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1. An isolated protein complex comprising a RING-SH3 polypeptide in combination with at least one polypeptide selected from the group consisting of: a Gag protein, a Gag late domain, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, RING-SH3, and a clathrin. 2. The isolated protein complex of claim 1, wherein said Gag protein is an HIV gag protein. 3. The isolated protein complex of claim 1, wherein said Gag protein comprises the Gag late domain. 4. The isolated protein complex of claim 3, wherein said Gag late domain is PTAP. 5. The isolated protein complex of claim 3, wherein said Gag late domain is PxxY. 6. The isolated protein complex of claim 3, wherein said Gag late domain is PxxL. 7. The isolated protein complex of claim 3, wherein said Gag late domain is PPxY. 8. The isolated protein complex of claim 3, wherein said Gag late domain is YxxL. 9. The isolated protein complex of claim 3, wherein said Gag late domain is PxxP. 10. A host cell comprising a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a recombinant RING-SH3 nucleic acid, and wherein the second nucleic acid comprises a recombinant nucleic acid encoding a Gag protein. 11. The host cell of claim 10, wherein said Gag protein is an HIV gag protein. 12. The host cell of claim 11, wherein said Gag protein comprises the Gag late domain. 13. The host cell of claim 12, wherein said Gag late domain is PTAP. 14. The host cell of claim 12, wherein said Gag late domain is PxxY. 15. The host cell of claim 12, wherein said Gag late domain is PxxL. 16. The host cell of claim 12, wherein said Gag late domain is PPxY. 17. The host cell of claim 12, wherein said Gag late domain is YxxL. 18. The host cell of claim 12, wherein said Gag late domain is PxxP. 19. A method for identifying modulators of protein complexes, comprising: (i) forming a reaction mixture comprising (a) a RING-SH3; and (b) a second polypeptide selected from the group consisting of: RING-SH3, a gag protein, a Gag late domain, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, and a clathrin; (ii) contacting said reaction mixture with a test agent, and (iii) determining the effect of said test agent for one or more activities selected from the group comprising (a) a change in the level of the protein complex, (b) a change in the enzymatic activity of the complex, or (c) where the reaction mixture is a whole cell, a change in the plasma membrane localization of the complex or a component thereof. 20. A method for identifying a test compound which inhibits or potentiates complex formation, comprising: (i) forming a reaction mixture comprising (a) a RING-SH3; and (b) a second polypeptide selected from the group consisting of: RING-SH3, a gag protein, a Gag late domain, P13K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, and a clathrin; (ii) contacting said reaction mixture with a test agent, and (iii) detecting binding of said RING-SH3 to said second polypeptide; wherein a change in the binding of said RING-SH3 to said second polypeptide in the presence of the test compound, relative to binding in the absence of the test compound, indicates that said test compound potentiates or inhibits complex formation between said RING-SH3 and said second polypeptide. 21. A method for inhibiting infection in a subject in need thereof, comprising administering an effective amount of an agent that inhibits the binding of a RING-SH3 polypeptide to an gag protein. 22. The method of claim 21, wherein said agent is selected from the group comprising a small molecule, a antibody, and a peptide. 23. The method of claim 22, wherein the Gag protein is an HIV Gag protein. 24. The method of claim 22, wherein the Gag polypeptide is HIV p24. 25. An isolated antibody, or fragment thereof, specifically immunoreactive with an epitope of a RING-SH3 polypeptide, which disrupts the interaction between said RING-SH3 and a RING-SH3-associating polypeptide (RING-SH3-AP). 26. The antibody of claim 25, wherein said antibody is a monoclonal antibody. 27. The antibody of claim 25, wherein said antibody is a Fab fragment. 28. The antibody of claim 25, wherein said antibody is labeled with a detectable label. 29. The antibody of claim 25, wherein said RING-SH3-AP is a gag polypeptide. 30. The antibody of claim 25, wherein said RING-SH3-AP is an HIV gag polypeptide. 31. A kit for detecting a RING-SH3 polypeptide protein comprising (i) isolated anti-RING-SH3 antibodies, or fragment thereof, specifically immunoreactive with an epitope of an RING-SH3, which epitope interacts with an RING-SH3-AP, and (ii) a detectable label for detecting said anti-RING-SH3 antibody in immunoclomplexes with said RING-SH3 polypeptide. 32. A host cell comprising a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a recombinant RING-SH3 nucleic acid, and wherein the second nucleic acid comprises a recombinant nucleic acid encoding a HIV Gag protein. 33. A method for inhibiting infection in a subject in need thereof, comprising administering an effective amount of an agent that inhibits the binding of a RING-SH3 polypeptide to an HIV gag protein. 34. An anti-viral therapeutic composition comprising a double stranded oligoribonucleotide molecule that inhibits expression of a nucleic acid molecule encoding a RING-SH3 polypeptide |
<SOH> BACKGROUND <EOH>Viral maturation requires the proteolytic processing of viral proteins, such as Gag, and the activity of the host proteins. It is believed that cellular machineries for exo/endocytosis and for ubiquitin conjugation may be involved in the maturation. In particular, the assembly and subsequent budding of retroviruses, rhabdoviruses, and filoviruses depends on the Gag polyprotein. After its synthesis, Gag is targeted to the plasma membrane where it induces budding of nascent virus particles. The role of ubiquitin in virus assembly was suggested by Dunigan et al. (1988, Virology 165, 310, Meyers et al. 1991, Virology 180, 602), who observed that mature virus particles were enriched in unconjugated ubiquitin. More recently, it was shown that proteasome inhibitors suppress the release of HIV-1, HV-2 and virus-like particles derived from SIV and RSV Gag. Also, inhibitors affect Gag processing and maturation into infectious particles (Schubert et al 2000, PNAS 97, 13057, Harty et al. 2000, PNAS 97, 13871, Strack et al. 2000, PNAS 97, 13063, Patnaik et al. 2000, PNAS 97, 13069). It is well known in the art that ubiquitin-mediated proteolysis is the major pathway for the selective, controlled degradation of intracellular proteins in eukaryotic cells. Ubiquitin modification of a variety of protein targets within the cell appears to be important in a number of basic cellular functions such as regulation of gene expression, regulation of the cell-cycle, modification of cell surface receptors, biogenesis of ribosomes, and DNA repair. One major function of the ubiquitin-mediated system is to control the half-lives of cellular proteins. The half-life of different proteins can range from a few minutes to several days, and can vary considerably depending on the cell-type, nutritional and environmental conditions, as well as the stage of the cell-cycle. Targeted proteins undergoing selective degradation, presumably through the actions of a ubiquitin-dependent proteosome, are covalently tagged with ubiquitin through the formation of an isopeptide bond between the C-terminal glycyl residue of ubiquitin and a specific lysyl residue in the substrate protein. This process is catalyzed by a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), and in some instances may also require auxiliary substrate recognition proteins (E3s). Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin may be attached to lysine side chains of the previously conjugated moiety to form branched multi-ubiquitin chains. The conjugation of ubiquitin to protein substrates is a multi-step process. In an initial ATP requiring step, a thioester is formed between the C-terminus of ubiquitin and an internal cysteine residue of an E1 enzyme. Activated ubiquitin is then transferred to a specific cysteine on one of several E2 enzymes. Finally, these E2 enzymes donate ubiquitin to protein substrates. Substrates are recognized either directly by ubiquitin-conjugated enzymes or by associated substrate recognition proteins, the E3 proteins, also known as ubiquitin ligases. |
<SOH> SUMMARY <EOH>It is proposed that a variety of proteins, including ubiquitin protein ligases and proteins involved in membrane trafficking, are recruited for the process of viral maturation (including, for example, assembly, budding and release) by direct or indirect interaction with viral proteins, for example Gag proteins. The ligase then ubiquitinates viral and/or cellular proteins that are part of the membrane remodeling machinery. For example, a number of Gag protein motifs such as PxxP, PxxY, PPXY and YxxL, are known to recruit proteins involved in viral maturation. To this end, in certain embodiments, the invention provides a family of nucleic acid sequences and proteins encoded thereby that play a role in viral maturation: the Alternate Viral Maturation Scaffolding Protein, or the AVMSP family of proteins. Broadly, AVMSP polypeptides comprise a first domain or functional role and a second domain. The first domain or functional role is selected from the following: SH2, SH3, or membrane spanning (“membrane”), or functions as a receptor. A preferred AVMSP also comprises a second domain that is a RING domain. Accordingly, different categories of AVMSPs may be referred to as RING-SH3 proteins and nucleic acids, RING-SH2 proteins and nucleic acids, RING-membrane proteins and nucleic acids and RING-receptor polypeptides and nucleic acids. The first domain and second domain may be found in any order within the AVMSP sequence (i.e. the first domain need not be N-terminal to the second domain). It is understood that polypeptides that function as receptors will often have membrane or other domains. In certain embodiments AVMSP proteins comprise a C2 domain. In further aspects, in cells infected with viruses that utilize a Gag-dependent pathway for assembly, budding and/or release, AVMSPs, act to assemble complexes of proteins that mediate release. AVMSP complexes may, for example, stimulate, ubiquitylation of certain proteins, stimulate membrane fusion, stimulate assembly of viral particles, or a combination of the preceding. As one of skill in the art can readily appreciate, any single AVMSP may form multiple different complexes at different times. In additional aspects, the invention provides nucleic acid sequences and proteins encoded thereby, as well as probes derived from the nucleic acid sequences, antibodies directed to the encoded proteins, diagnostic methods for detecting cells infected with a virus, and assays for identifying agents having an antiviral activity. In one aspect, the invention provides a RING-SH3 nucleic acid, such as an isolated nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to a sequence encoding a RING-SH3 protein, such as a sequence of SEQ ID Nos: 1-3, or a sequence complementary thereto. In a related embodiment, the nucleic acid is at least about 80%, 90%, 95%, or 97-98%, or 100% identical to a sequence corresponding to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive nucleotides up to the full length of one of SEQ ID Nos. 40-99 or a sequence complementary thereto or up to the full length of the gene of which said sequence is a fragment. In a further embodiment, the RING-SH3 nucleic acid comprises a nucleic acid encoding an amino acid sequence as set forth in SEQ ID Nos. 1-39 or a nucleic acid complement thereof. In a related embodiment, the encoded amino acid sequence is at least about 80%, 90%, 95%, or 97-98%, or 100% identical to a sequence corresponding to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive amino acids up to the full length of one of SEQ ID Nos: 1-39. In yet another embodiment, the RING-SH3 nucleic acid is an isolated nucleic acid encoding a polypeptide comprising a RING domain and an SH3 domain. In a preferred embodiment, the RING-SH3 nucleic acid is a PRT3 nucleic acid of SEQ ID NOs:40-44 or a functional variant thereof. In a further aspect, the invention provides a RING-SH2 nucleic acid, such as an isolated nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to a sequence encoding a RING-SH2 protein, such as a sequence of SEQ ID Nos: 45-46, or a sequence complementary thereto. In a related embodiment, the nucleic acid is at least about 80%, 90%, 95%, or 97-98%, or 100% identical to a sequence corresponding to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive nucleotides up to the full length of one of SEQ ID Nos.45-46, or a sequence complementary thereto or up to the full length of the gene of which said sequence is a fragment. In a further embodiment, the RING-SH2 nucleic acid comprises a nucleic acid encoding an amino acid sequence as set forth in SEQ ID Nos. 4-5, or a nucleic acid complement thereof. In a related embodiment, the encoded amino acid sequence is at least about 80%, 90%, 95%, or 97-98%, or 100% identical to a sequence corresponding to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive amino acids up to the full length of one of SEQ ID NOS: 4-5. In yet another embodiment, the RING-SH2 nucleic acid is an isolated nucleic acid encoding a polypeptide comprising a RING domain and an SH2 domain. In a further aspect, the invention provides a RING-membrane nucleic acid, such as an isolated nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to a sequence encoding a RING-membrane protein, such as a sequence of SEQ ID Nos: 47-56, or a sequence complementary thereto. In a related embodiment, the nucleic acid is at least about 80%, 90%, 95%, or 97-98%, or 100% identical to a sequence corresponding to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive nucleotides up to the full length of one of SEQ ID Nos. 47-56, or a sequence complementary thereto or up to the fill length of the gene of which said sequence is a fragment. In a further embodiment, the RING-membrane nucleic acid comprises a nucleic acid encoding an amino acid sequence as set forth in SEQ ID Nos. 6-15, or a nucleic acid complement thereof. In a related embodiment, the encoded amino acid sequence is at least about 80%, 90%, 95%, or 97-98%, or 100% identical to a sequence corresponding to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive amino acids up to the full length of one of SEQ ID NOS: 6-15. In yet another embodiment, the RING-membrane nucleic acid is an isolated nucleic acid encoding a polypeptide comprising a RING domain and a membrane domain. In a further aspect, the invention provides a RING-receptor nucleic acid, such as an isolated nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to a sequence encoding a RING-receptor protein, such as a sequence of SEQ ID Nos: 57-72, or a sequence complementary thereto. In a related embodiment, the nucleic acid is at least about 80%, 90%, 95%, or 97-98%, or 100% identical to a sequence corresponding to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive nucleotides up to the full length of one of SEQ ID Nos. 57-72, or a sequence complementary thereto or up to the full length of the gene of which said sequence is a fragment. In a further embodiment, the RING-receptor nucleic acid comprises a nucleic acid encoding an amino acid sequence as set forth in SEQ ID Nos. 16-31, or a nucleic acid complement thereof. In a related embodiment, the encoded amino acid sequence is at least about 80%, 90%, 95%, or 97-98%, or 100% identical to a sequence corresponding to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive amino acids up to the full length of one of SEQ ID NOS: 16-31. In yet another embodiment, the RING-receptor nucleic acid is an isolated nucleic acid encoding a polypeptide comprising a RING domain and a receptor domain. In one embodiment, the invention provides an expressible RING-SH3, RING-SH2, RING-membrane or RING-receptor nucleic acid operably linked to a transcriptional regulatory sequence, rendering the expressible nucleotide sequence suitable for use as an expression vector. In another embodiment, the nucleic acid may be included in an expression vector capable of replicating in a prokaryotic or eukaryotic cell. In a related embodiment, the invention provides a host cell transfected with the expression vector. In yet another embodiment, the invention provides a substantially pure RING-SH3, RING-SH2, RING-membrane or RING-receptor nucleic acid which hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive nucleotides up to the full length of one of SEQ ID Nos. 40-72, or a sequence complementary thereto or up to the full length of the gene of which said sequence is a fragment. The invention also provides an antisense oligonucleotide analog which hybridizes under stringent conditions to at least 12, at least 25, or at least 50 consecutive nucleotides of one of SEQ ID NOS 40-72, or a sequence complementary thereto. In another embodiment, the invention provides a probe/primer comprising a substantially purified RING-SH3 oligonucleotide, said oligonucleotide containing a region of nucleotide sequence which hybridizes under stringent conditions to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive nucleotides of sense or antisense sequence selected from SEQ ID Nos.40-44, or a sequence complementary thereto. In another embodiment, the invention provides a probe/primer comprising a substantially purified RING-SH2 oligonucleotide, said oligonucleotide containing a region of nucleotide sequence which hybridizes under stringent conditions to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive nucleotides of sense or antisense sequence selected from SEQ ID Nos.4546, or a sequence complementary thereto. In another embodiment, the invention provides a probe/primer comprising a substantially purified RING-membrane oligonucleotide, said oligonucleotide containing a region of nucleotide sequence which hybridizes under stringent conditions to at least about 12 , at least about 15, at least about 25, or at least about 40 consecutive nucleotides of sense or antisense sequence selected from SEQ D Nos.47-56, or a sequence complementary thereto. In another embodiment, the invention provides a probe/primer comprising a substantially purified RING-receptor oligonucleotide, said oligonucleotide containing a region of nucleotide sequence which hybridizes under stringent conditions to at least about 12, at least about 15, at least about 25, or at least about 40 consecutive nucleotides of sense or antisense sequence selected from SEQ ID Nos.57-72, or a sequence complementary thereto. In preferred embodiments, a probe as described above selectively hybridizes with a target nucleic acid. In another embodiment, the probe may include a label group