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External skin compositions |
The external skin care composition of the present invention comprises N-acetylglucosamine and at least one member selected from the group consisting of retinoid and pro-vitamin A. The external skin care composition of the present invention has an effect of promoting the production of epidermal hyaluronic acid and can retain firmness and moisture of the skin. |
1-6. (canceled) 7. An external skin care composition comprising N-acetylglucosamine and at least one member selected from the group consisting of retinoid and pro-vitamin A. 8. The external skin care composition according to claim 7, wherein said retinoid is at least one member selected from the group consisting of retinoic acid, retinal, retinol, fatty acid retinyl ester, dehydroretinal, dehydroretinol and fatty acid dehydroretinyl ester. 9. The external skin care composition according to claim 7, wherein said retinoid is at least one member selected from the group consisting of retinoic acid, retinol and fatty acid retinyl. 10. The external skin care composition according to claim 8 or 9, wherein said retinoic acid is at least one member selected from the group consisting of all-trans-retinoic acid, 13-cis-retinoic acid, 11-cis-retinoic acid, 9-cis-retinoic acid and 3,4-dehydro-retinoic acid. 11. The external skin care composition according to claim 8 or 9, wherein said retinoic acid is at least one member selected from the group consisting of all-trans-retinoic acid and 13-cis retinoic acid. 12. The external skin care composition according to claim 8 or 9, wherein said retinol is at least one member selected from the group consisting of all-trans-retinol, 13-cis-retinol, 11-cis-retinol, 9-cis-retinol and 3,4-dehydro-retinol. 13. The external skin care composition according to claim 8 or 9, wherein said retinol is at least one member selected from the group consisting of all-trans-retinol and 13-cis-retinol. 14. The external skin care composition according to claim 8 or 9, wherein said fatty acid retinyl ester is at least one member selected from the group consisting of retinyl palmitate, retinyl formate, retinyl acetate, retinyl propionate, butyric acidretinyl, retinyl valerate, retinyl isovalerate, retinyl hexanoate, retinyl heptanoate, retinyl octanoate, retinyl nonanoate, retinyl decanoate, retinyl undecanoate, retinyl laurate, retinyl tridecanoate, retinyl myristate, retinyl pentadecanoate, retinyl heptadecanoate, stearic acidretinyl, isostearic acidretinyl, retinyl nonadecanoate, retinyl arachidonate, retinyl arachidonate, retinyl linoleate and retinyl oleate. 15. The external skin care composition according to claim 8 or 9, wherein said fatty acid retinyl ester is at least one member selected from the group consisting of retinyl palmitate, retinyl acetate and retinyl propionate. 16. The external skin care composition according to claim 7, wherein said pro-vitamin A is selected from the group consisting of αcarotene, βcarotene, γcarotene, cryptoxanthin and echinenone. 17. The external skin care composition according to claim 7, which contains N-acetylglucosamine in the amount of 0.001 to 10% by mass based on the total amount of said composition. 18. The external skin care composition according to claim 7, which contains N-acetylglucosamine in the amount of 0.01 to 5% by mass based on the total amount of said composition. 19. The external skin care composition according to claim 7, which contains retinoid and/or pro-vitamin A in the amount of 0.0001 to 10% by mass based on the total amount of said composition. 20. The external skin care composition according to claim 7, which contains retinoid and/or pro-vitamin A in the amount of 0.01 to 1% by mass based on the total amount of said composition. 21. The external skin care composition according to claim 7, which contains N-acetylglucosamine in the amount of 0.001 to 10% by mass based on the total amount of said composition and retinoid and/or pro-vitamin A in the amount of 0.0001 to 10% by mass based on the total amount of said composition. 22. The external skin care composition according to claim 7, which contains N-acetylglucosamine in the amount of 0.01 to 5% by mass based on the total amount of said composition and retinoid and/or pro-vitamin A the amount of 0.01 to 1% by mass based on the total amount of said composition. 23. The external skin care composition according to claim 7, which is a cosmetic composition. 24. A makeup method for preventing or improving wrinkled skin, dry skin, tanned skin or aged skin, which comprises applying the external skin care composition according to claim 7 onto the skin. |
<SOH> BACKGROUND ART <EOH>Hyaluronic acid has various functions such as retention of moisture in intercellular spaces, retention of cell structures by formation of a jelly-like matrix, retention of humidity and elasticity of the skin, resistance to an external force such as mechanical disorder, and prevention of bacterial infection (BIO INDUSTRY, Vol. 8, page 346, 1991). It has been reported that the intensity of the staining signal of hyaluronic acid in the epidermis is reduced with aging (J. Invest. Dermatol., 102, 385, 1994), and that hyaluronic acid at solar elastosis site under irradiation with ultraviolet light is scarcely detected (Clin. Dermatol, (special number 5) 51, 53, 1997; Nagoya Med. J., 41, 27, 1997), thus causing dry skin and deterioration of firmness and elasticity of the skin, resulting in increase of wrinkles. To improve these skin conditions, a method of retaining moisture on the surface of the skin by applying a cosmetic composition containing hyaluronic acid formulated therein has been employed. However, since hyaluronic acid, as a large polymer, can not penetrate into the skin, a drastic improvement can not be expected. Therefore, it is expected to develop a substance capable of drastically improving cutaneous functions by promoting a cellular ability of production of hyaluronic acid. As a substance capable of promoting the production of hyaluronic acid in epidermis, retinoic acid has been known so far. Retinoic acid is an essential substance which intrinsically exists in the epidermis and plays an important roles in the growth and differentiation of epidermal cells. Retinoic acid has widely used as an agent for restoring skin characteristics and an agent for reintegration of the skin in foreign countries in order to treat various dermatopathies, for example, acne vulgaris, fine wrinkles, psoriasis and age spots. Various reports with respect to the effect of retinoic acid on (photo)aging have been made and its improving effect on the formation of fine wrinkles is recognized (Plastic Surgery, 42: 801, 1999; J. Dematol., 122, 91, 1990). Also it has been reported that deposition of mucopolysaccharides such as hyaluronic acid increases and the histological change of the photoaged skin is improved by applying retinoic acid (J. Dermatol. Sci., 11, 177, 1996). Therefore, it is considered that the deposition of hyaluronic acid, as an epidermal matrix component, and an increase in moisture achieved thereby may contribute remarkably to the effect of smoothing the skin surface of retinoic acid (The Japanese Journal of Dermatology, Vol. 110, No. 12, 1878, 2000) and an epidermal hyaluronic acid production promoting ingredient is useful for anti-wrinkling (prevention of formation of wrinkles or improvement of wrinkles) (FRAGRANCE JOURNAL, 4, 49, 1998). However, retinoic acid causes skin irritation and it is required to formulate an external preparation containing low-concentration retinoic acid in order to prevent skin irritation. On the other hand, retinol or retinyl ester with less irritation must be metabolized in vivo into retinoic acid, as an activator, and it has exerts a smaller effect as compared with retinoic acid when the skin is benefitted. Therefore, it has been required to develop an external skin care ingredient which does not cause side effect such as skin irritation while maintaining the effect of retinoic acid. The present invention is based on such finding that a combination of retinoid and N-acetylglucosamine gives a synergistic improvement in the synthesis of hyaluronic acid of keratinocytes (epidermal cells). Under these circumstances, an object of the present invention is to provide an external skin care ingredient which exerts a synergistic effect of promoting the production of hyaluronic acid by using in combination with retinoid. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a graph showing a synergistic effect of promoting the production of hyaluronic acid in human keratinocytes by using N-acetylglucosamine in combination with various retinoids. FIG. 2 is a graph showing a synergistic effect of promoting the hyaluronic acid production in human keratinocytes by using N-acetylglucosamine in combination with â-carotene. detailed-description description="Detailed Description" end="lead"? |
Tread pattern for car tire |
A tyre for a motor car includes a tread and two shoulders. The tread includes at least one circumferential row of blocks disposed between first and second circumferential grooves. Each of the blocks is delimited by a section of the first circumferential groove and by first and second transverse grooves that extend from the first circumferential groove and converge at a common vertex spaced from the second circumferential groove. The common vertex is separated from the second circumferential groove by a continuous circumferential tread rib. A second transverse groove that delimits a first block is separated from a first transverse groove that delimits a circumferentially adjacent block by a solid tread portion. The solid tread portion extends from the first circumferential groove to the circumferential tread rib and forms a single body with the circumferential tread rib, spacing the first block and the circumferentially adjacent block from each other. |
1-18. (canceled) 19. A tyre for a motor car, comprising: a tread; and two shoulders; wherein the tread comprises: at least one circumferential row of blocks disposed between first and second circumferential grooves; wherein each of the blocks is delimited by a section of the first circumferential groove and by first and second transverse grooves that extend from the first circumferential groove and converge at a common vertex spaced from the second circumferential groove, wherein the common vertex is separated from the second circumferential groove by a continuous circumferential tread rib, wherein a second transverse groove that delimits a first block is separated from a first transverse groove that delimits a circumferentially adjacent block by a solid tread portion, wherein the solid tread portion extends from the first circumferential groove to the circumferential tread rib, and wherein the solid tread portion forms a single body with the circumferential tread rib, spacing the first block and the circumferentially adjacent block from each other. 20. The tyre of claim 19, wherein at least one of the first and second transverse grooves comprises an increasing width in a direction from the common vertex toward the first circumferential groove. 21. The tyre of claim 19, wherein at least one of the first and second transverse grooves further comprises: a first section comprising a direction substantially parallel to an equatorial plane of the tyre; a second section comprising a predefined inclination with respect to the first circumferential groove; and a linking section joining together the first and second sections. 22. The tyre of claim 19, wherein at least one of the first and second transverse grooves is sickle-shaped. 23. The tyre of claim 19, wherein at least one of the first and second transverse grooves comprises a median line formed by an arc of a circle comprising a predefined radius of curvature. 24. The tyre of claim 23, wherein the median line comprises: a first section comprising a direction substantially parallel to an equatorial plane of the tyre; a second section comprising a predefined inclination with respect to the first circumferential groove; and a linking section joining together the first and second sections. 25. The tyre of claim 19, wherein the first and second transverse grooves of the at least one row of blocks extend beyond the first circumferential groove and into an axially internal region of one of the shoulders. 26. The tyre of claim 19, wherein each block of the at least one row of blocks is shaped in a form of a shark's fin. 27. The tyre of claim 19, wherein the common vertices of the first and second transverse grooves of the at least one row of blocks comprise a same orientation in a longitudinal direction of the tyre. 28. The tyre of claim 19, wherein the tread comprises: a central circumferential row of blocks; and first and second lateral circumferential rows of blocks. 29. The tyre of claim 28, wherein common vertices of the first and second transverse grooves of the first lateral row of blocks comprise an orientation opposite to that of common vertices of the first and second transverse grooves of the central row and second lateral row of blocks. 30. The tyre of claim 28, wherein common vertices of the first and second transverse grooves of the three rows of blocks comprise a same orientation. 31. The tyre of claim 28, wherein the first and second transverse grooves of at least one of the first and second lateral rows of blocks extend beyond a respective first circumferential groove and into an axially internal region of one of the shoulders. 32. The tyre of claim 19, wherein the tread comprises: first and second lateral circumferential row of blocks; and two central circumferential ribs separated by a middle circumferential groove. 33. The tyre of claim 32, wherein common vertices of the first lateral row of blocks comprise an orientation opposite to that of common vertices of the second lateral row of blocks. 34. The tyre of claim 32, wherein the first and second transverse grooves of at least one of the first and second lateral rows of blocks extend beyond a respective first circumferential groove and into an axially internal region of one of the shoulders. 35. The tyre of claim 19, wherein at least one of the shoulders comprises, in an axially external region, pairs of additional transverse grooves converging toward a second common vertex. 36. The tyre of claim 35, wherein the additional transverse grooves are separated from the first circumferential groove by solid portions of elastomeric material. 37. The tyre of claim 36, wherein an axially internal region of a first shoulder comprises a void/solid ratio smaller than that of an axially internal region of a second shoulder, and wherein the first shoulder is positioned on an outer side of the motor car when the tyre is mounted on the motor car. 38. The tyre of claim 35, wherein the additional transverse grooves extend from the first circumferential groove. 39. The tyre of claim 38, wherein an axially internal region of a first shoulder comprises a void/solid ratio smaller than that of an axially internal region of a second shoulder, and wherein the first shoulder is positioned on an outer side of the motor car when the tyre is mounted on the motor car. |
Microchip pileup type chemical reaction system |
A microchip pileup type chemical reaction system characterized in that a specified number of microchips, each having a reaction material liquid introducing section, a reaction product liquid discharge section and a reaction region, i.e. microchannels, interconnected therewith, are laid integrally in layers, the same kind of reaction material is introduced from the reaction material liquid introducing section into each microchip and the same kind of reaction product is collected from the reaction product liquid discharge section. The novel system for high efficiency chemical reaction makes the most use of the feature of microspace where general organic synthesis reaction is performed in the microchips while enabling mass synthesis. |
1. A microchip pileup type chemical reaction system comprising a specified number of integrally laminated microchips each having reaction material liquid introducing sections, a reaction product liquid discharge section, and microchannels as regions communicating therewith, wherein the same kind of reaction materials are introduced from the reaction material liquid introducing sections into each microchip to perform the same kind of reaction in each reaction region microchannel, and the same kind of reaction product is collected from the reaction product liquid discharge section. 2. The microchip pileup type chemical reaction system according to claim 1, wherein the reaction material liquid introducing section of each microchip directly communicates with the reaction material liquid introducing section of the microchip laminated thereon or thereunder, or with the reaction material liquid introducing sections laminated thereon and thereunder. 3. The microchip pileup type chemical reaction system according to claim 1, wherein the reaction product liquid discharge section of each microchip directly communicates with the reaction product liquid discharge section of a microchip laminated thereon or thereunder, or with the reaction product liquid discharge sections laminated thereon and thereunder. 4. The microchip pileup type chemical reaction system comprising a specified number of integrally laminated microchip pileup members according to claim 1 integrally assembled in parallel. 5. The microchip pileup type chemical reaction system according to claim 2, wherein the reaction product liquid discharge section of each microchip directly communicates with the reaction product liquid discharge section of a microchip laminated thereon or thereunder, or with the reaction product liquid discharge sections laminated thereon and thereunder. 6. The microchip pileup type chemical reaction system comprising a specified number of integrally laminated microchip pileup members according to claim 2 integrally assembled in parallel. 7. The microchip pileup type chemical reaction system comprising a specified number of integrally laminated microchip pileup members according to claim 3 integrally assembled in parallel. |
<SOH> BACKGROUND ART <EOH>It has been actively developed in recent years to form microchannels as fine grooves having an width of 500 μm or less on a several centimeters square substrate, and to use these microchannels as chemical reaction regions. The present inventors have also noticed that the microchannel involves various advantages for highly efficient chemical reactions such as short molecular diffusion distances, large specific interface areas and small heat capacity, when the microspace of the liquid phase in the microchannel is considered to be a chemical reaction field. Accordingly, the present inventors have applied the microchannel reaction system to various intermolecular chemical reactions such as complexing reactions, solvent extraction, immunological reactions, enzyme reactions and ion-pair extraction reactions. Highly efficient chemical reactions are expected to be proceeded in such reaction field since substance transfer time is shortened, solid-liquid or liquid-liquid interface reactions become predominant, and heat energies are promptly transferred from or to the reaction system. However, few basic researches systematically investigating basic chemical reactions in the liquid phase in the micro-spaces have been reported today. While large scale synthesis rather than high efficiency should be particularly considered in usual organic synthesis reactions, substantially no relations between minute quantity of reactions in the microchip and large scale synthesis have been investigated. Accordingly, the object of the invention is to enable large scale synthesis in the usual organic synthesis reactions carried out in the microchip, while realizing highly efficient chemical reactions by taking advantage of the features of the microspace. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a perspective view showing an example of the system of the invention. FIG. 2 illustrates the modes of introduction of the material liquid and discharge of the product liquid in the example shown in FIG. 1 . FIG. 3 is a perspective view showing an example different from the example shown in FIGS. 1 and 2 . FIG. 4 is a perspective view showing a further different example. detailed-description description="Detailed Description" end="lead"? |
Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins, and uses thereof |
The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the transporter peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the transporter peptides, and methods of identifying modulators of the transporter peptides. |
1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids. 2. An isolated peptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids. 3. An isolated antibody that selectively binds to a peptide of claim 2. 4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in. SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e), a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d). 5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d). 6. A gene chip comprising a nucleic acid molecule of claim 5. 7. A transgenic non-human animal comprising a nucleic acid molecule of claim 5. 8. A nucleic acid vector comprising a nucleic acid molecule of claim 5. 9. A host cell containing the vector of claim 5. 10. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence. 11. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence. 12. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide. 13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample. 14. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide. 15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide. 16. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide. 17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor. 18. A method for treating a disease or condition mediated by a human transporter protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim 16. 19. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide. 20. An isolated human transporter peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence shown in SEQ ID NO:2. 21. A peptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence shown in SEQ ID NO:2. 22. An isolated nucleic acid molecule encoding a human transporter peptide, said nucleic acid molecule sharing at least 80percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or 3. 23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or 3. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Transporters Transporter proteins regulate many different functions of a cell, including cell proliferation, differentiation, and signaling processes, by regulating the flow of molecules such as ions and macromolecules, into and out of cells. Transporters are found in the plasma membranes of virtually every cell in eukaryotic organisms. Transporters mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of molecules and ion across cell membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, transporters, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122. Transporters are generally classified by structure and the type of mode of action. In addition, transporters are sometimes classified by the molecule type that is transported, for example, sugar transporters, chlorine channels, potassium channels, etc. There may be many classes of channels for transporting a single type of molecule (a detailed review of channel types can be found at Alexander, S. P. H. and J. A. Peters: Receptor and transporter nomenclature supplement. Trends Pharmacol. Sci., Elsevier, pp. 65-68 (1997) and http://www-biology.ucsd.edu/˜msaier/transport/titlepage2.html. The following general classification scheme is known in the art and is followed in the present discoveries. Channel-Type Transporters. Transmembrane channel proteins of this class are ubiquitously found in the membranes of all types of organisms from bacteria to higher eukaryotes. Transport systems of this type catalyze facilitated diffusion (by an energy-independent process) by passage through a transmembrane aqueous pore or channel without evidence for a carrier-mediated mechanism. These channel proteins usually consist largely of a-helical spanners, although b-strands may also be present and may even comprise the channel. However, outer membrane porin-type channel proteins are excluded from this class and are instead included in class 9. Carrier-Type Transporters. Transport systems are included in this class if they utilize a carrier-mediated process to catalyze uniport (a single species is transported by facilitated diffusion), antiport (two or more species are transported in opposite directions in a tightly coupled process, not coupled to a direct form of energy other than chemiosmotic energy) and/or symport (two or more species are transported together in the same direction in a tightly coupled process, not coupled to a direct form of energy other than chemiosmotic energy). Pyrophosphate Bond Hydrolysis-Driven Active Transporters. Transport systems are included in this class if they hydrolyze pyrophosphate or the terminal pyrophosphate bond in ATP or another nucleoside triphosphate to drive the active uptake and/or extrusion of a solute or solutes. The transport protein may or may not be transiently phosphorylated, but the substrate is not phosphorylated. PEP-Dependent, Phosphoryl Transfer-Driven Group Translocators. Transport systems of the bacterial phosphoenolpyruvate:sugar phosphotransferase system are included in this class. The product of the reaction, derived from extracellular sugar, is a cytoplasmic sugar-phosphate. Decarboxylation-Driven Active Transporters. Transport systems that drive solute (e.g., ion) uptake or extrusion by decarboxylation of a cytoplasmic substrate are included in this class. Oxidoreduction-Driven Active Transporters. Transport systems that drive transport of a solute (e.g., an ion) energized by the flow of electrons from a reduced substrate to an oxidized substrate are included in this class. Light-Driven Active Transporters. Transport systems that utilize light energy to drive transport of a solute (e.g., an ion) are included in this class. Mechanically-Driven Active Transporters. Transport systems are included in this class if they drive movement of a cell or organelle by allowing the flow of ions (or other solutes) through the membrane down their electrochemical gradients. Outer-Membrane Porins (of b-Structure). These proteins form transmembrane pores or channels that usually allow the energy independent passage of solutes across a membrane. The transmembrane portions of these proteins consist exclusively of b-strands that form a b-barrel. These porin-type proteins are found in the outer membranes of Gram-negative bacteria, mitochondria and eukaryotic plastids. Methyltransferase-Driven Active Transporters. A single characterized protein currently falls into this category, the Na+-transporting methyltetrahydromethanopterin:coenzyme M methyltransferase. Non-Ribosome-Synthesized Channel-Forming Peptides or Peptide-like Molecules. These molecules, usually chains of L- and D-amino acids as well as other small molecular building blocks such as lactate, form oligomeric transmembrane ion channels. Voltage may induce channel formation by promoting assembly of the transmembrane channel. These peptides are often made by bacteria and fungi as agents of biological warfare. Non-Proteinaceous Transport Complexes. Ion conducting substances in biological membranes that do not consist of or are not derived from proteins or peptides fall into this category. Functionally Characterized Transporters for which Sequence Data are Lacking. Transporters of particular physiological significance will be included in this category even though a family assignment cannot be made. Putative Transporters in which no Family Member is an Established Transporter. Putative transport protein families are grouped under this number and will either be classified elsewhere when the transport function of a member becomes established, or will be eliminated from the TC classification system if the proposed transport function is disproven. These families include a member or members for which a transport function has been suggested, but evidence for such a function is not yet compelling. Auxiliary Transport Proteins. Proteins that in some way facilitate transport across one or more biological membranes but do not themselves participate directly in transport are included in this class. These proteins always function in conjunction with one or more transport proteins. They may provide a function connected with energy coupling to transport, play a structural role in complex formation or serve a regulatory function. Transporters of unknown Classification. Transport protein families of unknown classification are grouped under this number and will be classified elsewhere when the transport process and energy coupling mechanism are characterized. These families include at least one member for which a transport function has been established, but either the mode of transport or the energy coupling mechanism is not known. Ion Channels An important type of transporter is the ion channel. Ion channels regulate many different cell proliferation, differentiation, and signaling processes by regulating the flow of ions into and out of cells. Ion channels are found in the plasma membranes of virtually every cell in eukaryotic organisms. Ion channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ion across epithelial membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, ion channels, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122. Ion channels are generally classified by structure and the type of mode of action. For example, extracellular ligand gated channels (ELGs) are comprised of five polypeptide subunits, with each subunit having 4 membrane spanning domains, and are activated by the binding of an extracellular ligand to the channel. In addition, channels are sometimes classified by the ion type that is transported, for example, chlorine channels, potassium channels, etc. There may be many classes of channels for transporting a single type of ion (a detailed review of channel types can be found at Alexander, S. P. H. and J. A. Peters (1997). Receptor and ion channel nomenclature supplement. Trends Pharmacol. Sci., Elsevier, pp. 65-68 and http://www-biology.ucsd.edu/˜msaier/transport/toc.html. There are many types of ion channels based on structure. For example, many ion channels fall within one of the following groups: extracellular ligand-gated channels (ELG), intracellular ligand-gated channels (ILG), inward rectifying channels (INR), intercellular (gap junction) channels, and voltage gated channels (VIC). There are additionally recognized other channel families based on ion-type transported, cellular location and drug sensitivity. Detailed information on each of these, their activity, ligand type, ion type, disease association, drugability, and other information pertinent to the present invention, is well known in the art. Extracellular ligand-gated channels, ELGs, are generally comprised of five polypeptide subunits, Unwin, N. (1993), Cell 72: 31-41; Unwin, N. (1995), Nature 373: 37-43; Hucho, F., et al., (1996) J. Neurochem. 66: 1781-1792; Hucho, F., et al., (1996) Eur. J. Biochem. 239: 539-557; Alexander, S. P. H. and J. A. Peters (1997), Trends Pharmacol. Sci., Elsevier, pp.4-6; 36-40; 42-44; and Xue, H. (1998) J. Mol. Evol. 47: 323-333. Each subunit has 4 membrane spanning regions: this serves as a means of identifying other members of the ELG family of proteins. ELG bind a ligand and in response modulate the flow of ions. Examples of ELG include most members of the neurotransmitter-receptor family of proteins, e.g., GABAI receptors. Other members of this family of ion channels include glycine receptors, ryandyne receptors, and ligand gated calcium channels. The Voltage-Gated Ion Channel (VIC) Superfamily Proteins of the VIC family are ion-selective channel proteins found in a wide range of bacteria, archaea and eukaryotes Hille, B. (1992), Chapter 9: Structure of channel proteins; Chapter 20: Evolution and diversity. In: Ionic Channels of Excitable Membranes, 2nd Ed., Sinaur Assoc. Inc., Pubs., Sunderland, Mass.; Sigworth, F. J. (1993), Quart. Rev. Biophys. 27: 1-40; Salkoff, L. and T. Jegla (1995), Neuron 15: 489-492; Alexander, S. P. H. et al., (1997), Trends Pharmacol. Sci., Elsevier, pp. 76-84; Jan, L. Y. et al., (1997), Annu. Rev. Neurosci. 20: 91-123; Doyle, D. A., et al., (1998) Science 280: 69-77; Terlau, H. and W. Stühmer (1998), Naturwissenschaften 85: 437-444. They are often homo- or heterooligomeric structures with several dissimilar subunits (e.g., a1-a2-d-b Ca 2+ channels, ab 1 b 2 Na + channels or (a) 4 -b K + channels), but the channel and the primary receptor is usually associated with the a (or a1) subunit. Functionally characterized members are specific for K + , Na + or Ca 2+ . The K + channels usually consist of homotetrameric structures with each a-subunit possessing six transmembrane spanners (TMSs). The a1 and a subunits of the Ca 2+ and Na + channels, respectively, are about four times as large and possess 4 units, each with 6 TMSs separated by a hydrophilic loop, for a total of 24 TMSs. These large channel proteins form heterotetra-unit structures equivalent to the homotetrameric structures of most K + channels. All four units of the Ca 2+ and Na + channels are homologous to the single unit in the homotetrameric K + channels. Ion flux via the eukaryotic channels is generally controlled by the transmembrane electrical potential (hence the designation, voltage-sensitive) although some are controlled by ligand or receptor binding. Several putative K + -selective channel proteins of the VIC family have been identified in prokaryotes. The structure of one of them, the KcsA K + channel of Streptomyces lividans , has been solved to 3.2 Å A resolution. The protein possesses four identical subunits, each with two transmembrane helices, arranged in the shape of an inverted teepee or cone. The cone cradles the “selectivity filter” P domain in its outer end. The narrow selectivity filter is only 12 Å long, whereas the remainder of the channel is wider and lined with hydrophobic residues. A large water-filled cavity and helix dipoles stabilize K + in the pore. The selectivity filter has two bound K + ions about 7.5 Å apart from each other. Ion conduction is proposed to result from a balance of electrostatic attractive and repulsive forces. In eukaryotes, each VIC family channel type has several subtypes based on pharmacological and electrophysiological data. Thus, there are five types of Ca 2+ channels (L, N, P, Q and T). There are at least ten types of K + channels, each responding in different ways to different stimuli: voltage-sensitive [Ka, Kv, Kvr, Kvs and Ksr], Ca 2+ -sensitive [BK Ca , IK Ca and SK Ca ] and receptor-coupled [K M and K ACh ]. There are at least six types of Na + channels (I, II, III, μ1, H1 and PN3). Tetrameric channels from both prokaryotic and eukaryotic organisms are known in which each a-subunit possesses 2 TMSs rather than 6, and these two TMSs are homologous to TMSs 5 and 6 of the six TMS unit found in the voltage-sensitive channel proteins. KcsA of S. lividans is an example of such a 2 TMS channel protein. These channels may include the K Na (Na + -activated) and K Vol (cell volume-sensitive) K + channels, as well as distantly related channels such as the Tok1 K + channel of yeast, the TWIK-1 inward rectifier K + channel of the mouse and the TREK-1 K + channel of the mouse. Because of insufficient sequence similarity with proteins of the VIC family, inward rectifier K + IRK channels (ATP-regulated; G-protein-activated) which possess a P domain and two flanking TMSs are placed in a distinct family. However, substantial sequence similarity in the P region suggests that they are homologous. The b, g and d subunits of VIC family members, when present, frequently play regulatory roles in channel activation/deactivation. Experimental evidence indicates that voltage gated Ca 2+ channels may be implicated in diseases such as Lambert-Eaton myasthenic syndrome, a paraneoplastic neuromuscular disorder in which an autoimmune response directed against a small-cell lung tumor crossreacts with antigens in the neuromuscular junction. For more information, see Rosenfeld, M. R., et al., Ann. Neurol. 33: 113-120, 1993, PubMed ID : 8494331; and Taviaux, S., et al., Hum. Genet. 100: 151-154, 1997, PubMed ID: 9254841. The Epithelial Na + Channel (ENaC) Family The ENaC family consists of over twenty-four sequenced proteins (Canessa, C. M., et al., (1994), Nature 367: 463-467, Le, T. and M. H. Saier, Jr. (1996), Mol. Membr. Biol. 13: 149-157; Garty, H. and L. G. Palmer (1997), Physiol. Rev. 77: 359-396; Waldmann, R., et al., (1997), Nature 386: 173-177; Darboux, I., et al., (1998), J. Biol. Chem. 273: 9424-9429; Firsov, D., et al., (1998), EMBO J. 17: 344-352; Horisberger, J. -D. (1998). Curr. Opin. Struc. Biol. 10: 443-449). All are from animals with no recognizable homologues in other eukaryotes or bacteria. The vertebrate ENaC proteins from epithelial cells cluster tightly together on the phylogenetic tree: voltage-insensitive ENaC homologues are also found in the brain. Eleven sequenced C. elegans proteins, including the degenerins, are distantly related to the vertebrate proteins as well as to each other. At least some of these proteins form part of a mechano-transducing complex for touch sensitivity. The homologous Helix aspersa (FMRF-amide)-activated Na + channel is the first peptide neurotransmitter-gated ionotropic receptor to be sequenced. Protein members of this family all exhibit the same apparent topology, each with N- and C-termini on the inside of the cell, two amphipathic transmembrane spanning segments, and a large extracellular loop. The extracellular domains contain numerous highly conserved cysteine residues. They are proposed to serve a receptor function. Mammalian ENaC is important for the maintenance of Na + balance and the regulation of blood pressure. Three homologous ENaC subunits, alpha, beta, and gamma, have been shown to assemble to form the highly Na + -selective channel. The stoichiometry of the three subunits is alpha 2 , beta1, gamma1 in a heterotetrameric architecture. The Glutamate-Gated Ion Channel (GIC) Family of Neurotransmitter Receptors Members of the GIC family are heteropentameric complexes in which each of the 5 subunits is of 800-1000 amino acyl residues in length (Nakanishi, N., et al, (1990), Neuron 5: 569-581; Unwin, N. (1993), Cell 72: 31-41; Alexander, S. P. H. and J. A. Peters (1997) Trends Pharmacol. Sci., Elsevier, pp. 3640). These subunits may span the membrane three or five times as putative a-helices with the N-termini (the glutamate-binding domains) localized extracellularly and the C-termini localized cytoplasmically. They may be distantly related to the ligand-gated ion channels, and if so, they may possess substantial b-structure in their transmembrane regions. However, homology between these two families cannot be established on the basis of sequence comparisons alone. The subunits fall into six subfamilies: a, b, g, d, e and z. The GIC channels are divided into three types: (1) a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-, (2) kainate- and (3) N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Subunits of the AMPA and kainate classes exhibit 35-40% identity with each other while subunits of the NMDA receptors exhibit 22-24% identity with the former subunits. They possess large N-terminal, extracellular glutamate-binding domains that are homologous to the periplasmic glutamine and glutamate receptors of ABC-type uptake permeases of Gram-negative bacteria. All known members of the GIC family are from animals. The different channel (receptor) types exhibit distinct ion selectivities and conductance properties. The NMDA-selective large conductance channels are highly permeable to monovalent cations and Ca 2+ . The AMPA- and kainate-selective ion channels are permeable primarily to monovalent cations with only low permeability to Ca 2+ . The Chloride Channel (CIC) Family The CIC family is a large family consisting of dozens of sequenced proteins derived from Gram-negative and Gram-positive bacteria, cyanobacteria, archaea, yeast, plants and animals (Steinmeyer, K., et al., (1991), Nature 354: 301-304; Uchida, S., et al., (1993), J. Biol. Chem. 268: 3821-3824; Huang, M.-E., et al., (1994), J. Mol. Biol. 242: 595-598; Kawasaki, M., et al, (1994), Neuron 12: 597-604; Fisher, W. E., et al., (1995), Genomics. 29:598-606; and Foskett, J. K. (1998), Annu. Rev. Physiol. 60: 689-717). These proteins are essentially ubiquitous, although they are not encoded within genomes of Haemophilus influenzae, Mycoplasma genitalium , and Mycoplasma pneumoniae . Sequenced proteins vary in size from 395 amino acyl residues ( M. jannaschii ) to 988 residues (man). Several organisms contain multiple ClC family paralogues. For example, Synechocystis has two paralogues, one of 451 residues in length and the other of 899 residues. Arabidopsis thaliana has at least four sequenced paralogues, (775-792 residues), humans also have at least five paralogues (820-988 residues), and C. elegans also has at least five (810-950 residues). There are nine known members in mammals, and mutations in three of the corresponding genes cause human diseases. E. coli, Methanococcus jannaschii and Saccharomyces cerevisiae only have one ClC family member each. With the exception of the larger Synechocystis paralogue, all bacterial proteins are small (395-492 residues) while all eukaryotic proteins are larger (687-988 residues). These proteins exhibit 10-12 putative transmembrane a-helical spanners (TMSs) and appear to be present in the membrane as homodimers. While one member of the family, Torpedo ClC-O, has been reported to have two channels, one per subunit, others are believed to have just one. All functionally characterized members of the ClC family transport chloride, some in a voltage-regulated process. These channels serve a variety of physiological functions (cell volume regulation; membrane potential stabilization; signal transduction; transepithelial transport, etc.). Different homologues in humans exhibit differing anion selectivities, i.e., ClC4 and ClC5 share a NO 3 − >Cl − >Br − >I − conductance sequence, while ClC3 has an I − >Cl − selectivity. The ClC4 and ClC5 channels and others exhibit outward rectifying currents with currents only at voltages more positive than +20 mV. Animal Inward Rectifier K + Channel (IRK-C) Family IRK channels possess the “minimal channel-forming structure” with only a P domain, characteristic of the channel proteins of the VIC family, and two flanking transmembrane spanners (Shuck, M. E., et al., (1994), J. Biol. Chem. 269: 24261-24270; Ashen, M. D., et al., (1995), Am. J. Physiol. 268: H506-H511; Salkoff, L. and T. Jegla (1995), Neuron 15: 489-492; Aguilar-Bryan, L., et al., (1998), Physiol. Rev. 78: 227-245; Ruknudin, A., et al., (1998), J. Biol. Chem. 273: 14165-14171). They may exist in the membrane as homo- or heterooligomers. They have a greater tendency to let K + flow into the cell than out. Voltage-dependence may be regulated by external K + , by internal Mg 2+ , by internal ATP and/or by G-proteins. The P domains of IRK channels exhibit limited sequence similarity to those of the VIC family, but this sequence similarity is insufficient to establish homology. Inward rectifiers play a role in setting cellular membrane potentials, and the closing of these channels upon depolarization permits the occurrence of long duration action potentials with a plateau phase. Inward rectifiers lack the intrinsic voltage sensing helices found in VIC family channels. In a few cases, those of Kir1.1a and Kir6.2, for example, direct interaction with a member of the ABC superfamily has been proposed to confer unique functional and regulatory properties to the heteromeric complex, including sensitivity to ATP. The SUR1 sulfonylurea receptor (spQ09428) is the ABC protein that regulates the Kir6.2 channel in response to ATP, and CFTR may regulate Kir1.1a. Mutations in SUR1 are the cause of familial persistent hyperinsulinemic hypoglycemia in infancy (PHHI), an autosomal recessive disorder characterized by unregulated insulin secretion in the pancreas. ATP-Gated Cation Channel (ACC) Family Members of the ACC family (also called P2X receptors) respond to ATP, a functional neurotransmitter released by exocytosis from many types of neurons (North, R. A. (1996), Curr. Opin. Cell Biol. 8: 474-483; Soto, F., M. Garcia-Guzman and W. Stühmer (1997), J. Membr. Biol. 160: 91-100). They have been placed into seven groups (P2X 1 -P2X 7 ) based on their pharmacological properties. These channels, which function at neuron-neuron and neuron-smooth muscle junctions, may play roles in the control of blood pressure and pain sensation. They may also function in lymphocyte and platelet physiology. They are found only in animals. The proteins of the ACC family are quite similar in sequence (>35% identity), but they possess 380-1000 amino acyl residues per subunit with variability in length localized primarily to the C-terminal domains. They possess two transmembrane spanners, one about 30-50 residues from their N-termini, the other near residues 320-340. The extracellular receptor domains between these two spanners (of about 270 residues) are well conserved with numerous conserved glycyl and cysteyl residues. The hydrophilic C-termini vary in length from 25 to 240 residues. They resemble the topologically similar epithelial Na + channel (ENaC) proteins in possessing (a) N- and C-termini localized intracellularly, (b) two putative transmembrane spanners, (c) a large extracellular loop domain, and (d) many conserved extracellular cysteyl residues. ACC family members are, however, not demonstrably homologous with them. ACC channels are probably hetero- or homomultimers and transport small monovalent cations (Me + ). Some also transport Ca 2+ ; a few also transport small metabolites. The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca 2+ Channel (RaR-CaC) Family Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate (IP3)-sensitive Ca 2+ -release channels function in the release of Ca 2+ from intracellular storage sites in animal cells and thereby regulate various Ca 2+ -dependent physiological processes (Hasan, G. et al., (1992) Development 116: 967-975; Michikawa, T., et al., (1994), J. Biol. Chem. 269: 9184-9189; Tunwell, R. E. A., (1996), Biochem. J. 318: 477-487; Lee, A. G. (1996) Biomembranes , Vol. 6, Transmembrane Receptors and Channels (A. G. Lee, ed.), JAI Press, Denver, Colo.., pp 291-326; Mikoshiba, K., et al., (1996) J. Biochem. Biomem. 6: 273-289). Ry receptors occur primarily in muscle cell sarcoplasmic reticular (SR) membranes, and IP3 receptors occur primarily in brain cell endoplasmic reticular (ER) membranes where they effect release of Ca 2 +into the cytoplasm upon activation (opening) of the channel. The Ry receptors are activated as a result of the activity of dihydropyridine-sensitive Ca 2+ channels. The latter are members of the voltage-sensitive ion channel (VIC) family. Dihydropyridine-sensitive channels are present in the T-tubular systems of muscle tissues. Ry receptors are homotetrameric complexes with each subunit exhibiting a molecular size of over 500,000 daltons (about 5,000 amino acyl residues). They possess C-terminal domains with six putative transmembrane a-helical spanners (TMSs). Putative pore-forming sequences occur between the fifth and sixth TMSs as suggested for members of the VIC family. The large N-terminal hydrophilic domains and the small C-terminal hydrophilic domains are localized to the cytoplasm. Low resolution 3-dimensional structural data are available. Mammals possess at least three isoforms that probably arose by gene duplication and divergence before divergence of the mammalian species. Homologues are present in humans and Caenorabditis elegans. IP 3 receptors resemble Ry receptors in many respects. (1) They are homotetrameric complexes with each subunit exhibiting a molecular size of over 300,000 daltons (about 2,700 amino acyl residues). (2) They possess C-terminal channel domains that are homologous to those of the Ry receptors. (3) The channel domains possess six putative TMSs and a putative channel lining region between TMSa 5 and 6. (4) Both the large N-terminal domains and the smaller C-terminal tails face the cytoplasm. (5) They possess covalently linked carbohydrate on extracytoplasmic loops of the channel domains. (6) They have three currently recognized isoforms (types 1, 2, and 3) in mammals which are subject to differential regulation and have different tissue distributions. IP 3 receptors possess three domains: N-terminal IP 3 -binding domains, central coupling or regulatory domains and C-terminal channel domains. Channels are activated by IP 3 binding, and like the Ry receptors, the activities of the IP 3 receptor channels are regulated by phosphorylation of the regulatory domains, catalyzed by various protein kinases. They predominate in the endoplasmic reticular membranes of various cell types in the brain but have also been found in the plasma membranes of some nerve cells derived from a variety of tissues. The channel domains of the Ry and IP 3 receptors comprise a coherent family that in spite of apparent structural similarities, do not show appreciable sequence similarity of the proteins of the VIC family. The Ry receptors and the IP 3 receptors cluster separately on the RIR-CaC family tree. They both have homologues in Drosophila . Based on the phylogenetic tree for the family, the family probably evolved in the following sequence: (1) A gene duplication event occurred that gave rise to Ry and IP 3 receptors in invertebrates. (2) Vertebrates evolved from invertebrates. (3) The three isoforms of each receptor arose as a result of two distinct gene duplication events. (4) These isoforms were transmitted to mammals before divergence of the mammalian species. The Organellar Chloride Channel (O-ClC) Family Proteins of the O-ClC family are voltage-sensitive chloride channels found in intracellular membranes but not the plasma membranes of animal cells (Landry, D, et al., (1993), J. Biol. Chem. 268: 14948-14955; Valenzuela, S et al., (1997), J. Biol. Chem. 272: 12575-12582;. and Duncan, R. R., et al., (1997), J. Biol. Chem. 272: 23880-23886). They are found in human nuclear membranes, and the bovine protein targets to the microsomes, but not the plasma membrane, when expressed in Xenopus laevis oocytes. These proteins are thought to function in the regulation of the membrane potential and in transepithelial ion absorption and secretion in the kidney. They possess two putative transmembrane a-helical spanners (TMSs) with cytoplasmic N- and C-termini and a large luminal loop that may be glycosylated. The bovine protein is 437 amino acyl residues in length and has the two putative TMSs at positions 223-239 and 367-385. The human nuclear protein is much smaller (241 residues). A C. elegans homologue is 260 residues long. Transporter proteins, particularly members of the calcium channel subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown transport proteins. The present invention advances the state of the art by providing previously unidentified human transport proteins. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based in part on the identification of amino acid sequences of human transporter peptides and proteins that are related to the calcium channel transporter subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate transporter activity in cells and tissues that express the transporter. Experimental data as provided in FIG. 1 indicates expression in humans in the liver, adrenal gland, normal and tumorous nervous tissue, adult amygdala, brain meningioma tissue, denis-drash, adult and fetal brain, placenta, testis and kidney. |
