proteins or DNA encoding them. 61. Method according to claim 60 wherein Tat delivers one or more antigens to induce an immune response. 62. Method according to claim 61 wherein antigens are selected among antigens from intracellular pathogens such as viruses, mycobacterium tuberculosis, candida, malaria, or from tumor cells such as those from lung, colon, breast, prostatic cancer, but specifically excluding HIV antigens Gag, Nef, Rev. 63. Method according to claim 59 wherein Tat is fused to one or more compounds selected among proteins, peptides or DNA encoding them in order to deliver such compound (s), in vitro and in vivo, intracellularly or to the cell membrane. 64. Method according to claim 63 wherein compound is one or more antigens selected among antigens from intracellular pathogens such as viruses, mycobacterium tuberculosis, candida, malaria, or from tumor cells such as those from lung, colon, breast, prostatic cancer, but specifically excluding HIV antigens Gag, Nef, Rev. 65. Method according to claim 63 wherein the compound to be fused with Tat is one or more therapeutic molecules selected among antiviral compounds, anti-inflammatory drugs, anti-angiogenic molecules, cytotoxic anti-tumor drugs, immunomodulating molecules such as chemokines or cytokines, antibodies and corresponding mixtures. 66. Method according to claim 57 wherein Tat is bound, alone or in combination with other compounds in the form of proteins, peptides or DNA encoding them, to particles such as microparticles, nanoparticles, liposomes and other particulated inert carriers and mixtures thereof. 67. Method according to claim 66 wherein compound is one or more antigens selected among antigens from intracellular pathogens such as viruses, mycobacterium tuberculosis, candida, malaria, or from tumor cells such as those from lung, colon, breast, prostatic cancer, but specifically excluding HIV antigens Gag, Nef, Rev. 68. Method according to claim 66 wherein compound is one or more therapeutic molecules selected among antiviral compounds, anti-inflammatory drugs, anti-angiogenic molecules, cytotoxic anti-tumor drugs, immunomodulating molecules such as chemokines or cytokines, antibodies and corresponding mixtures. 69. Method according to claim 66 wherein compound is one or more expression vectors. 70. Method according to claim 69 wherein the expression vector is selected among plasmid DNA, bacterial or virus vectors expressing one or more antigens. 71. Method according to claim 58 to selectively target antigen presenting cells expressing α5β1 and αvβ3 integrins or other integrins, including, but not limited to, dendritic cells, endothelial cells and macrophages capable of taking up Tat via the integrin-mediated pathway and/or other uptake pathways conferring a selective uptake, in order to deliver one or more antigens to enhance an immune response and to induce Th-1 type immune responses against infectious diseases and tumors. 72. Method according to claim 71 wherein Tat delivers antigens in the form of peptides, proteins or DNA encoding them. 73. Method according to claim 72 wherein Tat delivers one or more antigens to induce an immune response. 74. Method according to claim 71 wherein Tat is fused to one or more compounds selected among proteins, peptides or DNA encoding them in order to deliver such compound (s), in vitro and in vivo, intracellularly or to the cell membrane. 75. Method according to claim 74 wherein compound is one or more immunomodulating molecules, such as chemokines or cytokines, antibodies and corresponding mixtures. 76. Method according to claim 74 wherein compound is one or more antigens selected among antigens from intracellular pathogens such as viruses, mycobacterium tuberculosis, candida, malaria, or from tumor cells such as those from lung, colon, breast, prostatic cancer, but specifically excluding HIV antigens Gag, Nef, Rev. 77. Method according to claim 58 wherein Tat is bound, alone or in combination with other compounds in the form of proteins peptides or DNA encoding them, to particles such as microparticles, nanoparticles, liposomes and other particulated inert carriers and mixtures thereof. 78. Method according to claim 77 wherein compound is one or more antigens selected among antigens from intracellular pathogens such as viruses, mycobacterium tuberculosis, candida, malaria, or from tumor cells such as those from lung, colon, breast, prostatic cancer, but specifically excluding HIV antigens Gag, Nef, Rev. 79. Method according to claim 77 wherein compound is one or more immunomodulating molecules, such as chemokines or cytokines, antibodies and corresponding mixtures. 80. Method according to claim 77 wherein compound is one or more expression vectors. 81. Method according to claim 80 wherein the expression vector is selected among plasmid DNA, bacterial or virus vectors expressing one or more antigens. 82. Method for preventive and therapeutic vaccination against tumors infectious diseases said method comprising the step of administering to a subject in need an effective amount of HIV-1 Tat or tat DNA according to claim 55. 83. Method for the treatment of tumors or infectious diseases or inflammatory and angiogenic diseases said method comprising the step of administering to a subject in need an effective amount of HIV-1 Tat or tat DNA according to claim 55. 84. Method according to claim 82 wherein tat DNA is in combination with antigens selected among antigens from intracellular pathogens such as viruses, mycobacterium tuberculosis, candida, malaria, or from tumor cells such as those from lung, colon, breast, prostatic cancer, but specifically excluding HIV antigens Gag, Nef, Rev. 85. Method according to claim 83 wherein tat DNA is in combination with antigens selected among antigens from intracellular pathogens such as viruses, mycobacterium tuberculosis, candida, malaria, or from tumor cells such as those from lung, colon, breast, prostatic cancer, but specifically excluding HIV antigens Gag, Nef, Rev. 86. Method according to claim 82 wherein tat DNA is bound, alone or in combination with other antigens DNA or therapeutic compounds to particles such as microparticles, nanoparticles, liposomes and other particulated inert carriers and mixtures thereof. 87. Method according to claim 83 wherein tat DNA is bound, alone or in combination with other antigens DNA or therapeutic compounds to particles such as microparticles, nanoparticles, liposomes and other particulated inert carriers and mixtures thereof. 88. Method according to claim 56 wherein tat DNA comprises the nucleotide sequence according to SEQ ID NO. 1, 99, or 101. 89. Method according to claim 56 wherein Tat comprises the amino acid sequence according to SEQ ID NO. 2, 100, or 102. 90. Method according to claim 56 wherein the fragments of biologically active Tat or tat DNA are selected among Tat peptides or corresponding tat DNA comprising, alone or associated, the RGD domain, the cystein-rich domain, the basic domain. 91. Method according to claim 90 wherein the fragments are combined with other HIV-1 Tat peptides or corresponding tat DNA comprising the core domain: aa 38 to 47 in the HTLV-IIIB, clone BH-10, and/or the amminoterminal region aa 1 to 20 in the HTLV-IIIB, clone BH-10. 92. Method according to claim 90 wherein the RGD domain comprises: aa 73 to 86 in the HTLV-IIIB, clone BH-10, aa 74 to 84, aa 75 to 83, aa 76 to 82, aa 77 to 81, aa 77 to 82, aa 77 to 83, aa 76 to 83; the cystein-rich domain comprises: aa 22 to 37 in the HTLV-IIIB, clone BH-10; the basic domain comprises: aa 48 to 61 in the HTLV-IIIB, clone BH-10, and all their corresponding nucleotide sequences. 93. Method according to claim 56 wherein the fragments of biologically active Tat or tat DNA are selected among any HIV variant (HIV-1, HIV-2 and other HIV types and subtypes) that contain one or more T-cell epitopes in their amino acid sequences or corresponding nucleotide sequence HTLV-IIIB, clone BH-10 or 89.6. 94. Method according to claim 56 wherein the derivatives of Tat or tat DNA comprise Tat mutants of the HTLV-IIIB, clone BH-10, variant, selected among that ones comprising the amino acid sequence or corresponding nucleotide sequences, of cys22 and/or lys41. 95. Pharmaceutical composition comprising as active principle an effective amount of biologically active HIV Tat or tat DNA, fragments or derivative thereof, combined or fused with at least one of the following: antigens, therapeutic compounds, adjuvants, support particles, for preventive and therapeutic vaccination against infectious diseases and tumors or for the treatment of a disease selected among infectious diseases, inflammatory and angiogenic diseases, tumors. 96. Pharmaceutical composition according to claim 95 wherein the antigen is selected among antigens from intracellular pathogens such as viruses, mycobacterium tuberculosis, candida, malaria, or from tumor cells such as those from lung, colon, breast, prostatic cancer, but specifically excluding HIV antigens Gag, Nef, Rev. 97. Pharmaceutical composition according to claim 95 wherein the therapeutic compound is selected among antiviral compounds, anti-inflammatory drugs, anti-angiogenic molecules, cytotoxic anti-tumor drugs, immunomodulating molecules such as chemokines or cytokines, antibodies and corresponding mixtures. 98. Pharmaceutical composition according to claim 95 wherein the support particles are selected among: microparticles, nanoparticles, liposomes and other particulated delivery systems and mixtures thereof. 99. Pharmaceutical composition according to claim 95 wherein the adjuvant is selected among Alum, RIBI, ISCOMS, CpG sequence, Lipopeptides and corresponding mixtures. 100. Pharmaceutical composition according to claim 95 wherein the infectious disease is selected among those infections caused by human or animal viruses, bacteria or other intracellular and extracellular pathogens, including sexual infectious diseases, endocarditis, urinary tract infections, osteomyelitis, cutaneous infections, or streptococcus and staphylococcus infections, pneumococcus infections, tetanus, meningococcus infections, tuberculosis, malaria, candidosis, infections by Helicobacter, salmonella, syphilis, herpetic infections, including varicella, mononucleosis and Epstein-Barr-derived infections, human herpesvirus-8 infection, cytomegalovirus, herpes labialis and genitalis, hepatitis virus infection (A, B, C, D, G), papilloma virus-derived infections, influenza, lysteria, vibrio cholerae. 101. Pharmaceutical composition according to claim 95 wherein the inflammatory disease is an allergy or inflammation associated or not with a viral, bacterial or parasitic infection, including immune-mediated cutaneous diseases, Lupus erythematous systemic, rheumatoid arthritis, systemic sclerosis, dermatomiositis, Sjögren syndrome, Goodpasture syndrome, vasculitis, sarcoidosis, osteoarthrosis, infectious arthritis, psoriasis, Chron disease, rectocolitis ulcerosus, tyroiditis, scleroderma, allergic diseases. 102. Pharmaceutical composition according to claim 95 wherein the angiogenic disease is selected among non-neoplastic angioproliferative diseases including diabetic retinopathy, retrolental fibroplasia, trachoma, vascular glaucoma, immune inflammation, non-immune inflammation, atherosclerosis, excessive wound repair, angiodermatitis, colon angiodisplasia, angioedema and angiofybromas. 103. Pharmaceutical composition according to claim 95 wherein the tumor is selected among benign and malignant tumors including tumors of soft tissues, bones, cartilages and blood, such as, but not limited to, Kaposi's sarcoma and other neoplasia of the skin, lung, breast, gut, liver, pancreas, endocrine system, uterus, ovary, sarcomas, acute and chronic leukemia, and neoplasia of lymphatic cells. 104. Pharmaceutical composition according to claim 95 further comprising adjuvants, diluents, eccipients, carriers. 105. Pharmaceutical composition according to claim 95 in the form of tablets, pills, sprays, injectable solutions, suspensions, powders, creams, ointments. 106. Pharmaceutical composition according to claim 95 administered by the parenteral: subcute, intramuscular, intradermic; or mucosal: vaginal, rectal, oral, nasal; or topic route.
<SOH> BACKGROUND OF THE INVENTION <EOH>Tat is a regulatory protein of human immunodeficiency virus type 1 (HIV-1) produced very early after infection and essential for virus gene expression, replication and infectivity (Arya 1985; Fisher 1986; Chang 1995). During acute infection of T cells by HIV, Tat is also released in the extracellular milieu and taken-up by neighbour cells (Frankel 1988; Ensoli 1990; Ensoli 1993; Chang 1997) where, according to the concentration, can increase virus infectivity. Specifically, upon uptake Tat can enhance, in infected cells, virus gene expression and replication (Frankel 1988; Ensoli 1993; Chang 1997), and, in uninfected cells, the expression of the β-chemokines receptors CCR5 and CXCR4 favouring transmission of both macrophage and T lymphocyte-tropic HIV-1 strains (Huang 1998; Secchiero 1999). Extracellular HIV-1 Tat protein is also responsible for the increased frequency and aggressiveness of Kaposi's sarcoma (KS) a vascular tumor particularly frequent in HIV-infected individuals (Friedman-Kien 1981; Safai 1985). In particular, previous work from our and other groups indicated that Tat cooperates with angiogenic and inflammatory cytokines that are highly expressed in KS patients (Samaniego 1998; Ensoli 1994) in inducing new blood vessels formation (angiogenesis) and the growth and locomotion of spindle shaped cells of endothelial cell origin (KS cells) and of activated endothelial cells (Barillari 1992; Albini 1995; Ensoli 1994). Moreover, the sequence comprised between residues 21 and 40 (core domain) in the HIV-1 BH-10 Tat protein has been shown to act as a transactivator, to induce HIV replication and to trigger angiogenesis (International Patent number WO 00/78969 A1). In particular, our data have shown that biologically active Tat binds through its RGD region the integrin receptors α5β1 and αvβ3 and that this interaction mediates the adhesion, growth and locomotion induced by Tat on KS cells and endothelial cells activated by inflammatory cytokines (Barillari 1993; Barillari 1999a and 1999b). In addition, Tat acts also as a chemotactic factor for these cell types as well as for monocytes and dendritic cells (DC) (Albini 1995; Benelli 1998; Lafrenie 1996; Mitola 1997). Finally, our data demonstrated that KS and HUVE cell migration and invasion are toward the Tat protein is mediated by the binding of the Tat RGD region to the α 5 β 1 and αvβ 3 integrins (Barillari, 1999b). Consistent with these findings, the immune response to Tat has been shown to play a key role in controlling the progression of AIDS and AIDS-associated diseases. In fact, a Tat-specific immune response is present in HIV-1 infected subjects and simian immunodeficiency virus (SIV)-infected monkeys, and correlates inversely with progression to the symptomatic stage of the infection (Reiss 1990; Venet 1992; Rodman 1993; Froebel 1994; Re 1995; Van Baalen 1997; Zagury 1998; Addo 2001). Moreover, vaccination with biologically active Tat protein or tat DNA induces protection against SHIV89.6P virus replication and disease onset which correlates with the presence of Th-1 responses including specific cytotoxic T lymphocytes (CTLs) (Cafaro 1999; Cafaro 2000; Cafaro 2001, and PCT WO99/27958). The same protection data have been more recently observed with a tat-rev vaccine delivered with viral vectors in macaques (Osterhaus 2001). In contrast, a limited containment of the infection has been observed in monkeys vaccinated with inactivated Tat or Tat peptides, in which antibodies and T helper specific responses but no CTLs nor Th-1 responses had been induced (Goldstein 2000; Pauza 2000). Again, the repeated intradermal (i.d.) inoculation of monkeys with native and active Tat protein alone (in the absence of any adjuvant) at low doses (5-6 μg) selectively induced a Th-1 response and specific CTLs in the absence of any significant antibody production (Cafaro 1999 and PCT WO99/27958). These immunological results were recently confirmed in a new vaccination protocol in which native Tat alone was repeatedly inoculated i.d. in 4 monkeys (unpublished data), and are comparable to those induced by i.m. vaccination with tat DNA in a published (Cafaro 2001 and PCT WO99/27958) and in an ongoing study. Similarly, recent work performed in SIV-infected macaques indicate that anti-Tat CTLs are key to control early virus replication after primary infection and exert a selective immune pressure on the virus leading to the appearance of slowly replicating, less pathogenic escape mutants (Allen 2000). Finally, Tat is presented with major histocompatibility complex (MHC) class I antigen (Moy 1996; Kim 1997), hence inducing anti-Tat CTL (Cafaro 1999). Micromolar concentrations of recombinant Tat protein (often of unknown biological activity) or peptides encompassing the basic region of Tat have been shown to enter many different cell types (Frankel 1988; Mann 1991; Ensoli 1993; Chang 1997; Fawell 1994; Moy 1996; Kim 1997). The highly basic charge of Tat residues 48-57, in fact, enables the protein to bind to heparan sulphate proteoglycans (HSPG) that are present on the membrane of all cell types (Chang 1997; Rusnati 1998). After release from acutely infected cells, a fraction of extracellular Tat binds, through its basic residues, to the HSPG (Chang 1997). This protects extracellular Tat from proteolytic degradation, as previously found for several growth factors (reviewed in Raines and Ross, 1992). Upon the binding of its basic region to cell surface HSPG, Tat is internalised through a receptor-independent pathway (Frankel 1988; Rusnati 1998; Tyagi 2001). In fact, Tat residues 49-57 (in the BH-10 Tat sequence) have been indicated to be able to translocate an OVA peptide into the cytosol of DC and to sensitize CD8+ T cells to this peptide (Kim, 1997). Furthermore, the 47-57 Tat sequence (from the BH-10 variant), fused with several effector proteins, has been suggested to be able to deliver them to cells (International patent number WO 01/19393 A1). However, this internalization mechanism requires high (micromolar) concentrations of Tat, occurs with any cell type and it is not sequence-specific. In fact, it has been shown that mutations of this region, which do not change its basic charge, do not affect the properties of the Tat basic region (Barillari 1999b). Similarly, the substitution of the Tat basic region with that of HIV rev or other genes does not change Tat properties. In this regard, the basic region of Tat has been shown to be very similar to the arginin-rich region carried by the members of the small family of proteins known as penetratins , that are all capable of entering many cell types (Derossi 1998). In fact, arginin homopolymers have been shown to enter cells even more efficiently than Tat basic region (Derossi 1998). The property of the Tat basic region of being internalized by cells has been exploited to deliver foreign proteins to a variety of cell types (Fawel 1994; Wender 2000; and WO 01/19393). To this purpose, foreign proteins have been conjugated or fused to the Tat basic region which has been used as a carrier for the protein to be transduced (Fawel 1994; Wender 2000; and WO 01/19393). However, the inventor believes that due to the ubiquitous expression of HSPG, Tat basic region cannot be used for selective targeting, delivery and/or uptake of Tat by specific primary cell types, including antigen presenting cells (APC). APC initiate and drive the type of immune response upon encountering foreign molecules (Bancherau 1998; Bell 1999). Typical APC include monocyte-derived DC (MDDC), T cell blasts (TCB), B-lymphoblastoid cell lines (BLCL) and monocytes-macrophages (Bancherau 1998; Bell 1999). In addition, when activated by inflammatory cytokines also endothelial cells acquire APC functions (Pober 1988). Among these inflammatory cytokines, interleukin (IL)-1, tumor necrosis factor (TNF) and interferon (IFN)γ are key for endothelial cell activation (Pober 1988). Exposure to these cytokines increases in endothelial cells the expression of α5β1 and αvβ3, that are among the several cell surface receptors binding Tat (Barillari 1993, Fiorelli 1999; Benelli 1998; Kolson 1993; Sabatier 1991; Vogel 1993; Boykins 1999; Ganju 1998; Milani 1996; Mitola 1997 and 2000; Weeks 1993; Albini 1996 and 1998; Chang 1997; Lafrenie 1996; Morini 2000; Rusnati 1998). Among all these APC, DC are the most efficient APC and are key to the induction of immune responses against infections and tumors (Banchereau 1998; Bell 1999). Their function is associated with a high expression of MHC and costimulatory molecules (CD40, CD80, CD86) and with the production of cytokines known to activate T lymphocytes, and β-chemokines. Upon encountering the antigens, DC undergo a maturation process characterized by an increase of costimulatory molecules expression and by a reduction of their phagocytic and pinocytic capability (Banchereau 1998; Bell 1999). Further, due to the upregulation of the homing receptor CCR7 and to the downregulation of CCR5, mature DC migrate to lymph nodes where they present antigens to T lymphocytes (Banchereau 1998; Bell 1999). Prior art indicates that the addition of Tat protein to DC blocks in these cells the extracellular calcium influx, the production of interleukin-12, and the uptake of apoptotic bodies (Zocchi 1997; Rubartelli 1997). As a result, it is predicted that profound impairment of important DC functions including antigen uptake, processing and presentation and induction of Th-1 responses should occur. Further, impairment of phagolysosomal fusion has been reported in peripheral blood monocytes upon exposure to Tat, suggesting impairment in this cell type of both microbicidal and antigen processing (and presentation) functions (Pittis 1996). Moreover, Tat has been reported to induce both monocytes/macrophages and lymphocytes to secrete IL-10 (Masood 1994; Badou 2000), while inhibiting IL-12 production in monocytes (Ito 1998). Finally, exposure of APC to Tat has been reported to impair their capability to organize cell clusters and to properly activate T cells (Mei 1997). Moreover, prior art indicates that Tat profoundly impairs also T cell functions including suppression of responses to mitogens anti-CD3 or specific antigens (Viscidi 1989; Benjouad 1993; Subramanyam 1993; Chirmule 1995; Wrenger 1996; Wrenger 1997; Zagury 1998), T cell hyperactivation (Ott 1997; Li 1997), and T cell apoptosis (Westendorp 1995; Li 1995; McCloskey 1997). Further, inoculation of biologically active Tat has been reported to be immunosuppressive in vivo (Cohen 1999). Part of the effects of Tat on the immune system have been related to upregulation by Tat of the chemokines receptors CCR5 and CXCR4 (Huang 1998; Secchiero 1999), or the direct interaction of Tat with the chemokine receptors CCR2 and CCR3 (Albini 1998a) or with other receptors including CD26 (Gutheil 1994), Flt-1 (Mitola 1997), KDR (Albini 1996; Morini 2000), that are expressed by immune cells, as well as by endothelial cells. Therefore, according to this previous art Tat is expected to drive a Th-2 type of immune response and/or to interfere with or abolish proper APC function and T cell activation. By contrast, our novel and unexpected finding, supported by experimental evidence exhibited in this patent application, indicate that: (i) APC are specifically targeted by Tat that selectively recognises and enters these cells at pico-nanomolar concentrations, but that this requires the interaction of native, substantially monomeric, biologically active Tat with α5β1, αvβ3 integrins, through the Tat RGD sequence; (ii) and that native, substantially monomeric, biologically active Tat activates, rather than inhibiting, APC function and induces, rather than suppressing, Th-1 type immune responses against itself and, most notably, other antigens. Specifically, our data show that Tat acts not only as an antigen but also as an adjuvant with potent immunomodulatory properties. These properties of Tat, namely of being selectively internalised as biologically active protein by APCs at picomolar-nanomolar concentrations and to act as an adjuvant, are strictly related each other. In particular, we have found, that Tat RGD sequence is key for the internalisation of active Tat by these cells through the α5β1 and αvβ3 integrin receptors. In fact, antibodies or competitor ligands blocking these integrins completely abolish or greatly reduce the uptake of picomolar-nanomolar concentrations of Tat, respectively. This uptake is very rapid, is dose-, cell maturation/differentiation- and time-dependent. Even more unexpectedly, we did not obtain similar results with other APC including monocytes, T cell blasts, or B cell blasts or non-activated endothelial cells. Therefore, these findings are completely novel since prior art indicates that Tat is taken up only at much higher concentrations (micromolar range), through its basic region, by a non-receptor-mediated pathway (Frankel 1988; Mann 1991; Rusnati 1998; Tyagi 2001). This internalization pathway occurs with any cell type, and it is not maturation/differentiation-dependent. Further, we have found that Tat in its native, substantially monomeric, and biologically active form is absolutely required to observe all the above novel effects which do not occur when Tat is oxidized and inactivated. In fact, Tat has 7 cysteines and it is extremely sensitive to oxidation which, when occurring, causes the loss of native protein conformation and consequent loss of biological activity (Frankel 1989). Therefore, Tat is likely to lose its native conformation and activity when purified with procedures that are not specifically designed at maintaining this protein in its native form. Although established concepts in the field claim that biologically active Tat is toxic (Gallo 1999; Sabatier 1991; Kolson 1993; Westendrop 1995; Purvis 1995), by contrast, the highly purified, biologically active preparations of recombinant Tat utilized by the inventor has no cytotoxic nor pro-apoptotic effects on endothelial cells, DC, macrophages, other cell type tested, nor in vivo in mice or monkeys (Ensoli 1994; Barillari 1999a; Zauli 1993, 1995a and 1995b; Cafaro 1999, 2000 and 2001). Thus, the inventor believes that full-length, wild type, native, substantially monomeric, and biologically active Tat from any HIV variant or its fragments or derivates containing the RGD region can be used as a highly efficient system for the selective targeting and delivery of molecules to specific cell types expressing the integrins recognized by the Tat RGD region (Barillari 1993, 1999a and 199b; Ensoli 1994). Given the very large amount and ubiquitous distribution of DC, macrophages and endothelial cells in the human body, the inventor believes that the capability of biologically active Tat or its fragments or derivatives containing the RGD sequence of targeting these APC and of driving Th-1 type cellular responses will offer a unique opportunity to, 1) to deliver cargo molecules to these cell types which represent a specific target for Tat and are recruited and activated in infections, pathologic angiogenesis, inflammatory diseases and tumors in the delivery system embodiment of the present invention; 2) induce a potent immune response against not only Tat but also against other antigens delivered by or with Tat, in the vaccine-adjuvant and immunomodulatory embodiment of the present invention. This belief is strongly supported by the successful previous work of the inventor with biologically active Tat as a vaccine to control HIV replication and to block disease onset (Cafaro 1999; Cafaro 2000; Cafaro 2001, and PCT WO99/27958) as opposed to inactivated Tat protein (Goldstein. 2000; Pauza 2000). The present patent application is substantially different and innovative as compared to our previous patent application WO 99/27958 in many aspects. In fact, the above mentioned application claimed biologically active Tat or Tat encoding DNA to be effective as a vaccine against HIV/AIDS. At the time when said patent application was filed it was not known to us that low (picomolar-nanomolar) amounts of biologically active Tat, or its fragments or derivatives containing the RGD region, (i) specifically target APC and thus we could have not claimed its use as a carrier to selectively deliver cargo molecules to them; (ii) cause EC and DC cell maturation and activation and induce Th-i type immune responses against different antigens, and thus we could have not claimed its use as a adjuvant and immunomodulator. Thus, in this invention, biologically active Tat is proposed, in the first embodiment, as a delivery system to deliver to APC (i) different antigens or combinations of antigens for vaccination against different infectious diseases (not only HIV/AIDS) and tumors, or for multivalent vaccination against one or more infectious diseases, and (ii) therapeutic molecules for the treatment of infectious, inflammatory and angiogenic diseases and tumor growth and metastasis; and in the second embodiment, biologically active Tat is proposed as an adjuvant to drive T-cell mediated immune responses against different antigens, and in particular to enhance the immunogenicity of poorly immunogenic antigens, such as those expressed by certain intracellular pathogens as well as tumor cells, by combining or fusing them with biologically active Tat or its fragments or derivatives containing the RGD region. In summary, the most important innovative aspect which makes the difference with the prior art is that here native, substantially monomeric, and biologically active Tat is claimed as a molecule which exerts different functions, i.e. it is a carrier to selectively deliver antigens to APCs or active compounds to specific tissues, and an adjuvant stimulating immune responses to other antigens. This unexpected properties make native, substantially monomeric, and biologically active Tat suitable for different applications in different infectious diseases (not only AIDS), inflammatory and angiogenic diseases and tumors. Thus, the inventor believes that native, substantially monomeric, and biologically active Tat, fragments or derivatives thereof, containing the RGD sequence, acts with at least one of the following actions: as delivery system to specific APC or as an adjuvant, and claims that it can be exploited for preventive and therapeutic vaccination and/or drug delivery for the prevention and treatment of HIV/AIDS, other infectious, inflammatory, and angiogenic diseases.
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention the use of native, substantially monomeric, and biologically active HIV-1 Tat, fragments or derivatives thereof, to selectively target antigen-presenting cells expressing α5β1 and αvβ3 integrins. It is another object of the present invention the use of native, substantially monomeric, and biologically active HIV-1 Tat, fragments or derivatives thereof, to selectively target α5β1 and αvβ3 integrins expressed by antigen presenting cells, including dendritic cells, endothelial cells and macrophages, for the uptake of Tat, fragments or derivatives thereof, by these cells. It is another object of the present invention the use of native, substantially monomeric, and biologically active HIV-1 Tat, fragments or derivatives thereof, to selectively target antigen presenting cells expressing the α5β1 and αvβ3 integrins, including dendritic cells, endothelial cells and macrophages, to induce the maturation and/or the antigen presenting functions of these cells by Tat, fragments or derivatives thereof. Another object is the use of native, substantially monomeric, and biologically active HIV-1 Tat, fragments or derivatives thereof, combined with one or more antigens, including, but not limited to, antigens from intracellular pathogens (such as viruses, mycobacterium tuberculosis, candida, malaria) and from tumor cells, (such as those from lung, colon, breast, prostatic cancer) in the form of peptides, proteins or DNA encoding them, to selectively target in vitro and in vivo antigen-presenting cells expressing the α5β1 and αvβ3 integrins, including dendritic cells, endothelial cells and macrophages, for preventive and therapeutic vaccination or treatment against infectious diseases and tumors. Another object is the use of native, substantially monomeric, and biologically active Tat, fragments or derivatives thereof, to selectively deliver in vitro and in vivo one or more antigens to antigen-presenting cells expressing the α5β1 and αvβ3 integrins, including dendritic cells, endothelial cells and macrophages in order to induce immune responses for preventive and therapeutic vaccination or treatment of infectious diseases, inflammatory and angiogenic diseases and tumors. Another object is the use of native, substantially monomeric, and biologically active Tat, fragments or derivatives thereof, to selectively deliver, intracellularly or to the cell membrane, in vitro and in vivo, to antigen-presenting cells expressing the α5β1 and αvβ3 integrins, including dendritic cells, endothelial cells and macrophages, one or more antigens or therapeutic compounds (such as, but not limited to, antiviral compounds, anti-inflammatory drugs, anti-angiogenic molecules, cytotoxic anti-tumor drugs or immunomodulating molecules such as, for example chemokines or cytokines, or antibodies) with or without the presence of support particles (such as, but not limited to, microparticles, nanoparticles, liposomes and other particulated delivery systems such as the ones described in Speiser 1991 and Takeuchi, 2001) for preventive and therapeutic vaccination or treatment of infectious diseases, inflammatory and angiogenic diseases and tumors. Another object is the use of native, substantially monomeric, and biologically active Tat, fragments or derivatives thereof, fused to other proteins or peptides or support particles (as defined in the above) to selectively deliver in vitro and in vivo antigens or therapeutic compounds (as defined in the above) to antigen presenting cells expressing β5β1 and αvβ3 integrins including dendritic cells, endothelial cells and macrophages for combined preventive and therapeutic vaccination or treatment of infectious diseases, inflammatory and angiogenic diseases and tumors. Another object is the use of native, substantially monomeric, and biologically active Tat, fragments or derivatives thereof, to selectively target in vitro and in vivo cells expressing RGD-binding integrin receptors such as antigen-presenting cells and other cell types capable of taking up Tat via the integrin-mediated pathway, and/or other uptake pathways upon the binding to integrin receptors, in order to deliver antigens or therapeutic molecules (as defined in the above) for preventive and therapeutic vaccination or treatment of infectious diseases, inflammatory and angiogenic diseases and tumors. Another object is the use of native, substantially monomeric, and biologically active Tat, fragments or derivatives thereof, combined with antigens, adjuvants (such as, but not limited to, Alum, RIBI, ISCOMS, CpG sequences, Lipopeptides) or therapeutic molecules or support particles (as defined in the above) administered by the parenteral (subcute, intramuscular, intradermic) or mucosal (vaginal, rectal, oral, nasal) or topic route for preventive and therapeutic vaccination or treatment against infectious diseases inflammatory, and angiogenic diseases and tumors. Another object is the use of native, substantially monomeric, and biologically active Tat, fragments or derivatives thereof to selectively deliver in vitro and in vivo antigens or therapeutic molecules (as defined in the above) within or attached to support particles (as defined in the above), to antigen-presenting cells expressing RGD-binding integrin receptors including dendritic cells, endothelial cells and macrophages, for preventive and therapeutic vaccination or treatment against infectious diseases, inflammatory and angiogenic diseases and tumors. Another object is the use of native, substantially monomeric, and biologically active Tat, fragments or derivatives thereof, to selectively deliver in vitro and in vivo expression vectors including plasmid DNA and bacterial or virus vectors expressing one or more antigens, in the presence or absence of support particles (as defined in the above), to antigen presenting cells expressing RGD-binding integrin receptors, including dendritic cells, endothelial cells and macrophages for preventive and therapeutic vaccination or treatment against infectious diseases, inflammatory and angiogenic diseases and tumors. Another object is the use of tat DNA or native, substantially monomeric, and biologically active Tat protein, fragments or derivatives thereof, fused or combined with DNA coding for antigens, with or without support particles (as defined in the above), for combined preventive and therapeutic vaccination of infectious diseases, inflammatory and angiogenic diseases and tumors. Another object is native, substantially monomeric, and biologically active HIV Tat or tat DNA, fragments or derivative thereof, combined or fused with antigens, therapeutic molecules (as defined in the above), adjuvants (as defined in the above), or support particles (as defined in the above) such combination or fusion being defined as the association by means of chemical or physical interactions, or any other interactions, in any combination, such as, for example, but not limited to, the absorption of Tat and a DNA plasmid on nanoparticles; the inclusion of Tat and a synthetic drug in the same pharmaceutical preparation; the association of Tat or a fragment or a derivative thereof with a peptide by chemical crosslinking or by other means; the fusion of Tat, fragment or derivative thereof, with another protein or another peptide upon their expression in bacteria or eucariotic cells through chimeric DNA, where the DNA sequences encoding for the above polypeptides have been fused together using recombinant DNA technologies. Another object is the use of native, substantially monomeric, and biologically active HIV-1 Tat, fragments or derivatives thereof, as adjuvant to activate or enhance in vitro and in vivo the antigen-presenting function of cells expressing RGD-binding integrin receptors including dendritic cells, endothelial cells and macrophages and to induce Th-1 type immune responses against HIV/AIDS, other infectious diseases and tumors. Another object is the use of native, substantially monomeric, and biologically active Tat protein, tat DNA, fragments or derivates thereof, as in the above for vaccination or therapeutic treatment by the parentheral (intradermic, intramuscular, subcute), mucosal (oral, nasal, vaginal, rectal) or topic route. Another object are fragments of native, substantially monomeric, and biologically active Tat, defined as Tat peptides from any HIV variant (HIV-1, HIV-2 and other types and subtypes) comprising, alone or associated, the RGD domain (aa 73 to 86 in the HTLV-IIIB, clone BH-10; aa 74 to 84; aa 75 to 83; aa 76 to 82; aa 77 to 81; aa 77 to 82; aa 77 to 83; aa 76 to 83); the cystein-rich domain (aa 22 to 37 in the HTLV-IIIB, clone BH-10); the basic domain (aa 48 to 61 in the HTLV-IIIB, clone BH-10), combined or not with other HIV-1 Tat peptides including the core domain (aa 38 to 47 in the HTLV-IIB, clone BH-10) and/or the amminoterminal region (aa 1 to 20 in the HTLV-IIIB, clone BH-10). Another object are fragments of native, substantially monomeric, and biologically active Tat are defined as nucleotide sequences from any HIV variant (HIV-1, HIV-2 and other types and subtypes) comprising, alone or associated, the RGD domain (sequence coding for aa 73 to 86 in the HTLV-IIIB, clone BH-10; sequence coding for aa 74 to 84 in the HTLV-IIIB, clone BH-10; sequence coding for aa 75 to 83 in the HTLV-IIIB, clone BH-10; sequence coding for aa 76 to 82 in the HTLV-IIIB, clone BH-10; sequence coding for aa 77 to 81 in the HTLV-IIIB, clone BH-10; sequence coding for aa 77 to 82 in the HTLV-IIIB, clone BH-10; sequence coding for aa 77 to 83 in the HTLV-IIIB, clone BH-10; sequence coding for aa 76 to 83 in the HTLV-IIIB, clone BH-10) the cystein-rich domain (sequence coding for aa 22 to 37 in the HTLV-IIIB, clone BH-10), the basic domain (sequence coding for aa 48 to 61 in the HTLV-IIIB, clone BH-10), combined or not with other HIV-1 Tat peptides including the core domain (sequence coding for aa 38 to 47 in the HTLV-IIB, clone BH-10) and/or the amminoterminal region (sequence coding for aa 1 to 20 in the HTLV-IIIB, clone BH-10). Another object are fragments of Tat from any HIV variant (HIV-1, HIV-2 and other HIV types and subtypes) that comprise one or more T-cell epitopes in their amino acid sequences (HTLV-IIIB, clone BH-10 or 89.6). Another object are fragments of Tat from any HIV variant (HIV-1, HIV-2 and other HIV types and subtypes) that comprise one or more T-cell epitopes in their nucleotide sequences (HTLV-IIIB, clone BH-10 or 89.6). Another object are derivatives of Tat which comprise Tat mutants of the HTLV-IIIB, clone BH-10, variant, selected among that ones having the following nucleotide sequences, or part of them: Nucleotide sequence of cys22 mutant and nucleotide sequence of lys41. Another object are derivatives of Tat which comprise Tat mutants of the HTLV-IIIB, clone BH-10, variant, selected among that ones having the following aminoacid sequences, or part of them: Amino acid sequence of cys22 mutant and amino acid sequence of lys41. Another object of the present invention is the use of native, substantially monomeric, and biologically active Tat protein acting and combined as above described to produce medicaments to cure affections in the group of infectious diseases, inflammatory and angiogenic diseases, tumors. Further objects will be evident from the detailed description of the invention.