attached thereto and able to be detected. The label group may be selected from radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. The invention further provides arrays of at least about 10, at least about 25, at least about 50, or at least about 100 different probes as described above attached to a solid support. In another aspect, the invention provides polypeptides. In one embodiment, the invention pertains to a RING-SH3 polypeptide including an amino acid sequence encoded by a nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to a sequence of SEQ ID Nos. 40-44, or a sequence complementary thereto, or a fragment comprising at least about 25, or at least about 40 amino acids thereof. In another aspect, the invention provides polypeptides. In one embodiment, the invention pertains to a RING-SH2 polypeptide including an amino acid sequence encoded by a nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to a sequence of SEQ ID Nos. 45-46, or a sequence complementary thereto, or a fragment comprising at least about 25, or at least about 40 amino acids thereof. In another aspect, the invention provides polypeptides. In one embodiment, the invention pertains to a RING-membrane polypeptide including an amino acid sequence encoded by a nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to a sequence of SEQ ID Nos. 47-56, or a sequence complementary thereto, or a fragment comprising at least about 25, or at least about 40 amino acids thereof. In another aspect, the invention provides polypeptides. In one embodiment, the invention pertains to a RING-receptor polypeptide including an amino acid sequence encoded by a nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to a sequence of SEQ ID Nos. 57-72, or a sequence complementary thereto, or a fragment comprising at least about 25, or at least about 40 amino acids thereof. In a preferred embodiment, the polypeptide is identical with or homologous to a RING-SH3, RING-SH2, RING-membrane or RING-receptor protein represented by SEQ ID Nos: 1-31. For instance, a polypeptide preferably has an amino acid sequence at least 70% homologous to a polypeptide represented by any of SEQ ID Nos: 1-31, though polypeptides with higher sequence homologies of, for example, 80%, 90% or 95% are also contemplated. The polypeptide can comprise a full length protein, such as represented in the sequence listings, or it can comprise a fragment of, for instance, at least 5, 10, 20, 50, 100, 150 or 200 amino acids in length. In another preferred embodiment, the invention features a purified or recombinant polypeptide fragment of a RING-SH3, RING-SH2, RING-membrane or RING-receptor polypeptide, which polypeptide has the ability to modulate, e.g., mimic or antagonize, an activity of a wild-type RING-SH3, RING-SH2, RING-membrane or RING-receptor polypeptide. Preferably, the polypeptide fragment comprises a sequence identical or homologous to an amino acid sequence designated in one of SEQ ID Nos: 1-31. Moreover, as described below, the RING-SH3, RING-SH2, RING-membrane or RING-receptor polypeptide can be either an agonist (e.g. mimics), or alternatively, an antagonist of a biological activity of a naturally occurring form of the protein, e.g., the polypeptide is able to modulate the intrinsic biological activity of a RING-SH3, RING-SH2, RING-membrane or RING-receptor complex, such as an enzymatic activity, binding to other cellular components, cellular compartmentalization, and the like. The subject proteins can also be provided as chimeric molecules, such as in the form of fusion proteins. For instance, the AVMSP can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the AVMSP, e.g. the second polypeptide portion is glutathione-S-transferase, e.g. the second polypeptide portion is an enzymatic activity such as alkaline phosphatase, e.g. the second polypeptide portion is an epitope tag, e.g. the second polypeptide is an affinity purification tag. Yet another aspect of the present invention concerns an immunogen comprising an AVMSP in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for said AVMSP; e.g. a humoral response, e.g. an antibody response; e.g. a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g. a unique determinant, from a protein represented by one of SEQ ID Nos. 1-39. In yet another aspect, this invention provides antibodies immunoreactive with one or more AVMSPs. In one embodiment, antibodies are specific for a RING domain, an SH3 domain, a SH2 domain, or a receptor domain and preferably the domain is part of an AVMSP. In a more specific embodiment, the domain is part of an amino acid sequence set forth in SEQ ID Nos. 1-39. In another embodiment, the antibodies are immunoreactive with one or more proteins having an amino acid sequence that is at least 80% identical to an amino acid sequence as set forth in SEQ ID Nos. 1-39. In other embodiments, an antibody is immunoreactive with one or more proteins having an amino acid sequence that is 85%, 90%, 95%, 98%, 99% or identical to an amino acid sequence as set forth in SEQ ID Nos. 1-39. In an additional aspect, the invention provides complexes comprising an AVMSP and an AVMSP associated protein (an “AVMSP-AP”). In one embodiment, the invention provides an isolated protein complex comprising a RING-SH3, RING-SH2, RING-membrane or RING-receptor polypeptide in combination with at least one polypeptide selected from the group consisting of: a RING-SH3, a RING-SH2, a RING-membrane, a RING-receptor, a Gag protein, a Gag late domain, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin and a clathrin. In another embodiment, the isolated protein complex comprises a RING-SH3, RING-SH2, RING-membrane or RING-receptor polypeptide and a Gag protein in combination with a polypeptide selected from the group consisting of: a RING-SH3, a RING-SH2, a RING-membrane, a RING-receptor, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin and a clathrin. In yet another embodiment, the invention provides an isolated protein complex comprising an AVMSP polypeptide and a HIV Gag protein in combination with a polypeptide selected from the group consisting of: RING-SH3, RING-SH2, RING-membrane, RING-receptor, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin and a clathrin. The invention also provides an isolated protein complex comprising a RING-SH3, RING-SH2, RING-membrane or RING-receptor polypeptide and an HIV Gag protein in combination with a polypeptide selected from the group consisting of: RING-SH3, RING-SH2, RING-membrane or RING-receptor, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin and a clathrin. In yet another aspect, the invention provides an assay for screening test compounds for inhibitors, or alternatively, potentiators, of an interaction between an AVMSP and an AVMSP-AP. In the case of a RING-SH3 polypeptide, exemplary associated proteins (“RING-SH3-AP”) include RING-SH3 proteins, E2 proteins (e.g. tsg101), Gag proteins, proteins comprising an L-domain, phosphatidylinositol-3-kinases, as well as proteins involved in endocytosis such as clathrins, actins, myosins, HSP60, HSP70, HSP90, STAM1, STAM2A, and STAM2B. An exemplary method includes the steps of (i) combining AVMSP-AP (e.g. a RING-SH3-AP, RING-SH2-AP, RING-membrane-AP or RING-receptor-AP), an AVMSP, and a test compound, e.g., under conditions (including, as desired, the addition of additional proteins) wherein, but for the test compound, the AVMSP and an AVMSP-AP are able to interact; and (ii) detecting the formation of a complex which includes the AVMSP and an AVMSP-AP. A statistically significant change, such as a decrease, in the formation of the complex in the presence of a test compound (relative to what is seen in the absence of the test compound) is indicative of a modulation, e.g., inhibition, of the interaction between the AVMSP and an AVMSP-AP. Similar assays may employ preformed AVMSP-AVMSP-AP complexes to assess the ability of the test compound to destabilize or stabilize the complex. In yet another aspect, the invention provides cells carrying a recombinant form of an AVMSP nucleic acid, often included on a vector. In further embodiments, cells carry a recombinant form of an AVMSP nucleic acid and a recombinant form of a nucleic acid encoding a Gag protein and/or a polypeptide comprising an L domain motif, such as P(T/S)AP, PPxY or YxxL. In certain aspects, the cells are bacterial, and in other aspects the cells are eukaryotic cells, preferrably a mammalian cell line. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning , Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195 ; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology , Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology , Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo , (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. |
Dna molecules conferring tolerance to herbicidal compounds |
The present invention relates to tolerance to inhibitors of ALS/AHAS activity in plants, in particular to DNA molecules encoding herbicide tolerant proteins having ALS/AHAS activity. The present invention also relates to molecular markers used to detect tolerance to the herbicide and to methods using such molecular markers. |
1-47. (canceled) 48. An isolated DNA molecule comprising about 15 successive nucleotides of the nucleotide sequence set forth in SEQ ID NO:1, wherein said DNA molecule comprises a nucleotide polymorphism, wherein said polymorphism allows to differentiate between an allele conferring tolerance to an inhibitor of ALS/AHAS activity and an allele sensitive to said inhibitor in a sunflower plant, wherein said polymorphism is at a position corresponding to any one of positions 1566, 1229, 1232 or 1235 in SEQ ID NO:1. 49. The DNA molecule according to claim 48, wherein said polymorphism allows to differentiate between a nucleotide sequence encoding a imidazolinone-sensitive ALS/AHAS and a nucleotide sequence encoding a imidazolinone-tolerant ALS/AHAS in a sunflower plant. 50. The DNA molecule according to claim 48, wherein DNA molecule comprises about 20 successive nucleotides of the nucleotide sequence set forth in SEQ ID NO:1. 51. The DNA molecule according to claim 48, wherein said DNA molecule is an amplified PCR fragment. 52. An oligonucleotide capable of hybridizing to a DNA molecule comprising a nucleotide sequence set forth in SEQ ID NO:1, wherein said oligonucleotide comprises a nucleotide corresponding to any one of positions 1566, 1229, 1232 or 1235 of SEQ ID NO:1 or a complement thereto. 53. The oligonucleotide according to claim 52, wherein said oligonucleotide further comprises a detectable label. 54. A method of identifying a plant tolerant to an inhibitor of ALS/AHAS activity comprising the steps of: a) obtaining a sample from a plant; b) detecting in said sample a DNA molecule according to claim 48, the presence of said DNA molecule being indicative of an allele conferring tolerance to an inhibitor of ALS/AHAS activity in said plant, wherein said plant is tolerant to an inhibitor of ALS/AHAS activity. 55. A method of selecting a plant tolerant to an inhibitor of ALS/AHAS activity from a population of plants comprising the steps of: a) providing a population of plants; b) obtaining a sample of a plant of said population; c) detecting in said sample a DNA molecule according to claim 48, the presence of said DNA molecule being indicative of an allele conferring tolerance to an inhibitor of ALS/AHAS activity in said plant; d) selecting said plant, wherein said plant is tolerant to an inhibitor of ALS/AHAS activity. 56. A method for introgressing tolerance to an inhibitor of ALS/AHAS activity into a plant comprising the steps of: a) obtaining a plant tolerant to inhibitor of ALS/AHAS activity; b) crossing said plant of step a) with a plant which sensitive or less tolerant to said inhibitor; c) detecting in a plant resulting from the cross in step b) a DNA molecule according to claim 48, the presence of said DNA molecule being indicative of an allele conferring tolerance to an inhibitor of ALS/AHAS activity in said plant; d) selecting a plant of step c) for further breeding, wherein said plant is tolerant to an inhibitor of ALS/AHAS activity. 57. The method according to claim 56, further comprising repeating steps b) to d) until the tolerance to said inhibitor is introgressed into said plant which sensitive or less tolerant to said inhibitor. 58. A method to determine whether a plant is homozygous or heterozygous for an allele conferring tolerance to an inhibitor of ALS/AHAS activity comprising: a) obtaining a sample of a plant; b) detecting in said sample a DNA molecule according to claim 48, wherein said step of detecting is carried out using a co-dominant marker; c) determining whether a nucleotide sequence encoding an A protein having ALS/AHAS activity tolerant to an inhibitor of ALS/AHAS activity is heterozygous or homozygous in said plant. 59. The method according to claim 54, wherein said inhibitor of ALS/AHAS activity is an imidazolinone herbicide. 60. The method according to any one of claims 54, wherein said plant is sunflower. 61. A kit for detecting a single nucleotide polymorphism indicative for tolerance or sensitivity to an inhibitor of ALS/AHAS activity in a sunflower plant comprising an oligonucleotide according to claim 52. 62. The kit according to claim 61, wherein said oligonucleotide is any one of the oligonucleotides set forth in SEQ ID NO:8 or SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:11 or SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17. |
<SOH> BRIEF DESCRIPTION OF THE SEQUENCE LISTING <EOH>SEQ ID NO:1 nucleotide sequence encoding a protein having ALS/AHAS activity SEQ ID NO:2 amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:1 SEQ ID NO:3 nucleotide sequence encoding an herbicide-tolerant protein having ALS/AHAS activity SEQ ID NO:4 amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3 SEQ ID NO:5 Oligonucleotide HiNK366 SEQ ID NO:6 Oligonucleotide HiNK369 SEQ ID NO:7 Oligonucleotide HiNK370 SEQ ID NO:8 Oligonucleotide HiNK379 SEQ ID NO:9 Oligonucleotide HiNK415 SEQ ID NO:10 Oligonucleotide HiNK451 SEQ ID NO:11 Oligonucleotide HiNK414 SEQ ID NO:12 Oligonucleotide HiNK452 SEQ ID NO:13 Oligonucleotide HiNK415 SEQ ID NO:14 Oligonucleotide HiNK702 SEQ ID NO:15 Oligonucleotide HiNK703 SEQ ID NO:16 Oligonucleotide HiNK700 SEQ ID NO:17 Oligonucleotide HiNK701 The present invention discloses a new mechanism of tolerance to inhibitors of ALS/AHAS activity in plants. The inventors of the present invention have determined that a novel amino acid substitution in the acetolactate synthase (ALS) or acetohydroxyacid synthase (AHAS) protein underlies the tolerance to an inhibitor. The inventors of the present invention have also determined the nucleotide and amino acid sequence of a sunflower ALS/AHAS. The DNA molecules of the present invention are used to confer tolerance to inhibitors of ALS/AHAS activity in a wide variety of crops. Molecular markers based on these DNA molecules and on polymorphisms between herbicide tolerant and herbicide sensitive alleles are also disclosed. These markers are used in breeding new varieties tolerant to herbicides and in seeds production. The ALS/AHAS catalyzes the first common step in the biosynthetic pathway of the amino acids valine, leucine and isoleucine, and is known under both names ALS and AHAS (EC 4.1.3.18). ALS/AHAS is the target for various classes or herbicidal compounds, such as the imidazolinones, sulfonylureas and triazolopyrimidines. Mutations conferring tolerance to such inhibitors of ALS/AHAS activity in plants have been described in several crops, but unexpectedly, the inventors of the present invention disclose here a novel mutation conferring tolerance to inhibitors of ALS/AHAS activity. Accordingly, in one aspect, the inventors of the present invention disclose herein the isolation and identification of the nucleotide sequence and amino acid sequence of an acetolactate synthase (ALS) or acetohydroxyacid synthase (AHAS) from sunflower ( Helianthus annuus ). The nucleotide sequence encoding the sunflower ALS/AHAS is set forth in SEQ ID NO:1, and the corresponding amino acid sequence is set forth in SEQ ID NO:2. The procedure leading to isolation of the nucleotide and amino acid sequence of the sunflower ALS/AHAS are described in example 2. A nucleotide sequence encoding a sunflower ALS/AHAS is for example expressed in a transgenic plant to confer tolerance to said plant to an inhibitor of the ALS/AHAS activity naturally occurring in said plant. According to this embodiment, plants, plant tissue, plant seeds, or plant cells are transformed, preferably stably transformed, with a recombinant DNA molecule comprising a suitable promoter functional in plants operatively linked to a nucleotide sequence encoding a sunflower ALS/AHAS. The transgenic plants, plant tissue, plant seeds, or plant cells thus created are then selected using conventional techniques disclosed herein or well-known in the art, whereby herbicide tolerant lines are isolated, characterized, and developed. Increased expression of the nucleotide sequence results in a level of ALS/AHAS activity at least sufficient to overcome growth inhibition caused by an herbicide when applied in amounts sufficient to inhibit normal growth of control plants. The level of expressed protein generally is at least two times, preferably at least five times, and more preferably at least ten times the natively expressed amount. A nucleotide sequence encoding a sunflower ALS/AHAS is also expressed recombinantly in a host cell and used to screen for chemicals that selectively inhibit a sunflower ALS/AHAS. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E. coli , yeast, and insect cells (see, e.g., Luckow and Summers, Bio/Technol. 