Isolated human ras- like proteins, nucleic acid molecules encoding these human ras-like proteins , and uses thereof |
The present invention provides amino acid sequences of polypeptides that are encoded by genes within the human genome, the Ras-like protein polypeptides of the present invention. The present invention specifically provides isolated polypeptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the Ras-like protein polypeptides, and methods of identifying modulators of the Ras-like protein polypeptides. |
1. An isolated polypeptide consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids. 2. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids. 3. An isolated antibody that selectively binds to a polypeptide of claim 2. 4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d). 5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d). 6. A gene chip comprising a nucleic acid molecule of claim 5. 7. A transgenic non-human animal comprising a nucleic acid molecule of claim 5. 8. A nucleic acid vector comprising a nucleic acid molecule of claim 5. 9. A host cell containing the vector of claim 8. 10. A method for producing any of the polypeptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the polypeptides are expressed from the nucleotide sequence. 11. A method for producing any of the polypeptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the polypeptides are expressed from the nucleotide sequence. 12. A method for detecting the presence of any-of the polypeptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the polypeptide in the sample and then detecting the presence of the polypeptide. 13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample. 14. A method for identifying a modulator of a polypeptide of claim 2, said method comprising contacting said polypeptide with an agent and determining if said agent has modulated the function or activity of said polypeptide. 15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said polypeptide. 16. A method for identifying an agent that binds to any of the polypeptides of claim 2, said method comprising contacting the polypeptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the polypeptide. 17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor. 18. A method for treating a disease or condition mediated by a human Ras-like protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim 16. 19. A method for identifying a modulator of the expression of a polypeptide of claim 2, said method comprising contacting a cell expressing said polypeptide with an agent, and determining if said agent has modulated the expression of said polypeptide. 20. An isolated human Ras-like protein polypeptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence shown in SEQ ID NO:2. 21. A polypeptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence shown in SEQ ID NO:2. 22. An isolated nucleic acid molecule encoding a human Ras-like protein polypeptide, said nucleic acid molecule sharing at least 80 percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or 3. 23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or 3. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Ras-like proteins, particularly members of the Rab subfamilies, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of Ras-like proteins. The present invention advances the state of the art by providing a previously unidentified human Ras-like proteins that have homology to members of the Rab subfamilies. Ras Protein Ras proteins are small regulatory GTP-binding proteins, or small G proteins, which belong to the Ras protein superfamily. They are monomeric GTPases, but their GTPase activity is very slow (less than one GTP molecule per minute). Ras proteins are key relays in the signal-transducing cascade induced by the binding of a ligand to specific receptors such as receptor tyrosine kinases (RTKs), since they trigger the MAP kinase cascade. The ligand can be a growth factor (epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin, an interleukin (IL), granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF). Ras proteins contain sequences highly conserved during evolution. Their tertiary structure includes ten loops connecting six strands of beta-sheet and five alpha helices. In mammalians, there are four Ras proteins, which are encoded by Ha-ras, N-ras, Ki-rasA and Ki-rasB genes. They are composed of about 170 residues and have a relative molecular mass of 21 kD. Ras proteins contain covalently attached modified lipids allowing these proteins to bind to the plasma membrane. Ha-Ras has a C-terminal farnesyl group, a C-terminal palmitoyl group and a N-terminal myristoyl group. In Ki-Ras(B), a C-terminal polylysine domain replaces the palmitoyl group. Ras proteins alternate between an inactive form bound to GDP and an active form bound to GTP. Their activation results from reactions induced by a guanine nucleotide-exchange factor (GEF). Their inactivation results from reactions catalyzed by a GTPase-activating protein (GAP). When a Ras protein is activated by a GEF such as a Sos protein, the N-terminal region of a serine/threonine kinase, called “Raf protein”, can bind to Ras protein. The C-terminal region of the activated Raf thus formed binds to another protein, MEK, and phosphorylates it on both specific tyrosine and serine residues. Active MEK phosphorylates and activates, in turn, a MAP kinase (ERK1 or ERK2), which is also a serine/threonine kinase. This phosphorylation occurs on both specific tyrosine and threonine residues of MAP kinase. MAP kinase phosphorylates many different proteins, especially nuclear transcription factors (TFs) that regulate expression of many genes during cell proliferation and differentiation. Recent researches suggest that, in mammalians, phosphatidyl inositol 3′-kinase (PI3-kinase) might be a target of Ras protein, instead of Raf protein. In certain mutations, the translation of ras genes may produce oncogenic Ras proteins. Ras-Like Protein Guanine nucleotide-binding proteins (GTP-binding proteins, or G proteins) participate in a wide range of regulatory functions including metabolism, growth, differentiation, signal transduction, cytoskeletal organization, and intracellular vesicle transport and secretion. These proteins control diverse sets of regulatory pathways in response to hormones, growth factors, neuromodulators, or other signaling molecules. When these molecules bind to transmembrane receptors, signals are propagated to effector molecules by intracellular signal transducing proteins. Many of these signal-transducing proteins are members of the Ras superfamily. The Ras superfamily is a class of low molecular weight (LMW) GTP-binding proteins that consist of 21-30 kDa polypeptides. These proteins regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. In particular, the LMW GTP-binding proteins activate cellular proteins by transducing mitogenic signals involved in various cell functions in response to extracellular signals from receptors (Tavitian, A. (1995) C. R. Seances Soc. Biol. Fil. 189:7-12). During this process, the hydrolysis of GTP acts as an energy source as well as an on-off switch for the GTPase activity of the LMW GTP-binding proteins. The Ras superfamily is comprised of five subfamilies: Ras, Rho, Ran, Rab, and ADP-ribosylation factor (ARF). Specifically, Ras genes are essential in the control of cell proliferation. Mutations in Ras genes have been associated with cancer. Rho proteins control signal transduction in the process of linking receptors of growth factors to actin polymerization that is necessary for cell division. Rab proteins control the translocation of vesicles to and from membranes for protein localization, protein processing, and secretion. Ran proteins are localized to the cell nucleus and play a key role in nuclear protein import, control of DNA synthesis, and cell-cycle progression. ARF and ARF-like proteins participate in a wide variety of cellular functions including vesicle trafficking, exocrine secretion, regulation of phospholipase activity, and endocytosis. Despite their sequence variations, all five subfamilies of the Ras superfamily share conserved structural features. Four conserved sequence regions (motifs I-IV) have been studied in the LMW GTP-binding proteins. Motif I is the most variable but has the conserved sequence, GXXXXGK. The lysine residue is essential in interacting with the .beta.- and .gamma.-phosphates of GTP. Motif II, III, and IV contain highly conserved sequences of DTAGQ, NKXD, and EXSAX, respectively. Specifically, Motif II regulates the binding of gamma-phosphate of GTP; Motif III regulates the binding of GTP; and Motif IV regulates the guanine base of GTP. Most of the membrane-bound LMW GTP-binding proteins generally require a carboxy terminal isoprenyl group for membrane association and biological activity. The isoprenyl group is added posttranslationally through recognition of a terminal cysteine residue alone or a terminal cysteine-aliphatic amino acid-aliphatic amino acid-any amino acid (CAAX) motif. Additional membrane-binding energy is often provided by either internal palmitoylation or a carboxy terminal cluster of basic amino acids. The LMW GTP-binding proteins also have a variable effector region, located between motifs I and II, which is characterized as the interaction site for guanine nucleotide exchange factors (GEFs) or GTPase-activating proteins (GAPs). GEFs induce the release of GDP from the active form of the G protein, whereas GAPs interact with the inactive form by stimulating the GTPase activity of the G protein. The ARF subfamily has at least 15 distinct members encompassing both ARF and ARF-like proteins. ARF proteins identified to date exhibit high structural similarity and ADP-ribosylation enhancing activity. In contrast, several ARF-like proteins lack ADP-ribosylation enhancing activity and bind GTP differently. An example of ARF-like proteins is a rat protein, ARL184. ARL184 has been shown to have a molecular weight of 22 kDa and four functional GTP-binding sites (Icard-Liepkalns, C. et al. (1997) Eur. J. Biochem. 246: 388-393). ARL184 is active in both the cytosol and the Golgi apparatus and is closely associated with acetylcholine release, suggesting that ARL184 is a potential regulatory protein associated with Ca.sup.2+-dependent release of acetylcholine. A number of Rho GTP-binding proteins have been identified in plasma membrane and cytoplasm. These include RhoA, B and C, and D, rhoG, rac 1 and 2, G25K-A and B, and TC10 (Hall, A. et al. (1993) Philos. Trans. R. Soc. Lond. (Biol.) 340:267-271). All Rho proteins have a CAAX motif that binds a prenyl group and either a palmitoylation site or a basic amino acid-rich region, suggesting their role in membrane-associated functions. In particular, RhoD is a protein that functions in early endosome motility and distribution by inducing rearrangement of actin cytoskeleton and cell surface (Murphy, C. et al. (1996) Nature 384:427-432). During cell adhesion, the Rho proteins are essential for triggering focal complex assembly and integrin-dependent signal transduction (Hotchin, N. A. and Hall, A. (1995) J. Cell Biol. 131:1857-1865). The Ras subfamily proteins already indicated supra are essential in transducing signals from receptor tyrosine kinases (RTKs) to a series of serine/threonine kinases which control cell growth and differentiation. Mutant Ras proteins, which bind but cannot hydrolyze GTP, are permanently activated and cause continuous cell proliferation or cancer. TC21, a Ras-like protein, is found to be highly expressed in a human teratocarcinoma cell line Drivas, G. T. et al. (1990) Mol. Cell. Biol. 10: 1793-1798). Rin and Rit are characterized as membrane-binding, Ras-like proteins without the lipid-binding CAAX motif and carboxy terminal cysteine (Lee, C.-H. J. et al. (1996) J. Neurosci. 16: 6784-6794). Further, Rin is shown to localize in neurons and have calcium-dependant calmodulin-binding activity. Rab Proteins The novel human protein, and encoding gene, provided by the present invention is related to the Rab family of Ras-like proteins and the Rab2B isoform in particular. Furthermore, the protein of the present invention may be an alternative splice form of a Rab2B protein provided in published patent applications WO200058464 and EP1074617.12472 (see the amino acid sequence alignment in FIG. 2 ). Specifically, the protein of the present invention differs from the art-known protein in the first exon. RAB proteins are important for regulating the targeting and fusion of membranous vesicles during organelle assembly and transport. RAB proteins undergo controlled exchange of GTP for GDP, and they hydrolyze GTP in a reaction that may regulate the timing and unidirectional nature of these assemblies. Generally, known RAB proteins terminate in sequences such as cys-X-cys (e.g., RAB3A), cys-cys (e.g., RAB1A), or a similar sequence, and generally all are geranylgeranylated. Rab GTP-binding proteins are similar to YPT1/SEC4 in Saccharomyces cerevisiae, which are critical for transport along the exocytic route (Chavrier et al., Mol Cell Biol 1990 December; 10(12):6578-85). Different Rab proteins are presumed to control different steps in membrane traffic, leading to a high level of diversity and complexity within the Rab family (Chavrier et al., Mol Cell Biol 1990 December; 10(12):6578-85). The Rab1 gene maps in close viscinity to the ‘wobbler’ spinal muscular atrophy gene. Rab1 and Rab2 from the snail Lymnaea stagnalis share a very high degree (95-97%) of sequence identity with mammalian Rab1 and Rab2 over the first 178-191 N-terminal amino acids; however, the C-terminal region is almost completely divergent, except for the final 2-4 amino acids at the extreme ends. Rab1 was found to be highly expressed in the albumin gland of Lymnaea stagnalis, suggesting an important role in that gland (Agterberg et al., Eur. J. Biochem. 217 (1), 241-246 (1993)). The tethering factor p115 is a RAB1 effector that binds directly to activated RAB1. It is thought that RAB1-regulated assembly of functional effector-SNARE complexes serves as a conserved molecular mechanism for regulating recognition between different subcellular compartments such as endoplasmic reticulum and Golgi apparatus (Allan et al., Science 289: 444-448, 2000). GTPases play important roles in a wide variety of cell functions such as signal transduction, cytoskeletal organization, and membrane trafficking. Rab GTPases are particularly important for regulating cellular membrane dynamics by modulating the activity of effector proteins that then regulate vesicle trafficking. The Rab8 GTPase plays important roles in Golgi to plasma membrane vesicle trafficking. Studies have suggested that Rab37 plays an important role in mast cell degranulation. Thus, novel human Rab GTPases may be valuable as potential therapeutic targets for the development of allergy treatments (Masuda et al., FEBS Lett 2000 Mar. 17; 470). Rab15 may act, together with Rab3A, to regulate synaptic vesicle membrane flow within nerve terminals, thereby regulating neurotransmitter release. Rab15 and Rab3A are low molecular weight GTP-binding proteins. Rab proteins are generally comprised of four conserved structural domains necessary for GTP binding, as well as additional domains for membrane localization and effector protein interactions. Rab15 is expressed primarily in neural tissues such as the brain and is localized to synaptic vesicles (Elferink et al., J. Biol. Chem. 267 (9), 5768-5775 (1992)). For a further review of Rab proteins, see Wedemeyer et al., Genomics 32: 447-454, 1996 and Zahraoui et al., J. Biol. Chem. 264: 12394-12401, 1989. Due to their importance in human physiology, particularly in regulating membrane trafficking, novel human Rab proteins/genes, such as provided by the present invention, are valuable as potential targets for the development of therapeutics to treat a wide variety of diseases/disorders caused or influenced by defects in membrane trafficking. Furthermore, SNPs in Rab genes are valuable markers for the diagnosis, prognosis, prevention, and/or treatment of such diseases/disorders. The discovery of new human Ras-like proteins and the polynucleotides that encode them satisfies a need in the art by providing new compositions that are useful in the diagnosis, prevention, and treatment of inflammation and disorders associated with cell proliferation and apoptosis. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based in part on the identification of amino acid sequences of a novel human Rab protein alternative splice form, as well as other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate Ras-like protein activity in cells and tissues that express the Ras-like protein. Experimental data as provided in FIG. 1 indicates expression in humans in germ cell tumors, retina, testis, liver/spleen, colon, melanotic melanoma, brain, and leukocytes. |
System and method for management of remote devices in a network |
A system and method of managing devices connected to a network by restricted bandwidth links, where a restricted bandwidth link comprises a wireless or other link which has a relatively limited amount of bandwidth available and/or a link wherein bandwidth is relatively expensive. The network has one or more master agents executing which communicate with proxy agents upstream of the restricted bandwidth links and which maintain management information about the managed objects that is obtained from both network management services for the restricted links and from relatively simple servers running at the managed objects. Communication between the proxy agents and the servers is by way of a simple protocol and the master agent performs translations to and from more complicated management protocols used in other parts of the network and this simple protocol. |
1. A wireless local loop system comprising at least one base station and a plurality of customer premises equipment devices communicating with said base station by a shared radio link, comprising: a radio resource manager monitoring the operating conditions of the shared radio link and obtaining usage and historical usage information for said radio link; a customer premises server in each of said plurality of customer premises equipment devices; a proxy agent at said base station to communicate with said customer premises servers over said shared radio link to request information from said customer premises equipment devices, to transmit management data to said customer premises equipment devices and to maintain a management information base for said customer premises equipment devices including usage and historical information obtained from said radio resource manager, information obtained from said customer premises equipment devices over said shared radio link and information requested from said customer premises equipment devices; a master agent, associated with said base station, and operable to receive management information requests from a network and to obtain requested data from said management information base. 2. The wireless local loop system as claimed in claim 1 wherein said master agent and said proxy agent are combined and are the same element. 3. A method of managing devices connected to a network by a restricted bandwidth link, comprising the steps of: (i) collecting and storing in a proxy agent, operating upstream of said restricted bandwidth link, statistical information relating to operation of the restricted bandwidth link connecting a managed device to the network; (ii) polling, at selected times, the managed device over said restricted bandwidth link for information with respect to the operation of the managed device and storing the information returned in said proxy agent; (iii) receiving at a master agent a request for information about the managed device, said master agent identifying a proxy agent associated with the managed device and forwarding a request for the information to the identified proxy agent; (iv) upon receipt of said request, the identified proxy agent determining if the requested information is stored in the proxy agent and responding to the master agent with said information if present and requesting said information from said managed device over said restricted bandwidth link if not present in said proxy agent and receiving and forwarding a response from said managed device to said master agent. 4. The method of claim 3 further comprising the step of said master agent translating said received request from a first protocol to a second protocol in which the request is forwarded to said identified proxy and said master agent translating information received from said proxy agent in said second protocol into said first protocol to respond to said request for information. 5. The method of claim 4 wherein said first protocol is SNMP. 6. The method of claim 3 wherein said information stored in said proxy is stored in a management information base. 7. The method of claim 3 wherein said master agent and said proxy agent are combined and are the same element. 8. A system for managing a plurality of devices connected to a network by restricted bandwidth links, comprising: at least one master agent upstream of said restricted bandwidth links and operable to receive and respond to requests for management information about at least one of said plurality of devices; at least one proxy agent, upstream of said restricted bandwidth links, storing management information about at least one of said plurality of devices, said management information including information relating to the operation of said link obtained from said network, information relating to operation of said device obtained by polling said device at pre-selected intervals and information obtained from said device upon request; a client executing on said at least one device and responsive to polling and other requests from said proxy agent to obtain and forward information to said proxy agent over said restricted bandwidth link. 9. The system of claim 8 wherein said proxy agent translates requests received from said master agent in a first protocol into a second protocol for transmission to said device over said restricted bandwidth link, said client receiving and responding to said requests in said second protocol and said proxy translating responses received in said second protocol into said first protocol for responding to said master agent. 10. The system as claimed in claim 8 wherein said master agent and said proxy agent are combined and are the same element. |
<SOH> BACKGROUND OF THE INVENTION <EOH>As networks, and particularly TCP/IP networks such as the Internet, have grown in use and complexity, various systems and methods have been developed to manage the diverse components which make up such networks. One popular system and method for managing such networks is SNMP (Simple Network Management Protocol) which was developed by the Internet Engineering Task Force (IETF) and has been widely used since about 1993. SNMP employs a client/server type relationship wherein each physical or logical device managed with SNMP, (typically referred to as a “managed object”), executes a server program (also referred to as the “SNMP agent”) that a management tool (typically referred to as the “SNMP client”) can communicate with. Managed objects can include almost any device connected to the network, such as routers, gateways, firewalls, concentrators, computers, terminals, etc. Each SNMP server maintains a management information base (“MIB”) about the object it manages and the MIB contains at least a minimum set of defined statistical and control values for the managed object. The SNMP client communicates as needed, usually from a remote location, with the SNMP server to obtain status information, set alarm conditions, etc. for the managed object. Due to its widespread use, most network devices support SNMP and SNMP servers are available for them. While SNMP is widely used, some network and/or device developments were not foreseen by its creators and thus its operation/suitability may be less than desired in some circumstances. For example, wireless telecommunication networks, or telecommunication networks which include wireless links, have limited and/or expensive bandwidth which SNMP may make inefficient use of, by requiring relatively large amounts of information to be exchanged between the SNMP server and SNMP client over the links and/or requiring many exchanges of data from the server to the client on an ongoing, or on-demand, basis in normal use. Such exchanges can utilize a significant proportion of the total capacity of a wireless, or other bandwidth limited, link and/or can be expensive. |
<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide a novel system and method for the management of remote devices in a network which obviates or mitigates at least some of the above-identified disadvantages of the prior art. According to a first aspect of the present invention, there is provided a wireless local loop system comprising at least one base station and a plurality of customer premises equipment devices communicating with said base station by a shared radio link, comprising: a radio resource manager monitoring the operating conditions of the shared radio link and obtaining usage and historical usage information for said radio link; a customer premises server in each of said plurality of customer premises equipment devices; a proxy agent at said base station to communicate with said customer premises servers over said shared radio link to request information from said customer premises equipment devices, to transmit management data to said customer premises equipment devices and to maintain a management information base for said customer premises equipment devices including usage and historical information obtained from said radio resource manager, information obtained from said customer premises equipment devices over said shared radio link and information requested from said customer premises equipment devices; a master agent, associated with said base station, and operable to receive management information requests from a network and to obtain requested data from said management information base. According to another aspect of the present invention, there is provided a method of managing devices connected to a network by a restricted bandwidth link, comprising the steps of: (i) collecting and storing in a proxy agent operating upstream of said restricted bandwidth link, statistical information relating to operation of the restricted bandwidth link connecting a managed device to the network; (ii) polling, at selected times, the managed device over said restricted bandwidth link for information with respect to the operation of the managed device and storing the information returned in said proxy agent; (iii) receiving at a master agent a request for information about the managed device, said master agent identifying a proxy agent associated with the managed device and forwarding a request for the information to the identified proxy agent; (iv) upon receipt of said request, the identified proxy agent determining if the requested information is stored in the proxy agent and responding to the master agent with said information if present and requesting said information from said managed device over said restricted bandwidth link if not present in said proxy agent and receiving and forwarding a response from said managed device to said master agent. According to another aspect of the present invention, there is provided a system for managing a plurality of devices connected to a network by restricted bandwidth links, comprising: at least one master agent upstream of said restricted bandwidth links and operable to receive and respond to requests for management information about at least one of said plurality of devices; at least one proxy agent, upstream of said restricted bandwidth links, storing management information about at least one of said plurality of devices, said management information including information relating to the operation of said link obtained from said network, information relating to operation of said device obtained by polling said device at pre-selected intervals and information obtained from said device upon request; a client executing on said at least one device and responsive to polling and other requests from said proxy agent to obtain and forward information to said proxy agent over said restricted bandwidth link. |
Cartilaginous neo-tissue capable of being grafted |
A cartilaginous neo-tissue that is capable of being grafted is constituted by rows of approximately parallel cells with a cell maturation gradient orientated from a predetermined zone towards its periphery. During preparation of the cartilaginous neo-tissue with a chitosan hydrogel, the predetermined zone corresponds to the junction of cells with the chitosan hydrogel in contact with which the neo-tissue develops. |
1. A cartilaginous neo-tissue that is capable of being grafted, the neo-tissue being constituted by rows of approximately parallel cells with a cell maturation gradient orientated from a predetermined zone towards its periphery. 2. A neo-tissue according to claim 1, wherein the predetermined zone corresponds to the junction of cells with a chitosan hydrogel in contact with which the neo-tissue develops during its preparation. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Cartilage is a tissue of mesenchymal origin constituted by a small percentage of chondrocytes distributed in an extracellular matrix which is renewed by them. That matrix is composed of a network of collagen fibers, in particular type II fibers, and glycosaminoglycans associated with structure proteins to form proteoglycans. The amphiphilic nature and ionic sites of the ensemble produce a physical gel ensuring the viscoelastic properties of the cartilaginous tissue. Cartilaginous tissue disappears in adulthood apart from at the joints, where it ensures that articular surfaces can move and tolerate large compressive loads. However, articular cartilage is not capable of spontaneous regeneration. For that reason, graft techniques such as mosaic grafts or autologous cell grafts are used in the event of cartilaginous lesions. Mosaic grafts consist of removing bone covered in cartilage from non bearing regions and grafting them into the lesion. Autologous cell grafts consist of removing healthy cartilage, carrying out enzymatic digestion to release chondrocytes from the extracellular matrix and multiplying the chondrocytes ex vivo to obtain a sufficient number of chondrocytes, which are then re-implanted into the cartilaginous lesion. Since the chondrocytes are in the form of a cell suspension in an aqueous medium (dispersion in a liquid medium), the excised lesion must first be covered with a membrane formed from periosteum securely sutured to the edge of the cartilage, then the chondrocyte suspension (dispersion containing the culture) is injected into the cavity that is created. After a certain period, those cells produce an extracellular matrix which, however, does not have the tissue organization of normal articular cartilage. It should be noted that the mode of multiplication of the chondrocytes to be implanted must be determined so as to avoid cell dedifferentiation. In particular, if chondrocytes are proliferated on a support (synthetic polymer) such as the bottom of cell culture dishes, chondrocytes dedifferentiate into fibroblast cells. They are then fusiform instead of being polygonal, like chondrocytes, and synthesize collagen I instead of collagen II. International patent document WO-A-00/56251 proposes multiplying cells, including human chondrocytes, on biodegradable polysaccharide beads cross-linked by polyamines. The polysaccharides are selected from the following compounds: dextran, cellulose, arabinogalactan, pullulan, and amylase. The cross-linking agent is glutamic acid, lysine, albumin or gelatin, for example. According to that document, after bringing the chondrocytes into contact with said polymer beads with mechanical agitation, the chondrocytes multiply, retaining their form and phenotype; more particularly, they synthesize collagen II. After said multiplication, the chondrocytes are recovered by digesting the polysaccharide beads using specific enzymes, for example dextranase, which does not alter chondrocyte cells. Those cells are then detached for inclusion into a chitosan matrix. To this end, chondrocytes are added to an acid chitosan solution, then the mixture is agitated until a three-dimensional structure is formed, which is placed in a 1N sodium hydroxide solution to precipitate out the chitosan over several minutes. After polymerization, the sodium hydroxide is rapidly eliminated, then the polymerized conglomerate of chitosan and cells is cultured at 37° C. under 5% CO 2 for a predetermined period. Thus, according to WO-A-00/56251, the chondrocytes mixed with the chitosan are incorporated into the three-dimensional structure of precipitated chitosan, which structure should have a firm consistency resembling the texture of cartilage. WO-A-00/56251 describes a further possible variation in the first chondrocyte multiplication step, namely multiplying said cells on a chitosan film. The two other steps remain the same; the second step consists in extracting the multiplied chondrocytes by enzymatic digestion using collagenase or trypsin and the third step consists in including said chondrocytes in a three-dimensional chitosan matrix under the conditions described above. Chitosan is obtained by deacetylating chitin, the most common biopolymer to be found in nature after cellulose. Chitin can be extracted from the exoskeleton of certain crustaceans such as the lobster or crab, or from the squid endoskeleton, for example. Chitin and chitosan are constituted by the same two monomer units, N-acetyl-D-glucosamine and D-glucosamine. When the polymer is highly acetylated, i.e. when it comprises more than 60% of N-acetyl-D-glucosamine, it is known as chitin. Both are biodegradable, bioresorbable and compatible with living tissue. Chitosan is known to have a biostimulating activity on tissue reconstitution. However, it is generally used in association with other elements. As an example, in WO-A-96/02259, chitosan is combined with another polysaccharide to form an agent for stimulating and regenerating hard tissue at an integration site for an implant, for example a titanium implant. In WO-A-99/47186, for example, chitosan is cross-linked with glycosaminoglycan to constitute a biochemical environment that is close to cartilaginous tissue, stimulating cell growth. The methods described in WO-A-00/56521 and WO-A-99/47186 are based on the “scaffold” technique in which the cells which are incorporated and included in a three-dimensional structure which forms a scaffold or framework. Said three-dimensional structure, constituted by chitosan alone in WO-A-00/56521 or associated with other constituents in WO-A-99/47186, forms an integral part of the material intended to be grafted. |
<SOH> OBJECTS AND SUMMARY OF THE INVENTION <EOH>The cartilaginous neo-tissue that is capable of being grafted of the present invention differs from the disclosure of the prior art documents in that it does not comprise a component forming a three-dimensional scaffold type structure. In accordance with the present invention, said cartilaginous neo-tissue is constituted by rows of approximately parallel cells with a cell maturation gradient orientated from a predetermined zone towards its periphery. In particular, said cartilaginous neo-tissue is obtained by a method consisting in: a) culturing chondrogenic cells, which are either autologous chondrocytes or chondrocyte precursor cells prepared in vitro from pluripotent stem cells; b) bringing said chondrogenic cells into contact with a chitosan hydrogel having amphiphilic properties and a degree of acetylation such that said cells adhere naturally to the outer surface of said hydrogel; c) covering the hydrogel/cell ensemble obtained with a culture medium; and d) allowing a cartilaginous neo-tissue to develop in contact with the chitosan hydrogel for a minimum period of two weeks, frequently renewing the culture medium. Thus, in contrast to that which is proposed in WO-A-00/56251, the chondrogenic cell amplification method is carried out either spontaneously in the presence of the chitosan hydrogel, or after prior amplification under conventional high density culture conditions, and the extra-cellular matrix is formed simultaneously in the presence of the chitosan hydrogel. The natural adhesion of cells to the outer surface of the chitosan hydrogel can produce very good distribution of said cells and prevents the loss of cells during the operation, for example when it is carried out in culture wells. The chitosan hydrogel acts as an inducer on the chondrogenic cell phenotype, which proliferate without dedifferentiating. It should be noted that the chondrogenic cells do not penetrate directly into the hydrogel, which has a pore size that is insufficient compared with the size of said cells. The chitosan hydrogel is progressively metabolized and/or replaced and/or invaded by cartilage type matrix proteins, which are neo-synthesized by the chondrocytes. After at least two weeks of culture, the ensemble produces cartilaginous neo-tissue which can be grafted as is; the chitosan hydrogel, which serves as a temporary support for said cartilaginous neo-tissue, is partially or completely biodegraded. The degree of acetylation of the chitosan used to prepare the hydrogel is in the range 30% to 70%, preferably in the range 40% to 60%. In a first variation, the chondrogenic cells are brought into contact with the outer surface of the chitosan hydrogel which is in the form of small particles with a size of several millimetres. In a second variation, the chondrogenic cells are spread in the form of at least one sheet between at least two layers of chitosan hydrogel, each layer being of the order of a few millimeters thick. This particular disposition can very readily produce cartilaginous neo-tissue of large size after complete disappearance of the chitosan hydrogel. The cartilage neo-tissue formed using the method of the invention is constituted by rows of approximately parallel cells, with a cell maturation gradient orientated from a predetermined zone to its periphery, the predetermined zone corresponding to the junction of the cells with the chitosan hydrogel. When said neo-tissue is analyzed histologically, its morphological appearance is close to that of normal cartilaginous tissue. detailed-description description="Detailed Description" end="lead"? |
Materials, methods, and uses for photochemical generation of acids and/or radical species |
The present invention provides compounds and compositions, which include: at least one chromophore having strong simultaneous two-photon or multi-photon absorptivity; at least one acid- or radical-generator in close proximity to the chromophore; such that the single- or multi-photon excitation of the chromophore results in the generation of an acid and/or redical that is capable of activating chemistry; and such that compositions of matter based on the componds and compositions of the invention can be photo-patterned by one- or multiphoton excitation. |