Workpiece forming
A method of forming a workpiece (18) comprises: holding the workpiece adjacent a mould (20); using a laser (30) to heat at least a part of the workpiece to a temperature sufficient to induce superplasticity; and applying a fluid pressure to the workpiece, so that it takes the shape of the mould. This has the advantage that the superplastic properties of the material can be used to form the workpiece precisely to the required shape, without needing to heat all of the processing chamber to the superplastic temperature. Before using the laser to heat the workpiece to its superplastic temperature, the laser can be used to heat the whole of the workpiece to a substantially uniform temperature to anneal it. Similarly, after using the laser to superplastically form the workpiece, the laser is used to heat the whole of the workpiece to a substantially uniform temperature to remove any residual stresses. This has the advantage that the whole of the forming can be carried out as a single process, in a single processing apparatus.
1. A method of forming a workpiece, comprising: holding the workpiece adjacent a mould; using a laser to heat at least a part of the workpiece to a temperature sufficient to induce superplasticity therein; applying a fluid pressure to the workpiece so that it takes the shape of the mould. 2. A method as claimed in claim 1, wherein the step of holding the workpiece comprises: clamping the workpiece adjacent the mould; using the laser to heat the whole of the workpiece to a substantially uniform temperature to anneal it; and reducing the temperature of the workpiece to below the superplastic temperature thereof. 3. A method as claimed in claim 1, further comprising, after shaping the workpiece: using the laser to heat the whole of the workpiece to a substantially uniform temperature to remove residual stresses therein. 4. A method as claimed in claim 1, wherein the mould comprises first and second halves, and the workpiece comprises first and second sheets, the method comprising: holding the first and second sheets of the workpiece adjacent the first and second halves of the mould, respectively; using the laser to heat at least parts of the first and second sheets of the workpiece; and applying fluid pressure to the first and second sheets of the workpiece so that they take the respective shapes of the first and second halves of the mould. 5. A method as claimed in claim 4, wherein the step of applying fluid pressure comprises applying an increased fluid pressure between the first and second sheets of the workpiece. 6. A method as claimed in claim 4, wherein at least one of the first and second halves of the mould is transparent to the laser, and the step of using the laser to heat at least parts of the first and second sheets of the workpiece comprises heating at least one of the first and second sheets of the workpiece through said transparent half of the mould. 7. A method as claimed in claim 1, wherein the mould comprises a plurality of pillars, which are individually movable, such that distal ends of the pillars form a mould surface. 8. A method as claimed in claim 7, wherein at least some of the plurality of pillars include means for directing a laser beam at a workpiece held adjacent thereto. 9. A method as claimed in claim 7, wherein at least some of the plurality of pillars include means for directing a coolant at a workpiece held adjacent thereto. 10. A forming apparatus, comprising: means for retaining a mould; means for clamping a workpiece adjacent the mould; a laser source, suitable for heating at least a part of a workpiece held in the clamping means; and means for applying a fluid pressure to the workpiece so that it takes the shape of the mould. 11. A forming apparatus as claimed in claim 10, wherein the means for applying a fluid pressure to the workpiece comprises means for introducing a fluid on the side of the workpiece away from the mould. 12. A forming apparatus as claimed in claim 10, wherein the means for applying a fluid pressure to the workpiece comprises means for evacuating a region between the workpiece and the mould. 13. A forming apparatus as claimed in claim 10, wherein the means for retaining the mould comprises means for retaining first and second mould halves, and the means for clamping the workpiece comprises means for clamping first and second workpiece sheets adjacent the first and second mould halves, respectively. 14. A forming apparatus as claimed in claim 13, wherein the means for applying fluid pressure comprises means for applying an increased fluid pressure between the first and second workpiece sheets. 15. A forming apparatus as claimed in claim 13, comprising a mould wherein at least one of the first and second halves of the mould is transparent to the laser. 16. A forming apparatus as claimed in claim 10, wherein the mould comprises a plurality of pillars, which are individually movable, such that distal ends of the pillars form a mould surface. 17. A forming apparatus as claimed in claim 16, wherein at least some of the plurality of pillars include means for directing a laser beam at a workpiece held adjacent thereto. 18. A forming apparatus as claimed in claim 16, wherein at least some of the plurality of pillars include means for directing a coolant at a workpiece held adjacent thereto. 19. A product manufactured using a method in accordance with claim 1.



Flight for a conveyor
A flight (5) for use on a flight conveyor has an elongated core (20) and a sound absorbing material (30) secured to the core (20). The sound absorbing material (30) also has high wear resistance. A method of producing such a flight (5) involves securing to the core (20) a sound absorbing material (30).
1. A flight adapted for use on the deck of a flight conveyor, wherein the flight is moved along the deck by a drive mechanism, said flight comprised of: a) an elongated core having 1) one end adapted to be attached to a drive mechanism; 2) a bottom surface; and 3) a front face for urging material along the deck; and b) a sound absorbing material secured to the core. 2. The flight according to claim 1, wherein the sound absorbing material is also high wear resistance. 3. The flight according to claim 1, wherein the bottom surface of the core has a sound absorbing material secured thereto. 4. The flight according to claim 3, wherein the material secured to the bottom surface of the core provides a flight bottom surface which is flat. 5. The flight according to claim 1, wherein the core is generally T-shaped with a horizontal base leg and a vertical leg extending upwardly therefrom, wherein the vertical leg of the core has sound absorbing material secured thereto. 6. The flight according to claim 1, wherein the front face of the flight is angled rearwardly to force the flight against the deck when displacing material. 7. The flight according to claim 1, wherein the flight is generally T-shaped. 8. The flight according to claim 1, wherein the end of the core adapted to be attached to a drive mechanism is exposed and the remainder of the core has sound absorbing material secured thereto. 9. The flight according to claim 1, wherein the sound absorbing material is secured to the core to define a predetermined shape of the flight. 10. The flight according to claim 1, wherein the sound absorbing material in the form of a sheet and is secured to and conforms to a predetermined core shape. 11. The flight according to claim 1, wherein the sound absorbing material is polyurethane. 12. The flight according to claim 11, wherein the polyurethane is MDI. 13. The flight according to claim 11, wherein the polyurethane has a Shore Durometer Hardness of between 80 and 85. 14. The flight according to claim 1, wherein the combination of the core and material secured thereto has an external shape similar to that of a flight without such material. 15. The flight according to claim 1, wherein the sound absorbing material is secured to the core by molding onto the core. 16. The flight according to claim 1, wherein the sound absorbing material is secured to the core by clamping. 17. The flight according to claim 1, wherein the sound absorbing material is secured to the core by mechanical fastening. 18. The flight according to claim 1, wherein the flight is used for a flight conveyor in a long wall mining operation. 19. A method of producing a flight used on the deck of a flight conveyor wherein the flight is moved along the deck by a drive mechanism, wherein the flight has a core and said method comprising the step of securing to the core a sound absorbing material. 20. The method according to claim 19, wherein the sound absorbing material is polyurethane. 21. The method according to claim 19, wherein the core has a bottom and the sound absorbing material is secured to the bottom of the core. 22. The method according to claim 19, wherein the core has a top surface and the sound absorbing material is secured to the top of the core. 23. The method according to claim 19, wherein the core has an end surface adapted to be secured to a drive mechanism on a flight conveyor and the sound absorbing material is secured to the remainder of the core. 24. The method according to claim 19, wherein the core is reduced in size prior to securing the sound absorbing material such that the combined core and sound absorbing material provide a flight having the external shape of a conventional flight. 25. The method according to claim 19, wherein the step of securing the sound absorbing material is comprised of molding the material to the core. 26. The method according to claim 18, wherein the step of securing the sound absorbing material is comprised of clamping the material to the core. 27. The method according to claim 18, wherein the step of securing the sound absorbing material is comprised of mechanically fastening the material to the core.
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention This invention relates to a conveyor device and more particularly to a flight conveyor having a flight with a core and a sound absorbing material about the core. 2. Description of Related Art Flight conveyors employing a central sprocket driven chain with elongated flights extending laterally in opposite directions from spaced sides of the chain are extensively used for the conveying of material. U.S. Pat. Nos. 3,103,275; 4,238,028; and 5,000,310 illustrate general designs for flight conveyors. These types of flight conveyors travel over flat surfaces or decks, wherein the laterally extending flights cooperate with the flat deck to move material therealong. Where such sprocket driven flight conveyors are used on loading machines, the flights are required to move heavy materials, such as large masses of coal, rock and iron along the deck plates. In such structures, the flights travel closely to the deck plates. Typically, the deck plates and the flights are made of steel such that the motion of the flights over the deck plates is inordinately loud. Flight conveyors are used extensively in coal mines, and recent MSHA standards limit the intensity of noise to which individuals working in coal mines may be exposed. Flight conveyors in mines are a large contributor to such noise. A design is required that will reduce the noise produced by flight conveyors.
<SOH> SUMMARY OF THE INVENTION <EOH>One embodiment of the subject invention is directed to a flight adapted for use on the deck of a flight conveyor wherein the flight is moved along the deck by a drive mechanism, said flight comprised of a) an elongated core having 1) one end adapted to be attached to the drive mechanism; 2) a bottom surface; and 3) a front face for urging material along the deck; and b) a sound absorbing material secured to the core. A second embodiment of the subject invention is directed to a method of producing a flight used on the deck of a flight conveyor wherein the flight is moved along the deck by a drive mechanism, wherein the flight has a core and said method comprises the step of securing to the core a sound absorbing material.

