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Cytokinin response regulators and uses thereof |
The invention generally features methods for increasing yield, shoot formation, and delaying senesence in plants with the use of transgenes that regulate the cytokinin response. The invention also features plants and plant components that harbor the transgene(s). |
1. A method for increasing yield in a plant, said method comprising the steps of: (a) introducing into plant cells a transgene comprising DNA encoding a B-type response regulator operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from said transformed cells, wherein said B-type response regulator is expressed in the cells of said transgenic plant, thereby increasing yield in said plant. 2. The method of claim 1, wherein said B-type response regulator is a crucifer B-type response regulator. 3. The method of claim 2, wherein said crucifer B-type response regulator is selected from the group consisting of ARR1, ARR2, and ARR10. 4. The method of claim 1, wherein said DNA encoding said B-type response regulator is constitutively expressed, inducibly expressed, expressed in a cell-specific, tissue-specific, or organ-specific manner, or expressed under cycling conditions. 5. A method for increasing yield in a plant, said method comprising the steps of: (a) introducing into plant cells a transgene operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from said transformed cells, wherein expression of said transgene reduces expression of an A-type response regulator in the cells of said plant, thereby increasing yield in said plant. 6. The method of claim 5, wherein said A-type response regulator is a crucifer A-type response regulator. 7. The method of claim 6, wherein said crucifer A-type response regulator is selected from the group consisting of ARR4, ARR5, ARR6, and ARR7. 8. The method of claim 5, wherein said transgene expresses antisense A-type response regulator RNA. 9. The method of claim 5, wherein said transgene expresses a dominant negative A-type response regulator. 10. The method of claim 5, wherein said transgene co-suppresses expression of A-type response regulator. 11. The method of claim 5, wherein said DNA encoding said transgene is constitutively expressed, inducibly expressed, expressed in a cell-specific, tissue-specific, or organ-specific manner, or expressed under cycling conditions. 12. A method for increasing shoot formation in a plant, said method comprising the steps of: (a) introducing into plant cells a transgene comprising DNA encoding a B-type response regulator operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from said transformed cells, wherein said B-type response regulator is expressed in the cells of said plant, thereby increasing shoot formation in said plant. 13. The method of claim 12, wherein said B-type response regulator is a crucifer B-type response regulator. 14. The method of claim 13, wherein said crucifer B-type response regulator is selected from the group consisting of ARR1, ARR2, and ARR10. 15. The method of claim 12, wherein said DNA encoding said B-type response regulator is constitutively expressed, inducibly expressed, expressed in a cell-specific, tissue-specific, or organ-specific manner, or expressed under cycling conditions. 16. A method for increasing shoot formation in a plant, said method comprising the steps of: (a) introducing into plant cells a transgene operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from said transformed cells, wherein expression of said transgene reduces expression of an A-type response regulator in the cells of said plant, thereby increasing shoot formation in said plant. 17. The method of claim 16, wherein said A-type response regulator is a crucifer A-type response regulator. 18. The method of claim 17, wherein said crucifer A-type response regulator is selected from the group consisting of ARR4, ARR5, ARR6, and ARR7. 19. The method of claim 16, wherein said transgene expresses antisense A-type response regulator RNA. 20. The method of claim 16, wherein said transgene expresses a dominant negative A-type response regulator. 21. The method of claim 16, wherein said transgene co-suppresses expression of A-type response regulator. 22. The method of claim 16, wherein said transgene is constitutively expressed, inducibly expressed, expressed in a cell-specific, tissue-specific, or organ-specific manner, or expressed under cycling conditions. 23. A method for delaying senescence in a plant, said method comprising the steps of: (a) introducing into plant cells a transgene comprising DNA encoding a B-type response regulator operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from said transformed cells, wherein said B-type response regulator is expressed in the cells of said plant, thereby delaying senesence in said plant. 24. The method of claim 23, wherein said B-type response regulator is a crucifer B-type response regulator. 25. The method of claim 24, wherein said crucifer B-type response regulator is selected from the group consisting of ARR1, ARR2, and ARR10. 26. The method of claim 23, wherein said DNA encoding said B-type response regulator is constitutively expressed, inducibly expressed, expressed in a cell-specific, tissue-specific, or organ-specific manner, or expressed under cycling conditions. 27. A method for delaying senescence in a plant, said method comprising the steps of: (a) introducing into plant cells a transgene operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from said transformed cells, wherein expression of said transgene reduces expression of an A-type response regulator in the cells of said plant, thereby delaying senescence in said plant. 28. The method of claim 27, wherein said A-type response regulator is a crucifer A-type response regulator. 29. The method of claim 28, wherein said crucifer A-type response regulator is selected from the group consisting of ARR4, ARR5, ARR6, and ARR7. 30. The method of claim 27, wherein said transgene expresses antisense A-type response regulator RNA. 31. The method of claim 27, wherein said transgene expresses a dominant negative A-type response regulator. 32. The method of claim 27, wherein said transgene co-suppresses expression of A-type response regulator. 33. The method of claim 27, wherein said transgene is constitutively expressed, inducibly expressed, expressed in a cell-specific, tissue-specific, or organ-specific manner, or expressed under cycling conditions. 34. A method for increasing yield in a plant, said method comprising the steps of: (a) introducing into plant cells a transgene comprising DNA encoding a histidine kinase operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from said transformed cells, wherein histidine kinase is expressed in the cells of said plant, thereby increasing yield in said plant. 35. The method of claim 34, wherein said histidine kinase is a crucifer histidine kinase. 36. The method of claim 35, wherein said crucifer histidine kinase is CKI1 or CRE1. 37. The method of claim 34, wherein said DNA encoding said histidine kinase is constitutively expressed, inducibly expressed, expressed in a cell-specific, tissue-specific, or organ-specific manner, or expressed under cycling conditions. 38. A method for increasing shoot formation in a plant, said method comprising the steps of: (a) introducing into plant cells a transgene comprising DNA encoding a histidine kinase operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from said transformed cells, wherein histidine kinase is expressed in the cells of said plant, thereby increasing shoot formation in said plant. 39. The method of claim 38, wherein said histidine kinase is a crucifer histidine kinase. 40. The method of claim 39, wherein said crucifer histidine kinase is CKI1 or CRE1. 41. The method of claim 38, wherein said DNA encoding said histidine kinase is constitutively expressed, inducibly expressed, expressed in a cell-specific, tissue-specific, or organ-specific manner, or expressed under cycling conditions. 42. A method for delaying senescence in a plant, said method comprising the steps of: (a) introducing into plant cells a transgene comprising DNA encoding a histidine kinase operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from said transformed cells, wherein histidine kinase is expressed in the cells of said plant, thereby delaying senescence in said plant. 43. The method of claim 42, wherein said histidine kinase is a crucifer histidine kinase. 44. The method of claim 43, wherein said crucifer histidine kinase is CKI1 or CRE1. 45. The method of claim 42, wherein said DNA encoding said histidine kinase is constitutively expressed, inducibly expressed, expressed in a cell-specific, tissue-specific, or organ-specific manner, or expressed under cycling conditions. 46. A plant or plant component comprising at least one transgene encoding (i) an A-type response regulator polypeptide, (ii) an antisense A-type response regulator RNA, (iii) a dominant negative A-type response regulator polypeptide, (iv) a B-type response regulator polypeptide, (v) an antisense B-type response regulator RNA, (vi) a dominant negative B-type response regulator, (vii) an antisense HK RNA, (viii) a dominant negative HK polypeptide, or any combination of (i)-(viii). 47. The plant or plant component of claim 48, further comprising a transgene encoding a histidine kinase polypeptide. 48. A plant or plant component comprising: (a) a first transgene encoding (i) an A-type response regulator polypeptide, (ii) an antisense A-type response regulator RNA, or (iii) a dominant negative A-type response regulator polypeptide or combination thereof, (b) a second transgene encoding (iv) a B-type response regulator polypeptide, (v) an antisense B-type response regulator RNA, (vi) a dominant negative B-type response regulator or combination thereof, and (c) a third transgene encoding (vii) a HK polypeptide; (viii) an antisense HK RNA, (ix) a dominant negative HK polypeptide or combination thereof. 49. The plant or plant component of claim 48, wherein said plant or plant component is selected from the group consisting of wheat, rice, maize, barley, potato, soybean, tomato, oats, cotton, and sunflower. 50. The plant or plant component of claim 46, wherein said plant or plant component is selected from the group consisting of wheat, rice, maize, barley, potato, soybean, tomato, oats, cotton, and sunflower. 51. The plant or plant component of claim 47, wherein said plant or plant component is selected from the group consisting of wheat, rice, maize, barley, potato, soybean, tomato, oats, cotton, and sunflower. |
<SOH> BACKGROUND OF THE INVENTION <EOH>This invention relates to genetically-engineered plants having increased yield and productivity, shoot, leaf and meristem formation, enhanced photosynthesis, and delayed senescence. Despite long recognition of cytokinins as essential plant hormones involved in diverse processes of plant growth and development, including cell division, shoot initiation, leaf and root differentiation, chloroplast biogenesis, apical dominance, and senescence, the molecular and biochemical mechanisms underlying cytokinin actions have not been elucidated (Davies, Plant Hormones: Physiology, Biochemistry and Molecular Biology, 1995, Kluwer Academic Publishers, Dordrecht; Meijer and Murray, Curr. Opin. Plant Biol. 4: 44-9, 2001; Mok and Mok, Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 89-118, 2001; Quilino et al., Trends Plant Sci. 5: 278-82, 2000). Recent genetic identification of Arabidopsis hybrid histidine protein kinases (AHKs), CKI1 and CRE1, in cytokinin signalling (Kakimoto, Science 274: 982-5, 1996; Suzuki et al., Plant Cell. Physiol. 42: 107-13, 2001; Inoue et al., Nature 409: 1060-3, 2001), the characterization of Arabidopsis response regulators (ARRs) as cytokinin primary response genes (D'Agostino et al., Plant Physiol. 124: 1706-17, 2000; Kiba et al., Plant Cell Physiol. 40: 767-71, 1999), and the existence of Arabidopsis histidine-containing phosphotransmitters (AHPs; Suzuki, Imamura, et al., Plant Cell l Physiol. 39: 1258-68, 1998), have implicated the involvement of a two-component phosphorelay mechanism in cytokinin signal transduction. However, cellular and molecular evidence is lacking in supporting a complete cytokinin signalling circuit in plant cells. Two-component circuitry, consisting of a histidine kinase (HK) sensor and a response regulator (RR) output, are responsible for signal transduction in most prokaryotic and some eukaryotic systems. The signalling pathway is initiated by a HK sensor and mediated by phosphotransfer between a conserved histidine (His) residue in HKs or histidine-containing phosphotransmitters (HPs) and a conserved aspartate (Asp) residue in RRs (Wurgler-Murphy and Saito, Trends Biochein. Sci. 22: 172-6, 1997; Stock et al., Annu. Rev. Biochem. 69: 183-215, 2000). Since there are only one HK, one HP, and two RRs in S. cerevisiae , it was previously believed that two-component signal transduction has limited function in eukaryotes (Stock et al., supra). However, the completion of the Arabidopsis genome has revealed over 40 genes encoding putative two-component signal transducers, AHKs, AHPs and ARRs, suggesting a significant involvement of the ancient and conserved signalling mechanism in many facets of plant cell regulation (Urao, et al., Trends Plant Sci. 5: 67-74, 2000). The identification of conserved HK signature motifs and/or activity in the photoreceptor phytochrome, putative osmosensor, and the ethylene and cytokinin receptors in Arabidopsis further supports this view (Inoue et al., supra; Urao, et al., supra; Yeh and Lagarias, Proc. Natl. Acad. Sci. USA 95: 13976-81, 1998; Bleecker and Kende, Annu. Rev. Cell Dev. Biol. 16: 1-18, 2000). In eukaryotic signal transduction, the two-component circuit often provides a link between the HK sensor to a MAP kinase (MAPK) signaling cascade. For example, the osmosensing signal transduction pathway in yeast is mediated by the SLN1/PD1/SSK1 phosphorelay. The RR SSK1 then activates the HOG1 MAPK cascade in the cytosol to control gene expression (Maeda et al., Nature 369: 242-5, 1994; Posas et al., Cell 86: 865-75, 1996; Posas and Saito, EMBO J. 17: 1385-94, 1998). It has been speculated that plant HKs such as the ethylene receptor (ETR1) and a putative osmosensor (AHK1) can initiate a phosphorelay and transmit the signal through a MAPK cascade (Bleecker and Kende, supra; Gamble et al., Proc. Natl. Acad. Sci. USA 95: 7825-9, 1998: Clark et al., Proc. Natl. Acad. Sci. USA 95: 5401-6, 1998; Urao et al., Plant Cell 11: 1743-54, 1999). Due to the lack of physiological plant cell assays, the mechanisms of HK action and the signal transduction pathways of any HK-mediated plant responses, including cytokinin signalling, remain obscure. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention capitalizes on the discovery that manipulation of the expression of cytokinin response regulators increases plant yield growth, and productivity. In one aspect, the invention features a method for increasing yield in a plant, the method including the steps of: (a) introducing into plant cells a transgene including DNA encoding a B-type response regulator operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from the transformed cells, wherein the B-type response regulator is expressed in the cells of the transgenic plant, thereby increasing yield in the plant. In preferred embodiments, the B-type response regulator is a crucifer B-type response regulator (for example, ARR1 (SEQ ID NO.: 2); ARR2 (SEQ ID NO.: 3); and-ARR10 (SEQ ID NO.: 9)). In a second aspect, the invention features a method for increasing yield in a plant, the method including the steps of: (a) introducing into plant cells a transgene operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from the transformed cells, wherein expression of the transgene reduces expression of an A-type response regulator in the cells of the plant, thereby increasing yield in the plant. In preferred embodiments, the A-type response regulator is a crucifer A-type response regulator (for example, ARR 4 (SEQ ID NO.: 5); ARR 5 (SEQ ID NO.: 6); ARR 6 (SEQ ID NO.: 7); and ARR 7 (SEQ ID NO.: 8)). In other preferred embodiments, the transgene expresses an antisense molecule of A-type response regulator; a dominant negative gene product of A-type response regulator; or expression of the transgene results in the co-suppression of the A-type response regulator. In a third aspect, the invention features a method for increasing shoot formation in a plant, the method including the steps of: (a) introducing into plant cells a transgene including DNA encoding a B-type response regulator operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from the transformed cells, wherein the B-type response regulator is expressed in the cells of the plant, thereby increasing shoot formation in the plant. In preferred embodiments, the method includes the use of a B-type response regulator that is a crucifer B-type response regulator. In a fourth aspect, the invention features a method for increasing shoot formation in a plant, the method including the steps of: (a) introducing into plant cells a transgene operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from the transformed cells, wherein expression of the transgene reduces expression of an A-type response regulator in the cells of the plant, thereby increasing shoot formation in the plant. In a fifth aspect, the invention features a method for delaying senescence in a plant, the method including the steps of: (a) introducing into plant cells a transgene including DNA encoding a B-type response regulator operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from the transformed cells, wherein the B-type response regulator is expressed in the cells of the plant, thereby delaying senescence in the plant. In a sixth aspect, a method for delaying senescence in a plant, the method including the steps of: (a) introducing into plant cells a transgene operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from the transformed cells, wherein expression of the transgene reduces expression of an A-type response regulator in the cells of the plant, thereby delaying senescence in the plant. In a seventh aspect, the invention features a method for increasing yield in a plant, the method including the steps of: (a) introducing into plant cells a transgene including DNA encoding a histidine kinase operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from the transformed cells, wherein histidine kinase is expressed in the cells of the plant, thereby increasing yield in the plant. In preferred embodiments, the histidine kinase is a crucifer histidine kinase (for example, CKI1(SEQ ID NO.: 13) and CRE1 (SEQ ID NO.: 18)). In an eighth aspect, the invention features a method for increasing shoot formation in a plant, the method including the steps of: (a) introducing into plant cells a transgene including DNA encoding a histidine kinase operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from the transformed cells, wherein histidine kinase is expressed in the cells of the plant, thereby increasing shoot formation in the plant. In a ninth aspect, a method for delaying senescence in a plant, the method including the steps of: (a) introducing into plant cells a transgene including DNA encoding a histidine kinase operably linked to a promoter functional in plant cells to yield transformed plant cells; and (b) regenerating a plant from the transformed cells, wherein histidine kinase is expressed in the cells of the plant, thereby delaying senescence in the plant. The invention further includes plants and plant components expressing one, or a combination of two or more, of the aforementioned transgenes. The invention also encompasses methods of generating these plants or plant components, which may involve introducing the transgenes individually to separate plants or plant components, and then crossing the appropriate genotypes to get the desired transgene combination. Alternatively, one or more constructs expressing a desired combination of transgenes may be generated and then introduced into the plant or plant component. For example, the invention also relates to a plant or plant component comprising at least one transgene encoding (i) an A-type response regulator polypeptide, (ii) an antisense A-type response regulator RNA, (iii) a dominant negative A-type response regulator polypeptide, (iv) a B-type response regulator polypeptide, (v) an antisense B-type response regulator RNA, (vi) a dominant negative B-type response regulator, (vii) an antisense HK RNA, (viii) a dominant negative HK polypeptide, or any combination of (i) lviii). In addition, the invention includes any of the aforementioned plants further including a transgene encoding a histidine kinase polypeptide. The invention further includes a plant or plant component that includes: (a) a first transgene encoding (i) an A-type response regulator polypeptide, (ii) an antisense A-type response regulator RNA, or (iii) a dominant negative A-type response regulator polypeptide or combination thereof; (b) a second transgene encoding (iv) a B-type response regulator polypeptide, (v) an antisense B-type response regulator RNA, (vi) a dominant negative B-type response regulator or combination thereof, and (c) a third transgene encoding (vii) a HK polypeptide; (viii) an antisense HK RNA, (ix) a dominant negative HK polypeptide or combination thereof. Exemplary plants which are useful in the methods of the invention, as well as for generating the plants (or plant cells, plant components, plant tissues, or plant organs) of the invention, include dicots and monocots, such as sugar cane, wheat, rice, maize, sugar beet, barley, manioc, crucifer, mustard, potato, soybean, sorghum, cassava, banana, grape, oats, tomato, millet, coconut, orange, rye, cabbage, apple, eggplant, watermelon, canola, cotton, carrot, garlic, onion, pepper, strawberry, yam, papaya, peanut, onion, legume, bean, pea, mango, and sunflower. By “operably linked” is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) are bound to the regulatory sequence(s). By “plant cell” is meant any self-propagating cell bounded by a semi-plant component expression control permeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired. Plant cell, as used herein, includes, without limitation, algae, cyanobacteria, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. By “plant component” is meant a part, segment, or organ obtained from an intact plant or plant cell. Exemplary plant components include, without limitation, somatic embryos, leaves, stems, roots, flowers, tendrils, fruits, scions, and rootstocks. By “transgene” is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. By “transgenic” is meant any cell which includes a nucleic acid sequence (e.g., a recombinant DNA sequence) which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic plants and the DNA (transgene) is inserted by artifice into the nuclear or plastidic genome. By “yield” or “plant yield” is meant increased growth (e.g., crop growth) or increased biomass. For example, increased yield results from increased shoot growth or meristem formation. Plants expressing the genes disclosed herein exhibiting increased yield can be selected by visual observation, for example by comparison with a wild-type plant. By “reducing expression” or “reduces expression” is meant a decrease in the level of gene expression (for example, expression of a gene encoding a A-type response regulator) by at least 30-50%, preferably by at least 50-80%, and more preferably by at least 80-95% or greater relative to the level in a control plant (for example, a wild-type plant). Reduction of such expression levels may be accomplished by employing standard methods which are known in the art including, without limitation, antisense and co-suppression technologies, expression of a dominant negative gene product, or through the generation of mutated genes using standard mutagenesis techniques. Levels of negative regulator polypeptide or transcript are monitored according to any standard technique including, but not limited to, northern blotting, RNase protection, or immunoblotting. By “crucifer” is meant any plant that is classified within the Cruciferae family. The Cruciferae include many agricultural crops, including, without limitation, rape (for example, Brassica campestris and Brassica napus ), broccoli, cabbage, brussel sprouts, radish, kale, Chinese kale, kohlrabi, cauliflower, turnip, rutabaga, mustard, horseradish, and Arabidopsis. By “a promoter functional in a plant cell” is meant any minimal sequence sufficient to direct transcription in a plant cell. Included in the invention are promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell-, tissue-, or organ-specific gene expression, or elements that are inducible by external or internal agents (for example, cytokinins), or elements that are capable of cycling gene transcription; such elements may be located in the 5′ or 3′ regions of the native gene or engineered into a transgene construct. The invention provides a number of important advances and advantages for improving and enhancing agronomically important traits such as photosynthesis, productivity, yield, leaf, shoot, and meristem formation, and delaying senescence. In particular, the invention provides for increased production efficiency, as well as for improvements in quality and yield of crop plants and ornamentals. Thus, the invention contributes to the production of high quality and high yield agricultural products; for example, fruits, ornamentals, vegetables, cereals, and field crops. Genetically-improved seeds and other plant products that are produced using plants expressing the genes and methods described herein also render farming possible in areas previously unsuitable for agricultural production The mechanisms disclosed herein for increasing plant yield and productivity is expected to be ubiquitous throughout the plant kingdom. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. |
Radiative cooling surface coatings |
A surface coating composition which exhibits low solar absorption and preferential emission at wavelengths corresponding to atmospheric windows in the infra-red regions is provided by the addition of microspheres to a composition containing a solar reflective pigment. |
1. A surface coating composition for the provision of a surface coating having radiative cooling properties, the composition including a solar reflective pigment and an effective quantity of microspheres. 2. A surface coating composition according to claim 1 in which the microspheres are silica microspheres. 3. A surface coating composition according to claim 2 in which the microspheres have a shell of silica-alumina. 4. A composition according to claim 1 in which the diameters of the microspheres lie substantially between 45 μm and 150 μm. 5. A composition according to claim 4 wherein the average diameter of the microspheres is approximately 50 μm. 6. A composition according to claim 3 in which the microspheres have shell thicknesses substantially between 0.3 μm and 1 μm. 7. A composition according to claim 1 containing from substantially 60 to 150 Kg of microspheres per 600 l. 8. A composition according to claim 7 containing from substantially 60 to 140 Kg of microspheres per 600 l. 9. A composition according to claim 7 containing from substantially 60 to 70 Kg of microspheres per 600 l. 10. A method of reducing the temperature within a building relative to ambient temperature, including the step of applying to the roof of the building a coating containing a solar reflective pigment and an effective quantity of microspheres. 11. A method according to claim 10 in which the coating preferentially emits thermal energy in the wavelength range 8-13 μm. 12. A method according to claim 11 in which the surface has a radiative emittance greater than 85% at thermal wavelengths. 13. A method according to claim 12 in which the surface has a solar reflectance greater than 80%. 14. A surface coating composition for providing a solar selective surface preferentially emitting thermal energy in the wavelength range 8-13 μm containing a solar selective pigment and an effective quantity of silica microspheres. 15. A surface coating according to claim 14 in which the surface has a radiative emittance greater than 85% at thermal wavelengths. 16. A surface coating according to claim 15 in which the surface has a solar reflectance greater than 80%. 17. A building having a roof substantially exposed to the sky, the roof having a surface coating containing a solar reflective pigment and an effective quantity of microspheres. |
<SOH> BACKGROUND TO THE INVENTION <EOH>This invention relates to surface coatings having radiative cooling properties and in particular to the use of such coatings on external surfaces of buildings to reduce the heat load to those buildings. Radiative cooling refers to the process whereby a body will emit as radiation heat energy absorbed through normal convection and conduction processes. The physics of black body radiation states that the wavelength at which a body will emit radiation is dependent on its temperature. For terrestrial temperatures, emission occurs in the infra-red (IR) wavelengths with a peak emission at approximately 11.4 μm. Incident solar radiation, on the other hand, corresponds to a black body temperature of 6000° K and is concentrated in the ultra-violet, visible and near IR wavelengths. Not all of the radiation emitted by the Earth passes through to space. A significant portion of this radiation is absorbed in the Earth's atmosphere, particularly by the so-called “greenhouse gases” water vapour, carbon dioxide and ozone, and re-emitted back to the Earth's surface. FIG. 1 shows atmospheric absorption as a function of wavelength. The species responsible for the various absorption peaks are identified on the horizontal axis. There is a low absorption “atmospheric window” in the region of 8-13 μm where the atmosphere is relatively transparent. A similar window exists for some wavelengths within the 1-5 μm band. Radiation from the Earth's surface within these wavelengths is likely to pass through these atmospheric windows to space rather than absorbed by the atmosphere and returned to the Earth's surface. For the wavelengths having high atmospheric absorption there will be significant amounts of radiation in the atmosphere as that radiation is absorbed and re-emitted back to Earth. Conversely, for the wavelengths corresponding to these atmospheric windows there will be little radiation in the atmosphere as the majority of radiation emitted by the Earth at these wavelengths is allowed to pass through the atmosphere to space. A “selective surface” is one that exploits the atmospheric window by preferentially emitting thermal energy at wavelengths corresponding to these atmospheric windows where there is reduced incident radiation which may be absorbed by the surface, that allow rapid transfer of that radiation to space and by being non-absorptive of radiation outside these wavelengths. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention resides in a curable formulation for use as a radiative cooling surface coating for an external surface of a building characterised by low absorption at wavelengths of incident solar radiation and high radiative emittance at wavelengths of reduced incident radiation. The invention is based on the discovery that such a coating may be obtained by the inclusion of a dispersion of gas or vacuum filled microspheres in a coating composition containing a solar reflective pigment. The microspheres employed are preferably of the silica type, most preferably having a shell of silica-alumina. Coatings according to the invention, incorporating effective amounts of such microspheres, have in fact demonstrated the ability to achieve below-ambient temperatures in the interior of coated structures, The microspheres employed in such coatings may be gas filled, for example with CO 2 or N 2 , or they may be vacuum filled. Preferably the coating is not only non-absorptive of solar radiation but is reflective, rather than transmissive, of these wavelengths. Preferably the coating has a solar reflectance greater than 80% and more preferably greater than 84%. Preferably the coating is characterised by radiative emittance (ε) greater than 85%, more preferably greater than 90% and still more preferably greater than 95% at thermal wavelengths. The combined effect of the solar absorption and radiative emittance characteristics of the coating are such that the coating is able to absorb heat energy from within an internal air space and re-radiate that energy as thermal radiation to achieve net radiative cooling of the air space. The coating of the invention, when applied to a sheet steel surface exposed to solar radiation at Air Mass 1.5 Solar Spectrum and ambient temperature of 20-28° C. the formulation displays a net radiative cooling effect to the air space below the steel sheet. Preferably, the formulation of the invention is curable and may be applied to an existing building using a large area spray process. |
Milking plant and method for reducing sound emissions in a milking plant |
The invention relates to a milking plant wherein the vibrations and sound emissions thereof are significantly reduced. In said inventive milking plant, a vacuum control valve (500) is directly connected to a vacuum compensation tank (200). The vacuum control valve (500) leads into the vacuum compensation tank (200) via a diffusor (501). The vacuum control valve (500) is enclosed in a cylindrical container (503). Preferably, the vacuum pump (110) and the vacuum compensation tank (200) are connected to each other via a main line (150) which is embodied in the form of two bent, flexible hoses. Preferably, the main line (150) leads tangentially into the vacuum compensation tank (200). The invention also relates to a method for reducing sound emissions in one such milking plant. According to said method, the flow speed of the air in the air-guiding parts (150, 200, 300, 302, 403, 420) of the milking plant is reduced. A buffer tank (300) can be used in the air line (30), enabling a stable operating vacuum to be obtained in addition to a reduction in structure-borne noise. |
1. Milking installation with: a minimum of one vacuum pump unit; a minimum of one vacuum equalization tank; at least one vacuum control valve; at least one air pipe or one drive with a variable speed of rotation for regulation of the delivery output of the vacuum pump unit; at least one pulsator; at least one milking device, which is attached to the air pipe via a pulse hose and is attached to a milk pipe via a milk hose; at least one milk pipe; at least one milk collection vessel, and at least one safety separator, whereby the vacuum control valve (500) is attached directly to the vacuum equalization tank (200), characterized in that the vacuum control valve (500) discharges via a diffuser (501) into the vacuum equalization tank (200). 2. Milking installation in accordance with Patent claim 1, characterized in that the vacuum control valve (500) is enclosed by a cylindrical container (503). 3. Milking installation in accordance with one of the foregoing Patent claims, characterized in that the vacuum control valve (500) exhibits an external air supply, in particular via a hose (502). 4. Milking installation in accordance with one of the foregoing Patent claims, characterized in that the vacuum pump (110) and the vacuum equalization tank (200) are connected together via a main pipe (150), which in particular exhibits the form of two curved flexible hoses (150a, 150b). 5. Milking installation in accordance with Patent claim 4, characterized in that the main pipe (150) discharges tangentially into the vacuum equalization tank (200). 6. Milking installation in accordance with one of the foregoing Patent claims, characterized in that the vacuum equalization tank (200) exhibits a dividing wall (201). 7. Milking installation in accordance with one of the foregoing Patent claims, characterized in that the vacuum equalization tank (200) exhibits a filter element (202). 8. Milking installation in accordance with one of the foregoing Patent claims, characterized in that the pipe (420) arriving from the safety separator (90) and the vacuum equalization tank (200) are separated from one another. 9. Milking installation in accordance with Patent claim 8, characterized in that a number of dividing elements (403), in particular five to twenty hoses, are provided between the pipe (420) and the vacuum equalization tank (200), which elements discharge into the vacuum equalization tank (200) via a dome (401). 10. Milking installation in accordance with one of the foregoing Patent claims, characterized in that the pulsator (60) is flexibly attached, in particular suspended by means of elastic materials (601), to the fabric of the building (ceiling) or to the milking parlour framework. 11. Milking installation in accordance with one of the foregoing Patent claims, characterized in that at least one flexible, hose-formed adapter (302), in particular a non-fibrous rubber hose, is provided between the buffer tank (300) and the vacuum equalization tank (200) as an air pipe, which is arranged preferably in a curve with a radius of at least 30 cm and an angle of about 90°. 12. Milking installation in accordance with one of the foregoing Patent claims, characterized in that the vacuum pump unit (100) and/or the vacuum equalization tank (200) is arranged on flexible feet (141, 142; 205, 206). 13. Milking installation in accordance with one of the foregoing Patent claims, characterized in that the length of the vacuum equalization tank (200) corresponds approximately to its diameter. 14. Method for the reduction of noise emissions in a milking installation, in particular in accordance with Patent claim 1, characterized in that the flow velocity of the air in the air-carrying parts (150, 200, 300, 302, 403, 420) of the milking installation is reduced. 15. Method in accordance with Patent claim 14, characterized in that the air from the vacuum equalization tank (200) is led away tangentially to the principal direction of flow, in particular at an angle of 0 to 30°. 16. Method in accordance with Patent claim 14, characterized in that the air from the vacuum equalization tank (200) is led away in particular via two main pipes (150a, 150b). 17. Method in accordance with one of Patent claims 14 to 16, characterized in that the air supply from the vacuum control valve (500) to the vacuum equalization tank (200) takes place via a diffuser (501). 18. Method in accordance with one of Patent claims 14 to 17, characterized in that separation from the vacuum-generating system and the milking system is undertaken, in particular via an adapter comprising hose-formed dividing elements (403) between the vacuum equalization tank (200) and the pipe (420) from the safety separator (90). 19. Method in accordance with one of Patent claims 14 to 18, characterized in that a buffer tank (300) is separated from the vacuum equalization tank (200), in particular via an adapter comprising at least one flexible air-carrying hose (302). 20. Method in accordance with one of Patent claims 14 to 19, characterized in that the air from the diffuser (501) flows into the vacuum equalization tank (200) tangentially to the principal direction of flow. |