1. A compound or composition, comprising: at least one chromophore having a simultaneous two-photon or multi-photon absorptivity; and at least one photoacid or radical generator in close proximity to said chromophore; wherein said chomophore has a two-photon absorption cross-section of >50×10−50 cm4 s/photon. 2. The compound or composition of claim 1, wherein said generator comprises at least one sulfonium, selenonium, or iodonium group, or other acid- or radical generating group. 3. The compound or composition of claim 1, wherein upon simultaneous absorption of two or more photons, said chromophore adopts an electronically excited state, and therefrom activates said generator to generate a Brnsted or Lewis acid and/or radical. 4. The compound or composition of claim 1, wherein said generator comprises at least one anion selected from the group consisting of F−, Cl−, Br−, I−, CN−, SO42−, PO43−, CH3CO2−, CF3SO3−, NO2−, NO3−, BF4−, PF6−, SbF6−, AsF6−, SbCl4−, ClO3−, ClO4−, and B(aryl)4−, where aryl is an aryl group containing 25 or fewer carbon atoms that may be optionally substituted with one or more alkyl groups, aryl groups or halogens. 5. The compound or composition of claim 1, wherein said generator is brought into close proximity with said chromophore by at least one mechanism selected from the group consisting of covalent linkage, ion-pairing, hydrogen-bonding, charge-transfer complex formation, perfluoroaryl-aryl electrostatic interaction, π-stacking association, coordinative-bond formation, dipole-dipole pairing, and combinations thereof. 6. The compound or composition of claim 1, wherein the chromophore and the generator are covalently linked, and wherein the chromophore is a molecule having a structure selected from the group consisting of D-π-D, D-A-D, A-π-A and A-D-A, where D is an electron donor and A is an electron acceptor. 7. The compound or composition of claim 1, wherein the sulfonium group has the formula —(CH2)γ—(C6H4)δ—SRa5Ra6, wherein Ra5 and Ra6 are each independently alkyl, aryl, or monomer groups, and wherein γ=0 to 25, and δ=0 to 5. 8. The compound or composition of claim 1, wherein the selenonium group has the formula —(CH2)γ—(C6H4)δ—SeRa5Ra6, wherein Ra5 and Ra6 are each independently alkyl, aryl, or monomer groups, and wherein γ=0 to 25, and δ=0 to 5. 9. The compound or composition of claim 1, wherein the iodonium group has the formula —(CH2)γ—(C6H4)δ—IRa7, wherein Ra7 is alkyl, aryl, or monomer group, and wherein γ=0 to 25, and δ=0 to 5. 10. The compound or composition of claim 1, which has the structure: wherein X═S or Se and n=0, 1, 2, 3, 4, or 5, wherein Ar1 and Ar2 are each independently a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein each of Ar1 and Ar2 are optionally independently substituted with one or more H, alkyl group, alkoxy group, or aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein R1, R2, R3, and R4 are each independently alkyl or aryl groups, which groups may be optionally independently substituted one or more sulfonium, selenoniumn, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein Y is an anion selected from the group consisting of F−, Cl−, Br−, I−, CN−, SO42−, PO43−, CH3CO2−, CF3SO3−, NO2−, NO3−, BF4−, PF6−, SbF6−, AsF6−, SbCl4−, ClO331, ClO4−, and B(aryl)4−, where aryl is an aryl group containing 25 or fewer carbon atoms that may be optionally substituted with one or more alkyl groups, aryl groups or halogens, wherein z is an integer equal to the charge of the chromophore portion of the compound, wherein p is an integer equal to the charge on the anion, and wherein q and Q are integers such that the relationship zQ=pq is satisfied. 11. The compound or composition of claim 1, which has the structure: wherein X═I and n=0, 1, 2, 3, 4, or 5, wherein Ar1 and Ar2 are each independently a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein each of Ar1 and Ar2 are optionally independently substituted with one or more H, alkyl group, alkoxy group, or aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein R1 and R2 are each independently alkyl, or aryl groups, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein Y is an anion selected from the group consisting of F−, Cl−, Br−, I−, CN−, SO42−, PO43−, CH3CO2−, CF3SO3−, NO2−, NO3−, BF4−, PF6−, SbF6−, AsF6−, SbCl4−, ClO3−, ClO4−, and B(aryl)4−, where aryl is an aryl group containing 25 or fewer carbon atoms that may be optionally substituted with one or more alkyl groups, aryl groups or halogens, wherein z is an integer equal to the charge of the chromophore portion of the compound, wherein p is an integer equal to the charge on the anion, and wherein q and Q are integers such that the relationship zQ=pq is satisfied. 12. The compound or composition of claim 1, which has the structure: wherein X is O and n=1, 2, 3, 4, or 5, wherein Ar1 and Ar2 are each independently a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein each of Ar1 and Ar2 are optionally independently substituted with one or more H, alkyl group, alkoxy group, or aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein R1 and R2 are each independently H, alkyl, or aryl groups, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein at least one of Ar1, Ar2, R1 or R2 is substituted with one or more sulfonium, selenonium, or iodonium groups, or other acid- or radical generating groups, wherein Y is an anion selected from the group consisting of F−, Cl−, Br−, I−, CN−, SO42−, PO43−, CH3CO2−, CF3SO3−, NO2−, NO3−, BF4−, PF6−, SbF6−, AsF6−, SbCl4−, ClO3−, ClO4−, and B(aryl)4−, where aryl is an aryl group containing 25 or fewer carbon atoms that may be optionally substituted with one or more alkyl groups, aryl groups or halogens, wherein z is an integer equal to the charge of the chromophore portion of the compound, wherein p is an integer equal to the charge on the anion, and wherein q and Q are integers such that the relationship zQ=pq is satisfied. 13. The compound or composition of claim 1, which has the structure: wherein X is N and n=0, 1, 2, 3, 4, or 5, wherein Ar1 and Ar2 are each independently a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein each of Ar1 and Ar2 are optionally independently substituted with one or more H, alkyl group, alkoxy group, or aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein R1, R2, R3, and R4 are each independently H, alkyl group, or aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein at least one of Ar1, Ar2, R1, R2, R3, or R4 is substituted with one or more sulfonium, selenonium, or iodonium groups, or other acid- or radical generating groups, wherein Y is an anion selected from the group consisting of F−, Cl−, Br−, I−, CN−, SO42−, PO43−, CH3CO2−, CF3SO3−, NO2−, NO3−, BF4−, PF6−, SbF6−, AsF6−, SbCl4−, ClO3−, ClO4−, and B(aryl)4−, where aryl is an aryl group containing 25 or fewer carbon atoms that may be optionally substituted with one or more alkyl groups, aryl groups or halogens, wherein z is an integer equal to the charge of the chromophore portion of the composition, wherein p is an integer equal to the charge on the anion, and wherein q and Q are integers such that the relationship zQ=pq is satisfied. 14. The compound or composition of claim 1, which has the structure: wherein X is I, n=0, 1, 2, 3, 4, or 5, and n′=0, 1, 2, 3, 4 or 5, wherein Ar1 and Ar2 are each independently a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein Ar3 is a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein each of Ar1 and Ar2 are optionally independently substituted with one or more H, alkyl group, alkoxy group, or aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein Ar3 is optionally substituted with one or more H, acceptor group, alkyl group, alkoxy group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein R1 and R2 are each independently alkyl group, or aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein Y is an anion selected from the group consisting of F−, Cl−, Br−, I−, CN−, SO42−, PO43−, CH3CO2−, CF3SO3−, NO2−, NO3−, BF4−, PF6−, SbF6−, AsF6−, SbCl4−, ClO3−, ClO4−; and B(aryl)4−; where aryl is an aryl group containing 25 or fewer carbon atoms that may be optionally substituted with one or more alkyl groups, aryl groups or halogens, wherein z is an integer equal to the charge of the chromophore portion of the compound, wherein p is an integer equal to the charge on the anion, and wherein q and Q are integers such that the relationship zQ=pq is satisfied. 15. The compound or composition of claim 1, which has the structure: wherein X is S or Se, n=0, 1, 2, 3, 4, or 5, and n′=0,1,2,3,4 or 5, wherein Ar1 and Ar2 are each independently a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein Ar3 is a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein each of Ar1 and Ar2 are optionally independently substituted with H, alkyl group, alkoxy group, or aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein Ar3 is optionally independently substituted with one or more H, acceptor group, alkyl group, alkoxy group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein R1, R2, R3, and R4 are each independently alkyl group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein Y is an anion selected from the group consisting of F−, Cl−, Br−, I−, CN−, SO42−, PO43−, CH3CO2−, CF3SO3−, NO2−, NO3−, BF4−, PF6−, SbF6−, AsF6−, SbCl4−, ClO3−, ClO4−; and B(aryl)4−, where aryl is an aryl group containing 25 or fewer carbon atoms that may be optionally substituted with one or more alkyl groups, aryl groups or halogens, wherein z is an integer equal to the charge of the chromophore portion of the compound, wherein p is an integer equal to the charge on the anion, and wherein q and Q are integers such that the relationship zQ=pq is satisfied. 16. The compound or composition of claim 1, which has the structure: wherein X is O, n=0, 1, 2, 3, 4, or 5, and n′=0, 1, 2, 3, 4 or 5, wherein Ar1 and Ar2 are each independently a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein Ar3 is a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein each of Ar1 and Ar2 are optionally independently substituted with one or more H, alkyl group, alkoxy group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein Ar3 is optionally substituted with one or more H, acceptor group, alkyl group, alkoxy group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein R1 and R2 are each independently H, alkyl group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein at least one of Ar1, Ar2, Ar3, R1 or R2 is substituted with one or more sulfonium, selenonium, or iodonium groups, or other acid- or radical generating groups, wherein Y is an anion selected from the group consisting of F−, Cl−, Br−, I−, CN−, SO42−, PO43−, CH3CO2−, CF3SO3−, NO2−, NO3−, BF4−, PF6−, SbF6−, AsF6−, SbCl4−, ClO3−, ClO4−, and B(aryl)4−, where aryl is an aryl group containing 25 or fewer carbon atoms that may be optionally substituted with one or more alkyl groups, aryl groups or halogens, wherein z is an integer equal to the charge of the chromophore portion of the compound, wherein p is an integer equal to the charge on the anion, and wherein q and Q are integers such that the relationship zQ=pq is satisfied. 17. The compound or composition of claim 1, which has the structure: wherein X is N, n=0, 1, 2, 3, 4 or 5, n′=0, 1, 2, 3, 4, or 5 and n′″=0, 1, 2, 3, 4, or 5, wherein Ar1 and Ar2 are each independently a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein Ar3 is a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein each of Ar1 and Ar2 are optionally independently substituted with one or more H, alkyl group, alkoxy group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein Ar3 is optionally substituted with one or more H, acceptor group, alkyl group, alkoxy group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein R1, R2, R3, and R4 are each independently H, alkyl group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein at least one of Ar1, Ar2, Ar3, R1, R2, R3, or R4 is substituted with one or more sulfonium, selenonium, or iodonium groups, or other acid- or radical generating groups, wherein Y is an anion selected from the group consisting of F−, Cl−, Br−, I−, CN−, SO42−, PO43−, CH3CO2−, CF3SO3−, NO2−, NO3−, BF4−, PF6−, SbF631 , AsF6−, SbCl4−, ClO3−, ClO4−, and B(aryl)4−, where aryl is an aryl group containing 25 or fewer carbon atoms that may be optionally substituted with one or more alkyl groups, aryl groups or halogens, wherein z is an integer equal to the charge of the chromophore portion of the compound, wherein p is an integer equal to the charge on the anion, and wherein q and Q are integers such that the relationship zQ=pq is satisfied. 18. The compound or composition of claim 1, which has the structure: wherein X is N, n=0, 1, 2, 3, 4, or 5, n′=0, 1, 2, 3, 4 or 5 and n′″=0, 1, 2, 3, 4, or 5, wherein Ar1 and Ar2 are each independently a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein Ar3 is a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein each of Ar1 and Ar2 are optionally independently substituted with one or more H, alkyl group, alkoxy group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein Ar3 is optionally substituted with one or more H, acceptor group, alkyl group, alkoxy group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein R1, R2, R3, and R4 are each independently H, alkyl group, aryl group, which groups may be optionally independently substituted with one or more sulfonium, selenonium, or iodonium groups; other acid- or radical-generating species; or monomer or pre-polymer groups, wherein at least one of Ar1, Ar2, Ar3, R1, R2, R3, or R4 is substituted with one or more sulfonium, selenonium, or iodonium groups, or other acid- or radical generating groups, wherein Y is an anion selected from the group consisting of F−, Cl−, Br−, I−, CN−, SO42−, PO43−, CH3CO2−, CF3SO3−, NO2−, NO3−, BF4−, PF6−, SbF6−, AsF6−, SbCl4−, ClO3−, ClO4−, and B(aryl)4−, where aryl is an aryl group containing 25 or fewer carbon atoms that may be optionally substituted with one or more alkyl groups, aryl groups or halogens, wherein z is an integer equal to the charge of the chromophore portion of the compound, wherein p is an integer equal to the charge on the anion, and wherein q and Q are integers such that the relationship zQ=pq is satisfied. 19. The compound or composition of claim 1, wherein said chromophore and said generator are each present in concentrations of 0.001 M to 2 M. 20. The compound or composition of claim 1, wherein said chromophore is an anion. 21. The compound or composition of claim 1, further comprising at least one polymerizable or cross-linkable monomer, oligomer, or prepolymer, or acid-modifiable medium. 22. The compound or composition of claim 1, wherein the chromophore is a molecule having a structure selected from the group consisting of D-π-D, D-A-D, A-π-A and A-D-A, where D is an electron donor and A is an electron acceptor. 23. A method for making an article, comprising: contacting the compound or composition of claim 1 with at least one polymerizable or cross-linkable monomer, oligomer, or prepolymer, or acid-modifiable medium; irradiating said compound or composition to cause a simultaneous two-photon or multi-photon absorption in said chomophore; and polymerizing said monomer, oligomer, or prepolymer or cleaving a group from said acid-modifiable medium. 24. An article, produced by the method of claim 23. 25. A method for generating a Bronsted or Lewis acid and/or radical, comprising irradiating said compound or composition of claim 1 to cause a simultaneous two-photon or multi-photon absorption in said chomophore. 26. A compound or composition, comprising: a first means for simultaneously absorbing two or more photons; a second means for producing an electronically excited state upon simultaneous absorption of two or more photons; and a third means for generating a Bronsted or Lewis acid and/or radical upon reaction with said excited state; wherein said third means comprises at least one sulfonium, selenonium, or iodonium group, or other acid- or radical generating group. 27. The compound or composition of claim 26, wherein said first and second means are comprised within a single molecule. 28. The compound or composition of claim 26, wherein said first, second and third means are comprised within a single molecule. 29. An apparatus, comprising: a compound or composition, comprising: a first means for simultaneously absorbing two or more photons; a second means for producing an electronically excited state upon simultaneous absorption of two or more photons; and a third means for generating a Bronsted or Lewis acid and/or radical upon reaction with said excited state; wherein said third means comprises at least one sulfonium, selenonium, or iodonium group, or other acid- or radical-generating group; and a means for irradiating said compound or composition. 30. The apparatus of claim 29, wherein said means for irradiating comprises one or more laser beams. 31. The apparatus of claim 30, wherein said one or more laser beams are pulsed laser beams. 32. A compound or composition, which has the structure: wherein X is N, n=0, 1, 2, 3, 4 or 5, n′=0, 1, 2, 3, 4, or 5 and n′″=0, 1, 2, 3, 4, or 5, wherein Ar1 and Ar2 are each independently a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein Ar3 is a 5-membered heteroaromatic ring; a 6-membered aromatic ring; or a 6-membered heteroaromatic ring, wherein each of Ar1 and Ar2 are optionally independently substituted with one or more H, alkyl, alkoxy, aryl, thioalkoxy, thioaryloxy, selenoalkoxy, or selenoaryloxy groups, which groups may be optionally independently substituted with monomer or pre-polymer groups, wherein Ar3 is optionally independently substituted with one or more H, acceptor, alkyl, alkoxy, aryl, thioalkoxy, thioaryloxy selenoalkoxy, or selenoaryloxy groups, which groups may be optionally independently substituted with monomer or pre-polymer groups, wherein R1, R2, R3, and R4 are each independently H, alkyl, or aryl groups, which groups may be optionally independently substituted with one or more thioalkoxy, thioaryloxy selenoalkoxy, selenoaryloxy or groups, and wherein at least one of Ar1, Ar2, Ar3, R1, R2, R3, or R4 is substituted with one or more thioalkoxy, thioaryloxy selenoalkoxy, selenoaryloxy groups. 33. The compound or composition of claim 32, wherein the thioether fragment has the formula —(CH2)γ—(CH4H4)δ—SRa5, wherein Ra5 is an alkyl group, and wherein γ=0 to 25, and δ=0 to 5. 34. The compound or composition of claim 32, wherein the thioether fragment has the formula —(CH2)γ—(C6H4)δ—SRa5, wherein Ra5 is an aryl group, and wherein γ=0 to 25, and δ=0 to 5. 35. The compound or composition of claim 32, wherein the selenoether fragment has the formula —(CH2)γ—(C6H4)δ—SeRa5, wherein Ra5 is an alkyl group, and wherein γ=0 to 25, and δ=0 to 5. 36. The compound or composition of claim 32, wherein the selenoether fragment has the formula —(CH2)γ(C6H4)δ—SeRa5, wherein Ra5 is an aryl group, and wherein γ=to 25, and δ=0 to 5. 37. A composition of a form selected from the group consisting of: wherein R is methyl or benzyl, and wherein X is F−, Cl−, Br−, I−, CN−, SO42−, PO43−, CH3CO2−, CF3SO3−, NO2−, NO3−, BF4−, PF6−, SbF6−, AsF6−, SbCl4−, ClO3−, ClO4−, or B(aryl)4−, where aryl is an aryl group containing 25 or fewer carbon atoms that may be optionally substituted with one or more alkyl groups, aryl groups or halogens. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The invention relates to compositions and compounds that have large two-photon or higher-order absorptivities, which, after excitation, generate Lewis or Brønsted acids, radicals or a combination thereof The invention also relates to methods of making and using the compositions and compounds. 2. Discussion of the Background Two-photon or higher-order absorption refers to the initial simultaneous absorption of two or more photons (also referred to as multi-photon absorption) without the actual population of an excited state by the absorption of a single photon. Molecular two-photon absorption was predicted in Göppert-Mayer, M., Ann. Phys. 1931, 9, 273. Upon the invention of pulsed ruby lasers in 1960, experimental observation of two-photon absorption became reality. In the years since, multi-photon excitation has found application in biology and optical data storage, as well as in other fields. Although interest in multi-photon excitation has exploded, there is a paucity of two-photon absorbing dyes with adequately strong two-photon absorption in the correct spectral region for many applications. There are two key advantages of two-photon (or higher-order) induced processes relative to single-photon induced processes. Whereas single-photon absorption scales linearly with the intensity of the incident radiation, two-photon absorption scales quadratically. Higher-order absorptions will scale with yet a higher power of incident intensity. As a result, it is possible to perform multi-photon induced processes with three dimensional spatial resolution. Further, because these processes involve the simultaneous absorption of two or more photons, the chromophore is excited with a number of photons whose total energy equals the energy of a multi-photon absorption transition, although each photon individually has insufficient energy to excite the chromophore. Because the exciting light is not attenuated by single-photon absorption in this case, it is possible to excite selectively molecules at a greater depth within a material than would be possible via single-photon excitation by use of a beam that is focused to that depth in the material. These two advantages also apply to, for example, excitation within tissue or other biological materials. In multi-photon lithography or stereolithography, the nonlinear scaling of absorption with intensity can lead to the ability to write features of a size below the diffraction limit of light, and the ability to write features in three dimensions, which is also of interest for holography. The ability to realize many of the possible applications of two-photon or higher-order absorption by molecules rests on the availability of chromophores with large two-photon or higher-order absorption cross sections. We have taught in U.S. Pat. No. 6,267,913, which is incorporated herein by reference, that certain classes of molecules exhibit enhanced two-photon or multi-photon absorptivities. These molecules can be categorized as follows: a) molecules in which two donors are connected to a conjugated π-electron bridge (abbreviated “D-π-D” motif); b) molecules in which two donors are connected to a conjugated π-electron bridge which is substituted with one or more electron accepting groups (abbreviated “D-A-D” motif); c) molecules in which two acceptors are connected to a conjugated π-electron bridge (abbreviated “A-π-A” motif); and d) molecules in which two acceptors are connected to a conjugated π-electron bridge which is substituted with one or more electron donating groups (abbreviated “A-D-A” motif). Accordingly, molecules from the aforementioned classes can be excited efficiently by simultaneous two-photon (or higher-order) absorption, leading to efficient generation of electronically excited states. These excited state species can be exploited in a great variety of chemical and physical processes, with the advantages enabled by multiphoton excitation. For example, by employing polymerizable resin formulations containing cross-linkable acrylate containing monomers and D-π-D molecules as two-photon initiators of radical polymerization, complex three-dimensional objects can be prepared using patterned two-photon excitation. Most two-photon induced photopolymerization processes involve radical reactions in which there is some volume decrease upon polymerization (Cumpston et al. Nature 398, (1999) 51; Belfield, K. D. et al. J. Am. Chem. Soc. 122, (2000) 1217). The applications that depend upon two-photon or multi-photon excitation also require that the two-photon or multiphoton excited states cause a chemical or physical change in the exposed region of the materials. Such changes can result from the generation of a Brønsted or Lewis acid and/or radical species and subsequent further reactions of that species with other components in the material, for example, resulting in cleavage of a functional group from a polymer or initiation of a polymerization, as is well known to one skilled in the art of lithography. Under one photon excitation conditions it has been shown that sulfonium and iodonium salts are effective for the generation of Brønsted acids. Methods for the synthesis of sulfonium salts are well documented in J. L. Dektar and N. P. Hacker, “Photochemistry of Triarylsulfonium Salts”, J. Am. Chem. Soc. 112, (1990) 6004-6015 which are incorporated herein by reference. Additional methods for synthesizing onium salts of the the general type described in the invention can be prepared conveniently from aryl aliphatic sulfides and primary aliphatic halides or benzyl halides, by well known methods such as those described in Lowe, P. A., “Synthesis of Sulfonium Salts”, The Chemistry of the Sulfonium Group (Part 1), ed. C. J. M. Sterling, John Wiley & Sons, Ltd., (1981), p 267 et seq and as described in U.S. Pat. Nos. 5,302,757, 5,274,148, 5,446,172, 5,012,001, 4,882,201, 5,591011, and 2,807,648, which are all incorporated herein by reference. Methods for the synthesis of iodonium salts are well documented in C. Herzig and S. Scheiding, DE 4,142,327, CA 119,250,162 and C. Herzig, EP 4,219,376, CA 120,298,975 and U.S. Pat. Nos. 5,079,378, 4,992,571, 4,450,360, 4,399,071, 4,310,469, 4,151,175, 3,981,897, and 5,144,051 which are incorporated herein by reference. It is known to those skilled in the art that epoxide-containing monomers exhibit relatively small shrinkage upon polymerization. It is also known that expoxide monomers as well as others, such as vinyl ether monomers, can be photo-polymerized under one photon excitation conditions using iodonium salts and sulfonium salts as photoacid generating initiators as described by: Crivello, J. V.; Lam, J. H. W. Macromolecules, 1977, 10, 1307; DeVoe, R. J.; Sahyn, M. R. V.; Schmidt, E. Can. J. Chem. 1988, 66, 319; Crivello, J. V.; Lee, J. J. Polym. Sci. Polym. Chem. Ed. 1989, 27 3951; Dektar, J.; Hacker, N. P. J. Am. Chem. Soc. 1990, 112, 6004; Crivello, J. V.; Lam, J. H. W.; Volante, C. N. J. Rad. Curing, 1977, 4, 2; Pappas, S. P.; Pappas, B. C.; Gatechair, L. R.; Jilek, J. H. Polym. Photochem. 1984, 5, 1; Welsh, K. M.; Dektar, J. L.; Garcia-Garibay, M. A.; Hacker, N. P.; Turro, N. J. J. Org. Chem. 1992, 57, 4179; Crivello, J. V.; Kong, S. Macromolecules, 2000, 33, 825, which are incorporated herein by reference. It is known that dialkyl aryl sulfonium ions—as described by Saeva, F. D.; Morgan, B. P. J. Am. Chem. Soc., 1984, 106, 4121; Saeva, F. D. Advances in Electron Transfer Chem. 1994, 4, 1, which are incorporated herein by reference—and iodonium salts can be sensitized through electron transfer by the addition of other molecules. These include Class I and Class II photoacid generating species, as described by Saeva et al. (cited above). |
<SOH> SUMMARY OF THE INVENTION <EOH>One object of the invention is to provide compounds and compositions which can be efficiently photoactivated by two- or multi-photon excitation to yield acid and/or radical species and which consequently overcome the limitations associated with conventional compounds and compositions. This and other objects have been achieved by the present invention, the first embodiment of which provides a compound or composition, which includes: at least one chromophore having a simultaneous two-photon or multi-photon absorptivity; at least one photoacid or radical generator in close proximity to the chromophore; wherein the generator may be a sulfonium, selenonium, or iodonium group, or other acid- or radical generating group. The present invention is not restricted to acid-generators consisting of only sulfonium, selenonium or iodonium groups. Another embodiment of the present invention provides a method for making an article, which includes contacting the above compound or composition with at least one polymerizable or cross-linkable monomer, oligomer, or prepolymer, or acid-modifiable medium (such as ester-functionalized chemically amplified resins); irradiating the compound or composition to cause a simultaneous two-photon or multiphoton absorption in the chomophore; and polymerizing the monomer, oligomer, or prepolymer, or affecting a chemical change in an acid-modifiable medium. The invention can be used for the fabrication of articles by scanning of a focused laser beam or by multiple-beam interference. Another embodiment of the present invention provides an article, produced by the above process. Another embodiment of the present invention provides a method for generating a Brønsted or Lewis acid and/or radical, which includes irradiating the above compound or composition to cause a simultaneous two-photon or multiphoton absorption in the chomophore. Another embodiment of the present invention provides a compound or composition, which includes: a first means for simultaneously absorbing two or more photons; a second means for producing an electronically excited state upon simultaneous absorption of two or more photons; a third means for generating a Brønsted or Lewis acid and/or radical upon reaction with the excited state; wherein the third means includes at least one sulfonium, selenonium, or iodonium group, or other acid- or radical generating group. Another embodiment of the present invention provides an apparatus, which includes: a compound or composition, which includes: a first means for simultaneously absorbing two or more photons; a second means for producing an electronically excited state upon simultaneous absorption of two or more photons; a third means for generating a Brønsted or Lewis acid and/or radical upon reaction with the excited state; wherein the third means includes at least one sulfonium, selenonium, or iodonium group, or other acid- or radical generating group; and a means for irradiating the compound or composition. |
Automatic number plate recognition system |
A handheld automatic number plate recognition system comprising a handheld personal computer and a camera, the camera being operable to capture images and the computer including a processor to identify a number plate from an image captured by the camera. |
1. A handheld automatic number plate recognition system comprising a handheld personal computer and a camera, the camera being operable to capture images and the computer comprising a processor to identify a number plate from an image captured by the camera, wherein the processor comprises means to identify rectangular areas, each such identified area comprising a possible number plate to be interpreted. 2. A system according to claim 1, wherein the computer is operable to interpret the image of the plate and output a string of recognized characters in response thereto. 3. A system according to claim 1 or 2, wherein the computer is operable to power down the camera when not in use. 4. A system according to any preceding claim, wherein the processor includes means to identify rectangular areas, each such identified area comprising a possible number plate to be interpreted. 5. A system according to any preceding claim, wherein the processor sets a plate size threshold range outside of which a captured image will not be processed. 6. A system according to claim 5, wherein the threshold range is between 1,000 and 10,000 pixels. 7. A system according to claim 6, wherein the threshold range is between 1,800 and 5,000 pixels. 8. A system according to any preceding claim, wherein the computer has a memory in which is stored a plurality of known number plates. 9. A system according to any preceding claim, wherein the system is portable. 10. A system according to any preceding claim, wherein the camera is attached to the computer. 11. A system according to any preceding claim, wherein a single housing holds the camera and the computer. 12. A system according to any preceding claim, wherein the computer includes a display operable to indicate a recognized number plate. 13. A system according to any preceding claim, wherein the computer includes a speaker and a synthesizer to enunciate the characters making up a recognized number plate. 14. A system according to any preceding claim, wherein the camera is operable in the infrared spectrum and the system has at least one infrared source. 15. A system according to claim 14, wherein the or each infrared source is located adjacent the camera such that a path of incident light from the infrared source is within 5° of a return path of light reflected from a subject. 16-25. (Canceled). |
Modification of organelle metabolism by unc-51-like kinases roma1 or 2tm proteins |
The invention discloses polypeptides (Unc-51 kinase, ROMA1, and/or 2TM protein) affecting the activity of Uncoupling Proteins (UCPs), thereby leading to an altered mitochondrial activity and thus contributing to membrane stability and/or function of organelles, preferably mitochondria. This invention relates to the use of these polypepetides in the diagnosis, study, prevention, and treatment of diseases and disorders, for example, but not limited to, metabolic diseases such as obesity, adipositas, eating disorders, wasting syndromes (cachexia), pancreatic dysfunctions (for example diabetes), disorders related to ROS production, and others. |
1. A pharmaceutical composition comprising a nucleic acid molecule of the Unc-51, regulator of mitochondrial activity 1 (ROMA1), and/or mitochondrial 2TM gene family or a polypeptide encoded thereby or a fragment or a variant of said nucleic acid molecule or said polypeptide or an antibody, an aptamer or another receptor recognizing said nucleic acid molecule or polypeptide encoded thereby together with pharmaceutically acceptable carriers, diluents and/or adjuvants. 2. The composition of claim 1, wherein the nucleic acid molecule is a vertebrate or insect Unc-51, ROMA1, and/or 2TM nucleic acid, particularly a human nucleic acid such as human Unc-51-like kinase 1 (ULK-1) (Genbank Accession No. NM 003565) or human KIAA 0623 gene (ULK-2) (Genbank Accession No. NM.01 4683) or human BAP37 (Genbank Accession No. XP 006639.1) or human mitochondrial 2TM (SEQ ID NO:7, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:9) or a mouse nucleic acid such as mouse Unc-51-like kinase 1 (ULK-1) (Genbank Accession No. NM 009469) or mouse Unc-51-like kinase 2 (ULK-2) (Genbank Accession No. AB 019577) or mouse BAP37 (Genbank Accession No. NP 031557.1) or mouse 2TM (Genbank accession number BAB26124), or an insect nucleicacid such as Drosophila melanogaster Unc-51 (GadFly Accession Number CG 10967), ROMA1 (GadFly Accession Number CG15081), and/or 2TM (GadFly Accession Number CG7620), or a fragment thereof or a variant thereof. 3. The composition of claim 1, wherein said nucleic acid molecule (a) comprises a nucleotide sequence encoding one of the proteins mentioned in claim 2 or the complement thereof; (b) a nucleotide sequence which hybridizes at 65 or 66° C. in a solution containing 0.2×SSC and 0.1% SDS to the complementary strand of a nucleic acid molecule encoding one of the amino acid sequences of claim 2; (c) is degenerate with respect to the nucleic acid molecule of (a) and/or (b); (d) encodes a polypeptide which is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99.6% identical to one of the proteins of claim 2; (e) differs from the nucleic acid molecule of (a) to (d) by mutation and wherein said mutation causes an alteration, deletion, duplication or premature stop in the encoded polypeptide or (f comprises a partial sequence of any of the nucleotide sequences of (a) to (e) having a length of at least 15 bases. 4. The composition of claim 1, wherein the nucleic acid molecule is a DNA molecule, particularly a cDNA or a genomic DNA. 5. The composition of claim 1, wherein said nucleic acid encodes a polypeptide contributing to membrane stability and/or function of organelles. 6. The composition of claim 1, wherein said nucleic acid encodes a polypeptide which is a regulator of a transporter molecule. 7. The composition of claim 1, wherein said nucleic acid encodes a polypeptide which is a modifier of mitochondrial proteins. 8. The composition of claim 1, wherein said nucleic acid molecule is a recombinant nucleic acid molecule. 9. The composition of claim 1, wherein said nucleic acid molecule is a DNA or an RNA. 10. The composition of claim 1, wherein the nucleic acid molecule is a vector, particularly an expression vector. 11. The composition of claim 1, wherein the polypeptide is a recombinant polypeptide. 12. The composition of claim 11, wherein said recombinant polypeptide is a fusion polypeptide. 13. The composition of claim 1, wherein said nucleic acid molecule is selected from hybridization probes, primers and 15 anti-sense oligonucleotides. 14. The composition of claim 1 which is a diagnostic composition. 15. The composition of claim 1 which is a therapeutic composition. 16. Use of the composition of claim 1 for the manufacture of an agent for detecting and/or verifying, for the diagnosis, for the treatment, alleviation and/or prevention of a disorder, wherein such disorder is a metabolic disorder or a mitochondrial disorder such as obesity, adipositas, eating/body weight disorders (bulimia nervosa, anorexia nervosa), cachexia (wasting), pancreatic dysfunction(diabetes), mitochondrial disorders, and/or a disorder related to ROS production and others, in cells, cell masses, organs and/or subjects. 17. Use of a nucleic acid molecule of the Unc-51, ROMA1, and/or 2TM gene family or a polypeptide encoded thereby or a fragment or a variant of said nucleic acid molecule or said polypeptide or an antibody, an aptamer or another receptor recognizing said nucleic acid molecule of or a polypeptide encoded thereby for controlling the function of a gene and/or a gene product which is influenced and/or modified by an Unc-51, ROMA1, and/or 2TM polypeptide. 18. The use of claim 17, wherein said gene and/or gene product is a 10 gene and/or gene product expressed in organelles, wherein said organelle is a mitochondrium, a peroxisome or a chloroplast. 19. Use of a nucleic acid molecule of the Unc-51, ROMA 1, and/or 2TM gene family or a polypeptide encoded thereby or a fragment or a variant of said nucleic acid molecule or said polypeptide or an antibody, an aptamer or another receptor recognizing said nucleic acid molecule or a polypeptide encoded thereby for identifying substances capable of interacting with an Unc-51-kinase-like, ROMA1, and/or 2TM polypeptide. 20. The use of claim 19, wherein said substances capable of interacting with said polypeptide are antagonists or agonists. 21. A non-human transgenic animal exhibiting a modified expression of an Unc-51-kinase-like, ROMA 1, and/or 2TM polypeptide. 22. The animal of claim 21, wherein the expression of the Unc-51 kinase-like, ROMA1, and/or 2TM polypeptide is increased and/or reduced. 23. A recombinant host cell exhibiting a modified expression of an Unc-51-kinase-like, ROMA1, and/or 2TM polypeptide. 24. The cell of claim 23 which is a human cell. 25. A method of identifying a polypeptide or a substance involved in cellular metabolism in an animal or capable of modifying homeostasis comprising the steps of: (a) testing a collection of polypeptides or substances for interaction with an Unc-51, ROMA1, and/or 2TM polypeptide or a fragment thereof using a readout system; and (b) identifying polypeptides or substances which test positive for interaction in step (a), (c) repeating steps (a) and (b) with the polypeptides identified one or more times wherein the newly identified polypeptide replaces the previously identified polypeptide as a bait for the identification of a further interacting polypeptide. 26. The method of claim 25 further comprising the step of identifying the nucleic acid molecule(s) encoding the one or more interacting (poly)peptides. 27. A method of identifying a polypeptide involved in the regulation of body weight in a mammal comprising the steps of (a) contacting a collection of (poly)peptides with an Unc-51, ROMA1, and/or 2TM like polypeptide or a fragment thereof under conditions that allow binding of said (poly)peptides; (b) removing (poly)peptides from said collection of (poly)peptides that did not bind to said Unc-51, ROMA 1, and/or 2TM polypeptide in step (a); and (c) identifying (poly)peptides that bind to said Unc-51, ROMA1, and/or 2TM polypeptide. 28. The method of claim 27 further comprising the step of identifying the nucleic acid molecule(s) encoding the one or more binding (poly)peptides. 29. A method of identifying a compound influencing the expression of a nucleic acid molecule of the Unc-51, ROMA1, and/or 2TM gene family or the activity of an Unc-51, ROMA 1, and/or 2TM polypeptide comprising the steps of (a) contacting a host carrying an expression vector comprising a nucleic acid molecule of Unc-51, ROMA 1, and/or 2TM or a nucleic acid molecule identified by the method of claim 26 or 28 operatively linked to a readout system with a compound or a collection of compounds; (b) assaying whether said contacting results in a change of signal intensity provided by said readout system; and, optionally, (c) identifying a compound within said collection of compounds that induces a change of signal in step (b); wherein said change in signal intensity correlates with a change of expression of said nucleic acid molecule. 30. A method of assessing the impact of the expression of one or more Unc-51, ROMA1, and/or 2TM polypeptides in a non-human animal comprising the steps of (a) overexpressing or underexpressing a nucleic acid molecule of the Unc-51, ROMA1, and/or 2TM gene family or a nucleic acid molecule obtainable according to the method of claim 26 in said animal; and (d) determining whether the weight of said animal has increased, decreased, whether metabolic changes are induced and/or whether the eating behaviour is modified. 31. A method of screening for an agent which modulates the interaction of an Unc-51, ROMA1, and/or 2TM polypeptide with a binding target/agent, comprising the steps of (a) incubating a mixture comprising (aa) an Unc-51, ROMA1, and/or 2TM polypeptide, or a fragment thereof or a fusion protein or a fragment thereof; (ab) a binding target/agent of said (poly)peptide or fusion protein or fragment thereof; and (ac) a candidate agent under conditions whereby said (poly)peptide, fusion protein or fragment thereof specifically binds to said binding target/agent at a reference affinity; (b) detecting the binding affinity of said (poly)peptide, fusion protein or fragment thereof to said binding target to determine an (candidate) agent-biased affinity; and (c) determining a difference between (candidate) agent-biased affinity and the reference affinity. 32. A method for producing a composition comprising the polypeptide identified by the method of claim 25 with a pharmaceutically acceptable carrier, diluent and/or adjuvant. 33. The method of claim 32 wherein said composition is a pharmaceutical composition for preventing, alleviating or treating obesity, adipositas, eating disorders, wasting syndromes (cachexia), mitochondrial disorders, pancreatic dysfunctions (for example diabetes), disorders related to ROS production. 34. A composition comprising (a) an inhibitor or stimulator of an Unc-51, ROMA1, and/or 2TM (poly)peptide or of a (poly)peptide identified by the method of claim 25. 35. The composition of claim 34 which is a pharmaceutical composition. 36. Use of (a) an inhibitor or stimulator of the (poly)peptide identified by the method of claim 25; (b) a modulator of the expression of the gene identified by the method of claim 25; for the preparation of a pharmaceutical composition for the treatment of obesity, adipositas, eating disorders, wasting syndromes (cachexia), mitochondrial disorders, pancreatic dysfunctions (for example diabetes), disorders related to ROS production. 37. Use of an agent as identified by the method of claim 31 for the preparation of a pharmaceutical composition for the treatment, alleviation and/or prevention of obesity, adipositas, eating disorders, wasting syndromes (cachexia), mitochondrial disorders, pancreatic dysfunctions (for example diabetes), disorders related to ROS production. 38. Use of a nucleic acid molecule of Unc-51, ROMA1, and/or 2TM or fragment thereof for the preparation of a non-human animal which over-or underexpresses the Unc-51, ROMA 1, and/or 2TM gene product. 39. Kit comprising at least one of (a) an Unc-51, ROMA 1, and/or 2TM nucleic acid molecule, or a fragment thereof, (b) a vector comprising the nucleic acid of (a); (c) a host cell comprising the nucleic acid of (a) or the vector of (b); (d) a polypeptide encoded by the nucleic acid of (a); (e) a fusion polypeptide encoded by the nucleic acid of (a); (f) an antibody or a fragment or derivative thereof or an antiserum, an aptamer or another receptor against the nucleic acid of (a) or the polypeptide of (d) or (e); and (g) an anti-sense oligonucleotide, a hybridization probe or a primer for the nucleic acid of (a). 40. A method for producing a composition comprising the polypeptide identified by the method of claim 27 with a pharmaceutically acceptable carrier, diluent and/or adjuvant. 41. A method for producing a composition comprising the polypeptide identified by the method of claim 31 with a pharmaceutically acceptable carrier, diluent and/or adjuvant. 42. A composition comprising (a) an inhibitor or stimulator of an Unc-51, ROMA1, and/or 2TM (poly)peptide or of a (poly)peptide identified by the method of claim 27. 43. A composition comprising an inhibitor of the expression of a gene identified by the method of claim 26. 44. A composition comprising an inhibitor of the expression of a gene identified by the method of claim 26. 45. A composition comprising a compound identified by the method of claim 29. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 . Nucleotide and amino acid sequences of the Drosophila Uncoupling protein (UCPy) FIG. 1A : Full length cDNA of the Drosophila Uncoupling protein (UCPy) (SEQ ID NO: 1) FIG. 1B : Deduced open reading frame of the Drosophila Uncoupling protein (UCPy) (SEQ ID NO: 2) FIG. 1C . Amino acid sequence (one letter code) encoding the Drosophila Uncoupling protein (UCPy) (SEQ ID NO:3) FIG. 2A -E. CLUSTAL X (1.8) multiple amino acid sequence alignment of Unc-51-like protein from Drosophila melanogaster , mouse, and human. The alignment was produced using the multisequence alignment program of Clustal V software (Higgins, D. G. and Sharp, P. M. (1989). CABIOS, vol. 5, no. 2, 151-153. hsNP — 003556 is the human ULK-1 protein, mmNP — 033495 refers to mouse ULK-1, hsNP — 055498 is the human ULK-2 protein, mmBAA77341 refers to mouse ULK-2, dmAAF49878 refers to the Drosophila Unc-51 like protein. Identical amino acid residues are marked with a star. FIG. 3 . Expression of Unc51 in mammalian tissues. FIG. 3A : Real-time PCR analysis of unc51-like kinase 1 in wildtype mouse tissues. The relative RNA-expression is shown on the left hand side, the tissues tested are given on the horizontal line. WAT=white adipose tissue, BAT=brown adipose tissue FIG. 3B : Real-time PCR mediated comparison of unc51-like kinase 1 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes. The relative RNA-expression is shown on the left hand side, the days of differention are shown on the horizontal line (d0=day 0, start of the experiment, until d10=day 10) FIG. 3C : Real-time PCR mediated comparison of unc51-like kinase 1 expression during the differentiation of 3T3-F442A from preadipocytes to mature adipocytes. The relative RNA-expression is shown on the left hand side, the days of differention are shown on the horizontal line (d0=day 0, start of the experiment, until d10=day 10) FIG. 4 . Nucleotide and protein sequences encoding Roma1 FIG. 4A . Nucleotide sequence of the open reading frame encoding the Drosophila melanogaster Roma1 gene (GADFLY Accession Number CG15081, pp-CT34956) (SEQ ID NO: 4) FIG. 4B . Deduced amino acid sequence (shown in the one-letter-code) of the Drosophila melanogaster Roma1 protein (GADFLY Accession Number CG15081, pp-CT34956) (SEQ ID NO: 5) FIG. 5 . shows the the amino acid sequence alignments among the ROMA1 protein (line 1; SEQ ID NO:5), human BAP37 protein (line 2, Genbank Accession Number XP — 006639.1), mouse BAP37 protein (line 3, Genbank Accession Number NP — 031557.1). The alignment was produced using the multisequence alignment program of Clustal V software (Higgins, D. G. and Sharp, P. M. (1989). CABIOS, vol. 5, no. 2, 151-153.) FIG. 6 . Expression of ROMA1 in mammalian tissues. FIG. 6A : Real-time PCR analysis of ROMA expression in wildtype mouse tissues. The relative RNA-expression is shown on the left hand side, the tissues tested are given on the horizontal line. WAT=white adipose tissue, BAT=brown adipose tissue FIG. 6B : Real-time PCR mediated comparison of ROMA expression during the differentiation of 3T3-L1 cells from preadipocytes to adipocytes. The relative RNA-expression is shown on the left hand side, the days of differention are shown on the horizontal line (d0=day 0, start of the experiment, until d10=day 10) FIG. 7A -D shows the amino acid sequences (one-letter code) of human 2TM homologous proteins FIG. 8 shows the amino acid sequence alignments among the 2TM proteins from Drosophila melanogaste (GadFly accession number CG7620), mouse (GenBank accession number BAB26124), and human (Accession numbers ENSP00000242518-SEQ ID NO:7, BG432914-SEQ ID NO:6, ENSP00000243785-SEQ ID NO:8, and ENSP00000250594-SEQ ID NO:9). The alignment was produced using the multisequence alignment program of Clustal V software (Higgins, D. G. and Sharp, P. M. (1989). CABIOS, vol. 5, no. 2, 151-153.) FIG. 9 shows a transmembrane domain plot of Drosophila ( FIG. 9A ) and human ( FIG. 9B ; SEQ ID NO:7) 2TM proteins. Calculated following: J. Glasgow et al., Proc. Sixth Int. Conf. on Intelligent Systems for Molecular Biology. 175-182, AAAI Press, 1998. FIG. 10 shows the mitochondrial localisation of tagged 2TM protein in transfected mammalian NIH3T3 cells. NIH3T3 cells were transiently transfected with an expression vector for mouse 2TM protein, specifically labeled with a FLAG-tag, fixed, and immunostained with an antisera against the FLAG-tag (see Examples). detailed-description description="Detailed Description" end="lead"? |