Fragrance device
A fragrance-emitting device for the release of fragrances or other volatile materials, e.g. insecticides employing the heat of a light bulb, comprising a hollow container filled with a fragrance-containing material; the hollow container having a central orifice permitting the container to be fitted over the light bulb.
1. A fragrance-emitting device for releasing fragrances or other volatile materials employing the heat of a light bulb, comprising: a hollow container filled with a fragrance-containing material; the hollow container having a central orifice; and a base member, the upper surface of which is adapted with electrical connection means to receive a light bulb such that the light bulb projects upwardly from said upper surface, wherein the hollow container sits on the base member supported by the upper surface of the base member, and the light bulb is accommodated within the central orifice. 2. A device according to claim 1 wherein the base member contains therein a battery for supplying power to the light bulb. 3. A device according to claim 2 wherein the light bulb is a flicker-bulb designed to imitate the flickering effect of a candle. 4. (cancelled) 5. A device according to claim 1 wherein the fragrance-containing material is a gel. 6. A device according to claim 5 wherein the gel is translucent or transparent. 7. A device according to claim 1 wherein the hollow container is provided with vent holes which permit egress of fragrance from inside the hollow container to the surrounding air. 8. A device according to claim 1 wherein the hollow container is formed from translucent or transparent material. 9. A process for the delivery of a fragrance or volatile material which comprises the step of: providing a fragrance-emitting device according to claim 1; and operating the device to emit to surrounding air a fragrance and/or an insecticide. 10. A hollow container having a central orifice and filled with a fragrance-containing material, adapted for use with the fragrance-emitting device according to claim 1.



Neutral glycosphingolipids and glycosyl-sphingosines and methods for isolating the same
In vitro/cell-free process of preparing a sialylated oligosaccharides are described. The sialylated oligosaccharides include gangliosides. The oligosaccharides linked to various moieties including sphingoids and ceramides. Novel compounds that comprise sphingoid groups are disclosed. The compounds include sialylated oligosaccharides including gangliosides as well as various sphingoids and ceramides.
1. A method of separating a first electronically neutral lipid from a second electronically neutral lipid, said method comprising: (a) cleaving an ester moiety of said second electronically neutral lipid, forming a cleaved lipid mixture; (b) contacting said cleaved lipid mixture with a mixed bed ion exchange resin and a solvent, forming a resin-bound species and a solution of said first electronically neutral lipid; and (c) separating said resin-bound species from said solution of said first electronically neutral lipid, thereby separating said first electronically neutral lipid from said second electronically neutral lipid. 2. The method according to claim 1, wherein said first electronically neutral lipid is a glycolipid. 3. The method according to claim 2, wherein said solution of said first electronically neutral lipid has a conductivity between about 500 μS/cm and about 1.0 μS/cm. 4. The method according to claim 1, wherein said first electronically neutral lipid is a glycosylceramide and said second electronically neutral lipid is phosphatidyl choline. 5. The method according to claim 1, further comprising: (d) cleaving an amide moiety of said first electronically neutral lipid into its amine and carboxylate consitutents. 6. The method according to claim 5, further comprising: (e) submitting said amine constituent to a member selected from cation exchange chromatography, reverse-phase chromatography and silica gel chromatography, thereby separating said amine from said carboxylate constituent. 7. The method according to claim 1, further comprising: (f) following said separating, contacting said first electronically neutral lipid from step (c) with a glycosyltransferase and a glycosyl donor under conditions appropriate to transfer said glycosyl donor to said electronically neutral lipid from step (c). 8. The method of claim 6, wherein said amine is a glycosyl sphingosine. 9. The method of claim 8, wherein said glycosyl sphingosine is a glucosyl sphingosine that is a member selected from d18:2, d18:1, t18:1, d18:1:1 and combinations thereof. 10. A composition comprising a mixture of two or more of glycosyl sphingosine d18:2, glycosyl sphingosine d18:1, glycosyl sphingosine d18:1:1 and glycosyl sphingosine t18:1, wherein said mixture is essentially free of charged lipid.
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention This invention relates to the area of isolated lipid components, lipid component analysis and lipid purification. 2. Background Gangliosides are a class of glycosphingolipids that have a structure containing a carbohydrate moiety linked to a ceramide. The carbohydrate moiety includes at least one monosaccharide and a sialic acid moiety. The sialic acid moiety is composed of one or more sialic acid groups (N-acetyl or N-glycolyl neuraminic acid). Gangliosides are classified according to the number of monosaccharides in the sugar moiety and the number of sialic acid groups present in the structure. Gangliosides are known as mono-, di-, tri- or poly-sialogangliosides, depending upon the number of sialic acid residues. Abbreviations employed to identify these molecules include “GM1”, “GD3”, “GT1”, etc., with the “G” standing for ganglioside, “M”, “D” or “T”, etc. referring to the number of sialic acid residues, and the number or number plus letter (e.g., “GT1a”), referring to the elution order in a TLC assay observed for the molecule. See, Lelminger, Biochemistry, pg. 294-296 (Worth Publishers, 1981); Wiegandt, Glycolipids: New Comprehensive Biochemistry (Neuberger et al., ed., Elsevier, 1985), pp. 199-260. For example, the international symbol GM 1a designates one of the more common gangliosides, which has been extensively studied. The “M” in the symbol indicates that the ganglioside is a monosialoganglioside and “1” defines its position in a TLC elution profile. The subscripts “a”, “b” or “c” also indicate the positions in a TLC assay of the particular ganglioside. The terminal saccharide is the saccharide, which is located at the end of the carbohydrate moiety, which is opposite to the end that is attached to the ceramide moiety. Hundreds of glycosphingolipids (GSLs) are derived from glucosylceramide (GlcCer), which is enzymatically formed from ceramide and UDP-glucose. The enzyme involved in GlcCer formation is UDP-glucose:N-acylsphingosine glucosyltransferase (GlcCer synthase). The rate of GlcCer formation under physiological conditions may depend on the tissue level of UDP-glucose, which in turn depends on the level of glucose in a particular tissue (Zador, I. Z. et al., J. Clin. Invest. 91:797-803 (1993)). In vitro assays based on endogenous ceramide yield lower synthetic rates than mixtures containing added ceramide, suggesting that tissue levels of ceramide are also normally rate-limiting (Brenkert, A. et al., Brain Res. 36: 183-193 (1972)). The level of GSLs controls a variety of cell functions, such as growth, differentiation, adhesion between cells or between cells and matrix proteins, binding of microorganisms and viruses to cells, and metastasis of tumor cells. In addition, the GlcCer precursor, ceramide, may cause differentiation or inhibition of cell growth (Bielawska, A. et al., FEBS Letters 307: 211-214 (1992)) and be involved in the functioning of vitamin D 3 , tumor necrosis factor-α, interleukins, and apoptosis (programmed cell death). The sphingols (sphingoid bases), precursors of ceramide, and products of ceramide catabolism, have also been shown to influence many cell systems, possibly by inhibiting protein kinase C (PKC). Gangliosides are known to be functionally important in the nervous system and it has been claimed that gangliosides are useful in the therapy of peripheral nervous system disorders. Numerous gangliosides and derivatives thereof have been used to treat a wide variety of nervous system disorders including Parkinson's disease (Ganglioside GM 1 is currently being used in phase II clinical development for the treatment of Parkinson's Disease (FIDIA, Italy)), and cerebral ischemic strokes (see, U.S. Pat. Nos. 4,940,694; 4,937,232; and 4,716,223). Gangliosides have also been used to affect the activity of phagocytes (U.S. Pat. No. 4,831,021) and to treat gastrointestinal disease-producing organisms (U.S. Pat. No. 4,762,822). The gangliosides GM 2 and GD 2 , purified from animal brain, have been conjugated to keyhole limpet hemacyanin (KLH) and mixed with adjuvant QS21, and used to elicit immune responses to these gangliosides, as the basis of a cancer vaccine in phase Il and III trials (Progenics, Tarrytown, N.Y.). Ganglioside GM 3 is being investigated for use as an anti-cancer agent (WO 98/52577; Nole et al., Exp. Neurology 168:. 300-9 (2001)). Glycolipids are also of interest in the treatment of inflammatory bowel disease. See, Tubaro et al., Naunyx - Schmiedebergg's Arch. Pharmacol. 348: 670-678 (1993). Gangliosides are generally isolated via purification from tissue, particularly from animal brain (G LYCOLIPID M ETHODOLOGY , Lloyd A. Witting Ed., American Oil Chemists Society, Champaign, Ill. 187-214 (1976); U.S. Pat. Nos. 5,844,104; 5,532,141; Soinino et al., J. Lipid Res. 33: 1221-1226 (1992); Sonnino et al., Ind. J. Biochem. Biophys., 25: 144-149 (1988); Svennerholm, Adv. Ed. Med. Biol. 125: 533-44 (1980)). Gangliosides have been isolated from bovine buttermilk (Ren et al., J. Bio. Chem. 267: 12632-12638 (1992); Takamizawa et al., J. Bio. Chem. 261: 5625-5630(1986)). Even under optimum conditions, the yields of pure gangliosides, e.g., GM2 and GM3, are vanishingly small. Moreover, purification from mammalian tissue carries with it the risk of transmitting contaminants such as viruses, prion particles, and so forth. Alternate methodologies for securing ganglioside specific antibodies are thus highly desirable. Glycosyl-sphingosines are useful as precursors for the synthesis of gangliosides. Moreover, neutral glycosphingolipids, including glycosyl-ceramides, are useful for the preparation of products in the cosmetic industry, neutraceutical, and the pharmaceutical industry. A number of techniques have been employed in the isolation of lipid components. Silicic acid (silica gel) column chromatography using sequential elutions with different solvents is used to fractionate lipid extracts into a neutral lipid fraction (chloroform elution), a glycolipid fraction (acetone elution) and a phospholipid fraction (methanol elution) (Rouser et al., Lipids 2: 3740 (1967)). Glycolipid factions have been further fractionated to yield a crude cerebroside fraction by silicic acid chromatography and elution with chloroform/acetone (Ito & Fujino, Nippon Nogeikagaku Kaishi 49: 205-212 (1972)). Cerebrosides (ceramide monoglycosides) are sphingolipids found in various organisms, including plants and animals. Cerebrosides have been isolated from lipid extracts of marine animals and from the green leaves of higher plants by silicic acid chromatography (Hayashi & Matsuura, Adv. Mass Spectrom. 7B: 1567-1571 (1978)). Cerebrosides have also been isolated from total lipid extracts of Aspergillus oryzae by silicic acid chromatography, where the crude cerebroside fraction was further purified by mild alkali treatment, rechromatography on a silicic acid column with chloroform/methanol and precipitation from ether (Fujino & Ohnishi, Biochim. Biophys. Acta 486: 161-171 (1977)). Further, cerebroside has been isolated from alfalfa ( Medicago saliva ) leaves by solvent extraction, mild alkaline hydrolysis, and silicic acid chromatography (Ito & Fujino, Can. J. Biochem. 51: 957-961 (1973)). Pure cerebroside has also been isolated from crude lipid extracts of hay and concentrate (extracted oilseed used for cattle fee), by mild alkaline hydrolysis, followed by silicie acid column chromatography to isolate the alkali-stable polar lipid fraction, followed acetylation with acetic anhydride-pyridine and thin-layer chromatography and mild alkaline hydrolysis (Morrison, Chem. Phys Lipids 11: 99-102 (1973)). Ceramides and ceramide monohexosides have been isolated from lipid extracts of rice bran (Fujino & Ohnishi, Chem. Phys. Lipids 17: 275-289 (1976)). Two rounds of silicic acid chromatography were carried out; the first to separate the neutral lipid, glycolipid, and phospholipid fractions, and the second to fractionate the glycolipid faction into ceramide and ceramide monohexoside fractions. The ceramide and ceramide monohexoside fractions were subsequently further purified by thin-layer chromatography, mild alkaline treatment, re-chromatography on silicic acid columns, and precipitation from methanol or ether. Ceramide and cerebroside have been isolated from Azuld bean ( Phaseolus angularis ) by a protocol where total lipid extracts were subjected to silicic acid column chromatography (to separate the nonpolar and polar lipid fractions), the polar fraction was treated with mild alkali, and the ceramide and cerebroside were isolated from the alkali-stable lipid fraction and purified by a combination of column chromatography and thin-layer chromatography (Ohnishi & Fujino, Agric. Biol. Chem. 45: 1283-1284 (1981)). Ceramides and cerebrosides have been isolated, similarly using silicic acid chromatography, from lipid extracts of immature and mature soybeans (Ohnishi & Fujino, Lipids 17: 803-810 (1982)). Ceramide has also been isolated from the glycolipid fraction of lipids from alfalfa leaves by silicic acid column chromatography (Fujino & Ito, Biochim. Biophys. Acta 231: 242-243 (1971)). Ceramide monohexosides have been isolated from lipid mixtures prepared from various species of Aspergillus by silica gel chromatography followed by chromatography on Iatrobeads 6RS-8060 (Boas et al., Chem. Phys. Lipids 70: 11-19 (1994)). Galactosyl- and glucosyl-ceramides have been prepared from pig brain chloroform/methinol extracts using sodium sulfate to absorb the water, tdiodide to cleave the ether linkage of plasmalogens, and alkaline methanolysis to cleave the ester linkages of the glycerolipids, followed by separation of the lipids on silica gel chromatography (Radin, J. Lipid Res. 17: 290-293 (1976)). Synthetic isomers and analogs of the ceramide (4E, 8E, 2S, 3R, 2′R)-N-2′-hydroxyhexadecanoyl-9-methyl-4,8-sphingadienineglucosyl-sphingosine are described by Funaki et al., Agric. Biol. Chem. 50: 615-623 (1986)). Mori & Kishino, Liebigs Ann. Chem. 807-814 (1998), which is incorporated herein by reference, synthesized glucosyl-ceramide d18:2 with a fatty acid moiety of alpha-hydroxy palmitic acid. Other references describing sphingolipids of plants are Sullards et al., J. Mass Spec. 35: 347-353 (2000); Sastry & Kates, Biochemistry 3: 1271-1280 (1964); Shibuya et al., Chem. Pharm. Bull. 38: 2933-2938 (1990); and Carter & Koob, J. Lipid Res. 10: 363-369 (1969)). Other references related to isolation and/or analysis of glycosyl-ceramides are Norberg et al., Biochim. Biophys. Acta 1066: 257-260 (1991); Norberg et al., Biochim. Biophys. Acta 1299: 80-86 (1996); Kawaguchi et al., Biosci. Biotech. Biochem. 64: 1271-1273 (2000); Ohnishi et al., Bio. Biophys. Acta 752: 416-422 (1983). There is a need for improved methods of obtaining neutral glycospbingolipids and glycosyl-sphingosines from natural sources. The methods described above rely upon the extraction of an aqueous fraction containing the desired compound with an organic solvent in which the desired compound is soluble. The extraction processes are time-consuming and often result in the formation of complex emulsions that are either intractable or resolved with great difficulty. Moreover, the extraction processes require the use of large amounts of organic solvents that must be disposed of. Once the material is successfully extracted into an organic solvent, it must be fractionated to obtain the desired constituent. The methods presently used for fractionation, which are based upon silica gel chromatography are cumbersome and expensive. The methods are only truly useful for the small-scale fractionation of enriched extracts, and cannot be used for the large-scale fractionation of crude mixtures that include one or more glycolipid of interest. To scale up silica gel-based procedure would require the use of columns of impractical size and expense; relative to the cost of the bulk materials being fractionated (e.g., lecithin, dairy products, etc.), silica gel is exorbinantly expensive. Moreover, after use, silica gel cannot be readily and reactivated. Consequently, the once-used silica gel is disposed of, which is both economically unattractive and creates waste disposal problems. The waste disposal problems are exacerbated in that the silica gel is generally saturated with one or more organic solvents, which create waste disposal issues themselves. Moreover, silica gel is known to be an inhalation hazard. Despite the difficulties and disadvantages attending the techniques utilizing organic solvent extraction followed by silica gel fractionation of glycolipids, it is the standard in the art, with little or nothing proposed to replace the technique.
<SOH> SUMMARY OF INVENTION <EOH>It has now, quite surprisingly, been discovered that ion-exchange chromatography of glycolipid mixtures presents a simple and inexpensive alternative to silica gel chromatography that eliminates many of the disadvantages of silica gel-based methods. Ion-exchange chromatography is an art-recognized means to separate charged species from electronically neutral species, or to separate species of opposite charges. The inventors have unexpectedly discovered that ion-exchange methods can be used to separate a first electronically neutral lipid from a second electronically neutral lipid. The present invention provides an ion-exchange chromatography-based method of fractionating a lipid mixture, separating a first electronically neutral glycolipid from a second electronically neutral glycolipid contained the lipid mixture. The first electronically neutral lipid is recovered from the mixture in good to excellent yields, using water and relatively benign organic solvents, such as alcohols. The method of the invention does not require preparing an extract of the desired component in an organic solvent prior to fractionation as is currently necessary with silica gel chromatography, however, the method is flexible enough to encompass such a step if desired. Moreover, ion exchange media is less expensive than silica gel and it is readily re-activated following its use, allowing it to be used repeatedly in sequential separation procedures. The ability to re-use the separation medium dramatically reduces the cost incurred to isolate the neutral lipids and also reduces the amount of waste produced by the process. The invention provides a method isolating a glycolipid on a commercially viable scale. For example, the methods of the invention can be used to process large volumes of starting lipid preparation to isolate one or more selected glycolipid. In an exemplary embodiment, the method of the invention is used to process from about 1 gram to about 1000 kilograms of starting lipid preparation. Amounts of starting lipid preparation of from about 5 kilograms to about 50 kilograms are routinely processed by the method of the invention. Moreover, the present invention provides a method that avoids many of the difficulties associated with the large-scale extractive methods presently used. The extraction of glycolipids with an organic solvent from an aqueous lipid preparation is attended by the formation of virtually intractable emulsions. The present method avoids the organic solvent extraction of glycolipids, thereby preventing the formation of complex and wasteful emulsions. The invention is exemplified by a method of separating a first electronically neutral lipid from a second electronically neutral lipid. The method includes: (a) cleaving an ester moiety of the second electronically neutral lipid, forming a cleaved lipid mixture; (b) contacting the cleaved lipid mixture with a mixed bed ion exchange resin and a solvent, forming a resin-bound species and a solution of the St electronically neutral lipid; and (c) separating the resin-bound species from the solution of the first electronically neutral lipid, thereby separating the first electronically neutral lipid from the second electronically neutral lipid. The resin-bound species includes the second electronically neutral lipid from which the ester group was cleaved. As will be appreciated by those of skill in the art, step (a) is optionally performed. The invention also provides a method in which the ester of the second electronically neutral lipid is not hydrolyzed. The invention also provides novel compounds that have been discovered using the methods of the invention. Additional compounds are provided that are derivatives of liposaccharide compounds isolated by methods of the invention. Additional objects and advantages of the present invention will be apparent from the detailed description that follows.

Reading unit and marking card for optical mark reader
The present invention relates to a reading unit and a marking card for optical mark reader. The reading unit comprises a plurality of optical sensing devices (R1, R2, R3, R4 . . . ) corresponding to a plurality of marks (M1, M2, M3, M4 . . . ); and a plurality of counters (CT1, CT2, CT3, CT4 . . . ) for converting output signals to output values corresponding to an amount of light sensed, and for sending the output values to a central processing unit (CPU). The CPU performs an answer checking process by a logical calculation based on the output values from the counters (CT1, CT2, CT3, CT4 . . . ).
1. A marking card read by an optical mark reader, wherein a plurality of marking areas are formed in each of answering marks of each questions in said marking card. 2. The marking card of claim 1, wherein the plurality of marking areas are divided by a plurality of circle marks having diameters different from each other. 3. A reading unit for an optical mark reader, the reading unit comprising: an optical sensing device for sensing the light which is transmitted through or reflected from an answering mark; and a counter for converting an output signal from the optical sensing device to an output value corresponding to an amount of light sensed by the optical sensing device, and sending the output value to a CPU, wherein the CPU checks a correct answer by programmatically processing a logical calculation based on a predetermined number of values selected according to an order of magnitude thereof among the output values from the counters. 4. The reading unit for an optical mark reader according to claim 3, wherein a plurality of marking areas are formed in each of said answering marks. 5. The reading unit for an optical mark reader according to claim 3, wherein said optical sensing device is an image sensing device including either of a CCD device (CD) or a CMOS device having a plurality of photo cells for performing a binary process. 6. (Cancelled) 7. The reading unit for an optical mark reader according to claim 3, wherein said counter counts a number of times that a marking of the mark is sensed, based on the output signals of the optical sensing device, and said CPU determines a size of the marked area based on the number counted by the counters, and checks a correct answer based on the determined size.
<SOH> TECHNICAL FIELD <EOH>The present invention relates to a reading unit and marking card for optical mark reader, and more particularly to a marking card having a plurality of marking areas for each of answering marks for each question and a reading unit reading thereof.
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention tries to resolve the aforementioned conventional problems and its object is to provide a reading unit for an optical mark reader that recognizes correction or re-correction of the answers made by examinees more effectively and accurately. The other object of the present invention is to save cost by simplifying the hardware circuit structure of the reading unit in checking the correct/incorrect answers for multiple-choice type, and to provide a reading unit for the optical mark reader applicable to various types of answer. In order to achieve the object of the invention, the present invention provides a reading unit that checks the correct answer by programmatically processing a logical calculation based on predetermined number of values selected according to an order of magnitude thereof among the data input by the central processing unit (CPU) of the optical process reader, etc. thereof. Also, in order to achieve the other object of the present invention, the present invention furnishes a marking card read by an optical mark reader, wherein a plurality of marking areas is formed in each of a plurality of answering marks for a question in the card. Moreover, in order to achieve another object of the present invention, the present invention provides a reading unit (U) for an optical mark reader, which reads a marking card formed with a plurality of marks (M 1 , M 2 , M 3 , M 4 . . . ) in each of a plurality of answering marks for a question, the reading unit comprising a plurality of optical sensing devices (R 1 , R 2 , R 3 , R 4 . . . ) corresponding to the plurality of marks (M 1 , M 2 , M 3 , M 4 . . . ), and responding to a transmitted or reflected marks (M 1 , M 2 , M 3 , M 4 . . . ); and a plurality of counters (CT 1 , CT 2 , CT 3 , CT 4 . . . ) for converting output signals from the optical sensing devices (R 1 , R 2 , R 3 , R 4 . . . ) to an output values corresponding to an amount of light sensed by the optical sensing devices, and send the output values to a CPU; wherein the CPU performs an answer checking process by a logical calculation based on the output values from the counters (CT 1 , CT 2 , CT 3 , CT 4 . . . ).