6: 47 (1988), and baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus (AcMNPV). A preferred baculoviris/insect system is pAcHLT (Pharmingen, San Diego, Calif.) used to transfect Spodoptera frugiperda Sf9 cells (ATCC) in the presence of linear Autographa californica baculovirus DNA (Pharmigen, San Diego, Calif.). The resulting virus is used to infect HighFive Tricoplusia ni cells (Invitrogen, La Jolla, Calif.). Recombinantly produced protein is isolated and purified using a lines of standard techniques. The actual techniques that may be used will vary depending upon the host organism used, whether the protein is designed for secretion, and other such factors familiar to the skilled artisan (see, e.g. chapter 16 of Ausubel, F. et al., “Current Protocols in Molecular Biology”, pub. by John Wiley & Sons, Inc. (1994)). Accordingly, the present invention also discloses recombinant DNA molecules, expression cassettes and recombinant vectors comprising a nucleotide sequence encoding a sunflower ALS/AHAS. The present invention also discloses cells, e.g. plant, bacterial and insect cells, plant tissue and plants including the seeds and progeny thereof comprising such nucleotide sequences, which are preferably tolerant to an inhibitor of ALS/AHAS activity. The present invention also discloses an amino acid substitution in the amino acid sequence of a protein having ALS/AHAS activity, wherein the amino acid substitution confers tolerance to an inhibitor of a corresponding protein which does not comprise the amino acid substitution. The amino acid substitution is at a position corresponding to position 475 in the comparative alignment shown in Table 3. Preferably, a tyrosine (Y) at said position is replaced by a histidine (H). Preferably, a tyrosine at a position corresponding to position 475 in Table 3 is substituted in a plant protein having ALS/AHAS activity, and confers to said protein tolerance to the inhibitor. Thus, the present invention discloses an isolated DNA molecule comprising a nucleotide sequence that encodes a protein having ALS/AHAS activity, wherein said protein comprises an amino acid substitution occurring at a position corresponding to position 475 in the comparative alignment shown in Table 3, wherein said amino acid substitution confers to said protein tolerance to an inhibitor of a protein having ALS/AHAS activity which does not comprise said amino acid substitution. In another preferred embodiment, the present invention discloses an isolated DNA molecule comprising a nucleotide sequence that encodes a protein having ALS/AHAS activity, wherein the protein comprises any one of the amino acid sub-sequences PQΔ 1 AI, PΔ 1 AI or PQΔ 1 A, wherein Δ 1 is an amino acid other than tyrosine. Preferably, Δ 1 is histidine. A preferred protein having ALS/AHAS activity of the present invention is an ALS/AHAS protein from a dicotyledonous plant or from a monocotyledonous plant. Preferably, the ALS/AHAS protein is from Amaranthus sp., Arabidopsis thaliana, Bassia scoparia, Brassica napus, Gossypium hirsutum, Nicotiana tabacum , maize, rice or xanthium. The amino acid sequences of the ALS/AHAS proteins of these crops are shown at Table 3. Also encompassed in the present invention are nucleotide sequences encoding herbicide-tolerant proteins having ALS/AHAS activity of the present invention. In a preferred embodiment, a codon encoding a substituted amino acid is at a position corresponding to positions 1415-1417 in Table 1 or corresponding to positions 1566-1568 in SEQ ID NO:1. Preferably, a triplet “TAT” or TAC” is substituted, preferably to a “CAT” or “CAC” triplet, coding for a histidine. One skilled in the art has no difficulties to find and modify the corresponding codons in further nucleotide sequences encoding proteins having ALS/AHAS activity, so that they encode an herbicide-tolerant protein. A particularly preferred tolerant protein having ALS/AHAS activity of the present invention is a sunflower ALS/AHAS protein. The position of the amino acid substitution corresponds to position 445 in SEQ ID NO:2. Preferably, the tolerant sunflower ALS/AHAS protein comprises the amino acid sequence set forth in SEQ ID NO:4. A preferred nucleotide sequence encoding such amino acid sequence comprises the nucleotide sequence set forth in SEQ ID NO:1 with a “T” to “C” substitution at position 1566 of SEQ ID NO:1. A particularly preferred nucleotide sequence encoding a tolerant sunflower ALS/AHAS protein is set forth in SEQ ID NO:3. Nucleotide sequences encoding a tolerant sunflower ALS/AHAS protein are used to confer tolerance to inhibitors of naturally-occurring ALS/AHAS activity. In a preferred embodiment, such nucleotide sequences are transformed into plants or plant cells using methods and techniques disclosed herein or well-known in the art. Accordingly, the present invention also discloses recombinant DNA molecules, expression cassettes and recombinant vectors comprising a nucleotide sequence encoding an herbicide-tolerant protein having ALS/AHAS activity of the present invention. The present invention also discloses cells, e.g. plant, bacterial and insect cells, plant tissue and plants, including the seeds and progeny thereof, comprising such nucleotide sequence. The transgenic plants, plant tissue, plant seeds, or plant cells thus created are then selected using conventional techniques disclosed herein or well-known in the art, whereby herbicide tolerant lines are isolated, characterized, and developed. Expression of the nucleotide sequence results in tolerance at least sufficient to overcome growth inhibition caused by an herbicide when applied in amounts sufficient to inhibit normal growth of control plants. In another preferred embodiment, an herbicide-tolerant allele encoding an herbicide-tolerant ALS/AHAS protein of the present invention is obtained by direct selection in plants, plant cells or plant tissue. Determination of the lowest dose of herbicide used in the selection experiments is routine in the art. Mutagenesis of plant material is preferably utilized to increase the frequency at which tolerant alleles occur in the selected population. Mutagenized seed material is derived from a variety of sources, including chemical or physical mutagenesis of seeds or pollen (Neuffer, In Maize for Biological Research Sheridan, ed. Univ. Press, Grand Forks, N.Dak., pp. 61-64 (1982)), which is then used to fertilize plants and the resulting M 1 mutant seeds collected. Preferred chemical agents for mutagenesis include ethyl methane sulfonate or methyl methane sulfonate. Preferred physical agents for mutagenesis include gamma rays, UV light or fast neutrons. Typically for Arabidopsis , M 2 seeds (Lehle Seeds, Tucson, Ariz.), which are progeny seeds of plants grown from seeds mutagenized with chemicals or with physical agentsare plated at densities of up to 10,000 seeds/plate (10 cm diameter) on minimal salts medium containing an appropriate concentration of inhibitor to select for tolerance. Seedlings that continue to grow and remain green 7-21 days after plating are transplanted to soil and grown to maturity and seed set. Progeny of these seeds are tested for tolerance to the herbicide. If the tolerance trait is dominant, plants whose seed segregate 3:1/tolerant:sensitive are presumed to have been heterozygous for the tolerance at the M 2 generation. Plants that give rise to all tolerant seed are presumed to have been homozygous for the resistance at the M 2 generation. Such mutagenesis on intact seeds and screening of their M2 progeny seed can also be carried out on other species, for instance soybean (see, e.g. U.S. Pat. No. 5,084,082). Alternatively, mutant seeds to be screened for herbicide tolerance are obtained as a result of fertilization with pollen mutagenized by chemical or physical means. Another method of obtaining herbicide-tolerant alleles is by selection in plant cell cultures. Explants of plant tissue, e.g. embryos, leaf disks, etc. or actively growing callus or suspension cultures of a plant of interest are grown on medium in the presence of increasing concentrations of the inhibitory herbicide or an analogous inhibitor suitable for use in a laboratory environment. Varying degrees of growth are recorded in different cultures. In certain cultures, fast-growing variant colonies arise that continue to grow even in the presence of normally inhibitory concentrations of inhibitor. The frequency with which such faster-growing variants occur can be increased by treatment with a chemical or physical mutagen before exposing the tissues or cells to the inhibitor. Putative tolerance-conferring alleles encoding the ALS/AHAS protein are isolated and tested as described herein or using methods well known in the art. Those alleles identified as conferring herbicide tolerance may then be engineered for optimal expression and transformed into the plant. Alternatively, plants can be regenerated from the tissue or cell cultures containing these alleles. A nucleotide substitution encoding an amino acid substitution of the present invention can also be introduced by directed mutagenesis techniques, such as homologous recombination and selected for based on the resulting herbicide-tolerance phenotype (see, e.g. Example 10, Paszkowski et al., EMBO J. 7. 4021-4026 (1988), and U.S. Pat. No. 5,487,992, particularly columns 18-19 and Example 8) or by oligonucleotide-based mutagenesis (see for example U.S. Pat. No. 5,563,350 or 5,756,325). Accordingly, the present invention also discloses methods of obtaining a plant, plant cell or plant tissue tolerant to an inhibitor of ALS/AHAS activity. In a preferred embodiment, the method comprises introducing into said plant, plant cell or plant tissue a DNA molecule comprising a nucleotide sequence of the present invention, wherein said plant, plant cell or plant tissue is tolerant to said inhibitor. In another preferred embodiment, the method comprises introducing a nucleotide substitution of the present invention in a nucleotide sequence of said plant, plant cell or plant tissue which encodes a protein having ALS/AHAS activity, wherein the nucleotide substitution codes for an amino acid substitution conferring tolerance to an inhibitor of ALS/AHAS activity. Preferably, the nucleotide substitution is introduced in said nucleotide sequence by mutagenesis, preferably by chemical or physical mutagenesis, or by homologous recombination or oligonucleotide-based mutagenesis. The present invention thus also encompasses plants, plant cells or tissues comprising a nucleotide sequence encoding a protein having ALS/AHAS activity comprising an amino acid substitution of the present invention, wherein the plant, plant cell or tissue is obtained as described immediately above. Transformation or mutagenesis of virtually any type of plant, both monocot and dicot is contemplated in the present invention. Among the crop plants for which transformation to herbicide tolerance is particularly contemplated are corn, wheat, rice, millet, oat, barley, sorghum, sunflower, sweet potato, alfalfa, sugar beet, Brassica species, tomato, pepper, soybean, tobacco, melon, squash, potato, peanut, pea, cotton, or cacao. The nucleotide sequences of the present invention may also be used to transform ornamental species, such as rose, and woody species, such as pine and poplar. Various chemicals groups, such as the imidazolinones, sulfonylureas and triazolopyrimidines, are known as inhibitors of ALS/AHAS activity. In the present invention, preferred inhibitors of ALS/AHAS activity are imidazolinone herbicides (Hart et al. 1991, CRC Press. Pp. 247-256). Examples of imidazolinone herbicides are imazapyr (Orwick et al., Proc. South. Weed Sci. Soc., Annu. Mtg., 36 th , 1983, p. 291), imazaquin (U.S. Pat. No. 4,798,619) and imazethapyr (U.S. Pat. No. 4,798,619). As described in the “Herbicide Handbook of the Weed Science Society of America”, 6th Ed., (1989), imazapyr (2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridinecarboxylic acid), is a non-specific, broad-spectrum herbicide, whereas both imazaquin (2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-quinolinecarboxylic acid), and imazethapyr (2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl-3-pyridinecarboxylic acid) are crop-specific herbicides. Other imidazolinone herbicides suitable for the present invention are for example Imazapic (Wixson et al. (1992), Proc. South. Weed Sci. Soc. 45, 341), Imazamox ((+)-5-methoxymethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) nicotinic acid), Imazamethabenz-Me (U.S. Pat. No. 4,188,487). Examples of sulfonylurea herbicides include 1-(2-chloro-phenylsulfonyl)-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea (chlorsulfuron), 1-[(2-methoxycarbonylphenyl)-sulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin yl-2-yl)urea (metsulfuron-methyl), 1-[(2-methoxycarbonyl-phenyl)sulfonyl]-3-methyl-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea (DPX-L5300), 1-[(2-methoxycarbonylphenylmethyl)-sulfonyl]-3-(4,6-dimethoxypyrimid-2-yl) urea (bensulfuronmethyl), 1-[(2-methoxycarbonylphenyl)sulfonyl]-3-(di-methylpyrimid-2-yl)urea (sulfometuron-methyl), 1-[(2-methoxycarbonylthienyl)sulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea (thiameturon-methyl), 1-[(2-ethoxycarbonylphenyl)sulfonyl]-3-(4-chloro-6-methoxypyrimid-2-yl)urea (chlorimuron-ethyl), 1-[(3-(N,N-dimethylamino-carbonyl)-pyrid-2-yl)sulfonyl]-3-(4,6-dimethoxypyrimid-2-yl)urea (nicosulfuron, SL 950), 1-[3-(ethylsulfonyl)-pyrid-2-yl)sulfonyl]-3-(4,6-dimethoxypyrimid-2-yl)urea (DPX-E9636), 1-[(2-(2-chloroethyl)-phenyl)sulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea (triasulfuron), 1-[(2-methoxycarbonylphenyl)-sulfonyl]-3-(4,6-bis-(difluoromethoxy)pyrimid-2-yl)urea (pirimisulfuron), 1-[(4-ethoxycarbonyl-1-methyl-1,2-imidazol-5-yl)-sulfonyl]-3-(4,6-dimethoxypyrimid-2-yl)urea (pyrazosulfuron-methyl), 1-[(2-methoxycarbonylphenyl)sulfonyl]-3-(4-ethoxy-6-methylamino-1,3,5-triazin-2-yl)urea (DPX-A7881), cinosulfuron (CGA 142464), 1-[(3-trifluoroethylpyrid-2-yl)sulfonyl]-3-(4,6-dimethoxypyrimid-2-yl)urea (flazasulfuron, SL 160), 1-[(N-methyl-N-methylsulfonylamo)sulfonyl]-3-(4,6-dimethoxypyrimd-2-yl) urea (amidosulfuron, Hoe 75032),1-[(N-ethylsulfonyl-N-methylamino)sulfonyl]-3-(4,6-dimethoxypyrimid-2-yl)urea (SH-1),1-[(N-ethyl-N-ethylsulfonylamino)sulfonyl]-3-(4,6-dimethoxypyrimid-2-yl)urea (SH-2),1-[(2-ethoxy-phenoxy)sulfonyl]-3-(4,6-dimethoxypyrimid-2-yl)urea (SH-3),1-[(N-(dimethylaminosulfonyl)-N-methylamino)sulfonyl]-3-(4,6-dimethoxypyrimid-2-yl)urea (SH-4). An amino acid substitution of the present invention preferably confers tolerance to an imidazolinone-type herbicide, as for example an imidazolinone herbicide listed above. The amino acid substitution may also confer cross-tolerance to other chemical groups of ALS/AHAS inhibitors, e.g. to sulfonylureas. Alternatively, an amino acid substitution of the present invention confers imidazolinone-specific tolerance, under which it is understood that the amino acid substitution does not confer substantial cross-tolerance to other chemical groups of ALS/AHAS inhibitors. Plants according to the present invention, which are tolerant to an inhibitor of ALS/AHAS activity, are planted in a field and are used for the control of the growth of undesired vegetation in the field. Such methods of control comprise applying to a population of a plant disclosed above, or to the locus where such plants are grown, an effective amount of an inhibitor of ALS/AHAS activity. Preferably, the inhibitor of ALS/AHAS activity is an imidazolinone herbicide. Preferably, the plant is selected from the group consisting of sunflower, sugar cane, soybean, barley, cotton, tobacco, sugar beet, oilseed rape, maize, wheat, sorghum, rye, oats, turf and forage grasses, millet, forage and rice. In another preferred embodiment, the dose of inhibitor used is between 5 g and 1000 g active ingredient (a.i.) per hectare. Preferably, the dose is between 10 g and 200 g a.i. per hectare. Herbicide tolerant plants or lines obtained using the present invention are particularly useful for the control of parasitic weed, e.g. broomrape, in sunflower, when an effective amount of an inhibitor of ALS/AHAS activity is applied thereto. The present invention also discloses polymorphisms between herbicide-sensitive and herbicide-tolerant alleles. Using these polymorphisms, the inventors of the present invention have developed molecular markers used to breed tolerant plants, to follow the presence of the tolerance trait during breeding of plants or lines, and to control the presence of the tolerance trait in a commercial seeds lot. Such polymorphisms and markers are disclosed below. The polymorphisms described herein are preferably used in sunflower breeding, but may in some cases also used in other species. In a preferred embodiment, a polymorphism of the present invention is within less than one centi-Morgan (cM) from the locus corresponding to the amino acid substitution conferring tolerance to an herbicide, more preferably within less than 0.1 cM, even more preferably within less than 0.05 cM, and thus tightly co-segregates with the amino acid substitution. Preferably, a polymorphism is located within the promoter region or termination region of a nucleotide sequence encoding an ALS/AHAS protein. Preferably, a polymorphism is located within the coding region of a nucleotide sequence encoding an ALS/AHAS protein. In a preferred embodiment, the inventors of the present invention disclose three consecutive silent nucleotide substitutions located at positions encoding amino acids 332 to 334 in SEQ ID NO:2 in a nucleotide sequence encoding a herbicide-tolerant ALS/AHAS. Particularly preferred polymorphism are nucleotide substitutions located at third-base positions in such codons, preferably at any one of positions 1229, 1232 or 1235 in SEQ NO: 1 (see e.g. example 5). Accordingly, the present invention discloses detections methods and kits based on such polymorphisms. In a preferred embodiment, the polymorphisms are used in sunflower. In another preferred embodiment, a polymorphism disclosed by the inventors of the present invention is located within the codon encoding the amino acid substitution, which confers tolerance to herbicides. Such polymorphism comprises a nucleotide substitution at any position corresponding to positions 1566-1568 in SEQ ID NO:1. Particularly preferred polymorphisms are at positions corresponding to positions 1566-1567 in SEQ ID NO:1, more particularly at a position corresponding to position 1566 in SEQ ID NO:1. A particularly preferred polymorphism is a substitution of a “T” by a “C” at this position. A preferred marker disclosed in the present invention is a CAPS marker taking advantage of the creation of a new NspI restriction site by such “T” to “C” substitution. Accordingly, the present invention discloses detections methods and kits based on such polymorphisms, in particular based on an altered restriction digestion pattern (see for example example 4). Such polymorphism and molecular markers based thereupon, are used preferably in sunflower but may in somes cases also be used in other species. In another preferred embodiment, the present invention discloses an isolated DNA molecule comprising about 15 successive nucleotides, preferably about 20 successive nucleotides, preferably about 50 successive nucleotides, of the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, wherein said DNA molecule comprises a nucleotide polymorphism, wherein said polymorphism allows to differentiate between an allele conferring tolerance to an inhibitor of ALS/AHAS activity and an allele sensitive to said inhibitor. Such DNA molecules are preferably used as molecular markers. Preferably, the polymorphism is located at any one of positions 1229, 1232 or 1235 in SEQ NO: 1. Preferably, in this case, the DNA molecule comprises any one of the nucleotide sequences “GACTGTC” or “TACGGTT”, or a sequence complementary thereto. In another