Demodulator and receiver using same |
A high performance demodulator able to realize a further wide band property, low distortion characteristics, and low power consumption in comparison with a conventional multi-port demodulator and having a small fluctuation in characteristics with respect to temperature fluctuations and aging and comprising a five-port junction circuit 101 receiving a received signal Sr and a local signal Slo generated at a local signal generation circuit 102, generating three signals having a phase difference, detecting signal levels (amplitude components) of these signals to obtain three power detection signals (baseband signals) P1, P2, and P3; a first multiplier 103 for multiplying the power detection signal P1 output from a first power detector of the five-port junction circuit 101 by a coefficient A1 (=(κ21/κ11)2) for canceling square components of an interference signal and a local signal; a second multiplier 104 for multiplying the power detection signal P1 output from the first power detector by a coefficient A2 (=(κ31/κ11)2) for canceling square components of an interference signal and a local signal; a first subtractor 105 for subtracting a multiplication result of the first multiplier 103 from the power detection signal P2 output from a second power detector of the five-port junction circuit 101; a second subtractor 106 for subtracting the multiplication result of the second multiplier 104 from the power detection signal P3 output from a third power detector of the five-port junction circuit 101; and a multi-port signal-to-IQ signal conversion circuit 109 for converting the result to an In-phase signal I and a quadrature signal Q as demodulated signals based on the output signals of the first subtractor 105 and the second subtractor 106. |
1. A demodulator comprising: a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating at least two signals having a phase difference, and a plurality of power detectors for detecting signal levels of signals generated by said generating means; at least one multiplier for multiplying an output signal of one power detector among said plurality of power detectors by a coefficient for canceling an unnecessary component included in the output signal of other power detector; at least one subtractor for subtracting the output signal of said one power detector multiplied by a coefficient at said multiplier from the output signal of said other power detector; and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the output signal of said subtractor. 2. A demodulator as set forth in claim 1, comprising a removing means for removing a DC offset from the output of said subtractor. 3. A demodulator as set forth in claim 2, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for measuring a DC offset amount from the output of said offset removal subtractor and feeding back a signal for canceling the DC offset amount to the offset removal subtractor. 4. A demodulator as set forth in claim 2, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for taking an average of outputs of said offset removal subtractor and feeding back the average result as a signal for canceling the DC offset amount to the offset removal subtractor. 5. A demodulator as set forth in claim 1, comprising a channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature, component signal Q based on the output signal of said channel selecting means,and predetermined circuit constants. 6. A demodulator as set forth in claim 3, comprising a channel selecting means for selecting a desired channel from the output signal of said offset removal subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signal of said channel selecting means and predetermined circuit constants. 7. A demodulator as set forth in claim 4, comprising a channel selecting means for selecting a desired channel from the output signal of said offset removal subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signal of said channel selecting means and predetermined circuit constants. 8. A demodulator as set forth in claim 5, wherein said channel selecting means includes a low-pass filter. 9. A demodulator as set forth in claim 6, wherein said channel selecting means includes a low-pass filter. 10. A demodulator as set forth in claim 7, wherein said channel selecting means includes a low-pass filter. 11. A demodulator comprising: a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating at least two signals having a phase difference, and a plurality of power detectors for detecting signal levels of signals generated by said generating means; at least one multiplier for multiplying an output signal of one power detector among said plurality of power detectors by a coefficient for canceling an unnecessary component included in the output signal of other power detector; at least one subtractor for subtracting the output signal of said one power detector multiplied by a coefficient at said multiplier from the output signal of said other power detector; at least one variable gain amplifier for adjusting the level of the output signal of said subtractor with a gain in accordance with a control signal; an analog/digital converter for converting the output signal of said variable gain amplifier from an analog signal to a digital signal; and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the digital signal from said analog/digital converter, then outputting said control signal to said variable gain amplifier so as to adjust the level of the output signal of said subtractor to a level suitable for a dynamic range of the analog/digital converter. 12. A demodulator as set forth in claim 11, comprising a removing means for removing a DC offset from the output of said subtractor. 13. A demodulator as set forth in claim 12, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for measuring a DC offset amount from the output of said offset removal subtractor and feeding back a signal for canceling the DC offset amount to the offset removal subtractor. 14. A demodulator as set forth in claim 12, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for taking an average of outputs of said offset removal subtractor and feeding back the average result as a signal for canceling the DC offset amount to the offset removal subtractor. 15. A demodulator as set forth in claim 11, comprising a channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said variable gain amplifier, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the digital signal from said analog/digital converter and predetermined circuit constants. 16. A demodulator as set forth in claim 13, comprising a channel selecting means for selecting a desired channel from the output signal of said offset removal subtractor and inputting the same to said variable gain amplifier, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the digital signal from said analog/digital converter and predetermined circuit constants. 17. A demodulator as set forth in claim 14, comprising a channel selecting means for selecting a desired channel from the output signal of said offset removal subtractor and inputting the same to said variable gain amplifier, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the digital signal from said analog/digital converter and predetermined circuit constants. 18. A demodulator as set forth in claim 15, wherein said channel selecting means includes a low-pass filter. 19. A demodulator as set forth in claim 16, wherein said channel selecting means includes a low-pass filter. 20. A demodulator as set forth in claim 17, wherein said channel selecting means includes a low-pass filter. 21. A demodulator as set forth in claim 15, wherein said conversion circuit outputs a control signal to said variable gain amplifier and calibrates the gain of the variable gain amplifier so that the digital signal from said analog/digital converter becomes a desired level at the time of no reception of a signal. 22. A demodulator as set forth in claim 16, wherein said conversion circuit outputs a control signal to said variable gain amplifier and calibrates the gain of the variable gain amplifier so that the digital signal from said analog/digital converter becomes a desired level at the time of no reception of a signal. 23. A demodulator as set forth in claim 17, wherein said conversion circuit outputs a control signal to said variable gain amplifier and calibrates the gain of the variable gain amplifier so that the digital signal from said analog/digital converter becomes a desired level at the time of no reception of a signal. 24. A demodulator comprising: a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating at least two signals having a phase difference, and a plurality of power detectors for detecting signal levels of signals generated by said generating means; a plurality of variable gain amplifiers for adjusting levels of the output signals of said plurality of power detectors with a gain in accordance with a control signal; a plurality of analog/digital converters for converting the output signals of said plurality of variable gain amplifiers from analog signals to digital signals; at least one multiplier for multiplying the output signal of one power detector among said plurality of power detectors converted to a digital signal by said analog/digital converter by a coefficient for canceling unnecessary components included in the output signal of the other power detector; at least one subtractor for subtracting the output signal of said one power detector multiplied by a coefficient at said multiplier from the output signal of said other power detector converted to a digital signal by said analog/digital converter; and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the digital signal from said subtractor, then outputting said control signal to said variable gain amplifier so as to adjust the level of the output signal of said power detector to a level suitable for a dynamic range of said analog/digital converter. 25. A demodulator as set forth in claim 24, comprising a removing means for removing a DC offset from the output of said subtractor. 26. A demodulator as set forth in claim 25, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for measuring a DC offset amount from the output of said offset removal subtractor and feeding back a signal for canceling the DC offset amount to the offset removal subtractor. 27. A demodulator as set forth in claim 25, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for taking an average of outputs of said offset removal subtractor and feeding back the average result as a signal for canceling the DC offset amount to the offset removal subtractor. 28. A demodulator as set forth in claim 24, wherein: the demodulator has a channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said variable gain amplifier, and said conversion circuit demodulates an In-phase component signal I and a quadrature component signal Q based on the digital signal from said channel selecting means and predetermined circuit constants. 29. A demodulator as set forth in claim 26, comprising a channel selecting means for selecting a desired channel from the output signal of said offset removal subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the digital signal from said channel selecting means and predetermined circuit constants. 30. A demodulator as set forth in claim 27, comprising a channel selecting means for selecting a desired channel from the output signal of said offset removal subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the digital signal from said channel selecting means and predetermined circuit constants. 31. A demodulator as set forth in claim 28, wherein said channel selecting means includes a low-pass filter. 32. A demodulator as set forth in claim 29, wherein said channel selecting means includes a low-pass filter. 33. A demodulator as set forth in claim 30, wherein said channel selecting means includes a low-pass filter. 34. A demodulator as set forth in claim 28, wherein said conversion circuit outputs a control signal to said variable gain amplifier and calibrates the gain of the variable gain amplifier so that the digital signal from said channel selecting means becomes a desired level at the time of no reception of signal. 35. A demodulator as set forth in claim 29, wherein said conversion circuit outputs a control signal to said variable gain amplifier and calibrates the gain of the variable gain amplifier so that the digital signal from said channel selecting means becomes a desired level at the time of no reception of signal. 36. A demodulator as set forth in claim 30, wherein said conversion circuit outputs a control signal to said variable gain amplifier and calibrates the gain of the variable gain amplifier so that the digital signal from said channel selecting means becomes a desired level at the time of no reception of signal. 37. A demodulator comprising: a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating a first signal, a second signal, and a third signal having a phase difference, and a first power detector for detecting a signal level of the first signal generated by said generating means and outputting a first power detection signal, a second power detector for detecting the signal level of said second signal and outputting a second power detection signal, and a third power detector for detecting the signal level of said third signal and outputting a third power detection signal; a first multiplier for multiplying the first power detection signal from said first power detector by a coefficient for canceling an unnecessary component included in the second power detection signal from said second power detector; a second multiplier for multiplying the first power detection signal from said first power detector by a coefficient for canceling an unnecessary component included in the third power detection signal from said third power detector; a first subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said first multiplier from the second power detection signal from said second power detector; a second subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said second multiplier from the third power detection signal from said third power detector; and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the output signals of said first and second subtractors. 38. A demodulator as set forth in claim 37, comprising a removing means for removing a DC offset from the output of said subtractor. 39. A demodulator as set forth in claim 38, wherein said removing means includes first and second offset removal subtractors connected to the latter stage of said first and second subtractors and a circuit for measuring the DC offset amount from the outputs of said offset removal subtractors and feeding back a signal for canceling the DC offset amount to the offset removal subtractors. 40. A demodulator as set forth in claim 38, wherein said removing means includes first and second offset removal subtractors connected to the latter stage of said first and second subtractors and a circuit for taking averages of outputs of said offset removal subtractors and feeding back the average results as a signal for canceling the DC offset amount to the offset removal subtractors. 41. A demodulator as set forth in claim 37, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first subtractor and inputting the same to said conversion circuit and a second channel selecting means for selecting a desired channel from the output signal of said second subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second channel selecting means and predetermined circuit constants. 42. A demodulator as set forth in claim 41, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=α1x1+β1x2+γ1 Q=α2x1+β2x2+γ2 where, x1 is the output signal of the first channel selecting means, x2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 43. A demodulator as set forth in claim 39, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first offset removal subtractor and inputting the same to said conversion circuit and a second channel selecting means for selecting a desired channel from the output signal of said second offset removal subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second channel selecting means and predetermined circuit constants. 44. A demodulator as set forth in claim 43, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=α1x1+β1x2+γ1 Q=α2x1+β2x2+γ2 where, x1 is the output signal of the first channel selecting means, x2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 45. A demodulator as set forth in claim 40, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first offset removal subtractor and inputting the same to said conversion circuit and a second channel selecting means for selecting a desired channel from,the output signal of said second offset removal subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second channel selecting means and predetermined circuit constants. 46. A demodulator as set forth in claim 45, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=α1x1+β1x2+γ1 Q=α2x1+β2x2+γ2 where, x1 is the output signal of the first channel selecting means, x2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 47. A demodulator as set forth in claim 41, wherein at least one of said first and second channel selecting means includes a low-pass filter. 48. A demodulator as set forth in claim 43. wherein at least one of said first and second channel selecting means includes a low-pass filter. 49. A demodulator as set forth in claim 45, wherein at least one of said first and second channel selecting means includes a low-pass filter. 50. A demodulator comprising: a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating a first signal, a second signal, and a third signal having a phase difference, a first power detector for detecting a signal level of the first signal generated by said generating means and outputting a first power detection signal, a second power detector for detecting the signal level of said second signal and outputting a second power detection signal, and a third power detector for detecting the signal level of said third signal and outputting a third power detection signal; a first multiplier for multiplying the first power detection signal from said first power detector by a coefficient for canceling an unnecessary component included in the second power detection signal from said second power detector; a second multiplier for multiplying the first power detection signal from said first power detector by a coefficient for canceling an unnecessary component included in the third power detection signal from said third power detector; a first subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said first multiplier from the second power detection signal from said second power detector; a second subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said second multiplier from the third power detection signal from said third power detector; a first variable gain amplifier for adjusting the level of the output signal of said first subtractor with a gain in accordance with a control signal; a second variable gain amplifier for adjusting the level of the output signal of said second subtractor with a gain in accordance with a control signal; a first analog/digital converter for converting the output signal of said first variable gain amplifier from an analog signal to a digital signal; a second analog/digital converter for converting the output signal of said second variable gain amplifier from an analog signal to a digital signal; and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the digital signals from said first and second analog/digital converters, then outputting said control signal to said first and second variable gain amplifiers so as to adjust the levels of the output signals of said first and second subtractors to levels suitable for the dynamic range of said first and second analog/digital converters. 51. A demodulator as set forth in claim 50, comprising a removing means for removing a DC offset from the output of said subtractor. 52. A demodulator as set forth in claim 51, wherein said removing means includes first and second offset removal subtractors connected to the latter stage of said first and second subtractors and a circuit for measuring the DC offset amount from the outputs of said offset removal subtractors and feeding back a signal for canceling the DC offset amount to the offset removal subtractors. 53. A demodulator as set forth in claim 51, wherein said removing means includes first and second offset removal subtractors connected to the latter stage of said first and second subtractors and a circuit for taking averages of outputs of said offset removal subtractors and feeding back the average results as a signal for canceling the DC offset amount to the offset removal subtractors. 54. A demodulator as set forth in claim 50, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first subtractor and inputting the same to said first variable gain amplifier and a second channel selecting means for selecting a desired channel from the output signal of said second subtractor and inputting the same to said second variable gain amplifier, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second analog/digital converters and predetermined circuit constants. 55. A demodulator as set forth in claim 54, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=α1X1+β1X2+γ1 Q=α2X1+β2X2+γ2 where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 56. A demodulator as set forth in claim 52, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first offset removal subtractor and inputting the same to said first variable gain amplifier and a second channel selecting means for selecting a desired channel from the output signal of said second offset removal subtractor and inputting the same to said second variable gain amplifier, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second analog/digital converters and predetermined circuit constants. 57. A demodulator as set forth in claim 56, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=α1X1+β1X2+γ1 Q=α2X1+β2X2+γ2 where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, and α1, α2,β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 58. A demodulator as set forth in claim 53, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first offset removal subtractor and inputting the same to said first variable gain amplifier and a second channel selecting means for selecting a desired channel from the output signal of said second offset removal subtractor and inputting the same to said second variable gain amplifier, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second analog/digital converters and predetermined circuit constants. 59. A demodulator as set forth in claim 58, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=α1X1+β1X2+γ1 Q=α2X1+β2X2+γ2 where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, and α1, α2, β1, β2 1, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 60. A demodulator as set forth in claim 54, wherein at least one of said first and second channel selecting means includes a low-pass filter. 61. A demodulator as set forth in claim 56, wherein at least one of said first and second channel selecting means includes a low-pass filter. 62. A demodulator as set forth in claim 58, wherein at least one of said first and second channel selecting means includes a low-pass filter. 63. A demodulator as set forth in claim 55, wherein said conversion circuit outputs the control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second analog/digital converters become levels obtained from the following equations at the time of no reception of signal. X=(−γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ1α2−α1γ2)/(α1β2−α2β2) where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 64. A demodulator as set forth in claim 57, wherein said conversion circuit outputs the control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second analog/digital converters become levels obtained from the following equations at the time of no reception of signal. X1=(−γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ1α2−α1γ2)/(α1β2−α2β1) where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found -from circuit elements of the demodulator. 65. A demodulator as set forth in claim 59, wherein said conversion circuit outputs the control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second analog/digital converters become levels obtained from the following equations at the time of no reception of signal. X1=(−γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ1α2−α1γ2)/(α1β2−α2β1) where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 66. A demodulator comprising: a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating a first signal, a second signal, and a third signal having a phase difference, a first power detector for detecting a signal level of the first signal generated by said generating means and outputting a first power detection signal, a second power detector for detecting the signal level of said second signal and outputting a second power detection signal, and a third power detector for detecting the signal level of said third signal and outputting a third power detection signal; a first variable gain amplifier for adjusting the level of the first power detection signal from said first power detector with a gain in accordance with a control signal; a second variable gain amplifier for adjusting the level of the second power detection signal from said second power detector with a gain in accordance with a control signal; a third variable gain amplifier for adjusting the level of the third power detection signal from said third power detector with a gain in accordance with a control signal; a first analog/digital converter for converting the output signal of said first variable gain amplifier from an analog signal to a digital signal; a second analog/digital converter for converting the output signal of said second variable gain amplifier from an analog signal to a digital signal; a third analog/digital converter for converting the output signal of said third variable gain amplifier from an analog signal to a digital signal; a first multiplier for multiplying the first power detection signal from said first power detector converted to a digital signal at said first analog/digital converter by a coefficient for canceling an unnecessary component included in the second power detection signal from said second power detector; a second multiplier for multiplying the first power detection signal from said first power detector converted to a digital signal at said first analog/digital converter by a coefficient for canceling an unnecessary component included in the third power detection signal from said third power detector; a first subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said first multiplier from the second power detection signal from said second power detector converted to a digital signal at said second analog/digital converter; a second subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said second multiplier from the third power detection signal from said third power detector converted to a digital signal at said third analog/digital converter; and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the digital signals from said first and second subtractors, and then outputting said control signal to said second and third variable gain amplifiers so as to adjust the levels of the output signals of said second and third power detectors to levels suitable for the dynamic range of at least said second and third analog/digital converters. 67. A demodulator as set forth in claim 66, comprising a removing means for removing a DC offset from the output of said first and second subtractors. 68. A demodulator as set forth in claim 67, wherein said removing means includes first and second offset removal subtractors connected to the latter stage of said first and second subtractors and a circuit for measuring a DC offset amount from the outputs of said offset removal subtractors and feeding back a signal for canceling the DC offset amount to the offset removal subtractors. 69. A demodulator as set forth in claim 67, wherein said removing means includes first and second offset removal subtractors connected to the latter stage of said first and second subtractors and a circuit for taking averages of outputs of said offset removal subtractors and feeding back the average results as a signal for canceling the DC offset amount to the offset removal subtractors. 70. A demodulator as set forth in claim 66, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first subtractor and inputting the same to said conversion circuit and a second channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said second conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second channel selecting means and predetermined circuit constants. 71. A demodulator as set forth in claim 70, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=α1X1+β1X2+γ1 Q=α2X1+β2X2+γ2 where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, and α1, α2, β1, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 72. A demodulator as set forth in claim 68, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first offset removal subtractor and inputting the same to said conversion circuit and a second channel selecting means for selecting a desired channel from the output signal of said second offset removal subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second channel selecting means and predetermined circuit constants. 73. A demodulator as set forth in claim 72, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=α1X1+β1X2+γ1 Q=α2X1+β2X2+γ2 where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, and α1, α2, β1, β2,γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 74. A demodulator as set forth in claim 69, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first offset removal subtractor and inputting the same to said conversion circuit and a second channel selecting means for selecting a desired channel from the output signal of said second offset removal subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second channel selecting means and predetermined circuit constants. 75. A demodulator as set forth in claim 74, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=α1X1+β2X2+γ1 Q=α2X1+β2X2+γ2 where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 76. A demodulator as set forth in claim 70, wherein at least one of said first and second channel selecting means includes a low-pass filter. 77. A demodulator as set forth in claim 72, wherein at least one of said first and second channel selecting means includes a low-pass filter. 78. A demodulator as set forth in claim 74, wherein at least one of said first and second channel selecting means includes a low-pass filter. 79. A demodulator as set forth in claim 71, wherein said conversion circuit outputs the control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second channel selecting means become levels obtained from the following equations at the time of no reception of signal. X1=(−γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ1α2−α1γ2)/(α1β2−α2β1) where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 80. A demodulator as set forth in claim 73, wherein said conversion circuit outputs the control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second channel selecting means become levels obtained from the following equations at the time of no reception of signal. X1=(−γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ1α2−αaγ2)/(α1β2−α2β1) where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 81. A demodulator as set forth in claim 75, wherein said conversion circuit outputs the control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second channel selecting means become levels obtained from the following equations at the time of no reception of signal. X1=(−γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ1α2−α1γ2)/(α1β2−α2β1) where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 82. A receiver comprising: a demodulator having a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating at least two signals having a phase difference, and a plurality of power detectors for detecting signal levels of signals generated by said generating means, at least one multiplier for multiplying an output signal of one power detector among said plurality of power detectors by a coefficient for canceling an unnecessary component included in the output signal of the other power detector, at least one subtractor for subtracting the output signal of said one power detector multiplied by a coefficient at said multiplier from the output signal of said other power detector, and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the output signal of said subtractor; a gain control circuit for adjusting the level of the received signal to a desired level and supplying the result to the generating means of said multi-port junction circuit; and a local signal generation circuit for generating a local signal of a desired level at a desired oscillation frequency and supplying the same to the generating means of said multi-port junction circuit. 83. A receiver as set forth in claim 82, wherein said gain control circuit: receives a gain control signal to be controlled in gain and includes a gain control signal generation circuit for outputting said gain control signal to said gain control circuit so that the received signal level becomes constant based on the output signal of one power detector among said plurality of power detectors. 84. A receiver as set forth in claim 82, comprising: a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 85. A receiver as set forth in claim 83, comprising: a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 86. A receiver as set forth in claim 85, comprising a channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signal of said channel selecting means and predetermined circuit constants. 87. A receiver as set forth in claim 82, comprising: a variable circuit for adjusting the level of the local signal by said local signal generation circuit to a level in accordance with a level control signal and a level control circuit for outputting said level control signal to said variable circuit so that said multi-port junction circuit becomes a level enabling operation at an optimum level in accordance with the received signal level obtained at said conversion circuit. 88. A receiver as set forth in claim 87, comprising a channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said conversion circuit, and said conversion circuit being given the local signal level and demodulating an In-phase component signal I and a quadrature component signal Q based on the given local signal level, the output signal of said channel selecting means, and predetermined circuit constants. 89. A receiver as set forth in claim 87, comprising: a channel selecting means for selecting a desired channel , from the output signal of said subtractor and inputting the same to said conversion circuit and a level measurement circuit for measuring and calculating the local signal level from the output signal of one power detector among said plurality of power detectors at the time of no reception of signal and holding the calculated local signal level, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the held local signal level, the output signal of said channel selecting means, and predetermined circuit constants. 90. A receiver as set forth in claim 86, comprising a removing means for removing the DC offset from the output of said subtractor. 91. A receiver as set forth in claim 90, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for measuring the DC offset amount from the output of said offset removal subtractor and feeding back a signal for canceling the DC offset amount to the offset removal subtractor. 92. A receiver as set forth in claim 90, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for taking an average of outputs of said offset removal subtractor and feeding back the average result to the offset removal subtractor as a signal for canceling the DC offset amount. 93. A receiver as set forth in claim 88, comprising a removing means for removing the DC offset from the output of said subtractor. 94. A receiver as set forth in claim 93, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for measuring the DC offset amount from the output of said offset removal subtractor and feeding back a signal for canceling the DC offset amount to the offset removal subtractor. 95. A receiver as set forth in claim 93, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for taking an average of outputs of said offset removal subtractor and feeding back the average result to the offset removal subtractor as a signal for canceling the DC offset amount. 96. A receiver comprising: a demodulator having a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating at least two signals having a phase difference, and a plurality of power detectors for detecting signal levels of signals generated by said generating means, at least one multiplier for multiplying an output signal of one power detector among said plurality of power detectors by a coefficient for canceling an unnecessary component included in the output signal of the other power detector, at least one subtractor for subtracting the output signal of said one power detector multiplied by a coefficient at said multiplier from the output signal of said other power detector, at least one variable gain amplifier for adjusting the level of the output signal of said subtractor with a gain in accordance with a control signal, an analog/digital converter for converting the output signal of said variable gain amplifier from an analog signal to a digital signal, and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the digital signal from said analog/digital converter and then outputting said control signal to said variable gain amplifier so as to adjust the level of the output signal of said subtractor to a level suitable for a dynamic range of the analog/digital converter; a gain control circuit for adjusting the level of the received signal to a desired level and supplying the result to the generating means of said multi-port junction circuit; and a local signal generation circuit for generating a local signal of the desired level at a desired oscillation frequency and supplying the same to the generating means of said multi-port junction circuit. 97. A receiver as set forth in claim 96, wherein said gain control circuit: receives a gain control signal to be controlled in gain and includes a gain control signal generation circuit for outputting said gain control signal to said gain control circuit so that the received signal level becomes a constant level based on the output signal of one power detector among said plurality of power detectors. 98. A receiver as set forth in claim 96, comprising a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 99. A receiver as set forth in claim 97, comprising a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 100. A receiver as set forth in claim 99, comprising a channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said variable gain amplifier, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signal of said analog/digital converter and predetermined circuit constants. 101. A receiver as set forth in claim 96, comprising: a variable circuit for adjusting the level of the local signal from said local signal generation circuit to a level in accordance with a level control signal and a level control circuit for outputting said level control signal to said variable circuit so that said multi-port junction circuit becomes a level enabling operation at the optimum level in accordance with the received signal level obtained at said conversion circuit. 102. A receiver as set forth in claim 101, comprising: a channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said variable gain amplifier, and said conversion circuit being given the local signal level and demodulating an In-phase component signal I and a quadrature component signal Q based on the given local signal level, the output signal of said analog/digital converter, and predetermined circuit constants. 103. A receiver as set forth in claim 101, comprising: a channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said conversion circuit and a level measurement circuit for measuring and calculating the local signal level from the output signal of one power detector among said plurality of power detectors at the time of no reception of signal and holding the calculated local signal level, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the held local signal level, the output signal of said analog/digital converter, and predetermined circuit constants. 104. A receiver as set forth in claim 100, comprising a removing means for removing the DC offset from the output of said subtractor. 105. A receiver as set forth in claim 104, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for measuring the DC offset amount from the output of said offset removal subtractor and feeding back a signal for canceling the DC offset amount to the offset removal subtractor. 106. A receiver as set forth in claim 104, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for taking an average of outputs of said offset removal subtractor and feeding back the average result to the offset removal subtractor as a signal for canceling the DC offset amount. 107. A receiver as set forth in claim 102, comprising a removing means for removing the DC offset from the output of said subtractor. 108. A receiver as set forth in claim 107, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for measuring the DC offset amount from the output of said offset removal subtractor and feeding back a signal for canceling the DC offset amount to the offset removal subtractor. 109. A receiver as set forth in claim 107, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for taking an average of outputs of said offset removal subtractor and feeding back the average result to the offset removal subtractor as a signal for canceling the DC offset amount. 110. A receiver as set forth in claim 100, wherein said conversion circuit outputs the control signal to said variable gain amplifier and calibrates the gain of the variable gain amplifier so that the digital signal from said analog/digital converter becomes the desired level at the time of no reception of signal. 111. A receiver as set forth in claim 102, wherein said conversion circuit outputs the control signal to said variable gain amplifier and calibrates the gain of the variable gain amplifier so that the digital signal from said analog/digital converter becomes the desired level at the time of no reception of signal. 112. A receiver as set forth in claim 103, wherein said conversion circuit outputs the control signal to said variable gain amplifier and calibrates the gain of the variable gain amplifier so that the digital signal from said analog/digital converter becomes the desired level at the time of no reception of signal. 113. A receiver comprising: a demodulator having a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating at least two signals having a phase difference, and a plurality of power detectors for detecting signal levels of signals generated by said generating means, a plurality of variable gain amplifiers for adjusting levels of the output signals of said plurality of power detectors with a gain in accordance with a control signal, a plurality of analog/digital converters for converting the output signals of said plurality of variable gain amplifiers from analog signals to digital signals, at least one multiplier for multiplying the output signal of one power detector among said plurality of power detectors converted to a digital signal by said analog/digital converter by a coefficient for canceling an unnecessary component included in the output signal of the other power detector, at least one subtractor for subtracting the output signal of said one power detector multiplied by a coefficient at said multiplier from the output signal of said other power detector converted to a digital signal by said analog/digital converter, and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the digital signal from said subtractor and then outputting said control signal to said variable gain amplifier so as to adjust the level of the output signal of said power detector to a level suitable for the dynamic range of said analog/digital converter; a gain control circuit for adjusting the level of the received signal to a desired level and supplying the same to the generating means of said multi-port junction circuit; and a local signal generation circuit for generating a local signal of a desired level at a desired oscillation frequency and supplying the same to the generating means of said multi-port junction circuit. 114. A receiver as set forth in claim 113, wherein said gain control circuit: receives a gain control signal to be controlled in gain and includes a gain control signal generation circuit for outputting said gain control signal to said gain control circuit so that the received signal level becomes constant based on the output signal of one power detector among said plurality of power detectors. 115. A receiver as set forth in claim 113, comprising: a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 116. A receiver as set forth in claim 114, comprising: a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 117. A receiver as set forth in claim 116, comprising: a channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signal of said channel selecting means and predetermined circuit constants. 118. A receiver as set forth in claim 113, comprising: a variable circuit for adjusting the level of the local signal by said local signal generation circuit to a level in accordance with a level control signal and a level control circuit for outputting said level control signal to said variable circuit so that said multi-port junction circuit becomes a level enabling operation at the optimum level in accordance with the received signal level obtained at said conversion circuit. 