Electrolytic processing apparatus and method
This invention relates to an electrolytic processing apparatus and method useful for processing a conductive material formed in the surface of a substrate, or for removing impurities adhering to the surface of a substrate. An electrolytic processing apparatus, including, a processing electrode that can come close to a workpiece, a feeding electrode for feeding electricity to the workpiece, an ion exchanger disposed in the space between the workpiece and the processing and the feeding electrodes, a fluid supply section for supplying a fluid between the workpiece and the ion exchanger, and a power source, wherein the processing electrode and/or the feeding electrode is electrically divided into a plurality of parts, and the power source applies a voltage to each of the divided electrode parts and can control voltage and/or electric current independently for each of the divided electrode parts.
1. An electrolytic processing apparatus, comprising: a processing electrode that can come close to or into contact with a workpiece; a feeding electrode for feeding electricity to the workpiece; an ion exchanger disposed in at least either between the workpiece and the processing electrode, or between the workpiece and the feeding electrode; a fluid supply section for supplying a fluid between the workpiece and the ion exchanger; and a plurality of power sources, each for applying a voltage between the processing electrode and the feeding electrode; wherein at least one of the processing electrode and the feeding electrode is electrically divided into a plurality of parts, and the power sources apply each voltage to each of the divided electrode parts and can control at least one of voltage and electric current independently for each of the divided electrode parts. 2. The electrolytic processing apparatus according to claim 1, wherein the fluid is ultrapure water, pure water, a liquid having an electric conductivity of not more than 500 μS/cm. 3. The electrolytic processing apparatus according to claim 1, wherein the power sources apply respectively different constant voltages at least one time to each of the divided electrode parts. 4. The electrolytic processing apparatus according to claim 1, wherein each of the power sources applies an electric current or a voltage, which changes with time, to each of the divided electrode parts. 5. An electrolytic processing method, comprising: providing a processing electrode and a feeding electrode, at least one of which is electrically divided into a plurality of parts; allowing a workpiece to be close to or in contact with the processing electrode; feeding electricity from the feeding electrode to the workpiece; disposing an ion exchanger between at least either between the workpiece and the processing electrode, or between the workpiece and the feeding electrode; supplying a fluid between the workpiece and the ion exchanger; applying a voltage to each of the divided electrode parts; and controlling at least one of voltage and electric current independently for each of the divided electrode parts. 6. The electrolytic processing method according to claim 5, wherein the fluid is ultrapure water, pure water, a liquid having an electric conductivity of not more than 500 μS/cm. 7. The electrolytic processing method according to claim 5, wherein the control is effected such that respectively different constant voltages are applied at least one time to each of the divided electrode portions. 8. An electrolytic processing apparatus, comprising: a processing electrode; a feeding electrode for feeding electricity to the workpiece; a holder for holding the workpiece that can come close to or into contact with the processing electrode; an ion exchanger disposed in at least either between the workpiece and the processing electrode, or between the workpiece and the feeding electrode; a power source for applying a voltage between the processing electrode and the feeding electrode; a fluid supply section for supplying a fluid between the workpiece and at least one of the processing electrode and the feeding electrode, in which the ion exchanger is disposed; and a drive section for allowing the workpiece held by the holder and the processing electrode to make a relative movement; wherein a dummy member, at least the surface of which has an electric conductivity, is disposed outside of the periphery of the workpiece. 9. The electrolytic processing apparatus according to claim 8, wherein the dummy member makes the area of the portion of the workpiece facing the processing electrode constant during the relative movement of the workpiece and the processing electrode. 10. The electrolytic processing apparatus according to claim 8, wherein the conductive portion of the dummy member is formed of an electrochemically inactive material. 11. The electrolytic processing apparatus according to claim 8, wherein the conductive portion of the dummy member is formed of the same material as the workpiece. 12. The electrolytic processing apparatus according to claim 8, wherein a buffering member is disposed between the workpiece and the dummy member. 13. An electrolytic processing apparatus, comprising: a processing electrode having a larger diameter than a workpiece; a feeding electrode for feeding electricity to the workpiece; a holder for holding the workpiece that can come close to or into contact with the processing electrode; an ion exchanger disposed in at least either between the workpiece and the processing electrode, or between the workpiece and the feeding electrode; a power source for applying a voltage between the processing electrode and the feeding electrode; a fluid supply section for supplying a fluid to the space between the workpiece and at least one of the processing electrode and the feeding electrode, in which the ion exchanger is disposed; and a drive section for allowing the workpiece held by the holder and the processing electrode to make a relative movement in such a state that the center of movement of the processing electrode lies within the range of the workpiece. 14. An electrolytic processing apparatus, comprising: a processing electrode having a larger diameter than a workpiece; a feeding electrode for feeding electricity to the workpiece; a holder for holding the workpiece that can come close to or into contact with the processing electrode and the feeding electrode; a power source for applying a voltage between the processing electrode and the feeding electrode; a fluid supply section for supplying a fluid between the workpiece and the processing and feeding electrodes; and a drive section for allowing the workpiece held by the holder and the processing and feeding electrodes to make a relative movement in such a state that the center of movement of the processing electrode lies within the range of the workpiece. 15. An electrolytic processing apparatus, comprising: a processing electrode having a larger diameter than a workpiece; a plurality of feeding electrodes disposed in a peripheral portion of the processing electrode; a holder for holding the workpiece that can come close to or into contact with the processing electrode; an ion exchanger disposed in at least either between the workpiece and the processing electrode or between the workpiece and the feeding electrodes; a power source for applying a voltage between the processing electrode and the feeding electrodes; a fluid supply section for supplying a fluid to the space between the workpiece and at least one of the processing electrode and the feeding electrodes, in which the ion exchanger is disposed; and a drive section for allowing the workpiece held by the holder and the processing electrode to make a relative movement in such a state that at least one of the feeding electrodes always feeds electricity to the workpiece. 16. The electrolytic processing apparatus according to claim 15, wherein the processing electrode comprises an outer processing electrode defined by the peripheral portion in which the feeding electrodes are disposed, and an inner processing electrode positioned on the inner side of the outer processing electrode. 17. The electrolytic processing apparatus according to claim 16, wherein the power source controls independently the respective voltages or electric currents applied to the outer processing electrode and to the inner processing electrode. 18. An electrolytic processing apparatus, comprising: a processing electrode having a larger diameter than a workpiece; a plurality of feeding electrodes disposed in a peripheral portion of the processing electrode; a holder for holding the workpiece that can come close to or into contact with the processing electrode and the feeding electrodes; a power source for applying a voltage between the processing electrode and the feeding electrodes; a fluid supply section for supplying a fluid between the workpiece and the processing and feeding electrodes; and a drive section for allowing the workpiece held by the holder and the processing and feeding electrodes to make a relative movement in such a state that at least one of the feeding electrodes always feeds electricity to the workpiece. 19. The electrolytic processing apparatus according to claim 18, wherein the processing electrode comprises an outer processing electrode defined by the peripheral portion in which the feeding electrodes are disposed, and an inner processing electrode positioned on the inner side of the outer processing electrode. 20. The electrolytic processing apparatus according to claim 19, wherein the power source controls independently the respective voltages or electric currents applied to the outer processing electrode and to the inner processing electrode. 21. An electrolytic processing method, comprising: providing a processing electrode having a larger diameter than a workpiece and a feeding electrode for feeding electricity to the workpiece; disposing an ion exchanger between the workpiece and at least one of the processing electrode and the feeding electrode; applying a voltage between the processing electrode and the feeding electrode; allowing the workpiece to be close to or into contact with the processing electrode; supplying a fluid between the workpiece and at least one of the processing electrode and the feeding electrode, in which the ion exchanger is disposed; and allowing the workpiece and the processing electrode to make a relative movement in such a state that the center of movement of the processing electrode always lies within the range of the workpiece, thereby processing the surface of the workpiece. 22. An electrolytic processing method, comprising: providing a processing electrode having a larger diameter than a workpiece and a feeding electrode for feeding electricity to the workpiece; applying a voltage between the processing electrode and the feeding electrode; allowing the workpiece to be close to or into contact with the processing electrode and the feeding electrode; supplying a fluid between the workpiece and the processing electrode and feeding electrode; and allowing the workpiece and the processing and feeding electrodes to make a relative movement in such a state that the center of movement of the processing electrode always lies within the range of the workpiece, thereby processing the surface of the workpiece. 23. An electrolytic processing method comprising: providing a processing electrode having a larger diameter than a workpiece and a plurality of feeding electrodes disposed in a peripheral portion of the processing electrode; disposing an ion exchanger between the workpiece and at least one of the processing electrode and the feeding electrodes; applying a voltage between the processing electrode and the feeding electrodes; allowing the workpiece to be close to or into contact with the processing electrode; supplying a fluid between the workpiece and at least one of the processing electrode and the feeding electrodes, in which the ion exchanger is disposed; and allowing the workpiece and the processing electrode to make a relative movement in such a state that at least one of the feeding electrodes always feeds electricity to the workpiece, thereby processing the surface of the workpiece. 24. An electrolytic processing method, comprising: providing a processing electrode having a larger diameter than a workpiece and a plurality of feeding electrodes disposed in a peripheral portion of the processing electrode; applying a voltage between the processing electrode and the feeding electrodes; allowing the workpiece to be close to or into contact with the processing electrode and the feeding electrodes; supplying a fluid between the workpiece and the processing and feeding electrodes; and allowing the workpiece and the processing and feeding electrodes to make a relative movement in such a state that at least one of the feeding electrodes always feeds electricity to the workpiece, thereby processing the surface of the workpiece. 25. An electrolytic processing method, comprising: allowing a workpiece to be close to or into contact with a plurality of processing electrodes; applying a voltage between the processing electrodes and a feeding electrode for feeding electricity to the workpiece; supplying a fluid between the workpiece and at least one of the processing electrodes and the feeding electrode; and allowing the processing electrodes and the workpiece to make a relative movement so that a plurality of processing electrodes, which are uneven in the processing amount per unit time, pass every point in the to-be-processed surface of the workpiece, thereby processing the surface of the workpiece. 26. The electrolytic processing method according to claim 25, wherein the feeding electrode comprises a plurality of electrodes. 27. The electrolytic processing method according to claim 25, wherein the plurality of processing electrodes are disposed such that the presence frequencies of processing electrodes at every points in the to-be-processed surface of the workpiece become substantially equal during the relative movement. 28. The electrolytic processing method according to claim 26, wherein the plurality of feeding electrodes are disposed such that the presence frequencies of feeding electrodes at every points in the to-be-processed surface of the workpiece become substantially equal during the relative movement. 29. The electrolytic processing method according to claim 25, wherein the plurality of processing electrodes are of the same shape. 30. The electrolytic processing method according to claim 25, wherein the relative movement is one of a rotational movement, a reciprocating movement, an eccentric rotational movement and a scroll movement, or a combination thereof. 31. The electrolytic processing method according to claim 25, wherein an ion exchanger is disposed between the workpiece and at least one of the processing electrodes and the feeding electrode. 32. The electrolytic processing method according to claim 25, wherein the fluid is ultrapure water, pure water, a liquid having an electric conductivity of not more than 500 ìS/cm. 33. An electrolytic processing method, comprising: allowing a workpiece to be close to or into contact with a processing electrode; applying a voltage between the processing electrode and a feeding electrode for feeding electricity to the workpiece; supplying a fluid between the workpiece and at least one of the processing electrode and the feeding electrode; and allowing the processing electrode and the workpiece to make a relative movement so that a plurality of points in the processing electrode, which are uneven in the processing amount per unit time, pass every point in the to-be-processed surface of the workpiece, thereby processing the surface of the workpiece. 34. The electrolytic processing method according to claim 33, wherein the feeding electrode is disposed such that the presence frequencies thereof at every points in the to-be-processed surface of the workpiece become substantially equal during the relative movement. 35. The electrolytic processing method according to claim 33, wherein the feeding electrode comprises a plurality of electrodes, and the plurality of feeding electrodes are of the same shape. 36. The electrolytic processing method according to claim 33, wherein the relative movement is one of a rotational movement, a reciprocating movement, an eccentric rotational movement and a scroll movement, or a combination thereof. 37. The electrolytic processing method according to claim 33, wherein an ion exchanger is disposed between the workpiece and at least one of the processing electrode and the feeding electrode. 38. The electrolytic processing method according to claim 33, wherein the fluid is ultrapure water, pure water, a liquid having an electric conductivity of not more than 500 μS/cm. 39. An electrolytic processing apparatus, comprising: a plurality of processing electrodes; a feeding electrode for feeding electricity to the workpiece; a holder for holding the workpiece that can come close to or into contact with the processing electrodes; a power source for applying a voltage between the processing electrodes and the feeding electrode; a fluid supply section for supplying a fluid between the workpiece and one of the processing electrodes and the feeding electrode; and a drive section for allowing the processing electrodes and the workpiece to make a relative movement so that a plurality of processing electrodes, which are uneven in the processing amount per unit time, pass every point in the to-be-processed surface of the workpiece held by the holder. 40. The electrolytic processing apparatus according to claim 39, wherein the feeding electrode comprises a plurality of electrodes. 41. The electrolytic processing apparatus according to claim 39, wherein the plurality of processing electrodes are disposed in the feeding electrode such that the presence frequencies of processing electrodes at every points in the to-be-processed surface of the workpiece become substantially equal during the relative movement. 42. The electrolytic processing apparatus according to claim 40, wherein the plurality of feeding electrodes are disposed such that the presence frequencies of feeding electrodes at every points in the to-be-processed surface of the workpiece become substantially equal during the relative movement. 43. The electrolytic processing apparatus according to claim 39, wherein the plurality of processing electrodes are of the same shape. 44. The electrolytic processing apparatus according to claim 42, wherein the relative movement is one of a rotational movement, a reciprocating movement, an eccentric rotational movement and a scroll movement, or a combination thereof. 45. The electrolytic processing apparatus according to claim 42, wherein an ion exchanger is disposed between the workpiece and at least one of the processing electrodes and the feeding electrodes. 46. The electrolytic processing apparatus according to claim 42, wherein the fluid is ultrapure water, pure water, a liquid having an electric conductivity of not more than 500 μS/cm. 47. An electrolytic processing apparatus comprising: a processing electrode; a feeding electrode for feeding electricity to a workpiece; a holder for holding the workpiece that can come close to or into contact with the processing electrodes; a power source for applying a voltage between the processing electrode and the feeding electrode; a fluid supply section for supplying a fluid between the workpiece and at least one of the processing electrode and the feeding electrode; and a drive section for allowing the processing electrode and the workpiece to make a relative movement so that a plurality of points in the processing electrode, which are uneven in the processing amount per unit time, pass every point in the to-be-processed surface of the workpiece held by the holder. 48. The electrolytic processing apparatus according to claim 47, wherein the feeding electrode is disposed such that the presence frequencies thereof at every points in the to-be-processed surface of the workpiece become substantial equal during the relative movement. 49. The electrolytic processing apparatus according to claim 47, wherein the feeding electrode comprises a plurality of electrodes, and the plurality of feeding electrodes are of the same shape. 50. The electrolytic processing apparatus according to claim 47, wherein the relative movement is one of a rotational movement, a reciprocating movement, an eccentric rotational movement and a scroll movement, or a combination thereof. 51. The electrolytic processing apparatus according to claim 47, wherein an ion exchanger is disposed between the workpiece and at least one of the processing electrode and the feeding electrode. 52. The electrolytic processing apparatus according to claim 47, wherein the fluid is ultrapure water, pure water, a liquid having an electric conductivity of not more than 500 μS/cm, or an electrolytic solution. 53. A fixing method for fixing an ion changer for use in electrolytic processing on an electrode, comprising: positioning an ion exchanger between an electrode support, which supports an electrode with its surface exposed, and a fixing jig engageable with the periphery of the electrode support; and engaging the fixing jig with the electrode support, thereby fixing the ion exchanger with its peripheral portion sandwiched in between the fixing jig and the electrode support. 54. The fixing method according to claim 53, wherein the fixing jig consists of a pair of divided jigs, and the pair of divided jigs, with the ion exchanger at its peripheral portion sandwiched therebetween, is pressed into engagement with the electrode support. 55. A method for fixing an ion exchanger for use in electrolytic processing on an electrode, comprising: disposing an ion exchanger-fixing jig outside of an electrode; holding the ion exchanger by the ion exchanger-fixing jig; and attaching the ion exchanger-fixing jig to the electrode while allowing the ion exchanger to be supported in a tense state on the electrode. 56. A fixing structure for fixing an ion exchanger for use in electrolytic processing on an electrode, comprising: an electrode support that supports an electrode with its surface exposed; and a fixing jig engageable with the periphery of the electrode support; wherein the electrode support and the fixing jig fix an ion exchanger by sandwiching therebetween a peripheral portion of the ion exchanger and stretching the ion exchanger over the surface of the electrode. 57. The fixing structure according to claim 56, wherein the fixing jig consists of a pair of divided jigs, and an outer peripheral portion of the ion exchanger, outside of the portion covering the electrode support, is sandwiched in between the fixing jigs. 58. An electrolytic processing apparatus comprising an ion exchanger-fixing device, said ion exchanger-fixing device including; an electrode support that supports an electrode with its surface exposed; and a fixing jig engageable with the periphery of the electrode support; wherein the ion exchanger-fixing device fixes an ion exchanger by sandwiching a peripheral portion of the ion exchanger in between the electrode support and the fixing jig. 59. The electrolytic processing apparatus according to claim 58, wherein the electrode support and the fixing jig are allowed to move relatively to fix the ion exchanger by sandwiching the peripheral portion of the ion exchanger between the electrode support and the fixing jig. 60. The electrolytic processing apparatus according to claim 58, wherein the ion exchanger, disposed between the electrode support and the fixing jig, is capable of traveling. 61. The electrolytic processing apparatus according to claim 60, wherein the ion exchanger has an endless form and is capable of traveling in one direction, and a regeneration section for regenerating the ion exchanger is provided in a traveling route of the ion exchanger. 62. The electrolytic processing apparatus according to claim 60, wherein the ion exchanger is capable of traveling in two directions, and two regeneration sections for regenerating the ion exchanger are provided on both sides of the electrode support in the traveling direction of the ion exchanger.
<SOH> BACKGROUND ART <EOH>In recent years, instead of using aluminum or aluminum alloys as a material for forming interconnection circuits on a substrate such as a semiconductor wafer, there is an eminent movement towards using copper (Cu) which has a low electric resistivity and high electromigration resistance. Copper interconnects are generally formed by filling copper into fine recesses formed in the surface of a substrate. There are known various techniques for forming such copper interconnects, including CVD, sputtering, and plating. According to any such technique, a copper film is formed in the substantially entire surface of a substrate, followed by removal of unnecessary copper by chemical mechanical polishing (CMP) FIGS. 1A through 1C illustrate, in sequence of process steps, an example of forming such a substrate W having copper interconnects. As shown in FIG. 1A , an insulating film 2 , such as an oxide film of SiO 2 or a film of low-k material, is deposited on a conductive layer 1 a in which semiconductor devices are formed, which is formed on a semiconductor base 1 . Contact holes 3 and trenches 4 for interconnects are formed in the insulating film 2 by the lithography/etching technique. Thereafter, a barrier layer 5 of TaN or the like is formed on the entire surface, and a seed layer 7 as an electric supply layer for electroplating is formed on the barrier layer 5 . Then, as shown in FIG. 1B , copper plating is performed onto the surface of the substrate W to fill the contact holes 3 and the trenches 4 with copper and, at the same time, deposit a copper film 6 on the insulating film 2 . Thereafter, the copper film 6 and the barrier layer 5 on the insulating film 2 are removed by chemical mechanical polishing (CMP) so as to make the surface of the copper film 6 filled in the contact holes 3 and the trenches 4 for interconnects and the surface of the insulating film 2 lie substantially on the same plane. An interconnection composed of the copper film 6 as shown in FIG. 1C is thus formed. Components in various types of equipments have recently become finer and have required higher accuracy. As sub-micro manufacturing technology has commonly been used, the properties of materials are largely influenced by the processing method. Under these circumstances, in such a conventional machining method that a desired portion in a workpiece is physically destroyed and removed from the surface thereof by a tool, a large number of defects may be produced to deteriorate the properties of the workpiece. Therefore, it becomes important to perform processing without deteriorating the properties of the materials. Some processing methods, such as chemical polishing, electrolytic processing, and electrolytic polishing, have been developed in order to solve this problem. In contrast with the conventional physical processing, these methods perform removal processing or the like through chemical dissolution reaction. Therefore, these methods do not suffer from defects, such as formation of an altered layer and dislocation, due to plastic deformation, so that processing can be performed without deteriorating the properties of the materials. An electrolytic processing method that utilizes an ion exchanger has been developed. According to this method, an ion exchanger mounted on a processing electrode and an ion exchanger mounted on a feeding electrode are allowed to be close to or into contact with the surface of a workpiece. A voltage is applied from a power source to between the processing electrode and the feeding electrode while a liquid, such as ultrapure water, is supplied from a fluid supply section to between the processing and feeding electrodes and the workpiece, thereby carrying out removal processing of the surface layer of the workpiece. FIG. 2 schematically shows a conventional electrolytic processing apparatus generally employed for such electrolytic processing. The electrolytic processing apparatus includes a processing electrode 52 and an ion exchanger 54 that is mounted on the processing electrode 52 . Depending upon the material of a workpiece W, the processing electrode 52 is connected to the cathode or the anode of a power source 56 , and the workpiece W is connected to the opposite pole, and the workpiece W is utilized as a feeding electrode. FIG. 2 shows the case where the processing electrode 52 is connected to the cathode of the power source 56 and the workpiece W is connected to the anode of the power source 56 . The processing electrode 52 concentrates e.g. OH − ions, in an electrolytic solution capable of dissolving the atoms of the to-be-processed surface WA of the workpiece W, at the to-be-processed surface WA closed to the processing electrode 52 to cause a reaction between the atoms of the workpiece W and OH − ions, thereby processing the workpiece W. In the case of a semiconductor substrate W, a film of a conductive material formed in the substrate surface WA is removed by the processing electrode 52 in order to form semiconductor interconnects or contacts. According to the conventional electrolytic processing apparatus, an ion exchanger for use in such electrolytic processing is tight on the exposed surface of a processing electrode or a feeding electrode, and is fixed on the electrode or at a peripheral portion of e.g. a support that supports the electrode, usually by screwing or using an adhesive tape or the like at a peripheral portion of the ion exchanger. In recent years, as interconnects of the circuit to be formed in a semiconductor substrate has become finer with higher integrated density of the semiconductor device, it is desired to improve the flatness of the processed surface of the semiconductor substrate. Therefore, there is a demand for a technique that can improve the uniformity of the processing rate over the entire to-be processed surface. Metals of the platinum group or their oxides have become candidates for an electrode material for use in forming a capacitor, which utilizes a high dielectric material, on a semiconductor substrate. Among them ruthenium, because of its good film-forming properties and good processibility for patterning, is being progressively studied as a feasible material. A ruthenium film can be formed on a substrate generally by sputtering or CVD. In either method, deposition of the ruthenium film on the entire front surface of a substrate, including the peripheral region, is carried out. As a result, a ruthenium film is formed also in the peripheral region of the substrate and, in addition, the back surface of the substrate is unavoidably contaminated with ruthenium. The ruthenium film formed on or adhering to the peripheral region or back surface of a substrate, i.e. the non-circuit region of the substrate, is not only unnecessary, but can also cause cross-contamination during later transfer, storage and various processing steps of the substrate whereby, for instance, the performance of a dielectric material can be lowered. Accordingly, during the process for forming a ruthenium film or after carrying out some treatments of the formed ruthenium film, it is necessary to completely remove the unnecessary ruthenium film. Further, in the case of using ruthenium as an electrode material for forming a capacitor, a step for removing part of a ruthenium film formed on the circuit region of a substrate is needed. According to the conventional electrolytic processing apparatus as shown in FIG. 2 , however, because of unevenness of the electric current value due to the shape of the processing electrode 52 or to the influence of the reaction products or gas bubbles generated during processing, the processing rate is likely to be uneven in the to-be-processed surface WA. On the other hand, chemical mechanical polishing (CMP), for example, generally necessitates a complicated operation and control, and needs a considerably long processing time. In addition, a sufficient cleaning of a substrate must be conducted after the polishing treatment. This also imposes a considerable load on the slurry or cleaning liquid waste disposal. Accordingly, there is a strong demand for omitting CMP entirely or reducing a load upon CMP. Also in this connection, it is to be pointed out that though a low-k material, which has a low dielectric constant, is expected to be predominantly used in the future as a material for the insulating film of a semiconductor substrate, the low-k material has a low mechanical strength and therefore is hard to endure the stress applied during CMP processing. Thus, also from this standpoint, there is a demand for a technique that enables the flattering of a substrate without giving any stress thereto. Further, a method has been reported which performs CMP processing simultaneously with plating, viz. chemical mechanical electrolytic polishing. According to this method, the mechanical processing is carried out to the growing surface of a plating film, causing the problem of denaturing of the resulting film. Further, though it is desired that an ion exchange for use in electrolytic processing be tightly fixed on the exposed surface of a processing electrode or a feeding electrode, as described above, in order to ensure evenness of the processing accuracy, it has practically been difficult to keep the ion exchanger fixed tightly on the electrode. Thus, when continuing electrolytic processing while an ion exchanger is fixed on an electrode by screwing or with an adhesive tape, the fixing of ion exchanger is likely to become incomplete, so that the ion exchanger can move easily, impairing evenness of the processing accuracy.
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIGS. 1A through 1C are diagrams illustrating, in sequence of process steps, an example of the formation of copper interconnects; FIG. 2 is a cross-sectional view schematically showing a conventional electrolytic processing apparatus; FIG. 3 is a diagram illustrating the principle of electrolytic processing according to the present invention as carried out by allowing a processing electrode and a feeding electrode, both having an ion exchanger mounted thereon, to be close to or into contact with a substrate (workpiece), and supplying a liquid between the processing and feeding electrodes and the substrate (workpiece); FIG. 4 is a diagram illustrating the principle of electrolytic processing according to the present invention as carried by mounting an ion exchanger only on the processing electrode and supplying a liquid between the processing electrode and the substrate (workpiece); FIG. 5A is a diagram illustrating “face-to-face” area in the case of not providing a dummy member, and FIG. 5B is a diagram illustrating “face-to-face” area in the case of providing a dummy member; FIG. 6A is a diagram illustrating the electric field intensity in the case of not providing a dummy member, FIG. 6B is a diagram illustrating a workpiece after processing in the case of FIG. 6A , and FIG. 6C is a diagram illustrating the electric field intensity in the case of providing a dummy member; FIG. 7 is a cross-sectional view schematically showing an electrolytic processing apparatus according to a first embodiment of the present invention; FIG. 8 is a diagram showing the connection between the processing electrode and the wiring, and the connection between the substrate and the wiring in the electrolytic processing apparatus of FIG. 7 ; FIG. 9 is a plan view of the electrolytic processing apparatus of FIG. 7 ; FIG. 10 is a cross-sectional view schematically showing an electrolytic processing apparatus according to a second embodiment of the present invention; FIG. 11 is a plan view of the electrode holder of the electrolytic processing apparatus of FIG. 10 ; FIG. 12 is a diagram showing the connection between the processing electrode and the wiring, and the connection between the substrate and the wiring in the electrolytic processing apparatus of FIG. 10 ; FIG. 13 is a plan view showing the processing electrode and the feeding electrode of an electrolytic processing apparatus according to a third embodiment of the present invention; FIG. 14 is a diagram showing the connection between the processing electrode and the wiring, and the connection between the substrate and the wiring in the electrolytic processing apparatus of FIG. 13 ; FIG. 15 is a plan view illustrating a case of division of an ion exchanger; FIG. 16 is a plan view of a substrate processing apparatus provided with an electrolytic processing apparatus according to the present invention; FIG. 17 is a cross-sectional view schematically showing an electrolytic processing apparatus according to a fourth embodiment of the present invention; FIG. 18A is a plan view showing the rotation-preventing mechanism of the electrolytic processing apparatus of FIG. 17 , and FIG. 18B is a sectional view taken along the line A-A of FIG. 18A ; FIG. 19 is a cross-sectional view schematically showing the substrate holder and the electrode section of the electrolytic processing apparatus of FIG. 17 ; FIG. 20 is a plan view showing the relationship between the substrate holder and the electrode section of FIG. 17 ; FIG. 21A is a graph showing the relationship between the electric current and time in electrolytic processing of the surface of a substrate, the substrate having in the surface a laminated film composed of two different materials, and FIG. 21B is a graph showing the relationship between the voltage and time in electrolytic processing of the surface of a substrate, the substrate having in the surface a laminated film composed of two different materials; FIG. 22 is a perspective view schematically showing an electrolytic processing apparatus according to a fifth embodiment of the present invention; FIG. 23 is a plan view of the electrolytic processing apparatus of FIG. 22 ; FIG. 24 is a cross-sectional view schematically showing an electrolytic processing apparatus according to a sixth embodiment of the present invention; FIG. 25 is a cross-sectional view schematically showing the substrate holder and the electrode section of the electrolytic processing apparatus of FIG. 24 ; FIG. 26 is a plan view showing the relationship between the electrode section and the substrate of FIG. 25 ; FIG. 27 is a cross-sectional view schematically showing a substrate holder and an electrode according to another embodiment of the present invention; FIG. 28 is a plan view of an electrode section according to still another embodiment of the present invention; FIG. 29 is a perspective view of an electrode section according to still another embodiment of the present invention; FIG. 30 is a plan view showing an electrode section according to still another embodiment of the present invention together with a substrate; FIG. 31 is a cross-sectional view schematically showing an electrolytic processing apparatus according to a seventh embodiment of the present invention; FIG. 32 is a plan view of the electrolytic processing apparatus of FIG. 31 ; FIG. 33 is a plan view showing the electrode section of the electrolytic processing apparatus of FIG. 31 ; FIG. 34 is an enlarged view of a portion of the electrode section of FIG. 33 ; FIG. 35 is a plan view showing an electrode section according to an eighth embodiment of the present invention; FIG. 36 is an enlarged view of a portion of the electrode section of FIG. 35 ; FIG. 37 is a plan view showing an electrode section according to a ninth embodiment of the present invention; FIG. 38 is an enlarged view of a portion of the electrode section of FIG. 37 ; FIG. 39 is a cross-sectional view schematically showing an electrolytic processing apparatus according to a tenth embodiment of the present invention, which is provided with a fixing structure for ion exchanger; FIG. 40 is a cross-sectional view showing the state of the fixing structure before fixing an ion exchanger to the electrode support; FIG. 41 is a cross-sectional view showing the main portion of a fixing structure for ion exchanger according to another embodiment; FIGS. 42A and 42B are cross-sectional views showing the main portion of a fixing structure for ion exchanger according to still another embodiment; FIG. 43 is a cross-sectional view schematically showing an electrolytic processing apparatus according to eleventh embodiment of the present invention; FIG. 44 is a cross-sectional view of the main portion of the ion exchanger-fixing device provided in the electrolytic processing apparatus of FIG. 43 , showing the state of the device before fixing of an ion exchanger; FIG. 45 is a cross-sectional view showing the main portion of an electrolytic processing apparatus according to a twelfth embodiment of the present invention; FIG. 46 is a cross-sectional view showing the main portion of an electrolytic processing apparatus according to a thirteenth embodiment of the present invention; FIG. 47 is a cross-sectional view showing an example of a regeneration section; FIG. 48 is a cross-sectional view showing another example of a regeneration section; and FIG. 49 is a plan view schematically showing a variation of the ion exchanger-fixing device. detailed-description description="Detailed Description" end="lead"?