preferred embodiment, the polymorphism is located at any position corresponding to positions 1566-1568 in SEQ ID NO:1, more preferably at positions corresponding to positions 1566-1567 in SEQ ID NO:1, even more preferably at a position corresponding to position 1566 in SEQ ID NO:1. Preferably, in this case, the DNA molecule comprises any one of the nucleotide sequences “GCATGC” or “GTATGC”, or a sequence complementary thereto, or any one of the nucleotide sequences “CAGCATG”, “CAGTATG”, or a sequence complementary thereto. Methods for detection of polymorphisms, which are well known in the art, are applied in the context of the present invention. Some of such methods or part thereof are described below. In particular, methods using the PCR amplification technique are contemplated within the scope of the present invention. A number of oligonucleotides are disclosed which are particularly useful in carrying out the present invention. For example, SEQ ID NO:8 (HiNK379) or SEQ ID NO:9 (HiNK415) is used to detect a polymorphism at the site of the amino acid substitution conferring tolerance to an herbicide. SEQ ID NO:10 (HiNK451), SEQ ID NO:11 (HiNK414), SEQ ID NO:12 (HiNK452) or SEQ ID NO:13 (HiNK415) are used to detect silent polymorphisms at positions corresponding to positions 1229, 1232 or 1235 in SEQ ID NO:1. Variations of the sequences of such nucleotides are however also contemplated and also form part of the instant invention. For example, addition or removal of a few nucleotides, for example 1-10 nucleotides (nt), preferably 1-5 nt, even more preferably 1-3 nt, at either end of the oligonucleotide are also contemplated. Also, a shift of the nucleotide on the nucleotide sequence of SEQ ID NO:1, for example by 1-10 nt, preferably 1-5 nt is also contemplated. Other preferred oligonucleotides of the present invention comprises about 6 successive nt from about position 1536 to about position 1596 in the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, or in a sequence complementary thereto, or about 6 successive nt from about position 1199 to about position 1265 in the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, or in a sequence complementary thereto. Preferred oligonucleotides are about 8 to 50 nt long, more preferably 10 to 30 nt long, even more preferably 15 to 25 nt long. DNA molecules comprising a site comprising a polymorphism of the present invention are preferably about 100-3,000 nt long, more preferably about 200 to 2,000 nt long, even more preferably about 500 to 1,500. Preferably, such DNA molecules are amplified PCR fragments. In another preferred embodiment, the molecular markers of the present invention allow to differentiate between plants heterozygous or homozygous for the herbicide-tolerance allele. Examples of such co-dominant markers are described in examples 4 and 5. In another preferred embodiment, the present invention provides methods for mapping the tolerance trait in tolerant plants, for determining whether a herbicide-tolerant allele is present in a plant, and for transferring the tolerance to a susceptible or less tolerant plant. For example, the methods are used in breeding new plants or lines tolerant to herbicides or to control the quality of a seeds lot. The molecular markers disclosed herein allow for quicker release of herbicide tolerant lines to the market and for a better quality control of commercial seeds lots. Spraying of plants with the herbicide is avoided or greatly reduced. Such methods are preferably used in sunflower breeding, but can also be applied for other crops. In a preferred embodiment, a molecular marker of the present invention is beneficially used when herbicide-tolerant plants are selected from herbicide sensitive plants and is for example used on seeds originating from any self-pollination or cross-pollination of an herbicide tolerant plant. Herbicide-sensitive seeds originated from the cross and not carrying the herbicide tolerant allele or seeds contaminations accidentally present in a populations of seeds obtained from the cross are thus detected and can be set aside. The molecular markers and methods of the present invention are also particularly beneficial in the various steps leading to a commercial variety, such as in breeding programs and in the seeds production process. For example, the present invention is used to introgress the herbicide tolerance allele and, optionally, to also introgress another trait that co-segregates with the herbicide tolerance trait, into a plant, for example an elite inbred line. The introgression of herbicide tolerance into the elite line is for example achieved by recurrent selection breeding, for example by backcrossing. In this case, the elite line (recurrent parent) is first crossed to a donor inbred (the non-recurrent parent) that carries the appropriate herbicide tolerance trait. The progeny of this cross is then mated back to the recurrent parent followed by selection in the resultant progeny for herbicide tolerance. After three, preferably four, more preferably five or more generations of backcrosses with the recurrent parent with selection for herbicide tolerance, the progeny is heterozygous for the locus harboring herbicide tolerance, but is like the recurrent parent for most or almost all other genes (see, for example, Poehlman & Sleper (1995) Breeding Field Crops, 4th Ed., 172-175; Fehr (1987) Principles of Cultivar Development, Vol. 1: Theory and Technique, 360-376, incorporated herein by reference). Selection for herbicide tolerance after each cross is carried out using the present invention, i.e. by testing seeds resulting from each cross with the molecular markers and methods of the present invention. In a preferred embodiment, the present invention therefore encompasses methods of breeding a herbicide tolerance allele into an elite line sensitive or less tolerant to the herbicide using the teachings of the present invention and comprising the following steps: crossing a first plant with a second plant, wherein one of the plants is tolerant to the herbicide, harvesting the seeds resulting from the cross, obtaining a sample of the seed or of a plant grown therefrom, detecting in said sample a DNA molecule of the present invention, the presence of said DNA molecule being indicative of an allele conferring tolerance to a herbicide, wherein said plant is tolerant to a herbicide and comprise the herbicide tolerant allele. The present invention is also conveniently used in the production of stable homogenous inbred lines or cultivars (also sometimes called varieties), whereby a particular line is self-pollinated until satisfactory purity and homogeneity of the line is reached. The present invention is similarly used for the commercial production of seeds of a particular inbred line or cultivar. Here again, after each cross a method of the present invention is applied to the seeds resulting from the cross and only herbicide tolerant plants are selected. In a preferred embodiment, the present invention therefore encompasses methods of producing herbicide tolerant seeds using the teachings of the present invention and comprising the following steps: crossing a herbicide tolerant plant with itself, harvesting the seeds resulting from the cross, obtaining a sample of the seed or of a plant grown therefrom, detecting in said sample a DNA molecule of the present invention, the presence of said DNA molecule being indicative of an allele conferring tolerance to a herbicide. In a preferred embodiment, the absence of a DNA molecule of the present invention in the sample, shows the absence of a herbicide-sensitive allele in said plant. A co-dominant marker of the present invention is particularly useful for determining whether a plant to be screened is homozygous or heterozygous for the tolerant allele. Preferably, only homozygous plant are further used in the breeding or seeds production program. Such difference between homozygous or heterozygous plants cannot be detected by chemical sprays. Current methods indeed require an additional cross to be able to differentiate between hetero- and homozygocity. The present invention therefore saves one cross which is of great advantage. Similarly, the present invention is used in hybrid seed production. In this case, the present invention is used to assure that all hybrid seeds that germinate and grow in the field are herbicide tolerant. Preferably, molecular markers of the present invention also also used in quality assurance to ensure that the herbicide-tolerance trait is present in the hybrid seeds, and if desired, to ensure that the tolerance allele is in the homozygous state in hybrids seeds. In a preferred embodiment, the present invention therefore encompasses methods producing herbicide tolerance seeds using the teachings of the present invention and comprising the following steps: crossing a first plant with a second plant, wherein one of the plants is tolerant to the herbicide, harvesting the seeds resulting from the cross, obtaining a sample of the seed or of a plant grown therefrom, detecting in said sample a DNA molecule of the present invention, the presence of said DNA molecule being indicative of an allele conferring tolerance to a herbicide, wherein said sunflower plant is tolerant to a herbicide and comprise the herbicide tolerant allele. Co-dominant markers are preferably used. The present invention therefore also disclose methods to determine whether a plant is homozygous or heterozygous for a herbicide-tolerant allele comprising obtaining a sample of a plant, detecting in said sample a DNA molecule of the present invention using a co-dominant marker, determining whether a nucleotide sequence encoding a herbicide tolerance ALS/AHAS of the present invention is heterozygous or homozygous in said plant. The present invention thus provides a significant advancement to commercial breeding and seeds production processes using herbicide tolerance. Using the present invention large commercial quantities of herbicide tolerant seeds are produced with low impact on the environment and reduced cost. Plant Transformation A DNA molecule of the present invention is incorporated in plant or bacterial cells using conventional recombinant DNA technology. Preferably, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e., not normally present) using standard cloning procedures known in the art. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences in a host cell containing the vector. A large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses. The components of the expression system may also be modified to increase expression. For example, truncated sequences, nucleotide substitutions, nucleotide optimization or other modifications may be employed. Expression systems known in the art can be used to transform virtually any crop plant cell under suitable conditions. A DNA molecule of the present invention is preferably stably transformed and integrated into the genome of the host cells. In another preferred embodiment, it is located on a self-replicating vector. Examples of self-replicating vectors are viruses, in particular gemini viruses. Transformed cells are regenerated into whole plants such that the DNA molecule confers herbicide tolerance in the transgenic plants. A. Requirements for Construction of Plant Expression Cassettes Gene sequences intended for expression in transgenic plants is first assembled in expression cassettes behind a suitable promoter expressible in plants. The expression cassettes may also comprise any further sequences required or selected for the expression of the heterologous DNA sequence. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns and sequences intended for the targeting of the gene product to specific organelles and cell compartments. These expression cassettes can then be easily transferred to the plant transformation vectors described infra. The following is a description of various components of typical expression cassettes. 1. Promoters The selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the heterologous DNA sequence in the plant transformed with this DNA sequence. Selected promoters will express heterologous DNA sequences in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters known in the art can be used. For example, for constitutive expression, the CaMV 35S promoter, the rice actin promoter, or the ubiquitin promoter may be used. For regulatable expression, the chemically inducible PR-1 promoter from tobacco or Arabidopsis may be used (see, e.g., U.S. Pat. No. 5,689,044). 2. Transcriptional Terminators A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the heterologous DNA sequence and its correct polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35 S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledonous and dicotyledonous plants. 3. Sequences for the Enhancement or Regulation of Expression Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants. For example, various intron sequences such as introns of the maize Adhl gene have been shown to enhance expression, particularly in monocotyledonous cells. In addition, a number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. 4. Coding Sequence Optimization The coding sequence of the selected gene may be genetically engineered by altering the coding sequence for optimal expression in the crop species of interest. Methods for modifying coding sequences to achieve optimal expression in a particular crop species are well known (see, e.g. Perlak et al., Proc. Natl. Acad. Sci. USA 88: 3324 (1991); and Koziel et al., Bio/technol. 11: 194 (1993)). 5. Targeting of the Gene Product within the Cell Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (e.g. Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol. 13: 411-418 (1989)). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous products encoded by DNA sequences to these organelles. In addition, sequences have been characterized which cause the targeting of products encoded by DNA sequences to other cell compartments. Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)). By the fusion of the appropriate targeting sequences described above to heterologous DNA sequences of interest it is possible to direct this product to any organelle or cell compartment. B. Construction of Plant Transformation Vectors Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptII gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), the manA gene, which allows for positive selection in the presence of mannose (Miles and Guest (1984) Gene, 32:41-48; U.S. Pat. No. 5,767,378), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), and the EPSPS gene, which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642). 1. Vectors Suitable for Agrobacterium Transformation Many vectors are available for transformation using Agrobacterium tumefaciens . These typically carry at least one T-DNA border sequence and include vectors such as pBIN 19 (Bevan, Nucl. Acids Res. (1984)). Typical vectors suitable for Agrobacterium transformation include the binary vectors pCIB200 and pCIB2001, as well as the binary vector pCIB10 and hygromycin selection derivatives thereof. (See, for example, U.S. Pat. No. 5,639,949). 2. Vectors Suitable for Non- Agrobacterium Transformation Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Typical vectors suitable for non- Agrobacterium transformation include pCIB3064, pSOG19, and pSOG35. (See, for example, U.S. Pat. No. 5,639,949). C. Transformation Techniques Once the coding sequence of interest has been cloned into an expression system, it is transformed into a plant cell. Methods for transformation and regeneration of plants are well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro-injection, and microprojectiles. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant cells. Transformation techniques for dicotyledons are well known in the art and include Agrobacterium -based techniques and techniques that do not require Agrobacterium . Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG- or electroporation-mediated uptake, particle bombardment-mediated delivery, or microinjection. In each case the transformed cells are regenerated to whole plants using standard techniques known in the art. Transformation of most monocotyledon species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, particle bombardment into callus tissue, as well as Agrobacterium -mediated transformation. D. Plastid Transformation In another preferred embodiment, a nucleotide sequence encoding a polypeptide having 1917, 2092, or 7724 activity is directly transformed into the plastid genome. Plastid expression, in which genes are inserted by homologous recombination into the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein. In a preferred embodiment, the nucleotide sequence is inserted into a plastid-targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplasmic for plastid genomes containing the nucleotide sequence are obtained, and are preferentially capable of high expression of the nucleotide sequence. Plastid transformation technology is for example extensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818, and 5,877,462 in PCT application no. WO 95/16783 and WO 97/32977, and in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305, all incorporated herein by reference in their entirety. The basic technique for plastid transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the nucleotide sequence into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45). The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub, J. M., and Maliga, P. (1993) EMBO J. 12, 601-606). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the invention. Methods of Screening/Detecting Polymorphisms Methods for screening for polymorphisms in nucleic acids for example include polymerase chain reaction (PCR), direct sequencing of nucleic acids, single strand polymorphism assay, ligase chain reaction, enzymatic cleavage, and southern hybridization. Screening for nucleic acids can be accomplished by direct sequencing of nucleic acids. In fact, putative mutants identified by other methods may be sequenced to determine the exact nature of the mutation. Nucleic acid sequences can be determined through a number of different techniques which are well known to those skilled in the art. In order to sequence the nucleic acid, sufficient copies of the material must preferably be first amplified. Amplification of a selected, or target, nucleic acid sequence may be carried out by any suitable means. (See generally Kwoh, D. and Kwoh, T., Am Biotechnol Lab, 8, 14 (1990)) Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction, ligase chain reaction (see Barany, Proc Natl Acad Sci USA 88, 189 (1991)), strand displacement amplification (see generally Walker, G. et al., Nucleic Acids Res. 20, 1691 (1992); Walker. G. et al., Proc Natl Acad Sci USA 89, 392 (1992)), transcription-based amplification (see Kwoh, D. et al., Proc Natl Acad Sci USA, 86, 1173 (1989)), self-sustained sequence replication (or “3SR”) (see Guatelli, J. et al., Proc Natl Acad Sci USA, 87, 1874 (1990)), the Q.beta. replicase system (see Lizardi, P. et al., Biotechnology, 6, 1197 (1988)), nucleic acid sequence-based amplification (or “NASBA”) (see Lewis, R., Genetic Engineering News, 12(9), 1 (1992)), the repair chain reaction (or “RCR”) (see Lewis, R., Genetic Engineering News, 12(9), 1 (1992)), and boomerang DNA amplification (or “BDA”) (see Lewis, R., Genetic Engineering News, 12(9), 1 (1992)). Polymerase chain reaction is currently preferred. The present invention provides several methods for detecting Cleaved Amplified Polymorphic Sequences (CAPS; Konieczny et al., The Plant Journal 4(2):403-410, 1993) and for detecting Single Nucleotide Polymorphisms (SNPs) with a method termed “CAMPS” for Cleaved Amplified Modified Polymorphic Sequences, also known as dCAPS (Neff et al, 1998. Plant J. 14: 387-392; Michaels and Amasino, 1998. Plant J 14: 381-385). In the CAPS method, a nucleic acid containing a polymorphic restriction site is amplified using primers flanking the restriction site. The resulting PCR product is digested with the restriction endonuclease corresponding to the polymorphic restriction site, and the digested products are analyzed by gel electrophoresis. In the CAMPS method, a nucleic acid molecule containing a single nucleotide polymorphism is mutagenized during PCR amplification to create a restriction endonuclease recognition site which includes the single nucleotide polymorphism. The resulting PCR product is digested with the corresponding restriction endonuclease, and the restriction endonuclease-treated products are analyzed for cleavage in a rapid high through-put assay. The primers and oligonucleotides used in the methods of the present invention are preferably DNA, and can be synthesized using standard techniques and, when appropriate, detectably labeled using standard methods (Ausubel et al., supra). Detectable labels that can be used to tag the primers and oligonucleotides used in the methods of the invention include, but are not limited to, digoxigenin, fluorescent labels (e.g., fluorescein and rhodamine), enzymes (e.g., horseradish peroxidase and alkaline phosphatase), biotin (which can be detected by anti-biotin specific antibodies or enzyme-conjugated avidin derivatives), radioactive labels (e.g., .sup.32 P and sup. 125 I), calorimetric reagents, and chemiluminescent reagents. The labels used in the methods of the invention are detected using standard methods. The specific binding pairs useful in the methods of the invention include, but are not limited to, avidin-biotin, streptavidin-biotin, hybridizing nucleic acid pairs, interacting protein pairs, antibody-antigen pairs, reagents containing chemically reactive groups (e.g., reactive amino groups), and nucleic acid sequence-nucleic acid binding protein pairs. The solid supports useful in the methods of the invention include, but are not limited to, agarose, acrylamide, and polystyrene beads; polystyrene microtiter plates (for use in, e.g., ELISA); and nylon and nitrocellulose membranes (for use in, e.g., dot or slot blot assays). Some methods of the invention employ solid supports containing arrays of nucleic acid probes. In these cases, solid supports made of materials such as glass (e.g., glass plates), silicon or silicon-glass (e.g., microchips), or gold (e.g., gold plates) can be used. Methods for attaching nucleic acid probes to precise regions on such solid surfaces, e.g., photolithographic methods, are well known in the art, and can be used to make solid supports for use in the invention. (For example, see, Schena et al., Science 270:467-470, 1995; Kozal et al., Nature Medicine 2(7):753-759, 1996; Cheng et al., Nucleic Acids Research 24(2):380-385, 1996; Lipshutz et al., BioTechniques 19(3):442-447, 1995; Pease et al., Proc. Natl. Acad. Sci. USA 91:5022-5026, 1994; Fodor et al., Nature 364:555-556, 1993; Pirrung et al., U.S. Pat. No. 5,143,854; and Fodor et al., WO 92/10092.) In general, DNA amplification techniques such as the foregoing involve the use of a probe, a pair of probes, or two pairs of probes which specifically bind to DNA encoding the gene of interest, but do not bind to DNA which does not encode the gene, under the same hybridization conditions, and which serve as the primer or primers for the amplification of the gene of interest or a portion thereof in the amplification reaction. Nucleic acid sequencing can be performed by chemical or enzymatic methods. The enzymatic method relies on the ability of DNA polymerase to extend a primer, hybridized to the template to be sequenced, until a chain-terminating nucleotide is incorporated. The most common methods utilize didoexynucleotides. Primers may be labelled with radioactive or fluorescent labels. Various DNA polymerases are available including Klenow fragment, AMV reverse transcriptase, Thermus aquaticus DNA polymerase, and modified T7 polymerase. Recently, single strand polymorphism assay (“SSPA”) analysis and the closely related heteroduplex analysis methods have come into use as effective methods for screening for single-base polymorphisms (Orita, M. et al., Proc Natl Acad Sci USA, 86, 2766 (1989)). In these methods, the mobility of PCR-amplified test DNA from test sources is compared with the mobility of DNA amplified from control sources by direct electrophoresis of samples in adjacent lanes of native polyacrylamide or other types of matrix gels. Single-base changes often alter the secondary structure of the molecule sufficiently to cause slight mobility differences between the normal and mutant PCR products after prolonged electrophoresis. Ligase chain reaction is yet another recently developed method of screening for mutated nucleic acids. Ligase chain reaction (LCR) is also carried out in accordance with known techniques. LCR is especially useful to amplify, and thereby detect, single nucleotide differences between two DNA samples. In general, the reaction is called out with two pairs of oligonucleotide probes: one pair binds to one strand of the sequence to be detected; the other pair binds to the other strand of the sequence to be detected. The reaction is carried out by, first, denaturing (e.g., separating) the strands of the sequence to be detected, then reacting the strands with the two pairs of oligonucleotide probes in the presence of a heat stable ligase so that each pair of oligonucleotide probes hybridize to target DNA and, if there is perfect complementarity at their junction, adjacent probes are ligated together. The hybridized molecules are then separated under denaturation conditions. The process is cyclically repeated until the sequence has been amplified to the desired degree. Detection may then be carried out in a manner like that described above with respect to PCR. Southern hybridization is also an effective method of identifying differences in sequences. Hybridization conditions, such as salt concentration and temperature can be adjusted for the sequence to be screened. Southern blotting and hybridizations protocols are described in Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience), pages 2.9.1-2.9.10. Probes can be labelled for hybridization with random oligomers (primarily 9-mers) and the Klenow fragment of DNA polymerase. Very high specific activity probe can be obtained using commercially available kits such as the Ready-To-Go DNA Labelling Beads (Pharmacia Biotech), following the manufacturer's protocol. Briefly, 25 ng of DNA (probe) is labelled with .sup.32 P-dCTP in a 15 minute incubation at 37.degree. C. Labelled probe is then purified over a ChromaSpin (Clontech) nucleic acid purification column. Possible competition of probes having high repeat sequence content, and stringency of hybridization and washdown will be determined individually for each probe used. Alternatively, fragments of a candidate gene may be generated by PCR, the specificity may be verified using a rodent-human somatic cell hybrid panel, and subcloning the fragment. This allows for a large prep for sequencing and use as a probe. Once a given gene fragment has been characterized, small probe preps can be done by gel- or column-purifying the PCR product. These mismatch detection protocols use samples generated by PCR and thus require use of very little genomic template. All of these methods can provide very good clues regarding the location of the sequence change which leads to the appearance of anomalous bands, hence facilitating subsequent cloning and sequencing strategies. Methods used in the present invention to detect polymorphisms also include Taqman (Livak, 1999, Genet Anal 14: 143-149), FRET (Chen et al, 1998, Genome Res 8:549-546) and Pyrosequencing (Ahmadian et al, 2000, Anal Biochem 280: 103-110; Alderborn et al, 2000, Genome Res 10: 1249-1258; Nordstrom et al., 2000, Biotechnol Appl Biochem 31: 107-112). Methods of screening for mutated nucleic acids can be carried out using either deoxyribonucleic acids (“DNA”) or messenger ribonucleic acids (“mRNA”) isolated from the biological sample. During periods when the gene is expressed, mRNA may be abundant and more readily detected. However, these genes are temporally controlled and, at most stages of development, the preferred material for screening is DNA. Alternatively, the detection of a mutated gene is carried out by collecting a biological sample and testing for the presence or form of the protein produced by the gene. The mutation in the gene may result in the production of a mutated form of the peptide or the lack of production of the gene product. In this embodiment, the determination of the presence of the polymorphic form of the protein can be carried out, for example, by isoelectric focusing, protein sizing, or immunoassay. In an immunoassay, an antibody that selectively binds to the mutated protein can be utilized. Such methods for isoelectric focusing and immunoassay are well known in the art, and are discussed in further detail below. Changes in the size or charge of the polypeptide can be identified by isoelectric focusing or protein sizing techniques. Changes resulting in amino acid substitutions, where the substituted amino acid has a different charge than the original amino acid, can be detected by isoelectric focusing. Isoelectric focusing of the polypeptide through a gel having an ampholine gradient at high voltages separates proteins by their pI. The pH gradient gel can be compared to a simultaneously run gel containing the wild-type protein. Protein sizing techniques such as protein electrophoresis and sizing chromatography can also be used to detect changes in the size of the product. As an alternative to isoelectric focusing or protein sizing, the step of determining the presence of the mutated polypeptides in a sample may be carried out by an antibody assay with an antibody which selectively binds to the mutated polypeptides (i.e., an antibody which binds to the mutated polypeptides but exhibits essentially no binding to the wild-type polypeptide without the polymorphism in the same binding conditions). Antibodies used to bind selectively the products of the mutated genes can be produced by any suitable technique. For example, monoclonal antibodies may be produced in a hybridoma cell line according to the techniques of Kohler and Milstein, Nature, 265, 495 (1975), which is hereby incorporated by reference. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody. The mutated products of genes which are associated with autism may be obtained from a human patient, purified, and used as the immunogen for the production of monoclonal or polyclonal antibodies. Purified polypeptides may be produced by recombinant means to express a biologically active isoform, or even an immunogenic fragment thereof may be used as an immunogen. Monoclonal Fab fragments may be produced in Escherichia coli from the known sequences by recombinant techniques known to those skilled in the art. (See, e.g., Huse, W., Science 246, 1275 (1989)) (recombinant Fab techniques). The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. detailed-description description="Detailed Description" end="lead"? |
Alerting inhaler for inhalation therapy |
The alerting inhaler has a smaller air flow tube, with a front air inlet closed by a cover with a hole for slow, deep inhalation. An air inlet leads to alert giving whistle or vibrator or rotating vanes and longer mouth piece. The long spray rod of the drug vial with drug(s) in solvent, propellant, enters to air tube and ends in a novel jet spray nozzle at back of mouth piece. A handle for support, helps easy pressing of drug vial on the body. The long mouth piece is kept in mouth, air is sucked, alert is heard, drug vial is pressed, the spray is released at back of throat for easy flow into wind pipes and lungs for good relief. The inhaler gives an alert to time the spray, with delivery of spray at the back of throat and not outside mouth as now, has no mouth wastage, and reliably delivers drugs to lungs for good effect even for children and elderly persons! |
1) an alerting inhaler comprising of an air inlet guarded by a stopper with an appropriate hole to develop deep and longer inspiration, the said air inlet fixed to a whistle or vibrator, the said whistle gives sound alert for the drug spray trigger, the said whistle is fixed to a drug spray path, formed by spray rod of the drug vial with a fine jet nozzle to back spray into the adjustable mouth piece, the said mouth piece directs the spray into wind passages, such that the said drug spray with the inspired air flows into the lungs. 2) an alerting inhaler as claimed in claim 1, wherein the said air inlet has a cover at the front, the said cover having a hole preferably adjustable for entry of air. 3) an alerting inhaler as claimed in claim 1, wherein the said whistle or vibrator is placed before the spray, the said whistle is activated by the inspired air, the said whistle alerts to time the spray. 4) an alerting inhaler as claimed in claim 1, wherein the said spray path comprises of a drug spray vial with a spray rod, the said spray rod fixed behind the said whistle, the said rod fitting a jet nozzle for the spray to be formed, the said spray is directed back through the mouthpiece into lungs. 5) an alerting inhaler as claimed in claim 1, wherein the said mouthpiece comprises of an outer tubes sliding on the inner tube on a screw or slot to adjust lip to throat distance for correct spray. 6) an alerting inhaler as claimed in claim 1, wherein the said drug vial is placed along the long axis to the said air path. 7) an alerting inhaler as claimed in claim 1, wherein the said drug vial is placed at perpendicular to the air path. 8) an alerting inhaler as claimed in claim 1, wherein the vibrator is placed instead of the whistle before the said spray to alert the user by vibrations. 9) an alerting inhaler as claimed in claim 1, wherein a coloured rotating wheel is placed in a transparent enclosure is placed instead of whistle before the said spray to alert the user by visual clue. 10) an alerting inhaler as claimed in claim 1, wherein the inhaler is made of plastic or metal. 11) an alerting inhaler as claimed in claim 1, wherein the drug vial comprises of strong metal can with a, crimped cap covering the mouth of the can and a drug metering valve in the cap. 12) an alerting inhaler as claimed in claim 1, wherein the drug formulation comprises of an active agent for the medical need, as a micronised particle, in a solvent, with a propellant with or without surfactants or exceipients the said drug when sprayed reaches the lung for rapid relief. 13) an alerting inhaler as claimed in claim 1, wherein a handle is fixed to the body to steady and easily press the drug vial. 14) an alerting inhaler as claimed in claim 1, wherein the inhalation drug may be single or combination of active pharmacological agents as described in the specification. 15) A method of spraying the contents of drug vial for inhalation therapy in the mouth comprising of (a) Placing the inhaler jet spray at the depth of mouth so that spray is released at the back of the mouth, (b) Use an alert mechanism for effective spray using alert trigger which gives more effective spray in correct path of inhalation. (c) Effecting slow and deep inhalation of air through a smaller hole of inhaler air inlet such that the inspired air flows into lungs with drugs for relief. 16) An alerting alerting inhaler for inhalation therapy as described in the specification and as illustrated by way of drawings. 17) A method of spraying the contents of vial in the mouth as described in the specification. |
<SOH> TECHNICAL FIELD <EOH>This invention in general relates to human necessities. More particularly this invention relates to medical equipments. Precisely this invention relates to a novel kind of inhaler for successfully delivering drugs to lungs where the drug is absorbed and works, producing relief. |
<SOH> SUMMARY OF THE INVENTION <EOH>An inhaler comprising of a smaller air inlet guarded by a stopper with an appropriate hole, the said air inlet leads to a whistle (or vibrator or rotating wheel), the said whistle leads to a spray rod with jet spray nozzle, the said spray path leads to a mouth piece for the spray with the inspired air to flow into the lungs. The drug vial with the desired drugs in solvent and propellants is fixed in a body incorporated with the air path for easy pressing and release of spray. |
Human cdnas and proteins and uses thereof |
The invention concerns GENSET polynucleotides and polypeptides. Such GENSET products may be used as reagents in forensic analyses, as chromosome markers, as tissue/cell/organelle-specific markers, in the production of expression vectors. In addition, they may be used in screening and diagnosis assays for abnormal GENSET expression and/or biological activity and for screening compounds that may be used in the treatment of GENSET-related disorders. |