Textual data storage method |
A hand-held electronic device for use in accessing and displaying textual information. The device may include: a display for displaying information; a processor; a memory; and a word dictionary table stored in the memory, the word dictionary table may include a word list of unique words that are contained in the textual information. The word dictionary table may also include a set of word identification tokens, where each word identification token represents one of the unique words in the word list. The memory may also include a phrase dictionary table, which may include a phrase list of word identification token groups, each word identification token group representing a phrase that is contained in the textual information. The phrase dictionary table may further include a set of phrase identification tokens, each phrase identification token representing one of the phrases in the textual information. The memory may include removable module. |
1. A method for storing data comprising: storing a word dictionary in a memory, the word dictionary including a first list of the unique words that are contained in the data and that also includes a set of word identification tokens, each word identification token representing one of the unique words in the first list; and storing a phrase dictionary in the memory, the phrase dictionary including a second list of word identification token groups, each word identification token group representing a phrase that is contained in the data, the second list further including a set of phrase identification tokens, each phrase identification token representing one of the word identification token groups. 2. The method of claim 1, further comprising the step of: determining an optimum range of the length of the phrases that are to be included in the phrase dictionary and including only phrases of optimum length in the phrase dictionary. 3. The method of claim 1, wherein the data comprises textual information. 4. The method of claim 3, wherein the textual information comprises automotive repair and servicing information. 5. The method of claim 1, wherein the memory comprises at least one removable module. |
<SOH> BACKGROUND <EOH>1. Technical Field A method and apparatus relating to the field of data storage, and, more particularly, a method and apparatus for storing, accessing, and presenting technical information for use in automotive maintenance and repair, is disclosed. 2. Description of Related Art Recent advances in data storage techniques and the development of portable digital assistants (PDAs) and similar devices have made it possible for users to have immediate access to large amounts of data, literally at their fingertips. Such data may include names, phone numbers, addresses, date books, documents, specialized wireless web pages, financial information, personal to-do lists, or calendars. In addition to providing built-in functions, some PDAs include expansion slots for inserting modules. These modules allow for virtually unlimited functions to be performed by the devices, such as digital photography, MP3 music, memory expansion, games, modems, universal remote controls, or global positioning systems. Some specialized hand-held devices (i.e., units that are not general-purpose PDAs) have made limited amounts of technical data for use in servicing and repairing automobiles available to users. One such hand-held device provides specifications-dedicated information, such as battery, ignition system, starter, belt tension, engine torque, wheel alignment, and wheel nut torque specifications for a range of vehicles and model years. However, while specialized devices may save a technician a trip to a shop manual for a specification, it is not a replacement for the comprehensive repair information contained in a bound set of shop manuals, such as the manuals published by the Mitchell Repair Information Company (MRIC). Specifically, MRIC illustrates the steps in addition to the raw specifications needed to complete a repair or other operation. Also, specialized handhelds typically don't have any provision for a technician to enter his own information to help him keep track of (or share) what he learns through experience, or to maintain an inventory of his tools, for example. In addition, updating a similar dedicated device is inconvenient and error-prone: it requires some disassembly of the unit and the removal and replacement, by a user, of an internal memory component that may be sensitive to electrostatic discharge or other damage. Finally, by definition, specialized hand-held references do not provide general-purpose functionality, such as a calculator, date book, or to-do list to help justify their purchase. General purpose PDAs, on the other hand, do provide a wide range of functions, but due to memory limitations (and limitations of current data compression techniques), they can not store the comprehensive amounts of data needed to make them viable alternatives to hardbound service manuals. Thus, a better solution is desired. |
<SOH> SUMMARY <EOH>A hand-held electronic device for use in accessing and displaying textual information, such as automotive repair specifications and procedures, is disclosed. The device may (or may not be) a general-purpose PDA. If a general purpose PDA is used, the PDA can, of course, be used for its other included functions when it is not being used as a technician's reference tool. The device may comprise: a display for displaying information to a user; a processor; a memory; and at least one word dictionary table stored in the memory, the word dictionary table comprising a first list of unique words that are contained in the textual information, and further comprising a set of word identification tokens, each word identification token representing one of the unique words in the first list. The device may also include at least one phrase dictionary table stored in the memory, the phrase dictionary table comprising a second list of word identification token groups. Each word identification token group in turn represents a phrase that is contained in the textual information. The phrase dictionary may further comprise a set of phrase identification tokens, each phrase identification token representing one of the phrases in the textual information. A user may select various menu items (by, for example, using a touchscreen) to cause the device to display the desired information. In response to the selection of a menu item, the device may display (in uncompressed, human-readable form) a portion of the textual information stored in the memory. In another embodiment, the memory may comprise one or more user removable memory modules. Through the use of proven, rugged memory modules that may be inserted in an external expansion slot, the data stored in memory can be easily updated. For example, if the device is used to store automotive reference data in accordance with one disclosed embodiment, modules containing specifications for other models of cars can be added. Moreover, modules with data of a type not contained on previously available modules may be supplied to users, greatly expanding the functionality and flexibility of the device. For example, modules could be developed to record and store operating temperatures of various components of a racecar, and the device could then be used to predict failure or improve the performance of the racecar. By first converting a set of textual information (such as repair information) into word tokens representing unique words in the text and then screening the resulting list of tokens for repeating phrases, very high compression ratios may be realized, especially where certain phrases are repeated in the text frequently. Because of this high compression efficiency, much more data can be stored in a memory of a given size. This compression efficiency, in turn, allows a significant amount of repair information, detailed procedures, specifications, technical service bulletins (TSBs), electrical component locators, to be stored, accessed and displayed from a single, hand-held device. Using such a device, a technician could greatly reduce or even eliminate his reliance on (and the inconvenience of) hardbound shop manuals for repair information. Further, using an efficient compression technique can free up enough memory (either module-based or built-in) to allow a user to store his own notes and tool inventory in the device for quick reference. |
Method for characterizing polypeptides |
Provided is a method for characterising a polypeptide, which method comprises the steps of; (a) optionnally reducing cysteine disulphide bridges in the polypeptide to form free thiols, and capping the free thiols; (b) cleaving the polypeptide with a sequence specific cleavage reagent to form peptide fragments; (c) optionally deactivating the cleavage reagent; (d) capping one or more ε-amino groups that are present with a lysine reactive agent; (e) analysing peptide fragments by mass spectrometry to form a mass fingerprint for the polypeptide; and (f) determining the identity of the polypeptide from the mass fingerprint. |
1. A method for characterising a polypeptide, which method comprises the steps of: (a) optionally reducing cysteine disulphide bridges in the polypeptide to form free thiols, and capping the free thiols; (b) cleaving the polypeptide with a sequence specific cleavage reagent to form peptide fragments; (c) optionally deactivating the cleavage reagent; (d) capping one or more ε-amino groups that are present with a lysine reactive agent, wherein the lysine reactive agent comprises a hindered Michael reagent; (e) analysing peptide fragments by mass spectrometry to form a mass fingerprint for the polypeptide; and (f) determining the identity of the polypeptide from the mass fingerprint. 2. A method for characterising a population of polypeptides, which method comprises the steps of: (a) optionally reducing cysteine disulphide bridges in one or more polypeptides to form free thiols, and capping the free thiols; (b) separating one or more polypeptides from the population; (c) cleaving one or more polypeptides with a sequence specific cleavage reagent to form peptide fragments; (d) optionally deactivating the cleavage reagent; (e) capping one or more E-amino groups that are present with a lysine reactive agent, wherein the lysine reactive agent comprises a hindered Michael reagent; (f) analysing the peptide fragments by mass spectrometry to form a mass fingerprint for one or more of the polypeptides; and (g) determining the identity of one or more polypeptides from the mass fingerprint. 3. A method for comparing a plurality of samples, each sample comprising one or more polypeptides, which method comprises the steps of: (a) optionally reducing cysteine disulphide bridges and capping the free thiols in one or more polypeptides from the samples; (b) separating one or more polypeptides from each of the samples; (c) cleaving the polypeptides with a sequence specific cleavage reagent to form peptide fragments; (d) optionally deactivating the cleavage reagent; (e) capping one or more ε-amino groups that are present with a lysine reactive agent, wherein the lysine reactive agent comprises a hindered Michael reagent; (f) analysing the peptide fragments by mass spectrometry to form a mass fingerprint for one or more polypeptides in the samples; and (g) determining the identity of one or more polypeptides in the samples from one or more mass fingerprints. 4. A method according to claim 1, wherein the lysine-reactive agent is a labelled lysine-reactive agent. 5. A method according to claim 3, for comparing a plurality of samples, each sample comprising one or more polypeptides, which method comprises the steps of: (a) optionally reducing cysteine disulphide bridges and capping the free thiols in one or more polypeptides from the samples; (b) capping one or more ε-amino groups that are present in each sample with a labelled lysine reactive agent; (c) pooling the samples; (d) separating one or more polypeptides from the pooled samples; (e) cleaving the polypeptides with a sequence specific cleavage reagent to form peptide fragments; (f) optionally deactivating the cleavage reagent; (g) analysing the peptide fragments by mass spectrometry to form a mass fingerprint for one or more polypeptides in the samples; and (h) determining the identity of one or more polypeptides in the samples from one or more mass fingerprints. wherein the same label is employed for polypeptides or peptides from the same sample, and different labels are employed for polypeptides or peptides from different samples, such that the sample from which a polypeptide or peptide originates can be determined from its label. 6. A method according to claim 1, wherein the sequence specific cleavage agent cleaves the one or more polypeptides on the C-terminal side of a lysine residue. 7. A method according to claim 1, wherein the specific cleavage reagent comprises Lys-C or Trypsin. 8. A method according to claim 1, wherein the peptide fragments having capped ε-amino groups are removed by affinity capture, and wherein the lysine reactive agent comprises biotin. 9. A method according to claim 1, wherein the hindered Michael agent comprises a compound having the following structure: wherein X is an electron withdrawing group that is capable of stabilising a negative charge; the R groups independently comprise a hydrogen, a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support. 10. A method according to claim 9, wherein one R comprises a methyl or phenyl group. 11. A method according to claim 9 wherein at least one R comprises an electron withdrawing group. 12. A method according to claim 9, wherein at least one R comprises a cyclic or heterocylic aromatic ring or fused ring. 13. A method according to claim 9, wherein X comprises an —S02R1 group, wherein R1 comprises an alkyl group or an aryl group, including aromatic groups cyclic groups, fused cyclic groups, and heterocyclic groups. 14. A method according to claim 13, wherein R1 comprises an electron withdrawing group. 15. A method according to claim 13, wherein the ring comprises a phenyl, pyridyl, naphthyl, quinolyl, pyrazine, pyrimidine or triazine ring structure. 16. A method according to claim 9 wherein the X group is substituted with an electron withdrawing group. 17. A method according to claim 16, wherein the electron withdrawing group is selected from halogens, such as fluorine chlorine, bromine or iodine, and nitro and nitrile groups. 18. A method according to claim 9, wherein the X group comprises a structure capable of promoting water solubility. 19. A method according to claim 1, wherein the polypeptide, population of polypeptides or samples comprise a sub-cellular fraction. 20. A method according to claim 1, which further comprises preparing the polypeptide, population of polypeptides or samples by liquid chromatography. 21. A method for assaying for one or more specific target polypeptides in a test sample, which comprises performing a method according to claim 1, wherein the sequence of the target polypeptide is determined by assaying the one or more mass fingerprints for a predetermined mass fingerprint specific to the target polypeptide. 22. A method for determining the expression profile of one or more samples, which method comprises characterising one or more polypeptides from one or more samples, according to a method as defined in claim 1. 23. A method according to claim 22, which method comprises identifying the quantity of each of the polypeptides detected by mass spectrometry. |
<SOH> BACKGROUND TO THE ART <EOH>The identification of proteins in biological samples is an essential activity of biochemical analysis, particularly the determination of the sequence of a protein, since the sequence determines the structure of a protein, which, in turn, determines the function of the protein. Traditional techniques for protein identification are cumbersome and relatively slow. The mainstay of protein identification techniques has been chemical sequencing of peptides using the Edman degradation, which can sequentially identify amino acids in a peptide from the N-terminus. This sequencing technique is typically used in conjunction with enzymatic digestion of a protein or polypeptide. Typically, an unidentified polypeptide is digested and its component peptides are separated from each other by chromatography. The individual peptides are then subjected to Edman degradation. The sequences of the peptides can be ordered by comparing the sequences of peptides from digestion of the polypeptide with different sequence specific cleavage reagents. This process allows the complete sequence of a polypeptide to be determined. While this has been a highly successful technique for the identification of proteins, it is quite laborious. New technologies have made rapid protein identification more feasible such as Matrix Assisted Laser Desorption lonisation Time-of-Flight (MALDI-TOF) mass spectrometry. This technique has permitted the development of peptide mass fingerprinting as a relatively rapid procedure for protein identification. A typical peptide mass fingerprinting protocol involves determining the mass of the unidentified protein followed by digestion of the protein with trypsin. Trypsin cleaves polypeptides selectively at arginine and lysine residues, leaving either arginine or lysine at the C-temmini of the product peptides. The positions of lysine and arginine in the sequence of a polypeptide determine where the polypeptide is cut giving rise to a characteristic series of peptides. The pattern of peptides can be easily detected by MALDI-TOF mass spectrometry. This mass spectrometric technique has a large mass range, can readily ionise large biomolecules, will preferentially produce singly charged ions and competition for ionisation with this technique is not severe, although competition can be problematic. This means that there is generally one peak in the mass spectrum for each peptide, the mass-to-charge ratio for each peak has essentially the same value as the mass of the peptide, with an added proton to ionise the peptide, and most (and sometimes all) the peptides from the tryptic digest of an unidentified protein can be analysed simultaneously. In effect the mass spectrum is a ‘bar-code’ in which the lines in the spectrum represent the masses of the characteristic cleavage peptides of the protein. For any given protein, there may be some peptides, which have the same mass as a peptide from another protein but it is very unlikely that two different proteins will give rise to peptides that all have identical masses. This means that the pattern of masses of the tryptic digest of a protein is a fairly unique identifier of that protein and is called a Peptide Mass Fingerprint (PMF). The relative uniqueness of PMF means that databases of predicted PMFs, determined from known protein sequences or sequences that have been predicted from genomic DNA or expressed sequence tags (ESTs), can be used to identify proteins in biological samples (Pappin D J C, Höjrup P and Bleasby A J, Current Biology 3: 327-332, “Rapid identification of proteins by peptide-mass fingerprinting.” 1993; Mann M, Hojrup P, Roepstorff P. Biol Mass Spectrom 22(6): 338-345, “Use of mass spectrometric molecular weight information to identify proteins in sequence databases.” 1993; Yates J R 3rd, Speicher S, Griffin P R, Hunkapiller T, Anal Biochem 214(2): 397-408, “Peptide mass maps: a highly informative approach to protein identification.” 1993). The PMF for an unknown protein can be compared with all of the PMFs in a database to find the best match, thereby identifying the protein. Searches of this kind can be constrained by determining the mass of the protein prior to digestion. In this way the pattern of masses of an unidentified polypeptide can be related to its sequence, which in turn can help to determine the role of a protein in a particular sample. There are, however, many technical difficulties involved in determining the PMF for a protein. A typical protein will give rise to twenty to thirty peptides after cleavage with trypsin, but not all of these peptides will appear in the mass spectrum. The precise reasons for this are not fully understood. One factor that is believed to cause incomplete spectra is competition for protonation during the ionisation process, resulting in preferential ionisation of arginine containing peptides (Krause E. & Wenschuh H. & Jungblut P. R., Anal Chem. 71(19): 4160-4165, “The dominance of arginine-containing peptides in MALDI-derived tryptic mass fingerprints of proteins.” 1999). In addition, there are surface effects that result from the process of preparing MALDI targets. The targets are prepared by dissolving the peptide digest in a saturated solution of the matrix material. Small droplets of the peptide/matrix solution are dropped onto a metal target and left to dry. Differences in solubility of peptides will mean that some peptides will preferentially crystallise near the top surface of the matrix where they will be desorbed more readily. Sensitivity is also a problem with conventional protocols for identifying proteins from their PMF. To be an effective tool, it should be possible to determine a PMF for as small a sample of protein as possible to improve the dynamic range of the analysis of protein samples. Some attempts have been made to improve the ionisation of peptides that do not contain arginine. Conversion of lysine to homo-arginine is one approach that has met with some success (V. Bonetto et al., Journal of Protein Chemistry 16(5): 371 -374, “C-terminal Sequence Determination of Modified Peptides by MALDI MS”, 1997: Brancia et 3l.: Electrophoresis 22: 552-559, “A combination of chemical derivitisation and improved bioinformatics tools optimises protein identification for proteomics”, 2001). The conversion of lysine to homo-arginine introduces guanidino functionalities into all of the peptides from a tryptic digest, with the exception of C-terminal peptides, greatly improving the representation of lysine containing peptides in the MALDI-TOF mass spectra. Conventional techniques for determining the expression of proteins in biological samples depend on protein identification. The goal of protein expression profiling is to identify as many proteins in a sample as possible and, preferably, to determine the quantity, of the protein in the sample. A typical method of profiling a population of proteins is by two-dimensional electrophoresis (R. A. Van Bogelen., E. R. Olson, “Application of two-dimensional protein gels in biotecnology.”, Biotechnol Annu Rev, 1:69-103, 1995). In this method a protein sample extracted from a biological sample is separated by two independent electrophoretic procedures. This first separation usually separates proteins on the basis of their iso-electric point using a gel-filled capillary or gel strip along which a pH gradient exists. Proteins migrate electrophoretically along the gradient until the pH is such that the protein has no net charge, referred to as the iso-electric point, from which the protein can migrate no further. After all of the proteins in the sample have reached their iso-electric point, the proteins are separated further using a second electrophoretic procedure. To perform the second procedure, the entire iso-electric focussing gel strip is then laid against one edge of a rectangular gel. The separated proteins in the strip are then electrophoretically separated in the second gel on the basis of their size. The proteins are thus resolved into a 2-dimensional array of spots in a rectangular slab of acrylamide. However, after separating the proteins in a sample from each other, there remains the problem of detecting and then identifying the proteins. The currently favoured approach to identify proteins is to analyse the protein in specific spots on the gel by peptide mass fingerprinting using MALDI-TOF mass spectrometry (Jungblut P, Thiede B. “Protein identification from 2-DE gels MALDI mass spectrometry.” Mass Spectrom Rev. 16:145-162, 1997). 2-DE technology is therefore limited by the detection capabilities of the peptide mass fingerprinting methods used in the identification of proteins in gel spots. The existing technology cannot easily compare the expression levels of two or more samples and there are sensitivity problems with such a complex process due to sample losses during the separation of the proteins and their subsequent recovery from the 2-D gel. In addition, proteins extracted from a 2-D gel are generally in buffers containing solutes that are incompatible with mass spectrometric analysis. It is an aim of this invention to solve the problems associated with the known methods described above. It is thus an aim of this invention to provide improved methods for producing peptide mass fingerprints, using labels (tags). It is a further aim of this invention to provide methods to determine peptide mass fingerprints using protein reactive reagents that are stable in water, selective for lysine and that work under mild reaction conditions without degradation of the reagents. |
Characterising polypeptides |
Provided is a method for characterising a polypeptide or a population of polypeptides, which method comprises the steps of: (a) contacting a sample comprising one or more polypeptides with a lysine reactive agent to cap ε-amino groups; (b) optionally reacting the sample of polypeptides with an amine reactive reagent to block α-amino groups; (c) digesting the sample of polypeptides with a cleavage reagent to produce peptide fragments; (d) optionally deactivating the cleavage reagent; (e) removing those peptides having uncapped or unblocked amino groups; and (f) recovering the N-terminal peptides. |
1. A method for characterising a polypeptide or a population of polypeptides, which method comprises the steps of: (a) contacting a sample comprising one or more polypeptides with a lysine selective agent to cap ε-amino groups; (b) optionally reacting the sample of polypeptides with an amine reactive reagent to block α-amino groups; (c) digesting the sample of polypeptides with a cleavage reagent to produce peptide fragments; (d) optionally deactivating the cleavage reagent; (e) removing those peptide fragments having uncapped or unblocked amino groups; and (f) recovering the N-terminal peptide fragment or fragments. 2. A method according to claim 1, wherein the polypeptide or polypeptides comprise one or more N-terminal amine groups that are naturally unblocked, which method comprises reacting the sample of polypeptides with an amine reactive reagent to block α-amino groups according to step (b). 3. A method according to claim 1, wherein the polypeptide or polypeptides comprise N-terminal amine groups that are naturally blocked, which method does not comprise reacting the sample of polypeptides with an amine reactive reagent to block α-amino groups according to step (b). 4. A method according to claim 1, wherein non-N-terminal peptides and/or naturally unblocked N-terminal peptides are removed by capturing them on a solid phase and N-terminal peptides are recovered in solution. 5. A method for characterising a polypeptide or a population of polypeptides, which method comprises the steps of: (a) contacting a sample comprising one or more polypeptides with a lysine selective agent to cap ε-amino groups; (b) contacting the resultant capped polypeptides with an amine reactive agent which reacts with the unblocked α-amino groups at the N-termini of the polypeptides; (c) digesting the sample of polypeptides with a cleavage agent to produce peptide fragments; (d) optionally deactivating the cleavage reagent; and (e) recovering N-terminal peptides that have reacted with the amine reactive agent. 6. A method according to claim 5, wherein N-terminal peptides are recovered by capturing them on a solid phase and non-N-terminal peptides are removed in solution. 7. A method according to claim 6, wherein the amine reactive agent or the lysine selective agent is attached to a solid phase. 8. A method according to claim 6, wherein the amine reactive agent comprises biotin and the solid phase is an avidinated solid phase. 9. A method according to claim 6, wherein two or more samples are reacted with differently labelled amine reactive agents, and subsequently the samples are pooled and analysed simultaneously. 10. A method according to claim 9, wherein at least one of the amine reactive agents is labelled with deuterium and the samples are analysed by mass spectrometry. 11. A method according to claim 1, wherein only one molecule of the lysine selective agent reacts with each ε-amine group available in the peptides or polypeptides. 12. A method according to claim 1, wherein the lysine selective agent comprises a hindered Michael reagent 13. A method according to claim 1, wherein the hindered Michael agent comprises a compound having the following structure: wherein X is an electron withdrawing group that is capable of stabilising a negative charge; the R groups independently comprise a hydrogen, a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group. 14. A method according to claim 13, wherein one R comprises a methyl or phenyl group. 15. A method according to claim 13, wherein at least one R comprises an electron withdrawing group. 16. A method according to claim 13, wherein at least one R comprises a cyclic or heterocylic aromatic ring or fused ring. 17. A method according to claim 13, wherein X comprises an —SO2R1 group, wherein R1 comprises an alkyl group or an aryl group, including aromatic groups cyclic groups, fused cyclic groups, and heterocyclic groups. 18. A method according to claim 17, wherein R1 comprises an electron withdrawing group. 19. A method according to claim 17, wherein the ring comprises a phenyl, pyridyl, naphthyl quinolyl, pyrazine, pyrimidine or triazine ring structure. 20. A method according to claim 13, wherein the X group is substituted with an electron withdrawing group. 21. A method according to claim 20, wherein the electron withdrawing group is selected from halogens, such as fluorine chlorine, bromine or iodine, and nitro and nitrile groups. 22. A method according to claim 13, wherein the X group comprises a structure capable of promoting water solubility. 23. A method according to claim 1, wherein the cleavage agent comprises a sequence-specific cleavage agent. 24. A method according to claim 1, wherein the cleavage agent comprises a peptidase, cyanogen bromide or BNPS-Skatole. 25. A method according to claim 24, wherein the peptidase comprises trypsin, Lys-C or Arg-C. 26. A method according to claim 1, Wherein the sample of step (a) comprises a sub-cellular fraction. 27. A method according to claim 1, which further comprises preparing the sample of step (a) by liquid chromatography. 28. A method for assaying for one or more specific polypeptides in a test sample, which comprises performing a method according to claim 1, wherein the sequence of the specific polypeptide is determined by assaying the resulting N-termini for a predetermined N-terminal sequence of amino acid residues. 29. A method of characterising one or more mixtures of polypeptides, which method comprises the following steps: (a) recovering one or more N-terminal peptides from the mixtures by employing one or more of the methods as defined in claim 1; (b) detecting the peptides by mass spectrometry. 30. A method for determining the expression profile of a sample, which method comprises characterising one or more mixtures of polypeptides according to a method as defined in claim 26. 31. A method according to claim 29, which method comprises determining the identity of each of the peptides detected by mass spectrometry. 32. A method according to claim 28, which method comprises identifying the quantity of each of the peptides detected by mass spectrometry. 33. A method for characterising a polypeptide or a population of polypeptides, which method comprises contacting a sample comprising one or more polypeptides with a lysine selective agent to attach the agent to ε-amino groups, wherein the lysine selective agent comprises a hindered Michael reagent. 34. A method according to claim 33, wherein the hindered Michael agent is a compound having the following structure: wherein X is an electron withdrawing group that is capable of stabilising a negative charge; the B. groups independently comprise a hydrogen, a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group. 35. A compound having the following structure: wherein R1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure and the R groups independently comprise a hydrogen, a halogen, or an alkyl or aryl group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group. 36. A compound according to claim 35, wherein at least one R group comprises a methyl or phenyl group. 37. A compound according to claim 35, wherein at least one R group comprises an electron-withdrawing group. 38. A compound according to claim 37, wherein at least one R group comprises a halogen atom or a halogenated alkyl group, or a phenyl ring with one or more electron withdrawing substituents. 39. A kit for characterising a polypeptide or a population of polypeptides, which kit comprises: (a) a lysine selective agent for capping ε-amino groups; (b) a means for recovering or isolating N-terminal peptides; (c) optionally an amine reactive reagent for blocking α-amino groups; (d) optionally a cleavage reagent for producing peptide fragments. 40. A kit according to claim 39, wherein the lysine selective agent comprises a compound having the following structure: wherein X is an electron withdrawing group that is capable of stabilising a negative charge; the R groups independently comprise a hydrogen, a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group. 41. A kit according to claim 40, wherein the lysine selective agent comprises a compound having the following structure: wherein R1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure and the R groups independently comprise a hydrogen, a halogen, or an alkyl or aryl group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group. 42. A kit according to claim 40, wherein the a means for recovering or isolating N-terminal peptides comprises a solid phase adapted for capturing peptides comprising free α-amino groups. 43. A method for protecting ε-amino groups in peptides and polypeptides comprising using a compound having the following structure: wherein R1 comprises an alkyl group or an aryl group, including aromatic groups cyclic groups, fused cyclic groups, and heterocyclic groups, and the R groups independently comprise a hydrogen, a halogen, or an alkyl or aryl group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group. 44. The method according to claim 43, wherein R1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure. 45. The method according to claim 43, wherein at least one R group comprises a methyl or phenyl group. 46. The method according to claim 43, wherein at least one R group comprises an electron-withdrawing group. 47. The method according to claim 46, wherein at least one R group comprises a halogen atom or a halogenated alkyl group, or a phenyl ring with one or more electron withdrawing substituents. 48. The method according to claim 41, wherein the protection is against further reaction of the ε-amino groups with Edman agents, capture agents and agents which are capable of reacting with α-amino groups. 49. The method according to claim 48, wherein the Edman agent comprises an isothiocyanate or an isocyanate, the capture agent comprises N-hydroxysuccinimidyl biotin and the agent which is capable of reacting with α-amino groups comprises acetic acid N-hydroxysuccinimide ester. |