Butyric acid derivatives
The present invention relates to a compound of the formula I: (Formula I) in which: A, B, R1, Z, n and R2 are as defined in claim 1. These compounds are useful in the treatment of dyslipidaemia, atherosclerosis and diabetes.
1. Compound of the formula I: in which: A represents carboxyl; (C6-C18)aryloxycarbonyl in which the aryl group is optionally substituted; (C1-C14)alkoxycarbonyl in which the alkyl group is optionally substituted; —CO—NHOH; -tetrazolyl; B represents an optionally substituted ethylene group —CH2—CH2—; R1 represents a hydrogen atom; optionally substituted (C1C14)alkyl; optionally substituted (C6-C18)aryl; optionally substituted heteroaryl; (C6-C18)aryl(C1-C14)alkyl in which each of the aryl and/or alkyl radicals are optionally substituted; and heteroaryl(C1-C14)alkyl in which each of the heteroaryl and/or alkyl radicals are optionally substituted; Z represents S or Se; n is an integer equal to 0, 1 or 2; R2 represents optionally substituted (C6-C18)aryl; optionally substituted heteroaryl; or optionally substituted heterocycle containing an aromatic moiety; and when R1 represents optionally substituted (C6-C18)aryl, then R2 can also represent (C1-C14)alkyl; it being understood that when R1 represents naphthyl or 4-methoxyphenyl, A represents carboxyl or methoxycarbonyl, B represents ethylene, n represents 0, and P represents S or Se, then R2 does not represent phenyl, the stereoisomers thereof and the addition salts thereof with acids or bases. 2. Compound of the formula I according to claim 1, in which A represents —COOH. 3. Compound of the formula I according to claim 1, in which B represents ethylene. 4. Compound of the formula I according to claim 1, in which R1 represents benzyl optionally substituted on the phenyl nucleus; optionally substituted phenyl; or optionally substituted pyridyl; the substituents on the phenyl nuclei and on the pyridyl nucleus preferably being chosen from halogen atoms and cyano groups, trifluoromethyl groups, (C1-C6)alkyl groups or (C1-C6)alkoxy groups or a (C6-C18)aryl group itself optionally substituted by halogen, (C1-C6)alkyl, (C1-C6)alkoxy, CF3 or CN. 5. Compound of the formula I according to claim 1, in which R2 represents a radical chosen from optionally substituted phenyl; optionally substituted benzopyridine; optionally substituted benzothiazole; optionally substituted quinolyl; optionally substituted naphthyl; optionally substituted triazole; and the radical which is optionally substituted, the substituents on these radicals preferably being chosen from halogen atoms and —CN, —CF3, (C1-C6)alkyl or (C1-C6)alkoxy groups or a (C6-C18)aryl group optionally substituted by halogen, (C1-C6)alkyl, (C1-C6)alkoxy, CF3 or —CN. 6. Compound of the formula I according to claim 1, characterised in that Z represents S. 7. Process for preparing a compound of the formula I according to claim 1, in which A represents —COOH, Z represents S and n=0, characterised in that a compound of the formula II: in which B and R1 are as defined in claim 1, is reacted with a thiol of the formula III: R2—SH III in which R2 is as defined in claim 1, in the presence of a base. 8. Process according to claim 7, for the preparation of an enantiomer of the formula Ib in which: A, B, Z, R1, n and R2 are as defined in above and * denotes an asymmetric carbon, characterised in that the compound of the formula II is the enantiomer of the formula: in which B and R1 are as defined for formula Ib and * denotes an asymmetric carbon of the same configuration as the equivalent carbon of the formula Ib. 9. Process for preparing a compound of the formula I according to claim 1, in which A represents —COOH, Z represents Se and n=0, characterised in that a selenium compound of the formula IV: R2—Se—Se—R2 IV in which R2 is as defined in claim 1, is reacted with an organic or mineral base, and the resulting compound is then reacted with a compound of the formula II: in which B and R1 are as defined in claim 1 for formula I. 10. Process for preparing a compound of the formula I according to claim 1, in which A represents —COOH and n≠0, characterised in that a compound of the formula I in which n=0: in which R1, B, Z and R2 are as defined in claim 1 and A represents —COOH, is reacted with an oxidising agent such as m-chloroperbenzoic acid. 11. Pharmaceutical composition comprising an effective amount of at least one compound chosen from a compound of the formula I according to claim 1 and the compounds of the formula I for which R1 represents naphthyl or 4-methoxyphenyl; A represents carboxyl or methoxycarbonyl; B represents ethylene; n represents 0; Z represents S or Se and R2 represents phenyl, in combination with at least one pharmaceutically acceptable vehicle. 12. Use of a compound of the formula I according to claim 1 for the preparation of a medicament intended for preventing or treating dyslipidaemia, atherosclerosis and diabetes.