1. An isolated polynucleotide, comprising a nucleic acid sequence selected from the group consisting of: a) a polynucleotide of an odd SEQ ID NO., or of a human cDNA of a deposited clone, encoding at least any single integer from 6 to 776 amino acids of any one even SEQ ID NO., b) a polynucleotide of an odd SEQ ID NO., or of a human cDNA of a deposited clone, encoding the signal peptide sequence of any one even SEQ ID NO., c) a polynucleotide of an odd SEQ ID NO., or of a human cDNA of a deposited clone, encoding a mature polypeptide sequence of any one even SEQ ID NO., d) a polynucleotide of an odd SEQ ID NO., or of a human cDNA of a deposited clone, encoding a full length polypeptide sequence of any one even SEQ ID NO., e) a polynucleotide of an odd SEQ ID NO., or of a human cDNA of a deposited clone, encoding a polypeptide sequence of a biologically active fragment of any one even SEQ ID NO., f) a polynucleotide encoding a polypeptide sequence of at least any single integer from 6 to 776 amino acids of any one even SEQ ID NO. or of a polypeptide encoded by a human cDNA of a deposited clone, g) a polynucleotide encoding a polypeptide sequence of a signal peptide of any one even SEQ ID NO. or of a signal peptide encoded by a human cDNA of a deposited clone, h) a polynucleotide encoding a polypeptide sequence of a mature polypeptide of any one even SEQ ID NO. or of a mature polypeptide encoded by a human cDNA of a deposited clone, i) a polynucleotide encoding a polypeptide sequence of a full length polypeptide of any one even SEQ ID NO. or of a mature polypeptide encoded by a human cDNA of a deposited clone, j) a polynucleotide encoding a polypeptide sequence of a biologically polypeptide of any one even SEQ ID NO., or of a biologically polypeptide encoded by a human cDNA of a deposited clone, k) a polynucleotide of any one of a) through j) further comprising an expression vector, l) a host cell recombinant for a polynucleotide of a) through k) above, m) a non-human transgenic animal comprising the host cell of k), n) a polynucleotide of a) through j) further comprising a physiologically acceptable carrier. 2. A polypeptide comprising an amino acid sequence selected from the group consisting of: a) any single integer from 6 to 776 amino acids of any one even SEQ ID NO. or of a polypeptide encoded by a human cDNA of a deposited clone; b) a signal peptide sequence of any one even SEQ ID NO. or encoded by a human cDNA of a deposited clone; c) a mature polypeptide sequence of any one even SEQ ID NO. or encoded by a human cDNA of a deposited clone; d) a full length polypeptide sequence of any one even SEQ ID NO. or encoded by a human cDNA of a deposited clone; e) a polypeptide of a) through d) further comprising a physiologically acceptable carrier. 3. A method of making a polypeptide, said method comprising a) providing a population of host cells comprising the polynucleotide of claim 1; b) culturing said population of host cells under conditions conducive to the production of a polypeptide of claim 2 within said host cells; and c) purifying said polypeptide from said population of host cells. 4. A method of making a polypeptide, said method comprising: a) providing a population of cells comprising a polynucleotide encoding the polypeptide of claim 2, operably linked to a promoter; b) culturing said population of cells under conditions conducive to the production of said polypeptide within said cells; and c) purifying said polypeptide from said population of cells. 5. An antibody that specifically binds to the polypeptide of claim 2. 6. A method of binding a polypeptide of claim 2 to an antibody of claim 5, comprising contacting said antibody with said polypeptide under conditions in which antibody can specifically bind to said polypeptide. 7. A method of determining whether a GENSET gene is expressed within a mammal, said method comprising the steps of: a) providing a biological sample from said mammal b) contacting said biological sample with either of: i) a polynucleotide that hybridizes under stringent conditions to the polynucleotide of claim 1; or ii) a polypeptide that specifically binds to the polypeptide of claim 2; and c) detecting the presence or absence of hybridization between said polynucleotide and an RNA species within said sample, or the presence or absence of binding of said polypeptide to a protein within said sample; wherein a detection of said hybridization or of said binding indicates that said GENSET gene is expressed within said mammal. 8. The method of claim 7, wherein said polynucleotide is a primer, and wherein said hybridization is detected by detecting the presence of an amplification product comprising the sequence of said primer. 9. The method of claim 7, wherein said polypeptide is an antibody. 10. A method of determining whether a mammal has an elevated or reduced level of GENSET gene expression, said method comprising the steps of: a) providing a biological sample from said mammal; and b) comparing the amount of the polypeptide of claim 2, or of an RNA species encoding said polypeptide, within said biological sample with a level detected in or expected from a control sample; wherein an increased amount of said polypeptide or said RNA species within said biological sample compared to said level detected in or expected from said control sample indicates that said mammal has an elevated level of said GENSET gene expression, and wherein a decreased amount of said polypeptide or said RNA species within said biological sample compared to said level detected in or expected from said control sample indicates that said mammal has a reduced level of said GENSET gene expression. 11. A method of identifying a candidate modulator of a GENSET polypeptide, said method comprising. a) contacting the polypeptide of claim 2 with a test compound; and b) determining whether said compound specifically binds to said polypeptide; wherein a detection that said compound specifically binds to said polypeptide indicates that said compound is a candidate modulator of said GENSET polypeptide. 12. The method of claim 11, further comprising testing the biological activity of said GENSET polypeptide in the presence of said candidate modulator, wherein an alteration in the biological activity of said GENSET polypeptide in the presence of said compound in comparison to the activity in the absence of said compound indicates that the compound is a modulator of said GENSET polypeptide. 13. A method for the production of a pharmaceutical composition comprising a) identifying a modulator of a GENSET polypeptide using the method of claim 1; and b) combining said modulator with a physiologically acceptable carrier. |
<SOH> BACKGROUND OF THE INVENTION <EOH>cDNAs encoding secreted proteins or fragments thereof represent a particularly valuable source of therapeutic agents. Thus, there is a need for the identification and characterization of secreted proteins and the nucleic acids encoding them. In addition to being therapeutically useful themselves, secretory proteins include short peptides, called signal peptides, at their amino termini which direct their secretion. These signal peptides are encoded by the signal sequences located at the 5′ ends of the coding sequences of genes encoding secreted proteins. Because these signal peptides will direct the extracellular secretion of any protein to which they are operably linked, the signal sequences may be exploited to direct the efficient secretion of any protein by operably linking the signal sequences to a gene encoding the protein for which secretion is desired. In addition, fragments of the signal peptides called membrane-translocating sequences may also be used to direct the intracellular import of a peptide or protein of interest. This may prove beneficial in gene therapy strategies in which it is desired to deliver a particular gene product to cells other than the cells in which it is produced. Signal sequences encoding signal peptides also find application in simplifying protein purification techniques. In such applications, the extracellular secretion of the desired protein greatly facilitates purification by reducing the number of undesired proteins from which the desired protein must be selected. Thus, there exists a need to identify and characterize the 5′ fragments of the genes for secretory proteins which encode signal peptides. Sequences coding for secreted proteins may also find application as therapeutics or diagnostics. In particular, such sequences may be used to determine whether an individual is likely to express a detectable phenotype, such as a disease, as a consequence of a mutation in the coding sequence for a secreted protein. In instances where the individual is at risk of suffering from a disease or other undesirable phenotype as a result of a mutation in such a coding sequence, the undesirable phenotype may be corrected by introducing a normal coding sequence using gene therapy. Alternatively, if the undesirable phenotype results from overexpression of the protein encoded by the coding sequence, expression of the protein may be reduced using antisense or triple helix based strategies. The secreted human polypeptides encoded by the coding sequences may also be used as therapeutics by administering them directly to an individual having a condition, such as a disease, resulting from a mutation in the sequence encoding the polypeptide. In such an instance, the condition can be cured or ameliorated by administering the polypeptide to the individual. In addition, the secreted human polypeptides or fragments thereof may be used to generate antibodies useful in determining the tissue type or species of origin of a biological sample. The antibodies may also be used to determine the cellular localization of the secreted human polypeptides or the cellular localization of polypeptides which have been fused to the human polypeptides. In addition, the antibodies may also be used in immunoaffinity chromatography techniques to isolate, purify, or enrich the human polypeptide or a target polypeptide which has been fused to the human polypeptide. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a purified or isolated polynucleotide comprising, consisting of, or consisting essentially of a nucleotide sequence selected from the group consisting of: (a) the sequences of the odd SEQ ID NOs:1-23; (b) the sequences of clone inserts of the deposited clone pool; (c) the coding sequences of the odd SEQ ID NOs:1-23; (d) the coding sequences of the clone inserts of the deposited clone pool; (e) the sequences encoding one of the polypeptides of the even SEQ ID NOs:2-24; (f) the sequences encoding one of the polypeptides encoded by the clone inserts of the deposited clone pool; (g) the genomic sequences coding for the GENSET polypeptides; (h) the 5′ transcriptional regulatory regions of GENSET genes; (i) the 3′ transcriptional regulatory regions of GENSET genes; () the polynucleotides comprising the nucleotide sequence of any combination of (g)-(i); (k) the variant polynucleotides of any of the polynucleotides of (a)-(j); (l) the polynucleotides comprising a nucleotide sequence of (a)-(k), wherein the polynucleotide is single stranded, double stranded, or a portion is single stranded and a portion is double stranded; (m) the polynucleotides comprising a nucleotide sequence complementary to any of the single stranded polynucleotides of (l). The invention further provides for fragments of the nucleic acids and polypeptides of (a)-(m) described above. Further embodiments of the invention include purified or isolated polynucleotides that comprise, consist of, or consist essentially of a nucleotide sequence at least 70% identical, more preferably at least 75%, and even more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, to any of the nucleotide sequences in (a)-(m) above, e.g. over a region of contiguous nucleotides at least about any one integer between 10 and the last integer representing the last integer representing the last nucleotide of a specified sequence of the sequence listing, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide of the present invention including (a) through (m) above. The present invention also relates to recombinant vectors, which include the purified or isolated polynucleotides of the present invention, and to host cells recombinant for the polynucleotides of the present invention, as well as to methods of making such vectors and host cells. The present invention further relates to the use of these recombinant vectors and recombinant host cells in the production of GENSET polypeptides. The present invention further relates to a polynucleotide of the present invention operably linked to a regulatory sequence including promoters, enhancers, etc. The invention further provides a purified or isolated polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of: (a) the full length polypeptides of even SEQ ID NOs:2-24; (b) the full length polypeptides encoded by the clone inserts of the deposited clone pool; (c) the epitope-bearing fragments of the polypeptides of even SEQ ID NOs:2-24; (d) the epitope-bearing fragments of the polypeptides encoded by the clone inserts contained in the deposited clone pool; (e) the domains of the polypeptides of even SEQ ID NOs:2-24; (f) the domains of the polypeptides encoded by the clone inserts contained in the deposited clone pool; (g) the signal peptides of the polypeptides of even SEQ ID NOs:2-24 or encoded by the human cDNAs of the deposited clone pool; (h) the mature polypeptides of even SEQ ID NOs:2-24 or encoded by the human cDNAs of the deposited clone pool; and (i) the allelic variant polypeptides of any of the polypeptides of (a)-(h). The invention further provides for fragments of the polypeptides of (a)-(i) above, such as those having biological activity or comprising biologically functional domain(s). The present invention further includes polypeptides with an amino acid sequence with at least 70% similarity, and more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity to those polypeptides described in (a)-(i), or fragments thereof, as well as polypeptides having an amino acid sequence at least 70% identical, more preferably at least 75% identical, and still more preferably 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to those polypeptides described in (a)-(i), or fragments thereof, e.g. over a region of amino acids at least any one integer between 6 and the last integer representing the last amino acid of a specified polypeptide sequence of the sequence listing. The invention further relates to methods of making the polypeptides of the present invention. The present invention further relates to transgenic plants or animals, wherein said transgenic plant or animal is transgenic for a polynucleotide of the present invention and expresses a polypeptide of the present invention. The invention further relates to antibodies that specifically bind to GENSET polypeptides of the present invention and fragments thereof as well as to methods for producing such antibodies and fragments thereof. The invention also provides kits, uses and methods for detecting GENSET gene expression and/or biological activity in a biological sample. One such method involves assaying for the expression of a GENSET polynucleotide in a biological sample using the polymerase chain reaction (PCR) to amplify and detect GENSET polynucleotides or Southern and Northern blot hybridization to detect GENSET genomic DNA, cDNA or mRNA. Alternatively, a method of detecting GENSET gene expression in a test sample can be accomplished using a compound which binds to a GENSET polypeptide of the present invention or a portion of a GENSET polypeptide. The present invention also relates to diagnostic methods and uses of GENSET polynucleotides and polypeptides for identifying individuals or non-human animals having elevated or reduced levels of GENSET gene products, which individuals are likely to benefit from therapies to suppress or enhance GENSET gene expression, respectively, and to methods of identifying individuals or non-human animals at increased risk for developing, or at present having, certain diseases/disorders associated with GENSET polypeptide expression or biological activity. The present invention also relates to kits, uses and methods of screening compounds for their ability to modulate (e.g. increase or inhibit) the activity or expression of GENSET polypeptides including compounds that interact with GENSET gene regulatory sequences and compounds that interact directly or indirectly with a GENSET polypeptide. Uses of such compounds are also within the scope of the present invention. The present invention also relates to pharmaceutical or physiologically acceptable compositions comprising, an active agent, the polypeptides, polynucleotides or antibodies of the present invention, as well as, typically, a physiologically acceptable carrier. The present invention also relates to computer systems containing cDNA codes and polypeptide codes of sequences of the invention and to computer-related methods of comparing sequences, identifying homology or features using GENSET polypeptides or GENSET polynucleotide sequences of the invention. In another aspect, the present invention provides an isolated polynucleotide, the polynucleotide comprising a nucleic acid sequence encoding a polypeptide of the present invention including the polypeptide of (a) through (i) above. In another aspect, the present invention provides a non-human transgenic animal comprising the host cell. In another aspect, the present invention provides a method of making a GENSET polypeptide, the method comprising a) providing a population of host cells comprising a herein-described polynucleotide and b) culturing the population of host cells under conditions conducive to the production of the polypeptide within said host cells. In one embodiment, the method further comprises purifying the polypeptide from the population of host cells. In another aspect, the present invention provides a method of making a GENSET polypeptide, the method comprising a) providing a population of cells comprising a polynucleotide encoding a herein-described polypeptide; b) culturing the population of cells under conditions conducive to the production of the polypeptide within the cells; and c) purifying the polypeptide from the population of cells. In another aspect, the present invention provides a biologically active polypeptide encoded by any of the herein-described polynucleotides. In one embodiment, the polypeptide is selectively recognized by an antibody raised against an antigenic polypeptide, or an antigenic fragment thereof, the antigenic polypeptide comprising any one of the sequences shown as even SEQ ID NOs:2-24 or any one of the sequences of polypeptides encoded by the human cDNAs of the deposited clone pool. In another aspect, the present invention provides an antibody that specifically binds to any of the herein-described polypeptides and methods of binding antibody to said polypeptide. In another aspect, the present invention provides a method of determining whether a GENSET gene is expressed within a mammal, the method comprising the steps of: a) providing a biological sample from said mammal; b) contacting said biological sample with either of: (i) a polynucleotide that hybridizes under stringent conditions to any of the herein-described polynucleotides; or (ii) a polypeptide that specifically binds to any of the herein-described polypeptides; and c) detecting the presence or absence of hybridization between the polynucleotide and an RNA species within the sample, or the presence or absence of binding of the polypeptide to a protein within the sample; wherein a detection of the hybridization or of the binding indicates that the GENSET gene is expressed within the mammal. In one embodiment, the polynucleotide is a primer, and the hybridization is detected by detecting the presence of an amplification product comprising the sequence of the primer. In another embodiment, the polypeptide is an antibody. In another aspect, the present invention provides a method of determining whether a mammal has an elevated or reduced level of GENSET gene expression, the method comprising the steps of: a) providing a biological sample from the mammal; and b) comparing the amount of any of the herein-described polypeptides, or of an RNA species encoding the polypeptide, within the biological sample with a level detected in or expected from a control sample; wherein an increased amount of the polypeptide or the RNA species within the biological sample compared to the level detected in or expected from the control sample indicates that the mammal has an elevated level of the GENSET gene expression, and wherein a decreased amount of the polypeptide or the RNA species within the biological sample compared to the level detected in or expected from the control sample indicates that the mammal has a reduced level of the GENSET gene expression. In another aspect, the present invention provides a method of identifying a candidate modulator of a GENSET polypeptide, the method comprising: a) contacting any of the herein-described polypeptides with a test compound; and b) determining whether the compound specifically binds to the polypeptide; wherein a detection that the compound specifically binds to the polypeptide indicates or inhibits or activates of a specified biological activity that the compound is a candidate modulator of the GENSET polypeptide. |