<SOH> BACKGROUND IN THE ART <EOH>Techniques for profiling proteins, that is to say cataloguing the identities and quantities of proteins in a tissue, are not well developed in terms of automation or high throughput. A typical method of profiling a population of proteins is by two-dimensional electrophoresis (R. A. Van Bogelen., E. R. Olson, “Application of two-dimensional protein gels in biotechnology”, Biotechnol Annu. Rev., 1, 69-103, 1995). In this method, a protein sample extracted from a biological sample is separated on a narrow gel strip. This first separation usually separates proteins on the basis of their iso-electric point. The entire gel strip is then laid against one edge of a rectangular gel. The separated proteins in the strip are then electrophoretically separated in the second gel on the basis of their size. This technology is slow and very difficult to automate. It is also relatively insensitive in its simplest embodiments. A number of improvements have been made to increase resolution of proteins by 2-D gel electrophoresis and to improve the sensitivity of the system. One approach to improve the sensitivity of 2-D gel electrophoresis and its resolution is to analyse the protein in specific spots on the gel by mass spectrometry (Jungblut P, Thiede B. “Protein identification from 2-D gels by MALDI mass spectrometry.” Mass Spectrom. Rev. 16, 145-162, 1997. One example of a mass spectrometry method is in-gel tryptic digestion followed by analysis of the tryptic fragments by mass spectrometry to generate a peptide mass fingerprint. If sequence information is required, tandem mass spectrometry analysis can be performed. More recently attempts have been made to exploit mass spectrometry to analyse whole proteins that have been fractionated by liquid chromatography or capillary electrophoresis (Dolnik V. “Capillary zone electrophoresis of proteins.”, Electrophoresis 18, 2353-2361, 1997). In-line systems exploiting capillary electrophoresis mass spectrometry have been tested. The analysis of whole proteins by mass spectrometry, however, suffers from a number of difficulties. The first difficulty is the analysis of the complex mass spectra resulting from multiple ionisation states accessible by individual proteins. The second major disadvantage is that the mass resolution of mass spectrometers is at present quite poor for high molecular weight species, i.e. for ions that are greater than about 4 kilodaltons (kDa) in mass, so resolving proteins that are close in mass is difficult. A third disadvantage is that further analysis of whole proteins by tandem mass spectrometry is difficult as the fragmentation patterns for whole proteins are extremely complex and difficult to interpret. As a result of the difficulties of analysing whole proteins, techniques that rely on the analysis of peptides from proteins are preferred. Peptide mass fingerprinting has been used in the analysis of gel separated proteins as described above. However, this process is adequate only for the analysis of individual proteins or very simple mixtures of proteins. A typical protein will give rise to from twenty to thirty peptides after cleavage with trypsin. The pattern of peptide masses is useful for identifying single proteins, but the complexity of the mass spectrum of the trypsin digest of a mixture of proteins rapidly rises in complexity as the number of proteins in the mixture increases. This increases the chance that a peptide mass is assigned incorrectly to a protein, thus limiting the number of proteins that may be analysed simultaneously. As a result new protein characterisation methods are being developed in which specific peptides are isolated from each protein in a mixture. Nature Biotechnology 17, 994-999 (1999) discloses the use of ‘isotope encoded affinity tags’ for the capture of peptides from proteins, to allow protein expression analysis. In this article, the authors describe the use of a biotin linker, which is reactive to thiols, for the capture peptides with cysteine in them. A sample of protein from one source is reacted with the biotin linker and cleaved with an endopeptidase. The biotinylated cysteine-containing peptides can then be isolated on avidinated beads for subsequent analysis by mass spectrometry. Two samples can be compared quantitatively by labelling one sample with the biotin linker and labelling the second sample with a deuterated form of the biotin linker. Each peptide in the samples is then represented as a pair of peaks in the mass spectrum where the relative peak heights indicate their relative expression levels. This ‘isotope encoding’ method has a number of limitations. A first is the reliance on the presence of thiols in a protein—many proteins do not have thiols while others have several. In a variation on this method, linkers may be designed to react with other side chains, such as amines. However, since many proteins contain more than one lysine residue, multiple peptides per protein would generally be isolated in this approach It is likely that this would not reduce the complexity of the sample sufficiently for analysis by mass spectrometry. A sample that contains too many species is likely to suffer from ‘ion suppression’, in which certain species ionise preferentially over other, species which would normally appear in the mass spectrum in a less complex sample. In general, capturing proteins by their side chains is likely to give either too many peptides per protein or certain proteins will be missed altogether. The second limitation of this approach is the method used to compare the expression levels of proteins from different samples. Labelling each sample with a different isotope variant of the affinity tag results in an additional peak in the mass spectrum for each peptide in each sample. This means that if two samples are analysed together there will be twice as many peaks in the spectrum Similarly, if three samples are analysed together, the spectrum will be three times more complex than for one sample alone. It is clear that this approach will be limited, since the ever increasing numbers of peaks will increase the likelihood that two different peptides will have overlapping peaks in the mass spectrum. A further limitation, which is reported by the authors of the above paper, is the mobility change caused by the tags. The authors report that peptides labelled with the deuterated biotin tag elute slightly after the same peptide labelled with the undeuterated tag. Published international patent application WO 98/32876 discloses methods of profiling a population of proteins by isolating a single peptide from one terminus of each protein in the population. In a first aspect the invention comprises the steps of: 1. capturing a population of proteins onto a solid phase support by one terminus of each protein in the population; 2. cleaving the captured proteins with a sequence specific cleavage agent; 3. washing away peptides generated by the cleavage agent not retained on the solid phase support; 4. releasing the terminal peptides retained on the solid phase support; and 5. analysing the released terminal peptides, preferably identifying and quantifying each peptide in the mixture. The analysis is preferably performed by mass spectrometry. In this application, the C-terminus is discussed as being more preferable as the terminus by which to capture a population of proteins, since the N-terminus is often blocked. In order to capture a population of proteins by the C-terminus, the C-terminal carboxyl group must be distinguished from other reactive groups on a protein and must be reacted specifically with a reagent that can effect immobilisation. In many C-terminal sequencing chemistries the C-terminal carboxyl group is activated to promote formation of an oxazolone group at the C-terminus. During the activation of the C-terminal carboxyl, side chain carboxyls are also activated, but these cannot form an oxazolone group. It has been reported that the C-terminal oxazolone is less reactive to nucleophiles under basic conditions than the activated side-chain carboxyls, offering a method of selectively capping the side chain carboxyl groups (V. L. Boyd et al., Methods in Protein Structure Analysis: 109-118, Plenum Press, Edited M. Z. Atassi and E. Appella, 1995). Other more reactive side chains can be capped prior to the activation of the carboxyls using a variety of conventional reagents. In this way all reactive side chains can be capped and the C-terminus can be specifically labelled. EP A 0 594 164 and EP B 0 333 587 describe methods of isolating a C-terminal peptide from a protein in a method to allow sequencing of the C-terminal peptide using N-terminal sequencing reagents. In this method the protein of interest is digested with an endoprotease, which cleaves at the C-terminal side of lysine residues. The resultant peptides are reacted with diisothiocyanato (DITC) polystyrene which reacts with all free amino groups. N-terminal amino groups that have reacted with the DITC polystyrene can be cleaved with trifluoroacetic acid (TFA) thus releasing the N-terminus of all peptides. The epsilon-amino group of lysine is not cleaved however and all non-terminal peptide are thus retained on the support and only C-terminal peptides are released. According to this patent the C-terminal peptides are recovered for micro-sequencing. Anal. Biochem. 132: 384-388 (1983) and DE A 4344425 (1994) describe methods of isolating an N-terminal peptide from a protein by reacting the protein with a capping reagent which will cap any free amino groups in the protein. The protein is then cleaved, and if trypsin is used cleavage occurs only at arginine residues. Cleavage with trypsin thus exposes α-amino groups in the non-N-terminal peptides. In the first disclosure (Anal. Biochem.) the α-amino groups are reacted with dinitrofluorobenzene (DNF) which allows the non-N-terminal peptides to be captured by affinity chromatography onto a polystyrene resin while the N-terminal peptides flow through unimpeded. In DE A 4344425, the epsilon amino groups are reacted with an acylating agent prior to cleavage. After cleavage in this method, the α-amino groups on the non-N-terminal peptides are reacted with an amine reactive solid support such as diisothiocyanato glass, leaving the N-terminal peptides free in solution. The main drawback of all of these N-terminal isolation methods is the use of acylating reagents which tend to be unstable in aqueous conditions at the pH needed for lysine modification. As a result, large excesses of reagent need to be used which can lead to side-reactions particularly with histidine residues. The Anal. Biochem. method also requires that the DNF groups be removed from histidine and tyrosine by thiolysis prior to isolating the N terminal peptide, if the N terminal peptide contains these groups. This additional step requires extra effort and may not go to completion. In the Anal. Chem. disclosure the protein and terminal peptides are not analysed by mass spectrometry and so it is not possible to know whether the capping of the lysine epsilon amino groups goes to completion. It is an aim of this invention to solve the problems associated with the known methods described above. It is thus an aim of this invention to provide improved methods for isolating a single terminal peptide from each protein in a mixture of polypeptides using protein reactive reagents that are stable in water, selective for lysine and that work under mild reaction conditions without degradation of the reagents. |
Method and equipment for manufacturing aluminum nitride bulk single crystal |
The present invention provides a process for forming a bulk monocrystalline aluminum nitride by using a supercritical ammonia. The process comprises the steps of forming a supercritical solvent containing ions of an alkali metal in an autoclave; and dissolving a feedstock in this supercritical solvent to form a supercritical solution, and simultaneously or separately crystallizing aluminum nitride on the face of a crystallization seed. This process is carried out in the autoclave (1) which is provided with a convection-controller (2) arranged therein and which is to produce a supercritical solvent. The autoclave is set in a furnace unit (4) equipped with a heater (5) and/or a cooler (6). |
1. A process for preparing a bulk monocrystalline aluminum nitride, comprising the steps of forming a supercritical ammonia solvent containing ions of an alkali metal in an autoclave; dissolving an aluminum-containing feedstock in the supercritical ammonia solvent to form a supercritical solution in which the feedstock is dissolved; and crystallizing aluminum nitride on the face of a crystallization seed from the supercritical solution, under a condition of a higher temperature and/or a lower pressure than the temperature and/or the pressure found when the feedstock is dissolved in the supercritical solvent. 2. The process according to claim 1, wherein a step of moving the supercritical solution to a higher temperature and/or lower pressure zone is added, separately from the step of dissolving the feedstock. 3. The process according to claim 1, wherein at least two zones having a difference in temperature therebetween are concurrently formed in the autoclave, and wherein the aluminum-containing feedstock is set in the lower temperature dissolution zone, and the crystallization seed, in the higher temperature crystallization zone. 4. The process according to claim 3, wherein the difference in temperature between the dissolution zone and the crystallization zone is set within such a range that a chemical transport in the supercritical solution can be ensured. 5. The process according to claim 4, wherein the chemical transport in the supercritical solution is caused mainly by convection in the autoclave. 6. The process according to claim 4, wherein the difference in temperature between the dissolution zone and the crystallization zone is 1° C. or more. 7. The process according to claim 1, wherein the aluminum nitride may contain a donor, an acceptor or a magnetic dopant. 8. The process according to claim 1, wherein the supercritical solvent contains NH3 or its derivative. 9. The process according to claim 1, wherein the supercritical solvent contains at least ions of sodium or potassium. 10. The process according to claim 1, wherein the aluminum-containing feedstock is mainly composed of aluminum nitride or its precursor. 11. The process according to claim 10, wherein the precursor is selected from the group consisting of an azide, imide, amidoimide, amide and hydride, each of which contains aluminum. 12. The process according to claim 1, wherein the crystallization seed has a cryatalline layer of a nitride containing at least aluminum or other element of Group III. 13. The process according to claim 1, wherein the crystalline layer of aluminum nitride in the crystallization seed has a surface dislocation density of 106/cm2 or less. 14. The process according to claim 1, wherein the crystallization of aluminum nitride is carried out at a temperature of from 400 to 600° C. 15. The process according to claim 1, wherein the crystallization of aluminum nitride is carried under a pressure of from 1,000 to 5,500 bars, preferably from 1,500 to 3,000 bars. 16. The process according to claim 1, wherein the concentration of the ions of the alkali metal in the supercritical solvent is adjusted so that the specified solubilities of the feedstock and aluminum nitride can be ensured. 17. The process according to claim 1, wherein the molar ratio of the ions of the alkali metal to other components in the supercritical solvent is controlled within a range of from 1:200 to 1:2, preferably from 1:100 to 1:5, more preferably from 1:20 to 1:8. 18. An apparatus for preparing a bulk monocrystalline aluminum nitride, comprising an autoclave (1) for producing a supercritical solvent, a convection-controller (2) arranged in the autoclave, and a furnace unit (4) equipped with a heater (5) and/or a cooler (6), in which the autoclave is set. 19. The apparatus according to claim 18, wherein the furnace unit (4) has a higher temperature zone equipped with a heater (5) which corresponds to the crystallization zone (14) in the autoclave (1), and a lower temperature zone equipped with a heater (5) and/or a cooler (6) which corresponds to the dissolution zone (13) in the autoclave (1). 20. The apparatus according to claim 18, wherein the furnace unit (4) has a higher temperature zone equipped with a heater (5) and/or a cooler (6) which corresponds to the crystallization zone (14) in the autoclave (1), and a lower temperature zone equipped with a heater (5) and/or a cooler (6) which corresponds to the dissolution zone (13) in the autoclave (1). 21. The apparatus according to claim 18, wherein the convection-controller (2) is composed of one or more horizontal baffles (12) which partition the crystallization zone (14) from the dissolution zone (13), and each of which has a hole at the center or the periphery thereof. 22. The apparatus according to claim 18, wherein the feedstock (16) is set in the dissolution zone (13), and the crystallization seed (17), in the crystallization zone (14) in the autoclave (1), and wherein the convection of the supercritical solution between the zones (13) and (14) is controlled by the convection controller (2). 23. The apparatus according to claim 21, wherein the dissolution zone (13) is located above the horizontal baffle (12), and the crystallization zone (14), below the horizontal baffle (12). 24. A process for preparing a bulk monocrystalline aluminum nitride, comprising the steps of dissolving an aluminum-containing feedstock in a supercritical solvent containing ammonia and ions of an alkali metal in an autoclave, to form a supercritical solution of aluminum nitride whose solubility has a negative temperature coefficient; and selectively growing a crystal of aluminum nitride only on the face of a crystallization seed arranged in the autoclave, from the supercritical solution introduced, by taking advantage of the negative temperature coefficient of the solubility of aluminum nitride. 25. A process for preparing a bulk monocrystalline aluminum nitride, comprising the steps of dissolving an aluminum-containing feedstock in a supercritical solvent containing ammonia and ions of an alkali metal in an autoclave, to form a supercritical solution of aluminum nitride whose solubility has a positive pressure coefficient; and selectively growing a crystal of aluminum nitride only on the face of a crystallization seed arranged in the autoclave, from the supercritical solution introduced, by taking advantage of the positive pressure coefficient of the solubility of aluminum nitride. 26. The process according to claim 24 or 25, wherein the ions of the alkali metals are introduced in the form of an alkali metal or a mineralizer containing no halogen. 27. The process according to claim 26, wherein the ions of the alkali metal are of one or more selected from the group consisting of Li+, Na+ and K+. 28. The process according to claim 24 or 25, wherein the aluminum-containing feedstock dissolved in the supercritical solvent is composed of aluminum nitride or an aluminum precursor capable of producing an aluminum compound soluble in the supercritical solution. 29. The process according to claim 24 or 25, wherein the aluminum-containing feedstock is composed of AlN formed by HVPE or AlN formed by other chemical reaction, containing an element which does not hinder an ammonobasic supercritical reaction. 30. The process according to claim 24 or 25, wherein the aluminum-containing feedstock is composed of a combination of aluminum nitride which is dissolved in the supercritical ammonia solvent through an equilibrium reaction, and metallic aluminum which irreversibly reacts with the supercritical ammonia solvent. 31. The process according to claim 24 or 25, wherein the aluminum-containing feedstock is a sintered body of AlN. 32. The process according to claim 24 or 25, wherein the crystallization seed is a monocrystalline AlN. 33. A process for preparing a bulk monocrystalline aluminum nitride, comprising the steps of dissolving an aluminum-containing feedstock in a supercritical solvent containing ammonia and ions of an alkali metal to form a supercritical solution of aluminum nitride whose solubility has a negative temperature coefficient; adjusting the solubility of the supercritical solution below a level of concentration where no spontaneous crystallization occurs, while maintaining oversaturation of the supercritical solution relative to a crystallization seed by raising the temperature to a predetermined temperature or reducing the pressure to a predetermined pressure, at least, in a zone in the autoclave where the crystallization seed is arranged; and selectively growing a crystal of aluminum nitride only on the face of the crystallization seed arranged in the autoclave, from said supercritical solution. 34. The process according to claim 33, wherein two zones, i.e., a dissolution zone and a crystallization zone, are concurrently formed in the autoclave, and wherein the oversaturation of the supercritical solution relative to the crystallization seed is controlled by controlling the dissolution temperature and the crystallization temperature. 35. The process according to claim 34, wherein the temperature of the crystallization zone is set at a temperature from 400 to 600° C. 36. The process according to claim 34, wherein a difference in temperature between the two zones i.e., the dissolution zone and the crystallization zone, concurrently formed in the autoclave, is maintained to 150° C. or less, preferably 100° C. or less. 37. The process according to claim 34, wherein the oversaturation of the supercritical solution relative to the crystallization seed is adjusted by providing one or more baffles which partition the lower temperature dissolution zone from the higher temperature crystallization zone, and controlling the convection amount between the dissolution zone and the crystallization zone. 38. The process according to claim 34, wherein a specified difference in temperature is set between the two zones, i.e., the dissolution zone and the crystallization zone, formed in the autoclave, and wherein the oversaturation of the supercritical solution relative to the crystallization seed is adjusted by utilizing an aluminum-containing feedstock which is introduced as an AlN crystal having a total surface area larger than the total surface area of the crystallization seed. |
<SOH> BACKGROUND ART <EOH>Optoelectronic devices based on nitrides are generally fabricated on sapphire or silicon carbide substrates which differ from nitride layers deposited thereon (so-called heteroepitaxy). However, qualities of such devices that have epitaxially grown on heterogeneous substrates are not sufficiecnt. Therefore, there is a demand for a process for forming not only a bulk monocystalline GaN but also a bulk monocystalline AlN. The following are proposed as the process for forming bulk mono-crystalline GaN: Halide Vapor Phase Epitaxy (HVPE) employing halides in vapor phases [“Optical patterning of GaN films”, M. K. Kelly, O. Ambacher, Appl. Phys. Lett. 69 (12) (1996) and “Fabrication of thin-film InGaN light-emitting diode membranes” W. S. Wrong, T. Sands, Appl. Phys. Lett. 75 (10) (1999)]; the HNP process using high-pressure nitrogen [“Prospects for high-pressure crystal growth of III-V nitrides”, S. Porowski et al., Inst. Phys. Conf. Series, 137, 369 (1998)]; the ammono method using supercritical ammonia so as to lower the temperature and the pressure for a growing step [“ammono method of BN, AlN, and GaN synthesis and crystal growth” R. Dwilinski et al., Proc. EGW-3, Warsaw, Jun. 22-24, 1998, MRS Internet Journal of Nitride Semiconductor Research]; and [“Crystal Growth of gallium nitride in supercritical ammonia”, J. W. Kolis et al., J. Cryst. Growth 222, 431-434 (2001)]. On the other hand, as for bulk monocrystalline AlN, D. Peters has proposed a process for forming aluminum nitride crystals from metallic aluminum by using a supercritical ammonia (Journal of Crystal Growth 104 (1990), pp 411-418), however, the resultant crystals were very fine and only for use in packages. Recently, as an epitaxial growth method, Y. Shi et al. have succeeded in growing monocrystalline AlN on a SiC substrate through an AlN buffer layer by the sublimation method (MIJ-NSR, Vol. 6, Art. 5). However, improvement on the quality of crystals is limited, since the vapor phase growth is based on non-equilibrium chemical reaction. In the meantime, the lives of optics using semiconductors vary depending on mainly the crystallinity of the active layers, involving a dislocation density. In case of a laser diode employing an AlN substrate, it is preferable that the dislocation density thereof is decreased to 10 6 /cm 2 or lower, which is, however, very hard for the conventional processes to achieve. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 shows a graph illustrating a change in temperature in an autoclave with the passage of time, on condition that the pressure is kept constant, in case of Example (1). FIG. 2 shows a graph illustrating a change in pressure in an autoclave with the passage of time, on condition that the temperature is kept constant, in case of Example (2). FIG. 3 shows a graph illustrating a change in temperature in an autoclave with the passage of time, on condition that the volume is kept constant, in case of Example (3). FIG. 4 shows a graph illustrating a change in temperature in an autoclave with the passage of time, in case of Example (4). FIG. 5 shows a graph illustrating a change in temperature in an autoclave with the passage of time, in case of Example (5). FIG. 6 is a schematic sectional view of an autoclave and a furnace unit used in Examples. FIG. 7 is a schematic perspective view of an apparatus for forming a bulk monocrystalline aluminum nitride. FIG. 8 shows a graph illustrating a change in temperature in an autoclave with the passage of time, in case of Example (6). FIG. 9 shows a graph illustrating the temperature dependency of the dissolution limit and the spontaneous crystallization limit in the supercritical ammonia. detailed-description description="Detailed Description" end="lead"? |
Hybridization normalization methods |
The present invention includes methods of normalizing hybridization reactions that are designed to select normalization control genes, specifically 5′-3′-, and middle portions of the these genes, that hybridize similarly to a probe array and that produce the most consistently linear curve of hybridization signal over a range of normalization control gene segment concentrations. These methods have applicability across a broad spectrum of hybridization formats. |
1. A method of normalizing a hybridization reaction comprising a nucleic acid sample, comprising: a) adding at least one normalization control gene segment to the hybridization reaction corresponding to the 5′, middle or 3′ regions of at least one normalization control gene. 2. A method of claim 1, wherein the normalization control gene segment is not present in the nucleic acid sample. 3. A method of claim 2, wherein the normalization control genes are selected from the group consisting of: a) viral genes; b) prokaryotic genes; and c) eukaryotic genes. 4. A method of claim 2, wherein hybridization reaction is conducted on a solid substrate. 5. A method of claim 4, wherein the solid substrate is an oligonucleotide array. 6. A method of claim 5, wherein the array comprises oligonucleotide probes that are complementary to the normalization control gene segments. 7. A method of claim 6, wherein the oligonucleotide probes of the array are selected from the group consisting of: a) human nucleic acids; b) non-human nucleic acids; c) animal nucleic acids; d) microbial nucleic acids; e) bacterial nucleic acids; f) fungal nucleic acids; g) tissue specific nucleic acids; h) disease specific nucleic acids; and i) plant nucleic acids. 8. The method of claim 7, wherein the normalization control gene segments are selected by a method comprising determining the non-specific cross-hybridization of the nucleic acid sample to the normalization control gene segments. 9. The method of claim 8, wherein the normalization control gene segments that do not substantially cross-hybridize are selected. 10. The method of claim 7, wherein the normalization control gene segments that are added to the hybridization reaction are selected by a method comprising analyzing a series of hybridization reactions wherein each hybridization reaction of the series contains an increased concentration of the normalization control gene segment. 11. The method of claim 10, wherein the normalization control gene segments that produce the most consistently linear curve of hybridization signal over a range of normalization control gene segment concentrations are selected. 12. The method of claim 1, wherein the normalization control gene segments are the 5′, middle and 3′ fragments of at least one normalization control gene. 13. A method of normalizing a hybridization reaction comprising a nucleic acid sample, comprising: a) providing a normalization control comprising one or more normalization control gene segments, wherein said normalization control gene segments are mixed with the nucleic acid sample, and wherein said normalization control gene segments are prepared by a method comprising: i) selecting one or more candidate normalization control genes; ii) segmenting the candidate normalization control genes into 5′-, middle-, and 3′-segments, thereby producing candidate normalization control gene segments; iii) hybridizing said candidate normalization control gene segments to an oligonucleotide probe in the presence and absence of the nucleic acid sample; iv) determining the non-specific cross-hybridization of candidate normalization control gene segments to said oligonucleotide probe by determining the hybridization of candidate normalization control gene segments to probes other than those complementary to the candidate normalization control gene segments; v) repeating step (iii) at various concentrations of candidate normalization control gene segments; and vi) identifying and selecting those candidate normalization control gene segments that do not substantially cross-hybridize to said oligonucleotide probe. 14. The method of claim 13, wherein the normalization control gene segments are prepared by method further comprising the following steps: a) preparing individual mixtures of nucleic acid samples and candidate normalization control gene segments wherein each individual mixture contains a different concentration of the candidate normalization control gene segments identified in step (vi); b) hybridizing a mixture of step (a) to an oligonucleotide probe; c) repeating step (b) with mixtures containing different concentrations of candidate normalization control gene segments; d) identifying the candidate normalization control gene segments that produce the most consistently linear hybridization response over a range of candidate normalization control gene segment concentrations by measuring the hybridization of said candidate normalization control gene segments to oligonucleotide probes that are complementary to the normalization control gene segments over a range of candidate normalization control gene segment concentrations; and e) producing a solution containing one or more of the candidate normalization control gene segments of step (d) over a concentration range sufficient to produce a linear normalization curve. 15. The method of claim 14, further comprising the steps of: a) hybridizing a mixture of said nucleic acid sample and the solution of step (e) to said array; and b) quantifying the hybridization of said target or pool of nucleic acid sample to said array. 16. A method of claim 13, wherein the normalization control gene segment is not present in the nucleic acid sample. 17. A method of claim 16, wherein the normalization control genes are selected from the group consisting of: a) viral genes; b) prokaryotic genes; and c) eukaryotic genes. 18. A method of claim 16, wherein hybridization reaction is conducted on a solid substrate. 19. A method of claim 18, wherein the solid substrate is an oligonucleotide array. 20. A method of claim 19, wherein the nucleotide array comprises oligonucleotide probes that are complementary to the normalization control gene segments. 21. A method of claim 20, wherein the oligonucleotide probes of the oligonucleotide array are selected from the group consisting of: a) human nucleic acids; b) non-human nucleic acids; c) animal nucleic acids; d) microbial nucleic acids; e) bacterial nucleic acids; f) fungal nucleic acids; g) tissue specific nucleic acids; h) disease specific nucleic acids; and i) plant nucleic acids. 22. The method of claim 1, wherein the normalization control gene segments are labeled. 23. The method of claim 22, wherein the label is selected from one or more of the group consisting of: a) a fluorescent label; b) a chemiluminescent label; c) a bioluminescent label; d) a radioactive label; e) colorimetric label; and f) a light scattering label. 24. The method of claim 1, wherein the normalization control gene segments are produced by the polymerase chain reaction. 25. The method of claim 1, wherein the normalization control gene segments are produced by cloning into a vector and expressing said normalization control gene segments in a host cell. 26. The method of claim 1, wherein the normalization control gene segments are DNA or RNA. 27. The method of claim 26, wherein the normalization control gene segments are RNA. 28. The method of claim 1, further comprising fragmenting the normalization control gene segments. 29. The method of claim 1, wherein the normalization control genes are selected from one or more of the group consisting of: a) an Escherichia coli BioB gene; b) an Escherichia coli BioC gene; c) an Escherichia coli BioD gene; d) a P1 bacteriophage Cre gene; e) a Bacillus subtilis dap gene; f) a Bacillus subtilis thr gene; g) a Bacillus subtilis trp gene; h) a Bacillus subtilis phe gene; and i) a Bacillus subtilis lys gene. 30. The method of claim 1, wherein the nucleic acid sample is selected from the group consisting of: a) pooled nucleic acid samples; b) genomic DNA; c) cDNA; d) cRNA; e) mRNA; and f) polyA RNA. 31. The method of claim 5, wherein the oligonucleotide probe array is immobilized on a solid support selected from the group consisting of: a) filters; b) polyvinyl chloride dishes; c) silicon or glass beads; and d) glass wafers. 32. The method of claim 5, wherein the oligonucleotide probe array is a high density array or nucleic acid chip. 33. A method of claim 29, wherein the normalization control genes are selected from the group consisting of BioB, Dap, Cre, BioD, and BioC. 34. A method of claim 29, wherein the normalization control genes consist of BioB, Dap, Cre, BioD, and BioC. 35. A method of claim 34, wherein the normalization control gene segments comprise BioB 5′, Dap M, Dap 5′, Cre 5′, BioB 3′, BioB M, BioD 3′, BioC 5′, BioC 3′, Dap3′ and Cre 3′. 36. A method of claim 34, wherein the normalization control gene segments are a cocktail comprising BioB 5′, Dap M, Dap 5′, Cre 5′, BioB 3′, BioB M, BioD 3′, BioC 5′, BioC 3′, Dap 3′ and Cre 3′. 37. A method of claim 36, wherein the cocktail is cocktail 7211 in FIG. 4. 38. A method of claim 37, wherein the cocktail comprises normalization control gene fragments BioB 5′ at about 12.5 pM, Dap M at about 2 pM, Dap 5′ at about 1 pM, Cre 5′ at about 25 pM, BioB 3′ at about 100 pM, BioB M at about 50 pM, BioD 3′ at about 75 pM, BioC 5′ at about 1.5 pM, BioC 3′ at about 5 pM, Dap 3′ at about 0.5 pM and Cre 3′ at about 3 pM. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Nucleic acid hybridization-based methods have become prevalent in medical and biotechnological research and development, diagnostic testing, drug development and forensics. The reliability and utility of these nucleic acid hybridization-based methods depends on accurate and reliable methods for accounting for variations between analyses. For example, variations in hybridization conditions, label intensity, reading and detector efficiency, sample concentration and quality, background effects, and image processing effects each contribute to hybridization signal heterogeneity. Hegde et al. (2000) Biotechniques 29 (3): 548-562; Berger et al. (2000) WO 00/04188. Normalization of hybridization procedures such as Northern blot and Dot Blot analyses has often relied on control hybridizations to housekeeping genes such as (β-actin, glyceraldehyde-3-phosphate dehydrogenase, and the transferrin receptor gene. Eickhoff et al. (1999) Nucleic Acids Research 27 (22): e33; Spiess et al. (1999) Biotechniques 26 (1): 46-50. These methods, however, generally do not provide the linearity sufficient to detect small but significant changes in transcription or gene expression. Spiess et al. (1999) Biotechniques 26 (1): 46-50. In addition, the steady state levels of many housekeeping genes are susceptible to alterations in expression levels that are dependent on cell differentiation, nutritional state, specific experimental and stimulation protocols. Eickhoff et al. (1999) Nucleic Acids Research 27 (22): e33; Spiess et al. (1999) Biotechniques 26 (1): 46-50; Hegde et al. (2000) Biotechniques 29 (3): 548-562; and Berger et al. (2000) WO 00/04188. In addition to numerous assay-associated factors, such as variations in background, labeling, hybridization conditions and detection, characteristics of the hybridization control molecule itself, such as variations in base composition, probe length, secondary structure and ability to cross-hybridize with the probes or target nucleic acids, also contribute to the difficulty and imprecision of comparing results between analyses. The normalization of array format hybridizations has typically been conducted using full-length hybridization controls that are complementary to oligonucleotide probes contained on the array. (Affymetrix GeneChip® Expression Analysis Manual). Full-length hybridization controls, however, increase the likelihood of control-specific background effects as the normalization curves generated using full-length normalization controls may not achieve the linearity and reproducibility necessary for many of the emerging applications of array hybridization methodologies. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based on the surprising discovery of methods for optimizing the normalization of hybridization reactions comprising a nucleic acid sample, the method comprising the step of adding at least one normalization control gene segment to the hybridization reaction corresponding to the 3′, 5′ and middle regions of at least one normalization control gene. The normalization controls of the present invention are selected from nucleic acids that are not present in the nucleic acid sample. Preferably, the normalization controls are selected from, viral, prokaryotic or eukaryotic genes. In a preferred embodiment, the normalization control genes are selected from a Escherichia coli BioB, BioC, or BioD gene, a P1 bacteriophage cre gene, or a Bacillus subtilis dap, thr, trp, phe or lys gene. The normalization control gene segments of the present invention are typically either DNA or RNA and may be produced by the polymerase chain reaction or cloning of the normalization control genes or segments into a vector and expression of the normalization control genes or segments in a host cell. RNA normalization control gene segments may be produced, for example, by in vitro transcription of the cloned normalization control genes or segments. The methods of the present invention are applicable to any hybridization assay format. Preferred formats include formats where an oligonucleotide probe, complementary to the normalization control gene segments, is immobilized on a solid support such as filters, polyvinyl chloride dishes, silicon or glass beads or wafers in an array. Preferred arrays include high density or nucleic acid chip arrays. The oligonucleotide probes may be selected from nucleic acids isolated from human, non-humans, animals, microorganisms, bacteria, fungi, plants, and nucleic acids isolated from specific normal or diseased tissue. The nucleic acid samples compatible with the methods of the instant invention include pooled nucleic acid samples, genomic DNA, cDNA, cRNA, mRNA, and polyA RNA. The normalization control gene segments of the instant invention are selected by a method that comprises determining the non-specific cross-hybridization of the nucleic acid sample to the normalization control gene segments, wherein the normalization control gene segments that do not substantially cross-hybridize are selected. In another embodiment, the normalization controls of the present invention are selected by a method comprising analyzing a series of hybridization reactions, wherein each hybridization reaction of the series contains an increased concentration of the normalization control gene segment, and wherein the normalization control gene segments that produce the most consistently linear curve of hybridization signal over a range of normalization control gene segment concentrations are selected. In a preferred embodiment, the methods of normalizing a hybridization reaction of the present invention comprise the steps of: a) providing a normalization control comprising one or more normalization control gene segments, wherein said normalization control gene segments are mixed with the nucleic acid sample, and wherein said normalization control gene segments are prepared by a method comprising: i) selecting one or more candidate normalization control genes; ii) segmenting the candidate normalization control genes into 5′-, middle-, and 3′-segments, thereby producing candidate normalization control gene segments; iii) hybridizing said candidate normalization control gene segments to an oligonucleotide probe in the presence and absence of the nucleic acid sample; iv) determining the non-specific cross-hybridization of candidate normalization control gene segments to said oligonucleotide probe by determining the hybridization of candidate normalization control gene segments to probes other than those complementary to the candidate normalization control gene segments; v) repeating step (iii) at various concentrations of candidate normalization control gene segments; and vi) identifying and selecting those candidate normalization control gene segments that do not substantially cross-hybridize to said oligonucleotide probe. In a more preferred embodiment, the methods of normalizing a hybridization reaction of the present invention comprise steps wherein the normalization control gene segments are prepared by method further comprising the following steps: a) preparing individual mixtures of nucleic acid samples and candidate normalization control gene segments wherein each individual mixture contains a different concentration of the candidate normalization control gene segments identified in step (vi); b) hybridizing a mixture of step (a) to an oligonucleotide probe; c) repeating step (b) with mixtures containing different concentrations of candidate normalization control gene segments; d) identifying the candidate normalization control gene segments that produce the most consistently linear hybridization response over a range of candidate normalization control gene segment concentrations by measuring the hybridization of said candidate normalization control gene segments to oligonucleotide probes that are complementary to the normalization control gene segments over a range of candidate normalization control gene segment concentrations; and e) producing a solution or composition containing one or more of the candidate normalization control gene segments of step (d) over a concentration range sufficient to produce a linear normalization curve. In the most preferred embodiment, the methods of the present invention further comprise the steps of hybridizing a mixture of said nucleic acid sample and the solution of step (e) to said array, and quantifying the hybridization of said target or pool of nucleic acid sample to said array. The methods of the present invention also contemplate using normalization control gene segments that are labeled with either a fluorescent, chemiluminescent, bioluminescent, colorimetric, or a light scattering label. In another embodiment, the methods of the present invention further comprise the step of fragmenting the normalization control gene segments prior to use. |