Hybrid proteins with neuregulin heparin-binding domain for targeting to heparan sulfate proteoglycans
Polypeptides of the neuregulin (NRG) heparin binding domain (N-HBD) and nucleic acids coding therefor are disclosed. In particular, fusion polypeptides are produced that comprise, as a targeting structure, a N-HBD polypeptide, fragment, homologue or functional derivative and a protein to be targeted. This is fused to a polypeptide or peptide being targeted (Ptrg) to cell surfaces rich in heparan sulfate proteoglycans to either activate or inhibit interactions at tyrosine kinase receptors. Such products are used to treat diseases or conditions where either agonism or antagonism at tyrosine kinase receptors has beneficial effects, including cancer and a multitude of diseases of the nervous system.
1-58. (canceled) 59. An isolated nucleic acid molecule of no more than about 100 nucleotides that encodes an animal N-HBD polypeptide or a functional derivative of said polypeptide, which polypeptide or functional derivative is characterized in that it (a) is a member of the C2 subfamily of the immunoglobulin superfamily but has less than about 40% sequence identity to a C2 domain of an immunoglobulin heavy or light chain from the same animal species; and (b) binds to heparin and heparan sulfate proteoglycans with a Kd of 10−5M or lower when measured in a conventional heparin-binding or heparan sulfate-binding assay. 60. The nucleic acid molecule of claim 59 that encodes the N-HBD polypeptide comprising an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:1, (b) SEQ ID NO: 2, (c) SEQ ID NO:3, and (d) a functional derivative or homologue of (a), (b) or (c). 61. The nucleic acid molecule of claim 60 that encodes the polypeptide SEQ ID NO:1 or encodes said biologically active fragment, homologue or other functional derivative of SEQ ID NO:1. 62. The nucleic acid molecule of claim 59 that comprises a nucleotide sequence selected from the group consisting of the sequence SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 63. The nucleic acid molecule of claim 62 that comprises the nucleotide sequence SEQ ID NO:4. 64. An isolated nucleic acid molecule of no more than about 100 nucleotides that hybridizes with the nucleic acid molecule of claim 62 under stringent hybridization conditions. 65. A hybrid nucleic acid molecule encoding a N-HBD fusion polypeptide, which molecule comprises: (a) as a first nucleic acid sequence, the nucleic acid molecule of claim 1; (b) optionally, fused in frame with the first nucleic acid sequence, a linker nucleic acid sequence encoding a linker peptide; and (c) a second nucleic acid sequence that (i) is linked in frame to said first nucleic acid sequence or to said linker nucleic acid sequence, and (ii) encodes a second polypeptide Ptrg. 66. The hybrid nucleic acid molecule of claim 65 wherein the first nucleic acid sequence encodes an polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2 and SEQ ID NO:3. 67. The hybrid nucleic acid molecule of claim 65 wherein the first nucleic acid sequence is a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO5 and SEQ ID NO:6. 68. The nucleic acid molecule of claim 65 wherein said second polypeptide Ptrg is (a) a soluble form of a cell surface receptor that is capable, as part of said fusion polypeptide, of binding a ligand for the receptor, thereby acting as an antagonist for ligand activation of the receptor; (b) a ligand for a cell surface receptor that is capable, as part of the fusion polypeptide, of binding to said receptor and thereby acting as either an agonist or antagonist at the receptor. 69. The nucleic acid molecule of claim 68 wherein said receptor is a tyrosine kinase receptor, a G-protein-coupled receptor or a cell surface immunoglobulin. 70. The nucleic acid molecule of claim 69 wherein said receptor is a tyrosine kinase receptor. 71. The nucleic acid molecule of claim 70 wherein said tyrosine kinase receptor is an epidermal growth factor receptor. 72. The nucleic acid molecule of claim 68 wherein said ligand is a cytokine or growth factor. 73. The nucleic acid molecule of claim 70 wherein said ligand is an epidermal growth factor, a fibroblast growth factor, a neurotrophic factor, a vascular endothelial growth factor, a transforming growth factor, a netrin or an ephrin. 74. The nucleic acid molecule of claim 73 wherein said ligand is an epidermal growth factor 75. An expression vector comprising the nucleic acid of claim 59 operatively linked to (a) a promoter and (b) optionally, additional regulatory sequences that regulate expression of said nucleic acid in a eukaryotic cell, which vector can be expressed in a cell after delivery to said cell in vitro or in vivo. 76. An expression vector comprising the nucleic acid of claim 60 operatively linked to (a) a promoter; and (b) optionally, additional regulatory sequences that regulate expression of said nucleic acid in a eukaryotic cell, which vector can be expressed in the cell after delivery to the cell in vitro or in vivo. 77. An expression vector comprising the nucleic acid of claim 62 operatively linked to: (a) a promoter; and (b) optionally, additional regulatory sequences that regulate expression of said nucleic acid in a eukaryotic cell, which vector can be expressed in the cell after delivery to the cell in vitro or in vivo. 78. The expression vector of claim 75 which is a plasmid. 79. The expression vector of claim 75 which is a viral vector. 80. A vector composition comprising (a) a first recombinant expression vector that includes one or more nucleotide sequences encoding a N-HBD polypeptide or encoding a biologically active fragment, homologue or other functional derivative of said polypeptide; and (b) a second recombinant expression vector that includes a nucleotide sequence encoding a polypeptide Ptrg, which Ptrg is (i) a soluble form of a cell surface receptor that is capable of binding a ligand for the receptor, thereby acting as an antagonist for ligand activation of the receptor; (ii) a ligand for a cell surface receptor that is capable of binding to said receptor and thereby acting as either an agonist or antagonist at the receptor, wherein the first and second expression vectors are able to co-infect or co-transfect a host cell resulting in co-expression of Ptrg and the N-HBD polypeptide, fragment, homologue or derivative. 81. The vector composition of claim 80 wherein said first vector comprises SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. 82. The vector composition of claim 81 wherein said first vector comprises SEQ ID NO:4. 83. A cell transformed or transfected with the nucleic acid molecule of claim 59. 84. A cell transformed or transfected with the expression vector of claim 75. 85. A cell transformed or transfected with the composition of claim 80. 86. The cell of claim 85 which is a mammalian cell. 87. The cell of claim 86 which is a human cell. 88. An isolated mammalian cell transfected with an exogenous nucleic acid molecule encoding a mammalian N-HBD polypeptide or a biologically active fragment, homologue or other functional derivative thereof, such that said polypeptide, fragment, homologue or derivative is expressed by said cell, which polypeptide or functional derivative: (a) is a member of the C2 subfamily of the immunoglobulin superfamily but has less than about 40% sequence identity to a C2 domain of an immunoglobulin heavy or light chain from the same animal species; and (b) binds to heparin and heparan sulfate proteoglycans with a Kd of 10−5M or lower when measured in a conventional heparin-binding or heparan sulfate-binding assay. 89. The cell of claim 88 wherein said exogenous nucleic acid molecule comprises SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a homologue, fragment or functional derivative of said exogenous nucleic acid molecule. 90. A fusion polypeptide comprising (a) a first targeting polypeptide that binds heparan sulfate when said fusion polypeptide is permitted to contact cells or tissues, thereby localizing the fusion polypeptide to said heparan-sulfate-rich cell or tissue surface, wherein said fusion polypeptide binds to heparin or to a heparan sulfate proteoglycan with a Kd of 1 0-5M or lower when measured in a conventional heparin-binding or heparan-sulfate binding assay; and (b) a second targeted polypeptide Ptrg that is to be targeted and localized to the heparan sulfate-rich cell or tissue surface; wherein, optionally, said first targeting polypeptide is fused directly to the Ptrg or is fused to a linker peptide sequence that is fused to the Ptrg, and wherein said fusion polypeptide, through the action of said Ptrg, has enhanced biological activity in stimulating or blocking a target receptor compared to a native form of the Ptrg or the Ptrg that is not fused to said targeting polypeptide. 91. The fusion polypeptide of claim 90 wherein said first targeting polypeptide (i) comprises all or a part of a N-HBD or a homologue or functional derivative thereof, and (ii) is fused directly to the Ptrg or optionally, is fused to a linker peptide sequence that is fused to the Ptrg. 92. The fusion polypeptide of claim 90, wherein the targeting polypeptide sequence is encoded by: (a) a nucleic acid molecule of no more than about 100 nucleotides that encodes an animal N-HBD polypeptide or a functional derivative of said polypeptide, which polypeptide or functional derivative (i) is a member of the C2 subfamily of the immunoglobulin superfamily but has less than about 40% sequence identity to a C2 domain of an immunoglobulin heavy or light chain from the same animal species; and (ii) binds to heparin and heparan sulfate proteoglycans with a Kd of 10−5M or lower when measured in a conventional heparin-binding or heparan-sulfate binding assay, or (b) a fragment, homologue or equivalent of said nucleic acid molecule. 93. A fusion polypeptide comprising (a) a first targeting polypeptide that binds heparan sulfate when said fusion polypeptide is permitted to contact cells or tissues, thereby localizing the fusion polypeptide to a heparan sulfate-rich cell or tissue surface, wherein said fusion polypeptide binds to heparin or to a heparan sulfate proteoglycan with a Kd of 10−5M or lower when measured in a conventional heparin-binding or heparan sulfate-binding assay; and (b) a second targeted polypeptide Ptrg that is to be targeted and localized to the heparan sulfate-rich cell or tissue surface, wherein said fusion polypeptide, through the action of the Ptrg, has enhanced biological activity in stimulating or blocking a target receptor compared to a native form of the Ptrg or the Ptrg that is not fused to said targeting polypeptide, said fusion polypeptide being encoded by the hybrid nucleic acid molecule of claim 65 or a fragment, homologue or equivalent of said hybrid nucleic acid molecule. 94. The fusion polypeptide of claim 93 that is encoded in part by a first nucleic acid sequence comprising SEQ ID NO:4, SEQ ID NO5 or SEQ ID NO:6. 95. A fusion polypeptide or a biologically active fragment, homologue or other functional derivative of said polypeptide produced by recombinant expression of the expression vector of claim 75. 96. The fusion polypeptide of claim 90, wherein the targeting polypeptide comprises an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:1, (b) SEQ ID NO: 2, (c) SEQ ID NO:3, and (d) a heparin binding functional derivative or homologue of (a), (b) or (c). 97. The fusion polypeptide of claim 96 wherein said functional derivative is a fragment having the sequence KWFKNGNELNRKNKPQNIKIQKKPGK (SEQ ID NO: 7), KWFKNGNELNRKNKPENIKIQKKPGK (SEQ ID NO:8), or KWLKNGKEITKKNRPENVKIPKKQKK (SEQ ID NO:9). 98. The fusion polypeptide of claim 96 wherein said functional derivative is (i) a polypeptide having the sequence K-x-x-K-x-x-x-x-x-x-R-K-x-K-x-x-x-K-x-x-K-K-x-x-K (SEQ ID NO:10), wherein x is any amino acid, or a fragment of the sequence SEQ ID NO:10 that has at least six residues that are Lys or Arg. 99. The fusion polypeptide of claim 90 that comprises a linker peptide that is cleavable by a protease. 100. The fusion polypeptide of claim 99 wherein the linker peptide has the sequence VPRGSD (SEQ ID NO:11) or DDKDWH (SEQ ID NO:12). 101. The fusion polypeptide of claims 90 that comprises a tandemly linked dimer or trimer of the first targeting polypeptide fused to the second targeted polypeptide. 102. The fusion polypeptide of claim 90 wherein the Ptrg is: (a) a soluble form of a cell surface receptor that is capable, as part of said fusion polypeptide, of binding a ligand for the receptor, thereby acting as an antagonist for ligand activation of the receptor; or (b) a ligand for a cell surface receptor that is capable, as part of the fusion polypeptide, of binding to said receptor and thereby acting as either an agonist or antagonist at the receptor. 103. The fusion polypeptide of claim 102 wherein said receptor is a tyrosine kinase receptor, a G-protein coupled receptor or a surface immunoglobulin. 104. The fusion polypeptide of claim 103 wherein said tyrosine kinase receptor is an epidermal growth factor receptor. 105. The fusion polypeptide of claim 102 wherein said ligand is a cytokine or growth factor. 106. The fusion polypeptide of claim 105 wherein said ligand is an epidermal growth factor, a fibroblast growth factor, a neurotrophic factor, a vascular endothelial growth factor, a transforming growth factor, a netrin or an ephrin. 107. The fusion polypeptide of claim 106 wherein said cytokine or growth factor is a neurotrophic factor selected from the group consisting of brain derived neurotrophic factor, glial derived neurotrophic factor, neurotrophin 3, neurotrophin 4, and nerve growth factor. 108. A pharmaceutical composition useful for delivering a targeted polypeptide to a cell or tissue surface and thereby enhancing the biological activity of the targeted polypeptide, comprising: (a) the fusion polypeptide of claim 90; and (b) a pharmaceutically acceptable excipient or carrier. 109. A pharmaceutical composition useful for delivering a targeted polypeptide to a cell or tissue surface and thereby enhancing the biological activity of the targeted polypeptide, comprising: (a) the fusion polypeptide of claim 93; and (b) a pharmaceutically acceptable excipient. 110. A mammalian cell that expresses on its surface or secretes the fusion polypeptide of claim 90. 111. A mammalian cell that expresses on its surface or secretes the fusion polypeptide of claim 93. 112. A pharmaceutical composition useful for delivering a targeted polypeptide that is in a form expressed on the surface of, or secreted by, a recombinant cell, comprising: (a) the cell of claim 83; and (b) a pharmaceutically acceptable excipient or carrier. 113. A pharmaceutical composition useful for delivering a targeted polypeptide that is in a form expressed on the surface of, or secreted by, a recombinant cell, comprising: (a) the cell of claim 110; and (b) a pharmaceutically acceptable excipient or carrier. 114. A method for localizing a targeted polypeptide to a cell or tissue surface rich in heparan sulfate, and thereby enhancing the polypeptide's biological activity at said surface, comprising providing to said surface the fusion polypeptide of claim 90, whereby the Ptrg of the fusion polypeptide is localized to said surface, such that the biological activity of the Ptrg is increased compared to the activity of (i) a native form of the Ptrg or (ii) the Ptrg that is not fused to said targeting polypeptide. 115. A method for localizing a targeted polypeptide to a cell or tissue surface rich in heparan sulfate, and thereby enhancing the polypeptide's biological activity at said surface, comprising providing to said surface the fusion polypeptide of claim 93, whereby the Ptrg of the fusion polypeptide is localized to said surface, such that the biological activity of the Ptrg is increased compared to the activity of (i) native Ptrg or (ii) Ptrg that is not fused to said targeting polypeptide. 116. The method of claim 114 wherein said providing is in vivo. 117. A method for treating a disease or condition in a subject treatable by the action of a Ptrg in the subject, comprising administering to said subject an effective amount of the pharmaceutical composition of claim 108, whereby the biological activity of the Ptrg of the fusion polypeptide is increased compared to the activity of (i) a native form of the Ptrg or (ii) the Ptrg that is not fused to said targeting polypeptide, thereby treating said disease or condition. 118. A method for treating a disease or condition in a subject treatable by the action of a Ptrg in a subject, comprising administering to said subject an effective amount of the pharmaceutical composition of claim 112, whereby cells bearing or secreting said Ptrg are made available to said cell or tissue surface, and wherein, the biological activity of the Ptrg is increased compared to the activity of (i) a native form of the Ptrg or (ii) the Ptrg that is not fused to said targeting polypeptide, thereby treating said disease or condition. 119. The method of claim 117 wherein said disease or condition is a tumor or cancer. 120. The method of claim 118 wherein said disease or condition is a tumor or cancer. 121. The method of claim 117 wherein said disease or condition is a neurological disorder. 122. The method of claim 118 wherein said disease or condition is a neurological disorder. 123. The method of claim 121, wherein said neurological disorder is a neurodegenerative disease, multiple sclerosis, stroke, epilepsy or traumatic brain, spinal cord or peripheral nerve injury. 124. The method of claim 123, wherein said neurodegenerative disease is Alzheimer's disease, Parkinson's disease or amyotrophic lateral sclerosis. 125. The method of claim 122 wherein said neurological disorder is a neurodegenerative disease, multiple sclerosis, stroke, epilepsy or traumatic brain, spinal cord or peripheral nerve injury. 126. The method of claim 125 wherein said neurodegenerative disease is Alzheimer's disease, Parkinson's disease or amyotrophic lateral sclerosis.
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The invention in the field of biochemistry and neurosciences relates to compositions and methods based on the neuregulin heparin binding domain (N-HBD) use to target other polypeptides to cell surfaces and extracellular matrix rich in heparan sulfate proteoglycans (HSPGs) for the treatment of cancer or various nervous system diseases. 2. Description of the Backgound Art In order to carry out their diverse physiological functions, cells must adhere to cellular and extracellular components of their environment in a specific manner. Adherence-requiring functions in the nervous system include neurite extension, synapse formation and axon myelination. The ability to recognize multiple environmental cues and undergo specific adhesive reactions is critical to such complex cellular functions. Recognition and adhesion are mediated by cell adhesion molecules (“CAMs”) which bind to macromolecules expressed on neighboring cells or in the extracellular matrix (“ECM”). Three motifs present in adhesion molecules for which 3D structure is known are: immunoglobulin (Ig) superfamily domains, fibronectin type III (Fn-III) domains and the domains found in cadherins. In the nervous system, Ig superfamily members mediate Ca-independent homophilic and heterophilic binding. The extracellular regions of these molecules include one or more domains with sequence similarity to variable (V) or constant (C) domains of antibodies, i.e., Ig's (Williams, A F et al., Annu. Rev. Immunol. 6:381-405 (1988). Yoshihara, Y et al., Neurosci. Res. 10:83-105 (1991). Many Ig superfamily molecules consist of tandem Ig-like domains bonded in series with multiple copies of a second building block domain (e.g., an Fn-III repeat). Because two molecules that share detectable sequence similarity have been found to adopt the same folding topology, investigators have used structures of molecules discovered in studies of the immune system as “first order” models for the structures of Ig domains in neural CAMs. These molecules, in particular Ig-like domains, and their topology are reviewed in detail in Vaughn D E et al., Neuron 16:261-273 (1998), which is incorporated by reference in its entirety. Ig V domains are the prototype of the V-like domains of CAMs and their type of fold is found in antibody V H and V L domains and the N-terminal domains of the T cell receptor α Vaughn et al., supra, V-like domains are found in T cell surface molecules CD4 (first and third domains) and CD8, the N-terminal domains of the “immune system CAM” CD2, vascular cell adhesion molecule-1 (VCAM-1) and telokin, and the C-terminal domain of the myosin light chain kinase. Ig C1 domains consist of seven B strands arranged into two antiparallel sheets. The two sheets are connected by a disulfide bond between strands “B” and “F.” In an antibody, constant domains are found in the Fc regions and the C-terminal domains of the Fab Ig fragment. Constant-like or C1 set domains, are also found in the membrane proximal domains of MHC molecules and T cell receptors. The C2 and C1 folding topologies are similar, except for the “sheet switching” of one β strand and the fact that β strands in Ig C2 domains are somewhat shorter (˜6 vs˜9 residues) and lack many of the conserved sequence patterns at the N-terminal end of the β barrel. According to Vaughn et al., supra, C2 domains are present in three Ig superfamily members: CD2, the second domain of VCAM-1 and the second and fourth domains of CD4. The heparin binding domain of neuregulin, which is at the center of the present invention, is an Ig-C2 domain. An important means of intercellular communication is the release of growth and differentiation factors from one cell and their binding to and activation of membrane receptors on a nearby cell which ultimately changes its properties through changes in gene expression. Once released, many polypeptide factors have additional binding interactions with heparan-sulfate proteoglycans (HSPGs) situated in the ECM between cells. The functional consequences of this dual-binding interaction are not entirely clear, but may serve to concentrate these factors at sites where they are needed, to protect them from proteolysis, and to modulate their interactions with their receptors (Schlessinger, J. et al., (1995) Cell 83, 357-360). What is even less clear, is how these extracellular interactions modulate the intracellular events that ultimately change a cell's properties. The neuregulins (NRGs) are a family of heparin-binding growth and differentiation factors with multiple functions in (a) growth and development of the nervous system and heart, and (2) cancer (Fischbach, G. D. et al. (1997) Annu Rev Neurosci 20, 429-458). NRGs are released from motor nerve endings at neuromuscular synapses and activate members of the epidermal growth factor (EGF) family of tyrosine kinase receptors, erbB2, erbB3 and erbB4, in the postsynaptic muscle membrane (Loeb, J A et al., (1999) Development 126, 781-791; Goodearl, A D et al., (1995) J Cell Biol 130, 1423-1434; Moscoso, L M et al., (1995) Developmental Biology 172, 158-169; Zhu, X et al., (1995) EMBO Journal 14, 5842-5848). This trans-synaptic activation results in a dramatic up-regulation of muscle acetylcholine receptors (AChRs) needed to guarantee proper synaptic transmission. NRG also promotes the transition from embryonic to adult forms of mammalian AChRs by inducing the switch to the α-AChR subunit (Martinou, J C et al., (1991) Proc. Natl Acad Sci USA 88, 7669-7673) and the expression of voltage-gated sodium channels (Corfas, G et al., (1993) Journal of Neuroscience 13, 2118-2125). All of these effects are expected to increase the efficacy of synaptic transmission as the target muscle fiber increases in size and the neuromuscular junction matures. Consistently, mice in which the Type I NRG1 allele is disrupted by homologous recombination, exhibit (1) a 50% reduction in the density of postsynaptic AChRs, and (2) a reduced safety factor for neuromuscular transmission when challenged by low doses of curare (Sandrock, A W et al. (1997) Science 276, 599-603). A common feature shared by all NRGs is an epidermal growth factor-like (EGF-like) domain. Even when expressed by itself, this domain is sufficient for receptor binding and activation of homo- and heterodimers of erbB2, erbB3, and erbB4 receptors which are highly concentrated at the neuromuscular synapses in the postsynaptic muscle membrane (Moscoso et al.,supra; Xu et al., supra; Altiok, N. et al., v (1995) Embo J 14, 4258-4266). The rapid autophosphorylation of Tyr residues in these receptors initiates a signaling cascade that translates the initial binding event into the induction of AChR genes (Corfas, G. et al., (1993) Proc. Natl Acad Sci USA 90, 1624-1628). This signaling cascade involves a number of signaling pathways including both the mitogen-activated protein (MAP) kinase (Si, J. et al., Luo, Z., and Mei, L. (1996) J Biol Chem 271, 19752-19759; Tansey, M G et al., (1996) J. Cell Biol 134, 465-476; Altiok, N et al., (1997) EMBO Journal 16, 717-725) and phosphatidyl-inositol 3-kinase (PI3K) pathways (Si et al., supra). Most spliced forms of NRG also have an immunoglobulin-like (IG-like) domain N-terminal to the EGF-like domain ( FIG. 1 ). Because this domain is a heparin-binding domain (“HBD”) it is referred to herein as the neuregulin HBD (or “N-HBD”). The terms “IG-like domain” (from NRG) and “N-HBD” are meant to be interchangeable. The present inventor and others have shown that this domain interacts with HSPGs and may lead to the deposition of NRGs in the ECM of neuromuscular synapses and within the central nervous system (Loeb et al., supra; Loeb, J A et al., (1995) J Cell Biol 130, 127-135; Meier, T., et al., (1998) J Cell Biol 141, 715-726). HSPGs, including agrin, have been identified to play important roles in neuromuscular junction formation (Sanes, J R et al., (1999) Annu Rev Neurosci 22, 389-442). HSPGs may serve to “direct” the accumulation of NRG forms that include the N-HBD to the basal lamina of developing neuromuscular synapses and to other locations in the developing nervous system at key stages of development (Loeb et al., 1999, supra). The functional consequences of NRG-HSPG interactions on AChR expression, however, are not known. One feature of NRG that distinguishes it from other heparin-binding ligands is that it has distinct domains for heparan sulfate binding and receptor binding that are separated from one another by a glycosylated spacer region. Recognition of this fact led the present inventor to determine the direct effects of HSPG binding on receptor- and gene activation that would not be readily possible with other heparin-binding ligands. Rio, C et al., Neuron 19:39-50 (1997) described a 27 amino acid peptide of chick NRG that corresponded to the HBD. This peptide was made only for use as an immunogen for producing an antiserum in rabbits. Loeb, J A et al., 1995, supra, speculated that immobilization of NRGs to the ECM might be via their Ig-like domains binding to HSPGs. This was derived indirectly from the observation that heparin inhibited post-binding receptor tyrosine phosphorylation caused by recombinant NRGs. Since NRGs bind to heparin (Falls, D L et al., 1993, Cell 72:801-815), Meier T et al., J Cell Biol, 1998, 141:715-726, examined whether recombinant HRG (=NRG) cloned from a human cDNA library bound directly to recombinant chick agrin (a HSPG) by the negatively charged glycosaminoglycan (GAG) side chains as proposed by Loeb et al., supra. It was found that the Ig-like domain of NRGs mediated binding to these GAG chains. To test whether interaction of NRGs with components of the synaptic ECM could be mediated by the Ig-like domain, the investigators expressed a truncated HRG protein containing the Ig-like domain, HRGΔBbsI and discovered that the Ig-like domain, but not the EGF-like domain, bound to agrin. While there have been numerous disclosures of Ig-C regions or various parts of Ig molecules fused to other proteins for various purposes, these primarily derived from true Ig molecules. The N-HBD of the present invention has less than 40% homology or sequence similarity to these true Ig domains so as to be distinct structurally and functionally from those in the prior art. Examples of such disclosures include the following. U.S. Pat. Nos. 5,116,964 and 5,428,130, (Capon, et al) disclose a nucleic acid encoding a polypeptide fusion comprising a ligand binding partner protein containing more than one polypeptide chain one of which may be fused to an Ig C region through C-terminal carboxyl or the N-terminal amino groups. The lectin domain described in these documents, which is completely distinct from the NRG-HBD neuregulin IG domain of the present invention, is said to target active peptides to cell surfaces. Moreover, such targeting is not directed to, nor specific for, heparan sulfates at the cell surfaces. U.S. Pat. No. 5,565,335 (Capon, et al.) describes an “immunoadheson” comprising a fusion protein in which a polypeptide making up the adheson variable (V) region is fused at its C-terminus to the N-terminus of a polypeptide comprising an Ig C region. U.S. Pat. No. 6,018,026 and U.S. Pat. No. 5,155,027 (Sledziewski et al.) describe biologically active polypeptides (and their coding DNA), and, specifically, dimerized fusion products comprising a first and a second polypeptide chain, each of which comprises a non-Ig polypeptide and requires dimerization for biological activity, joined to a dimerizing protein of heterologous origin relative to the non-Ig polypeptide. Also described is a polypeptide chain of the non-Ig polypeptide dimer, joined to at least one Ig H chain C region domain (C H 1, C H 2, C H 3 or C H 4. The expressed, dimerized fusion polypeptide exhibits biological activity characteristic of the non-Ig polypeptide dimer. U.S. Pat. No. 5,541,087 (Lo, et al.) describes DNA encoding a fusion protein comprising a polynucleotide encoding an Ig Fc region which lacks at least the C H 1 domain and a target protein sequence. U.S. Pat. No. 5,869,046 (Presta, et al.) discloses a method for preparing a variant “polypeptide of interest” which is an Fab or a (Fab′) 2 fragment, the Ig domain (or an Ig-like domain) of which comprises at least one of a C H 1 or C L , region. U.S. Pat. No. 6,121,022 (Presta, et al.) discloses a modified polypeptide having an Ig C domain or an Ig-like C domain and an epitope that binds to a salvage receptor within the Ig- or Ig-like C domain. This epitope, absent from the unmodified polypeptide, is taken from two loops of the C H 2 domain of an Ig Fc region. The Ig-like domains described in these documents are clearly distinct from the N-HBD of the present invention. U.S. Pat. No. 6,121,415 describes a family of polypeptides, collectively called neuregulins (NRG1) that appear to result from alternate splicing of a single gene which was mapped to the short arm of human chromosome 8 by Orr-Urtreger et al (1993) Proc. Natl. Acad. Sci. USA 90:1867-1871. The NRG3s (murine and human) were disclosed as being about 713 and 720 amino acids in length, respectively, and to comprise an EGF-like domain, an N-terminal hydrophobic segment, the serine/threonine-rich portion, a predicted transmembrane domain, and a predicted intracellular domain. Three documents by Holmes et al. ( Science 256:1205-1210 (1992); WO 92/20798; and U.S. Pat. No. 5,367,060) describe isolation and cloning of a family of polypeptide activators for the HER2 receptor which they called heregulin-α (HRG-α), heregulin-β1 (HRG-β1), heregulin-β2 (HRG-β2), heregulin-β2-like (HRG-β2-like), and heregulin-β3 (HRG-β3). These documents describe (1) the ability of the purified HRG (=NRG) polypeptides to activate tyrosine phosphorylation of the HER2 receptor in MCF7 breast tumor cells and (2) the mitogenic activity of the HRG polypeptides on tumor cells expressing high levels of the HER2 receptor. Like other EGF family growth factors, soluble HRG polypeptides appear to be derived from a membrane bound precursor (pro-HRG) which is proteolytically processed to release the 45 kDa soluble form. Although substantially identical in the first 213 amino acid residues, the HRGs are classified into two major types, α and β, based on two variant EGF-like domains which differ in their C-terminal regions. Based on an amino acid sequence comparison, Holmes et al., supra found that between the first and sixth cysteines in the EGF-like domain, HRGs were 45% similar to heparin-binding EGF-like growth factor (HB-EGF), 35% identical to amphiregulin, 32% identical to TGF-α, and 27% identical to EGF. Falls et al. (1993) Cell 72:801-815 described another heregulin family member which termed “acetylcholine receptor inducing activity” (ARIA) polypeptide. The chicken-derived polypeptide stimulated synthesis of muscle AChR s. See also WO 94/08007. ARIA is a β type HRG and lacks the entire spacer region rich in glycosylation sites between the Ig-like domain and EGF-like domain of HRGα, and HRGβ1-β3. Marchionni et al. (1993) Nature 362:312-318, identified several bovine-derived proteins named glial growth factors (GGFs) which share the Ig-like domain and EGF-like domain with the other NRG/HRG proteins described above, but also have an amino-terminal kringle domain. See also WO 92/18627; WO 94/00140; WO 94/04560; WO 94/26298; and WO 95/32724. Ho et al.(1995) J. Biol. Chem. 270:14523-14532, described another member of the HRG family called sensory and motor neuron-derived factor (SMDF) which has an EGF-like domain characteristic of all other HRG polypeptides but a distinct N-terminal domain. The major structural difference between SMDF and the other HRG polypeptides is the lack of an Ig-like domain and the “glyco” spacer characteristic of all the other HRG polypeptides. Caraway et al. (1994) J Biol Chem. 269):14303-14306 subsequently demonstrated that ErbB3 is a receptor for HRG and mediates phosphorylation of intrinsic tyrosine residues as well as phosphorylation of ErbB2 receptor in cells which express both receptors. HRG was the only known member of the EGF-like family that could interact with several receptors (Carraway et al. (1994) Cell 78:5-8. A number of biological activities of the NRG/HRG proteins have been described: (1) myotube differentiation by acting on synthesis and concentration of neurotransmitter receptors in the postsynaptic muscle (Falls et al., supra); (2) increased number of sodium channels in chick muscle (Corfas et al., (1993) J. Neuroscience 13:2118-2125); (3) mitogenic stimulation of subconfluent quiescent human myoblasts and their differentiation to yield more myotubes (Sklar et al. (1994) J. Cell Biochem. Abstr. W462, 18D, 540); and WO 94/26298, Nov. 24, 1994); and (4) NRG1, expressed in endocardium, is an important ligand required for activation of myocardial ErbB2 and ErbB4 receptors (Ford, B D et al., Dev Biol. (1999) 214:139-150; Carraway, K L et al., Bioessays (1996) 18:263-266.
<SOH> SUMMARY OF THE INVENTION <EOH>Some of the ABBREVIATIONS used herein include: NRG, neuregulin; NRG, heregulin; HBD, heparin-binding domain; N-HBD, neuregulin heparin-binding domain; AChR, acetylcholine receptor, HSPG, heparan-sulfate proteoglycan; EGF, epidermal growth factor; IG or Ig, immunoglobulin; MAPK, mitogen-activated protein kinase; PI3-K, phosphatidylinositol 3-kinase; BSA, bovine serum albumin; MEM, minimum essential media; CEE, chick embryo extract; α-BTX, α-bungarotoxin; FGF, fibroblast growth factor; ECM, extracellular matrix; TGF-β transforming growth factor-β; CREB, cAMP response element-binding protein. The present inventor has discovered that the N-HBD (also referred to as the neuregulin IG-like domain) functions to keep the EGF-like domain at sufficiently high concentrations near erbB receptors for a sufficiently long period of time necessary to induce events downstream from -receptor binding such as AChR gene expression. The present inventor examined how NRG-HSPG interactions affect NRG-erbB receptor binding, erbB receptor auto-phosphorylation and downstream activation of AChR genes and newly-synthesized proteins in primary chick myotube cultures. Using recombinant NRG β1 isoforms with and without the HBD, it is demonstrated herein that the N-HBD potentiated the EGF-like domain's action on receptor phosphorylation by interacting with endogenous HSPGs. Through these HSPG interactions, the N-HBD induces sustained NRG-erbB receptor phosphorylation for over 8 hrs that is required, for example, to turn on AChR mRNA and protein expression. These results provide a molecular rationale for the high concentration of NRG in the ECM of neuromuscular synapses. The N-HBD contains two Cys residues separated by 55 amino acids with a Trp located 13 residues from the first Cys. This is characteristic of the Ig C2 subfamily of Ig gene superfamily. For example the native human N-HBD sequence had only 32% identity with a more “conventional” Ig C2 Ig domain found on CD4. The N-HBD, homologue or functional derivative of the present invention preferably has the above sequence characteristic and less than about 40% identity with an Ig C2 domain of an Ig H or L chain from the same animal species. The present invention is directed in particular to a novel hybrid or fusion polypeptide that includes at least two domains or peptidic structures: (1) a first “targeting” polypeptide domain whose role is to target the fusion polypeptide, and (2) a fusion partner referred to herein as a “targeted” polypeptide or “P trg. ” The targeting domain is preferably an animal N-HBD, more preferably a mammalian N-HBD, most preferably a human N-HBD. Some of the Examples below show results obtained using a chicken N-HBD, which has features in common with human N-HBD, including sequence identity or similarity. In one embodiment, DNA encoding the amino acid sequence corresponding to the N-HBD, containing or a biologically active fragment thereof is joined to DNA encoding a P trg , for example, by PCR, to form a construct that is expressed as an N-HBD/P trg fusion protein. The present invention is thus directed to an isolated nucleic acid molecule of no more than about 100 nucleotides that encodes an animal N-HBD polypeptide or a functional derivative of the polypeptide, which polypeptide or functional derivative is characterized in that it (a) is a member of the C2 subfamily of the immunoglobulin superfamily but has less than about 40% sequence identity to a C2 domain of an Ig H or L chain from the same animal species; and (b) binds to heparin or to a heparan sulfate proteoglycan with a K d of about 10 −5 M or lower when measured in a conventional heparin-binding or heparan-sulfate binding assay. In the above nucleic acid molecule, the N-HBD polypeptide preferably comprises an amino acid sequence selected from the group consisting of: (a) GSKLVLRCET SSEYSSLRFK WFKNGNELNR KNKPQNIKIQ KKPGKSELRI NKASLADSGE YMCKVISKLG (SEQ ID NO:1) from human NRG; (b) GSKLVLRCET SSEYSSLRFK WFKNGNELDN KNKPENIKIQ KKPGKSELRI NKASLADSGE YMCKVISKLG (SEQ ID NO: 2) from rat NRG; (c) GQKLVLRCET TSEYPALRKW LKNGKETTKK NRPENVKIPK KQKKYSELHI YRATLADAGE YACRVSSKLG SEQ ID NO:3 from avian NRG; and (d) a functional derivative or homologue of (a), (b) or (c). The above nucleic acid molecule may comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 or (SEQ ID NO:6). In another embodiment, the invention is an isolated nucleic acid molecule of no more than about 100 nucleotides that hybridizes with any of the above nucleic acid molecules under stringent hybridization conditions. The present invention is directed to a hybrid nucleic acid molecule encoding a N-HBD fusion polypeptide, which molecule comprises: (a) as a first nucleic acid sequence, any of the above nucleic acid molecules; (b) optionally, fused in frame with the first nucleic acid sequence, a linker nucleic acid sequence encoding a linker peptide; and (c) a second nucleic acid sequence that (i) is linked in frame to the first nucleic acid sequence or to the linker nucleic acid sequence and (ii) encodes a second polypeptide P trg . Preferably, in the above hybrid nucleic acid, the second polypeptide P trg is: (a) a soluble form of a cell surface receptor that is capable, as part of the fusion polypeptide, of binding a ligand for the receptor, thereby acting as an antagonist for ligand activation of the receptor; or (b) a ligand for a cell surface receptor that is capable, as part of the fusion polypeptide, of binding to the receptor and thereby acting as either an agonist or antagonist at the receptor, depending in part on affinity and concentration. The receptor is preferably a tyrosine kinase receptor, a G-protein-coupled receptor or an antibody, most preferably a tyrosine kinase receptor. A preferred receptor is an EGF receptor. The ligand may be a cytokine or growth factor, preferably an epidermal growth factor, a fibroblast growth factor, a neurotrophic factor, a vascular endothelial growth factor, a transforming growth factor, a netrin or an ephrin. Also provided is an expression vector comprising the above nucleic acid molecule operatively linked to (a) a promoter and (b) optionally, additional regulatory sequences that regulate expression of the nucleic acid in a eukaryotic cell, which vector can be expressed in a cell after delivery to the cell in vitro or in vivo. Preferred vectors are plasmids or viral vectors. A related embodiment is a vector composition, comprising (a) a first recombinant expression vector having incorporated in its nucleic acid one or more nucleotide sequences encoding a N-HBD polypeptide or a biologically active fragment, homologue or other functional derivative thereof; and (b) a second recombinant expression vector having incorporated in its nucleic acid a nucleotide sequence encoding a polypeptide P trg , which is (i) a soluble form of a cell surface receptor that is capable, as part of the fusion polypeptide, of binding a ligand for the receptor, thereby acting as an antagonist for ligand activation of the receptor; or (ii) a ligand for a cell surface receptor that is capable, as part of the fusion polypeptide, of binding to the receptor and thereby acting as either an agonist or antagonist at the receptor. wherein the expression vectors are able to co-infect or co-transfect a host cell resulting in co-expression of P trg and the N-HBD polypeptide, fragment, homologue or derivative. In the vector composition, the first vector preferably comprises SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. The present invention provides a cell transformed or transfected with any of the above nucleic acid molecules or vectors, preferably a mammalian cell, most preferably a human cell. One embodiment is an isolated mammalian cell transfected with an exogenous nucleic acid molecule encoding a mammalian N-HBD polypeptide or a biologically active fragment, homologue or other functional derivative thereof, such that the polypeptide, fragment, homologue or derivative is expressed by the cell, which polypeptide or functional derivative (a) is a member of the C2 subfamily of the immunoglobulin superfamily but has less than about 40% sequence identity to a C2 domain of an Ig H or L chain from the same animal species; and (b) binds to heparin or to heparan sulfate proteoglycans with a K d of about 10 −5 M or lower when measured in a conventional heparin-binding or heparan-sulfate binding assay. In the above cell, the exogenous nucleic acid molecule preferably comprises SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or a homologue, fragment or functional derivative thereof. This invention is also directed to a fusion polypeptide comprising (a) a first targeting polypeptide that binds heparan sulfate when the fusion polypeptide is permitted to contact cells or tissues, thereby localizing the fusion polypeptide to the heparan-sulfate rich cell or tissue surface; wherein the fusion protein binds to heparin or to a heparan sulfate proteoglycan with a K d of 10 −5 M or lower when measured in a conventional heparin-binding or heparan-sulfate binding assay; and (b) a second targeted polypeptide P trg that is to be targeted and localized to the heparan sulfate-rich cell or tissue surface; wherein the fusion polypeptide, through the action of the P trg , has enhanced biological activity in stimulating or blocking a target receptor compared to native P trg or P trg not fused to the targeting polypeptide. In the above fusion polypeptide, the first targeting polypeptide preferably comprises all or a part of a N-HBD or a homologue or functional derivative thereof which (i) is fused directly to the second targeted polypeptide or, (ii) optionally, is fused to a linker peptide sequence that is fused to the second targeted polypeptide. The targeting polypeptide sequence is preferably encoded by the a nucleic acid molecule as described above, or by a fragment, homologue or equivalent of the nucleic acid molecule. The above fusion polypeptide or biologically active fragment, homologue or other functional derivative of the polypeptide is preferably produced by recombinant expression of the above expression vector or vector composition. In the above fusion polypeptide, the targeting polypeptide preferably has the amino acid sequence SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a heparin binding functional derivative or homologue thereof. A preferred functional derivative is a fragment having the sequence KWFKNGNELNRtKNKPQNIKIQKKPGK (SEQ ID NO: 7), KWFKNGNELNRKNKPENIKIQKKPGK (SEQ ID NO:8) or KWLKNGKEITKKNRPENVKIPKKQKK (SEQ ID NO:9). In another embodiment the functional derivative is a polypeptide having the sequence K-x-x-K-x-x-x-x-x-x-R-K-x-K-x-x-x-K-x-x-K-K-x-x-K (SEQ ID NO:10), wherein x is any amino acid, or a fragment thereof having at least four, preferably at least six Lys or Arg residues. The fragment may have any consensus heparin binding sequence, for example Z-x-Z-Z, wherein Z is a basic amino acid. Preferably, the above fusion polypeptide binds to heparan sulfate with an affinity characterized by a K d of less than about 10 −5 M and has enhanced biological activity in stimulating or blocking a target receptor compared to native P trg or P trg not fused to the targeting polypeptide. In the above fusion polypeptides, the linker, if present, may be one cleavable by a protease, such as VPRGSD (SEQ ID NO:11) or DDKDWH (SEQ ID NO:12). The fusion polypeptide may be a linear multimer of two or more repeats of monomers of the first targeting polypeptide linked end to end (i) directly or (ii) with a linker sequence present between the monomer repeats. One example, comprises a tandemly linked dimer or trimer of the first targeting polypeptide fused to the second targeted polypeptide. The second “targeted” polypeptide P trg is preferably (a) a soluble form of a cell surface receptor that is capable, as part of the fusion polypeptide, of binding a ligand for the receptor, thereby acting as an antagonist for ligand activation of the receptor; (b) a ligand for a cell surface receptor that is capable, as part of the fusion polypeptide, of binding to the receptor and thereby acting as either an agonist or antagonist at the receptor. As indicated above, the receptor may be a tyrosine kinase receptor, a G-protein coupled receptor or an antibody, preferably an EGF receptor. The ligand is a cytokine or growth factor, preferably an epidermal growth factor, a fibroblast growth factor, a neurotrophic factor, a vascular endothelial growth factor, a transforming growth factor, a netrin or an ephrin. A preferred neurotrophic factor is brain derived neurotrophic factor, glial derived neurotrophic factor, neurotrophin 3, neurotrophin 4, or nerve growth factor. The present invention also provides a pharmaceutical composition useful for delivering a targeted polypeptide to a cell or tissue surface and enhancing the biological activity of the targeted polypeptide, comprising: (a) the fusion polypeptide described above; and (b) a pharmaceutically acceptable excipient or carrier. Also provided is a mammalian, preferably human, cell that expresses on its surface or secretes the above fusion polypeptide. Another pharmaceutical composition that is useful for delivering a targeted polypeptide that is in a form expressed on the surface of, or secreted by, a recombinant cell, comprises (a) a cell as described above and (b) a pharmaceutically acceptable excipient or carrier. This invention is further directed to a method for localizing a targeted polypeptide to a cell or tissue surface rich in heparan sulfate, and thereby enhancing its biological activity at the surface, comprising providing to the surface the fusion polypeptide of any of claims 28 - 46 whereby the P trg of the fusion polypeptide is localized to the surface, such that the biological activity of the P trg is increased compared to the activity of native P trg or P trg not fused to the targeting polypeptide. The providing is preferably in vivo. Also included is a method for treating a disease or condition in a subject treatable by the action of the P trg, comprising administering to the subject an effective amount of the above pharmaceutical composition, whereby the biological activity of the P trg of the fusion polypeptide is increased compared to the activity of native P trg or P trg not fused to the targeting polypeptide, thereby treating the disease or condition. The method for treating a disease or condition in a subject treatable by the action of the P trg, may comprise administering to the subject an effective amount of the above cellular pharmaceutical composition, whereby cells bearing or secreting the P trg are made available to the cell or tissue surface, and wherein, the biological activity of the P trg is increased compared to the activity of native P trg or P trg not fused to the targeting polypeptide, thereby treating the disease or condition. In the above method, the disease or condition may be a tumor or cancer. In another embodiment, the disease or condition is a neurological disorder, for example, a neurodegenerative disease, multiple sclerosis, stroke, epilepsy or traumatic brain, spinal cord or peripheral nerve injury. Neurodegenerative diseases treatable by this method include Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.