119. A receiver as set forth in claim 118, comprising: a channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said conversion circuit, and said conversion circuit being given the local signal level and demodulating an In-phase component signal I and a quadrature component signal Q based on the given local signal level, the output signal of said channel selecting means, and predetermined circuit constants. 120. A receiver as set forth in claim 118, comprising: a channel selecting means for selecting a desired channel from the output signal of said subtractor and inputting the same to said conversion circuit and a level measurement circuit for measuring and calculating the local signal level from the output signal of one power detector among said plurality of power detectors at the time of no reception of signal and holding the calculated local signal level, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the held local signal level, the output signal of said channel selecting means, and predetermined circuit constants. 121. A receiver as set forth in claim 117, comprising a removing means for removing the DC offset from the output of said subtractor. 122. A receiver as set forth in claim 121, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for measuring the DC offset amount from the output of said offset removal subtractor and feeding back a signal for canceling the DC offset amount to the offset removal subtractor. 123. A receiver as set forth in claim 121, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for taking an average of outputs of said offset removal subtractor and feeding back the average result to the offset removal subtractor as a signal for canceling the DC offset amount. 124. A receiver as set forth in claim 119, comprising a removing means for removing the DC offset from the output of said subtractor. 125. A receiver as set forth in claim 124, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for measuring the DC offset amount from the output of said offset removal subtractor and feeding back a signal for canceling the DC offset amount to the offset removal subtractor. 126. A receiver as set forth in claim 124, wherein said removing means includes an offset removal subtractor connected to the latter stage of said subtractor and a circuit for taking an average of outputs of said offset removal subtractor and feeding back the average result to the offset removal subtractor as a signal for canceling the DC offset amount. 127. A receiver as set forth in claim 117, wherein said conversion circuit outputs the control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signal from said analog/digital converter become a desired level at the time of no reception of signal. 128. A receiver as set forth in claim 119, wherein said conversion circuit outputs the control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signal from said analog/digital converter become a desired level at the time of no reception of signal. 129. A receiver as set forth in claim 120, wherein said conversion circuit outputs the control signal to said variable gain amplifier and calibrates the gain of the variable gain amplifier so that the digital signal from said analog/digital converter become a desired level at the time of not receiving a signal. 130. A receiver comprising: a demodulator having a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating a first signal, a second signal, and a third signal having a phase difference, a first power detector for detecting a signal level of the first signal generated by said generating means and outputting a first power detection signal, a second power detector for detecting the signal level of said second signal and outputting a second power detection signal, and a third power detector for detecting the signal level of said third signal and outputting a third power detection signal, a first multiplier for multiplying the first power detection signal from said first power detector by a coefficient for canceling an unnecessary component included in the second power detection signal from said second power detector, a second multiplier for multiplying the first power detection signal from said first power detector by a coefficient for canceling an unnecessary component included in the third power detection signal from said third power detector, a first subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said first multiplier from the second power detection signal from said second power detector, a second subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said second multiplier from the third power detection signal from said third power detector, and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the output signals of said first and second subtractors; a gain control circuit for adjusting the level of the received signal to a desired level and supplying the same to the generating means of said multi-port junction circuit; and a local signal generation circuit for generating a local signal of a desired level at a desired oscillation frequency and supplying the same to the generating means of said multi-port junction circuit. 131. A receiver as set forth in claim 130, wherein said gain control circuit: receives a gain control signal to be controlled in gain and includes a gain control signal generation circuit for outputting said gain control signal to said gain control circuit so that the received signal level becomes constant based on the output signal of one power detector among said plurality of power detectors. 132. A receiver as set forth in claim 130, comprising: a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 133. A receiver as set forth in claim 131, comprising: a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 134. A receiver as set forth in claim 133, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first subtractor and inputting the same to said conversion circuit and a second channel selecting means for selecting a desired channel from the output signal of said second subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second channel selecting means and predetermined circuit constants. 135. A receiver as set forth in claim 134, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=αlx1+β1x2+γ1 Q=α2xl+β2x2+γ2 where, x1 is the output signal of the first channel selecting means, x2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 136. A receiver as set forth in claim 130, comprising: a variable circuit for adjusting the level of the local signal by said local signal generation circuit to a level in accordance with a level control signal and a level control circuit for outputting said level control signal to said variable circuit so that said multi-port junction circuit becomes a level enabling operation at the optimum level in accordance with the received signal level obtained at said conversion circuit. 137. A receiver as set forth in claim 136, comprising: a first channel selecting means for selecting the desired channel from the output signal of said first subtractor and inputting the same to said conversion circuit and a second channel selecting means for selecting the desired channel from the output signal of said second subtractor and inputting the same to said conversion circuit, and said conversion circuit being given the local signal level and demodulating an In-phase component signal I and a quadrature component signal Q based on the given local signal level, the output signals of said first and second channel selecting means, and predetermined circuit constants. 138. A receiver as set forth in claim 137, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=a1x1/PLO+b1x2/PLO+γ1 Q=a2x1/PLO+b2x2/PLO+γ2 where, x1 is the output signal of the first channel selecting means, x2 is the output signal of the second channel selecting means, PLO is the local signal level, and a1, a2, b1, b2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 139. A receiver as set forth in claim 136, comprising: a first channel selecting means for selecting the desired channel from the output signal of said first subtractor and inputting the same to said conversion circuit, a second channel selecting means for selecting the desired channel from the output signal of said second subtractor and inputting the same to said conversion circuit, and a level measurement circuit for measuring and calculating the local signal level from the output signal of one power detector among said plurality of power detectors at the time of no reception of signal and holding the calculated local signal level, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the held local signal level, the output signals of said first and second channel selecting means, and predetermined circuit constants. 140. A receiver as set forth in claim 139, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I a1x1/PLO+b1x2/PLO+γ1 Q=a2x1/PLO+b2x2/PLO+γ2 where, x1 is the output signal of the first channel selecting means, x2 is the output signal of the second channel selecting means, PLO is the local signal level, and a‘, a2, b1, b2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 141. A receiver as set forth in claim 134, comprising a removing means for removing the DC offset from the output of said subtractor. 142. A receiver as set forth in claim 141, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for measuring the DC offset amount from the outputs of said offset removal subtractors and feeding back a signal for canceling the DC offset amount to the offset removal subtractors. 143. A receiver as set forth in claim 141, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for taking averages of outputs of said offset removal subtractors and feeding back the average results to the offset removal subtractors as a signal for canceling the DC offset amount. 144. A receiver as set forth in claim 137, comprising a removing means for removing the DC offset from the output of said subtractor. 145. A receiver as set forth in claim 144, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for measuring the DC offset amount from the outputs of said offset removal subtractors and feeding back a signal for canceling the DC offset amount to the offset removal subtractors. 146. A receiver as set forth in claim 144, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for taking averages of outputs of said offset removal subtractors and feeding back the average results to the offset removal subtractors as a signal for canceling the DC offset amount. 147. A receiver comprising: a demodulator having a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating a first signal, a second signal, and a third signal having a phase difference, a first power detector for detecting a signal level of the first signal generated by said generating means and outputting a first power detection signal, a second power detector for detecting the signal level of said second signal and outputting a second power detection signal, and a third power detector for detecting the signal level of said third signal and outputting a third power detection signal, a first multiplier for multiplying the first power detection signal from said first power detector by a coefficient for canceling an unnecessary component included in the second power detection signal from said second power detector, a second multiplier for multiplying the first power detection signal from said first power detector by a coefficient for canceling an unnecessary component included in the third power detection signal from said third power detector, a first subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said first multiplier from the second power detection signal from said second power detector, a second subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said second multiplier from the third power detection signal from said third power detector, a first variable gain amplifier for adjusting the level of the output signal of said first subtractor with a gain in accordance with a control signal, a second variable gain amplifier for adjusting the level of the output signal of said second subtractor with a gain in accordance with a control signal, a first analog/digital converter for converting the output signal of said first variable gain amplifier from an analog signal to a digital signal, a second analog/digital converter for converting the output signal of said second variable gain amplifier from an analog signal to a digital signal, and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the digital signals from said first and second analog/digital converters and then outputting said control signal to said first and second variable gain amplifiers so as to adjust the levels of the output signals of said first and second subtractors to levels suitable for the dynamic range of said first and second analog/digital converters; a gain control circuit for adjusting the level of the received signal to a desired level and supplying the result to the generating means of said multi-port junction circuit; and a local signal generation circuit for generating a local signal of a desired level at a desired oscillation frequency and supplying the same to the generating means of said multi-port junction circuit. 148. A receiver as set forth in claim 147, wherein said gain control circuit: receives a gain control signal to be controlled in gain and includes a gain control signal generation circuit for outputting said gain control signal to said gain control circuit so that the received signal level becomes constant based on the output signal of one power detector among said plurality of power detectors. 149. A receiver as set forth in claim 147, comprising: a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 150. A receiver as set forth in claim 148, comprising: a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 151. A receiver as set forth in claim 150, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first subtractor and inputting the same to said first variable gain amplifier and a second channel selecting means for selecting a desired channel from the output signal of said second subtractor and inputting the same to said second variable gain amplifier, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second analog/digital converters and predetermined circuit constants. 152. A receiver as set forth in claim 151, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=α1X1+β1X2+γ1 Q=α2X1+β2X2+Y2 where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 153. A receiver as set forth in claim 147, comprising: a variable circuit for adjusting the level of the local signal by said local signal generation circuit to a level in accordance with a level control signal and a level control circuit for outputting said level control signal to said variable circuit so that said multi-port junction circuit becomes a level enabling operation at the optimum level in accordance with the received signal level obtained at said conversion circuit. 153. A receiver as set forth in claim 152, comprising: a first channel selecting means for selecting the desired channel from the output signal of said first subtractor and inputting the same to said first variable gain amplifier and a second channel selecting means for selecting the desired channel from the output signal of said second subtractor and inputting the same to said second variable gain amplifier, and said conversion circuit being given the local signal level and demodulating an In-phase component signal I and a quadrature component signal Q based on the given local signal level, the output signals of said first and second analog/digital converters, and predetermined circuit constants. 154. A receiver as set forth in claim 153, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=a1X1/PLO+b1X2/PLO+γ1 Q=a2X1/PLO+b2X2/PLO+γ2 where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, PLO is the local signal level, and a1, a2, b1, b2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 155. A receiver as set forth in claim 152, comprising: a first channel selecting means for selecting the desired channel from the output signal of said first subtractor and inputting the same to said first variable gain amplifier, a second channel selecting means for selecting the desired channel from the output signal of said second subtractor and inputting the same to said second variable gain amplifier, and a level measurement circuit for measuring and calculating the local signal level from the output signal of one power detector among said plurality of power detectors at the time of no reception of signal and holding the calculated local signal level, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the held local signal level, the output signals of said first and second analog/digital converters, and predetermined circuit constants. 156. A receiver as set forth in claim 155, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computation based on the following equations: I=a1X1/PLO+b1X2/PLO+γ1 Q=a2X1/PLO+b2X2/PLO+γ2 where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, PLO is the local signal level, and a1, a2, b1, b2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 157. A receiver as set forth in claim 151, comprising a removing means for removing the DC offset from the output of said subtractor. 158. A receiver as set forth in claim 157, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for measuring the DC offset amount from the outputs of said offset removal subtractors and feeding back a signal for canceling the DC offset amount to the offset removal subtractors. 159. A receiver as set forth in claim 157, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for taking averages of outputs of said offset removal subtractors and feeding back the average results to the offset removal subtractor as a signal for canceling the DC offset amount. 160. A receiver as set forth in claim 153, comprising a removing means for removing the DC offset from the output of said subtractor. 161. A receiver as set forth in claim 160, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for measuring the DC offset amount from the outputs of said offset removal subtractors and feeding back a signal for canceling the DC offset amount to the offset removal subtractors. 162. A receiver as set forth in claim 160, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for taking averages of outputs of said offset removal subtractors and feeding back the average results to the offset removal subtractors as a signal for canceling the DC offset amount. 163. A receiver as set forth in claim 152, wherein said conversion circuit outputs a control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second analog/digital converters become levels obtained from the following equations: X1=(−γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ1α2−α1γ2)/(α1β2−α2β1) where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 164. A receiver as set forth in claim 154, wherein said conversion circuit outputs a control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second analog/digital converters become levels obtained from the following equations: I=(γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ1α2−α1γ2)/(α1β2−α2β1) where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 165. A receiver as set forth in claim 156, wherein said conversion circuit outputs a control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second analog/digital converters become levels obtained from the following equations: X1=(−γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ1α2−α1γ2)/(α1β2−α2β1) where, X1 is the output signal of the first analog/digital converter, X2 is the output signal of the second analog/digital converter, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 166. A receiver comprising: a demodulator having a multi-port junction circuit including a generating means receiving as input a received signal and a local signal and based on at least one signal, generating a first signal, a second signal, and a third signal having a phase difference, a first power detector for detecting a signal level of the first signal generated by said generating means and outputting a first power detection signal, a second power detector for detecting the signal level of said second signal and outputting a second power detection signal, and a third power detector for detecting the signal level of said third signal and outputting a third power detection signal, a first variable gain amplifier for adjusting the level of the first power detection signal from said first power detector with a gain in accordance with a control signal, a second variable gain amplifier for adjusting the level of the second power detection signal from said second power detector with a gain in accordance with a control signal, a third variable gain amplifier for adjusting the level of the third power detection signal from said third power detector with a gain in accordance with a control signal, a first analog/digital converter for converting the output signal of said first variable gain amplifier from an analog signal to a digital signal, a second analog/digital converter for converting the output signal of said second variable gain amplifier from an analog signal to a digital signal, a third analog/digital converter for converting the output signal of said third variable gain amplifier from an analog signal to a digital signal, a first multiplier for multiplying the first power detection signal from said first power detector converted to a digital signal at said first analog/digital converter by a coefficient for canceling an unnecessary component included in the second power detection signal from said second power detector, a second multiplier for multiplying the first power detection signal from said first power detector converted to a digital signal at said first analog/digital converter by a coefficient for canceling an unnecessary component included in the third power detection signal from said third power detector, a first subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said first multiplier from the second power detection signal from said second power detector converted to a digital signal at said second analog/digital converter, a second subtractor for subtracting the first power detection signal from said first power detector multiplied by a coefficient at said second multiplier from the third power detection signal from said third power detector converted to a digital signal at said third analog/digital converter, and a conversion circuit for converting the result to a plurality of signal components included in the received signal based on the digital signals from said first and second subtractors and then outputting said control signal to said second and third variable gain amplifiers so as to adjust the levels of the output signals of said second and third power detectors to levels suitable for the dynamic range of at least said second and third analog/digital converters; a gain control circuit for adjusting the level of the received signal to a desired level and supplying the result to the generating means of said multi-port junction circuit; and a local signal generation circuit for generating a local signal of a desired level at a desired oscillation frequency and supplying the same to the generating means of said multi-port junction circuit. 167. A receiver as set forth in claim 166, wherein said gain control circuit: receives a gain control signal to be controlled in gain and includes a gain control signal generation circuit for outputting said gain control signal to said gain control circuit so that the received signal level becomes constant based on the output signal of one power detector among said plurality of power detectors. 168. A receiver as set forth in claim 166, comprising: a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 169. A receiver as set forth in claim 167, comprising: a carrier reproduction circuit for reproducing a carrier based on a plurality of signal components obtained at said conversion circuit and outputting a reproduced signal, and said local signal generation circuit receiving said reproduced signal and setting an oscillation frequency of the local signal so as to become a frequency substantially equal to the carrier frequency of the received signal. 170. A receiver as set forth in claim 169, comprising: a first channel selecting means for selecting a desired channel from the output signal of said first subtractor and inputting the same to said conversion circuit and a second channel selecting means for selecting a desired channel from the output signal of said second subtractor and inputting the same to said conversion circuit, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the output signals of said first and second channel selecting means and predetermined circuit constants. 171. A receiver as set forth in claim 170, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=α1X1+β1X2+γ1 Q=α2X1+β2X2+γ2 where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 172. A receiver as set forth in claim 166, comprising: a variable circuit for adjusting the level of the local signal by said local signal generation circuit to a level in accordance with a level control signal and a level control circuit for outputting said level control signal to said variable circuit so that said multi-port junction circuit becomes a level enabling operation at the optimum level in accordance with the received signal level obtained at said conversion circuit. 173. A receiver as set forth in claim 172, comprising: a first channel selecting means for selecting the desired channel from the output signal of said first subtractor and inputting the same to said conversion circuit and a second channel selecting means for selecting the desired channel from the output signal of said second subtractor and inputting the same to said conversion circuit, and said conversion circuit being given the local signal level and demodulating an In-phase component signal I and a quadrature component signal Q based on the given local signal level, the output signals of said first and second channel selecting means, and predetermined circuit constants. 174. A receiver as set forth in claim 173, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=a1X1/PLO+b1X2/PLO+γ1 Q=a2X1/PLO+b2X2/PLO+γ2 where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, PLO is the local signal level, and a1, a2, b1, b2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 175. A receiver as set forth in claim 172, comprising: a first channel selecting means for selecting the desired channel from the output signal of said first subtractor and inputting the same to said conversion circuit, a second channel selecting means for selecting the desired channel from the output signal of said second subtractor and inputting the same to said conversion circuit, and a level measurement circuit for measuring and calculating the local signal level from the output signal of one power detector among said plurality of power detectors at the time of no reception of signal and holding the calculated local signal level, and said conversion circuit demodulating an In-phase component signal I and a quadrature component signal Q based on the held local signal level, the output signals of said first and second channel selecting means, and predetermined circuit constants. 176. A receiver as set forth in claim 175, wherein said conversion circuit obtains an In-phase component signal I and a quadrature component signal Q by computations based on the following equations: I=a1X1/PLO+b1X2/PLO+γ1 Q=a2X1/PLO+b2X2/PLO+γ2 where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, PLO is the local signal level, and a1, a2, b 1, b2, γ1,and γ2 are circuit constants found from circuit elements of the demodulator. 177. A receiver as set forth in claim 170, comprising a removing means for removing the DC offset from the output of said subtractor. 178. A receiver as set forth in claim 177, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for measuring the DC offset amount from the outputs of said offset removal subtractors and feeding back a signal for canceling the DC offset amount to the offset removal subtractors. 179. A receiver as set forth in claim 177, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for taking averages of outputs of said offset removal subtractors and feeding back the average results to the offset removal subtractors as a signal for canceling the DC offset amount. 180. A receiver as set forth in claim 173, comprising a removing means for removing the DC offset from the output of said subtractor. 181. A receiver as set forth in claim 180, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for measuring the DC offset amount from the outputs of said offset removal subtractors and feeding back a signal for canceling the DC offset amount to the offset removal subtractors. 182. A receiver as set forth in claim 180, wherein said removing means includes offset removal subtractors connected to the latter stage of said subtractors and a circuit for taking averages of outputs of said offset removal subtractors and feeding back the average results to the offset removal subtractors as a signal for canceling the DC offset amount. 183. A receiver as set forth in claim 171, wherein said conversion circuit outputs the control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second channel selecting means become levels obtained from the following equations: X1=(γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ1α2−α1γ2)/(α1β2−α2β1) where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 184. A receiver as set forth in claim 174, wherein said conversion circuit outputs the control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second channel selecting means become levels obtained from the following equations: X1=(−γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ2α2−α1γ2)/(α1β2−α2β1) where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. 185. A receiver as set forth in claim 176, wherein said conversion circuit outputs the control signal to said variable gain amplifiers and calibrates the gains of the variable gain amplifiers so that the digital signals from said first and second channel selecting means become levels obtained from the following equations: X1=(γ1β1+β2γ2)/(α1β2−α2β1) X2=(γ1α2−α1γ2)/(α1β2−α2β1) where, X1 is the output signal of the first channel selecting means, X2 is the output signal of the second channel selecting means, and α1, α2, β1, β2, γ1, and γ2 are circuit constants found from circuit elements of the demodulator. |
<SOH> BACKGROUND ART <EOH>FIG. 1 is a circuit diagram of the configuration of principal parts of a general demodulator. As shown in FIG. 1 , this demodulator 10 has a local signal generation circuit 11 , a +45 degree phase shifter 12 , a −45 degree phase shifter 13 , and RF mixers 14 and 15 as main components. In this demodulator 10 , a local signal Slo having a predetermined frequency generated by the local signal generation circuit 11 is shifted in phase by 45 degrees by the +45-degree phase shifter 12 and supplied to the RF mixer 14 or shifted in phase by −45 degrees by the −45 degree phase shifter 13 and supplied to the RF mixer 15 . Then, a signal Sr received via for example a not illustrated antenna element or a low noise amplifier is supplied to the RF mixers 14 and 15 , the received signal Sr and the local signal shifted in phase by exactly +45 degrees are multiplied at the RF mixer 14 to obtain an In-phase signal (I), and the received signal Sr and the local signal shifted in phase by exactly −45 degrees are multiplied at the RF mixer 15 to obtain a quadrature signal (Q). In the demodulator 10 using mixers as shown in FIG. 1 , however, it is difficult to broaden the band, so a high local level must be supplied to the mixer. Further, the mixers are in a nonlinear operating state due to the high local power, so there is the disadvantage that demodulation with a low distortion is difficult. Therefore, in recent years, a demodulator of an n-port system (n being an integer of 3 or more) based on a principle different from FIG. 1 using a power detection circuit (power detector) is proposed. In this multi-port system demodulator, the power detector easily broadens the band in comparison with the mixer used in the above demodulation system. Due to this, a multi-port demodulator can be tell to have a good compatability with a software wireless system in which a multi-band or a wide band characteristic is demanded. Further, recent wireless communication tends to use a higher frequency as the carrier frequency and can even meet the demand for higher frequency. Further, in a demodulation system using mixers, a high local level must be applied to the mixer. Contrary to this, in the multi-port system, the power detector operates in a linear region. Accordingly, with the multi-port system, demodulation is possible even with a low local signal power. Further, in a demodulation system using mixers, the mixers are in a nonlinear operating state due to the high local power. Contrary to this, in the multi-port system, the power detector operates in the linear region. Accordingly, with a multi-port system, demodulation with a low distortion is possible. FIG. 2 is a block diagram of an example of the configuration of an n-port demodulator (see for example WO99/33166 (PCT/EP98/08329)). Here, for simplification, an explanation will be given based on an ideal five-(n=5) port model shown in FIG. 2 . This five-port demodulator 1 has, as shown in FIG. 2 , a five-port junction circuit 2 , low-pass filters 3 to 5 , amplifiers 6 to 8 , analog/digital converters (ADCs) 9 to 11 , and a multi (n) port signal-to-IQ signal conversion circuit 12 . The five-port junction circuit 2 has a coupler 21 , branch circuits 22 and 23 , a phase shifter 24 , power detectors 25 to 27 , and a resistance element R 21 . In this five-port demodulator 1 , a received signal Sr is input to the branch circuit 22 by the coupler 21 . One part thereof is input to the power detector 25 . The received signal input to the branch circuit 22 is branched to two signals. One branched signal is input to the power detector 26 , and the other signal is input to the phase shifter 24 . The phase shifter 24 gives a phase shift e to the received signal from the branch circuit 22 , then inputs the signal receiving the phase shift action to the branch circuit 23 where it is branched into two signals. The branch circuit 23 inputs one branched signal to the power detector 27 . Further, a local signal Slo is input to the branch circuit 23 . The local signal input to the branch circuit 23 is branched into two signals. One branched signal is input to the power detector 27 , while the other signal is input to the phase shifter 24 . The phase shifter 24 gives a phase shift e to the local signal from the branch circuit 23 , then inputs the signal receiving the phase shift action to the branch circuit 22 where it is branched into two signals. The branch circuit 22 inputs one branched signal to the power detector 26 and supplies the other signal to the coupler 21 . The power detector 25 is supplied with the received signal. The power detector 25 detects an amplitude component of the supplied signal and outputs it as a signal P 1 to the low-pass filter 3 . The low-pass filter 3 removes for example a high frequency component, the amplifier 6 amplifies the result, then the ADC 9 converts the result from an analog signal to a digital signal and supplies the result to the conversion circuit 12 . The power detector 26 is supplied with the received signal and the local signal given the phase shift 0 . The power detector 26 detects the amplitude component of the supplied signal and outputs it as a signal P 2 to the low-pass filter 4 . The low-pass filter 4 removes for example a high frequency component, the amplifier 7 amplifies the result, then the ADC 10 converts the result from an analog signal to a digital signal and supplies the result to the conversion circuit 12 . Further, the power detector 27 is supplied with the local signal and the received signal given the phase shift e. The power detector 27 detects the amplitude component of the supplied signal and outputs it as a signal P 3 to the low-pass filter 5 . The low-pass filter 5 removes for example a high frequency component, the amplifier 8 amplifies the result, then the ADC 11 converts the result from an analog signal to a digital signal and supplies the result to the conversion circuit 12 . Then, the conversion circuit 12 performs the calculation indicated by the following equations based on the input digital signals P 1 , P 2 , and P 3 , converts the input signal to an In-phase signal (I) and a quadrature signal (Q) as demodulated signals and outputs the same. I ( t ) = - κ 21 κ 32 + κ 22 κ 31 4 κ 21 κ 31 cos θ - κ 21 κ 32 + κ 22 κ 21 4 κ 11 2 κ 22 κ 32 cos θ P 1 R 0 P lo + 1 4 κ 21 κ 22 cos θ P 2 R 0 P lo + 1 4 κ 31 κ 32 cos θ P 3 R 0 P lo ( 1 ) Q ( t ) = - κ 21 κ 32 - κ 22 κ 31 4 κ 21 κ 31 sin θ + κ 21 κ 32 - κ 22 κ 21 4 κ 11 2 κ 22 κ 32 sin θ P 1 R 0 P lo - 1 4 κ 21 κ 22 sin θ P 2 R 0 P lo + 1 4 κ 31 κ 32 sin θ P 3 R 0 P lo ( 2 ) Here, κ ij indicates a voltage transfer coefficient (i is an output terminal number, and j is a received signal input port when 1 and is a local signal input port when 2), P lo indicates a local signal power, R 0 indicates an impedance of a local signal source, and θ indicates the phase of the phase shifter. The low-pass filters 3 to 5 in the above five-port The low-pass filters 3 to 5 in the above five-port demodulator 1 are provided for the following two objects. The first object is to avoid aliasing at the following ADCs 9 to 11 and is for the case of a relatively high cut-off frequency (a cut-off frequency which is ½ of a sampling frequency or less and higher compared with the desired wave signal band). The second object is a desired frequency channel signal and is for the case where channel filtering is carried out. In the former case, in order to secure the reception performance when there is a strong interference signal in an adjacent channel of the desired received signal, the resolution of the ADCs must be made large. Increasing the resolution of the ADCs becomes disadvantageous in the point of increasing the speed of the ADCs or reducing the power consumption. As one means for alleviating this problem, the latter method may be mentioned. Namely, by removing the interference signal of the adjacent channel, the dynamic range of the ADCs can be reduced. However, the present method has room for improvement in the following point. The output signals P 1 , P 2 , and P 3 of the three power detectors are represented by the following equations: Here, an explanation will be given based on the ideal five-port model shown in FIG. 2 . Assume that the power detectors have ideal square characteristics and that the circuit constants are all 1. P 1 = LPF [ S 0 + X u 2 ] ( 3 ) P 2 = LPF [ S 0 + X u + S LO + 2 ] = LPF [ S 0 + X u 2 + S LO + 2 + 2 ( S 0 + X u ) · S LO + ] ( 4 ) P 3 = LPF [ S 0 + X u + S LO - 2 ] = LPF [ S 0 + X u 2 + S LO - 2 + 2 ( S 0 + X u ) · S LO - ] ( 5 ) Here, S 0 indicates a received desired signal, X u indicates an interference signal, S LO+ indicates a phase shifted local signal, and S LO− indicates a phase shifted local signal. Further, LPF[] means that only the low frequency component is extracted. As will be understood from equation (4) or (5), the third term is a term including the demodulated signals. The first term indicates the power level of the received signal including the interference signal, and the second term indicates the power level of the local signal. Further, these signals include the components of the desired received signal band. These unnecessary signals cannot be removed by the channel filtering. This causes the following problems. When the characteristics of the three power detectors do not completely match, the reception performance is deteriorated due to incompleteness of the circuit, for example, reflection of a local leakage signal. Further, it becomes necessary to obtain a large dynamic range of the ADCs for these unnecessary signals. Further, the number of bits of the variables in the digital signal processor after the ADCs becomes large. Further, the circuit of FIG. 2 requires three ADCs. This means there is room for improvement in the points of power consumption of the receiver, circuit size, and costs. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a circuit diagram of the configuration of a principal part of a general demodulator. FIG. 2 is a block diagram of an example of the configuration of a five-port demodulator. FIG. 3 is a block diagram of a demodulator of a direct conversion system according to a first embodiment of the present invention. FIG. 4 is a block diagram of an example of a concrete configuration of a five-port junction circuit according to the present invention. FIG. 5 is a circuit diagram of a concrete example of the configuration of a one-input three-output branch circuit according to the present invention. FIG. 6 is a circuit diagram of another concrete example of the configuration of a one-input three-output branch circuit according to the present invention. FIG. 7 is a circuit diagram of another concrete example of the configuration of a one-input three-output branch circuit according to the present invention. FIG. 8 is a circuit diagram of another concrete example of the configuration of a one-input three-output branch circuit according to the present invention. FIG. 9 is a circuit diagram of a concrete example of the configuration of a one-input two-output branch circuit according to the present invention. FIG. 10 is a circuit diagram of another concrete example of the configuration of a one-input two-output branch circuit according to the present invention. FIG. 11 is a circuit diagram of another concrete example of the configuration of a one-input two-output branch circuit according to the present invention. FIG. 12 is a circuit diagram of another concrete example of the configuration of a one-input two-output branch circuit according to the present invention. FIG. 13 is a circuit diagram of a concrete example of the configuration of a phase shifter according to the present invention. FIG. 14 is a circuit diagram of another concrete example of the configuration of a phase shifter according to the present invention. FIG. 15 is a circuit diagram of another concrete example of the configuration of a phase shifter according to the present invention. FIG. 16 is a circuit diagram of a concrete example of the configuration of a coupler circuit according to the present invention. FIG. 17 is a circuit diagram of an example of a power detector according to the present invention. FIG. 18 is a view of an example of detection characteristics of the power detector of FIG. 17 . FIG. 19 is a view of a high frequency input power Pin versus output detection voltage Vout when using a gate bias voltage as a parameter in the circuit of FIG. 17 . FIG. 20 is a block diagram of a demodulator of the direct conversion system according to a second embodiment of the present invention. FIG. 21 is a block diagram of a receiver employing a demodulator of the direct conversion system according to a third embodiment of the present invention. FIG. 22 is a block diagram of another embodiment of a five-port junction circuit according to the present invention. FIG. 23 is a block diagram of a receiver employing a demodulator of the direct conversion system according to a fourth embodiment of the present invention. FIG. 24 is a block diagram of a receiver employing a demodulator of the direct conversion system according to a fifth embodiment of the present invention. FIG. 25 is a block diagram of a receiver employing a demodulator of the direct conversion system according to a sixth embodiment of the present invention. FIG. 26 is a block diagram of a receiver employing a demodulator of the direct conversion system according to a seventh embodiment of the present invention. detailed-description description="Detailed Description" end="lead"? |
Protein modification and maintenance molecules |
The invention provides human protein modification and maintenance molecules (PMOD) and polynucleotides which identify and encode PMOD. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of PMOD. |