Characterising polypeptides |
Provided is a method for characterising a polypeptide or a population of polypeptides, which method comprises the steps of: (a) optionally reducing disulphide linkages in the polypeptides, if they are present and capping free thiols in the polypeptides, if they are present; (b) contacting a sample comprising one or more polypeptides with a cleavage reagent which cleaves one or more polypeptides on the C-terminal side of a lysine residue to produce peptide fragments; (c) optionally deactivating the cleavage reagent; (d) contacting the sample with a lysine reactive agent to cap ε-amino groups; (e) removing those peptides having capped ε-amino groups; and (f) recovering the C-terminal peptides. |
1. A method for characterising a polypeptide or a population of polypeptides, which method comprises the steps of: (a) contacting a sample comprising one or more polypeptides with a cleavage reagent which cleaves one or more polypeptides on the C-terminal side of a lysine residue to produce peptide fragments; (b) optionally deactivating the cleavage reagent; (c) contacting the sample with a lysine reactive agent to cap ε-amino groups; (d) removing those peptide fragments having capped ε-amino groups; and (e) recovering the C-terminal peptide fragments. 2. A method according to claim 1, wherein peptide fragments having capped ε-amino groups are removed by capturing them on a solid phase and C-terminal peptides are recovered in solution. 3. A method according to claim 2, wherein the lysine reactive agent is covalently attached to a solid phase. 4. A method according to claim 2, wherein the peptide fragments having capped ε-amino groups are removed by affinity capture and wherein the lysine reactive agent comprises biotin and the solid phase is an avidinated solid phase. 5. A method according to claim 1, wherein the lysine reactive agent comprises a hindered Michael reagent. 6. A method according to claim 1, wherein the hindered Michael agent comprises a compound having the following structure: wherein X is an electron withdrawing group that is capable of stabilising a negative charge; the R groups independently comprise a hydrogen, a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support. 7. A method according to claim 6, wherein one R comprises a methyl or phenyl group. 8. A method according to claim 6, wherein at least one R comprises an electron withdrawing group. 9. A method according claim 6, wherein at least one R comprises a cyclic or heterocylic aromatic ring or fused ring. 10. A method according to claim 6, wherein X comprises an —SO2R1 group, wherein R1 comprises an alkyl group or an aryl group, including aromatic groups cyclic groups, fused cyclic groups, and heterocyclic groups. 11. A method according to claim 10, wherein R1 comprises an electron withdrawing group. 12. A method according to claim 10, wherein the ring comprises a phenyl, pyridyl, naphthyl quinolyl, pyrazine, pyrimidine or triazine ring structure. 13. A method according to claim 6, wherein the X group is substituted with an electron withdrawing group. 14. A method according to claim 13, wherein the electron withdrawing group is selected from halogens, such as fluorine chlorine, bromine or iodine, and nitro and nitrile groups. 15. A method according to claim 6, wherein the X group comprises a structure capable of promoting water solubility. 16. A method according to claim 1, wherein the cleavage agent comprises a sequence-specific cleavage agent. 17. A method according to claim 1, wherein the cleavage agent comprises a peptidase, or cyanogen bromide. 18. A method according to claim 17, wherein the peptidase comprises Lys-C. 19. A method according to claim 1, wherein the sample of step (a) comprises a sub-cellular fraction. 20. A method according to claim 1, which further comprises preparing the sample of step (a) by liquid chromatography. 21. A method for assaying for one or more specific polypeptides in a test sample, which comprises performing a method according to claim 1, wherein the sequence of the specific polypeptide is determined by assaying the resulting C-termini for a predetermined C-terminal sequence of amino acid residues. 22. A method of characterising one or more mixtures of polypeptides, which method comprises the following steps: (a) recovering one or more C-terminal peptides from the mixtures by employing one or more of the methods as defined in claim 1; (b) detecting the peptides by mass spectrometry. 23. A method for determining the expression profile of a sample, which method comprises characterising one or more mixtures of polypeptides according to a method as defined in claim 22. 24. A method according to claim 22, which method comprises determining the identity of each of the peptides detected by mass spectrometry. 25. A method according to claim 22, which method comprises identifying the quantity of each of the peptides detected by mass spectrometry. 26. A method for characterising a polypeptide or a population of polypeptides, which method comprises contacting a sample comprising one or more polypeptides with a lysine reactive agent to attach the agent to ε-amino groups, wherein the lysine reactive agent comprises a hindered Michael reagent. 27. A method according to claim 26, wherein the hindered Michael agent is a compound having the following structure: wherein X is an electron withdrawing group that is capable of stabilising a negative charge: the R groups independently comprise a hydrogen, a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support. 28. A compound having the following structure: wherein R1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure and the R groups independently comprise a hydrogen, a halogen, or an alkyl or aryl group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support. 29. A compound according to claim 28, wherein at least one R group comprises a methyl, or phenyl group. 30. A compound according to claim 28, wherein at least one R group comprises an electron-withdrawing group. 31. A compound according to claim 30, wherein at least one R group comprises a halogen atom or a halogenated alkyl group, or a phenyl ring with one or more electron withdrawing substituents. 32. A kit for characterising a polypeptide or a population of polypeptides, which kit comprises: (a) a lysine reactive agent for capping ε-amino groups; (b) a means for recovering or isolating C-terminal peptides; (c) optionally an amine reactive reagent for labelling α-amino groups; (d) optionally a cleavage reagent for producing peptide fragments. 33. A kit according to claim 32, wherein the lysine reactive agent comprises a compound having the following structure: wherein X is an electron withdrawing group that is capable of stabilising a negative charge; the R groups independently comprise a hydrogen, a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support. 34. A kit according to claim 33, wherein the lysine reactive agent comprises a compound having the following structure: wherein R1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure and the R groups independently comprise a hydrogen, a halogen, or an alkyl or aryl group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support. 35. A kit according to claim 34, wherein the means for recovering or isolating C-terminal peptides comprises an affinity capture agent attached to the lysine reactive agent, or a solid phase covalently bound to the lysine reactive agent. 36. A method for protecting ε-amino groups in peptides and polypeptides comprising using the compound: wherein R1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure and the R groups independently comprise a hydrogen, a halogen, or an alkyl or aryl group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support. 37. The method according to claim 36, wherein R1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure. 38. The method according to claim 36, wherein at least one R group comprises a methyl or phenyl group. 39. The method according to claim 36, wherein at least one R group comprises an electron-withdrawing group. 40. The method according to claim 39, wherein at least one R group comprises a halogen atom or a halogenated alkyl group, or a phenyl ring with one or more electron withdrawing substituents. 41. The method according to claim 36, wherein the protection is against further reaction of the ε-amino groups with Edman agents, capture agents and agents which are capable of reacting with α-amino groups. 42. The method according to claim 41, wherein the Edman agent comprises an isothiocyanate or an isocyanate, the capture agent comprises N-hydroxysuccinimidyl biotin and the agent which is capable of reacting with α-amino groups comprises acetic acid N-hydroxysuccinimide ester. |
<SOH> BACKGROUND IN THE ART <EOH>Techniques for profiling proteins, that is to say cataloguing the identities and quantities of proteins in a tissue, are not well developed in terms of automation or high throughput. A typical method of profiling a population of proteins is by two-dimensional electrophoresis (R. A. Van Bogelen., E. R. Olson, “Application of two-dimensional protein gels in biotechnology”, Biotechnol Annu. Rev., 1, 69-103, 1995). In this method, a protein sample extracted from a biological sample is separated on a narrow gel strip. This first separation usually separates proteins on the basis of their iso-electric point. The entire gel strip is then laid against one edge of a rectangular gel. The separated proteins in the strip are then electrophoretically separated in the second gel on the basis of their size. This technology is slow and very difficult to automate. It is also relatively insensitive in its simplest embodiments. A number of improvements have been made to increase resolution of proteins by 2-D gel electrophoresis and to improve the sensitivity of the system. One approach to improve the sensitivity of 2-D gel electrophoresis and its resolution is to analyse the protein in specific spots on the gel by mass spectrometry (Jungblut P, Thiede B. “Protein identification from 2-D gels by MALDI mass spectrometry.” Mass Spectrom. Rev. 16, 145-162, 1997. One example of a mass spectrometry method is in-gel tryptic digestion followed by analysis of the tryptic fragments by mass spectrometry to generate a peptide mass fingerprint. If sequence information is required, tandem mass spectrometry analysis can be performed. More recently attempts have been made to exploit mass spectrometry to analyse whole proteins that have been fractionated by liquid chromatography or capillary electrophoresis (Dolnik V. “Capillary zone electrophoresis of proteins.”, Electrophoresis 18, 2353-2361, 1997). In-line systems exploiting capillary electrophoresis mass spectrometry have been tested. The analysis of whole proteins by mass spectrometry, however, suffers from a number of difficulties. The first difficulty is the analysis of the complex mass spectra resulting from multiple ionisation states accessible by individual proteins. The second major disadvantage is that the mass resolution of mass spectrometers is at present quite poor for high molecular weight species, i.e. for ions that are greater than about 4 kilodaltons (kDa) in mass, so resolving proteins that are close in mass is difficult. A third disadvantage is that further analysis of whole proteins by tandem mass spectrometry is difficult as the fragmentation patterns for whole proteins are extremely complex and difficult to interpret. As a result of the difficulties of analysing whole proteins, techniques that rely on the analysis of peptides from proteins are preferred. Peptide mass fingerprinting has been used in the analysis of gel separated proteins as described above. However, this process is adequate only for the analysis of individual proteins or very simple mixtures of proteins. A typical protein will give rise to from twenty to thirty peptides after cleavage with trypsin. The pattern of peptide masses is useful for identifying single proteins, but the complexity of the mass spectrum of the trypsin digest of a mixture of proteins rapidly rises in complexity as the number of proteins in the mixture increases. This increases the chance that a peptide mass is assigned incorrectly to a protein, thus limiting the number of proteins that may be analysed simultaneously. As a result new protein characterisation methods are being developed in which specific peptides are isolated from each protein in a mixture. Nature Biotechnology 17, 994-999 (1999) discloses the use of ‘isotope encoded affinity tags’ for the capture of peptides from proteins, to allow protein expression analysis. In this article, the authors describe the use of a biotin linker, which is reactive to thiols, for the capture peptides with cysteine in them. A sample of protein from one source is reacted with the biotin linker and cleaved with an endopeptidase. The biotinylated cysteine-containing peptides can then be isolated on avidinated beads for subsequent analysis by mass spectrometry. Two samples can be compared quantitatively by labelling one sample with the biotin linker and labelling the second sample with a deuterated form of the biotin linker. Each peptide in the samples is then represented as a pair of peaks in the mass spectrum where the relative peak heights indicate their relative expression levels. This ‘isotope encoding’ method has a number of limitations. A first limitation is the reliance on the presence of thiols in a protein—many proteins do not have thiols while others have several. In a variation on this method, linkers may be designed to react with other side chains, such as amines. However, since many proteins contain more than one lysine residue, multiple peptides per protein would generally be isolated in this approach. It is likely that this would not reduce the complexity of the sample sufficiently for analysis by mass spectrometry. A sample that contains too many species is likely to suffer from ‘ion suppression’, in which certain species ionise preferentially over other species which would normally appear in the mass spectrum in a less complex sample. In general, capturing proteins by their side chains is likely to give either too many peptides per protein or certain proteins will be missed altogether. The second limitation of this approach is the method used to compare the expression levels of proteins from different samples. Labelling each sample with a different isotope variant of the affinity tag results in an additional peak in the mass spectrum for each peptide in each sample. This means that if two samples are analysed together there will be twice as many peaks in the spectrum. Similarly, if three samples are analysed together, the spectrum will be three times more complex than for one sample alone. It is clear that this approach will be limited, since the ever increasing numbers of peaks will increase the likelihood that two different peptides will have overlapping peaks in the mass spectrum. A further limitation, which is reported by the authors of the above paper, is the mobility change caused by the tags. The authors report that peptides labelled with the deuterated biotin tag elute slightly after the same peptide labelled with the undeuterated tag. Published international patent application WO 98/32876 discloses methods of profiling a population of proteins by isolating a single peptide from one terminus of each protein in the population. In a first aspect the invention comprises the steps of: 1. capturing a population of proteins onto a solid phase support by one terminus of each protein in the population; 2. cleaving the captured proteins with a sequence specific cleavage agent; 3. washing away peptides generated by the cleavage agent not retained on the solid phase support; 4. releasing the terminal peptides retained on the solid phase support; and 5. analysing the released terminal peptides, preferably identifying and quantifying each peptide in the mixture. The analysis is preferably performed by mass spectrometry. In this application, the C-terminus is discussed as being more preferable as the terminus by which to capture a population of proteins, since the N-terminus is often blocked. In order to capture a population of proteins by the C-terminus, the C-terminal carboxyl group must be distinguished from other reactive groups on a protein and must be reacted specifically with a reagent that can effect immobilisation. In many C-terminal sequencing chemistries the C-terminal carboxyl group is activated to promote formation of an oxazolone group at the C-terminus. During the activation of the C-terminal carboxyl, side chain carboxyls are also activated, but these cannot form an oxazolone group. It has been reported that the C-terminal oxazolone is less reactive to nucleophiles under basic conditions than the activated side-chain carboxyls, offering a method of selectively capping the side chain carboxyl groups (V. L. Boyd et al., Methods in Protein Structure Analysis: 109-118, Plenum Press, Edited M. Z. Atassi and E. Appella, 1995). Other more reactive side chains can be capped prior to the activation of the carboxyls using a variety of conventional reagents. In this way all reactive side chains can be capped and the C-terminus can be specifically labelled. EP A 0 594 164 and EP B 0 333 587 describe methods of isolating a C-terminal peptide from a protein in a method to allow sequencing of the C-terminal peptide using N-terminal sequencing reagents. In this method the protein of interest is digested with an endoprotease, which cleaves at the C-terminal side of lysine residues. The resultant peptides are reacted with diisothiocyanato (DITC) polystyrene which reacts with all free amino groups. N-terminal amino groups that have reacted with the DITC polystyrene can be cleaved with trifluoroacetic acid (TFA) thus releasing the N-terminus of all peptides. The epsilon-amino group of lysine is not cleaved however and all non-terminal peptide are thus retained on the support and only C-terminal peptides are released. According to this patent the C-terminal peptides are recovered for micro-sequencing. Anal. Biochem. 132, 384-388 (1983) and DE A 4344425 (1994) describe methods of isolating an N-terminal peptide from a protein by reacting the protein with a capping reagent which will cap any free amino groups in the protein. The protein is then cleaved, and if trypsin is used cleavage occurs only at arginine residues. Cleavage with trypsin thus exposes α-amino groups in the non-N-terminal peptides. In the first disclosure (Anal. Biochem.) the α-amino groups are reacted with dinitrofluorobenzene (DNF) which allows the non-N-terminal peptides to be captured by affinity chromatography onto a polystyrene resin while the N-terminal peptides flow through unimpede. In DE A 4344425, the epsilon amino groups are reacted with an acylating agent prior to cleavage. After cleavage in this method, the α-amino groups on the non-N-terminal peptides are reacted with an amine reactive solid support such as diisothiocyanato glass, leaving the N-terminal peptides free in solution. The main drawback of all of these peptide isolation methods is the use of conventional amine modification reagents which tend to be unstable in aqueous conditions at the pH needed for lysine modification. As a result, large excesses of reagent need to be used which can lead to side-reactions particularly with histidine residues. The Anal. Biochem. method also requires that the DNF groups be removed from histidine and tyrosine by thiolysis prior to isolating the N terminal peptide, if the N terminal peptide contains these groups. This additional step requires extra effort and may not go to completion. In the Anal. Chem. disclosure the protein and terminal peptides are not analysed by mass spectrometry and so it is not possible to know whether the capping of the lysine epsilon amino groups goes to completion. It is an aim of this invention to solve the problems associated with the known methods described above. It is thus an aim of this invention to provide improved methods for isolating a single C-terminal peptide from each protein in a mixture of polypeptides using protein reactive reagents that are stable in water, selective for lysine and that work under mild reaction conditions without degradation of the reagents. It is a further aim that these reactions go substantially to completion in a relatively short time, e.g. in a few hours. |
Delivery system and method for low visibilty conditions |
An inventive system for delivering a package (e.g., plurality of packages) includes a transport vehicle for transporting the package to a destination, the transport vehicle including a first transceiver and a computer system, and an electronic positioning system for navigating the transport vehicle to the destination under a low-visibility condition. |
1. A system for delivering a package, comprising a transport vehicle for transporting said package to a destination, said transport vehicle comprising a first transceiver and a computer system; and an electronic positioning system for navigating said transport vehicle to said destination under a low-visibility condition. 2. The system according to claim 1, further comprising: a drop-box comprising a second transceiver for wirelessly communicating with said first transceiver, and a signaling device for locating said drop-box under a low-visibility condition. 3. The system according to claim 2, wherein during a low visibility condition, said first transciever wirelessly communicates with the said second transceiver, causing said drop-box to activate said signaling device. 4. The system according to claim 3, wherein said signaling device is activated when said transport vehicle is within a predetermined distance of said drop-box. 5. The system according to claim 1, further comprising: an electronic tag associated with said package, said electronic tag comprising: a signaling device which activates when said transport vehicle is within a predetermined distance of said destination; and a third transceiver, for wirelessly communicating with said first and second transceivers. 6. The system according to claim 1, wherein a low-visibility condition comprises at least one of a nighttime condition and an adverse weather condition. 7. The system according to claim 2, wherein said low-visibility condition comprises a condition under which said drop-box is obscured from a view of a person delivering said package. 8. The system according to claim 1, further comprising: a detecting device for detecting a low-visibility condition. 9. The system according to claim 8, wherein said detecting device comprises a photodiode. 10. The system according to claim 2, wherein said signaling device is manually activated using said computer system to regulate a signal transmitted from said first transceiver to said second transceiver. 11. The system according to claim 2, wherein a magnitude of a signal emitted by said signaling device is regulated by using said computer system to regulate a signal transmitted from said first transceiver to said second transceiver. 12. The system according to claim 2, wherein said computer system comprises a locator database for storing detailed location data for said drop-box. 13. The system according to claim 12, wherein data from said electronic positioning system is refined using said detailed location data. 14. A system for delivering packages, comprising: a transport vehicle comprising: a first transceiver; and a computer system; an electronic tag associated with at least one of said packages, comprising: a signaling device which activates when a package arrives at a destination; and a second transceiver, for wirelessly communicating with said transport vehicle; and an electronic positioning system for navigating said transport vehicle to said destination according to an optimum delivery route. 15. The system according to claim 14, wherein said electronic positioning system comprises a satellite-based global positioning system (GPS) comprising: at least one satellite for wirelessly transmitting signals; a ground based control station for uploading data to and receiving signals from said at least one satellite; and a user receiver located on said transport vehicle for receiving signals from said at least one satellite. 16. The system according to claim 14, wherein said electronic positioning system comprises a dead reckoning (DR) system which measures compass direction and a speed of said transport vehicle. 17. The system according to claim 16, wherein said electronic positioning system comprises a solid state gyroscope. 18. The system according to claim 15, wherein said global positioning system further comprises: a reference station which compares predicted pseudoranges to actually measured pseudoranges, computes correction data for each said satellite and broadcasts said correction data over a separate data link to said user receiver, wherein said user receiver applies said correction data to a pseudorange measurement to compute a position. 19. The system according to claim 15, wherein said electronic positioning system further comprises a dead reckoning (DR) system which measures compass direction and a speed of said transport vehicle, and wherein said dead reckoning system is used to augment said satellite-based global positioning system. 20. The system according to claim 14, further comprising: a base station comprising a third transceiver for wirelessly communicating with said transport vehicle and said electronic tag. 21. The system according to claim 20, further comprising: a drop-box located at said destination, comprising a fourth transceiver for wirelessly communicating with said transport vehicle, said electronic tag and said base station. 22. The system according to claim 14, wherein said signaling device is activated when said package arrives at said destination, and wherein said first and second transceivers wirelessly communicate with each other to minimize a delivery time. 23. A system for low-visibility package delivery, comprising: a transport vehicle comprising: a first transceiver; and a computer system; and an electronic positioning system for navigating said transport vehicle to said destination according to an optimum delivery route, wherein a low-visibility condition causes a location of said destination to be hardly visible to a person making a delivery. 24. The system according to claim 23, wherein said low-visibility condition comprises one of an adverse weather condition and a nighttime condition. 25. The system according to claim 23, further comprising: a drop-box located at said destination for receiving a package, wherein a low-visibility condition causes a drop-box located at said destination to be hardly visible to a person making a delivery. 26. A method for delivering packages, comprising: transporting said package to a destination using a transport vehicle; and navigating said transport vehicle to said destination under a low-visibility condition, using an electronic positioning system. 27. A method for delivering packages, comprising: associating said packages with electronic tags; inputting a destination address to a computer system to determine an optimum delivery route; placing said packages on a transport vehicle; and navigating said transport vehicle to a destination using an electronic positioning system. 28. The method according to claim 27, further comprising: activating a signaling device on a particular electronic tag when said transport vehicle arrives at a destination of a package associated with said particular electronic tag. 29. The method according to claim 27, wherein said electronic tag is temporarily affixed to one of said container and said package. 30. The method according to claim 27, wherein said packages are temporarily stored in a container and said electronic tag is affixed to said container. 31. A programmable storage medium tangibly embodying a program of machine-readable instructions executable by a digital processing apparatus to perform a method for delivering packages, said method comprising: transporting said package to a destination using a transport vehicle; and navigating said transport vehicle to said destination under a low-visibility condition, using an electronic positioning system. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to a system and method for delivering packages, and in particular, to a system and method for delivering packages which uses a positioning system to facilitate delivery, for example, in low visibility conditions. 2. Description of the Related Art In a conventional delivery system, packages are sorted at a distribution center according to a particular area or route corresponding to a particular driver or truck. The packages have been pre-sorted into the approximate hour of anticipated delivery, and then placed on the truck. The driver has a printed list of addresses and number of items for each address. The driver drives along the streets until he finds the address, parks the truck, and goes to the back of truck with the printed list. The driver then locates the package having the correct name and address, checks off a list taken to the door at the destination address and obtains a signature or some other indication that the package was delivered. However, such conventional delivery schemes are severely affected by low visibility conditions such as nighttime conditions and adverse weather conditions such as fog, snow or a hard rain. In such conditions, delivery drivers relying solely on a hand held paper map or their memory of a certain street, apartment complex or office complex often have a hard time locating the correct destination addresses for the packages. As a result, deliveries are often delayed during such conditions. |
<SOH> SUMMARY OF THE INVENTION <EOH>In view of the foregoing and other problems, disadvantages, and drawbacks of the conventional methods and structures, an object of the present invention is to provide a system and method for delivering packages under low-visibility conditions. An inventive system for delivering a package (e.g., plurality of package) includes a transport vehicle for transporting the package to a destination, the transport vehicle including first transceiver and a computer system, an electronic positioning system for navigating the transport vehicle to the destination under a low-visibility condition. The inventive system may also include a drop-box having a second transceiver for wirelessly communicating with the first transceiver, and a signaling device (e.g., light-emitting device such as a light-emitting diode, or an audible device) for locating the drop-box under a low-visibility condition. The system may also include a detecting device (e.g., a photodiode) for detecting a low-visibility condition. Further, the computer system may include a locator database for storing detailed location data for the drop-box. In addition, data from said electronic positioning system may be refined using such detailed location data. In addition, during a low visibility condition, the first transciever may wirelessly communicate with the the second transceiver, causing the drop-box to activate the signaling device. In addition, the signaling device is activated when the transport vehicle is within a predetermined distance of the drop-box. The inventive system may also include an electronic tag associated with the package. The electronic tag may include a signaling device which activates when the transport vehicle is within a predetermined distance of the destination, and a third transceiver, for wirelessly communicating with the first and second transceivers. Further, a low-visibility condition may include a nighttime condition and/or an adverse weather condition. A low-visibility condition may also include a condition under which the drop-box is obscured (e.g., hidden) from a view of a person delivering the package. Further, the signaling device may be manually activated using the computer system to regulate a signal transmitted from the first transceiver to the second transceiver. In addition, a magnitude of a signal emitted by the signaling device may be regulated by using the computer system to regulate a signal transmitted from the first transceiver to the second transceiver. In another aspect, the inventive system for delivering packages may include a transport vehicle having a first transceiver and a computer system, an electronic tag associated with the packages and having a signaling device which activates when a package arrives at a destination and a second transceiver for wirelessly communicating with the transport vehicle, and an electronic positioning system for navigating the transport vehicle to the destination according to an optimum delivery route. The electronic positioning system may include, for example, a satellite-based global positioning system (GPS) having at least one satellite for wirelessly transmitting signals, a ground based control station for uploading data to and receiving signals from the satellite, and a user receiver located on the transport vehicle, for receiving signals from the satellite. The electronic positioning system may also include a dead reckoning (DR) system which measures compass direction and a speed of the transport vehicle. The electronic positioning system may also include a solid state gyroscope. Further, the global positioning system may be a differential global positioning system which further includes a reference station which compares predicted pseudoranges to actually measured pseudoranges, computes correction data for each satellite and broadcasts correction data over a separate data link to the user receiver. In this case, the user receiver applies the correction data to a pseudorange measurement to compute a position. Further, the electronic positioning system could include a hybrid system including, for example, a global positioning system augmented by a dead reckoning (DR) system In addition, the inventive system may include a base station comprising a third transceiver for wirelessly communicating with the transport vehicle and the electronic tag. The inventive system may also include a drop-box located at a package destination, having a fourth transceiver for wirelessly communicating with the transport vehicle, the electronic tag and the base station. In the inventive system, the signaling device on the electronic tag may be activated when the package arrives at its destination. Further, the first and second transceivers wirelessly communicate with each other to minimize a delivery time. The present invention also includes an inventive method for delivering a package (e.g., plurality of packages). The inventive method includes transporting the package to a destination using a transport vehicle, and navigating the transport vehicle to the destination under a low-visibility condition, using an electronic positioning system. In another aspect, the inventive method may include associating the packages with electronic tags, inputting a destination address to a computer system to determine an optimum delivery route, placing the packages on a transport vehicle, and navigating the transport vehicle to a destination using an electronic positioning system. The inventive method may also include activating a signaling device on a particular electronic tag when the transport vehicle arrives at a destination of a package associated with that particular electronic tag. The present invention also includes a programmable storage medium tangibly embodying a program of machine-readable instructions executable by a digital processing apparatus to perform the inventive method. With its unique and novel aspects, the present invention provides a system and method for effectively and accurately delivering a package in a low-visibility condition. |