Method of sealing the join between a zip and a garment with a lining
A method of sealing the join between two tapes to which the teeth of a waterproof zip are attached and two edges of a garment composed of a first or waterproof layer and a second or fabric layer, which comprises: a) applying to a first side of each tape, a strip of heat-weldable textile material attached to it, by at least one weld line, such that each strip comprises a first and a second flap; b) welding the edge of the first layer to one face of the flap furthest from the teeth; c) attaching the edge of the second layer to the second side of each tape; d) folding down the flap nearest to the teeth of the zip; and e) forming another weld that hermetically joins the outline of the outer edges of the flap to the corresponding tape around the area.
1. Method of sealing the join between the two tapes (2,3) to which the teeth (4,5) of a waterproof zip (1) are attached and two edges of a garment composed of a first or waterproof layer (7) and a second or fabric layer (8), these layers being superimposed on each other, which method is characterized in that it comprises the following steps: a) applying, to a first side (2e, 3e) of each of the said two tapes (2,3), a strip (9) of heat-weldable textile material attached to it by at least one weld line (L) formed along a longitudinal line, in such a way that each strip (9) comprises a first unattached flap (9f) and a second unattached flap (9s), one on each side of the said weld line (L) and both parallel to the lines of teeth (4,5) of the zip (1); b) welding the edge of the said first or waterproof layer (7) to one face of the flap (9s) furthest from the teeth (4,5) of the zip (1); [C)] attaching the edge of the said second layer (8) to the second side (2i, 3i) of each of the said tapes (2,3) by means of a seam (K) in an area (Z) situated between the said weld line (L) and the zip (6); d) folding down the flap (9f) nearest to the teeth (4,5) of the zip (1) so that it lies parallel against the said first side (2e, 3e) of the corresponding tape (2,3) and entirely covers the said area (Z); and e) forming another weld (N) that hermetically joins the outline of the outer edges of the flap (9f) to the corresponding tape (2,3) around the said area [(Z).] 2. Method according to claim 1, in the case in which the said garment is composed of an impermeable outer layer (7) and a fabric lining (8), these two being superimposed on each other, which method is characterized in that it comprises the following steps: a) applying, to the outside [(2E, 3E)] of each of the said two tapes [(2, 3), A] strip (9) of heat-weldable textile material attached to it by at least one weld line (L) formed along a longitudinal line, in such a way that each strip (9) comprises a first unattached flap (9f) and a second unattached flap [(9S), ONE] on each side of the said weld line (L) and both parallel to the lines of teeth [(4, 5) OF&APOS;THE] zip (1); b) welding the edge of the said waterproof outer layer (7) to one face of the flap (9s) furthest from the teeth [(4, 5)] of the zip (1); [C)] attaching the edge of the said lining (8) to the inside (2i, 3i) of each of the said tapes (2,3) by means of a seam (K) in an area (Z) situated between the said weld line (L) and the zip (6); d) folding down the flap (9f) nearest to the teeth (4,5) of the zip (1) so that it lies parallel against the said outside (2e, 3e) of the corresponding tape (2,3) and entirely covers the said area (Z); and e) forming another weld (N) that hermetically joins the outline of the outer edges of the flap (9f) to the corresponding tape (2,3) around the said area [(Z).] 3. Method according to claim 1, in which the said welds are heat welds. 4. Method according to claim 1, in which the said welds are chemical welds. 5. Assembly of waterproof zip (1) and tapes (2,3) to which its teeth (4,5) are attached, characterized in that there is welded, to a first side (2e, 3e) of each tape (2,3) along a longitudinal weld line (L), a strip (9) of heat-weldable textile material which, after being welded, has a first unattached flap (9f) and a second unattached flap (9s) extending longitudinally on each side of the abovementioned weld line [(L)] and parallel with respect to the lines of the said teeth (4,5). 6. Method according to claim 2, in which the said welds are heat welds. 7. Method according to claim 2, in which the said welds are chemical welds.



Comminuting apparatus and rotor therefor
A rotor (1) for use in a comminuting apparatus (100) is described and claimed. The rotor (1) includes a hub (2) rotatable about a first axis (A) and one or more disc members (4) for comminuting material. The disc members (4) are secured to the hub (2) and rotatable about a second axis (B), removed from the first axis (A). Drive means is provided to simultaneously rotate the hub (2) disc members (4) about their respective axes. Guides (11) may be provided below the disc members (4) to remove comminuted material after it has passed through a screen (10). The screen (10) may include base portion (10A) and side wall portion C (10B), the side wall portion (10B) extending up to a level approximately level with the disc members (4). The guides (11) may include arms (11A) extending up beside the side wall portion (10B). A comminuting apparatus (100) including a rotor (1) is also claimed.
1. A comminuting apparatus, including: a) a receptacle to receive material to be comminuted and output comminuted material through an exit; b) a rotor including: i) a first member rotatable about a first axis, and ii at least one disc member for comminuting material and rotatable about a second axis removed from said first axis and coupled to said first member through a coupling; c) at least one drive means to rotate the first member about said first axis, thereby moving said second axis about said first axis through said coupling and to rotate said at least one disc members about said second axis; and d) a screen for comminuted material located between the at least one disc member and said exit. 2. The comminuting apparatus of claim 1, including a plurality of disc members along the second axis, each of the plurality of disc members rotatable about the second axis. 3. The comminuting apparatus of claim 1 including two or more sets of said at least one disc member, distributed about the first axis, the sets each rotatable about an axis removed from said first axis and coupled to the first member so as to move about said first axis when in use. 4. The comminuting apparatus of claim 1, wherein the drive means includes a first drive means for the first member and a second drive means, independently controllable from the first drive means, for the at least one disc members. 5. The comminuting apparatus of claim 1 including a sensor to detect jamming of the rotor and a controller in communication with the sensor to control the drive means to stop the rotation of the first member in response to the detection of a jam by the sensor. 6. The comminuting apparatus of claims 1 including a sensor to detect jamming of the rotor and a controller in communication with the sensor to control the drive means to reverse the direction the rotation of the first member in response to a detection of a jam by the sensor. 7. The comminuting apparatus of either claim 5 or claim 6, wherein the controller controls the drive means to resume the original rotation of the first member once the at least one disc member is rotating at or above a predetermined minimum speed. 8. The comminuting apparatus of claim 1, further including at least one guide for directing comminuted material, the at least one guide extending radially outward in relation to said first axis and rotatable about the first axis, below the screen. 9. The comminuting apparatus of claim 8, wherein said at least one guide is rotatable independently of the first member. 10. The comminuting apparatus of claim 1, further including a base portion extending across said receptacle below the at least one disc member and a side wall portion extending upwards from said base portion at the periphery of the receptacle. 11. The comminuting apparatus of claim 8, wherein the screen has a base portion extending across said receptacle below the at least one disc member and above the at least one guide and a side wall portion extending upwards from said base portion at the periphery of the receptacle. 12. The comminuting apparatus of claim 11, wherein the at least one guide includes a peripheral portion that extends towards the at least one disc members beside the side wall portion of the screen. 13. The comminuting apparatus of claim 12, wherein the at least one guide extends towards the at least one disc member beside the side wall portion so as to terminate at least approximately in the plane of the lowest of the one or more disc members. 14. The comminuting apparatus of claim 1, including a baffle below said screen, the baffle shaped to direct comminuted material radially outwards from said first axis. 15. The comminuting apparatus of claim 8, including a baffle below said screen, the baffle shaped to direct comminuted material radially outwards from said first axis, wherein said at least one guide extends radially outward from the outer periphery of said baffle. 16. (Cancelled) 17. (Cancelled)
<SOH> BACKGROUND <EOH>Apparatus for comminuting materials have application to a number of industries, which may involve shredding of wood, bales of hay or paper, comminuting bitumen, plastic, metal or other materials. An effective comminuting apparatus has a high throughput, does not jam often, is reliable, easy to maintain, is energy efficient and comminutes relatively uniformly Tub grinders are well known devices for grinding various materials. Vertical feed tub grinders have the advantage of using gravity to force the material onto the rotor, reducing or eliminating the need for further conveying means for the material other than that required to feed the material into the tub. However, one problem with tub grinders is their tendency to jam. Another problem with known tub grinders at present is apparent when they are used to comminute lighter material such as paper. The turbulence, vortices and movement of the rotor against the material tends to force the material upwards. Therefore, intervention may be required to force the material onto the rotor and through the screens. A comminuting apparatus including a rotating tub is described in the specifications of U.S. Pat. Nos. 5,379,951 and 5,927,624. Within the tub is mounted a toothed disc that preferably rotates in the opposite direction to the tub. The problem of potential jamming is stated to be overcome or reduced by reversing the direction of rotation of the toothed disc and/or by providing a reorienting attachment secured to the bottom of the chamber defined by the tub. However, such a comminuting apparatus may still be susceptible to jamming. It is an object of the present invention to provide a comminuting apparatus that may be freed from jamming, optionally automatically and/or has improved operation for lighter materials. It is a further or alternative object of the present invention to provide the public with a useful alternative. Further objects of the present invention may become apparent from the following description.
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 : Shows a partial cutaway view of a comminuting apparatus according to one aspect of the present invention. FIG. 2 : Shows a sectional view through the rotor of the comminuting apparatus of FIG. 1 . FIG. 3 : Shows a plan view of the rotor of FIG. 2 . detailed-description description="Detailed Description" end="lead"?