1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-8, SEQ ID NO:10-12, and SEQ ID NO:14-17, c) a polypeptide comprising a naturally occurring amino acid sequence at least 92% identical to the amino acid of SEQ ID NO:9, d) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and e) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. 3. An isolated polynucleotide encoding a polypeptide of claim 1. 4. An isolated polynucleotide encoding a polypeptide of claim 2. 5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3. 7. A cell transformed with a recombinant polynucleotide of claim 6. 8. CANCELED. 9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed. 10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. 11. An isolated antibody which specifically binds to a polypeptide of claim 1. 12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-25 and SEQ ID NO:27-34, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 92% identical to the polynucleotide sequence of SEQ ID NO:9, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), f) a polynucleotide complementary to a polynucleotide of c), and g) an RNA equivalent of a)-f). 13. CANCELED. 14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof. 15. CANCELED. 16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. 19. CANCELED. 20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample. 21. CANCELED. 22. CANCELED. 23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample. 24. CANCELED. 25. CANCELED. 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. 27. CANCELED. 28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. 29. A method of assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. 30-89. CANCELED. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Proteases cleave proteins and peptides at the peptide bond that forms the backbone of the protein or peptide chain. Proteolysis is one of the most important and frequent enzymatic reactions that occurs both within and outside of cells. Proteolysis is responsible for the activation and maturation of nascent polypeptides, the degradation of misfolded and damaged proteins, and the controlled turnover of peptides within the cell. Proteases participate in digestion, endocrine function, and tissue remodeling during embryonic development, wound healing, and normal growth. Proteases can play a role in regulatory processes by affecting the half life of regulatory proteins. Proteases are involved in the etiology or progression of disease states such as inflammation, angiogenesis, tumor dispersion and metastasis, cardiovascular disease, neurological disease, and bacterial, parasitic, and viral infections. Proteases can be categorized on the basis of where they cleave their substrates. Exopeptidases, which include aminopeptidases, dipeptidyl peptidases, tripeptidases, carboxypeptidases, peptidyl-di-peptidases, dipeptidases, and omega peptidases, cleave residues at the termini of their substrates. Endopeptidases, including serine proteases, cysteine proteases, and metalloproteases, cleave at residues within the peptide. Four principal categories of mammalian proteases have been identified based on active site structure, mechanism of action, and overall three-dimensional structure. (See Beynon, R. J. and J. S. Bond (1994) Proteolytic Enzymes: A Practical Aproach , Oxford University Press, New York N.Y., pp. 1-5.) Serine Proteases The serine proteases (SPs) are a large, widespread family of proteolytic enzymes that include the digestive enzymes trypsin and chymotrypsin, components of the complement and blood-clotting cascades, and enzymes that control the degradation and turnover of macromolecules within the cell and in the extracellular matrix. Most of the more than 20 subfamilies can be grouped into six clans, each with a common ancestor. These six clans are hypothesized to have descended from at least four evolutionarily distinct ancestors. SPs are named for the presence of a serine residue found in the active catalytic site of most families. The active site is defined by the catalytic triad, a set of conserved asparagine, histidine, and serine residues critical for catalysis. These residues form a charge relay network that facilitates substrate binding. Other residues outside the active site form an oxyanion hole that stabilizes the tetrahedral transition intermediate formed during catalysis. SPs have a wide range of substrates and can be subdivided into subfamilies on the basis of their substrate specificity. The main subfamilies are named for the residue(s) after which they cleave: trypases (after arginine or lysine), aspases (after aspartate), chymases (after phenylalanine or leucine), metases (methionine), and serases (after serine) (Rawlings, N. D. and A. J. Barrett (1994) Methods Enzymol. 244:19-61). Most mammalian serine proteases are synthesized as zymogens, inactive precursors that are activated by proteolysis. For example, trypsinogen is converted to its active form, trypsin, by enteropeptidase. Enteropeptidase is an intestinal protease that removes an N-terminal fragment from trypsinogen. The remaining active fragment is trypsin, which in turn activates the precursors of the other pancreatic enzymes. Likewise, proteolysis of prothrombin, the precursor of thrombin, generates three separate polypeptide fragments. The N-terminal fragment is released while the other two fragments, which comprise active thrombin, remain associated through disulfide bonds. The two largest SP subfamilies are the chymotrypsin (S1) and subtilisin (S8) families. Some members of the chymotrypsin family contain two structural domains unique to this family. Kringle domains are triple-looped, disulfide cross-linked domains found in varying copy number. Kringles are thought to play a role in binding mediators such as membranes, other proteins or phospholipids, and in the regulation of proteolytic activity (PROSITE PDOC00020). Apple domains are 90 amino-acid repeated domains, each containing six conserved cysteines. Three disulfide bonds link the first and sixth, second and fifth, and third and fourth cysteines (PROSITE PDOC00376). Apple domains are involved in protein-protein interactions. S1 family members include trypsin, chymotrypsin, coagulation factors IX-XII, complement factors B, C, and D, granzymes, kalikrein, and tissue- and urokinase-plasminogen activators. The subtilisin family has members found in the eubacteria, archaebacteria, eukaryotes, and viruses. Subtilisins include the proprotein-processing endopeptidases kexin and furin and the pituitary prohormone convertases PC1, PC2, PC3, PC6, and PACE4 (Rawlings and Barrett, supra). SPs have functions in many normal processes and some have been implicated in the etiology or treatment of disease. Enterokinase, the initiator of intestinal digestion, is found in the intestinal brush border, where it cleaves the acidic propeptide from trypsinogen to yield active trypsin (Kitamoto, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:7588-7592). Prolylcarboxypeptidase, a lysosomal serine peptidase that cleaves peptides such as angiotensin II and III and [des-Arg9] bradykinin, shares sequence homology with members of both the serine carboxypeptidase and prolylendopeptidase families (Tan, F. et al. (1993) J. Biol. Chem. 268:16631-16638). The protease neuropsin may influence synapse formation and neuronal connectivity in the hippocampus in response to neural signaling (Chen, Z.-L. et al. (1995) J. Neurosci. 15:5088-5097). Tissue plasminogen activator is useful for acute management of stroke (Zivin, J. A. (1999) Neurology 53:14-19) and myocardial infarction (Ross, A. M. (1999) Clin. Cardiol. 22:165-171). Some receptors (PAR, for proteinase-activated receptor), highly expressed throughout the digestive tract, are activated by proteolytic cleavage of an extracellular domain. The major agonists for PARs, thrombin, trypsin, and mast cell tryptase, are released in allergy and inflammatory conditions. Control of PAR activation by proteases has been suggested as a promising therapeutic target (Vergnolle, N. (2000) Aliment. Pharmacol. Ther. 14:257-266; Rice, K. D. et al. (1998) Curr. Pharm. Des. 4:381-396). Prostate-specific antigen (PSA) is a kallikrein-like serine protease synthesized and secreted exclusively by epithelial cells in the prostate gland. Serum PSA is elevated in prostate cancer and is the most sensitive physiological marker for monitoring cancer progression and response to therapy. PSA can also identify the prostate as the origin of a metastatic tumor (Brawer, M. K. and P. H. Lange (1989) Urology 33:11-16). The signal peptidase is a specialized class of SP found in all prokaryotic and eukaryotic cell types that serves in the processing of signal peptides from certain proteins. Signal peptides are amino-terminal domains of a protein which direct the protein from its nbosomal assembly site to a particular cellular or extracellular location. Once the protein has been exported, removal of the signal sequence by a signal peptidase and posttranslational processing, e.g., glycosylation or phosphorylation, activate the protein. Signal peptidases exist as multi-subunit complexes in both yeast and mammals. The canine signal peptidase complex is composed of five subunits, all associated with the microsomal membrane and containing hydrophobic regions that span the membrane one or more times (Shelness, G. S. and G. Blobel (1990) J. Biol. Chem. 265:9512-9519). Some of these subunits serve to fix the complex in its proper position on the membrane while others contain the actual catalytic activity. Thrombin is a serine protease with an essential role in the process of blood coagulation. Prodtrombin, synthesized in the liver, is converted to active thrombin by Factor Xa. Activated thrombin then cleaves soluble fibrinogen to polymer-forming fibrin, a primary component of blood clots. In addition, thrombin activates Factor XIIIa, which plays a role in cross-linking fibrin. Thrombin also stimulates platelet aggregation through proteolytic processing of a 41-residue amino-terminal peptide from protease-activated receptor 1 (PAR-1), formerly known as the thrombin receptor. The cleavage of the amino-terminal peptide exposes a new amino terminus and may also be associated with PAR-1 internalization (Stubbs, M. T. and Bode, W. (1994) Cuffent Opinion in Structural Biology 4:823-832 and Ofoso, F. A. et al. (1998) Biochem. J. 336:283-285). In addition to stimulating platelet activation through cleavage of the PAR-1 receptor, thrombin also induces platelet aggregation following cleavage of glycoprotein V, also on the surface of platelets. Glycoprotein V appears to be the major thrombin substrate on intact platelets. Platelets deficient for glycoprotein V are hypersensitive to thrombin, which is still required to cleave PAR-1. While platelet aggregation is required for normal hemostasis in mammals, excessive platelet aggregation can result in arterial thrombosis, atherosclerotic arteries, acute myocardial infarction, and stroke (Ramakrishnan, V. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:13336-41 and reference within). Another family of proteases which have a serine in their active site are dependent on the hydrolysis of ATP for their activity. These proteases contain proteolytic core domains and regulatory ATPase domains which can be identified by the presence of the P-loop, an ATP/GTP-binding motif (PROSITE PDOC00803). Members of this family include the eukaryotic mitochondrial matrix proteases, Clp protease and the proteasome. Clp protease was originally found in plant chloroplasts but is believed to be widespread in both prokaryotic and eukaryotic cells. The gene for early-onset torsion dystonia encodes a protein related to Clp protease (Ozelius, L. J. et al. (1998) Adv. Neurol. 78:93-105). The proteasome is an intracellular protease complex found in some bacteria and in all eukaryotic cells, and plays an important role in cellular physiology. Proteasomes are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins of all types, including proteins that function to activate or repress cellular processes such as transcription and cell cycle progression (Ciechanover, A. (1994) Cell 79:13-21). In the UCS pathway, proteins targeted for degradation are conjugated to ubiquitin, a small heat stable protein. The ubiquitinated protein is then recognized and degraded by the proteasome. The resultant ubiquitin-peptide complex is hydrolyzed by a ubiquitin carboxyl terminal hydrolase, and free ubiquitin is released for reutilization by the UCS. Ubiquitin-proteasome systems are implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes (p53), cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, supra). This pathway has been implicated in a number of diseases, including cystic fibrosis, Angelman's syndrome, and Liddle syndrome (reviewed in Schwartz, A. L. and A. Ciechanover (1999) Annu. Rev. Med. 50:57-74). A murine proto-oncogene, Unp, encodes a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation of NIH3T3 cells. The human homologue of this gene is consistently elevated in small cell tumors and adenocarcinomas of the lung (Gray, D. A. (1995) Oncogene 10:2179-2183). Ubiquitin carboxyl terminal hydrolase is involved in the differentiation of a lymphoblastic leukemia cell line to a non-dividing mature state (Maki, A. et al. (1996) Differentiation 60:59-66). In neurons, ubiquitin carboxyl terminal hydrolase (PGP 9.5) expression is strong in the abnorrnal structures that occur in human neurodegenerative diseases (Lowe, J. et al. (1990) J. Pathol. 161:153-160). The proteasome is a large (˜2000 kDa) multisubunit complex composed of a central catalytic core containing a variety of proteases arranged in four seven-membered rings with the active sites facing inwards into the central cavity, and terminal ATPase subunits covering the outer port of the cavity and regulating substrate entry (for review, see Schmidt, M. et al. (1999) Curr. Opin. Chem. Biol. 3:584-591). Cysteine Proteases Cysteine proteases (CPs) are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. Nearly half of the CPs kiown are present only in viruses. CPs have a cysteine as the major catalytic residue at the active site where catalysis proceeds via a thioester intermediate and is facilitated by nearby histidine and asparagine residues. A glutamine residue is also important, as it helps to form an oxyanion hole. Two important CP families include the papain-like enzymes (C1) and the calpains (C2). Papain-like family members are generally lysosomal or secreted and therefore are synthesized with signal peptides as well as propeptides. Most members bear a conserved motif in the propeptide that may have structural significance (Karrer, K. M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3063-3067). Three-dimensional structures of papain family members show a bilobed molecule with the catalytic site located between the two lobes. Papains include cathepsins B, C, H, L, and S, certain plant allergens and dipeptidyl peptidase (for a review, see Rawlings, N. D. and A. J. Barrett (1994) Methods Enzymol. 244:461-486). Some CPs are expressed ubiquitously, while others are produced only by cells of the immune system. Of particular note, CPs are produced by monocytes, macrophages and other cells which migrate to sites of inflammation and secrete molecules involved in tissue repair. Overabundance of these repair molecules plays a role in certain disorders. In autoimmune diseases such as rheumatoid arthritis, secretion of the cysteine peptidase cathepsin C degrades collagen, laminin, elastin and other structural proteins found in the extracellular matrix of bones. Bone weakened by such degradation is also more susceptible to tumor invasion and metastasis. Cathepsin L expression may also contribute to the influx of mononuclear cells which exacerbates the destruction of the rheumatoid synovium (Keyszer, G. M. (1995) Arthritis Rheum. 38:976-984). Calpains are calcium-dependent cytosolic endopeptidases which contain both an N-terminal catalytic domain and a C-terminal calcium-binding domain. Calpain is expressed as a proenzyme heterodimer consisting of a catalytic subunit unique to each isoform and a regulatory subunit common to different isoforms. Each subunit bears a calcium-binding EF-hand domain. The regulatory subunit also contains a hydrophobic glycine-rich domain that allows the enzyme to associate with cell membranes. Calpains are activated by increased intracellular calcium concentration, which induces a change in conformation and limited autolysis. The resultant active molecule requires a lower calcium concentration for its activity (Chan, S. L. and M. P. Mattson (1999) J. Neurosci. Res. 58:167-190). Calpain expression is predominantly neuronal, although it is present in other tissues. Several chronic neurodegenerative disorders, including ALS, Parkinson's disease and Alzheimer's disease are associated with increased calpain expression (Chan and Mattson, supra). Calpain-mediated breakdown of the cytoskeleton has been proposed to contribute to brain damage resulting from head injury (McCracken, E. et al (1999) J. Neurotrauma 16:749-761). Calpain-3 is predominantly expressed in skeletal muscle, and is responsible for limb-girdle muscular dystrophy type 2A (Minami, N. et al. (1999) J. Neurol. Sci. 171:31-37). Another family of thiol proteases is the caspases, which are involved in the initiation and execution phases of apoptosis. A pro-apoptotic signal can activate initiator caspases that trigger a proteolytic caspase cascade, leading to the hydrolysis of target proteins and the classic apoptotic death of the cell. Two active site residues, a cysteine and a histidine, have been implicated in the catalytic mechanism. Caspases are among the most specific endopeptidases, cleaving after aspartate residues. Caspases are synthesized as inactive zymogens consisting of one large (p20) and one small (p10) subunit separated by a small spacer region, and a variable N-terminal prodomain. This prodomain interacts with cofactors that can positively or negatively affect apoptosis. An activating signal causes autoproteolytic cleavage of a specific aspartate residue (D297 in the caspase-1 numbering convention) and removal of the spacer and prodomain, leaving a p10/p20 heterodimer. Two of these heterodimers interact via their small subunits to form the catalytically active tetramer. The long prodomains of some caspase family members have been shown to promote dimerization and auto-processing of procaspases. Some caspases contain a “death effector domain” in their prodomain by which they can be recruited into self-activating complexes with other caspases and FADD protein associated death receptors or the TNF receptor complex. In addition, two dimers from different caspase family members can associate, changing the substrate specificity of the resultant tetramer. Endogenous caspase inhibitors (inhibitor of apoptosis proteins, or IAPs) also exist. All these interactions have clear effects on the control of apoptosis (reviewed in Chan and Mattson, sunra; Salveson, G. S. and V. M. Dixit (1999) Proc. Natl. Acad. Sci. USA 96:10964-10967). Caspases have been implicated in a number of diseases. Mice lacking some caspases have severe nervous system defects due to failed apoptosis in the neuroepithelium and suffer early lethality. Others show severe defects in the inflammatory response, as caspases are responsible for processing IL-1b and possibly other inflammatory cytokines (Chan and Mattson, supra). Cowpox virus and baculoviruses target caspases to avoid the death of their host cell and promote successful infection. In addition, increases in inappropriate apoptosis have been reported in AIDS, neurodegenerative diseases and ischemic injury, while a decrease in cell death is associated with cancer (Salveson and Dixit, surra; Thompson, C. B. (1995) Science 267:1456-1462). Aspartyl Proteases Aspartyl proteases (APs) include the lysosomal proteases cathepsins D and E, as well as chymosin, renin, and the gastric pepsins. Most retroviruses encode an AP, usually as part of the pol polyprotein. APs, also called acid proteases, are monomeric enzymes consisting of two domains, each domain containing one half of the active site with its own catalytic aspartic acid residue. APs are most active in the range of pH 2-3, at which one of the aspartate residues is ionized and the other neutral. The pepsin family of APs contains many secreted enzymes, and all are likely to be synthesized with signal peptides and propeptides. Most family members have three disulfide loops, the first ˜5 residue loop following the first aspartate, the second 5-6 residue loop preceding the second aspartate, and the third and largest loop occurring toward the C terminus. Retropepsins, on the other hand, are analogous to a single domain of pepsin, and become active as homodimers with each retropepsin monomer contributing one half of the active site. Retropepsins are required for processing the viral polyproteins. APs have roles in various tissues, and some have been associated with disease. Renin mediates the first step in processing the hormone angiotensin, which is responsible for regulating electrolyte balance and blood pressure (reviewed in Crews, D. E. and S. R. Williams (1999) Hum. Biol. 71:475-503). Abnormal regulation and expression of cathepsins are evident in various inflammatory disease states. Expression of cathepsin D is elevated in synovial tissues from patients with rheumatoid arthritis and osteoarthritis. The increased expression and differential regulation of the cathepsins are linked to the metastatic potential of a variety of cancers (Chambers, A. F. et al. (1993) Crit. Rev. Oncol. 4:95-114). Metalloproteases Metaloproteases require a metal ion for activity, usually manganese or zinc. Examples of manganese metalloenzymes include aninopeptidase P and human proline dipeptidase (PEPD). Aminopeptidase P can degrade bradykinin, a nonapeptide activated in a variety of inflammatory responses. Aminopeptidase P has been implicated in coronary ischemia/reperfusion injury. Administration of aminopeptidase P inhibitors has been shown to have a cardioprotective effect in rats (Ersahin, C. et al (1999) J. Cardiovasc. Pharmacol. 34:604-611). Most zinc-dependent metalloproteases share a conmmon sequence in the zinc-binding domain. The active site is made up of two histidines which act as zinc ligands and a catalytic glutamic acid C-terminal to the first histidine. Proteins containing this signature sequence are known as the metzincins and include aminopeptidase N, angiotensin-converting enzyme, neurolysin, the matrix metalloproteases and the adamalysins (ADAMS). An alternate sequence is found in the zinc carboxypeptidases, in which all three conserved-residues—two histidines and a glutamic acid—are involved in zinc binding. A number of the neutral metalloendopeptidases, including angiotensin converting enzyme and the aminopeptidases, are involved in the metabolism of peptide hormones. High aminopeptidase B activity, for example, is found in the adrenal glands and neurohypophyses of hypertensive rats (Prieto, I. et al. (1998) Horm. Metab. Res. 30:246-248). Oligopeptidase M/neurolysin can hydrolyze bradykinin as well as neurotensin (Serizawa, A. et al. (1995) J. Biol. Chem 270:2092-2098). Neurotensin is a vasoactive peptide that can act as a neurotransmitter in the brain, where it has been implicated in limiting food intake (Tritos, N. A. et al. (1999) Neuropeptides 33:339-349). The matrix metalloproteases (MMPs) are a family of at least 23 enzymes that can degrade components of the extracellular matrix (ECM). They are Zn +2 endopeptidases with an N-terminal catalytic domain. Nearly all members of the family have a hinge peptide and C-terminal domain which can bind to substrate molecules in the ECM or to inhibitors produced by the tissue (TIMPs, for tissue inhibitor of metalloprotease; Campbell, I. L. et al. (1999) Trends Neurosci. 22:285). The presence of fibronectin-like repeats, transmembrane domains, or C-terminal hemopexinase-like domains can be used to separate MMPs into collagenase, gelatinase, stromelysin and membrane-type MMP subfamilies. In the inactive form, the Zn +2 ion in the active site interacts with a cysteine in the pro-sequence. Activating factors disrupt the Zn +2 -cysteine interaction, or “cysteine switch,” exposing the active site. This partially activates the enzyme, which then cleaves off its propeptide and becomes fully active. MMs are often activated by the serine proteases plasmin and furin. MMPs are often regulated by stoichiometric, noncovalent interactions with inhibitors; the balance of protease to inhibitor, then, is very important in tissue homeostasis (reviewed in Yong, V. W. et al. (1998) Trends Neurosci. 21:75). MMPs are implicated in a number of diseases including osteoarthritis (Mitchell, P. et al. (1996) J. Clin. Invest. 97:761), atherosclerotic plaque rupture (Sukhova, G. K. et al. (1999) Circulation 99:2503), aortic aneurysm (Schneiderman, J. et al. (1998) Am. J. Path. 152:703), non-healing wounds (Saarialho-Kere, U. K. et al. (1994) J. Clin. Invest. 94:79), bone resorption (Blavier, L. and J. M. Delaisse (1995) J. Cell Sci. 108:3649), age-related macular degeneration (Steen, B. et al. (1998) Invest. Ophthalmol. Vis. Sci. 39:2194), emphysema (Finlay, G. A. et al. (1997) Thorax 52:502), myocardial infarction (Rohde, L. E. et al. (1999) Circulation 99:3063) and dilated cardiomyopathy (Thomas, C. V. et al. (1998) Circulation 97:1708). MMP inhibitors prevent metastasis of mammary carcinoma and experimental tumors in rat, and Lewis lung carcinoma, hemangioma, and human ovarian carcinoma xenografts in mice (Eccles, S. A. et al. (1996) Cancer Res. 56:2815; Anderson et al. (1996) Cancer Res. 56:715-718; Volpert, O. V. et al. (1996) J. Clin. Invest. 98:671; Taraboletti, G. et al. (1995) J. NCI 87:293; Davies, B. et al. (1993) Cancer Res. 53:2087). MMPs may be active in Alzheimer's disease. A number of MMPs are implicated in multiple sclerosis, and administration of MMP inhibitors can relieve some of its symptoms (reviewed in Yong, supra). Another family of metalloproteases is the ADAMs, for A Disintegrin and Metalloprotease Domain, which they share with their close relatives the adamalysins, snake venom metalloproteases (SVMPs). ADAMs combine features of both cell surface adhesion molecules and proteases, containing a prodomain, a protease domain, a disintegrin domain, a cysteine rich domain, an epidermal growth factor repeat, a transmembrane domain, and a cytoplasmic tail. The first three domains listed above are also found in the SVMPs. The ADAMs possess four potential functions: proteolysis, adhesion, signaling and fusion. The ADAMs share the metzincin zinc binding sequence and are inhibited by some MMP antagonists such as TIMP-1. ADAMs are implicated in such processes as sperm-egg binding and fusion, myoblast fusion, and protein-ectodomain processing or shedding of cytokines, cytokine receptors, adhesion proteins and other extracellular protein domains (Schlöndorff, J. and C. P. Blobel (1999) 3. Cell. Sci. 112:3603-3617). The Kuzbanian protein cleaves a substrate in the NOTCH pathway (possibly NOTCH itself), activating the program for lateral inhibition in Drosophila neural development. Two ADAMs, TACE (ADAM 17) and ADAM 10, are proposed to have analogous roles in the processing of amyloid precursor protein in the brain (Schlöndorff and Blobel, supra). TACE has also been identified as the TNF activating enzyme (Black, R. A. et al. (1997) Nature 385:729). TNF is a pleiotropic cytokine that is important in mobilizing host defenses in response to infection or trauma, but can cause severe damage in excess and is often overproduced in autoimmune disease. TACE cleaves membrane-bound pro-TNF to release a soluble form. Other ADAMs may be involved in a similar type of processing of other membrane-bound molecules. The ADAMTS sub-family has all of the features of ADAM family metalloproteases and contain an additional thrombospondin domain (TS). The prototypic ADAMTS was identified in mouse, found to be expressed in heart and kidney and upregulated by proinflammatory stimuli (Kuno, K. et al. (1997) J. Biol. Chem. 272:556-562). To date eleven members are recognized by the Human Genome Organization (HUGO; http://www.gene.ucl.ac.uk/users/hester/adamts.html#Approved). Members of this family have the ability to degrade aggrecan, a high molecular weight proteoglycan which provides cartilage with important mechanical properties including compressibility, and which is lost during the development of arthritis. Enzymes which degrade aggrecan are thus considered attractive targets to prevent and slow the degradation of articular cartilage (See, e.g., Tortorella, M. D. (1999) Science 284:1664; Abbaszade, I. (1999) J. Biol. Chem. 274:23443). Other members are reported to have antiangiogenic potential (Kuno et al., supra) and/or procollagen processing (Colige, A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2374). Insertion of Trasnsposons into Gene-Coding Sequence Long interspersed nuclear elements (L1s or LINEs) are retro-transposons, many of which encode a reverse transcriptase activity, via which they transpose and insert themselves throughout the genome by reverse transcription of an RNA intermediate. This process is known as retrotransposition (Sassaman, D. M. et al. (1997) Nature Genet. 16 (1), 37-43). This event can be mutagenic with an evident phenotype such as certain disease conditions. Expression Profiling Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. The discovery of new protein modification and maintenance molecules, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of gastrointestinal, cardiovascular, autoimmunefmflammatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of protein modification and maintenance molecules. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention features purified polypeptides, protein modification and maintenance molecules, referred to collectively as “PMOD” and individually as “PMOD-1,” “PMOD-2,” “PMOD-3,” “PMOD-4,” “PMOD-5,” “PMOD-6,” “PMOD-7,” “PMOD-8,” “PMOD-9, ” “PMOD-10,” “PMOD-11,” “PMOD-12,” “PMOD-13,” “PMOD-14,” “PMOD-15,” “PMOD-16,” and “PMOD-17.” In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-17. The invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-17. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID NO:18-34. Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide. The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed. Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides. Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides. The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. The invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional PMOD, comprising administering to a patient in need of such treatment the composition. The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional PMOD, comprising administering to a patient in need of such treatment the composition. Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional PMOD, comprising administering to a patient in need of such treatment the composition. The invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide. The invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide. The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, iin) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary-to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:18-34, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. |
Peptides and antibodies to muc 1 proteins |
The invention relates to methods of inhibiting proliferation or growth of tumor cells and/or inducing cell death relates to the use of antibodies, hybridomas and pharmaceutical compositions containing same, for inhibiting growth or proliferation and inducing death in epithelial, colon, lung, breast and ovarian tumor cells or other cells which express MUC1 proteins. |
1. A method of inhibiting mammalian cell proliferation or cell growth comprising the step of administering to a subject in need, an effective amount of a ligand which specifically binds to an epitope in the extracellular region of a transmembrane isoform of MUC1 protein, thereby selectively inhibiting mammalian cell proliferation or cell growth. 2. A method of inducing mammalian cell death comprising the step of administering to a subject in need, an effective amount of a ligand which specifically binds to an epitope in the extracellular region of a transmembrane isoform of MUC1 protein, thereby selectively inducing mammalian cell death. 3. A method of treating a subject with a disease involving pathological proliferation of cells comprising the step of administering to a subject in need, an effective amount of a ligand which specifically binds to an epitope in the extracellular region of a transmembrane isoform of MUC1 protein, thereby treating the disease. 4. A method of selectively inhibiting cell proliferation or cell growth comprising the steps of contacting a cell which expresses MUC1 protein isoform with an effective amount of a ligand which specifically binds to an epitope in the extracellular region of the transmembrane isoform of the MUC1 protein, thereby selectively inhibiting the cell proliferation or cell growth. 5. A method of selectively inducing cell death comprising the step of contacting a cell which expresses MUC1 protein isoform with an effective amount of a ligand which specifically binds to an epitope in the extracellular region of the transmembrane isoform of MUC1 protein, thereby selectively inducing cell death. 6. The method according to claims 1 to 5, wherein said cell is a cancer cell. 7. The method according to claim 6, wherein said cancer cell is an epithelial cancer cell. 8. The method according to claim 7, wherein said epithelial cancer cell is a breast cancer cell, an ovarian cancer cell, a lung cancer cell or a colon cancer cell. 9. The method according to claims 1 to 5, wherein said MUC1 isoform is MUC1/Y, MUC1/REP, or MUC1/X. 10. The method according to claims 1 to 5, wherein said ligand is an antibody, a peptide, an antagonist, or an agonist. 11. The method according to claims 1 to 5, wherein said epitope is an amino acid sequence within a 59 amino acid sequence as set forth in SEQ ID No.1. 12. The method according to claims 1 to 5, wherein said ligand is conjugated to a cytotoxic drug. 13. The method according to claim 10, wherein said antibody is a monoclonal antibody, a synthetic antibody, a polyclonal antibody or a chimera. 14. A method of treating a subject with a disease involving pathological proliferation of cells comprising the step of administering to a subject in need, an effective amount of a peptide which comprises an amino acid sequence corresponding to the extracellular region of a transmembrane isoform of MUC1 protein, so as to induce an increase in the level of antibodies specific for said peptide in the subject, thereby treating the disease. 15. The method according to claim 14 wherein said MUC1 isoform is MUC1/Y, MUC1/REP, or MUC1/X. 16. The method according to claim 14 wherein said peptide comprises an amino acid sequence within a 59 amino acid sequence as set forth in SEQ ID No.1. 17. An isolated antibody which specifically binds to an epitope in the extracellular region of a transmembrane isoform of MUC1 protein. 18. The isolated antibody according to claim 17, wherein said isoform of MUC1 protein is MUC1/Y protein, MUC1/REP protein or MUC1/X protein. 19. The isolated antibody according to claim 17, wherein the antibody is a monoclonal antibody, a synthetic antibody, a polyclonal antibody or a chimera. 20. The antibody according to claim 17 wherein said epitope is an amino acid sequence within a 59 amino acid sequence as set forth in SEQ ID No.1. 21. A hybridoma cell producing a monoclonal antibody that binds to an epitope in the extracellular region of a transmembrane isoform of MUC1 protein. 22. The hybriodoma of claim 21, wherein said isoform of MUC1 protein is MUC1/Y protein, MUC1/REP protein or MUC1/X protein. 23. A pharmaceutical composition comprising an effective amount of a ligand which specifically binds to an epitope within a transmembrane isoform of MUC1 protein and a pharmaceutically acceptable carrier. 24. The pharmaceutical composition of claim 23 wherein said ligand is conjugated to a cell killing agent. 25. The pharmaceutical composition of claim 23, wherein said MUC1 isoform is MUC1/Y, MUC1/REP or MUC1/X. 26. The pharmaceutical composition of claim 23, wherein said epitope is an amino acid sequence within a 59 amino acid sequence as set forth in SEQ ID No.1 27. The pharmaceutical composition of claim 23, wherein said ligand is an antibody, a peptide, an antagonist or an agonist. 28. The pharmaceutical composition of claim 27, wherein the antibody is a monoclonal antibody, a synthetic antibody, a polyclonal antibody or a chimera. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Tumor markers are molecules that are associated with the transformation of a normal cell into a malignant cell. Tumor markers are either altered proteins which are different from the proteins expressed in normal cells or over-expression of proteins that are not expressed, or slightly expressed in normal cells. Mucins are high molecular weight glycoproteins, which are produced by normal epithelial cells. MUC1 is one of the four mucins known to date that are transmembrane molecules and while its function in adult life maybe lubrication, in fetal life development it is thought to play an important role in forming the lumen of the duct by keeping apart cells located opposite one another. The MUC1 gene was also shown to be expressed in hemopoietic tissues. It was found that MUC1 (also called H23-Ag, episialin, PEM-Polymorphic Epithelial Mucin, MCA-Mammary Carcinoma Antigen and EMA Epithelial Membrane Antigen) expression is elevated (10-50 fold) in breast cancer cells in comparison to normal resting mammary secretory epithelial cells. Moreover, immunohistochemical analyses using the H23 mAb (which recognizes MUC1), revealed that MUC1 is expressed in 91% of the breast cancer tissues and 100% of breast cancer metastases, whereas the non-malignant tissues were negative for the H23 mAb staining (1). Elevated levels of the MUC1 protein in serum and body fluids were reported in 7%, 17%, 64% and 67% of breast cancer patients presenting with stages I to IV, respectively. In addition, elevated levels of circulating MUC1 may be associated with a poor It was shown that MUC1 is over expressed in epithelial cancers other than breast cancer. MUC1 was shown to be over expressed in epithelial ovarian cancer cells as well as in all types of lung cancer cells and other cancers (2). In malignancy, the MUC1 oligosacharide chains are shorter and less dense comparing to MUC1 in normal cells. This results in the exposure of new epitopes of the core protein in the cancer-associated mucin. Isolation of MUC1 cDNAs revealed several protein isoforms: the MUC1/REP, MUC1/SEC MUC1/Y and MUC1/X proteins. A short description of four of the above-mentioned isoforms that are connected with the present invention is given below: The MUC1/REP isoform is a transmembrane protein that contains: a large extracellular domain consisting of a heavily glycosylated 20 amino acid repeat motif. The number of these repeats varies from 20 to 100 and thus is named a VNTR (Variable Number of Tandem Repeats); a transmembrane domain, which consists of a 28 amino acid hydrophobic sequence, and a 72 amino acid cytoplasmic domain. During the biosynthesis of the MUC1/REP protein it undergoes a proteolytic cleavage event. The cleavage takes place within the conserved sequence IKFRPGSVVV that is contained within the extracellular domain. Intriguingly, this cleavage site resides within a previously identified module, designated the “SEA” module (3), found in a number of highly O-linked glycosylated proteins that are invariably linked in one way or another to the cell membrane. The SEA module functions not only as a site for proteolytic cleavage, but also for subsequent re-association of the subunits. Consequently, the MUC1/REP protein is presented on the cell surface as a heterodimer which is composed of a large extracellular subunit (containing the repeat array) linked by non-covalent, SDS sensitive bonds to a smaller cell-anchored subunit which consists of a small extracellular fragment followed by the transmembrane and cytoplasmic domains. The large extracellular subunit can disconnect and reconnect with the small extracellular fragment of the cell-anchored subunit. The MUC1/Y isoform is a transmembrane protein that contains transmembrane and cytoplasmic domains identical to those of MUC1/REP protein. Unlike MUC1/REP, due to a differential splicing event that utilizes splicing sites located upstream and downstream to the repeat array, MUC1/Y protein is devoid of both the tandem repeat array and its flanking regions. Expression of MUC1/Y was demonstrated in various human secretory epithelial tumors. MUC1/Y was found to be expressed on the cell surface of various human epithelial tumor cells but is not detectable in the adjacent normal tissue. The MUC1/X isoform (4) is a transmembrane protein that contains transmembrane and cytoplasmic domains identical to those of MUC1/REP protein. Unlike MUC1/REP, due to a differential splicing event that utilizes splicing sites located upstream and downstream to the repeat array, MUC1/X protein is devoid of both the tandem repeat array and its flanking regions. The MUC1/SEC isoform: This isoform is generated by an alternative splicing mechanism. It is a secreted protein since it lacks the hydrophobic region that can attach the protein to the cell membrane. Its N-terminal sequences are identical to those of the MUC1/REP extracellular domain. Furthermore it has been shown that the soluble secreted MUC1/SEC protein may bind specifically to the extracellular domain of the MUC1/Y protein (5). Thus, it will be highly advantageous to develop a ligand which binds to a specific epitope in the MUC1 proteins and, more particularly, to a specific extracellular epitope in the MUC1/REP or MUC/Y proteins that will dramatically inhibit the proliferation or growth of cells, and will induce death in cells, such as, cancer cells and in particular cells which over express MUC1 proteins. |
<SOH> SUMMARY OF THE INVENTION <EOH>In one embodiment, this invention provides methods for inhibiting cell proliferation or growth and/or inducing cell death in cancer cells and in particular in epithelial, breast, colon, lung and ovarian tumor cells which express MUC1 proteins. In one embodiment, the invention provides a method of selectively inhibiting the proliferation or cell growth of cells, comprising the step of administering to a subject, an effective amount of a ligand which specifically binds to an epitope in the extracellular region of a transmembrane isoform of MUC1 protein, thereby selectively inhibiting proliferation or growth of such cells. In one embodiment, the invention further provides a method of inducing cell death comprising the step of administering to a subject, an effective amount of a ligand which specifically binds to an epitope in the extracellular region of a transmembrane isoform of MUC1 protein, thereby selectively inducing cell death. In one embodiment, the invention provides a method of selectively inhibiting cell proliferation or cell growth comprising the step of contacting a cell which expresses MUC1 protein isoform with an effective amount of a ligand which specifically binds to an epitope in the extracellular region of the transmembrane isoform of MUC1 protein, thereby selectively inhibiting the cell proliferation or cell growth. The invention provides a method of inducing cell death comprising the step of contacting a cell which expresses MUC1 protein isoform with an effective amount of a ligand which specifically binds to an epitope in the extracellular region of the transmembrane isoform of MUC1 protein, thereby selectively inducing cell death. In one embodiment, the invention further provides a method of treating a subject with a disease involving pathological proliferation of cells comprising the step of administering to a subject, an effective amount of a ligand which specifically binds to an epitope in the extracellular region of a transmembrane isoform of MUC1 protein, thereby treating the disease. In one embodiment, the invention further provides a method of treating a subject with a disease involving pathological proliferation of cells comprising the step of administering to a subject, an effective amount of a peptide which comprises an amino acid sequence corresponding to the extracellular region of a transmembrane isoform of MUC1 protein, so as to induce an increase in the level of antibodies specific for said peptide in the subject, thereby treating the disease. In one embodiment, the invention further provides an isolated antibody which specifically binds to an epitope in the extracellular region of an isoform of MUC1 protein wherein said epitope is located within a 59 amino acid sequence according to the amino acid sequence of SEQ ID No.1. In one embodiment, the invention further provides a pharmaceutical composition comprising an effective amount of a ligand, which specifically binds to a MUC1 protein isoform, and a pharmaceutically acceptable carrier. In one embodiment, the invention further provides a hybridoma cell producing monoclonal antibody that binds to an epitope in the extracellular region of an isoform of MUC1 protein. In one embodiment the epitope is located within a 59 amino acid sequence according to the amino acid sequence of SEQ ID No.1 and is located directly N′-terminal to the transmembrane domain of the protein. |
Method of making frozen products with a characteristic form from at least one flowable type of material, apparatus and use hereof |
The invention relates to a method of making frozen products with a characteristic form from at least one flowable type of material, the frozen products including edible ice products such as ice cream sticks, ice lollipops, ice dessert or the like. The method includes placing a first quantity of the flowable type of material in a first mould or moulding part and cooling the material of the at least one first mould or moulding part in order to create a basic frozen form of the product. Further, the method includes forming the frozen form of the product into a characteristic form, this forming including modification of the product in a second mould or moulding part, the second mould or moulding part basically having the contours of the characteristic form. |