Methods for treating disorders of the nervous and reproductive systems |
The invention features methods for treating, preventing or modulating a neurological disease or disorder, or for modulating an anaesthetic or a fertility process by administering compounds that modulate ABCA1 expression or activity. The invention also features methods for identifying compounds useful for such methods. |
1. A method for identifying an agent useful in treating an ABCA1-dependent neurological condition comprising: (a) administering to an animal exhibiting said neurological condition an agent that modulates ABCA1 biological activity, and (b) detecting a beneficial change in said neurological condition in said animal following said administering and compared to when said agent is not administered, thereby identifying an agent useful in treating said ABCA1-dependent neurological condition. 2. The method of claim 1 wherein said agent inhibits ABCA1 biological activity. 3. The method of claim 1 wherein said agent enhances ABCA1 biological activity. 4. The method of claim 1 wherein said ABCA1 biological activity is ABCA1-gene expression. 5. The method of claim 1 wherein said ABCA1 biological activity is selected from the group consisting of HDL-cholesterol transport, ion transport, ATP binding, ATP hydrolysis, and phospholipid transport. 6. The method of claim 1 wherein modulation of ABCA1 biological activity is due to a process selected from the group consisting of a change in stability of ABCA1 polypeptide, a change in ABCA1 membrane insertion and a change in ABCA1 membrane channel formation. 7. The method of claim 1 wherein said neurological condition is a member selected from the group consisting of a neurological condition of the central nervous system and a neurological condition of the peripheral nervous system. 8. The method of claim 7 wherein said neurological condition is characterized by neuronal loss or dysfunction. 9. The method of claim 7 wherein said neurological condition is a member selected from the group consisting of Alzheimer's Disease, dementia pugilistica, Parkinson's Disease, Huntington's Disease, Niemann-Pick disease, multiple sclerosis, a neuropathy and an ischemic condition. 10. The method of claim 9 wherein said ischemic condition is an ischemic condition of the central, peripheral, or compression type. 11. The method of claim 9 wherein said ischemic condition is a member selected from the group consisting of stroke and cerebral artery infarction. 12. The method of claim 7 wherein said neurological condition is caused by a defect in myelin repair or production. 13. The method of claim 7 wherein said defect in myelin repair or production is caused by a process selected from the group consisting of demyelination, the removal of myelin debris following injury and remyelination. 14. A method of treating a neurological condition comprising administering to an animal afflicted with such condition an effective amount of an agent that exhibits beneficial activity in a method of claim 1. 15. The method of claim 14 wherein said agent inhibits ABCA1 biological activity. 16. The method of claim 14 wherein said agent enhances ABCA1 biological activity. 17. The method of claim 14 wherein said ABCA1 biological activity is ABCA1-gene expression. 18. The method of claim 14 wherein said ABCA1 biological activity is selected from the group consisting of HDL-cholesterol transport, ion transport, ATP binding, ATP hydrolysis, and phospholipid transport. 19. The method of claim 14 wherein modulation of ABCA1 biological activity is due to a process selected from the group consisting of a change in stability of ABCA1 polypeptide, a change in ABCA1 membrane insertion and a change in ABCA1 membrane channel formation. 20. The method of claim 14 wherein said neurological condition is a member selected from the group consisting of a neurological condition of the central nervous system and a neurological condition of the peripheral nervous system. 21. The method of claim 20 wherein said neurological condition is characterized by neuronal loss or dysfunction, 22. The method of claim 21 wherein said neurological condition is a member selected from the group consisting of Alzheimer's Disease, dementia pugilistica, Parkinson's Disease, Huntington's Disease, Niemann-Pick disease, multiple sclerosis, a neuropathy and an ischemic condition. 23. The method of claim 22 wherein said ischemic condition is an ischemic condition of the central, peripheral, or compression type. 24. The method of claim 22 wherein said ischemic condition is a member selected from the group consisting of stroke and cerebral artery infarction. 25. The method of claim 20 wherein said neurological condition is caused by a defect in myelin repair or production. 26. The method of claim 20 wherein said defect in myelin repair or production is caused by a process selected from the group consisting of demyelination, the removal of myelin debris following injury and remyelination. 27. The method of claim 14 wherein said agent was first identified as having beneficial activity using a method of claim 1. 28. A method for identifying an agent useful in regulating fertility in a mammal comprising: (a) administering to a mammal an, agent that modulates ABCA1 biological activity, and (b) detecting a change in fertility of said mammal following said administering and compared to when said agent is not administered, thereby identifying an agent useful in regulating fertility of said mammal. 29. The method of claim 28 wherein said agent inhibits ABCA1 biological activity. 30. The method of claim 28 wherein said agent enhances ABCA1 biological activity. 31. The method of claim 28 wherein said ABCA1 biological activity is ABCA1-gene expression. 32. The method of claim 28 wherein said ABCA1 biological activity is selected from the group consisting of HDL-cholesterol transport, ion transport, ATP binding, ATP hydrolysis, and phospholipid transport. 33. The method of claim 28 wherein modulation of ABCA1 biological activity is due to a process selected from the group consisting of a change in stability of ABCA1 polypeptide, a change in ABCA1 membrane insertion and a change in ABCA1 membrane channel formation. 34. The method of claim 28 wherein said agent increases fertility. 35. The method of claim 28 wherein said agent decreases fertility. 36. The method of claim 28 wherein said mammal is male. 37. The method of claim 34 wherein said mammal is male and said agent increases capacitation of a sperm cell of said male. 38. The method of claim 35 wherein said mammal is male and said agent decreases capacitation of a sperm cell of said male. 39. The method of claim 28 wherein said mammal is female. 40. The method of claim 39 wherein said agent increases fertility. 41. The method of claim 39 wherein said agent decreases fertility. 42. The method of claim 40 wherein said agent promotes endometrial implantation. 43. The method of claim 40 wherein said agent interferes with endometrial implantation. 44. A method for identifying an agent useful in regulating fertility in a male mammal comprising: (a) contacting a sperm cell of said mammal with an agent that modulates ABCA1 biological activity and under conditions promoting said contacting and supporting viability of said sperm cell, and (b) detecting a change in the ability of said sperm cell to fertilize an ovum of a mammal of the same species as compared to when said sperm cell is not so contacted, thereby identifying an agent useful in regulating fertility of said male mammal. 45. The method of claim 44 wherein said agent inhibits ABCA1 biological activity. 46. The method of claim 44 wherein said agent enhances ABCA1 biological activity. 47. The method of claim 44 wherein said ABCA1 biological activity is ABCA1-gene expression. 48. The method of claim 44 wherein said ABCA1 biological activity is selected from the group consisting of HDL-cholesterol transport, ion transport, ATP binding, ATP hydrolysis, and phospholipid transport. 49. The method of claim 44 wherein modulation of ABCA1 biological activity is due to a process selected from the group consisting of a change in stability of ABCA1 polypeptide, a change in ABCA1 membrane insertion and a change in ABCA1 membrane channel formation. 50. The method of claim 44 wherein said agent increases the ability of said sperm cell to fertilize said ovum. 51. The method of claim 50 wherein said increase is due to an increase in capacitation of said sperm cells. 52. The method of claim 44 wherein said agent decreases the ability of said sperm cell to fertilize said ovum. 53. The method of claim 52 wherein said decrease is due to a decrease in capacitation of said sperm cells. 54. A method for regulating fertility in a mammal comprising administering to said mammal an agent having fertility-regulating ability using a method of claim 28. 55. The method of claim 54 wherein said regulating is an increase in ABCA1-biological activity. 56. The method of claim 54 wherein said regulation is a decrease in ABCA1-biological activity. 57. The method of claim 54 wherein said agent was first identified as having fertility-regulating ability using a method of claim 54. 58. The method of claim 54 wherein said mammal is male. 59. The method of claim 58 wherein said agent modulates said fertility by modulating the fertility of the sperm cells of said male mammal. 60. The method of claim 54 wherein said mammal is female. 61. The method of claim 60 wherein said agent modulates said fertility by modulating implantation into the endometrium of said male mammal. 62. A method for modulating the ability of a sperm cell to fertilize an ovum of an animal of the same species as said sperm cell comprising contacting said sperm cell with an agent that exhibits fertility-regulating ability using a method of claim 44. 63. The method of claim 62 wherein said modulation is an increase in the ability of said sperm cell to fertilize said ovum. 64. The method of claim 62 wherein said modulation is a decrease in the ability of said sperm cell to fertilize said ovum. 65. The method of claim 64 wherein said agent decreases or prevents capacitation of said sperm cell. 66. The method of claim 62 wherein said modulation is an increase in the ability of said sperm cell to fertilize said ovum. 67. The method of claim 64 wherein said agent promotes capacitation of said sperm cell. 68. A method of treating preeclampsia in a mammal comprising administering to a mammal afflicted therewith an effective amount of an agent that exhibits fertility-regulating ability using a method of claim 28. 69. A method of preventing preeclampsia in a mammal comprising administering to a mammal at risk thereof an effective amount of an agent that exhibits fertility-regulating ability using a method of claim 28. 70. The method of claim 68 or 69 wherein said mammal is a human being. 71. The method of claim 68, 69 or 70 wherein said agent was first identified as having fertility-regulating activity using a method of claim 28. 72. A method for producing a product comprising identifying an agent according to the process of claim 1, 28 or 44 wherein said product is the data collected with respect to said agent as a result of said process and wherein said data is sufficient to convey the chemical structure and/or properties of said agent. 73. A screening method for identifying an agent useful in treating an ABCA1-dependent neurological condition comprising: (a) administering to an animal exhibiting said neurological condition an agent that modulates ABCA1 biological activity, (b) detecting a beneficial change in said neurological condition in said animal following said administering as compared to when said agent is not administered, and thereby identifying an agent useful in treating an ABCA1-dependent neurological condition. 74. The screening method of claim 73 wherein said ABCA1-modulating agent was shown to modulate ABCA1-biological activity prior to use in said screening method. 75. The screening method of claim 73 wherein said ABCA1-modulating agent was shown to modulate ABCA1-biological activity after use in said screening method. 76. A method of treating an animal for an ABCA1-dependent neurological condition comprising administering to an animal afflicted with such condition an effective amount of an ABCA1-modulating agent. 77. The method of claim 76 wherein said agent has activity using a screening method of claim 73. 78. The method of claim 76 wherein said agent was first identified as useful in treating said neurological condition using a screening method of claim 73. 79. The method of claim 76 wherein said agent was not shown to have ABCA1-modulating activity prior to use in said treating method. 80. The method of claim 76 wherein said agent was shown to have ABCA1-modulating activity prior to use in said treating method. 81. A screening method for identifying an agent useful in negating the malfunctioning of a nervous system cell comprising: (a) contacting a malfunctioning nervous system cell, wherein said malfunctioning promotes the presence of said neurological condition, with an ABCA1-modulating agent under conditions promoting said contacting and otherwise supporting the normal functioning of said cell, (b) determining a beneficial change in one or more functions of said cell after said contacting wherein said beneficial change is not determined when said contacting does not occur, and thereby identifying an agent useful in negating malfunctioning of a neurological condition. 82. A screening method for identifying an agent useful in treating an ABCA1-dependent neurological condition comprising: (a) contacting a malfunctioning nervous system cell, wherein said malfunctioning promotes the presence of said neurological condition, with an ABCA1-modulating agent under conditions promoting said contacting and otherwise supporting the normal functioning of said cell, (b) determining a beneficial change in one or more functions of said cell after said contacting wherein said beneficial change is not determined when said contacting does not occur, and thereby identifying an agent useful in treating said neurological condition. 83. A screening method for identifying an agent useful in promoting myelin production in a connective tissue cell whose normal function includes myelin production, comprising: (a) contacting said connective tissue cell with an ABCA1-modulating agent under conditions promoting said contacting and otherwise supporting myelin production by said cell, (b) determining an increase in myelin production by said cell after said contacting wherein said increase is not determined when said contacting does not occur, and thereby identifying an agent useful in promoting myelin production by said cell. 84. The screening method of claim 83 wherein the cell of step (a) is deficient in myelin production. 85. The screening method of claim 83 wherein said cell is found in the central nervous system or the peripheral nervous system. 86. The method of claim 83 wherein said cell is a Schwann cell of an oligodendrocyte. 87. The screening method of claim 81, 82 or 83 wherein said contacting occurs in vitro. 88. The screening method of claim 81, 82 or 83 wherein said contacting occurs in vivo. 89. The screening method of claim 81, 82 or 83 wherein said ABCA1-modulating agent was shown to modulate ABCA1-biological prior to use in said screening method. 90. The screening method of claim 81, 82 or 83 wherein said ABCA1-modulating agent was not shown to modulate ABCA1-biological prior to use in said screening method. 91. A method of treating a neurological condition in an animal comprising administering to an animal afflicted with said condition an effective amount of an agent first identified as having therapeutic activity using a screening method of claim 81, 82 or 83. 92. A method for identifying an ABCA1-related cause of reduced fertility in a male patient afflicted with said reduced fertility comprising identifying in one or more sperm cells from said patient a reduced ABCA1-biological activity relative to a sperm cell from a patient without said ABCA1-related infertility. 93. The method of claim 92 wherein said reduced ABCA1-biological activity is a decrease in activity of an ABCA1-polypeptide in said one or more sperm cells. 94. The method of claim 92 wherein said reduced ABCA1-biological activity is a decreased amount of ABCA1-polypeptide in said one or more sperm cells. 95. The method of claim 92 wherein said reduced ABCA1-biological activity is a decreased expression of an ABCA1 gene in said one or more sperm cells. 96. The method of claim 95 wherein said decreased expression is due to a polymorphism in a promoter or other non-coding region of said ABCA1 gene. 97. The method of claim 92 wherein said reduced ABCA1-biological activity is due to a polymorphism in a coding region of an ABCA1 gene in said one or more sperm cells. 98. A method for identifying a male patient afflicted with reduced fertility as a candidate for treatment of said reduced fertility using an ABCA1-modulating agent comprising identifying in said male patient a reduced amount of ABCA1-biological activity using the method of claim 92. 99. The method of claim 98 wherein said ABCA1-modulator is a positive modulator of ABCA1-biological activity. 100. The method of claim 98 wherein said ABCA1-modulating agent was previously shown to modulate ABCA1-biological activity. 101. The method of claim 98 wherein said ABCA1-modulating agent was not previously shown to modulate ABCA1-biological activity. 102. A method for preventing capacitation of sperm cells during freezing and/or storage comprising freezing and/or storing sperm cells in a composition comprising an inhibitor of ABCA1 biological activity, thereby preventing or retarding capacitation during such freezing and/or storing. 103. A method for facilitating a process requiring sperm fertility, comprising addition of a positive ABCA1 modulator to a sample of sperm cells either not capacitated, or inadequately capacitated, or known to be comprised as to capacitation. 104. The method of claim 102 wherein said process to be facilitated is in vitro fertilization. 105. The method of claim 102 wherein said not capacitated, or inadequately capacitated, or known to be comprised as to capacitation is due to freezing and/or storage of said sperm cells. 106. A method to identify a therapeutic agent for modulating fertility in a mammal comprising (a) providing an assay which measures a biological activity of ABCA1, (b) contacting said assay with a compound, and (c) measuring whether said compound modulates said biological activity of ABCA1, wherein a compound which modulates said biological activity of ABCA1 is thereby identified as said therapeutic agent for modulating fertility in a mammal. 107. The method of claim 106 wherein said therapeutic agent is a contraceptive. 108. The method of claim 106 wherein said therapeutic agent promotes male fertility. 109. A method of modulating fertility in a mammal comprising administering to said mammal a compound which modulates the biological activity of ABCA1. 110. The method of claim 109 wherein said compound was identified according to the method of claim 105. 111. The method of claim 106 wherein said therapeutic agent enhances an ABCA1-dependent biological activity. 112. The method of claim 106 wherein said therapeutic agent inhibits an ABCA1-dependent biological activity. 113. A method to identify a therapeutic agent for treating a neurological disease or disorder in a mammal comprising (a) providing an assay which measures a biological activity of ABCA1, (b) contacting said assay with a compound, and (c) measuring whether said compound modulates said biological activity of ABCA1, wherein a compound which modulates said biological activity of ABCA1 is thereby identified as said therapeutic agent for treating a neurological disease or disorder in a mammal. 114. The method of claim 113 wherein said therapeutic agent enhances an ABCA1-dependent biological activity. 115. The method of claim 113 wherein said therapeutic agent inhibits an ABCA1-dependent biological activity. 116. A method of treating a neurological disease or disorder in a mammal comprising administering to said mammal a compound which modulates the biological activity of ABCA1. 117. The method of claim 116 wherein said compound was identified according to the method of claim 113. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Regulation of lipid homeostasis is an essential process for many cellular and bodily functions, and is controlled to a large extent by the regulated transport of cholesterol and phospholipids carried on lipoproteins. Almost all of the lipoproteins are formed in the liver, which is where most of the plasma cholesterol, phospholipids and triglycerides (except those obtained from dietary sources) are synthesized. Large quantities of phospholipids and cholesterol are present in both the plasma and internal cellular membranes of all cells, and the ratio of cholesterol to phospholipids is a critical determinant of membrane fluidity. Formation of membranes is critically dependent on the transport of the appropriate lipids in the form of lipoprotein particles. The primary function of the lipoproteins is to transport their lipid components in the blood. Aberrant regulation of lipid and lipoprotein homeostasis can lead to a variety of diseases including atherosclerosis. Major risk factors for atherosclerosis include elevated plasma LDL-cholesterol (LDL-C) and depressed HDL-cholesterol (HDL-C) levels. ABCA1 has recently been identified as a key regulator in the formation of HDL particles, acting as a crucial transporter of cholesterol and phosopholipids from cells to ApoA1. Mutations in the ABCA1 gene have been identified as the underlying cause of Tangier Disease (TD) and of a dominantly inherited form of hypoalphalipoproteinemia (FHA) associated with greatly reduced efflux of cholesterol and phospholipid to ApoA1. TD is a rare form of genetic HDL-C deficiency in which patients are homozygous or compound heterozygous for mutations in both alleles of ABCA1, resulting in nearly undetectable ApoA1-mediated cholesterol efflux and virtually no circulating HDL-C. The inability to remove excess cholesterol from peripheral tissues of TD patients often results in clinical manifestations of orange tonsils, hepatosplenomegaly, peripheral neuropathy, and premature coronary artery disease (CAD). TD patients are also observed to have small families, suggesting that aspects of reproductive fitness may be an additional effect of mutations with in ABCA1. Persons heterozygous for ABCA1 mutations have FHA, a less severe clinical outcome of deficient cholesterol efflux characterized with circulating HDL-C levels at or below the 5 th percentile for an age- and sex-matched population. ABCA1 belongs to the ATP-Binding-Cassette family of transporters and is a member of the full-size class A group of transporters. This highly conserved family of proteins mediates the ATP-dependent unidirectional transmembrane transport of numerous substrates. Full-size transporters consist of tandemly arranged ATP binding cassettes and two transmembrane domains consisting of 12 membrane spanning segments, whereas half-size transporters form dimers each containing one ATP-binding cassette and one transmembrane domain. ABC transporters have been localized to the plasma membrane, Golgi apparatus, endoplasmic reticulum, peroxisomes, and intracellular secretory vesicles. The vital role ABC transporters play in transport is underscored by the many human diseases resulting from ABC mutations including cystic fibrosis, Stardgardt disease, retinitis pigmentosa, cone-rod dystrophy, age-related macular degeneration, progressive familial intrahepatic cholestasis, Dubin-Johnson syndrome, adrenoleukodystrophy, and pseudoxanthoma elasticum. ABCA1 generates HDL by transferring cholesterol onto apoprotein-A1 (apoA1) in a process known as cholesterol efflux, which is also an essential component of male fertility. Sperm are made in the testes and released into the epididymis, where they await ejaculation into the female reproductive tract. Although differentiated, epididymal sperm are not capable of fertilization and must first complete their maturation in the female reproductive tract. This maturation process is known as capacitation and is known to require efflux of cholesterol from the sperm plasma membrane. How this occurs at the molecular level is not well understood. The present invention relies in part on the discovery that ABCA1 is highly expressed in the testes (being present in Sertoli and Leydig cells as well as in differentiating spermatids). ABCA1-deficient mice develop highly vacuolated and largely aspermic seminiferous tubules, indicating that ABCA1 is necessary for normal spermatogenesis. In accordance with the present invention, ABCA1 is also present in epididymal sperm where it can act to transport cholesterol during capacitation. In vivo, follicular HDL is an endogenous cholesterol acceptor for capacitating sperm, and HDL can stimulate capacitation in vitro. In accordance with the present invention, because cholesterol efflux through ABCA1 is dependent upon apoA1 the ability of apoA1 to stimulate capacitation of murine sperm in vitro was tested using a chlortetracycline assay. The results showed that de-lipidated apoA1 capacitates sperm in a dose-dependent manner, thereby showing ABCA1 to be a key transporter of cholesterol for capacitating sperm implicating it as a molecular link between cholesterol metabolism in the periphery and reproductive systems. The brain is the most cholesterol rich organ in the body, underscoring the essential nature of lipid homeostasis for brain physiology. Neuronal function in both the central and peripheral nervous systems depends heavily on appropriate regulation of membrane structure and composition. Membranes are needed for the plasma membrane itself, formation of synaptic vesicles, vesicular transport, and endocytosis and exocytosis of vesicles. As well, lipids are salvaged and recycled during synaptic remodeling, following neuronal lesions including neuroinflammatory reactions, and the ability to uptake and efflux lipids plays very important roles in these processes. The neuronal plasma membrane is highly compartmentalized, and specific regions of the membrane have very different protein compositions and functions, and it is not unreasonable to propose that the lipid composition of the different portions of the neuronal plasma membrane may vary as well. Virtually all functions of the neuron depend on membranes at some level. The high level of ABCA1 in the brain is consistent with multiple roles in aspects of lipid and lipoprotein metabolism in the brain. Because the brain and testis have highly developed membrane systems with functions requiring a tightly regulated membrane environment, the development of drugs for the treatment of infertility and neurological disorders has so far progressed slowly. The present invention solves this problem by identifying ABCA1 as a key molecule in regulating essential neurological- and fertility-related processes, thereby providing a means of screening agents for ability to advantageously affect such processes while simultaneously limiting the pool of compounds to be screened to those already known to be modulators of ABCA1. |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>In one aspect, the present invention relates to a method for identifying an agent useful in treating an ABCA1-dependent neurological condition comprising: (a) administering to an animal exhibiting said neurological condition an agent that modulates ABCA1 biological activity, and (b) detecting a beneficial change in said neurological condition in said animal following said administering and compared to when said agent is not administered, thereby identifying an agent useful in treating said ABCA1-dependent neurological condition. Such an agent is selected from those that either enhance or inhibit ABCA1 biological activity. In preferred embodiments, the ABCA1 biological activity affected by such agent may include ABCA1-gene expression and be some activity of the ABCA1-polypeptide, such as HDL-cholesterol transport, ion transport, ATP binding, ATP hydrolysis, and/or phospholipid transport. In other preferred embodiments, this may include ABCA1 biological activity that is due to a change in stability of ABCA1 polypeptide, a change in ABCA1 membrane insertion and a change in ABCA1 membrane channel formation. In other preferred embodiments, the neurological condition being screened for is a neurological condition of the central nervous system or of the peripheral nervous system and such neurological condition may be characterized by neuronal loss or dysfunction. In specific embodiments, the neurological condition is one or more of Alzheimer's Disease, dementia pugilistica, Parkinson's Disease, Huntington's Disease, Niemann-Pick disease, multiple sclerosis, a neuropathy and an ischemic condition. In a preferred embodiment, where the neurological condition involves an ischemic condition, this ischemic condition may be of the central, peripheral, or compression type. In specific embodiments, this ischemic condition is one of stroke and/or cerebral artery infarction. In other preferred embodiments, the neurological condition is caused by a defect in myelin repair or production, especially where said defect in myelin repair or production is caused by a process selected from demyelination, the removal of myelin debris following injury and remyelination. In another aspect, the present invention relates to a method of treating a neurological condition comprising administering to an animal afflicted with such condition an effective amount of an agent exhibiting beneficial activity using a screening method as disclosed herein, with similar embodiments as described herein. In one preferred embodiment, the agent used for such treatment is one that was initially identified as having beneficial activity using a screening method as disclosed herein. In an additional aspect the present invention relates to a method for identifying an agent useful in regulating fertility in a mammal comprising: (a) administering to a mammal an agent that modulates ABCA1 biological activity, and (b) detecting a change in fertility of said mammal following said administering and compared to when said agent is not administered, thereby identifying an agent useful in regulating fertility of said mammal. In specific embodiments, such agent may inhibit or enhance the activity of ABCA1 biological activity, wherein the latter may include ABCA1-gene expression or activities of the ABCA1-polypeptide, such as HDL-cholesterol transport, ion transport, ATP binding, ATP hydrolysis, and/or phospholipid transport, and also may include agents that modulate other activities of ABCA1, such as a change in stability of ABCA1 polypeptide, a change in ABCA1 membrane insertion and a change in ABCA1 membrane channel formation. Such agents may have the effect of either increasing or decreasing fertility in an animal, especially a mammal, most especially a human being. In one such embodiment, the mammal is male and ABCA1-modulating agent has the effect of increasing spermatogenesis and/or capacitation of one or more sperm cells of this male and thereby increase fertility. In a preferred embodiment of the latter effect, the agent is a positive modulator of one or more of the functions of ABCA1 genes or proteins and thus has the effect of enhancing ABCA1-biological activity. In another such embodiment, the mammal is male and the agent decreases spermatogenesis and/or capacitation of one or more sperm cells of this male, thereby reducing fertility. In a preferred embodiment of the latter effect, the agent is a negative modulator of one or more of the functions of ABCA1 genes or proteins and thus has the effect of decreasing ABCA1-biological activity. In another such embodiment, the agent decreases capacitation of one or more sperm cells collected from a male mammal, thereby protecting said sperm cells from the long-term decrease in fertilization potential by cryopreservation. In another such embodiment, the agent increases capacitation of one or more cyropreserved sperm cells originating from a male mammal, thereby increasing the fertilization potential of cryopreserved sperm. In another such embodiment, the mammal is female and the agent may increase fertility or decrease fertility. In one such embodiment, the ABCA1-modulating agent promotes implantation into the endometrium, thereby increasing fertility. In a preferred embodiment of the latter process, the agent is a positive modulator of ABCA1 gene expression or polypeptide activity and thus inhibits ABCA1-biological activity. In another such embodiment, the ABCA1-modulating agent inhibits implantation in the endometrium, thereby decreasing fertility. In a preferred embodiment of the latter process, the agent is a negative modulator of ABCA1 gene expression or polypeptide activity and thus inhibits ABCA1-biological activity. In a further aspect, the present invention relates to a method for identifying an agent useful in regulating fertility in a male mammal comprising: (a) contacting a sperm cell of said mammal with an agent that modulates ABCA1 biological activity and under conditions promoting said contacting and supporting viability of said sperm cell, and (b) detecting a change in the ability of said sperm cell to fertilize an ovum of a mammal of the same species as compared to when said sperm cell is not so contacted, thereby identifying an agent useful in regulating fertility of said male mammal. In specific embodiments thereof, the agent may inhibit ABCA1 biological activity or may enhance ABCA1 biological activity, which activity may include gene expression or any of the functions of the ABCA1-polypeptide, including HDL-cholesterol transport, ion transport, ATP binding, ATP hydrolysis, and/or phospholipid transport, or may include effects on stability of ABCA1 polypeptide, a change in ABCA1 membrane insertion and a change in ABCA1 membrane channel formation. Such agents may increase fertility by increasing the ability of said sperm cell to fertilize said ovum. In a specific embodiment, this increase is due to an increase in capacitation of the sperm cell(s), such as where the agent is a positive modulator of ABCA1-biological activity. Alternatively, the agent may decrease the ability of said sperm cell to fertilize said ovum, especially where this decrease is due to a decrease in capacitation of said sperm cells, such as where the agent is a negative modulator of ABCA1 biological activity. In a still further aspect, the present invention relates to a method for regulating fertility in a mammal comprising administering to said mammal an agent having fertility-regulating ability in one or more of the screening assays disclosed herein, especially where said agent was first identified as having such physiological activity using said screening assay. Where said mammal is male, the agent will preferably modulate fertility by modulating the fertility of the sperm cells of said male mammal, especially where the agent has the effect of regulating capacitation of the sperm cells of said male mammal. Where the mammal is female, the agent preferably modulates fertility by modulating implantation into the endometrium of said female mammal. In a still further aspect, the present invention relates to a method for modulating the ability of a sperm cell to fertilize an ovum of an animal of the same species as said sperm cell comprising contacting said sperm cell with an agent that exhibits fertility-regulating ability using a screening method as disclosed herein. In a preferred embodiment, said modulation is an increase in the