Pharmaceutical preparations containing aminobenzene-sulfonic acid derivatives as the active ingredient
A stable pharmaceutical preparation containing an aminobenzenesulfonic acid derivative represented by the following formula (I) known as a therapeutic agent for cardiac insufficiency: or a salt thereof, or a hydrate thereof or a solvate thereof as an active ingredient, wherein each production of substance A having a retention time of about 6.4 minutes in a high performance liquid chromatography, substance B having a retention time of about 15.6 minutes in the high performance liquid chromatography, and substance C having about 22.8 minutes in the high performance liquid chromatography is substantially suppressed, wherein said high performance liquid chromatography is performed at a controlled flow rate for elution so as to give a retention time of about 7 minutes of said active ingredient by using an ultraviolet absorptiometer at 220 nm, an octylsilylated silica gel packed column (4 mm ×250 mm) at 40° C., and a mobile phase prepared by dissolving 7.8 g of sodium dihydrogenphosphate dihydrate in 1000 mL of a mixture of water/acetonitrile (6:1) after said pharmaceutical preparation is extracted with a diluent prepared by dissolving 7.8 g of sodium dihydrogenphosphate dihydrate in 1000 mL of a mixture of water/acetonitrile (6:1) and adjusting a pH to 7.0 with addition of 8 mol/L sodium hydroxide solution.
1. A pharmaceutical preparation containing an aminobenzenesulfonic acid derivative represented by the following formula (I): wherein R1 represents hydrogen atom, a C1-C6 alkyl group, a C3-C7 cycloalkyl group, a halogenated C1-C4 alkyl group, a halogen atom or a C6-C12 aryl group; R2 represents hydrogen atom, a C1-C6 alkyl group or a C7-C12 aralkyl group which may have one or more substituents selected from the group consisting of cyano group, nitro group, a C1-C6 alkoxyl group, a halogen atom, a C1-C6 alkyl group and an amino group; and n represents an integer of 1 to 4 or a salt thereof, or a hydrate thereof or a solvate thereof as an active ingredient, wherein each production of substance A having a retention time of about 6.4 minutes in a high performance liquid chromatography, substance B having a retention time of about 15.6 minutes in the high performance liquid chromatography, and substance C having about 22.8 minutes in the high performance liquid chromatography is substantially suppressed, wherein said high performance liquid chromatography is performed at a controlled flow rate for elution so as to give a retention time of about 7 minutes of said active ingredient by using an ultraviolet absorptiometer at 220 nm, an octylsilylated silica gel packed column (4 mm ×250 mm) at 40° C., and a mobile phase prepared by dissolving 7.8 g of sodium dihydrogenphosphate dihydrate in 1000 mL of a mixture of water/acetonitrile (6:1) after said pharmaceutical preparation is extracted with a diluent prepared by dissolving 7.8 g of sodium dihydrogenphosphate dihydrate in 1000 mL of a mixture of water/acetonitrile (6:1) and adjusting a pH to 7.0 with addition of 8 mol/L sodium hydroxide solution. 2. The pharmaceutical preparation according to claim 1, wherein the coating layer consists of two layers. 3. The pharmaceutical preparation according to claim 1, wherein the coating layer consists of a first coating layer for substantially suppressing production of the substance A according to claim 1, and a second coating layer for substantially suppressing production of the substances B and C according to claim 1. 4. The pharmaceutical preparation according to claim 1, wherein the first coating layer does not contain titanium oxide. 5. The pharmaceutical preparation according to claim 1, wherein the second coating layer contains titanium oxide. 6. The pharmaceutical preparation according to claim 1, which is in the form of a tablet. 7. The pharmaceutical preparation according to claim 6, wherein an plain tablet is coated with the first coating layer and then coated with the second coating layer. 8. The pharmaceutical preparation according to claim 1, wherein the substitution of R1 is in the 5-position. 9. The pharmaceutical preparation according to claim 1, wherein n is 2. 10. The pharmaceutical preparation according to claim 1, wherein R2 is hydrogen atom, a C1-C3 alkyl group or a C7-C12 aralkyl group which may have one or more substituents selected from a C1-C3 alkyl group, a C1-C3 alkoxyl group and a halogen atom. 11. The pharmaceutical preparation according to claim 1, wherein R2 is hydrogen atom or a C7-C12 aralkyl group which may have one or more substituents selected from C1-C3 alkoxyl groups. 12. The pharmaceutical preparation according to claim 1, wherein R2 is hydrogen atom. 13. The pharmaceutical preparation according to claim 1, wherein R1 is hydrogen atom, a C1-C6 alkyl group, a C5-C6 cycloalkyl group, trifluoromethyl group, a halogen atom or phenyl group. 14. The pharmaceutical preparation according to claim 1, wherein R1 is a C1-C3 alkyl group, cyclohexyl group, trifluoromethyl group, chlorine atom, bromine atom or phenyl group. 15. The pharmaceutical preparation according to claim 1, wherein R1 is methyl group or propyl group. 16. The pharmaceutical preparation according to claim 1, wherein the active ingredient is selected from the following compounds: 5-trifluoromethyl-2-(1-piperazinyl)benzenesulfonic acid; 5-n-propyl-2-(1-piperazinyl)benzenesulfonic acid; 5-phenyl-2-(1-piperazinyl)benzenesulfonic acid; 5-chloro-2-(1-piperazinyl)benzenesulfonic acid; 5-bromo-2-(1-piperazinyl)benzenesulfonic acid; 5-iso-propyl-2-(1-piperazinyl)benzenesulfonic acid; 5-cyclohexyl-2-(1-piperazinyl)benzenesulfonic acid; 5-n-propyl-2-(1-homopiperazinyl)benzenesulfonic acid; 5-n-propyl-2-[4-(2,3,4-trimethoxybenzyl)-1-piperazinyl]benzenesulfonic acid; 5-n-propyl-2-[4-(3,4-dimethoxybenzyl)-1-piperazinyl]benzenesulfonic acid. 17. The pharmaceutical preparation according to claim 16, wherein the active ingredient is selected from the following compounds: 5-methyl-2-(1-piperazinyl)benzenesulfonic acid; 5-n-propyl-2-(1-piperazinyl)benzenesulfonic acid. 18. The pharmaceutical preparation according to claim 1, wherein the active ingredient is 5-methyl-2-(1-piperazinyl)benzenesulfonic acid monohydrate. 19. A tablet wherein a plain tablet containing an aminobenzenesulfonic acid derivative represented by the following formula (I): wherein R1 represents hydrogen atom, a C1-C6 alkyl group, a C3-C7 cycloalkyl group, a halogenated C1-C4 alkyl group, a halogen atom or a C6-C12 aryl group; R2 represents hydrogen atom, a C1-C6 alkyl group or a C7-C12 aralkyl group which may have one or more substituents selected from the group consisting of cyano group, nitro group, a C1-C6 alkoxyl group, a halogen atom, a C1-C6 alkyl group and an amino group; and n represents an integer of 1 to 4, or a salt thereof, or a hydrate thereof or solvate thereof as an active ingredient, is applied with two coating layers, which is characterized to comprise a first coating layer having a property of substantially suppressing production of a substance produced from a reaction of said active ingredient, titanium oxide, and light, and a second coating layer having a property of substantially suppressing production of a substance produced from a reaction of said active ingredient and light. 20. The tablet according to claim 19, wherein the first coating layer does not contain titanium oxide. 21. The tablet according to claim 19, wherein the second coating layer contains titanium oxide. 22. The tablet according to claim 19, wherein the plain tablet is coated with the first coating layer and then coated with the second coating layer.
<SOH> BACKGROUND ART <EOH>As methods for shielding lights for prevention of photodecomposition of pharmaceutical preparations, known methods include light-shielding coatings which shade a bulk of a drug itself, or means for shielding lights with packages such as light-shielding PTP packages, double-sided aluminum packages, light-shielding paper and the like. Among them, the methods of shielding lights with packages cannot guarantee stability after opening of the packages, and therefore it is considered that the method of shielding the bulk of a drug itself from lights is desirable. As a light-shielding ingredient in the light-shielding coatings, titanium oxide is commonly used, and variety of preparations are known. The aminobenzenesulfonic acid derivatives described in Japanese Patent Unexamined Publication (Kokai) Nos. 3-7263, 4-139127, 9-221479, 10-298077 and WO99/40919 are known as medicaments for treatment of cardiac insufficiency. These patent publications disclose general preparation methods and pharmaceutical formulations. When these compounds are made into tablets, light-shielding coatings are required, since plain tablets are found to generate photodecomposition products in a light irradiation test. However, when generally used titanium oxide is used as an ingredient of a light-shielding coating, the aforementioned aminobenzenesulfonic acid derivatives react with the titanium oxide to generate other photodecomposition products, and therefore sufficient stability cannot be guaranteed by ordinary light-shielding coatings. Therefore, an object of the present invention is to provide stable drugs comprising the aforementioned aminobenzenesulfonic acid derivatives.


Apparatus for continuously grouping on a conveyor
The invention relates to an apparatus for continuously grouping or marshalling objects (1) together on a conveyor. The conveyor is divided into a first (2) and a second (3) conveyor section, where the second conveyor section (3) has a higher speed than the first conveyor section (2). The conveyor sections (2, 3) have a common plane (4) on which the objects (1) are advanced. The conveyor sections (2, 3) each have two bending rollers (6, 7, 10, 11) in the common plane (4), of which each having a bending roller (6, 7) lying placed closely adjacent one another. Between the rollers (6, 7) placed adjacent one another, theme is disposed an arrest memeber (18). The arrest member (18) is movable through 90o in relation to the common plane (4). The arrest member (18) is moreover, together with the bending rollers (6,7), laterally displaceable along the common plane (4).
1. An apparatus for continuously grouping or marshalling of objects on a conveyor, the conveyor being divided into a first and second conveyor section, with a common plane on which the objects are advanced, both conveyor sections each having two bending rollers in the common plane, of which each having a bending roller lying placed closely adjacent one another, and the second conveyor section running at a higher speed than the first conveyor section, wherein between said bending rollers placed adjacent one another, there is disposed an arrest member which is movable through 90° in relation to the common plane; and wherein said bending rollers are, together with the arrest member, laterally displaceable along the common plane. 2. The apparatus as claimed in claim 1, wherein the speed of the second conveyor section is at least 10% higher than the speed of the first conveyor section. 3. The apparatus as claimed in claim 1, wherein the arrest member moves between an upper and a lower end position; and that the arrest member also moves between a forward end position and a rear end position. 4. The apparatus as claimed in claim 3, wherein the distance M between the forward and the rear end positions determines the number of objects which are grouped together.
<SOH> BACKGROUND ART <EOH>In the distribution of different objects, it is occasionally desirable to marshal or group together a given number of objects into a so-called distribution unit. The distribution unit may be held together in different ways; for example, the grouped or marshalled objects may be shrink-film wrapped, provided with a wrapping of paper or paperboard or be held together by other means as a unit. Also as regards the manufacture of single-use disposable packages for foods, there is often a need in the art, in particular for packages of smaller volumes, to marshal or group them together into distribution units. The single-use disposable packages may be manufactured from a paper laminate or alternatively from plastic and may have various configurations. A commonly occurring package shape is the parallelepipedic configuration. The packages are manufactured in a filling machine from a running material web, where the material web is formed into a product tube, is filled with product and severed to form individual packaging containers which are thereafter finally formed. The finished packaging containers depart from the filling machine normally on a conventional conveyor belt. There are many ways of marshalling or grouping together a given number of objects, such as packaging containers, before they are wrapped to form a distribution unit in a special machine. The commonest method is to cause a brake or an arrest device intermittently to release a predetermined number of objects from a long row of objects. The number of objects is normally monitored by photocells or the like. With filling machines that have constantly increasing capacity for manufacturing single use packages, the demands on the equipment or the machines that handle the packages before distribution are also increasing. The same applies to those machines that manufacture distribution units, such as shrink-film wrapped units. In order to be able to increase capacity output, it is necessary that the packages reach these machines already pre-grouped into the number desired. The apparatuses that marshal or group together the objects, i.e. the packages, must operate continuously.
<SOH> BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS <EOH>One preferred embodiment of the present invention will now be described in greater detail hereinbelow, with reference to the accompanying Drawings, in which: FIG. 1 shows an apparatus according to the present invention; and FIG. 2 schematically illustrates the function of the apparatus in steps A-E. The Drawings show only those parts and details essential to an understanding of the present invention and the positioning of the apparatus in its context has been omitted. detailed-description description="Detailed Description" end="lead"?

Portable wireless terminal
A portable wireless terminal has a hinge section provided type antenna suitable for a clamshell type which can maintain spacing from a head of a human body and alleviate influence of a hand of a user. The clamshell type portable wireless terminal having an upper housing (10) and a lower housing (20) connected to each other through a hinge has a projection section (30) projecting outward near a hinge section of the lower housing (20). In addition, a built-in antenna is housed in the projection section (30). The built-in antenna has an antenna element (50) in a linear or plate shape. At least a part of the antenna element on an open end side is disposed at a position which is hardly to be covered by the hand or finger of the user when the user uses the terminal.
1. A clamshell type portable wireless terminal having an upper housing and a lower housing connected to each other through a hinge, comprising: a projection section projecting outward near a hinge section of said lower housing; and a built-in antenna housed in said projection section and having an antenna element in a linear or plate shape, wherein at least a part of said antenna element on an open end side is disposed at a position which is hardly to be covered by a hand or finger of a user when the user uses the terminal. 2. The portable wireless terminal according to claim 1, wherein said antenna element is formed of a first element portion having a base end side connected to a feed point and a second element portion having a main surface forming a predetermined angle with a main surface of the first element portion, and said second element portion is connected to a side portion on an end side of said first element portion and forms a part of said open end side. 3. The portable wireless terminal according to claim 2, wherein said antenna element is formed by folding an intermediate portion of a conductive plate-shaped member substantially in a crank shape. 4. The portable wireless terminal according to claim 2, wherein said projection section has an outer surface extending in a housing width direction, said outer surface forming an acute angle with an outer main surface of said upper housing when said upper housing and said lower housing are opened, and said antenna element is disposed such that the main surface of said second element portion extends along an inner wall surface of this outer surface. 5. A clamshell type portable wireless terminal having an upper housing and a lower housing connected to each other through a hinge, comprising: an antenna element having a first element portion in a flat plate shape having a base end side connected to a feed point and a second element portion in a flat plate shape connected to a side portion on the end side of said first element portion and having a main surface forming a predetermined angle with a main surface of the first element portion, the antenna element being disposed within the housing near a hinge section along a housing width direction. 6. The portable wireless terminal according to claim 5, wherein said antenna element is formed by folding an intermediate portion of a conductive plate-shaped member substantially in a crank shape. 7. The portable wireless terminal according to claim 5, wherein said second element portion has a structure which is escaped to a position which is hardly to be covered by a hand or finger of a user. 8. A clamshell type portable wireless terminal having an upper housing and a lower housing connected to each other through a hinge, comprising: a built-in antenna disposed near a hinge portion along a housing width direction, the build-in antenna having an antenna element in a flat plate shape having a base end side connected to a feed point, in a state in which a hand or finger of a user is closest to an open end side of said antenna element from outside the housing in an open state, a main surface of said antenna element forming the right angle or an angle near the right angle with respect to a surface of said hand or finger at least over a predetermined length on the open end side. 9. The portable wireless terminal according to claim 8, wherein said antenna element is formed by folding an intermediate portion of a conductive plate-shaped member substantially in a crank shape. 10. The portable wireless terminal according to claim 1, further comprising a sound emitting port of a speaker generating ringtone on a back side of said lower housing, wherein, when the back side of said lower housing is placed on a placing surface in a plane shape, the sound emitting port of said speaker is lifted separately from said placing surface substantially uniformly at least on both sides of said lower housing by said projection section. 11. The portable wireless terminal according to claim 1, further comprising a sound emitting port of a speaker generating ringtone on a back side of said lower housing, wherein, when the back side of said lower housing is placed on a placing surface in a plane shape by said projection section, the sound emitting port of said speaker is lifted separated from said placing surface, and space opened substantially uniformly at least on both sides of said lower housing is formed.
<SOH> BACKGROUND ART <EOH>Portable wireless terminals for communication between mobile units such as a mobile phone (in the present specification, a terminal apparatus is simply referred to as a terminal) tend to have a reduced size and thickness. Thus, an antenna section mounted on the terminal is closer to a human body during a telephone call, so that concerns are rising that antenna radiation characteristics are degraded. In recent years, instead of terminals which employ an accommodation/extension type of whip antenna provided externally to the terminal, terminals which employ a so-called built-in antenna provided inside the terminal have come on the market as an antenna having advantages such as a low probability of breakage resulting from a drop or the like or the ability to improve design of the portable wireless terminal. It goes without saying that degradation of characteristics caused by a human body is also a concern in the terminal employing the built-in antenna. In addition, as a strong tendency in recent years, an increasing number of users use mobile phones not only for telephone calls by voice, but also for data communication such as electronic mails and connections to the Internet. In association therewith, terminals of a so-called clamshell type which is provided with a hinge in an intermediate section of the terminal have been on the market in increasing numbers due to the advantage that a liquid crystal display section can be increased in size. The clamshell type terminal generally has a receiver section and a liquid crystal display section provided in an upper housing unit and a keyboard section and a microphone section provided in a lower housing unit. In this case, a position where an antenna is provided is often (1) an upper portion of the upper housing or (2) an upper portion of the lower housing (that is, a hinge section). However, “provided in the upper portion of the upper housing” type generally must have a coaxial cable routed from a transmission/reception circuit (a wireless circuit) section provided within the lower housing to an antenna power supply section through the hinge section to cause the disadvantage that losses are produced corresponding thereto. In addition, since the upper housing unit tends to have a reduced thickness for design in recent years, there are disadvantages that the space for providing the antenna is not sufficient, the distance between the antenna and the head of a user is short to degrade antenna characteristics during a telephone call, and the like. Thus, the terminals employing “the hinge section provided” type antenna have grown in number. In the hinge section provided type antenna, however, while the distance between the antenna and the head of a human body is long during a telephone call, a user often holds the lower housing or the boundary between the upper and lower housings (that is, near the hinge section) for the call, so that the antenna section is close to a hand or fingers of the user. In addition, since the keyboard section is generally provided in the lower housing unit as described above, the antenna section is also closer to the hand of the user when the user manipulates the keyboard. Furthermore, depending on preferences of a user, the hinge section provided type antenna has the disadvantage in terms of the antenna characteristics that, when the portion of the housing including the antenna projects, the user conveniently uses the terminal by putting his finger on the projected portion since his hand is stable. On the other hand, the mobile phone generally has a speaker for generating ringtone on the back of a housing. Thus, when the terminal is placed on a desk or the like, the sound emitting port of the speaker faces the surface on which it is placed. It is thought that this is due to the issue of special allowance for disposing the speaker, no need to see the sound emitting port of the speaker in order to recognize sound such as ringtone, and the like. FIG. 11 shows a side view of a conventional clamshell type of mobile phone having an upper housing and a lower housing connected through a hinge section. When the phone folded as shown is placed on a placing surface such as a desk, a speaker section abuts on or is close to the placing surface. In this case, there are problems that radiation of sound emitted from a sound emitting port is prevented and sound quality is degraded. Similarly, the speaker section abuts on or is close to the placing surface, so that a magnetic substance may be absorbed due to magnetism of the speaker, or dust or the like may enter a number of holes provided in the sound emitting port. This problem applies to a so-called stick type of mobile phone. In a mobile phone in which a part of a housing is projected to accommodate an antenna section and the like, a speaker section may be lifted separately from a placing surface when the mobile phone is placed on a desk or the like. However, the size of open space formed between the housing and the placing surface for transmitting sound from the speaker is tilted in some directions, so that the problem occurs that sound is not necessarily radiated in the surroundings uniformly.
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1A and FIG. 1B are side views showing an opened state ( FIG. 1A ) and a closed (folded) state. ( FIG. 1B ) of a clamshell type portable wireless terminal to which the present invention is applied; FIG. 2A and FIG. 2B are views showing the external appearance of the terminal when the terminal in the opened state shown in FIG. 1A is viewed from an outward upper direction (1) ( FIG. 2A ), and the external appearance of the terminal when the terminal in the same state is viewed from an outward side direction (2) ( FIG. 2B ); FIG. 3A and FIG. 3B are views for explaining the different manners of holding the portable terminal by a user when the terminal is used for a telephone call; FIG. 4A and FIG. 4B are views for explaining the case where a finger is put on a projection section in an embodiment of the present invention; FIG. 5A and FIG. 5B are views for explaining a case where an open end has an escape from a finger and a case where it has no such an escape in the embodiment of the present invention; FIG. 6A and FIG. 6B are views for explaining the case where a thumb is put on a projection section, and FIG. 6C and FIG. 6D are diagrams for explaining the case where a finger other than the thumb (for example, a forefinger) is put on; FIG. 7 is a view showing the outline of a measuring method for seeing antenna radiation characteristics when a finger of a user is put on an antenna section of the terminal in the embodiment of the present invention; FIG. 8A and FIG. 8B show line graphs showing the antenna radiation characteristics based on the measurement in FIG. 7 ; FIG. 9 shows a bar graph showing comparison results of PAG values averaged in a used frequency band; FIG. 10A is a front view showing the structure of a mobile phone according to a first embodiment of the present invention, FIG. 10B is a rear view thereof, and FIG. 10C is a side view thereof; FIG. 11 is a side view showing a conventional clamshell type mobile phone having an upper housing and a lower housing connected through a hinge; FIG. 12A and FIG. 12B are side views showing the clamshell type mobile phone shown in FIG. 10A to FIG. 10C when the terminal is opened and closed; FIG. 13A is a front view showing the structure of a mobile phone according to a second embodiment of the present invention, FIG. 13B is a rear view thereof, and FIG. 13C is a side view thereof; FIG. 14A is a front view showing the structure of a mobile phone according to a third embodiment of the present invention, FIG. 14B is a rear view thereof, and FIG. 14C is a side view thereof; FIG. 15A is a front view showing the structure of a mobile phone according to a fourth embodiment of the present invention, FIG. 15B is a rear view thereof, and FIG. 15C is a side view thereof; FIG. 16A is a front view showing the structure of a mobile phone according to a fifth embodiment of the present invention, FIG. 16B is a rear view thereof, and FIG. 16C is a side view thereof; FIG. 17A is a front view showing the structure of a mobile phone according to a sixth embodiment of the present invention, FIG. 17B is a rear view thereof, and FIG. 17C is a side view thereof; FIG. 18A is a front view showing the structure of a mobile phone according to a seventh embodiment of the present invention, FIG. 18B is a rear view thereof, and FIG. 18C is a side view thereof; and FIG. 19A is a front view showing the structure of a mobile phone according to an eighth embodiment of the present invention, FIG. 19B is a rear view thereof, and FIG. 19C is a side view thereof. detailed-description description="Detailed Description" end="lead"?

Compositions and methods for the modulation of viral maturation
This application describes a family of nucleic acid sequences and proteins encoded thereby that play a role in viral maturation: the Alternate Viral Maturation Scaffolding Protein, or the AVMSP family of proteins.

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