1. Method of making frozen products with a characteristic form from at least one flowable type of material, said frozen products comprising edible ice products such as ice cream sticks, ice lollipops, ice dessert or the like, said method comprising: placing a first quantity of said flowable type of material in a first mould or moulding part in order to create a basic frozen form of the ice product, adding a surface coating to said basic frozen form, and forming said frozen form of the product into a characteristic form, said forming including modification of the product in a second mould or moulding part, said second mould or moulding part comprising contours of said characteristic form. 2. Method according to claim 1, where in said basic frozen form partly or substantially comprises the characteristics of the frozen product after said modification. 3. Method according to claim 1, wherein said surface is a liquid type of material added to said basic frozen form of the product and solidified before said modification. 4. Method according to, claim 1, wherein said modification includes cooling of the ice product in said first and/or second mould or moulding part(s) by cooling the surface of said first and/or second mould or moulding part(s). 5. Method according to, claim 4, wherein cooling is generated by means of a cryogenic fluid such as liquid nitrogen or dry ice. 6. Method according to, claim 4, wherein a temperature of said cooling is lower than minus 65 degrees Celsius. 7. Method according to, claim 4, wherein a temperature of said cooling is controllable. 8. Method according to claim, 1, wherein the first mould or moulding part is also used as the second mould or moulding part. 9. An apparatus for making frozen products with a characteristic form from at least one flowable type of material, said frozen products comprising edible ice products such as ice cream sticks, ice lollipops, ice dessert or the like, said apparatus comprising at least one first mould or moulding part adapted for a first quantity of the flowable type of material in order to create a basic frozen form of the product, at least one surface coating area to provide said basic frozen form of the product with a surface coating, and forming means to form said basic frozen form of the product with the surface coating into a characteristic form, said forming means including a second mould or moulding part which comprises contours of said characteristic form. 10. Apparatus according to claim 9, wherein said basic frozen form partly or substantially comprises the characteristics of the frozen product after forming. 11. Apparatus according to claim, wherein at least one of said moulds or moulding sections is coated with a Teflon® or a similar anti-stick surface. 12. Apparatus according to claims, wherein at least one of said moulds or mould sections comprises cooling means cooled by means of a cryogenic fluid comprising liquid nitrogen or dry ice. 13. Apparatus according to claims 9, wherein said forming means includes means for adding a surface of a liquid type of material to said basic frozen form of the product and means for solidifying said surface. 14. Apparatus according to claims 13, wherein said liquid type of material is chocolate, fruit juice or the like. 15. Apparatus according to, wherein said moulds or moulding parts comprise two halves with inner forming surfaces. 16. Apparatus according to claims 9, wherein said contours comprise different shapes, marks, stamps, patterns, and/or projections in order to create the frozen product with a three-dimensional characteristic form. 17. Apparatus according to claims 13, wherein an interior of said second mould or moulding part is partly or substantially similar in size to that of the basic form and the added surface. 18. Apparatus according to claims 9, wherein an interior of said second mould or moulding part is partly or substantially similar in size to that of the first mould or moulding part. 19. Apparatus according to claims 9, wherein said first mould or moulding part and said second mould or moulding part is one and the same mould or moulding part. 20. The method of claim 1, wherein said method is utilized in connection with forming a surface coating of ice products such as cream sticks, lollipops, dessert, bars, slices, logs, cakes, tubs or the like into characteristic three-dimensional forms or figures. 21. The method of claim 1, wherein said method is utilized in connection with characteristics including commercial messages such as brands, logos, trademarks etc. formed or embossed in the surface of said products. 22. The apparatus of claim 9, wherein said apparatus is utilized to form a surface coating of ice products such as cream sticks, lollipops, desserts, bars, slices, logs, cakes, tubs or the like into characteristic three-dimensional forms or figures 23. Method according to claim 6, wherein the temperature of said cooling between minus 75 and 179 degrees Celsius. 24. Method according to claim 23, wherein said cooling is between minus 75 and 80 degrees Celsius. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention relates to a method of making frozen products with a characteristic form from at least one flowable type of material, an apparatus and use hereof. In the area of creating frozen products, such as ice cream sticks or ice lollipops, it desirable to make frozen products with a characteristic form. So far, the characteristic form has been achieved by using a freezing mould with the desired characteristics. However, this method has the disadvantage of making frozen products with a somewhat blurred form. Further, the method does not allow quality surfaces with details to be made, as the method is too crude. With the method, it is also very difficult to mass fabricate the frozen products with a high degree of uniformity. If the frozen product is applied with a surface of another material, such as chocolate, it is especially difficult to achieve uniform and detailed products with the known method. The material will usually hide or smooth out any details under the surface. The invention provides a method and apparatus for making frozen products with a characteristic form from at least one flowable type of material without the above-mentioned disadvantages. Especially, the invention provides a method and apparatus for making frozen products with a characteristic form in which high-quality characteristics and details are produced, e.g. well-defined and distinct, regardless of whether they are simple or complicated in shape and have surfaces of another material. |
<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with the invention a method of making frozen products with a characteristic form from at least one flowable type of material is stated, said frozen products comprising edible ice products such as ice cream sticks, ice lollipops, ice dessert or the like, said method including the following steps: placing a first quantity of said flowable type of material in a first mould or moulding part in order to create a basic frozen form of an ice product, adding a surface coating to said basic frozen form, and forming said frozen form of the product into a characteristic form, said forming including modification of the product in a second mould or moulding part, said second mould or moulding part basically comprising the contours of said characteristic form. Hereby, it is possible to create a frozen product with a surface coating and a characteristic form, such as a 3D figure, that is well-defined and distinct in its details. The phrase “flowable type of material” may be understood as any type of edible fluid such as ice cream mix, fruit juice, lemonade or soft drink that may be frozen. The term “modification” is to be understood as a change of the basic form of the product, said basic form already comprising a form close to the finalized product. The modification may provide a more accurate defining or shaping of already existing details which have bee blurred by e.g. a surface coating. The phrase “mould or moulding part” is to be understood to include more than one moulding apparatus, each dedicated to a step in the production process or to include one moulding apparatus performing one or more formings of a frozen product during the production process. Further, the term “mould” is to be understood as any kind of process for creating a product in a form or forming part. The form may be a standard mould with one or more sections defining an interior chamber to be filled with the flowable product. In a more viscous version, the flowable product may be extruded before imposing an open mould on the product. Other possible types of forming methods and moulds are known by the skilled person. Further examples of ice products may be ice bars, slices, logs, cakes, tubs or the like. In an aspect of the invention, said basic frozen form partly or substantially comprises the characteristics of the frozen product after modification. In another aspect of the invention, a surface of a liquid type of material is added to said basic frozen form of the product and solidified prior to modification. With a solidified surface, it is possible to obtain a surface with the desired characteristics regardless of the liquid type of material used. Especially, embossing details into the surface may restore details in the basic form which would usually be hidden by thicker liquid types. In a further aspect of the invention, said modification includes cooling of the ice product in said first and/or second mould or moulding part(s), preferably by cooling the surface of said first and/or second mould or moulding part(s). In an even further aspect of the invention, cooling is generated by means of a cryogenic material such as liquid nitrogen or dry ice. By using a cryogenic fluid, it is possible to obtain the low temperatures necessary or at least to make it easy to remove an ice product from a mould. In another aspect of the invention, the temperature of said cooling is lower than minus 65 degrees Celsius, such as between minus 75 and 179 degrees Celsius, and preferably between minus 75 and 80 degrees Celsius. Hereby, a preferred relation between temperatures, cooling means, such as means based on a cryogenic fluid, and the ice products has been obtained. At the above-mentioned temperature intervals, it is especially easy to remove an ice product from a mould, as the surface gains an anti-stick-like characteristic in relation to the ice product. During production, the ice product itself usually has a temperature of minus 5 to minus 20 degrees Celsius. As it contains a lot of air and water, the ice product is frozen but still of a rather soft nature which allows it to be processed e.g. embossed or stamped. In a further aspect of the invention, the temperature of said cooling is controllable. Hereby, it is possible to create the optimal conditions during production for different types of ice products and surfaces as well as different types of modification. In particular, the temperature may be controlled just before or during modification when the surface of the ice product is chanced with the moulds. In an even further aspect of the invention, the first mould or moulding part is also used as the second mould or moulding part. Hereby, it is possible to create a compact and efficient system producing ice products with a surface coating comprising well-defined details. Further, in accordance with the invention an apparatus for making frozen products with a characteristic form from at least one flowable type of material is stated, said frozen products comprising edible ice products such as ice cream sticks, ice lollipops, ice dessert or the like, said apparatus comprising at least one first mould or moulding part adapted for a first quantity of a flowable type of material, cooling means for cooling the material of said at least one first mould or moulding part in order to create a basic frozen form of the product, and forming means to form said basic frozen form of the product into a characteristic form, said forming means including a second mould or moulding part which substantially comprises the contours of said characteristic form. Hereby, it is possible to create a frozen product, such as a 3D figure, with a characteristic form which is well-defined and distinct in its details. |
Site- targeted transformation using amplification vectors |
A process of causing a targeted integration of DNA of interest into a plynt cell nuclear genome, comprising; i) providing plant cells with an amplification vector, or a precursor thereof, capable of autonomous replication in plant cells, said vector comprising; a) DNA sequence(s) encoding an origin of replication functional in plant cells, b) DNA sequence(s) necessary for site-specific and/or homologous recombination between the vector and a host nuclear DNA, and c) optionally, further DNA of interest; ii) optionally providing conditions that facilitate vector amplification and/or cell to cell movement and/or site-specific and/or homologous reconbination, and iii) selecting cells having undergone recombination at a predetermined site in the plant nuclear DNA. |
1. A process of causing a targeted integration of DNA of interest into a plant cell nuclear genome, comprising: (i) providing plant cells with an amplification vector, or a precursor thereof, capable of replication in plant cells, said vector comprising: (a) DNA sequence(s) encoding an origin of replication functional in plant cells, (b) DNA sequence(s) necessary for site-specific and/or homologous recombination between the vector and a host nuclear DNA, and (c) optionally, further DNA of interest; whereby the replication of said amplification vector in said plant cells is transient; (ii) optionally providing conditions that facilitate vector amplification and/or cell to cell movement and/or site-specific and/or homologous recombination; and (iii) selecting cells having undergone recombination at a predetermined site in the plant nuclear DNA. 2. A process of causing a targeted integration of DNA of interest into a plant cell nuclear genome, comprising the following steps: (i) transfecting or transforming a plant cell with a first DNA comprising a sequence which, when integrated in the plant cell genome, provides a target site for site-specific and/or homologous recombination; (ii) selecting a cell which contains said target site for site-specific and/or homologous recombination in its nuclear genome; (iii) transfecting or transforming said selected cell with a second DNA comprising a region for recombination with said target site and a first sequence of interest; (iv) optionally providing enzymes for recombination; and (v) selecting cells which contain the sequence of interest from the second DNA integrated at the target site, whereby at least one of said first or said second DNA is delivered by an amplification vector, or a precursor thereof, capable of replication in a plant cell and comprising (a) DNA sequence(s) encoding an origin of replication functional in the plant cell, whereby the replication of said amplification vector in said plant cell is transient. 3. (cancelled) 4. The process according to claim 1 or 2, wherein said amplification vector comprises DNA sequence(s) necessary for homologous recombination. 5. The process according to claim 1 or 2, wherein said providing a plant cell with an amplification vector, or a precursor thereof, or said transfecting or transforming is done by Agrobacterium-mediated delivery. 6. The process according to claim 1 or 2, wherein said providing a plant cell with an amplification vector, or a precursor thereof, or said transfecting or transforming is done by direct viral transfection. 7. The process according to claim 1 or 2, wherein said providing a plant cell with an amplification vector, or a precursor thereof, or said transfecting or transforming is done by non-biological delivery. 8. The process according to claim 1 or 2, wherein said providing a plant cell with an amplification vector or said transfecting or transforming is done by conversion of a vector or a pro-vector DNA that was pre-integrated into a plant nuclear DNA to form an autonomously replicating plasmid. 9. The process according to claim 1 or 2, wherein said amplification vector is released from a precursor thereof which has two origins of replication. 10. The process according to claim 1 or 2, wherein said amplification vector is a DNA virus-derived vector. 11. The process according to claim 1 or 2, wherein said amplification vector is a DNA copy or a replication intermediate of an RNA virus-derived vector. 12. The process according to claim 1 or 2, wherein said amplification vector is of retrotransposon origin. 13. The process according to claim 1 or 2, wherein the amplification vector has additionally viral functions selected from the group consisting of: functions for reverse transcription, host infectivity, cell-to-cell and/or systemic movement, integration into a host chromosome, viral particle assembly, control of silencing by host, and control of host physiology. 14. The process according to claim 1 or 2, wherein said origin of replication functional in a plant cell is derived from a plant nuclear genome. 15. The process according to claim 1 or 2, wherein said origin of replication functional in a plant cell is derived from a ribosomal DNA intergenic spacer region. 16. The process according to claim 1 or 2, wherein said origin of replication functional in a plant cell is of synthetic nature. 17. The process according to claim 1 or 2, wherein said origin of replication is derived from a plant virus. 18. The process according to claim 1 or 2, wherein said homologous or site-specific recombination is one-sided. 19. The process according to claim 1 or 2, wherein said homologous or site-specific recombination is two-sided. 20. The process according to claim 1 or 2, wherein said site-specific recombination is promoted or facilitated by recombination enzymes selected from the group consisting of: site specific recombinases, restriction enzymes, integrases, and resolvases. 21. The process according to claim 1 or 2, wherein said homologous recombination is promoted or facilitated by recombination enzymes selected from the group consisting of: RecA-like proteins, rare-cutting endonuclease of HO type, and I-SceI endonuclease. 22. The process according to claim 1 or 2, wherein said amplification vector is assembled in a process of recombination. 23. The process according to claim 2, wherein said first DNA contains a selectable marker for the selection of step (ii). 24. The process according to claim 2, wherein said first DNA comprises additionally a second sequence of interest. 25. The process according to claim 2, wherein the recombination of said first and said second DNA establishes a functional sequence. 26. The process according to claim 2, wherein said first and said second DNA each additionally contains a fragment of a selection marker, which makes a selection marker as a result of said recombination. 27. The process according to claim 2, wherein said second DNA is delivered by an amplification vector or a precursor thereof. 28. The process according to claim 2, wherein the function of a sequence introduced into the plant in steps (i) and (ii) is destroyed in steps (III) to (v). 29. The process according to claim 1 or 2, wherein expression of genes involved in non-homologous recombination is inhibited or suppressed. 30. The process according to claim 1 or 2, wherein the end result of recombination is a site-directed mutation. 31. Plant cells, seeds and plants obtained by performing the process according to claim 1 or 2 one or more times. 32. Plant cells, seeds and plants obtained by performing the process of steps (i) and (ii) according to claim 2 one or more times. 33. Vector or pro-vector for performing the process according to claim 1 or 2. 34. Agrobacterium cells containing the vector or pro-vector according to claim 33. 35. Packaged viral particles containing the vector or pro-vector according to claim 33. 36. A kit-of-parts comprising (i) the plant cells, seeds or plants according to claim 32 and (ii) the vector or pro-vector according to claim 33, and/or the Agrobacterium cells according to claim 34, and/or the viral particles according to claim 35. 37. A kit-of-parts comprising a vector or a pro-vector for performing steps (i) and (ii) of claim 2, and a vector for performing steps (iii) and (iv) of claim 2. |
<SOH> BACKGROUND OF THE INVENTION <EOH>With current levels of research in the field of plant molecular genetics and functional genomics, plant transformation is likely to become an increasingly important tool for plant improvement. Limitations of current transformation procedures are numerous but one most important deficiency of currently used techniques is that they result in random insertions of target genes in host genomes, leading to uncontrolled delivery and unpredictable levels of transgene expression. As a result, existing methods require many independent transgenic plants to be generated and analyzed for several generations in order to find those with the desired level or pattern of expression. The vectors for such non-targeted transformation must necessarily contain full expression units, as the subsequent transformation to the same site is impossible, thus limiting engineering capability of the process. A number of different approaches have been investigated in an attempt to develop protocols for efficient targeting of DNA at specific sites in the genome. These efforts include: (i) attempts to improve the process of homologous recombination (that relies on the endogenous cellular recombination machinery) by over-expressing some of the enzymes involved in recombination/repair; (ii) attempts to decrease non-targeted recombination by down-regulating enzymes that contribute to non-specific recombination; (iii) use of heterologous recombinases of microbial origin; (iv) development of chimeraplasty for targeted DNA modification in plants. A brief description of these efforts is summarized below. Homologous recombination occurs readily in bacteria and yeast, where it is used for gene replacement experiments. More recently it has been developed as a tool for gene replacement in mammals (Mansour et al, 1988, Nature, 336, 348-336; Thomas et al, 1986, Cell, 44, 419-428; Thomas et al, 1987, Cell, 51, 503-512), and the moss Physcomytrella patens (Schaefer & Zryd, 1997 , Plant J., 11, 1195-1206). However, it is inefficient in plants. Targeted DNA modification by homologous recombination is accomplished by introducing into cells linear DNA molecules that share regions of homology with the target site. Homologous recombination occurs as a result of a repair mechanism induced by the double-strand breaks at the ends of the DNA fragment. Unfortunately, a competing repair mechanism called non-homologous end-joining (NHEJ) also takes place at a much higher frequency in many organisms and/or cell types, rendering selection of the desired site-targeted events difficult (Haber, 2000 , Curr. Op. Cell. Biol., 12, 286-292; Haber, 2000 , TIG, 16, 259-264; Mengiste & Paszkowski, 1999 , Bio.l Chem., 380, 749-758). In higher plants only a few cases of successful targeted transformation by homologous recombination have been reported, and all were obtained with efficiencies of targeted events over non-targeted events in the range of 10 −3 to 10 −5 (Paszkowski et al., 1988, EMBO J., 7, 4021-4026; Lee et al., 1990, Plant Cell; 2, 415-425; Miao & Lam, 1995 , Plant J., 7, 359-365; Offringa et al., 1990, EMBO J., 9, 3077-3084; Kempin et al., 1997, Nature, 389, 802-803). This means that the screening procedure will involve a very large number of plants and will be very costly in terms of time and money; in many cases this will be a futile effort. Attempts to increase homologous recombination frequencies have been made. Investigators have over-expressed some of the enzymes involved in double-strand break repair. For example, over-expression of either the E. coli RecA (Reiss et al., 1996, Proc Natl Acad Sci USA., 93, 3094-3098) or the E. coli RuvC (Shalev et al., 1999, Proc Natl Acad Sci USA., 96, 7398-402) proteins in tobacco has been tried. However, this has only led to an increase of intrachromosomal homologous recombination (of approximately 10 fold). There was no increase of gene targeting (Reiss et al., 2000, Proc Natl Acad Sci USA., 97, 3358-3363.). Using another approach to increase homologous recombination, investigators have induced double-strand breaks at engineered sites of the genome using rare cutting endonucleases such as the yeast HO endonuclease (Chiurazzi et al, 1996, Plant Cell, 8, 2057-2066; Leung et al., 1997, Proc. Natl. Acad. Sci., 94, 6851-6856) or the yeast I-Sce I endonuclease (Puchta et al., 1996, Proc. Natl. Acad. Sci., 93, 5055-5060). Site targeted frequency of 2×10 −3 to 18×10 −3 was obtained using the I-Sce I endonuclease. Although an improvement, this is still inefficient. In addition, many of the targeted events contained unwanted mutations or occurred by homologous recombination at one end of the break onyl. Incidentally, there is an interesting recent publication describing a hyperrecombinogenic tobacco mutant demonstrating three orders of magnitude increase of mitotic recombination between homologous chromosomes, but the gene(s) involved has not been identified yet (Gorbunova et al., 2000, Plant J., 24, 601-611) and targeted recombination is not involved. An alternative approach consists of decreasing the activity of enzymes (e.g. Ku70) involved in non-homologous end joining (U.S. Pat. No. 6,180,850) to increase the ratio of homologous/non-homologous recombination events. This approach has been far from being practically useful. A recently developed approach called chimeraplasty consists of using DNA/RNA oligonucleotides to introduce single-nucleotide mutations in target genes. This approach is highly efficient in mammalian cells (Yoon et al., 1996, Proc. Natl. Acad. Sc. USA., 93, 2071-2076; Kren et al., 1999, Proc. Natl. Acad. Sci. USA., 96, 10349-10354; Bartlett et al., 2000, Nature Biotech., 18, 615-622) with a success rate of more than 40%. Unfortunately, the efficiency is much lower in plants (Zhu et al., 1999, Proc. Natl. Acad. Sci. USA., 96, 8768-8773; Beetham et al., 1999, Proc. Natl. Acad. Sci. USA., 96, 8774-8778; Zhu et al., 2000, Nature Biotech., 18, 555-558; WO9925853) and reaches only a frequency of 10 −5 -10 −7 . A further severe drawback of using the chimeraplasty approach in plant systems is that it is limited to the introduction of single-nucleotide mutations and to the special case where the introduced mutation results in a selectable phenotype. Another approach has been to use heterologous site-specific recombinases of microbial origin. When these recombinases are used, specific recombination sites have to be included on each side not only of the DNA sequence to be targeted, but also of the target site. So far, this has been a severly limiting condition which gives this approach little practical usefulness. Examples of such systems include the Cre-Lox system from bacteriophage P1 (Austin et al., 1981, Cell, 25, 729-736), the Flp-Frt system from Saccharomyces cerevisiae (Broach et al., 1982, Cell, 29, 227-234), the R—RS system from Zygosaccharomyces rouxii (Araki et al., 1985, J. Mol. Biol., 182, 191-203) and the integrase from the Streptomyces phage PhiC31 (Thorpe & Smith, 1998 , Proc. Natl. Acad. Sci., 95, 5505-5510; Groth et al., 2000, Proc. Natl. Acad. Sci., 97, 5995-6000). Wild-type Lox sites (LoxP sites) consist of 13 bp inverted repeats flanking an 8 bp asymetrical core. The asymmetry of the core region confers directionality to the site. Recombination between LoxP sites is a reversible reaction that can lead to deletions, insertions, or translocations depending on the location and orientation of the Lox sites. In plants, the Cre-Lox system has been used to create deletions (Bayley et al, 1992, Plant Mol. Biol., 18, 353-361), inversions (Medberry et al., 1995, Nucl. Acids. Res., 23, 485-490), translocations (Qin et al., 1994, Proc. Natl. Aced. Sci., 91, 1706-1710); Vergunst et al, 2000, Chromosoma, 109, 287-297), insertion of a circular DNA into a plant chromosome (Albert et al., 1995, Plant J., 7, 649-659), interspecies translocation of a chromosome arm (Heather et al., 2000, Plant J., 23, 715-722), and removal of selection genes after transformation (Dale & Ow, 1991 , Proc. Natl. Acad. Sc., 88, 10558-62; Zuo et al., 2001, Nat Biotechnol., 19, 157-161). One problem encountered when the Cre-Lox system (or a similar recombination system) is used for targeted transformation is that insertion of DNA can be followed by excision. In fact, because the insertion of DNA is a bimolecular reaction while excision requires recombination of sites on a single molecule, excision occurs at a much higher efficiency than insertion. A number of approaches have been devised to counter this problem including transient Cre expression, displacement of the Cre coding sequence by insertion leading to its inactivation, and the use of mutant sites (Albert et al., 1995, Plant J., 7, 649-659; Vergunst et al., 1998, Plant Mol. Biol., 38, 393-406; U.S. Pat. No. 6,187,994). Some site-specific recombinases such as the Streptomyces phage PhiC31 integrase should not suffer from the same problem, theoretically, as recombination events are irreversible (the reverse reaction is carried out by different enzymes) (Thorpe & Smith, 1998 , Proc. Natl. Acad. Sci., 95, 5505-5510), but they are limited to animals), but the use of this recombination system in plant cells has not been confirmed yet. There are other flaws that render the site-specific recombination systems practically unattractive. First, one needs to engineer a landing or docking site in the recipient's genome, a procedure that is currently done by random insertion of recombination sites into a plant genome. This eliminates most of benefits of the site-specific integration. Second, the frequency of desired events is still very low, especially in economically important crops, thus limiting its use to tobacco and Arabidopsis . Expression of recombinant enzymes in plant cells leads to a toxicity problems, an issue that cannot be circumvented with commonly used systems such as Cre-lox or Flp-frt. WO 99/25855 and corresponding intermediate U.S. Pat. No. 6,300,545 disclose a method of mobilizing viral replicons from an Agrobacterium -delivered T-DNA by site-specific recombination-mediated excision for obtaining a high copy number of a viral replicon in a plant cell. It is speculated that said high copy number is useful for site-targeted integration of DNA of interest into a plant chromosome using site-specific recombination. However, the disclosure does not contain information on how to test this speculation. The examples given in the disclosure do not relate to site-targeted integration. Moreover, the examples cannot provide cells having undergone site-targeted integration, but only plants showing signs of viral infection such as appearance of yellow spots and stripes at the base of new leaves indicative of the decay of the infected cells. Therefore, the teaching of these references is limited to the infection of cells leading to the destruction of the cell by the viral vector. The teaching of these references neither allows the determination as to whether or not integration into the nuclear genome has taken place, let alone the selection of successful site-targeted integration events. This is underlined by the fact that the references do not contain a disclosure of selection methods for recovering site-targeted transformants. Selection and recovery of transgenic progeny cells containing said DNA of interest site-specifically integrated into the nuclear genome is simply impossible based on the teaching of these references. Moreover, WO 99/25855 and U.S. Pat. No. 6,300,545 are silent on this problem. Further, these documents are silent on homologous recombination. Moreover, the method is limited to replicon delivery by way of Agrobacterium. Therefore, it is the problem of the invention to provide a process for targeted transformation of plants which is sufficiently efficient for practical purposes. It is a further problem of the invention to provide a method of targeted integration of DNA of interest into a plant cell nuclear genome that allows recovery of integration events, i.e. selection of cells having undergone recombination in the plant nuclear DNA. It is a further problem of the invention to provide a method of targeted integration of DNA of interest into a plant cell nuclear genome by homologous recombination. It is therefore a further problem of the invention to provide a method of targeted integration of DNA of interest into a plant cell nuclear genome by delivery methods independent from Agrobacterium -mediated methods. |
<SOH> SUMMARY OF THE INVENTION <EOH>This problem is solved by a process of causing a targeted integration of DNA of interest into a plant cell nuclear genome, comprising: (i) providing plant cells with an amplification vector, or a precursor thereof, capable of autonomous replication in plant cells, said vector comprising: (a) DNA sequences encoding an origin of replication functional in plant cells, (b) DNA sequence(s) necessary for site-specific and/or homologous recombination between the amplification vector and a host nuclear DNA, and (c) optionally, a further DNA of interest; (ii) optionally providing conditions that facilitate vector amplification and/or cell to cell movement and/or site-specific and/or homologous recombination, and (iii) selecting cells having undergone recombination at a predetermined site in the plant nuclear DNA. Further, a process of causing a targeted integration of DNA of interest into a plant cell nuclear genome is provided, comprising the following steps: (i) transfecting or transforming a plant cell with a first DNA comprising a sequence which, when integrated in the plant cell genome, provides a target site for site-specific and/or homologous recombination; (ii) selecting a cell which contains said target site for site-specific and/or homologous recombination in its nuclear genome; (iii) transfecting or transforming said selected cell with a second DNA comprising a region for recombination with said target site and a first sequence of interest; (iv) optionally providing enzymes for recombination; and (v) selecting cells which contain the sequence of interest from the second DNA integrated at the target site, whereby at least one of said first or said second DNA is delivered by an amplification vector, or a precursor thereof, capable of autonomous replication in a plant cell and comprising DNA sequences encoding an origin of replication functional in the plant cell. Further, this invention provides plant cells, seeds and plants obtained or obtainable by performing these processes and a vector (amplification vector) or pro-vector (precursor) for performing these processes. Moreover, the invention provides Agrobacterium cells and packaged viral particles containing said vector or pro-vector. Finally, the invention provides a kit-of-parts comprising (i) plant cells, seeds or plants, notably according to steps (i) and (ii) of the above five-step process and (ii) a vector or pro-vector according to the invention and/or said Agrobacterium cells and/or said packaged viral particles. A further kit-of-parts is provided comprising a vector or a pro-vector for performing steps (i) and (ii) of the above five-step process and a vector for performing steps (iii) and (iv) of that process. It has been found that surprisingly the efficiency of site-targeted transformation of plant cells can be greatly improved by providing DNA sequences for site-specific and/or homologous recombination by an amplification vector. The exact reasons for this improvement are not yet known but it may be due to an increase of the copy number of the sequence(s) to be targeted. Examples are provided which demonstrate a strong increase of site-targeted insertion events by using amplification vectors as opposed to non-amplifying vectors. It is even more surprising that this increased copy number does not at the same time increase the frequency of non-targeted or random insertion of the sequence(s) to be targeted into the nuclear genome. As a result, the ratio of targeted to random insertion frequencies is highly increased by the processes of this invention. Most importantly, targeted transformation reaches a level of efficiency such that it may now become a routine method in plant biotechnology. Replication of the amplification vector, however, renders selection of integration events difficult or impossible since high copy numbers of an amplification vectors lead to disease symptoms, impediment of cell division and ultimately to death of affected cells. Consequently, progeny cells containing DNA of interest integrated into the nuclear genome cannot be obtained. The inventors were therefore faced with the following dilemma: on the one hand, efficient site-targeted integration requires replication of the vector. On the other hand, said replication prevents selection of cells having undergone recombination in the plant nuclear DNA. The inventors of the invention have surprisingly identified ways out of this dilemma. Preferably, the processes of the invention are designed such that the replication of said amplification vector in cells transformed or transfected with said amplification vector is transient. Transient replication means temporal replication, i.e. a replication that lasts for a limited period of time necessary to achieve or to detect homologous and/or site-specific recombination within said cells and integration of said DNA of interest into the nuclear genome. Transient replication of the amplification vector does preferably not prevent the ability of said cells to divide such that progeny cells are formed which can be selected. Preferably, the amplification vector disappears in progeny cells. Below, examples are provided which demonstrate successful selection of progeny cells according to the invention. Transient replication of the amplification vector may be achieved in several ways. One possibility is to provide the nucleic acid polymerase (replicase) involved in replicating the amplification vector transiently such that replication stops when said polymerase disappears. This may be done by providing the replicase gene to the plant cell on a non-replicating vector (cf. example 6). Preferably, selection pressure used for maintaining said non-replicating vector may be relieved to this end. Further, replication may stop or diminish as a result of the recombination event (cf. example 13), e.g. by rendering the replicase gene non-expressible. The invention further provides a process of causing targeted integration of DNA of interest into a plant cell nuclear genome comprising: (i) providing plant cells with an amplification vector, or a precursor thereof, capable of autonomous replication in plant cells, said vector comprising: (a) DNA sequences encoding an origin of replication functional in plant cells, (b) DNA sequence(s) necessary for homologous recombination between the amplification vector and a host nuclear DNA, and (c) optionally, a further DNA of interest; (ii) optionally providing conditions that facilitate vector amplification and/or cell to cell movement and/or site-specific and/or homologous recombination, and (iii) selecting cells having undergone recombination at a predetermined site in the plant nuclear DNA. In order to amplify in a plant cell, the amplification vector used in this invention has to have an origin of replication functional in a plant cell. The origin of replication may be derived from a plant nuclear genome, e.g. from a ribosomal DNA intergenic spacer region. Alternatively, the origin of replication may be of non-plant origin or of synthetic nature. Preferably, the origin of replication is derived from a plant virus, most preferably from a plant DNA virus. The origin of replication is functional in a plant cell if it is recognised by a replication enzyme (DNA or RNA polymerase) in said cell. The replication enzyme is preferably of the same origin as the origin of replication. If the replication enzyme originates from the plant species to be transformed, no foreign replication enzyme has to be provided to said plant cells. In order to facilitate vector amplification, a replication enzyme may be provided, notably if said origin of replication originates from a source different from said plant cells. This enzyme may be encoded on the amplification vector, on an additional vector or it may be incorporated into the plant nuclear genome. The amplification vector may be a plant virus-derived vector. It may be derived from an RNA virus. In this case it is preferably a DNA copy or a replication intermediate of an RNA virus-derived vector. Preferably however, the vector is derived from a DNA virus. A vector may be considered to be derived from an RNA or DNA virus, if it contains at least one functional element of such a virus. Preferably, such a functional element is an origin of replication which is recognized by a replication enzyme (polymerase) of that virus. Geminiviridae are particularly well-suited for the purpose of performing this invention. Preferably, the amplification vector has additionally other sequences encoding viral functions for host infectivity, cell-to-cell and/or systemic movement for spreading throughout the plant and for further increasing the frequency of targeted transformation. The amplification vector may have further sequences for functions such as integration into the host chromosome, viral particle assembly, control of gene silencing by the host, and/or control of host physiology. Alternatively, such additional viral functions may be provided on an additional vector. The additional vector may be a replicating vector as well. Preferably, the additional vector is a non-replicating vector such that the additional viral functions are only transiently expressed. This may reduce disease symptoms of the plant. Further, the amplification vector may be of retrotransposon origin. The amplification vector may further contain a DNA sequence of interest e.g. a gene to be expressed e.g. for conferring a useful trait, for performing mutagenesis etc. Said site-specific or homologous recombination takes place between the amplification vector and a host nuclear DNA. Said host nuclear DNA may belong to a nuclear chromosome of the host or it may belong to an episomal nuclear DNA. Preferably, said recombination takes place between the amplification vector and a DNA on a nuclear chromosome of the host. In order to facilitate site-specific or homologous recombination, suitable recombination enzymes such as site-specific recombinases, restriction enzymes or integrases may be provided from an additional vector or from a gene previously incorporated into said plant. Such an additional vector may be co-transformed with the amplification vector or it may be transformed separately. Expression of the recombination enzyme may be constitutive or inducible. Preferably, the recombination enzyme is only transiently expressed e.g. from a non-replicating vector. If the recombination enzyme is present at the target locus of the nuclear genome, its function may be destroyed as a result of the recombination event. In case of homologous recombination, a recombination enzyme may not have to be provided externally and the process may rely on an endogenous recombinase. However, the efficiency may be further increased by additionally providing a recombination enzyme for promoting homologous recombination. Such an enzyme may be an enzyme native to said plant, a heterologous enzyme or an engineered enzyme. If homologous recombination is used to target a DNA of interest into the nuclear genome of the plant, any site in the nuclear genome may be targeted as long as suitable selection means exist to select for the desired recombination event. Selection may be achieved by introducing a mutation conferring an antibiotic or inhibitor resistance or by providing a resistance marker gene. As more genome sequences become known, targeting of a desired site by homologous recombination becomes more broadly applicable. A preferred embodiment of targeted homologous recombination is site-directed mutagenesis of a gene of the plant nuclear genome. For this purpose, the amplification vector may contain the desired mutation flanked by homologous sequences of the target site. If site-specific recombination is used to target a DNA of interest into the nuclear genome of the plant, target site(s) recognizable by site-specific recominases are preferably pre-introduced into the plant according to the above five-step process. The above five-step process comprises two stages: in the first stage (step (i) and (ii)), a transgenic plant having pre-engineered target sites for site-specific recombination is produced. Preferably, the target sites are stably incorporated into the nuclear genome. Transfecting or transforming said first DNA in step (i) of the five-step process may be non-targeted. Many transgenic plants with target sites introduced in many different loci of the genome may be produced. Then a transgenic plant line with the target site at a desired location may be chosen for performing the steps of the second stage (steps (iii) to (v)). Integration of a DNA of interest in the second stage can then be targeted. According to this process, stable transgenic plant lines may be produced in the first phase. Each such transgenic plant line may then be used for various purposes according to the second stage, making this process highly versatile. At least one of said first or said second DNA is delivered by an amplification vector. Preferably, at least said second DNA is delivered by an amplification vector. Both said first and said second DNA may comprise a sequence of interest. Such a sequence of interest may be a selectable marker and/or a gene to be expressed e.g. for conferring the plant with a useful trait. Preferably, the recombination event may establish a functional sequence. An example for the establishment of a functional sequence is the placement of a DNA to be expressed under the control of a promoter, whereby the promoter may be provided by said first or said second DNA and the DNA to be expressed may be provided by said second or said first DNA, respectively. Further, other functions necessary for functional expression of a gene such as combination of two fragments of a coding sequence may be combined by said recombination event. The recombination event may also be used to destroy the function of a gene or to eliminate a sequence at the target site. Said plant cells may be provided with said amplification vector (e.g. a replicon) or with (a) precursor(s) thereof (a pre-replicon or pro-vector). If said plant cells are provided with said precursor, the precursor has to be adopted to be processed to said amplification vector in the plant cell. The amplification vector may e.g. be excised from a precursor by recombination. However, if an amplification vector is to be excised from a precursor, this is preferably achieved by providing the precursor with two origins of replication for allowing replicative release of the amplification vector. Excision of the amplification vector from a precursor is preferably done in combination with Agrobacterium transformation for excising the amplification vector out of the Ti-plasmid delivered by Agrobacterium . Further, the amplification vector may be assembled in plant cells from two or more precursors by recombination. Said plant cells may be provided with said amplification vector or its precursor by several methods. Preferred methods are Agrobacterium -mediated delivery, direct viral transfection, and non-biological delivery (e.g. particle bombardment). In direct viral transfection, infectious viral material is directly applied to plant tissue. Direct viral transfection should be distinguished from Agroinfection where viral DNA is delivered indirectly using Agrobacterium . In Agrobacterium -mediated delivery, Ti-plasmids are deliverd as precursors of amplification vectors, which are processed in the plant cell to generate said amplification vectors. Direct viral transfection and non-biological delivery methods are preferred. |
Secreted proteins |
The invention provides human secreted proteins (SECP) and polynucleotides which identify and encode SECP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of SECP. |