ability of said sperm cell to fertilize said ovum, especially where this agent acts to promote capacitation of said sperm cells. In an alternative embodiment, said modulation is a decrease in the ability of the sperm cell to fertilize the ovum, especially where this agent acts to inhibit, or prevent, capacitation of said sperm cells. In a yet still further aspect, the present invention provides a method of treating adverse conditions related to fertility processes, such as adverse conditions arising from pregnancy, especially conditions such as preeclampsia, involving such processes as hypertension and edema resulting from, or related to, pregnancy. In a preferred embodiment of such processes, the present invention provides a method of treating preeclampsia in a mammal comprising administering to a mammal afflicted therewith an effective amount of an agent that exhibits fertility-regulating ability using a method of claim 28 - 43 . In another such preferred embodiment, the present invention relates to a method of preventing preeclampsia in a mammal comprising administering to a mammal at risk thereof an effective amount of an agent that exhibits fertility-regulating ability using a screening method disclosed herein, most preferably where said method was used to first identify such agent as having this activity. In a preferred embodiment, the mammal to be treated is a human being. The present invention also relates to such screening methods wherein the identification of such an agent is itself a step of the procedure. In all of the screening methods of the invention, which may be either in vitro or in vivo methods, the ABCA1-modulating agent may or may not have been previously shown to modulate ABCA1-biological activity prior to use in the screening method. Thus, a compound may be screened that was not previously known to be such a modulator but the method of determining its ability to modulate ABCA1 may itself be part of the screen. Thus, the present invention also relates to a method of treating an animal for an ABCA1-dependent neurological condition comprising administering to an animal afflicted with such condition an effective amount of an ABCA1-modulating agent, preferably where the agent has activity using a screening method of the invention, most preferably where the agent was first identified as useful in treating said neurological condition using such screening method. In other preferred embodiments, the ABCA1-modulating agent used in the treatment had been previously shown to have ABCA1-modulating activity. Alternatively, it may not have been previously shown to have such activity. In a further aspect, the present invention relates to a screening method for identifying an agent useful in negating the malfunctioning of a nervous system cell comprising: (a) contacting a malfunctioning nervous system cell, wherein said malfunctioning promotes the presence of said neurological condition, with an ABCA1-modulating agent under conditions promoting said contacting and otherwise supporting the normal functioning of said cell, (b) determining a beneficial change in one or more functions of said cell after said contacting wherein said beneficial change is not determined when said contacting does not occur, and thereby identifying an agent useful in negating malfunctioning of a neurological condition. The present invention also relates to a screening method for identifying an agent useful in treating an ABCA1-dependent neurological condition comprising: (a) contacting a malfunctioning nervous system cell, wherein said malfunctioning promotes the presence of said neurological condition, with an ABCA1-modulating agent under conditions promoting said contacting and otherwise supporting the normal functioning of said cell, (b) determining a beneficial change in one or more functions of said cell after said contacting wherein said beneficial change is not determined when said contacting does not occur, and thereby identifying an agent useful in treating said neurological condition. The present invention also relates to a screening method for identifying an agent useful in promoting myelin production in a connective tissue cell whose normal function includes myelin production, comprising: (a) contacting said connective tissue cell with an ABCA1-modulating agent under conditions promoting said contacting and otherwise supporting myelin production by said cell, (b) determining an increase in myelin production by said cell after said contacting wherein said increase is not determined when said contacting does not occur, and thereby identifying an agent useful in promoting myelin production by said cell. In a preferred embodiment of the latter method the cell of step (a) is deficient in myelin production, such as where the cell is found in the central nervous system or the peripheral nervous system, most preferably wherein said cell is a Schwann cell of an oligodendrocyte. In other preferred embodiments, the contacting occurs in vitro or occurs in vivo. In a preferred embodiment of any of these screening methods, the ABCA1-modulating agent may have been previously shown to modulate ABCA1-biological prior to use in the screening method. Conversely, the agent may be novel and not previously shown to do so. The present invention preferably relates to a method of treating a neurological condition in an animal comprising administering to an animal afflicted with said condition an effective amount of an agent first identified as having therapeutic activity using a screening method of the invention. In another aspect, the present invention relates to a method for identifying an ABCA1-related cause of reduced fertility in a male patient afflicted with said reduced fertility comprising identifying in one or more sperm cells from said patient a reduced ABCA1-biological activity relative to a sperm cell from a patient without said ABCA1-related infertility. In a preferred embodiment, the reduced ABCA1-biological activity is a decrease in activity of an ABCA1-polypeptide in said one or more sperm cells, and/or a decreased amount of ABCA1-polypeptide in said one or more sperm cells, and/or decreased expression of an ABCA1 gene in said one or more sperm cells and/or the decreased expression is due to a polymorphism in a promoter or other non-coding region of said ABCA1 gene and/or the reduced ABCA1-biological activity is due to a polymorphism in a coding region of an ABCA1 gene in said one or more sperm cells. The present invention also relates to a method for identifying a male patient afflicted with reduced fertility as a candidate for treatment of said reduced fertility using an ABCA1-modulating agent comprising identifying in said male patient a reduced amount of ABCA1-biological activity using a screening method of the invention. In separate embodiments, the ABCA1-modulator is a positive modulator of ABCA1-biological activity and/or was previously shown to modulate ABCA1-biological activity. The present invention still further relates to a method of preventing capacitation of sperm cells during freezing and/or cryopreservation comprising preserving sperm cells by freezing/storing in a composition comprising a modulator, preferably a negative modulator, of ABCA1 biological activity, thereby preventing capacitation during such cryopreservation. The present invention yet further provides a method for facilitating processes requiring sperm fertility, such as in vitro fertilization, comprising addition of an ABCA1 modulator, preferably a positive modulator, to a sample of sperm cells either not capacitated, or inadequately capacitated, or known to be comprised as to capacitation, preferably following some form of cryopreservation, or other freezing and/or storage process, thereby enhancing and/or restoring the capacitation status of said sperm cells. |
Process for the production of a titanium alloy based composite material reinforced with titanium carbide, and reinforced composite material obtained thereby |
Object of the present invention is a process for the production of a Titanium alloy based composite material with satisfactory mechanical features at high temperature, characterised in that Titanium alloy powders and Titanium carbide powders are blended, hot-pressed and hot-rolled or extruded. The invention also encompasses a composite material obtainable with said process. |
1. A process for the production of a Titanium alloy based composite material having satisfactory mechanical features at high temperature, in which a Titanium alloy powder and a Titanium carbide powder are blended, hot-compacted and hot-rolled, or extruded, characterised in that the hot compacting is obtained by isostatic hot pressing at a temperature ranging from 850 to 950° C., at a pressure ranging from 80 to 130 MPa, for a time less than four hours, and the resulting material is heated to a temperature of about 1000° C. and pressed to provide a thickness reduction of from 5 to 50%. 2. The process for the production of a Titanium alloy based composite material having satisfactory mechanical features at high temperature according to claim 1, wherein the concentration of the Titanium carbide expressed in percent by weight ranges from 0.5 to 30%. 3. The process for the production of a Titanium alloy based composite material having satisfactory mechanical features at high temperature according to claim 1, wherein the particle size of the Titanium alloy is less than 250 μm. 4. The process for the production of a Titanium alloy based composite material having satisfactory mechanical features at high temperature according to claim 3, wherein the particle size of Titanium carbide is less than 5 μm. 5. The process for the production of a Titanium alloy based composite material having satisfactory mechanical features at high temperature according to claim 1, wherein the blending of the said two powders is carried out in the presence of 50% by volume acetone or of an anti-clumping agent, optionally added separately to each of the powders to be blended. 6. The process for the production of a Titanium alloy based composite material having satisfactory mechanical features at high temperature according to claim 1, wherein the blending of the two powders is carried out under inert gas, preferably Argon, atmosphere. 7. The process for the production of a Titanium alloy based composite material having satisfactory mechanical features at high temperature according to claim 6, wherein the blending is obtained by revolving a vessel, containing the two powders, at a high rate for a time ranging from 5 minutes to 8 hours. 8. The process for the production of a Titanium alloy based composite material having satisfactory mechanical features at high temperature according to claim 7, wherein the blended powders are dried under vacuum. 9. The process for the production of a Titanium alloy based composite material having satisfactory mechanical features at high temperature according to any of the preceding claims, wherein the resulting compound is hot-rolled in the range from 800 to 1000° C. with less than 5% reduction passages, until the desired total thickness is achieved. 10. The process for the production of a Titanium alloy based composite material having satisfactory mechanical features at high temperature according to claim 9, wherein the total thickness reduction is of about 80%. 11. A Titanium alloy based composite material reinforced with Titanium carbide, as obtained by the process of claim 1. |
Benzoisoselenazole derivatives with anti-inflammation, antivirus and antithrombosis activity and their use |
The present invention relates to new bisbenzisoselenazolonyl derivatives of the following general formulae (I), (II) or (III) and their pharmaceutically acceptable salts. The inventive derivatives have antineoplastic, anti-inflammatory and antithrombotic activities. |
1. Bisbenzisoselenazolonyl derivatives of general formulae (I)), (II) or (III) and their pharmaceutically acceptable salts; wherein: R is C1-6-alkylene, phenylidene, biphenyliden, triphenylidene, or the following group: wherein: M=Pt Pd or Rh; R′ is a saccharide residue or the following group: wherein R″ is Cl, H2O, OH, Br, or I, R′″ is —H , —CH2C6H5OH, —C2OH, —CH2CONH2, —CH2CH2COOH —CH2(CH2)4NH2, —CH2COOH, —CH2CH2CONH2, —(CH2)3CH, —(CH2)3NHC(NH)NH2, —(CH2)3CHCH2, —CH3, —CH2CH3, —CH2C6H5, —CH2SH, Or —CH2CH2SCH3, R″″ is —H, —CH2C6H5OH, —CH2OH, —CH2CONH2, —CH2CH2COOH —CH2(CH2)4NH2, —CH2COOH, —CH2CH2CONH2, —(CH2)3CH, —CH2)3NHC(NH)NH2, —(CH2)3CHCH2, —CH3, —CH2CH3, —CH2C6H5, —CH2SH, or —CH2CH2SCH30. 2. The bisbenzisoselenazolonyl derivatives according to claim 1, wherein R is C1-6-alkylene. 3. The bisbenzisoselenazolonyl derivatives according to claim 2, wherein R is ethylene. 4. The bisbenzisoselenazolonyl derivatives according to 1, wherein R is a phenylidene or biphenylidene group 5. The bisbenzisoselenazolonyl derivatives according to claim 4, wherein R is a biphenylidene group. 6. The bisbenzisoselenazolonyl derivatives according to claim 1, wherein R′ is a 1,3,4,6tetra-O-acetyl-2deoxy-D-glucopyranosyl group. 7. The bisbenzisoselenazolonyl derivatives according to claim 1, wherein R′″ and R″″ are independently —H, —CH3, —CH2CH3, —CH2C6H5, —CH2SH or —CH2CH2SCH3. 8. A pharmaceutical composition comprising as an active ingredient the. bisbenzisoselenazolonyl derivatives of general formulae (I), (II) or (III) according to claim 1 or their pharmaceutically acceptable salts and pharmaceutically acceptable excipient or carrier. 9. The pharmaceutical composition according to claim 8, further comprising other anti-inflammatory or antineoplastic agent or anti-thrombotics. 10. The pharmaceutical composition according to claim 9, wherein the other antineoplastic agent includes cisplatin, adriamycin, taxol or their combinations. 11. The pharmaceutical composition according to claim 9, wherein the other anti-inflammatory agent includes aspirin, indomethacin or their combinations. 12. The pharmaceutical composition according to claim 9, wherein the other anti-thrombotics includes aspirin. 13. Use of the bisbenzisoselenazolonyl derivatives of general formulae (I), (II) or (III) according to claim 1 or their pharmaceutically acceptable salt in manufacturing a medicine for the treatment of cancer and inflammatory diseases or preventing thrombosis. 14. A method for treating inflammatory and cancer diseases or preventing thrombosis in ma including human being, comprising the step of administering therapeutically effective dosage of the bisbenzisoselenazolonyl derivatives of general formulae (I), (II) or (III) according to claim 1 or their pharmaceutically acceptable salts to the patients in need of treatment. 15. A method for treating inflammatory and cancer diseases or preventing thrombosis in mammal including human being, comprising the step of administering therapeutically effective dosage of the bisbenzisoselenazolonyl derivatives of general formulae (I), (II) or (III) according to claim 1 or their pharmaceutically acceptable salts in combination with other anti-inflammatory or antineoplastic agents or anti-thrombotics to the patients in need of treatment 16. The method according to claim 15, wherein the bisbenzisoselenazolonyl derivatives of general formulae (I), (II) or (III) or their pharmaceutically acceptable salts and the other anti-inflammatory or antineoplastic agents or anti-thrombotics are administered at the same time. 17. The method according to claim 15, wherein the bisbenzisoselenazolonyl derivatives of general formulae (I), (II) or (III) or their pharmaceutically acceptable salts are administered firstly, and then the other anti-inflammatory or antineoplastic agents or anti-thrombotics. 18. The method according to claim 15, wherein the other anti-inflammatory or antineoplastic agents or anti-thrombotics are administered firstly, and then the bisbenzisoselenazolonyl derivatives of general formulae (I), (II) or (III) or their pharmaceutically acceptable salts. 19. The method according to any one of claims 15 to 18, wherein the other antineoplastic agent includes cisplatin, adriamycin, taxol or their combinations. 20. The method according to any one of claims 15 to 18, wherein the other anti-inflammatory agent includes aspirin, indomethacin or their combinations. 21. The method according to any one of claims 15 to 18, wherein the other anti-thrombotics includes aspirin. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Many researches focus on the remedy containing seleniun because the selenium element has important functions in biologic body. But problem is inorganic seleniun is difficult to absorb, and keeps a short time in blood, low activity and high toxicity. Compared with the characteristics of inorganic selenium, those of organoselenium compound have been improved very much. Selenium is an important trace element. Deficiencies of selenium (<0.1 ppm) for a long time may induce various diseases, including hepatonecrosis, cardiac muscle injury, cancer, and rheumatic diseases. So far, it has been known that Benzisoselenazolones (BISA), functioning in a GSH-Px-like way, inhibit in vitro the lipid peroxidation of microsome and have an effect in preventing the body from the peroxidation injuries. 2-phenyl-(1,2)-benzisoselenazol-3(2H)-on (Ebselen) of the following formula is the best one of GSH-Px-like compounds with high anti-oxidative activity and low toxicity (LD50>6810 mg/Kg, mice): Many researches are concentrated on modifying ebselen to improve its antineoplastic activity, but no successful antineoplastic active compound based thereon is reported up to now. Therefore, the object of the present invention is to modify ebselen to form new bisbenzisoselenazolonyl derivatives having higher anti-inflammatory activity, broader compatibility and lower toxicity. Meanwhile, antineoplastic organoselenium compounds having “biological response regulator” characteristic are obtained through modifying ebselen. |
<SOH> SUMMARY OF THE INVENTION <EOH>According to one aspect of the invention, there is provided bisbenzisoselenazolonyl derivatives of general formulae (I)), (II) or (III) and their pharmaceutically acceptable salts: wherein: R is C 1-6 -alkylene, phenylidene, biphenylidene, triphenylidene, or the following group: wherein: M=Pt, Pd or Rh; R′ is a saccharide residue or the following group: wherein: R″ is Cl, H 2 O, OH, Br, or I, R′″ is —H, —CH 2 C 6 H 5 OH, —CH 2 OH, —CH 2 CONH 2 , —CH 2 CH 2 COOH —CH 2 (CH 2 ) 4 NH 2 , —CH 2 COOH, —CH 2 CH 2 CONH 2 , —(CH 2 ) 3 CH, —(CH 2 ) 3 NHC(NH)NH 2 , —(CH 2 ) 3 CHCH 2 , —CH 3 , —CH 2 CH 3 , —CH 2 C 6 H 5 , —CH 2 SH, Or —CH 2 CH 2 SCH 3 , R″″ is —H, —CH 2 C 6 H 5 OH, —CH 2 OH, —CH 2 CONH 2 , —CH 2 CH 2 COOH —CH 2 (CH 2 ) 4 NH 2 , —CH 2 COOH, —CH 2 CH 2 CONH 2 , —(CH 2 ) 3 CH, —CH 2 ) 3 NHC(NH)NH 2 , —CH 2 ) 3 CHCH 2 , —CH 3 , —CH 2 CH 3 , —CH 2 C 6 H 5 , —CH 2 SH, or —CH 2 CH 2 SCH 3 . According to another aspect of the present invention, there is provided a pharmaceutical composition comprising as an active ingredient the above compounds (I), (II) or (III) or their pharmaceutically acceptable salts and any pharmaceutically acceptable excipient or carrier. According to still another aspect of the present invention, there is provided the use of the bisbenzisoselenazolonyl derivatives of general formulae (I), (II) or (III) or their pharmaceutically acceptable salt in manufacturing a medicine for the treatment of cancer and inflammatory diseases or preventing thrombosis. According to still another aspect of the present invention, there is provided a method for treating inflammatory and cancer diseases or preventing thrombosis in mammal including human being, comprising the step of administering therapeutically effective dosage of the bisbenzisoselenazolonyl derivatives of general formulae (I), (II) or (III) or their pharmaceutically acceptable salt to the patients in need of treatment. According to still another aspect of the present invention, there is provided a method for treating inflammatory and cancer diseases or preventing thrombosis in mammal including human being, comprising the step of administering therapeutically effective dosage of the bisbenzisoselenazolonyl derivatives of general formulae (I), (II) or (III) or their pharmaceutically acceptable salt in combination with other anti-inflammatory or antineoplastic agents to the patients in need of treatment. detailed-description description="Detailed Description" end="lead"? |
Pharmaceutical kit comprising anti-human seminal plasma protein single chain antibody/human carboxypeptidase fusion protein and prodrug |
This invention is in the field of antibody-directed enzyme prodrug therapy (ADEPT). More particularly, the invention relates to a kit comprising an anti-human γ-seminoprotein single-chain antibody (γ-Sm scFv)/human carboxypeptidase A(hCPA) fusion protein and a prodrug which is a methotrexate-α-peptide. The kit is useful in prostate cancer treatment with the ADEPT. The invention also relates to an anti-γ-seminoprotein single-chain antibody (γ-Sm scFv)/human carboxypeptidase (hCPA) fusion protein. |
1. A pharmaceutical kit for treating prostate cancer via the antibody-directed enzyme prodrug therapy, comprising several discrete containers containing, separately, a fusion protein of a human γ-seminoprotein single-chain antibody and human carboxypeptidase A (anti-γ-Sm scFv/hCPA fusion protein), a prodrug which is a methotrexate-α-peptide and pharmaceutically acceptable carriers, wherein the anti-γ-Sm scFv/hCPA fusion protein is administered at least 72 hours before the prodrug is administered. 2. The pharmaceutical kit according to claim 1, wherein said prodrug is methotrexate-α-phenylalanine. 3. A fusion protein of a human γ-seminoprotein single-chain antibody and human carboxypeptidase A, comprising an amino acid sequence as shown in FIG. 4. 4. A fusion gene encoding the fusion protein according to claim 3, comprising a nucleotide sequence as shown in FIG. 3. 5. Use of the pharmaceutical kit according to claim 1 in to the treatment of prostate cancer. |
<SOH> BACKGROUND OF INVENTION <EOH>Highly selective tumor chemotherapy is one of the important topics for tumor research at present. Most chemotherapeutic drugs in use are far from being highly selective towards tumor tissues. What is more, the drugs are toxic to normal tissues while taking effect in tumor tissues. Monoclonal antibodies have provided a foundation for selectively directed therapy of tumors. Despite the expectation that by means of monoclonal antibodies cytotoxic drugs can be directly carried towards tumor tissues, the toxic drugs cannot be concentrated within tumor sites as effectively as they are expected. This is due to several factors: 1) one antibody molecule can carry only a small amount of drug; 2) antigens in tumor tissues vary in their numbers; 3) it is relatively difficult for big antibody-drug complexes to access substantial tumor locations; 4) a host immune reaction to extrinsic antibodies may be stimulated. It was in such a situation, the antibody-directed enzyme prodrug therapy (ADEPT) proposed by Bagshawoe (Br J Cancer, 1987, 56(5): 531-532) aroused widespread concern in the field of chemotherapy. The strategy of the ADEPT is: by using a monoclonal antibody as a carrier, an enzyme which is specific for activating a prodrug (called activating enzyme) is selectively directed to a tumor site where it catalyzes the prodrug into activated cytotoxic molecules. This scenario can overcome many problems associated with chemical conjugates and immuno-toxins, since only a tiny amount of enzyme-antibody cross-linked complex is sufficient to convert a great lot of prodrug into activated toxic drug in the tumor site. In the first ADEPT proposed by Bagshawoe in 1987, a carboxypeptidase G2 gene was isolated from Bacillus pyocyaneus and cloned into E. coli . The enzyme thus produced in E. coli was initially employed to hydrolyze methtrexate and later was used to activate chlorethazine benzoate derivatives via removing their glutamic acid moiety. This is the most representative ADEPT strategy and has entered clinical trial on a small scale. The advantage of the strategy lies in that one enzyme molecule is able to convert many prodrug molecules. For example, one mole of carboxypeptidase G2 can degrade 800 moles of chlorethazine benzoate substrate in just one second and produce high concentration of active drug at tumor sites. This can compensate the lower affinity of immuno-conjugates in clinical applications. It has also been proved that high-concentration of drugs concentrated on the surface of tumor cells are more effective than the same concentration of active drugs which are systematically administered. Methotrexate (MTX) is an anti-metabolic drug that is widely used in clinical chemotherapy. However, it can inhibit normal cells while killing tumor cells and thus results in a serious toxic reaction. This has limited the clinical application of MTX at high concentration and large dose. As a result, the therapeutic effect of MTX is hindered. If an amino acid is linked to the α-carboxyl of the glutamate at the carboxyl terminal of MTX, MTX is converted to an MTX-α-peptide which has no toxicity to any cell. This compound can release cytotoxic MTX when the peptide bond at the carboxyl terminal is hydrolyzed by an appropriate carboxypeptidase. This result is desirable for the ADEPT strategy. There have been many rapid developments in the research and application of the ADEPT since it was first introduced by Bagshawoe in 1987. Some prodrugs have already entered Phase I clinical trial, however, the biggest obstacle to the in vivo application of the ADEPT goes to the heterology of a murine monoclonal antibody and/or the enzyme. Therefore, it is necessary to prepare a fusion protein of a humanized antibody and a human activating enzyme for better effects of the ADEPT. Heterology of antibody may be avoided by replacing a murine monoclonal antibody with an ScFv because ScFv only retains the variable region of an antibody that is responsible for the binding specificity of an antibody, and eliminates the constant region that has great diversity. ScFv takes up only ⅙ of an integral antibody and has good affinity and stability. The fusion protein of an ScFv and an activating enzyme is able to both bind specifically to tumor cells and hydrolyze the prodrug, with a dominant advantage that it can be prepared on a large scale and favorable for clinical application. Gama-Seminoprotein (γ-Sm) is a prostate-specific antigen secreted by prostate epithelium into semen, with a molecular weight of 23, 000-33,000. In the past it was used in forensic identification. Recently, by using histochemical staining method, it was found that γ-Sm is localized in both prostate cancer and their metastatic cells but not in other human tissues and malignant tumors. Just like other prostate-specific antigens (PSA), γ-Sm has been recognized as a specific marker for prostate cancer. Carboxypeptidase A (CP-A) is a carboxypeptidase secreted by some mammal tissues. The gene of CP-A has a total length of 1251 bp and encodes 417 amino acids. This enzyme can hydrolyze aliphatic and aromatic amino acids from the C terminal of a polypeptide successively. With this property, CP-A is the first enzyme that has been used in the ADEPT research. In view of above, starting from the anti-γ-seminoprotein single-chain antibody (γ-Sm scFv) which was constructed and expressed previously, the inventor has done further study on the treatment of prostate cancer using CP-A and methotrexate-α-peptides and finally accomplished the invention. Thus, it is an object of the invention to produce a pharmaceutical kit for the treatment of prostate cancer with the antibody-directed enzyme prodrug therapy (ADEPT). The kit comprises a fusion protein of anti-γ-seminoprotein single-chain antibody/human carboxypeptidase A, and a prodrug which is a methotrexate-α-peptide. Another object of the invention is to provide a fusion protein of anti-γ-seminoprotein single-chain antibody/human carboxypeptidase A (anti-γ-Sm scFv/hCPA) as well as a polynucleotide encoding the fusion protein. |
<SOH> SUMMARY OF THE INVENTION <EOH>In one aspect, the invention provides a pharmaceutical kit for the treatment of prostate cancer with the ADEPT. The kit comprises several containers which separately contains a fusion protein of an anti-γ-seminoprotein single-chain antibody and human carboxypeptidase A, a prodrug which is a methotrexate-α-peptide and pharmacologically acceptable carriers, wherein the fusion protein γ-Sm scFv/hCPA is administered at least 72 hours before the prodrug is administered. Preferably, the prodrug is methotrexate-α-phenylalanine. The amino acid sequence of the γ-Sm scFv/hCPA fusion protein is shown in FIG. 4 . In another aspect, the invention provides a fusion protein of an anti-γ-seminoprotein single-chain antibody and human carboxypeptidase A as well as a polynucleotide encoding the fusion protein. The amino acid sequence of the fusion protein is shown in FIG. 4 , while the polynucleotide sequence is shown in FIG. 3 . |
Optical frequency synthesizer |
Laser frequency locking apparatus (5), comprising: a slave laser (15), having associated with it means (14, 18) for coupling and/or means (18, 25) for coupling and propagating signals received and emitted; a phase lock loop (24); and a controller (16), operable to control the slave laser, wherein an output of a reference signal source (1) associated with a master source (2, 3), and receivable therefrom, is utilised in the phase lock loop to render the output frequency of the slave laser the same as an output frequency of the master source. The invention described relates to a technique for generating a set of highly stable optical frequency channels. There are provided methods and systems of locking laser frequencies and of synthesizing frequencies. |
1-33. (Cancelled) 34. Optical Frequency Synthesizer apparatus comprising an optical frequency comb generator, a single microwave reference source where the comb line frequency spacing is the same as the microwave reference source frequency, one of its harmonics or sub-harmonics, and a plurality of slave lasers whose principal output frequencies match selected comb line frequencies; locking of each laser being achieved using a control signal formed through mixing of the photo-detected heterodyne between a fraction of the slave laser principal output frequency and residual adjacent comb lines from the optical frequency comb generator, and the microwave reference source frequency, one of its harmonics or sub-harmonics. 35. Optical Frequency Synthesizer apparatus as claimed in claim 1, wherein the optical frequency comb generator incorporates an optical phase modulator. 36. Optical Frequency Synthesizer apparatus as claimed in claim 1, wherein the slave lasers are locked using optical phase lock loops. 37. Optical Frequency Synthesizer apparatus as claimed in claim 1, wherein the slave lasers are locked using an heterodyne optical injection phase lock loop. 38. Optical Frequency Synthesizer apparatus as claimed in claim 4, wherein the optical signal to drive the loop phase-detector is obtained by using the heterodyne between the slave laser principal output frequency with the residual adjacent comb lines from the optical frequency comb generator reflected from the slave laser facet. 39. Optical Frequency Synthesizer apparatus as claimed in claim 4, where the optical signal that drives the loop phase-detector is obtained by using the heterodyne between the slave laser principal output frequency with the residual adjacent comb lines from the optical frequency comb generator transmitted through the slave laser cavity. 40. Apparatus as claimed in claim 1, wherein the laser control circuit is operable to compensate for variations in the slave laser temperature and for disturbances to the equilibrium of carriers within the slave laser. 41. A method of locking a laser output frequency, comprising the steps of: combining a portion of a slave laser output with the output of an optical frequency comb generator in a photo-detector to generate an heterodyne signal; combining the heterodyne signal with the output of a microwave reference source associated with the optical frequency comb generator source; determining whether the frequency or phase of the heterodyne signal varies in relation to that of the microwave reference; if it does then generating an error correction signal to adjust the current and/or temperature of the slave laser, in order to retain the output frequency of the slave laser at a desired frequency. 42. A method of locking a laser output frequency, utilizing the apparatus of claim 1, the apparatus configured such that the time taken to lock the laser to a frequency is comparable to the reciprocal of the loop bandwidth of the phase lock loop or one cycle of the optical waveform when the laser is tuned within the injection locking range of an optical injection phase lock loop. |
Novel co-stimulatory molecules |
The invention provides polynucleotides and polypeptides encoded therefrom having advantageous properties, including an ability of the polypeptides to preferentially bind a human CD28 or CTLA-4 receptor at a level greater or less than the ability of human B7-1 to bind human CD28 or CTLA-4, or to induce or inhibit altered level of T cell proliferation response greater compared to that generated by human B7-1. The polypeptides and polynucleotides of the invention are useful in therapeutic and prophylactic treatment methods, gene therapy applications, and vaccines. |
1-258. (canceled) 259. An isolated or recombinant polypeptide comprising an amino acid sequence corresponding to an extracellular domain, wherein said amino acid sequence has at least about 92% amino acid sequence identity to the amino acid sequence corresponding to the extracellular domain of SEQ ID NO:66, and wherein said polypeptide has a hCD28/hCTLA-4 binding affinity ratio greater than the hCD28/hCTLA-4 binding affinity ratio of human B7-1. 260-268. (canceled) 269. An isolated or recombinant polypeptide variant comprising an amino acid sequence that differs from the polypeptide sequence of a primate B7-1, or a subsequence of a primate B7-1 comprising an extracellular domain, wherein the difference between the amino acid sequence of the variant and the polypeptide sequence of the primate B7-1 or subsequence of a primate B7-1 comprising an extracellular domain comprises a different amino acid at position 65 other than alanine, wherein the position corresponds to the position in the polypeptide sequence of human B7-1 of SEQ ID NO:278. 270. The isolated or recombinant polypeptide variant of claim 269, wherein the different amino acid is selected from the group of His, Arg, Lys, Pro, Phe, and Trp. 271. The isolated or recombinant polypeptide variant of claim 270, wherein the primate B7-1 is human B7-1 and the different amino acid is histidine. 272. The isolated or recombinant polypeptide variant of claim 271, wherein the variant has a hCTLA-4/hCD28 binding affinity ratio greater than the hCTLA-4/hCD28 binding affinity ratio of human B7-1. 273. The isolated or recombinant polypeptide variant of claim 271, wherein the variant has an ability to induce a T cell proliferation response that is about equal to or less than the T cell proliferation response induced by human B7-1. 274-305. (canceled) 306. An isolated or recombinant polypeptide comprising an amino acid sequence of an extracellular domain, wherein said extracellular domain (ECD) amino acid sequence has at least about 75% amino acid sequence identity to an extracellular domain amino acid sequence of at least one of SEQ ID NO:66, and is not a naturally-occurring extracellular domain amino acid sequence, and wherein said polypeptide is a soluble non-crosslinked polypeptide having, in the presence of a population of activated T cells, an ability to induce a T cell proliferation or T cell activation response that is less than that induced by a human B7-1 ECD amino acid sequence in the presence of a population of activated T cells. 307. The isolated or recombinant polypeptide of claim 306, wherein said polypeptide comprises a soluble ECD monomer. 308. The isolated or recombinant polypeptide of claim 307, wherein said soluble ECD monomer further comprises an Ig polypeptide. 309-310. (canceled) 311. A nucleic acid comprising a polynucleotide sequence that encodes an isolated or recombinant polypeptide of claim 306, or a complementary polynucleotide sequence thereof. 312-331. (canceled) 332. The isolated or recombinant polypeptide of claim 259, wherein said isolated or recombinant polypeptide comprises a soluble extracellular domain. 333. The isolated or recombinant polypeptide of claim 259, wherein said isolated or recombinant polypeptide comprises an extracellular domain monomer. 334. The isolated or recombinant polypeptide of claim 332, wherein the isolated or recombinant polypeptide has an ability to induce a T cell proliferation response that is about equal to or less than the T cell proliferation response induced by human B7-1. 335. The isolated or recombinant polypeptide of claim 332, wherein the polypeptide comprises a fusion protein comprising at least one additional amino acid sequence. 336. The isolated or recombinant polypeptide of claim 335, wherein the at least one additional amino acid sequence comprises at least one Ig polypeptide. 337. The isolated or recombinant polypeptide of claim 336, wherein the at least one Ig polypeptide comprises a human IgG polypeptide comprising an Fc domain. 338. The isolated or recombinant polypeptide of claim 308, wherein the at least one Ig polypeptide comprises a human IgG polypeptide comprising an Fc domain. 339. An isolated or recombinant nucleic acid comprising a polynucleotide sequence which encodes an isolated or recombinant polypeptide of claim 259. 340. An isolated or recombinant nucleic acid comprising a polynucleotide sequence which encodes an isolated or recombinant polypeptide of claim 269. |
<SOH> BACKGROUND OF THE INVENTION <EOH>T cells are a crucial component of the immune system. Not only is T cell activation required for all specific immune responses against infectious agents, but T cells also play an important role in tumor immunity and in autoimmune and allergic diseases. T cell activation is initiated when T cells recognize their specific antigen (Ag) in the context of major histocompatibility complex (MHC) molecules. T cell activation is well known by those of ordinary skill in the art and is characterized by such things as, e.g., cytokine synthesis, induction of various activation markers such as CD25 (interleukin-2 (IL-2) receptor), etc. CD4+ T cells recognize their immunogenic peptides in the context of MHC class II molecules, whereas CD8+ T cells recognize their immunogenic peptides in the context of MHC class I molecules. For induction of T cell activation, cytokine synthesis or effector function, a second signal, mediated through CD28, is required. Two ligands for CD28 are B7-1 (CD80) and B7-2 (CD86). B7-1 and B7-2 are termed co-stimulatory molecules and are typically expressed on professional antigen-presenting cells (APCs). In addition to binding the CD28 receptor, B7-1 and B7-2 also bind the CTLA-4 (CD152) receptor on T cells. B7 molecules mediate both positive and negative signals to T cells by binding to CD28 and CTLA-4 (CD152) molecules on T cells. CTLA-4 is a negative regulator of the immune system. In general, wild-type (WT) B7-1, e.g., human B7-1, preferentially binds CTLA-4 more strongly than it binds CD28. Typically, wild-type B7-1, e.g., human B7-1, binds CTLA-4 with about 100 times greater affinity than it binds CD28. Binding of B7-1 or B7-2 to CTLA-4 suppresses activation of T cells, resulting in reduced T cell proliferation and cytokine production (see, e.g., Walunas, T. L. et al. (1994) Immunity 1(5):405-413; Alegre, M. L. et al. (1998) J Immunol 161(7):3347-3356). Interaction between B7-1 or B7-2 and CTLA-4 expressed on T cells down-regulates T cell responses and raises thresholds required for activation by CD28. Blockade of CTLA-4/ligand interactions can also augment in vivo tumor immunity (Leach, D. et al. (1996) Science 271:1734-1736). Consequently, CD28 and CTLA-4 play a pivotal role in the regulation of T cell activation and both are essential for proper functioning of the immune system. For example, CD28 deficient mice are severely immunodeficient and show poor antigen specific T cell responses, while CTLA-4 deficient mice die of lymphoproliferative disease, show T cell expansion mediated by CD28 signaling and have a lack of down-regulation of T cell receptor signaling. Upon ligation by the co-stimulatory molecules B7-1 or B7-2, CD28 mediates a co-stimulatory signal that synergizes with T cell receptor signaling to induce, e.g., proliferation, cytokine production and effector functions by both CD4+ and CD8+ T cells (proliferation/activation). Ligation of CTLA-4 with B7-1 (CD80) or B7-2 (CD86), however, dampens the CD80 or CD86 activating signal through CD28, resulting in down-regulation of T cell activation. CD28 ligation reduces the inhibition mediated through the CTLA-4 signaling. CTLA-4 ligation mediates tolerance and anergy. CD28 and CTLA-4 are both involved in the generation of an immune response to genetic vaccinations (e.g., nucleic acid vaccinations (NAV), DNA vaccinations, and viral vectors). CD28 deficient mice are unable to mount T cell or antibody responses against Beta-galactosidase (Beta-gal) when immunized with a plasmid encoding the Beta-gal gene, and CTLA-4 ligation suppresses the antibody response to Beta-gal in immunized wild-type mice (Horspool, J. et al. (1998), J Immunol 160:2706-2714). Expression of B7-1 on human myeloma cells (Wendtner, C. et al. (1997) Gene Therapy 4(7):726-735), murine mammary tumors (Martin-Fontecha, A. et al.(2000) J Immunol 164(2):698-704) or murine sarcoma (Indrova et al. (1998) Intl J Onc 12(2):387-390) enhances anti-tumor immunity. Furthermore, transfection of human APCs with retroviral vectors encoding B7-1 and tumor antigens induces a stronger cytotoxic T-lymphocyte (CTL) response than transfection with similar vectors encoding the tumor antigens alone (Zajac, P. et al. (1998) Cancer Res 58(20):4567-4571). Anti-viral responses are also modulated by co-stimulatory molecules. For example, DNA vaccination of chimpanzees and mice with HIV antigens in conjunction with B7-2 augmented anti-viral responses (Kim, J. et al. (1998) Vaccine 16(19):1828-1835; Tsuji et al. (1997) Eur J Immunol 27(3):782-787). The binding properties of B7-1 and B7-2 have limited their usefulness in clinical applications. The present invention addresses needs for molecules having varied abilities to preferentially bind to and/or signal through either CD28 or CTLA-4 receptor and methods of using such molecules for selected and differential manipulation of T cell responses in vitro, ex vivo, and in vivo methods. Such molecules would be of beneficial use in a variety of applications, including, e.g., therapeutic and prophylactic treatments and vaccinations. The present invention fulfills these and other needs. |
<SOH> SUMMARY OF THE INVENTION <EOH>In one aspect, the present invention provides novel co-stimulatory molecules (abbreviated as “NCSM”) molecules, including polypeptides and proteins, related fusion polypeptide or fusion protein molecules, or functional equivalents thereof, homologues, and fragments of said polypeptide and protein molecules or equivalents, analogs, or derivatives thereof, and vectors, cells, and compositions comprising such NCSM molecules. The invention also provides nucleic acids encoding any of these polypeptides, proteins, fragments or variants thereof. In addition, the invention provides vectors, cells, and compositions comprising such nucleic acids, and uses of such NCSM polypeptides and NCSM nucleic acids; and other features are apparent upon further review. Generally speaking, a “co-stimulatory molecule” refers to a molecule that acts in association or conjunction with, or is involved with, a second molecule or with respect to an immune response in a co-stimulatory pathway. In one aspect, a co-stimulatory molecule may be an immunomodulatory molecule that acts in association or conjunction with, or is involved with, another molecule to stimulate or enhance an immune response. In another aspect, a co-stimulatory molecule is immunomodulatory molecule that acts in association or conjunction with, or is involved with, another molecule to inhibit or suppress an immune response. A “co-stimulatory molecule” need not act simultaneously with or by the same mechanism as the second molecule. The term “NCSM” in reference to a molecule is not intended to limit the molecule to only those molecules that have positive co-stimulatory properties (e.g., that stimulate or augment T cell proliferation). In the initial recombination procedures described below, libraries of recombinant molecules were generated by recombining nucleotide sequences of parental co-stimulatory molecules (CSM) as discussed herein. As shown by the data and analyses presented herein, novel recombinant molecules having a variety of properties were identified and selected. For example, polypeptide and nucleic acid molecules that enhance an immune response, such as by inducing T cell activation or proliferation (e.g., agonists), and molecules that down-regulate or inhibit an immune response, such as by inhibiting T cell activation or proliferation (e.g., antagonists) were identified and selected. Further, molecules that preferentially bind and/or signal through either or both the human CD28 and CTLA-4 receptors were identified and selected. Thus, the term “NCSM” refers to a co-stimulatory molecule and is not limited to molecules having the co-stimulatory properties of the parent sequences, but is intended to refer collectively to all polypeptides of the invention, and nucleic acids encoding them, and other embodiments as described herein, unless specifically noted otherwise. The terms CD28 and CTLA-4 are intended to refer to human CD28 receptor (“hCD28”) and human CTLA-4 receptor (“hCTLA-4”), respectively, as described herein and throughout the application, unless otherwise specifically noted. The term “NCSM” also includes variants, mutants, derivatives, and fragments of: 1) B7-1 and B7-2 polypeptides and nucleic acids, and 2) B7-1 and B7-2 polypeptides and nucleic acids of the Artiodactyla family (including, e.g., bovine B7-1 and B7-2), including all such polypeptide variants (and nucleic acids encoding such polypeptide variants) that exhibit properties similar or equivalent to the properties of the CD28BPs or CTLA-4BPs described herein. For example, the term includes B7-1, B7-2, and Artiodactyla (e.g., bovine) B7-1 and B7-2 polypeptide variants (and nucleic acids encoding such polypeptide variants) of the invention, wherein such polypeptide variants have a hCD28/hCTLA-4 binding affinity ratio about equal to, equal to, or greater than the hCD28/hCTLA-4 binding affinity ratio of human B7-1 (hB7-1) or human B7-2 (hB7-2) and/or an ability to induce a T-cell proliferation, and/or a T-cell activation response about equal to, equal to, or greater than that induced by hB7-1. “NCSM” also is intended to include B7-1, B7-2, and Artiodactyla (e.g., bovine) B7-1 & B7-2 polypeptide variants (and nucleic acids encoding such polypeptide variants), wherein such polypeptide variants have a hCTLA-4/hCD28 binding affinity ratio about equal to or greater than the hCTLA-4/hCD28 binding affinity ratio of hB7-1 or hB7-2, and/or an ability to induce T-cell proliferation and/or T-cell activation response about equal to or less than that induced by hB7-1 or hB7-2. In one aspect, the invention includes isolated or recombinant NCSM polypeptides, variants, homologues, derivatives, analogs, and fragments thereof The invention includes recombinant NCSM polypeptides having varied abilities to preferentially bind to and/or signal through hCD28 and/or hCTLA-4 receptor and provide for selected and differential manipulation of T cell responses in vitro, ex vivo, and in vivo. The invention also includes isolated or recombinant NCSM nucleic acids, variants, homologues, derivatives, analogs, and fragments thereof that encode polypeptides having varied abilities and uses described above. Such NCSM polypeptide and polynucleotides are useful in a variety of applications, including e.g., therapeutic and prophylactic treatment methods, vaccinations, and diagnostic assays described below. The invention also provides NCSM polypeptides, and polynucleotide encoding such polypeptides, that strongly or preferentially bind at least one of hCD28 or hCTLA-4, but do not effectuate signaling; such molecules are useful in methods as potential antagonists of endogenous molecules, such as e.g., endogenous co-stimulatory molecules. Further, the invention provides NCSM polypeptides, and polynucleotides encoding them, having improved or altered receptor/ligand binding affinities and methods of using such molecules, including in pharmaceutical, prophylactic, therapeutic, vaccine, and diagnostic applications. In one aspect is provided an isolated or recombinant polypeptide comprising an amino acid sequence of an extracellular domain (ECD), said ECD amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 94%, 93%, 95%, 96% or more sequence identity to: 1) a non-naturally-occurring ECD amino acid sequence of, or the full-length amino acid sequence of, at least one of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293, or 2) a naturally-occurring ECD amino acid sequence of, or the full-length sequence of, at least one of SEQ ID NOS:48-68, 174-182, 184-221, 283-285, and 290-293, wherein said polypeptide has a hCD28 receptor/hCTLA-4 receptor binding affinity ratio about equal to or greater than the hCD28/hCTLA-4 binding affinity ratio of human B7-1 and/or has an ability to induce a T-cell proliferation and/or T-cell activation response about equal to or greater than that induced by WT hB7-1. Some such polypeptides have an ability to induce T-cell proliferation or T-cell activation or both. In one aspect, the T-cell proliferation or activation response is at least about equal to or greater than that induced by WT hB7-1. Such polypeptides may have a binding affinity for hCD28 that is about equal to or greater than the binding affinity of hB7-1 for human CD28, and/or a binding affinity for hCTLA-4 less than that of hB7-1 for human CTLA-4, and are termed CD28 binding proteins (“CD28BP”). Some such CD28BP polypeptides that induce T-cell proliferation and/or activation response(s) about equal to or greater than that induced by hB7-1 are typically associated with or bound or linked to a cell membrane. Such polypeptides typically comprise a polypeptide sequence comprising at least: 1) substantially the entire length of a polypeptide sequence corresponding to an ECD of a CD28BP polypeptide or B7-1 polypeptide variant able to induce such response(s); and 2) substantially the entire length of a polypeptide sequence corresponding to a transmembrane domain (TMD) of a CD28BP polypeptide or B7-1 variant able to induce such response(s). A sufficient amino acid segment of the TMD (5, 10, 15, 20, amino acids in length) is usually needed to bind or link the polypeptide to the cell membrane upon expression of the polypeptide on the cell surface following transfection of the cell with a nucleic acid encoding the polypeptide. A signal peptide may also be included for proper expression of the polypeptide on the cell membrane. Often, such CD28BP polypeptides comprise an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or more sequence identity to substantially the entire length of a polypeptide sequence of any of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293 or B7-1 variant able to induce such response(s). Some such polypeptides comprise a sequence that is at least about 90% or 100% of the sequence length of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293 (or a B7-1 variant able to induce such response(s)) and following transfection and expression, remain bound to the cell. Soluble crosslinked or multimeric forms of CD28BP polypeptides also induce T-cell proliferation and/or activation response(s) about equal to or greater than that induced by hB7-1. Soluble crosslinked or multimeric polypeptides typically comprise a polypeptide sequence having at least about 75%, 80%, 90%, 92%, 93%, 95%, 96% or more sequence identity to an ECD sequence—or co-stimulatory fragment of an ECD sequence—of a CD28BP polypeptide, such as any of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293, or B7-1 polypeptide variant able to induce such response(s).CD28BP ECD polypeptides (or co-stimulatory fragments thereof) can be made to link to a cell membrane surface, without a TMD sequence, and thus to induce the T cell proliferation or activation responses described above by using known anchor molecules and conventional techniques to produce the anchored ECD. For example, glycosylphosphatidylinositol (GPI) anchor sequences are commonly used to anchor a polypeptide, including an ECD, to a cell membrane surface. See, e.g., K L Reid-Taylor et al., Biochem Cell Biol. 77(3):189-200 (1999) and C R Da Costa et al., J. Bio. Chem. 273(19):1 1874-80 (1998), each of which is incorporated herein by reference in its entirety for all purposes. In such formats, the presence of the TMD domain is not a prerequisite for expression of a NCSM polypeptide on a cell surface. A polypeptide sequence having at least about 75%, 80%, 90%, 92%, 93%, 95%, 96% or more amino acid sequence identity to an ECD sequence (e.g., from about residues 35-244) of any of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293 (comprising about, e.g., 200, 205, 206, 207, or 209 amino acid residues) can be anchored to a cell membrane using such conventional techniques. Some such CD28BP polypeptides of the invention may comprise an amino acid sequence having at least about 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more sequence identity to a full-length polypeptide sequence selected from any of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293. Such polypeptides may be expressed on the surface of a cell membrane (e.g., following transfection of the cell with a nucleic acid that encodes said polypeptide) or associated with or bound to a cell membrane, or form an integral membrane protein (e.g., by fuirther comprising a transmembrane domain amino acid sequence). Through such expression, such polypeptides typically become integral membrane proteins. Preferably, such cell-expressed polypeptides, membrane-associated, or membrane-bound polypeptides, have an ability to induce a T cell proliferation or activation response that is approximately equal to or greater than that induced by hB7-1. Polypeptides of the invention that have an ability to induce a T-cell proliferation or activation response at least about equal to or greater than that induced by hB7-1 include TMD/ECD CD28BP polypeptides. Such TMD/ECD CD28BP polypeptides may comprise an amino acid sequence substantially identical or identical to an amino acid sequence comprising at least the TMD and ECD subsequences of a full-length CD28BP polypeptide selected from the group of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293 (or a TMD/ECD of a B7-1 polypeptide variant, e.g., bovine B7-1 polypeptide variant, described below). A TMD/ECD CD28BP polypeptide may be generated by transfection of a cell with a nucleic acid encoding an amino acid sequence comprising at least the signal peptide, TMD, and ECD sequence of a full-length CD28BP polypeptide selected from the group of SEQ ID NOS:48-68, 174221, 283-285, and 290-293. Following expression, the signal peptide is typically cleaved, leaving the TMD/ECD CD28BP polypeptide which, due to the presence of the TMD, is typically bound or linked to the cell membrane. Crosslinked or multimeric CD28BP-ECD-Ig and mammalian B7-1 variant polypeptides also have an ability to induce a T cell proliferation or activation response (e.g., in the presence of soluble anti-CD3 monoclonal antibodies (mAb)) at least about equal to or greater than that induced by hB7-1-ECD-Ig or full-length polypeptide (e.g., in presence of soluble anti-CD3 mAbs). Such polypeptides may be crosslinked with another such polypeptide or a different polypeptide. Such CD28BP-ECD-Ig or B7-1 variant polypeptides may comprise an amino acid sequence substantially identical or identical to an ECD sequence of a full-length CD28BP polypeptide selected from any of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293 (or an ECD sequence of a mammalian B7-1 polypeptide variant, e.g., bovine, primate, or hB7-1 variant) fused to an Fc domain of a human IgG. SomeCD28BP and B7-1 variant polypeptides of the invention modulate T-cell activation, but do not induce proliferation of purified T-cells activated by soluble anti-CD3 mAbs. For example, soluble non-crosslinked monomeric CD28BP-ECD-Ig and/or mammalian B7-1 variant polypeptides (e.g., bovine or hB7-1 variants) of the invention typically inhibit the proliferation of purified T cells in the presence of soluble anti-CD3 mAbs compared to hB7-1 ECD-Ig polypeptide under similar conditions. In one embodiment, the isolated or recombinant polypeptide comprises an amino acid sequence that has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to an ECD sequence of at least one of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293. The ECD sequence typically comprises from about amino acid residue 35 to about amino acid residue 244 of a polypeptide sequence of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293. In another embodiment, the isolated or recombinant polypeptide comprises a non-naturally-occurring amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ECD sequence of at least one polypeptide of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293. In another embodiment, the isolated or recombinant polypeptide comprises a naturally- or non-naturally-occurring amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to substantially the entire length of a polypeptide sequence of at least one of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293. Some such polypeptides comprise a naturally- or non-naturally-occurring amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a co-stimulatory stimulatory subsequence of at least one of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293, wherein said subsequence comprises the: 1) signal peptide and ECD, 2) signal peptide, ECD, and transmembrane domain (TMD), 3) ECD and TMD, or 4) ECD, TMD, and cytoplasmic domain (CD) of at least one of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293, and has NCSM properties. The signal peptide, TMD, and CD typically comprise, respectively, from about amino acid residue 1 to about amino acid residue 34, from about amino acid residue 245 to about amino acid residue 265 or 268, and from about amino acid residue 266 or 269 to about amino acid residue 293, 296, 303, or 306 of a polypeptide sequence selected from the group of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293. In another embodiment, the isolated or recombinant polypeptide comprises a naturally- or non-naturally-occurring amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the entire length of a polypeptide sequence of at least one of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293. Such polypeptides typically comprise at least about 265, 268, 280, 293, or 303 amino acid residues in length. Some such isolated or recombinant polypeptides are soluble (e.g., not cell membrane-bound or membrane-associated or forming an integral part of a cell membrane). As discussed in detail below, soluble polypeptides typically comprise an amino acid sequence corresponding to an ECD and may additionally comprise a signal peptide (which may be subsequently cleaved), but do not include an amino acid sequence corresponding to a TMD or CD. The present invention includes monomeric and multimeric (or aggregated) forms of such polypeptides. A soluble monomer comprises one soluble polypeptide of the invention (e.g., one soluble NCSM-ECD), while a soluble multimer or soluble aggregate typically comprises at least two soluble polypeptides of the invention (e.g., two soluble NCSM-ECDs). The multimer or aggregate may be formed by cross-linking or using other means to link or immobilize polypeptide sequences. Such polypeptides may comprise a fuision protein comprising at least one additional amino acid sequence, such as an Ig polypeptide. As described below, multimeric or aggregate forms of selected CD28BP polypeptides have an ability to induce a T cell proliferation or activation response in the presence of a population of activated T cells that is at least about equal to or greater than that observed with multimeric or aggregate forms of hB7-1 polypeptide in the presence of a population of activated T cells. In contrast, some soluble monomeric CD28BP polypeptides have an ability to induce a T cell proliferation or activation response in the presence of a population of activated T cells that is less than the T cell proliferation or activation response induced by a soluble monomeric hB7-1 in the presence of a population of activated T cells. Some such isolated or recombinant CD28BP ECD polypeptides, or fragments thereof, further comprise at least one of a signal peptide domain, tmnsmembrane domain (TMD), and/or cytoplasmic domain (CD), including, e.g., wherein such domain is a WT, variant, or mutant domain of a co-stimulatory polypeptide, including, e.g., a recombinant domain derived from a mammalian B7-1 or B7-2. In one embodiment, the isolated or recombinant CD28BP ECD polypeptide (or fragment thereof) comprises an amino acid subsequence, including any of a signal peptide, TMD, or CD of any of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293. Nucleic acids encoding such polypeptides and domains are also provided. The invention also provides an isolated or recombinant polypeptide, which polypeptide comprises a naturally- or non-naturally-occurring amino acid sequence encoded by a nucleic acid comprising a polynucleotide sequence selected from the group of: (a) a polynucleotide sequence selected from SEQ ID NOS:1-21 and 95-142, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of a polypeptide selected from SEQ ID NOS:48-68, 174-221, 283-285, and 290-293, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which, but for the degeneracy of the genetic code, hybridizes under at least stringent or highly stringent conditions over substantially the entire length of polynucleotide sequence (a) or (b); (d) a polynucleotide sequence comprising all or co-stimulatory nucleotide fragment of (a), (b), or (c), wherein the nucleotide fragment encodes a co-stimulatory polypeptide (e.g., an ECD, signal peptide/ECD, or signal peptide/ECD/TD, ECD/TMD, or ECD/TMD/CD polypeptide) having a hCD28/hCTLA-4 binding affinity ratio about equal to or greater than the hCD28/hCTLA-4 binding affinity ratio of human B7-1, and/or has an ability to induce a T-cell proliferation and/or T-cell activation response about equal to or greater than induced by hB7-1; (e) a polynucleotide sequence encoding a polypeptide, the polypeptide comprising an amino acid sequence which is substantially identical over at least about 150 contiguous amino acid residues of any one of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293; and (f) a polynucleotide sequence encoding a polypeptide that has a hCD28/hCTLA-4 binding affinity ratio about equal to or greater than the hCD28/hCTLA-4 binding affinity ratio of human B7-1, and/or has an ability to induce T-cell proliferation or activation or both that is about equal to or greater than the ability of hB7-1 to induce such response, said polynucleotide sequence having at least about 70% amino acid sequence identity to at least one sequence of (a), (b), (c), or (d). Also provided is an isolated or recombinant polypeptide comprising an amino acid sequence according to the formula: MGHTM-X6-W-X8-SLPPK-X14-PCL-X18-X19-X20-QLLVLT-X27-LFYFCSGITPKSVTKRVKETVMLSCDY-X55-TSTE-X60-LTSLRIYW-X69-KDSKMVLAILPGKVQVWPEYKNRTITDMNDN-X101-RIVI-X106-ALR-110-SD-X113-GTYTCV-X120-QKP-X124-LKGAYKLEHL-X135-SVRLMIRADFPVP-X149-X150-X151-DLGNPSPNIRRLICS-X167-X168-X169-GFPRPHL-X177-WLENGEELNATNTT-X192-SQDP-X197-T-X199-LYMISSEL-X208-FNVTNN-X215-SI-X218-CLIKYGEL-X227-VSQIFPWSKPKQEPPIDQLPF-X249-VIPVSGALVL-X261-A-X263-VLY-X267-X268-ACRH-X2273-ARWKRTRRNEETVGTE RLSPIYLGSAQSSG (SEQ ID NO:284), or a subsequence thereof comprising an extracellular domain, wherein position X6 is Lys or Glu; position X8 is Arg or Gly; position X14 is Arg or Cys; position X18 is Trp or Arg; position X19 is Pro or Leu; position X20 is Ser or Pro; position X27 is Asp or Gly; position X55 is Asn or Ser; position X60 is Glu or Lys; position X69 is Gln or Arg; position X101 is Pro or Leu; position X106 is Leu or Gln; position X110 is Pro or Leu; position X113 is Lys or Ser; position X120 is Val or Ile; position X124 is Val or Asp; position X135 is Thr or Ala; position X149 is Thr, Ser, or del; position X150 is Ile or del; position X151 is Asn or Thr; position X167 is Thr or del; position X169 is Ser or del; position X169 is Gly or del; position X177 is Cys or Tyr; position X192 is Val or Leu; position X197 is Gly or Glu; position X199 is Glu or Lys; position X208 is Gly or Asp; position X215 is His or Arg; position X218 is Ala or Val; position X227 is Ser or Leu; position X249 is Trp, Leu, or Arg; position X261 is Ala or Thr; position X263 is Val, Ala, or lie; position X267 is Arg or Cys; position X268 is Pro or Leu; and position X273 is Gly or Val. Typically, the ECD comprises at least about amino acids 35 to 244 or amino acids 35 to 255 of SEQ ID NO:284. Some such polypeptides have a hCD28/hCTLA-4 binding affinity ratio about equal to or greater than the hCD28/hCTLA-4 binding affinity ratio of human B7-1, and/or an ability to induce T-cell proliferation or activation or both that is about equal to or greater than that induced by hB7-1. In yet another aspect, the invention provides an isolated or recombinant polypeptide comprising a subsequence of an amino acid sequence set forth in any of SEQ ID NOS:48-68, 174-182, 184-221, 283-285, and 290-293, wherein the subsequence is the extracellular domain of said amino acid sequence. A soluble form of an NCSM polypeptide or a human B7-1 polypeptide typically comprises a polypeptide, comprising at least one ECD, which is not expressed on and bound to the surface of a cell, is not embedded in a cell membrane, and/or is not associated with a lipid bilayer. Such soluble polypeptides may contain a TD or a fragment thereof such that the polypeptide remains soluble (e.g., is not embedded in or linked to the cell membrane as an integral membrane protein or associated with a lipid bilayer). Such soluble polypeptides may also include one or more additional amino acid sequences, such as an Fc portion of an Ig (e.g., IgG). Such soluble polypeptide may comprise an ECD monomer, ECD-Ig fusion protein monomer, ECD multimer or aggregate, or ECD-Ig fusion protein multimer or aggregate (see data and discussion below). Also provided is an isolated or recombinant polypeptide, which comprises an amino acid sequence of an extracellular domain, wherein said ECD amino acid sequence has at least about 75%, 80%, 90%, or 95% sequence identity to an extracellular domain amino acid sequence of at least one of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293, and is not a naturally-occurring ECD amino acid sequence, wherein such polypeptide is a soluble monomeric or non-crosslinked polypeptide having, in the presence of activated T cells, an ability to down-regulate or inhibit a T cell proliferation response to a greater degree than does soluble hB7-1 (e.g., hB7-1-ECD or hB7-1-ECD-Ig) in the presence of activated T cells. The polypeptide can be either a soluble ECD monomer or a soluble ECD-Ig monomeric fusion protein and is typically not crosslinked. Such polypeptides may comprise a fusion protein comprising at least one additional polypeptide, such as, e.g., an Ig polypeptide. Also included is an isolated or recombinant polypeptide comprising an ECD amino acid sequence that has at least about 92%, 93%, 95%, or 96% sequence identity to an ECD amino acid sequence of at least one polypeptide of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293 (or an ECD sequence of a B7-1 polypeptide variant described herein), where the polypeptide is a soluble monomeric or non-crosslinked (NCSM-ECD or NCSM-ECD-Ig) polypeptide that has an ability to more greatly inhibit or down-regulate T cell proliferation in the presence of activated T cells than soluble monomeric or non-crosslinked hB7-1-ECD or hB7-1-ECD-Ig. The invention also includes each nucleic acid comprising a polynucleotide sequence (including degenerate sequences) that encodes each such isolated or recombinant polypeptide comprising a soluble polypeptide as described above, and a complementary polynucleotide sequence thereof. Polynucleotide sequences that, but for the degeneracy of the genetic code, hybridizes under at least stringent conditions over substantially the entire length of each such nucleic acid are also included. The invention further provides isolated or recombinant polypeptides comprising an amino acid sequence having at least about 95% amino acid sequence identity to a full-length sequence of at least one of SEQ ID NOS:69-92, 222-252, 286-289, or to a subsequence thereof comprising the extracellular domain, wherein said sequence (a) is a non naturally-occurring sequence, and (b) comprises at least one of: Gly at position 2; Thr at position 4; Arg at position 5; Gly at position 8; Pro at position 12; Met at position 25; Cys at position 27; Pro at position 29; Leu at position 31; Arg at position 40; Leu at position 52; His at position 65; Ser at position 78; Asp at position 80; Tyr at position 87; Lys at position 120; Asp at position 122; Lys at position 129; Met at position 135; Phe at position 150; Ile at position 160; Ala at position 164; His at position 172; Phe at position 174; Leu at position 176; Asn at position 178; Asn at position 186; Glu at position 194; Gly at position 196; Thr at position 199; Ala at position 210; His at position 212; Arg at position 219; Pro at position 234; Asn at position 241; Leu at position 244; Thr at position 250; Ala at position 254; Tyr at position 265; Arg at position 266; Glu at position 273; Lys at position 275; Ser at position 276; an amino acid deletion at position 276; or Thr at position 279, wherein the position wherein said polypeptide has a hCTLA-4/hCD28 binding affinity ratio about equal to or greater than the hCTLA-4/hCD28 binding affinity ratio of human B7-1, and/or an ability to induce a T-cell proliferation or T-cell activation response about equal to or less than that induced by hB7-1. A subsequence comprises signal peptide, EDC, TMD, or CD. Each such polypeptide may further comprise at least one additional amino acid sequence, including, e.g., a sequence corresponding to a signal peptide, TMD, ECD, or CD. In another aspect, the invention provides isolated or recombinant polypeptides, each comprising an amino acid sequence that differs from the amino acid sequence of a primate (or mammalian) B7-1, wherein the difference between the amino acid sequence of the polypeptide and the amino acid sequence of the primate (or mammalian) B7-1 comprises a different amino acid at at least one amino acid residue position selected from the group consisting of 12, 25, 27, 29, 40, 52, 65, 122, 129, 135, 164, 174, 196, 199, 210, 219, 234, 241, 254, 275, 276, and 279, wherein the amino acid residue positions correspond to the amino acid residue positions in the amino acid sequence of human B7-1 of SEQ ID NO:278. For some such polypeptides, the different amino acid comprises at least one of: Pro at position 12; Met at position 25; Cys at position 27; Pro at position 29; Arg at position 40; Leu at position 52; His at position 65; Asp at position 122; Lys at position 129; Met at position 135; Ala at position 164; Phe at position 174; Gly at position 196; Thr at position 199; Ala at position 210; Arg at position 219; Pro at position 234; Asn at position 241; Ala at position 254; Lys at position 275; Ser at position 276; or Thr at position 279. Preferably, for some such polypeptides, the different amino acid is His at position 65. In another aspect, the invention provides isolated or recombinant polypeptides comprising an amino acid sequence that differs from a primate (or mammalian) B7-1 sequence in at least one mutation or substitution selected from: Ser 12 Pro; Leu 25 Met; Gly 27 Cys; Ser 29 Pro; Lys 40 Arg; His 52 Leu; Tyr 65 His; Glu 122 Asp; Glu 129 Lys; Thr 135 Met; Thr 164 Ala; Ser 174 Phe; Glu 196 Gly; Ala 199 Thr; Thr 210 Ala; Lys 219 Arg; Thr 234 Pro; Asp 241 Asn; Val 254 Ala; Arg 275 Lys; Arg 276 Ser; or Arg 279 Thr; the mutation or substitution being indicated relative to human B7-1 with the amino acid sequence shown in SEQ ID NO:278, wherein said sequence does not occur in nature, and wherein said polypeptide has a hCTLA-4/hCD28 binding affinity ratio about equal to or greater than the hCTLA-4/hCD28 binding affinity ratio of human B7-1, and/or an ability to induce a T-cell proliferation and/or T-cell activation response about equal to or less than the ability of hB7-1 to induce such response. Also included are isolated or recombinant polypeptides comprising an amino acid sequence having at least about 75% sequence identity to at least one of SEQ ID NOS:263-272, or a subsequence thereof comprising the extracellular domain, where the amino acid sequence is not naturally-occurring, and the polypeptide has a hCTLA-4/hCD28 binding affinity ratio about equal to or greater than the hCTLA-4/hCD28 binding affinity ratio of human B7-1, and/or an ability to induce a T-cell proliferation and/or activation response about equal to or less than the ability of hB7-1 to induce such response. In yet another aspect, the invention includes an isolated or recombinant polypeptides which comprises a non naturally-occurring amino acid sequence encoded by a nucleic acid comprising a polynucleotide sequence selected from: (a) a polynucleotide sequence selected from SEQ ID NOS:2245, 143-173, 253-262, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NOS:69-92, 222-247, 263-272, 286-289, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which, but for the degeneracy of the genetic code, hybridizes under highly stringent conditions over substantially the entire length of polynucleotide sequence (a) or (b); (d) a polynucleotide sequence comprising all or a fragment of (a), (b), or (c), wherein the fragment encodes a polypeptide having a hCTLA-4/hCD28 binding affinity ratio about equal to or greater than the hCTLA-4/hCD28 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation or activation response about equal to or less than that induced by hB7-1; (e) a polynucleotide sequence encoding a polypeptide, the polypeptide comprising an amino acid sequence which is substantially identical over at least about 150 contiguous amino acid residues of any one of SEQ ID NOS:69-92, 222-247, 263-272, 286-289, and (f) a polynucleotide sequence encoding a polypeptide that has a hCTLA-4/hCD28 binding affinity ratio about equal to or greater than the hCTLA-4hCD28 binding affinity ratio of human B7-1 or an ability to induce a T-cell proliferation or activation response about equal to or less than that of hB7-1, which polynucleotide sequence has at least about 70% identity to at least one polynucleotide sequence of (a), (b), (c), or (d). The invention also includes an isolated or recombinant polypeptide comprising an amino acid sequence according to the formula: MGHTRRQGTSP-X12-KCPYLKFFQLLV-X25-ACL-X29-HLCSGVIHVT-X40-EVKEVATLSCGLNVSVEELAQTRIHWQKEKKMVLTM MSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECWLKY-X122-KDAFKR-X129-HLAEVMLSVKAD FPTPSITDFEIPPSNIRRIICS-X164-SGGFPEPHLFWLENGEELNAINTTVSQDPET-X196-LYTVSSKLDFNM TANHSFMCLI-X219-YGHLRVNQTFNWNTPKQEHFP-X241-NLLPSWA ITLISANGIFVICCLTYRFAPRCRERKSNETLRRESVCPV (SEQ ID NO:287), or a subsequence thereof comprising the extracellular domain, wherein position X12 is Ser or Pro; position X25 is Leu or Met; position X29 is Ser or Pro; position X40 is Lys or Arg; position X122 is Glu or Asp; position X129 is Glu or Lys; position X164 is Thr or Ala; position X196 is Glu or Gly; position X219 is Lys or Arg; position X241 is Asp or Asn. Some such polypeptides have a hCTLA-4/hCD28 binding affinity ratio about equal to or greater than the hCTLA-4/hCD28 binding affinity ratio of hB7-1, and/or