1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2-24, c) a polypeptide comprising a naturally occurring amino acid sequence at least 96% identical to the amino acid sequence consisting of SEQ ID NO:1, d) a polypeptide comprising a naturally occurring amino acid sequence at least 95% identical to the amino acid sequence consisting of SEQ ID NO:25, e) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and f) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. 3. An isolated polynucleotide encoding a polypeptide of claim 1. 4. An isolated polynucleotide encoding a polypeptide of claim 2. 5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3. 7. A cell transformed with a recombinant polynucleotide of claim 6. 8. (canceled) 9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed. 10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. 11. An isolated antibody which specifically binds to a polypeptide of claim 1. 12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:27-50, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 96% identical to the polynucleotide sequence of SEQ ID NO:26, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), e) a polynucleotide complementary to a polynucleotide of c), and f) an RNA equivalent of a)-e). 13. (canceled) 14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof. 15. (canceled) 16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. 19. (canceled) 20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample. 21. (canceled). 22. (canceled) 23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample. 24. (canceled) 25. (canceled) 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. 27. (canceled) 28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. 29. A method of assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. 30-105. (canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>Protein transport and secretion are essential for cellular function. Protein transport is mediated by a signal peptide located at the amino terminus of the protein to be transported or secreted. The signal peptide is comprised of about ten to twenty hydrophobic amino acids which target the nascent protein from the ribosome to a particular membrane bound compartment such as the endoplasmic reticulum (ER). Proteins targeted to the ER may either proceed through the secretory pathway or remain in any of the secretory organelles such as the ER, Golgi apparatus, or lysosomes. Proteins that transit through the secretory pathway are either secreted into the extracellular space or retained in the plasma membrane. Proteins that are retained in the plasma membrane contain one or more transmembrane domains, each comprised of about 20 hydrophobic amino acid residues. Secreted proteins are generally synthesized as inactive precursors that are activated by post-translational processing events during transit through the secretory pathway. Such events include glycosylation, proteolysis, and removal of the signal peptide by a signal peptidase. Other events that may occur during protein transport include chaperone-dependent unfolding and folding of the nascent protein and interaction of the protein with a receptor or pore complex Examples of secreted proteins with amino terminal signal peptides are discussed below and include proteins with important roles in cell-to-cell signaling. Such proteins include transmembrane receptors and cell surface markers, extracellular matrix molecules, cytokines, hormones, growth and differentiation factors, enzymes, neuropeptides, vasomediators, cell surface markers, and antigen recognition molecules. (Reviewed in Alberts, B. et al. (1994) Molecular Biology of The Cell , Garland Publishing, New York, N.Y., pp. 557-560, 582-592.) Cell surface markers include cell surface antigens identified on leukocytic cells of the immune system. These antigens have been identified using systematic, monoclonal antibody (mAb)-based “shot gun,” techniques. These techniques have resulted in the production of hundreds of mAbs directed against unknown cell surface leukocytic antigens. These antigens have been grouped into “clusters of differentiation” based on common immunocytochemical localization patterns in various differentiated and undifferentiated leukocytic cell types. Antigens in a given cluster are presumed to identify a single cell surface protein and are assigned a “cluster of differentiation” or “CD” designation. Some of the genes encoding proteins identified by CD antigens have been cloned and verified by standard molecular biology techniques. CD antigens have been characterized as both transmembrane proteins and cell surface proteins anchored to the plasma membrane via covalent attachment to fatty acid-containing glycolipids such as glycosylphosphatidylinositol (GPI). (Reviewed in Barclay, A. N. et al. (1995) The Leukocyte Antigen Facts Book , Academic Press, San Diego, Calif., pp. 17-20.) Matrix proteins (MPs) are transmembrane and extracellular proteins which function in formation, growth, remodeling, and maintenance of tissues and as important mediators and regulators of the inflammatory response. The expression and balance of MPs may be perturbed by biochemical changes that result from congenital, epigenetic, or infectious diseases. In addition, MPs affect leukocyte migration, proliferation, differentiation, and activation in the immune response. MPs are frequently characterized by the presence of one or more domains which may include collagen-like domains, EGF-like domains, immunoglobulin-like domains, and fibronectin-like domains. In addition, MPs may be heavily glycosylated and may contain an Arginine-Glycine-Aspartate (RGD) tripeptide motif which may play a role in adhesive interactions. MPs include extracellular proteins such as fibronectin, collagen, galectin, vitronectin and its proteolytic derivative somatomedin B; and cell adhesion receptors such as cell adhesion molecules (CAMs), cadherins, and integrins. (Reviewed in Ayad, S. et al. (1994) The Extracellular Matrix Facts Book , Academic Press, San Diego, Calif., pp. 2-16; Ruoslahti, E. (1997) Kidney Int. 51:1413-1417; Sjaastad, M. D. and Nelson, W. J. (1997) BioEssays 19:47-55.) Mucins are highly glycosylated glycoproteins that are the major structural component of the mucus gel. The physiological functions of mucins are cytoprotection, mechanical protection, maintenance of viscosity in secretions, and cellular recognition. MUC6 is a human gastric mucin that is also found in gall bladder, pancreas, seminal vesicles, and female reproductive tract (Toribara, N. W. et al. (1997) J. Biol. Chem. 272:16398-16403). The MUC6 gene has been mapped to human chromosome 11 (Toribara, N. W. et al. (1993) J. Biol. Chem. 268:5879-5885). Hemomucin is a novel Drosophila surface mucin that may be involved in the induction of antibacterial effector molecules (Theopold, U. et al. (1996) J. Biol. Chem. 217:12708-12715). Tuftelins are one of four different enamel matrix proteins that have been identified so far. The other three known enamel matrix proteins are the amelogenins, enamelin and ameloblastin. Assembly of the enamel extracellular matrix from these component proteins is believed to be critical in producing a matrix competent to undergo mineral replacement. (Paine, C. T. et al. (1998) Connect Tissue Res. 38:257-267). Tuftelin mRNA has been found to be expressed inhuman ameloblastoma tumor, a non-mineralized odontogenic tumor (Deutsch, D. et al. (1998) Connect. Tissue Res. 39:177-184). Olfactomedin-related proteins are extracellular matrix, secreted glycoproteins with conserved C-terminal motifs. They are expressed in a wide variety of tissues and in broad range of species, from Caenorhabditis elegans to Homo sapiens . Olfactomedin-related proteins comprise a gene family with at least 5 family members in humans. One of the five, TIGR/myocilin protein, is expressed in the eye and is associated with the pathogenesis of glaucoma (Kulkarni, N. H. et al. (2000) Genet. Res. 76:41-50). Research by Yokoyama et al. (1996) found a 135-amino acid protein, termed AMY, having 96% sequence identity with rat neuronal olfactomedin-releated ER localized protein in a neuroblastoma cell line cDNA library, suggesting an essential role for AMY in nerve tissue (Yokoyama, M. et al (1996) DNA Res. 3:311-320). Neuron-specific olfactomedin-related glycoproteins isolated from rat brain cDNA libraries show strong sequence similarity with olfactomedin. This similarity is suggestive of a matrix-related function of these glycoproteins in neurons and neurosecretory cells (Danielson, P. E. et al. (1994) J. Neurosci. Res. 38:468-478). Mac-2 binding protein is a 90-kD serum protein (90K), a secreted glycoprotein isolated from both the human breast carcinoma cell line SK-BR-3, and human breast milk It specifically binds to a human macrophage-associated lectin, Mac-2. Structurally, the mature protein is 567 amino acids in length and is proceeded by an 18-amino acid leader. There are 16 cysteines and seven potential N-linked glycosylation sites. The first 106 amino acids represent a domain very similar to an ancient protein superfamily defined by a macrophage scavenger receptor cysteine-rich domain (Koths, K. et al. (1993) J. Biol. Chem. 268:14245-14249). 90K is elevated in the serum of subpopulations of AIDS patients and is expressed at varying levels in primary tumor samples and tumor cell lines. Ullrich et al. (1994) have demonstrated that 90K stimulates host defense systems and can induce interleukin-2 secretion. This immune stimulation is proposed to be a result of oncogenic transformation, viral infection or pathogenic invasion (Ullrich, A. et al. (1994) J. Biol. Chem. 269:18401-18407). Semaphorins are a large group of axonal guidance molecules consisting of at least 30 different members and are found in vertebrates, invertebrates, and even certain viruses. All semaphorins contain the sema domain which is approximately 500 amino acids in length. Neuropilin, a semaphorin receptor, has been shown to promote neurite outgrowth in vitro. The extracellular region of neuropilins consists of three different domains: CUB, discoidin, and MAM domains. The CUB and the MAM motifs of neuropilin have been suggested to have roles in protein-protein interactions and are thought to be involved in the binding of semaphorins through the sema and the C-terminal domains (reviewed in Raper, J. A. (2000) Curr. Opin. Neurobiol. 10:88-94). Plexins are neuronal cell surface molecules that mediate cell adhesion via a homophilic binding mechanism in the presence of calcium ions. Plexins have been shown to be expressed in the receptors and neurons of particular sensory systems (Ohta, K et al. (1995) Cell 14:1189-1199). There is evidence that suggests that some plexins function to control motor and CNS axon guidance in the developing nervous system. Plexins, which themselves contain complete semaphorin domains, may be both the ancestors of classical semaphorins and binding partners for semaphorins (Winberg, M. L. et al (1998) Cell 95:903-916). Human pregnancy-specific beta 1-glycoprotein (PSG) is a family of closely related glycoproteins of molecular weights of 72 KDa, 64 KDa, 62 KDa, and 54 KDa. Together with the carcinoembryonic antigen, they comprise a subfamily within the immunoglobulin superfamily (Plouzek, C. A. and Chou, J. Y. (1991) Endocrinology 129:950-958) Different subpopulations of PSG have been found to be produced by the trophoblasts of the human placenta, and the amnionic and chorionic membranes (Plouzek, C. A. et al. (1993) Placenta 14:277-285). Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration; therefore identification of the signaling pathway coupled with it has critical importance. Autocrine motility factor receptor (AMF) expression has been found to be associated with tumor progression in thymoma (Ohta Y. et al. (2000) Int. J. Oncol. 17:259-264). AMFR is a cell surface glycoprotein of molecular weight 78 KDa. Hormones are secreted molecules that travel through the circulation and bind to specific receptors on the surface of, or within, target cells. Although they have diverse biochemical compositions and mechanisms of action, hormones can be grouped into two categories. One category includes small lipophilic hormones that diffuse through the plasma membrane of target cells, bind to cytosolic or nuclear receptors, and form a complex that alters gene expression. Examples of these molecules include retinoic acid, thyroxine, and the cholesterol-derived steroid hormones such as progesterone, estrogen, testosterone, cortisol, and aldosterone. The second category includes hydrophilic hormones that function by binding to cell surface receptors that transduce signals across the plasma membrane. Examples of such hormones include amino acid derivatives such as catecholamines (epinephrine, norepinephrine) and histamine, and peptide hormones such as glucagon, insulin, gastrin, secretin, cholecystokinin adrenocorticotropic hormone, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and vasopressin. (See, for example, Lodish et al. (1995) Molecular Cell Biology , Scientific American Books Inc., New York, N.Y., pp. 856-864.) Pro-opiomelanocortin (POMC) is the precursor polypeptide of corticotropin (ACTH), a hormone synthesized by the anterior pituitary gland, which functions in the stimulation of the adrenal cortex. POMC is also the precursor polypeptide of the hormone beta-lipotropin (beta-LPH). Each hormone includes smaller peptides with distinct biological activities: alpha-melanotropin (alpha-MSH) and corticotropin-like intermediate lobe peptide (CLIP) are formed from ACTH; gamma-lipotropin (gamma-LPH) and beta-endorphin are peptide components of beta-LPH; while beta-MSH is contained within gamma-LPH. Adrenal insufficiency due to ACTH deficiency, resulting from a genetic mutation in exons 2 and 3 of POMC results in an endocrine disorder characterized by early-onset obesity, adrenal insufficiency, and red hair pigmentation (Chretien, M. et al. (1979) Can. J. Biochem. 57:1111-1121; Krude, H. et al. (1998) Nat. Genet. 19:155-157; Online Mendelian Inheritance in Man (OMIM) 176830). Growth and differentiation factors are secreted proteins which function in intercellular communication. Some factors require oligomerization or association with membrane proteins for activity. Complex interactions among these factors and their receptors trigger intracellular signal transduction pathways that stimulate or inhibit cell division, cell differentiation, cell signaling, and cell motility. Most growth and differentiation factors act on cells in their local environment (paracrine signaling). There are three broad classes of growth and differentiation factors. The first class includes the large polypeptide growth factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor, and platelet-derived growth factor. The second class includes the hematopoietic growth factors such as the colony stimulating factors (CSFs). Hematopoietic growth factors stimulate the proliferation and differentiation of blood cells such as B-lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils, basophils, neutrophils, macrophages, and their stem cell precursors. The third class includes small peptide factors such as bombesin, vasopressin, oxytocin, endothelin, transferrin, angiotensin II, vasoactive intestinal peptide, and bradykinin, which function as hormones to regulate cellular functions other than proliferation. Growth and differentiation factors play critical roles in neoplastic transformation of cells in vitro and in tumor progression in vivo. Inappropriate expression of growth factors by tumor cells may contribute to vascularization and metastasis of tumors. During hematopoiesis, growth factor misregulation can result in anemias, leukemias, and lymphomas. Certain growth factors such as interferon are cytotoxic to tumor cells both in vivo and in vitro. Moreover, some growth factors and growth factor receptors are related both structurally and functionally to oncoproteins. In addition, growth factors affect transcriptional regulation of both proto-oncogenes and oncosuppressor genes. (Reviewed in Pimentel, E. (1994) Handbook of Growth Factors , CRC Press, Ann Arbor, Mich., pp. 1-9.) The Slit protein, first identified in Drosophila, is critical in central nervous system midline formation and potentially in nervous tissue histogenesis and axonal pathfinding. Itoh et al. ((1998) Brain Res. Mol Brain Res. 62:175-186) have identified mammalian homologues of the slit gene human Slit-1, Slit-2, Slit-3 and rat Slit-1). The encoded proteins are putative secreted proteins containing EGF-like motifs and leucine-rich repeats, both of which are conserved protein-protein interaction domains. Slit-1, -2, and -3 mRNAs are expressed in the brain, spinal cord, and thyroid, respectively (Itoh, A. et al., supra). The Slit family of proteins are indicated to be functional ligands of glypican-1 in nervous tissue and it is suggested that their interactions may be critical in certain stages during central nervous system histogenesis (Liang, Y. et al. (1999) J. Biol. Chem. 274:17885-17892). Neuropeptides and vasomediators (NP/VM) comprise a large family of endogenous signaling molecules. Included in this family are neuropeptides and neuropeptide hormones such as bombesin, neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids, galanin, somatostatin, tachykinins, urotensin II and related peptides involved in smooth muscle stimulation, vasopressin, vasoactive intestinal peptide, and circulatory system-borne signaling molecules such as angiotensin, complement, calcitonin, endothelins, formyl-methionyl peptides, glucagon, cholecystokinin and gastrin. NP/VMs can transduce signals directly, modulate the activity or release of other neurotransmitters and hormones, and act as catalytic enzymes in cascades. The effects of NP/VMs range from extremely brief to long-lasting. (Reviewed in Martin, C. R. et al. (1985) Endocrine Physiology , Oxford University Press, New York, N.Y., pp. 57-62.) NP/VMs are involved in numerous neurological and cardiovascular disorders. For example, neuropeptide Y is involved in hypertension, congestive heart failure, affective disorders, and appetite regulation Somatostatin inhibits secretion of growth hormone and prolactin in the anterior pituitary, as well as inhibiting secretion in intestine, pancreatic acinar cells, and pancreatic beta-cells. A reduction in somatostatin levels has been reported in Alzheimer's disease and Parkinson's disease. Vasopressin acts in the kidney to increase water and sodium absorption, and in higher concentrations stimulates contraction of vascular smooth muscle, platelet activation, and glycogen breakdown in the liver. Vasopressin and its analogues are used clinically to treat diabetes insipidus. Endothelin and angiotensin are involved in hypertension, and drugs, such as captopril, which reduce plasma levels of angiotensin, are used to reduce blood pressure (Watson, S. and S. Arkinstall (1994) The G - protein Linked Receptor Facts Book , Academic Press, San Diego Calif., pp. 194; 252; 284; 55; 111). Neuropeptides have also been shown to have roles in nociception (pain). Vasoactive intestinal peptide appears to play an important role in chronic neuropathic pain. Nociceptin, an endogenous ligand for for the opioid receptor-like 1 receptor, is thought to have a predominantly anti-nociceptive effect, and has been shown to have analgesic properties in different animal models of tonic or chronic pain (Dickinson, T. and Fleetwood-Walker, S. M. (1998) Trends Pharmacol. Sci. 19:346-348). Other proteins that contain signal peptides include secreted proteins with enzymatic activity. Such activity includes, for example, oxidoreductase/dehydrogenase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, or ligase activity. For example, matrix metalloproteinases are secreted hydrolytic enzymes that degrade the extracellular matrix and thus play an important role in tumor metastasis, tissue morphogenesis, and arthritis (Reponen, P. et al (1995) Dev. Dyn. 202:388-396; Firestein, G. S. (1992) Curr. Opin. Rheumatol. 4:348-354; Ray, J. M. and Stetler-Stevenson, W. G. (1994) Eur. Respir. J. 7:2062-2072; and Mignatti, P. and Rifkin, D. B. (1993) Physiol. Rev. 73:161-195). Additional examples are the acetyl-CoA synthetases which activate acetate for use in lipid synthesis or energy generation (Luong, A. et al. (2000) J. Biol. Chem. 275:26458-26466). The result of acetyl-CoA synthetase activity is the formation of acetyl-CoA from acetate and CoA. Acetyl-CoA sythetases share a region of sequence similarity identified as the AMP-binding domain signature. Acetyl-CoA synthetase has been shown to be associated with hypertension (Toh, H. (1991) Protein Seq. Data Anal. 4:111-117; and Iwai, N. et al. (1994) Hypertension 23:375-380). A number of isomerases catalyze steps in protein folding, phototransduction, and various anabolic and catabolic pathways. One class of isomerases is known as peptidyl-prolyl cis-trans isomerases (PPIases). PPIases catalyze the cis to trans isomerization of certain proline imidic bonds in proteins. Two families of PPIases are the FK506 binding proteins (FKBPs), and cyclophilins (CyPs). FKBPs bind the potent immunosuppressants FK506 and rapamycin, thereby inhibiting signaling pathways in T-cells. Specifically, the PPIase activity of FKBPs is inhibited by binding of FK506 or rapamycin. There are five members of the FKBP family which are named according to their calculated molecular masses (FKBP12, FKBP13, FKBP25, FKBP52, and FKBP65), and localized to different regions of the cell where they associate with different protein complexes (Coss, M. et al. (1995) J. Biol. Chem. 270:29336-29341; Schreiber, S. L. (1991) Science 251:283-287). The peptidyl-prolyl isomerase activity of CyP may be part of the signaling pathway that leads to T-cell activation. CyP isomerase activity is associated with protein folding and protein trafficking, and may also be involved in assembly/disassembly of protein complexes and regulation of protein activity. For example, in Drosophila , the CyP NinaA is required for correct localization of rhodopsins, while a mammalian CyP (Cyp40) is part of the Hsp90/Hsc70 complex that binds steroid receptors. The mammalian CypA has been shown to bind the gag protein from human immunodeficiency virus 1 (HIV-1), an interaction that can be inhibited by cyclosporin. Since cyclosporin has potent anti-HIV-1 activity, CypA may play an essential function in HIV-1 replication. Finally, Cyp40 has been shown to bind and inactivate the transcription factor c-Myb, an effect that is reversed by cyclosporin. This effect implicates CyPs in the regulation of transcription, transformation, and differentiation (Bergsma, D. J. et al (1991) J. Biol. Chem. 266:23204-23214; Hunter, T. (1998) Cell 92:141-143; and Leverson, J. D. and Ness, S. A. (1998) Mol. Cell. 1:203-211). Most normal eukaryotic cells, after a certain number of divisions, enter a state of senescence in which cells remain viable and metabolically active but no longer replicate. A number of phenotypic changes such as increased cell size and pH-dependent beta-galactosidase activity, and molecular changes such as the upregulation of particular genes, occur in senescent cells (Shelton (1999) Current Biology 9:939-945). When senescent cells are exposed to mitogens, a number of genes are upregulated, but the cells do not proliferate. Evidence indicates that senescent cells accumulate with age in vivo, contributing to the aging of an organism In addition, senescence suppresses tumorigenesis, and many genes necessary for senescence also function as tumor suppressor genes, such as p53 and the retinoblastoma susceptibility gene. Most tumors contain cells that have surpassed their replicative limit, i.e. they are immortalized. Many oncogenes immortalize cells as a first step toward tumor formation. A variety of challenges, such as oxidative stress, radiation, activated oncoproteins, and cell cycle inhibitors, induce a senescent phenotype, indicating that senescence is influenced by a number of proliferative and anti-proliferative signals (Shelton supra). Senescence is correlated with the progressive shortening of telomeres that occurs with each cell division. Expression of the catalytic component of telomerase in cells prevents telomere shortening and immortalizes cells such as fibroblasts and epithelial cells, but not other types of cells, such as CD8+ T cells (Migliaccio et al. (2000) J Immunol 165:4978-4984). Thus, senescence is controlled by telomere shortening as well as other mechanisms depending on the type of cell. A number of genes that are differentially expressed between senescent and presenescent cells have been identified as part of ongoing studies to understand the role of senescence in aging and tumorigenesis. Most senescent cells are growth arrested in the G1 stage of the cell cycle. While expression of many cell cycle genes is similar in senescent and presenescent cells (Cristofalo (1992) Ann N Y Acad Sci 663:187-194), expression of others genes such as cyclin-dependent kinases p21 and p16, which inhibit proliferation, and cyclins D1 and E is elevated in senescent cells. Other genes that are not directly involved in the cell cycle are also upregulated such as extracellular matrix proteins fibronectin, procollagen, and osteonectin; and proteases such as collagenase, stromelysin, and cathepsin B (Chen (2000) Ann NY Acad Sci 908:111-125). Genes underexpressed in senescent cells include those that encode heat shock proteins, c-fos, and cdc-2 (Chen supra). Gamma-carboxyglutamic acid (Gla) proteins rich in proline (PRGPs) are members of a family of vitamin K-dependent single-pass integral membrane proteins. These proteins are characterized by an extracellular amino terminal domain of approximately 45 amino acids rich in Gla. The intracellular carboxyl terminal region contains one or two copies of the sequence PPXY, a motif present in a variety of proteins involved in such diverse cellular functions as signal transduction, cell cycle progression, and protein turnover (Kulman, J. D. et al. (2001) Proc. Natl. Acad. Sci. USA 98:1370-1375). The process of post-translational modification of glutamic residues to form Gla is Vitamin K-dependent carboxylation. Proteins which contain Gla include plasma proteins involved in blood coagulation. These proteins are prothrombin, proteins C, S, and Z, and coagulation factors VII, IX, and X. Osteocalcin (bone-Gla protein, BGP) and matrix Gla-protein (MGP) also contain Gla (Friedman, P. A. and C. T. Przysiecki (1987) Int. J. Biochem. 19:1-7; C. Vermeer (1990) Biochem. J. 266:625-636). Immunoglobulins Antigen recognition molecules are key players in the sophisticated and complex immune systems which all vertebrates have developed to provide protection from viral, bacterial, fungal, and parasitic infections. A key feature of the immune system is its ability to distinguish foreign molecules, or antigens, from “self” molecules. This ability is mediated primarily by secreted and transmembrane proteins expressed by leukocytes (white blood cells) such as lymphocytes, granulocytes, and monocytes. Most of these proteins belong to the immunoglobulin (Ig) superfamily, members of which contain one or more repeats of a conserved structural domain. This Ig domain is comprised of antiparallel β sheets joined by a disulfide bond in an arrangement called the Ig fold. The criteria for a protein to be a member of the Ig superfamily is to have one or more Ig domains, which are regions of 70-110 amino acid residues in length homologous to either Ig variable-like (V) or Ig constant-like (C) domains. Members of the Ig superfamily include antibodies (Ab), T cell receptors (TCRs), class I and II major histocompatibility (MHC) proteins and immune cell-specific surface markers such as the “cluster of differentiation” or CD antigens, CD2, CD3, CD4, CD8, poly-Ig receptors, Fc receptors, neural cell-adhesion molecule (NCAM) and platelet-derived growth factor receptor (PDGFR). Ig domains (V and C) are regions of conserved amino acid residues that give a polypeptide a globular tertiary structure called an immunoglobulin (or antibody) fold, which consists of two approximately parallel layers of β-sheets. Conserved cysteine residues form an intrachain disulfide-bonded loop, 55-75 amino acid residues in length, which connects the two layers of β-sheets. Each β-sheet has three or four anti-parallel β-strands of 5-10 amino acid residues. Hydrophobic and hydrophilic interactions of amino acid residues within the β-strands stabilize the Ig fold (hydrophobic on inward facing amino acid residues and hydrophilic on the amino acid residues in the outward facing portion of the strands). A V domain consists of a longer polypeptide than a C domain, with an additional pair of β-strands in the Ig fold. A consistent feature of Ig superfamily genes is that each sequence of an Ig domain is encoded by a single exon. It is possible that the superfamily evolved from a gene coding for a single Ig domain involved in mediating cell-cell interactions. New members of the superfamily then arose by exon and gene duplications. Modern Ig superfamily proteins contain different numbers of V and/or C domains. Another evolutionary feature of this superfamily is the ability to undergo DNA rearrangements, a unique feature retained by the antigen receptor members of the family. Many members of the Ig superfamily are integral plasma membrane proteins with extracellular Ig domains. The hydrophobic amino acid residues of their transmembrane domains and their cytoplasmic tails are very diverse, with little or no homology among Ig family members or to known signal-transducing structures. There are exceptions to this general superfamily description. For example, the cytoplasmic tail of PDGFR has tyrosine kinase activity. In addition Thy-1 is a glycoprotein found on thymocytes and T cells. This protein has no cytoplasmic tail, but is instead attached to the plasma membrane by a covalent glycophosphatidylinositol linkage. Another common feature of many Ig superfamily proteins is the interactions between Ig domains which are essential for the function of these molecules. Interactions between Ig domains of a multimeric protein can be either homophilic or heterophilic (i.e., between the same or different Ig domains). Antibodies are multimeric proteins which have both homophilic and heterophilic interactions between Ig domains. Pairing of constant regions of heavy chains forms the Fc region of an antibody and pairing of variable regions of light and heavy chains form the antigen binding site of an antibody. Heterophilic interactions also occur between Ig domains of different molecules. These interactions provide adhesion between cells for significant cell-cell interactions in the immune system and in the developing and mature nervous system. (Reviewed in Abbas, A. K. et al. (1991) Cellular and Molecular Immunology , W.B. Saunders Company, Philadelphia, Pa., pp. 142-145.) Antibodies MHC proteins are cell surface markers that bind to and present foreign antigens to T cells. MHC molecules are classified as either class I or class II. Class I MHC molecules (MHC I) are expressed on the surface of almost all cells and are involved in the presentation of antigen to cytotoxic T cells. For example, a cell infected with virus will degrade intracellular viral proteins and express the protein fragments bound to MHC I molecules on the cell surface. The MHC I/antigen complex is recognized by cytotoxic T-cells which destroy the infected cell and the virus within. Class II MHC molecules are expressed primarily on specialized antigen-presenting cells of the immune system, such as B-cells and macrophages. These cells ingest foreign proteins from the extracellular fluid and express MHC II/antigen complex on the cell surface. This complex activates helper T-cells, which then secrete cytokines and other factors that stimulate the immune response. MHC molecules also play an important role in organ rejection following transplantation. Rejection occurs when the recipient's T-cells respond to foreign MHC molecules on the transplanted organ in the same way as to self MHC molecules bound to foreign antigen. (Reviewed in Alberts, B. et al (1994) Molecular Biology of the Cell , Garland Publishing, New York, N.Y., pp. 1229-1246.) Antibodies are multimeric members of the Ig superfamily which are either expressed on the surface of B-cells or secreted by B-cells into the circulation. Antibodies bind and neutralize foreign antigens in the blood and other extracellular fluids. The prototypical antibody is a tetramer consisting of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds. This arrangement confers the characteristic Y-shape to antibody molecules. Antibodies are classified based on their H-chain composition. The five antibody classes, IgA, IgD, IgE, IgG and IgM, are defined by the α, δ, ε, γ, and μ H-chain types. There are two types of L-chains, κ and λ, either of which may associate as a pair with any H-chain pair. IgG, the most common class of antibody found in the circulation, is tetrameric, while the other classes of antibodies are generally variants or multimers of this basic structure. H-chains and L-chains each contain an N-terminal variable region and a C-terminal constant region. The constant region consists of about 110 amino acids in L-chains and about 330 or 440 amino acids in H-chains. The amino acid sequence of the constant region is nearly identical among H- or L-chains of a particular class. The variable region consists of about 110 amino acids in both H- and L-chains. However, the amino acid sequence of the variable region differs among H- or L-chains of a particular class. Within each H- or L-chain variable region are three hypervariable regions of extensive sequence diversity, each consisting of about 5 to 10 amino acids. In the antibody molecule, the H- and L-chain hypervariable regions come together to form the antigen recognition site. (Reviewed in Alberts, B. et al. supra, pp. 1206-1213 and 1216-1217.) Both H-chains and L-chains contain the repeated Ig domains of members of the Ig superfamily. For example, a typical H-chain contains four Ig domains, three of which occur within the constant region and one of which occurs within the variable region and contributes to the formation of the antigen recognition site. Likewise, a typical L-chain contains two Ig domains, one of which occurs within the constant region and one of which occurs within the variable region. The immune system is capable of recognizing and responding to any foreign molecule that enters the body. Therefore, the immune system must be armed with a full repertoire of antibodies against all potential antigens. Such antibody diversity is generated by somatic rearrangement of gene segments encoding variable and constant regions. These gene segments are joined together by site-specific recombination which occurs between highly conserved DNA sequences that flank each gene segment. Because there are hundreds of different gene segments, millions of unique genes can be generated combinatorially. In addition, imprecise joining of these segments and an unusually high rate of somatic mutation within these segments further contribute to the generation of a diverse antibody population. Expression Profiling Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. Breast Cancer There are more than 180,000 new cases of breast cancer diagnosed each year, and the mortality rate for breast cancer approaches 10% of all deaths in females between the ages of 45-54 (K. Gish (1999) AWIS Magazine 28:7-10). However the survival rate based on early diagnosis of localized breast cancer is extremely high (97%), compared with the advanced stage of the disease in which the tumor has spread beyond the breast (22%). Current procedures for clinical breast examination are lacking in sensitivity and specificity, and efforts are underway to develop comprehensive gene expression profiles for breast cancer that may be used in conjunction with conventional screening methods to improve diagnosis and prognosis of this disease (Perou C M et al. (2000) Nature 406:747-752). Breast cancer is a genetic disease commonly caused by mutations in cellular disease. Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a woman to breast cancer and may be passed on from parents to children (Gish, supra). However, this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to noninherited mutations that occur in breast epithelial cells. A good deal is already known about the expression of specific genes associated with breast cancer. For example, the relationship between expression of epidermal growth factor (EGF) and its receptor, BGFR, to human mammary carcinoma has been particularly well studied. (See Rhazaie et al., supra, and references cited therein for a review of this area.) Overexpression of EGFR, particularly coupled with down-regulation of the estrogen receptor, is a marker of poor prognosis in breast cancer patients. In addition, EGFR expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on cell functions related to metastatic potential, such as cell motility, chemotaxis, secretion and differentiation. Changes in expression of other members of the erbB receptor family, of which BGFR is one, have also been implicated in breast cancer. The abundance of erbB receptors, such as HER-2/neu, HER-3, and HER-4, and their ligands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S S et al. (1994) Am J Clin Pathol 102:S13-S24). Other known markers of breast cancer include a human secreted frizzled protein mRNA that is downregulated in breast tumors; the matrix G1a protein which is overexpressed is human breast carcinoma cells; Drg1 or RTP, a gene whose expression is diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaN19, a member of the S100 protein family, all of which are down regulated in mammary carcinoma cells relative to normal mammary epithelial cells (Zhou Z et al. (1998) Int J Cancer 78:95-99; Chen, L et al. (1990) Oncogene 5:1391-1395; Ulrix W et al (1999) FEBS Lett 455:23-26; Sager, R et al. (1996) Curr Top Microbiol Immunol 213:51-64; and Lee, S W et al (1992) Proc Natl Acad Sci USA 89:2504-2508). Cell lines derived from human mammary epithelial cells at various stages of breast cancer provide a useful model to study the process of malignant transformation and tumor progression as it has been shown that these cell lines retain many of the properties of their parental tumors for lengthy culture periods (Wistuba II et al. (1998) Clin Cancer Res 4:2931-2938). Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithelial cells at various stages of malignant transformation. Colon Cancer Colorectal cancer is the second leading cause of cancer deaths in the United States. Colon cancer is associated with aging, since 90% of the total cases occur in individuals over the age of 55 A widely accepted hypothesis is that several contributing genetic mutations must accumulate over time in an individual who develops the disease. To understand the nature of genetic alterations in colorectal cancer, a number of studies have focused on the inherited syndromes. The first known inherited syndrome, Familial Adenomatous Polyposis (FAP), is caused by mutations in the Adenomatous Polyposis Coli gene (APC), resulting in truncated or inactive forms of the protein. This tumor suppressor gene has been mapped to chromosome 5q. The second known inherited syndrome is hereditary nonpolyposis colorectal cancer (HNPCC), which is caused by mutations in mismatch repair genes. Although hereditary colon cancer syndromes occur in a small percentage of the population and most colorectal cancers are considered sporadic, knowledge from studies of the hereditary syndromes can be generally applied. For instance, somatic mutations in APC occur in at least 80% of indiscriminate colon tumors. APC mutations are thought to be the initiating event in the disease. Other mutations occur subsequently. Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in these genes lead to gene expression changes in colon cancer. Less is understood about downstream targets of these mutations and the role they may play in cancer development and progression. The discovery of new secreted proteins, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative, autoimmune/inflammatory, cardiovascular, neurological, and developmental disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of secreted proteins. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention features purified polypeptides, secreted proteins, referred to collectively as “SECP” and individually as “SECP-1,” “SECP-2,” “SECP-3,” “SECP-4,” “SECP-5,” “SECP-6,” “SECP-7,” “SECP-8,” “SECP-9,” “SECP-10,” “SECP-11,” “SECP-12,” “SECP-13,” “SECP-14,” “SECP-15,” “SECP-16,” “SECP-17,” “SECP-18,” “SECP-19,” “SECP-20,” “SECP-21,” “SECP-22,” “SECP-23,” “SECP-24,” and “SECP-25.” In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-25. The invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-25. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID NO:26-50. Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide. The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed. Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides. Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides. The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. The invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional SECP, comprising administering to a patient in need of such treatment the composition. The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional SECP, comprising administering to a patient in need of such treatment the composition. Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional SECP, comprising administering to a patient in need of such treatment the composition. The invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide. The invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide. The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. |
Gastrokines and derived peptides including inhibitors |
A novel group of gastrokines called Gastric Antrum Mucosal Protein is characterized. A member of the group is designated AMP-18. AMP-18 genomic DNA, cDNA and the AMP-18 protein are sequenced for human, mouse and pig. The AMP-18 protein and active peptides derived from it are cellular growth factors. Surprisingly, peptides capable of inhibiting the effects of the complete protein, are also derived from the AMP-18 protein. Control of mammalian gastro-intestinal tissues growth and repair is facilitated by the use of the proteins, making the proteins candidates for therapies. |
1. A group of isolated homologous cellular growth stimulating proteins designated gastrokines, said proteins produced by gastric epithelial cells and comprising an amino acid sequence selected from the group consisting of VKE(K/Q)KXXGKGPGG(P/A)PPK, (SEQ ID NO: 10) VKE(K/Q)KLQGKGPGG(P/A)PPK, (SEQ ID NO: 25) or VKE(K/Q)KGKGPGG(P/A)PPK. (SEQ ID NO: 26) 2. An isolated protein from the group of claim 1, said protein further characterized as comprising an amino acid sequence as in FIG. 8, present in pig gastric epithelia in a processed form lacking the 20 amino acids which constitute a signal peptide sequence, having 165 amino acids and an estimated molecular weight of approximately 18 kD as measured by polyacrylamide gel electophoresis, said protein capable of being secreted. 3. A protein from the group of claim 1, further characterized as comprising an amino acid sequence as in FIG. 3, said sequence deduced from a human cDNA. 4. A protein from the group of claim 1, further characterized as comprising an amino acid sequence as in FIG. 6, said sequence predicted from mouse RNA and DNA. 5. A growth stimulating peptide derived from a protein of claim 1. 6. A modified peptide produced by the method comprising the following steps: (a) eliminating major protease sites in an unmodified peptide amino acid sequence by amino acid substitution or deletion in the unmodified peptide derived from a protein of claim 1; and (b) optionally introducing amino acid analogs of amino acids in the unmodified peptide. 7. A synthetic growth stimulating peptide, having a sequence of amino acids from positions 78 to 119 as shown in FIG. 3. 8. The synthetic growth stimulating peptide of claim 7, said peptide having a 30 sequence of amino acids from position 97 to position 117 as shown in FIG. 3. 9. The synthetic growth stimulating peptide of claim 7, said peptide having a sequence of amino acids from position 97 to position 121 as shown in FIG. 3. 10. The synthetic growth stimulating peptide of claim 7, said peptide having a sequence of amino acids from position 104 to position 117 as shown in FIG. 3. 11. An isolated bioactive peptide comprising a sequence selected from the group consisting of KKLQGKGPGGPPPK, (SEQ ID NO: 11) LDALVKEKKLQGKGPGGPPPK, (SEQ ID NO: 12) or LDALVKEKKLQGKGPGGPPPKGLMY. (SEQ ID NO: 13) 12. An antibody to a protein of the group of claim 1, said antibody recognizing an epitope within a peptide of the protein that has an amino acid sequence from position 78 to position 119 as in FIG. 3. 13. An isolated genomic DNA molecule with the nucleotide sequence of a human as shown in FIG. 1. 14. An isolated cDNA molecule encoding a human protein, said protein having the amino acid sequence as shown in FIG. 3. 15. A method to stimulate growth of epithelial cells in the gastrointestinal tract of mammals, said method comprising: (a) contacting the epithelial cells with a composition comprising a protein from the group of claim 1 or a peptide derived from a protein of claim 1, and (b) providing environmental conditions for stimulating growth of the epithelial cells. |
<SOH> BACKGROUND <EOH>A novel group of Gastric Antrum Mucosal Proteins that are gastrokines, is characterized. A member of the gastrokine group is designated AMP-18. AMP-18 genomic DNA, and cDNA molecules are sequenced for human and mouse, and the protein sequences are predicted from the nucleotide sequences. The cDNA molecule for pig AMP-18 is sequenced and confirmed by partial sequencing of the natural protein. The AMP-18 protein and active peptides derived from its sequence are cellular growth factors. Surprisingly, peptides capable of inhibiting the effects of the complete protein, are also derived from the AMP-18 protein sequence. Control of mammalian gastro-intestinal tissues growth and repair is facilitated by the use of the protein or peptides, making the protein and the derived peptides candidates for therapies. Searches for factors affecting the mammalian gastro-intestinal (GI) tract are motivated by need for diagnostic and therapeutic agents. A protein may remain part of the mucin layer, providing mechanical (e.g., lubricant or gel stabilizer) and chemical (e.g against stomach acid, perhaps helping to maintain the mucus pH gradient and/or hydrophobic barrier) protection for the underlying tissues. The trefoil peptide family has been suggested to have such general cytoprotectant roles (see Sands and Podolsky, 1996). Alternatively, a cytokine-like activity could help restore damaged epithelia. A suggestion that the trefoil peptides may act in concert with other factors to maintain and repair the epithelium, further underlines the complexity of interactions that take place in the gastrointestinal tract (Podolsky, 1997). The maintenance of the integrity of the GI epithelium is essential to the continued well-being of a mammal, and wound closing after damage normally occurs very rapidly (Lacy, 1988), followed by proliferation and differentiation soon thereafter to reestablish epithelial integrity (Nusrat et al., 1992). Thus protection and restitution are two critical features of the healthy gastrointestinal tract, and may be important in the relatively harsh extracellular environment of the stomach. Searches for GI proteins have met with some success. Complementary DNA (cDNA) sequences to messenger RNAs (mRNA) isolated from human and porcine stomach cells were described in the University of Chicago Ph.D. thesis “Characterization of a novel messenger RNA and immunochemical detection of its protein from porcine gastric mucosa,” December 1987, by one of the present inventors working with the other inventors. However, there were several cDNA sequencing errors that led to significant amino acid changes from the AMP-18 protein disclosed herein. The protein itself was isolated and purified only as an aspect of the present invention, and functional analyses were performed to determine utility. Nucleic acid sequences were sought. |
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