ability to induce T-cell proliferation or activation or both about equal to or less than that induced by hB7-1. The invention also provides an isolated or recombinant polypeptide comprising a subsequence of an amino acid sequence set forth in any of SEQ ID NOS:69-92, 222-247, 263-272, and 286-289, wherein the subsequence is the extracellular domain or full-length sequence of such amino acid sequence. Furthermore, the invention includes the full-length polypeptide sequence and one or more subsequences thereof, e.g., signal peptide, extracellular domain (ECD), transmembrane domain (TMD), and/or cytoplasmic domain (CD) of any of SEQ ID NOS:66, 81, 85, 86, 88, 90, 91, 285, 288, 289, 291, and 294, and nucleic acid sequences encoding any of these amino acid sequences. The invention provides isolated or recombinant nucleic acids comprising a polynucleotide sequence selected from: (a) a polynucleotide sequence selected from SEQ ID NOS:1-21 and 95-142, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NOS:48-68, 174-221, 283-285, and 290-293, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which, but for codon degeneracy, hybridizes under at least stringent or highly stringent conditions over substantially the entire length of polynucleotide sequence (a) or (b); and (d) a polynucleotide sequence comprising all or a nucleotide fragment of (a), (b), or (c), wherein the fragment encodes a polypeptide having a hCD28/hCTLA-4 binding affinity ratio about equal to or greater than the hCD28/hCTLA-4 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation or activation response about equal to or greater than can be induced by hB7-1. In some such nucleic acids, the polynucleotide sequence of (d) encodes a nucleotide fragment of (a) or (b) that encodes a co-stimulatory ECD/TMD having a hCD28/hCTLA-4 binding affinity ratio or an ability to induce a T-cell proliferation or activation response about equal to or greater than that of hB7-1. Also included are isolated or recombinant nucleic acids comprising a polynucleotide sequence encoding a polypeptide, wherein the encoded polypeptide comprises an amino acid sequence which is (a) substantially identical over at least about 150 or 200 contiguous amino acid residues of any one of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293 and (b) is a non naturally-occurring sequence. In addition, the invention includes isolated or recombinant nucleic acids comprising a nucleotide sequence coding for a polypeptide comprising the amino acid sequence set forth in any of SEQ ID NOS:48-68, 174-221, 283-285, and 290-293, or a subsequence thereof, wherein the subsequence comprises at least one of: the signal sequence, extracellular domain, or transmembrane domain of said polypeptide, and the cytoplasmic domain of said polypeptide, and wherein the amino acid sequence or subsequence is a non naturally-occurring sequence. Similarly, fragments of the above nucleotides that encode a polypeptide that has a substantially equivalent or equivalent binding activity of a NCSM polypeptide molecule, produces a substantially equivalent or equivalent NCSM-polypeptide-mediated immune response, e.g., induction or inhibition of T cell activation or proliferation, or cytokine production are a feature. Also provided is an isolated or recombinant nucleic acid comprising a polynucleotide sequence selected from: (a) a polynucleotide sequence selected from SEQ ID NOS:2245, 143-173, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NOS:69-92, 222-247, 286-289, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which, but for the degeneracy of the genetic code, hybridizes under highly stringent conditions over substantially the entire length of polynucleotide sequence (a) or (b); and (d) a polynucleotide sequence comprising all or a fragment of (a), (b), or (c); wherein (c) or (d) encodes a polypeptide having a non naturally-occurring sequence comprising at least one of: Gly at position 2; Thr at position 4; Arg at position 5; Gly at position 8; Pro at position 12; Met at position 25; Cys at position 27; Pro at position 29; Leu at position 31; Arg at position 40; Leu at position 52; His at position 65; Ser at position 78; Asp at position 80; Tyr at position 87; Lys at position 120; Asp at position 122; Lys at position 129; Met at position 135; Phe at position 150; Ile at position 160; Ala at position 164; His at position 172; Phe at position 174; Leu at position 176; Asn at position 178; Asn at position 186; Glu at position 194; Gly at position 196; Thr at position 199; Ala at position 210; His at position 212; Arg at position 219; Pro at position 234; Asn at position 241; Leu at position 244; Thr at position 250; Ala at position 254; Tyr at position 265; Arg at position 266; Glu at position 273; Lys at position 275; Ser at position 276; an amino acid deletion at position 276; and Thr at position 279, wherein the position number corresponds to that of the human B7-1 amino acid sequence (SEQ ID NO:278), and wherein said polypeptide has a hCTLA-4/hCD28 binding affinity ratio about equal to or greater than the hCTLA-4/hCD28 binding affinity ratio of human B7-1. The invention further provides an isolated or recombinant nucleic acid comprising a polynucleotide sequence selected from: (a) a polynucleotide sequence selected from SEQ ID NOS:253-262, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NOS:263-272, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which, but for codon degeneracy, hybridizes under highly stringent conditions over substantially the entire length of polynucleotide sequence (a) or (b) and encodes a polypeptide having a non naturally-occurring sequence; and (d) a polynucleotide sequence comprising all or a fragment of (a), (b), or (c), wherein the fragment encodes a polypeptide having (i) a non naturally-occurring sequence and (ii) a hCTLA-4/hCD28 binding affinity ratio about equal to or greater than the hCTLA-4/hCD28 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation or activation response about equal to or less than that of hB7-1. Also included is an isolated or recombinant nucleic acid comprising a polynucleotide sequence encoding a polypeptide comprising an amino acid sequence which is substantially identical over at least about 150 contiguous amino acid residues of any one of SEQ ID NOS:69-92, 222-247, 263-272, and 286-289. The invention also provides an isolated or recombinant nucleic acid comprising a nucleotide sequence coding for a polypeptide comprising the amino acid sequence set forth in any of SEQ ID NOS:69-92, 222-247, 263-272, and 286-289, or a subsequence thereof, wherein the subsequence comprises at least one of the signal sequence, ECD, transmembrane domain, and cytoplasmic domain of said polypeptide, and the amino acid sequence or subsequence is a non naturally-occurring sequence. In another aspect, the invention provides an isolated or recombinant nucleic acid encoding a polypeptide that has a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation and/or activation response about equal to or greater than the ability of hB7-1 to induce such response, produced by mutating or recombining at least one nucleic acids described above. Also included is an isolated or recombinant polypeptide comprising an amino acid sequence having the formula: MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPK-SVTKRVKETVM-X50-SCDY-X55-X56-STEELTSLRIYWQKDSKMVL AILPGKVQVWPEYKNRTITD-MNDNPRIVILALRLSD-X113-GTYTCV-X120-QK-X123-X124-X125-X126-G-X128-X129-X130-X131-EHL-X135-SV-X138-L-X140-IRADFPVPSITDIGHPAPNVK RIRCSASG-X170-FPEPRLAWMEDGEEL-NAVNTRV-X193-X194-X195-LDTELYSVSSELD-X209-N-X211-TNNHSIVCUKYGELSVSQIFPWSKPK QEPPIDQLPFWVI-X252-X253-VSGALVLTAVVLYCLACRHVAR (SEQ ID NO:290), or a subsequence thereof comprising the extracellular domain, wherein position X50 is Leu or Pro; position X55 is Asn or Ser; position X56 is Ala or Thr, position XI 13 is Ser or Lys; position X120 is Ile or Val; position X123 is Pro or deleted; position X124 is Val, Asn, or Asp; position X125 is Leu or Glu; position X126 is Lys or Asn; position X128 is Ala or Ser; position X129 is Tyr or Phe; position X130 is Lys or Arg; position X131 is Leu or Arg; position X135 is Ala or Thr; position X138 is Arg or Thr; position X140 is Met or Ser; position X170 is Asp or Gly; position X193 is Asp or is deleted; position X194 is Gln or is deleted; position X195 is Asp or is deleted; position X211 is Val or Ala; position X252 is Ile or Val; and position X253 is Leu or Pro. The polypeptide may comprise a sequence of any of SEQ ID NOS:59, 62, 180, 184, 188, 195, 196, 200, 201, 204, 211, 213, 219, and 291. Some such polypeptides have a hCD28/hCTLA-4 binding affinity ratio about equal to or greater than that of hB7-1, and/or an ability to induce T-cell proliferation and/or activation about equal to or greater than that induced by hB7-1. Another feature of the invention is an isolated or recombinant polypeptide comprising an amino acid sequence according to the formula: MGHTMKWG-X9-LPPKRPCLWLSQLLVLTGLFYFCSG-X35-TPKSVTKRV KETVMLSCDY-X55-TSTEELTSLRIYWQKDSKMVLALPGKVQVW PEYKNRTITDMNDNPRIVILALR-X110-SDSGTYTCVIQKP-X124-LKGAYKLEHL-X135-SVRLMIRADFPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENG-X183-ELNATNTT-X192-SQDPETKLYMISSELDFN-X211-TSN-X215-X216-X217-LCLVKYGDLTVSQ-X231-FYWQESKPTPSANQHLTWTIPVSAFGISVIIAVI LTCLTCRNAAIRRQRRENEV-X288-M-X290-SCSQSP (SEQ ID NO:292), or a subsequence thereof comprising the extracellular domain, wherein position X9 is Thr or Ser; position X35 is lie or Thr; position X55 is Asn or Ser; position X110 is Leu or Pro; position X124 is Asp or Val; position X135 is Thr or Ala; position X183 is Lys or Glu; position X192 is Leu or Val; position X211 is Met or Thr; position X215 is His or is deleted; position X216 is Ser or is deleted; position X217 is Phe or is deleted; position X231 is Thr or Ser; position X288 is Lys or Glu; position X290 is Glu or Gln, and wherein said amino acid sequence is a non naturally-occurring sequence. Some such polypeptides have a hCD28/hCTLA-4 binding affinity ratio about equal to or greater than that of hB7-1, and/or an ability to induce T-cell proliferation and/or activation about equal to or greater than that induced by hB7-1. The invention includes an isolated or recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:93 or SEQ ID NO:94, or a subsequence thereof, wherein the subsequence comprises at least one of the signal sequence, ECD, transmembrane domain, and cytoplasmic domain of said polypeptide. Also provided is an isolated or recombinant nucleic acid comprising a polynucleotide sequence selected from: (a) a polynucleotide sequence selected from SEQ ID NO:46 or SEQ ID NO:47, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NO:93, SEQ ID NO:94, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence encoding a subsequence of a polypeptide selected from SEQ ID NO:93, SEQ ID NO:94, or a complementary polynucleotide sequence thereof, wherein the subsequence comprises at least one of: the signal peptide sequence, extracellular domain, transmembrane domain, and cytoplasmic domain of the polypeptide. Some such nucleic acids encode a polypeptide having a hCTLA-4/hCD28 binding affinity ratio about equal to or greater than that of hB7-1 and/or an ability to induce a T-cell proliferation or activation response about equal to or greater than that of hB7-1. In another aspect, the invention provides a polypeptide which is specifically bound by a polyclonal antisera raised against at least one antigen, the at least one antigen comprising the polypeptide sequence selected from any of SEQ ID NOS:48-94, 174-252, 263-272, 283-293, or a fragment thereof, wherein the antisera is subtracted with one or more (and optionally all) polypeptides encoded by one or more of the sequences set forth at GenBank Nucleotide Accession Nos: A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950. Some such polypeptides have a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1, and/or an ability to induce a T-cell proliferation or T-cell activation response about equal to or greater than the ability of hB7-1 to induce said response. The invention further includes an antibody or antisera produced by administering any NCSM polypeptide described above to a mammal, which antibody specifically binds at least one antigen, the antigen comprising a polypeptide comprising at least one amino acid sequence of any of SEQ ID NOS:48-94, 174-252, 263-272, and 283-293, which antibody does not specifically bind to a polypeptide encoded by at least one (optionally all) of the sequences at GenBank Nucleotide Accession Nos: A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950. The invention provides an antibody or antisera which specifically binds a polypeptide which comprises any sequence selected from any of SEQ ID NOS:48-94, 174-252, 263-272, and 283-293, wherein the antibody or antisera does not specifically bind to at least one (optionally all) polypeptide encoded by at least one of GenBank Nucleotide Accession Nos: A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950. The antibodies are, e.g., polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments, fragments produced by an Fab expression library, or the like. In another aspect, the invention provides a nucleic acid which comprises a unique subsequence in a nucleic acid selected from SEQ ID NOS:147, 95-173, and 253-262, wherein the unique subsequence is unique as compared to at least one (optionally all) nucleic acid corresponding to any of GenBank Nucleotide Accession Nos.: A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950. The invention also includes a polypeptide which comprises a unique subsequence in a polypeptide selected from: SEQ ID NOS:48-94, 174-252, 263-272, and 283-293, wherein the unique subsequence is unique as compared to at least one (optionally all) polypeptide encoded by any of GenBank Nucleotide Accession Nos. shown above. The invention includes a target nucleic acid which, but for nucleotide codon degeneracy, hybridizes under stringent conditions to a unique coding oligonucleotide that encodes a unique subsequence in a polypeptide selected from SEQ ID NOS:48-94, 174-252, 263-272, and 283-293, wherein the unique subsequence is unique as compared to at least one (optionally all) polypeptide encoded by any of GenBank Nucleot. Access. Nos.: A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950. The invention also includes compositions comprising any polypeptide and/or polynucleotide described herein in an excipient, preferably a pharmaceutically acceptable excipient. In one aspect, the invention provides compositions comprising an isolated or recombinant NCSM polypeptide comprising the amino acid sequence SEQ ID NOS:48-68, 174-221, 283-285, 290-293, or a costimulatory fragment thereof, wherein said costimulatory fragment has a hCD28/hCTLA-4 binding affinity ratio about equal to or greater than the hCD28/hCTLA-4 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation or activation response about equal to or greater than that of hB7-1, and a carrier or excipient. Compositions comprising an isolated or recombinant NCSM polypeptide comprising the amino acid sequence of SEQ ID NOS:69-92, 222-247, 263-272, 286-289, or a costimulatory fragment thereof, wherein said costimulatory fragment has a hCTLA-4/hCD28 binding affinity ratio about equal to or greater than the hCTLA-4/hCD28 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation or activation response about equal to or less than that of hB7-1, and a carrier are a feature of the invention. Also included are isolated or recombinant polypeptides, each comprising an amino acid sequence corresponding to an ECD sequence, the amino acid sequence having at least about 92%, 93%, 94%, 95% amino acid sequence identity to a sequence corresponding to the ECD sequence of SEQ ID NO:66, said polypeptide having a hCD28/hCTLA-4 receptor binding affinity ratio at least about equal to or greater than the hCD28/hCTLA-4 binding annnity ratio of WT human B7-1. Some such polypeptides comprise a sequence having at least about 80, 85, or 90% identity to substantially the entire length of SEQ NO:66, and/or further comprise at least one further amino acid sequence comprising a signal peptide (positoned N terminal) and/or TMD and/or CD (both positioned C terminal), and/or linked or bound to a cell membrane, crosslinked with another molecule, and/or more than one such polypeptide (multimer of the polypeptide). Also included are isolated or recombinant polypeptide variants, each comprising an amino acid sequence that differs from the amino acid sequence of a primate (or mammalian) B7-1, wherein the difference between the amino acid sequence of the variant and the amino acid sequence of the primate B7-1 is a different amino acid at amino acid residue position 65 other than Ala, wherein the position corresponds to the position in the amino acid sequence of hB7-1 (SEQ ID NO:278). The different amino acid may comprise His, Arg, Lys, Pro, Phe, or Trp, and the primate (mammalian) B7-1 may be hB7-1. Some such polypeptide variants have a CTLA-4/CD28 binding affinity ratio greater than that of hB7-1 and/or an ability to induce a T cell proliferation less than induced by hB7-1. The invention also includes an isolated or recombinant nucleic acid comprising a polynucleotide sequence encoding a polypeptide, where the polypeptide comprises an amino acid sequence which is substantially identical over at least 175 contiguous amino acids of any one of those NCSM polypeptide sequences listed. In various embodiments, the encoded polypeptide comprises at least about 100, 150, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 220, 225, 230, 240, 250, 260, 265, 270, 275, or 285 or more contiguous amino acid residues or substantially identical variants of any one of the polypeptide sequences listed, or encoded by any nucleic acid listed. These polypeptides can exist separately or as components of one of more fusion proteins. The invention also includes a cell comprising any nucleic acid described herein, or which expresses any polypeptide or nucleic acid noted herein. In one embodiment, the cell expresses a polypeptide encoded by the nucleic acids herein. The invention also includes a vector comprising any nucleic acid of the invention. The vector can comprise a plasmid, a cosmid, a phage, or a virus or a virus-like particle (VLP) (or virus fragment); the vector can be, e.g., an expression vector, a cloning vector, a packaging vector, an integration vector, or the like. The invention also includes a cell transduced by the vector. The invention also includes compositions comprising any nucleic acid described herein, and an excipient, preferably a pharmaceutically acceptable excipient. Cells and transgenic animals that include any polypeptide or nucleic acid herein, e.g., produced by transduction of the vector, are also a feature of the invention. The invention also includes compositions produced by digesting one or more of the nucleic acids described herein with a restriction endonuclease, an RNAse, or a DNAse; and, compositions produced by incubating one or more nucleic acids described herein in the presence of deoxyribonucleotide triphosphates and a nucleic acid polymerase, e.g., a thermostable polymerase. The invention also includes compositions comprising two or more nucleic acids described herein. The composition may comprise a library of nucleic acids, where the library contains at least 5, 10, 20 or 50 or more nucleic acids. In another aspect, the invention includes an isolated or recombinant polypeptide encoded by any nucleic acid described herein. In one embodiment, the polypeptide may comprise a sequence selected from any of SEQ ID NOS:48-94, 174-252, 263-272, and 283-293. These sequences and fragments thereof can be present separately or as components of larger proteins such as fusion proteins. Any polypeptide described herein optionally can effect or alter an immune response, e.g., either induce or inhibit proliferation or activation of T cells. In other embodiments, any polypeptide described above can bind preferentially either CD28 or CTLA-4 or both CD28 and CTLA-4 as described herein. In other embodiments, any polypeptide described herein optionally can enhance or limit cytokine production as described herein. Nucleotides encoding any such polypeptides having these properties are also a feature of the invention. In one class of embodiments, any polypeptide described herein may further include a secretion signal or localization signal sequence, e.g., a signal sequence, an organelle targeting sequence, a membrane localization sequence, and the like. Any polypeptide described herein may further include a sequence that facilitates purification, e.g., an epitope tag (such as, e.g., a FLAG epitope), a polyhistidine tag, a GST fusion, and the like. The polypeptide optionally includes a methionine at the N-terminus. Any polypeptide described herein optionally includes one or more modified amino acids, such as a glycosylated amino acid, a PEG-ylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, an acylated amino acid, or the like. Any polypeptide described herein further may be incorporated into a fusion protein, e.g., a fusion with an immunoglobulin (Ig) sequence. Methods for producing the polypeptides of the invention are also included. One such method comprises introducing into a population of cells any NCSM nucleic acid described herein, which is operatively linked to a regulatory sequence effective to produce the encoded polypeptide, culturing the cells in a culture medium to produce the polypeptide, and isolating the polypeptide from the cells or from the culture medium. Another such method comprises introducing into a population of cells a recombinant expression vector comprising any NCSM nucleic acid described herein; administering the expression vector into a mammal; and isolating the polypeptide from the mammal or from a byproduct of the mammal. Also included is a method of treating an autoimmune or allergic disorder in a subject in need of such treatment by administering to the subject an effective amount of any NCSM polypeptide (or polynucleotide or expression vector encoding such polypeptide) described herein. NCSM polypeptides and nucleic acids include, respectively, mammalian B7-1 and B7-2 polypeptide and nucleic variants described below. In various embodiments, the autoimmune disorder may be multiple sclerosis, rheumatoid arthritis, lupus erythematosus, type I diabetes, psoriasis and the like. The invention also includes a method of enhancing or reducing an immune response in a subject, such as either by inducing or inhibiting T cell proliferation or activation, by administration of at least one NCSM polypeptide and/or NCSM polynucleotide described herein to a population of cells. The population of cells to which the nucleic acid or polypeptide is administered can be in vivo, ex vivo, or in vitro (e.g., cultured cells). The invention includes a method of inducing, modifying, or inhibiting T-cell proliferation, the method comprising contacting a population of T cells with a polypeptide or nucleic acid of the invention, thereby inducing, modifying, or inhibiting, respectively, proliferation of the T cells (relative to the response generated by WT hB7-1). Polypeptides that induce such T cell proliferation include CD28BP polypeptides, including full-length and membrane-bound or associated CD28BP polypeptides and crosslinked or multimeric CD28BP-ECD polypeptides; polypeptides that inhibit T cell proliferation include the CTLA-4BP polypeptides and B7-1 and B7-2 polypeptide variants and soluble monomeric CD28BP-ECD-Ig and CD28BP-ECD polypeptides discussed herein. The invention also includes, in a method of treating a disorder or medical condition treatable by administration of NCSM polypeptides (or fragments thereof) or NCSM polynucleotides (or fragments thereof) to a subject, an improvement comprising administering to the subject an effective amount of a NCSM polypeptide and/or nucleic acid (or fragments thereof) described herein. The disorder, disease, or medical condition treatable by administration of NCSM polypeptides and/or nucleic acids (or fragments thereof, including soluble NCSMs, crosslinked soluble NCSMs, non-crosslinked NCSMs, and fusion proteins and vectors encoding them) may be, but is not limited to, e.g., chronic disease, autoimmune disorder, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, type I diabetes, psoriasis, AIDS or AIDS-related complexes, allogeneic or xendgeneic grafts or transplants, a variety of cancers, viral and/or bacterial infections, or the like. The type and form of the NCSM polypeptide or nucleic acid administered depends on the specific disorder, disease or condition to be treated as discussed in detail below. Also included is a method of therapeutic or prophylactic treatment of a disease or disorder in a subject in need of such treatment, comprising administering to the subject at least one of any NCSM polypeptide or nucleic acid described herein and at least one immunogen specific for said disease or disorder, wherein the combined amount of the at least one polypeptide or nucleic acid and the at least one immunogen is effective to prophylactically or therapeutically treat said disease or disorder. In yet another aspect, the invention includes a method of enhancing, diminishing, modifying, or potentiating an immune response in a subject, comprising: directly administering to the subject at least one polynucleotide comprising any NCSM nucleic acid sequence described herein, operably linked to a promoter sequence that controls the expression of said nucleic acid sequence, said polynucleotide being present in an amount sufficient that uptake of said polynucleotide into one or more cells of the subject occurs and sufficient expression of said nucleic acid sequence results to produce an amount of a polypeptide effective to enhance, diminish, or modify an immune response. Such some methods further comprise administering to the subject an antigen specific for the disease or disorder, wherein the at least one polynucleotide is administered to the subject in an amount sufficient to modulate the immune response induced in the subject by the antigen. In some such methods, the at least one polynucleotide further comprises a nucleotide sequence encoding for an antigen (e.g., cancer antigen, such as EpCam or an EpCam variant). The at least polynucleotide may comprise or be included in a vector. In some such methods, the at least one polynucleotide further comprises at least one additional nucleotide sequence encoding a cytokine (e.g., GM-CSF), adjuvant, co-stimulatory polypeptide, or at least one additional nucleotide sequence comprising a promoter or other regulatory sequence or marker sequence useful in a plasmid vector. In some such methods, the subject is a mammal, e.g., primate or human. In another aspect, the invention provides a method of modulating or altering a T-cell response specific to an antigen in a subject, the method comprising administering to the subject at least one first polynucleotide sequence encoding a CD28BP polypeptide (including, e.g., a polypeptide comprising any of SEQ ID NOS:48-94, 174-252, 263-272 and 283-293 or a B7-1 polypeptide variant, such as bovine B7-1 variant described herein), or co-stimulatory fragment thereof that has an ability to modulate or alter a T-cell response, and a second polynucleotide sequence encoding the antigen or antigenic fragment thereof, wherein each of the at least one first and second polynucleotide sequences is expressed in the subject in an amount effective to modulate or alter a T cell response. The antigen may comprise a cancer antigen, bacterial antigen, antigen of an infectious agent, viral antigen, allergen, parasitic antigen, etc. Following administration, the first and second polynucleotides transfect the subjects' cells and are expressed. The at least first polynucleotide sequence may comprise a polynucleotide sequence selected from the group of SEQ ID NOS:147, 95-173, and 253-262 or a polynucleotide sequence encoding a B7-1 variant of the invention, or a nucleotide fragment of any polynucleotide sequence of this group that encodes a polypeptide fragment able to modulate or alter T cell response. The first and second polynucleotide sequences may be administered separately on two plasmid monocistronic vectors or together via one bicistronic vector that includes the first and second polynucleotide sequences. The T cell proliferation or activation response induced by administration of the at least first polynucleotide (in conjunction with the antigen-encoding polynucleotide sequence) is typically greater than that induced by hB7-1 under similar conditions if the polypeptide encoded by the first polynucleotide sequence is expressed on the cell membrane and remains linked to or associated with the cell membrane (or, alternatively, administered as a multimer or crosslinked molecule). When bound to a cell membrane upon expression, the expressed polypeptide usually comprises at least a TMD and ECD, and often substantially the entire length of the NCSM polypeptide sequence, including the CD. In such instances, depending upon the dose given, the enhanced T cell response is usually sufficient to eliminate cells bearing the antigen or antigenic fragment thereof. The T cell proliferation or activation response induced by administration of the at least first polynucleotide (in conjunction with the antigen-encoding polynucleotide sequence) may be inhibited and thus less than that induced by hB7-1 under similar conditions if the polypeptide encoded by the first polynucleotide sequence is secreted from the cell and does not remain bound to or associated with the cell membrane (e.g., forms a soluble monomeric polypeptide). Also included is a method of modulating or altering an immune response in a subject, the method comprising introducing into cells of a tumor of the subject at least one polynucleotide sequence encoding a polypeptide comprising any of SEQ ID NOS:48-94, 174-252, 263-272 and 283-293, or co-stimulatory fragment thereof that is able to modulate or alter an immune response, wherein the polypeptide or fragment thereof interacts with or binds to a T cell receptor when expressed in a subject, and wherein the at least one polynucleotide sequence is operably linked to a promoter for expression in the subject and is present in an amount sufficient that when expressed is effective to modulate or alter a T cell response. In addition, the invention includes a vector comprising at least one first polynucleotide sequence encoding a polypeptide comprising any of SEQ ID NOS:48-94, 174-252, 263-272 and 283-293, or co-stimulatory fragment thereof capable of modulating or altering a T cell response, wherein the polypeptide or co-stimulatory fragment thereof interacts with or binds to a T cell receptor when expressed in a subject, wherein the at least one first polynucleotide sequence is operably linked to a promoter for expression in the subject and is present in an amount sufficient that when expressed is effective to modulate or alter a T cell response. The invention also provides a vector comprising at least one first polynucleotide sequence encoding a polypeptide comprising any of SEQ ID NOS:48-94, 174-252, 263-272 and 283-293, or co-stimulatory fragment thereof capable or modulating or altering a T cell response, and a second polynucleotide sequence encoding the antigen or antigenic fragment thereof, wherein the NCSM polypeptide or co-stimulatory fragment thereof interacts with or binds to a T cell receptor when expressed in a subject, and wherein each of the at least one first and second polynucleotide sequences is operably linked to a promoter for expression in the subject and is present in an amount sufficient that when expressed is effective to modulate or alter a T cell response or, optionally, to induce a sufficient T cell proliferation response such that one or more cells expressing the antigen or fragment thereof are eliminated or destroyed. Such at least one first polynucleotide sequence may comprise a sequence from the group of SEQ ID NOS:1-47, 95-173, and 253-262, or a nucleotide fragment of any nucleotide sequence of this group that encodes a co-stimulatory polypeptide able to induce a T cell proliferation or activation response equal to or greater than that induced by hB7-1. In general, nucleic acids and proteins derived by mutation, recursive sequence recombination (RSR) or other alterations of the sequences herein are a feature of the invention. Similarly, those produced by recombination, including recursive sequence recombination, are a feature of the invention. Mutation and recombination methods using the nucleic acids described herein are a feature of the invention. For example, one method of the invention includes recombining one or more nucleic acids described herein with one or more additional nucleic acids (including, but not limited to those noted herein), the additional nucleic acid encoding a NCSM polypeptide, co-stimulatory homologue or subsequence thereof. The recombining steps are optionally performed in vivo, ex vivo, or in vitro. Also included in the invention are a recombinant nucleic acid produced by this method, a cell containing the recombinant nucleic acid, a nucleic acid library produced by this method comprising recombinant polynucleotides, and a population of cells containing the library comprising recombinant polynucleotides. Also included is a method of designing or identifying agonists and antagonists of CD28 and CTLA-4 (which either enhance or inhibit signaling through CD28 or CTLA-4) based on the 3-dimensional structure of the polypeptides of the invention (e.g., SEQ ID NOS:48-94, 174-252, 263-272, and 283-293). The invention also includes soluble polypeptides and proteins (including fusion polypeptides and proteins) and nucleic acids encoding such soluble polypeptides and proteins. The invention also includes the use of such polypeptides and proteins as therapeutics, prophylactics, and diagnostics in therapeutic treatment and/or prevention of a variety of diseases and conditions. Soluble polypeptides and proteins (and nucleic acids encoding them) include, e.g., extracellular domain (ECD) amino acid sequences of each NCSM (e.g., each CTLA-4 binding protein and CD28 binding protein) described herein (or fragments thereof) and nucleic acids encoding same, as well as constructs comprising, e.g., each of said ECD, or fragments thereof, with an Ig polypeptide sequence (or fragment or variant thereof) (and nucleotide sequences encoding same) as fusion proteins. Some such soluble polypeptides and proteins exhibit a hCD28/hCTLA-4 binding affinity ratio that is greater than that of hB7-1 and/or have an ability to induce a T cell proliferation response, in the presence of activated T cells, that is less than that capable of being induced by soluble hB7-1 polypeptide in the presence of activate T cells. In another aspect, the invention provides a computer or computer readable medium comprising a database comprising a sequence record comprising one or more character strings corresponding to a nucleic acid or protein sequence selected from any of SEQ ID NOS:1-272 and 283-293. The invention further includes an integrated system comprising a computer or computer readable medium comprising a database comprising one or more sequence records, each comprising one or more character strings corresponding to a nucleic acid or protein sequence selected from any of SEQ ID NOS:1-272 and 283-293, the integrated system further comprising a user input interface allowing a user to selectively view one or more sequence records. Also provided are methods of using a computer system to present information pertaining to at least one of a plurality of sequence records stored in a database, said sequence records each comprising one or more character strings corresponding to any of SEQ ID NOS: 1-272 and 283-293. These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures. |
Beehive base device |
A device positioned beneath a beehive for use in controlling parasites in beehives including a base structure and a number of bars extending across the base structure. The bars are held spaced from one another by crosspieces engaged with the bars and the base structure. The bars are spaced from one another to enable waste and parasites to fall between the bars. The bars are formed of a low-adhesion material to prevent the parasites from being able to climb onto the bars and back into the beehive. |
1. A device (102) for a base for a beehive (101) comprising a framing (113, 114) wherein the section corresponds to the horizontal section of the beehive, the interior of the framing is hollow and comprises, under a honeycomb (108) of the beehive and parallel to this honeycomb, a bar (115), the lateral proximity of the bar being pierced, characterized in that the bar is fixed to the framing by at least one crosspiece (116, 117). 2. The device according to claim 1, characterized in that the bar is fixed to the framing by two crosspieces. 3. The device according to one of claims 1 or 2, characterized in that a lateral edge of the framing comprises a slot (201 to 204) for keeping a crosspiece in place. 4. The device according to one of claims 1 to 3, characterized in that a crosspiece comprises at least one aperture (304) corresponding to the section of a bar. 5. The device according to one of claims 1 to 4, characterized in that the device comprises several parallel bars. 6. The device according to claim 5, characterized in that the spacing between the bars is between 1 to 5 millimeters. 7. The device according to one of claims 1 to 6, characterized in that the section of a bar is circular. 8. The device according to one of claims 1 to 7, characterized in that the bars are manufactured of a low-adhesion material, preferably polyethylene. 9. The device according to one of claims 1 to 8, characterized in that the device comprises an alignment crosspiece (118), not fixed to the framing. 10. The device according to one of claims 1 to 9, characterized in that one of the edges (113) of the framing comprises a takeoff platform (112). 11. The device according to one of claims 1 to 10, characterized in that the device is placed under a beehive. |
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