Agonists and antagonists of moxifin for the treatment of metabolic disorders |
The present invention relates to the field of metabolic research, in particular the discovery of compounds effective for reducing body mass and useful for treating obesity-related diseases and disorders. The obesity-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to, hyperlipidemia, atherosclerosis, insulin resistance, diabetes, and hypertension. In particular, the invention provides for methods of identifying and using AGONISTS and ANTAGONISTS of MOXIFIN activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity. |
1-2. (canceled) 3. An AGONIST or an ANTAGONIST of MOXIFIN activity. 4. The AGONIST or the ANTAGONIST of claim 3, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, insulin-like activity, free fatty acid oxidation, and weight reduction. 5. A pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of the AGONIST or the ANTAGONIST of claim 3. 6. A method of preventing or treating an obesity-related disease or disorder comprising providing or administering to an individual in need of such treatment the composition of claim 5. 7. A method of screening of a candidate substance for interaction with a polypeptide comprising MOXIFIN extracellular domain, said method comprising the following steps: a) providing said polypeptide comprising MOXIFIN extracellular domain; b) obtaining a candidate substance; c) bringing into contact said polypeptide with said candidate substance; d) detecting the complexes formed between said polypeptide and said candidate substance. 8. The method according to claim 7, wherein said candidate substance is an AGONIST or ANTAGONIST of MOXIFIN activity. 9. The method according to claim 7, wherein said detecting step comprises assaying the activity or expression of the MOXIFIN polypeptide. 10. The method according to claim 9, wherein said activity is selected from the group consisting of lipid partition, lipid metabolism, insulin-like activity, free fatty acid oxidation, and weight reduction. 11. The method according to claim 8, wherein said candidate substance is an AGONIST. 12. The method according to claim 11, wherein said AGONIST is a composition consisting essentially of self-assembling homotrimers comprising gAPM1, gC2P, gZADJ2 or gZADJ-7 fragments. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention. Obesity is a public health problem that is serious, widespread, and increasing. In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634). Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643). Even modest weight loss ameliorates these associated conditions. Recently it was shown that particular carboxyl-terminal fragments of the full-length ACRP30 (mouse) and APM1 (human) polypeptides have unexpected effects in vitro and in vivo, including utility for weight reduction, prevention of weight gain, and control of blood glucose levels (Fruebis et al (2001) Proc Natl Acad Sci USA 98:2005-10). The effects of ACRP30 fragment administration in mammals also include reduction of elevated free fatty acid levels including elevated free fatty acid levels caused by administration of epinephrine, i.v. injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, and weight reduction in mammals consuming a normal or high fat/high sucrose diet. Throughout this application, various publications, patents and published patent applications are cited. The disclosures of these publications, patents and published patent specification referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains. |
<SOH> SUMMARY OF THE INVENTION <EOH>APM1 belongs to an expanding family of related secreted polypeptides that includes among others C2P, ZADJ-2 and ZADJ-7. These polypeptides have in common the structure: signal peptide, N-terminally disposed unique region, collagen-like region, and globular C-terminal C1q homology domain. APM1, C2P, ZADJ-2 and ZADJ-7 further share an NGLXXD amino acid motif C-terminally disposed within the globular domain within a loop implicated in receptor binding, wherein said receptor is MOXIFIN. Fragments of APM1, C2P, ZADJ-2 and ZADJ-7 polypeptide comprising the globular domain are herein referred to as gAPM1, gC2P, gZADJ-2 and gZADJ-7. It is further taken to be understood herein that LIGAND refers to a composition consisting essentially of or consisting of in vitro or in vivo self-assembling homotrimer comprised of gAPM1, gC2P, gZADJ-2, or gZADJ-7 polypeptide fragment. MOXIFIN is a member of the Tumor Necrosis Factor Receptor Super Family (TNFRSF) and is a Type I transmembrane protein. The instant invention is based on MOXIFIN as receptor for LIGAND that mediates effects, including utility for weight reduction, maintenance of weight loss, prevention of weight gain, increased insulin sensitivity, and control of blood glucose levels in humans and other mammals. These effects in mammals of MOXIFIN engagement by LIGAND also include reduction of elevated free fatty acid levels including elevated free fatty acid levels including elevated free fatty acid levels caused by administration of epinephrine, i.v. injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, and weight reduction in mammals consuming a normal or high fat/high sucrose diet. More specifically, the present invention is directed to MOXIFIN to which LIGAND binds and through which LIGAND mediates said effects. In particular, the invention provides for methods of identifying and using AGONISTS and ANTAGONISTS of MOXIFIN activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolisn, and insulin-like activity, as well as to pharmaceutical and physiologically acceptable compositions comprising said MOXIFIN AGONISTS or ANTAGONISTS and methods of administering said pharmaceutical and physiologically acceptable compositions in order to increase or reduce body weight, maintain weight loss, or to treat obesity-related diseases and disorders. Assays for identifying AGONISTS and ANTAGONISTS of obesity-related activity are also part of the invention. Preferably said MOXIFIN AGONIST or ANTAGONIST is a compound selected from the group consisting of polypeptide, polypeptide fragment, peptide, proein, antibody, carbohydrate, lipid, small molecular weight organic compound and small molecular weight inorganic compound. Preferably said MOXIFIN AGONIST or ANTAGONIST is a compound that selectively binds to the extracellular domain of MOXIFIN. In other embodiment, said MOXIFIN AGONIST or ANTAGONIST is a compound that selectively binds to the intracellular domain of a polypeptide comprising the extracellular domain of MOXIFIN. The present invention also provides a method of assaying test compounds to identify a test compound that binds to MOXIFIN polypeptide. The method comprises contacting MOXIFIN polypeptide with a test compound and to determine the extent of binding of the test compound to said MOXIFIN polypeptide. The method further comprises determining whether such test compounds are AGONISTS or ANTAGONISTS of MOXIFIN polypeptide. The present invention further provides a method of testing the impact of molecules on the expression of MOXIFIN polypeptide or on the activity of MOXIFIN polypeptide. The present invention also relates to diagnostic methods of identifying individuals or non-human animals having elevated or reduced levels of MOXIFIN products, which individuals are likely to benefit from therapies to suppress or enhance MOXIFIN expression, respectively, and to methods of identifying individuals or non-human animals at increased risk for developing, or present state of having, certain diseases/disorders associated with MOXIFIN abnormal expression or biological activity. The present invention provides for methods of identifying AGONISTS of MOXIFIN polypeptide biological activity comprising contacting a small molecule compound with MOXIFIN polypeptides and measuring MOXIFIN polypeptide biological activity in the presence and absence of these small molecules. The present invention further provides for methods of identifying ANTAGONISTS of MOXIFIN polypeptide biological activity comprising contacting a small molecule compound with MOXIFIN polypeptides and measuring MOXIFIN polypeptide biological activity in the presence and absence of these small molecules. These small molecules can be a naturally occurring medicinal compound or derived from combinatorial chemical libraries. The present invention also relates to pharmaceutical or physiologically acceptable compositions comprising, an active agent, including AGONIST or ANTAGONIST of the present invention. In a first aspect, the invention is directed to MOXIFIN AGONISTS, wherein said AGONIST is an antibody that specifically binds MOXIFIN, a compound excluding said MOXIFIN antibody (e.g., small organic or inorganic compound, protein, peptide, carbohydrate, lipid), or a LIGAND polypeptide or fragment thereof. In a further preferred embodiment, the invention is directed to a MOXIFIN AGONIST, wherein said AGONIST is an antibody that specifically binds MOXIFIN. More preferably the invention is directed to said MOXIFIN antibody, wherein said MOXIFIN antibody binds MOXIFIN and manifests LIGAND activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein. In a further preferred embodiment, the invention is directed to a MOXIFIN AGONIST, wherein said AGONIST is a compound excluding said MOXIFIN antibody. More preferably the invention is directed to said compound, wherein said compound binds MOXIFIN and manifests LIGAND activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein. Further more preferably the invention is directed to said compound, wherein said compound manifests LIGAND activity exclusive of binding to MOXIFIN, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein. Further more preferably the invention is directed to said compound, wherein said compound increases MOXIFIN expression. In a further preferred embodiment, the invention is directed to a MOXIFIN AGONIST that selectively binds to a polypeptide comprising the extracellular domain of MOXIFIN. In a further preferred embodiment, the invention is directed to a MOXIFIN AGONIST, wherein said AGONIST is LIGAND, and wherein it is understood that LIGAND refers to a composition consisting essentially of or consisting of in vitro or in vivo self-assembling homotrimer comprised of gAPM1, gC2P, gZADJ-2, or gZADJ-7 polypeptide fragment. More preferably the invention is directed to said LIGAND, wherein said LIGAND binds MOXIFIN and elicits biological activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein. More preferably the invention is directed to said LIGAND, wherein said LIGAND induces, enhances, or potentiates said biological activity exclusive of binding to MOXIFIN. In preferred embodiment, said homotrimer is comprised of preferred gAPM1, gC2P, gZADJ-2 or gZADJ-7 polypeptide fragment. APM1. Preferred gAPM1 polypeptide fragment is selected from amino acids 18-244, 34-244, 49-244, 56-244, 59-244, 66-244, 69-244, 78-244, 85-244, 93-244, 101-244, 102-244, 103-244, 104-244, 107-244, 110-244 or 113-244, wherein said numbering of said amino acids within APM1 amino acid sequence is understood to be taken from said APM1 amino acid sequence presented in Table 2. Less preferred gAPM1 fragments are indicated in bold. C2P. Preferred gC2P polypeptide fragment is selected from amino acids 20-333, 25-333, 43-333, 45-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333, 81-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, or 202-333, wherein said numbering of said amino acids within C2P amino acid sequence is understood to be taken from said C2P amino acid sequence presented in Table 2. Less preferred gC2P fragments are indicated in bold. ZADJ-2. Preferred gZADJ-2 polypeptide fragment is selected from amino acids 16-285, 25-285, 26-285, 29-285, 30-285, 91-285, 93-285, 97-285, 98-285, 99-285, 105-285, 109-285, 112-285, 120-285, 126-285, 127-285, 130-285, 132-285, 133-285, 134-285, or 150-285, wherein said numbering of said amino acids within ZADJ-2 amino acid sequence is understood to be taken from 10 said ZADJ-2 amino acid sequence presented in Table 2. Less preferred gZADJ-2 fragments are indicated in bold. ZADJ-7. Preferred gZADJ-7 polypeptide fragment is selected from amino acids 31-303, 39-303, 78-303, 81-303, 84-303, 85-303, 88-303, 91-303, 97-303, 99-303, 109-303, 117-303, 118-303, 127-303, 139-303, 142-303, 155-303, or 162-303, wherein said numbering of said amino acids within ZADJ-7 amino acid sequence is understood to be taken from said ZADJ-7 amino acid sequence presented in Table 2. Less preferred gZADJ-7 fragments are indicated in bold. More preferred LIGAND is APM1. In a further preferred embodiment, said AGONIST is able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides. Further preferred AGONISTS are those that significantly stimulate muscle lipid or free fatty acid oxidation as compared to untreated cells. Further preferred AGONISTS are those that cause C2C12 cells differentiated in the presence of said AGONISTS to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells. Further preferred AGONISTS are those that increase by at least 10%, 20%, 30%, 35%, or 40% leptin uptake in a liver cell line [preferably BPRCL mouse liver cells (ATCC CRL-2217)] as compared to untreated cells. Further preferred AGONISTS are those that significantly reduce the postprandial increase in plasma free fatty acids or triglycerides, particularly following a high fat meal. Further preferred AGONISTS are those that significantly reduce or eliminate ketone body production, particularly following a high fat meal. Further preferred AGONISTS are those that increase glucose uptake in skeletal muscle cells. Further preferred AGONISTS are those that increase glucose uptake in adipose cells. Further preferred AGONISTS are those that increase glucose uptake in neuronal cells. Further preferred AGONISTS are those that increase glucose uptake in red blood cells. Further preferred AGONISTS are those that increase glucose uptake in the brain. Further preferred AGONISTS are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal. Further preferred AGONISTS are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal. Further preferred AGONISTS are those that improve insulin sensitivity. Further preferred said AGONISTS are those that decrease body mass, wherein said decrease in body mass is comprised of a change in mass of the subcutaneous adipose tissue. Further preferred said AGONISTS are those that decrease body mass, wherein said decrease in body mass is comprised of a change in mass of the visceral (omental) adipose tissue. In a second aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said AGONIST described in the first aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent. In a third aspect, the invention features a method of reducing body mass comprising providing or administering to individuals in need of reducing body mass said pharmaceutical or physiologically acceptable composition described in the second aspect. In a fourth aspect, the invention features a method of preventing or treating an obesity-related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the second aspect. Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by said MOXIFIN AGONIST of the invention include hyperlipidemia and hyperuricemia. In preferred embodiments, said individual is a mammal, preferably a human. In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising AGONIST, wherein said biological response is selected from the group consisting of: (a) lowering circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids; (b) lowering circulating (either blood, serum or plasma) levels (concentration) of glucose; (c) lowering circulating (either blood, serum or plasma) levels (concentration) of triglycerides; (d) stimulating muscle lipid or free fatty acid oxidation; (c) increasing leptin uptake in the liver or liver cells; (e) reducing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; (f) reducing or eliminating ketone body production, particularly following a high fat meal; (g) increasing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain, and further wherein said biological response is significantly greater than, or at least 10%, 20%, 30%, 35%, or 40% greater than that observed in the absence of treatment; or alternatively wherein said biological response is greater than a transient response; or alternatively wherein said biological response is sustained. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy. In a further preferred embodiment, the present invention may be used in complementary therapy of NIDDM patients to improve their weight or glucose control in combination with an insulin secretagogue or an insulin sensitising agent. Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide. Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone. The present invention further provides a method of improving the body weight or glucose control of NIDDM patients alone, without an insulin secretagogue or an insulin sensitising agent. In a further preferred embodiment, the present invention may be used in complementary therapy of IDDM patients to improve their weight or glucose control in combination with an insulin secretagogue or an insulin sensitising agent. Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl) guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide. Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone. The present invention further provides a method of improving the body weight or glucose control of IDDM patients alone, without an insulin secretagogue or an insulin sensitising agent. In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent). Accordingly, the present invention further provides for a composition of pharmaceutical or physiologically acceptable composition and an insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose control in NIDDM or IDDM patients. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitiser. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) without insulin therapy. In a fifth aspect, the invention features a use of AGONIST described in the first aspect for treatment of obesity-related diseases and disorders and/or reducing body mass. Preferably, said obesity-related diseases and disorders are selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by said AGONIST of the invention include hyperlipidemia and hyperuricemia. In a sixth aspect, the invention features a use of AGONIST described in the second aspect for the preparation of a medicament for the treatment of obesity-related diseases and disorders and/or for reducing body mass. Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. In preferred embodiments, said individual is a mammal, preferably a human. In a seventh aspect, the invention provides AGONIST of the first aspect of the invention, or a composition of the second aspect of the invention, for use in a method of treatment of the human or animal body. In an eighth aspect, the invention features methods of reducing body weight comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the second aspect, or AGONIST described in the first aspect. Where the reduction of body weight is practiced for cosmetic purposes, the individual has a BMI of at least 20 and no more than 25. In embodiments for the treatment of obesity, the individual may have a BMI of at least 20. One embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 25. Another embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 30. Yet another embodiment provides for the treatment of individuals with BMI values of at least 40. In further embodiment, the invention features methods of maintaining weight loss comprising providing to an individual said pharmaceutical or physiologically acceptable composition. In a ninth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the second aspect for reducing body mass and/or for treatment or prevention of obesity-related diseases or disorders. Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. In preferred embodiments, said individual is a mammal, preferably a human. In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping LIGAND single nucleotide polymorphisms (SNPs) or measuring LIGAND polypeptide or mRNA levels in clinical samples from said individuals. Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva. In a tenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the second aspect for reducing body weight for cosmetic reasons. In an eleventh aspect, AGONIST of the invention is used in methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the second aspect, or AGONIST described in the first aspect. In a preferred aspect of the methods above and disclosed herein, the amount of AGONIST administered to an individual is sufficient to bring levels of MOXIFIN activation to their normal levels (levels in individuals without obesity-related disease or disorder). “Normal levels” of MOXIFIN activation may be followed using surrogate markers including circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides. In a twelfth aspect, the invention is directed to a MOXIFIN ANTAGONIST, wherein said ANTAGONIST is a soluble fragment of MOXIFIN polypeptide, an antibody that specifically binds MOXIFIN, a compound excluding said soluble fragment of MOXIFIN polypeptide and said MOXIFIN antibody (e.g., small molecular weight organic or inorganic compound, protein, peptide, carbohydrate, lipid), or a variant or fragment of LIGAND polypeptide. In a further preferred embodiment, the invention is directed to a MOXIFIN ANTAGONIST, wherein said ANTAGONIST is a soluble fragment of MOXIFIN polypeptide. More preferably the invention is directed to purified, isolated, or recombinant soluble fragments of MOXIFIN polypeptide. More preferably the invention is directed to said soluble fragment of MOXIFIN polypeptide, wherein said soluble fragment binds LIGAND and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said soluble fragment of MOXIFIN polypeptide does not activate MOXIFIN. Preferably said soluble fragment of MOXIFIN polypeptide blocks or inhibits LIGAND binding to MOXIFIN. In preferred embodiments, said soluble fragment of MOXIFIN polypeptide comprises, consists essentially of, or consists of, at least 6 and not more than 174 consecutive amino acids of SEQ ID NO:2, more preferably of amino acids comprising the extracellular domain of MOXIFIN. Preferred said soluble fragment of MOXIFIN comprises the extracellular domain of mature MOXIFIN polypeptide. Particularly preferred soluble fragment of MOXIFIN comprises amino acids 20-170, 20-171, 20-172, 20-173 or 20-175 of SEQ ID NO:2, where it is understood that amino acid 20 is predicted to be the N-terminal amino acid of the mature MOXIFIN polypeptide absent the putative signal peptide. In other preferred embodiments, said soluble fragment of MOXIFIN polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of SEQ ID NO:2. Further preferred embodiments include heterologous polypeptides comprising a MOXIFIN polypeptide of the invention. In further preferred embodiment, a MOXIFIN polypeptide of the invention is conjugated at its N- or C-terminus to an antibody Fc region or portion thereof. In a further preferred embodiment, the invention is directed to a MOXIFIN ANTAGONIST, wherein said ANTAGONIST is an antibody that specifically binds MOXIFIN. More preferably the invention is directed to said MOXIFIN antibody, wherein said MOXIFIN antibody binds MOXIFIN and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said MOXIFIN antibody does not activate MOXIFIN. Preferably said MOXIFIN antibody blocks or inhibits LIGAND binding to MOXIFIN. In a further preferred embodiment, the invention is directed to a MOXIFIN ANTAGONIST, wherein said ANTAGONIST is a compound excluding said soluble fragment of MOXIFIN polypeptide and said MOXIFIN antibody (e.g., small organic molecule, protein, peptide). More preferably the invention is directed to said compound, wherein said compound binds to MOXIFIN and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said compound does not activate MOXIFIN. Preferably said compound that binds to MOXIFIN blocks or inhibits LIGAND binding to MOXIFIN. Further more preferably the invention is directed to said compound, wherein said compound blocks or inhibits LIGAND activity exclusive of binding to MOXIFIN, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said compound does not activate MOXIFIN. Further more preferably the invention is directed to said compound, wherein said compound blocks or inhibits MOXIFIN expression and wherein said compound does not have LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said compound does not activate MOXIFIN. In a further preferred embodiment, the invention is directed to a MOXIFIN ANTAGONIST, wherein said ANTAGONIST is a variant or fragment of LIGAND polypeptide. More preferably the invention is directed to said variant of fragment of LIGAND polypeptide, wherein said variant or fragment of LIGAND polypeptide binds MOXIFIN and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said variant or fragment of LIGAND polypeptide does not activate MOXIFIN. Preferably said variant or fragment of LIGAND polypeptide blocks or inhibits LIGAND binding to MOXIFIN. More preferably the invention is directed to said variant or fragment of LIGAND polypeptide, wherein said variant or fragment of LIGAND polypeptide inhibits the induction, enhancement, or potentiation of said biological activity exclusive of binding to MOXIFIN. In a further preferred embodiment, the invention is directed to a MOXIFIN ANTAGONIST that selectively binds to a polypeptide comprising the extracellular domain of MOXIFIN. APM1. Preferred gAPM1 polypeptide fragment is selected from amino acids 18-244, 34-44, 49-244, 56-244, 59-244, 66-244, 69-244, 78-244, 85-244, 93-244, 101-244, 102-244, 103-244, 104-244, 107-244, 110-244, or 113-244, wherein said numbering of said amino acids within APM1 amino acid sequence is understood to be taken from said APM1 amino acid sequence presented in Table 2. C2P. Preferred gC2P polypeptide fragment is selected from amino acids 20-333, 25-333, 43-333, 45-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333, 81-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, or 202-333, wherein said numbering of said amino acids within C2P amino acid sequence is understood to be taken from said C2P amino acid sequence presented in Table 2. ZADJ-2. Preferred gZADJ-2 polypeptide fragment is selected from amino acids 16-285, 25-285, 26-285, 29-285, 30-285, 91-285, 93-285, 97-285, 98-285, 99-285, 105-285, 109-285, 112-285, 120-285, 126-285, 127-285, 130-285, 132-285, 133-285, 134-285, or 150-285, wherein said numbering of said amino acids within ZADJ-2 amino acid sequence is understood to be taken from said ZADJ-2 amino acid sequence presented in Table 2. ZADJ-7. Preferred gZADJ-7 polypeptide fragment is selected from amino acids 31-303, 39-303, 78-303, 81-303, 84-303, 85-303, 88-303, 91-303, 97-303, 99-303, 109-303, 117-303, 118-303, 127-303, 139-303, 142-303, 155-303, or 162-303, wherein said numbering of said amino acids within ZADJ-7 amino acid sequence is understood to be taken from said ZADJ-7 amino acid sequence presented in Table 2. Most preferred LIGAND is APM1 or C2P. Particularly most preferred LIGAND is APM1. In a further preferred embodiment, said ANTAGONIST is able to raise circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides. Further preferred said ANTAGONISTS are those that significantly inhibit muscle lipid or free fatty acid oxidation stimulated by its LIGAND. Further preferred said ANTAGONISTS are those that cause C2C12 cells differentiated in the presence of LIGAND to undergo at least 10%, 20%, 30%, 35%, or 40% less oleate oxidation as compared to untreated cells. Further preferred said ANTAGONISTS are those that inhibit by at least 10%, 20%, 30%, 35%, or 40% the increase in leptin uptake stimulated by LIGAND polypeptide in a liver cell line [preferably BPRCL mouse liver cells (ATCC CRL-2217)] as compared to untreated cells. Further preferred said ANTAGONISTS are those that significantly increase the postprandial increase in plasma free fatty acids, particularly following a high fat meal. Further preferred said ANTAGONISTS are those that significantly increase ketone body production, particularly following a high fat meal. Further preferred said ANTAGONISTS are those that decrease glucose uptake in skeletal muscle cells stimulated by LIGAND. Further preferred said ANTAGONISTS are those that decrease glucose uptake in adipose cells stimulated by LIGAND. Further preferred said ANTAGONISTS are those that decrease glucose uptake in neuronal cells stimulated by LIGAND. Further preferred said ANTAGONISTS are those that decrease glucose uptake in red blood cells stimulated by LIGAND. Further preferred said ANTAGONISTS are those that decrease glucose uptake in the brain stimulated by LIGAND. Further preferred said ANTAGONISTS are those that significantly increase the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal. Further preferred said ANTAGONISTS are those that significantly facilitate the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal. Further preferred said ANTAGONISTS are those that reduce the insulin sensitivity stimulated by LIGAND. Further preferred said ANTAGONISTS are those that increase body mass, wherein said increase in body mass is comprised of a change in mass of the subcutaneous adipose tissue. Further preferred said ANTAGONISTS are those that increase body mass, wherein said increase in body mass is comprised of a change in mass of the visceral (omental) adipose tissue. In a thirteenth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said ANTAGONIST described in the twelfth aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent. In a fourteenth aspect, the invention features a method of increasing body mass comprising providing or administering to individuals in need of increasing body mass said pharmaceutical or physiologically acceptable composition described in the thirteenth aspect. In a fifteenth aspect, the invention features a method of preventing or treating disorders associated with excessive weight loss comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the thirteenth aspect. Preferably said disorder is selected from the group consisting of cachexia, wasting, cancer-related weight loss, AIDS-related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia. Said disorders associated with excessive weight loss are comprised of those mediated by tumor necrosis factor (TNFalpha) alone, those mediated by TNFalpha plus one or more additional factors, and those mediated only by one or more factors exclusive of TNFalpha. Said factors include, but are not restricted to, macrophage migration inhibitory factor, interleukin 1, and interleukin 6. In preferred embodiments, said individual is a mammal, preferably a human. In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising ANTAGONIST, wherein said biological response is selected from the group consisting of: (a) raising circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids (FFA) or triglycerides (TG); (b) raising circulating (either blood, serum or plasma) levels (concentration) of glucose; (c) raising circulating (either blood, serum or plasma) levels (concentration) of triglycerides; (d) inhibiting muscle lipid or free fatty acid oxidation; (c) inhibiting leptin uptake in the liver or liver cells; (e) increasing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; and, (f) increasing or eliminating ketone body production, particularly following a high fat meal; (g) reducing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain, and further wherein said biological response is greater than a transient response; or alternatively wherein said biological response is sustained. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method of increasing body mass in some persons with cachexia, wasting, cancer-related weight loss, AIDS-related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin de-sensitiser, wherein the sensitivity of a cell or tissue to insulin is reduced. In a sixteenth aspect, the invention features a method of making the MOXIFIN polypeptide described in the twelfth aspect, wherein said method is selected from the group consisting of proteolytic cleavage, recombinant methodology and artificial synthesis. In a preferred embodiment, proteolytic cleavage is carried out using trypsin, plasmin, or collagenase. In a seventeenth aspect, the invention features a use of ANTAGONIST described in the twelfth aspect for the preparation of a medicament for the treatment of disorders associated with excessive weight loss and/or for increasing body mass. Preferably, said disorder is selected from the group consisting of cachexia, wasting, cancer-related weight loss, AIDS-related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia. In preferred embodiments, said individual is a mammal, preferably a human. In an eighteenth aspect, the invention provides ANTAGONIST of the twelfth aspect of the invention, or a composition of the thirteenth aspect of the invention, for use in a method of treatment of the human or animal body. In a nineteenth aspect, the invention features methods of increasing body weight comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the thirteenth aspect, or ANTAGONIST described in the twelfth aspect. Where the increase of body weight is practiced for cosmetic purposes, the individual has a BMI of no greater than 25 and at least 20. In embodiments for the treatment of disorders associated with excessive weight loss, the individual may have a BMI no greater than 20. One embodiment for the treatment of disorders associated with excessive weight loss provides for the treatment of individuals with BMI values of no greater than 15. Alternatively, for increasing the body weight of an individual, the BMI value should be at least 15 and no more than 20. In a twentieth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the thirteenth aspect for increasing body mass and/or for treatment of disorders associated with excessive weight loss. Preferably, said disorder is selected from the group consisting of cachexia, wasting, cancer-related weight loss, AIDS-related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia. In preferred embodiments, said individual is a mammal, preferably a human. In a twenty-first aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the thirteenth aspect for increasing body weight for cosmetic reasons. In a preferred aspect of the methods above and disclosed herein, the amount of ANTAGONIST administered to an individual is sufficient to bring levels of MOXIFIN activation to their normal levels (levels in healthy individuals). “Normal levels” of MOXIFIN activation may be followed using surrogate markers including circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides. |
Xobesin agonists and antagonists for the treatment of metabolic disorders |
The present invention relates to the field of metabolic research, in particular the discovery of compounds effective for reducing body mass and useful for treating obesity-related diseases and disorders. The obesity-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to, hyperlipidemia, atherosclerosis, insulin resistance, diabetes, and hypertension. In particular, the invention provides for methods of identifying and using AGONISTS and ANTAGONISTS of XOBESIN activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity. |
1-2 (canceled) 3. An AGONIST or an ANTAGONIST of XOBESIN activity. 4. The AGONIST or the ANTAGONIST of claim 3, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, insulin-like activity, free fatty acid oxidation, and weight reduction. 5. A pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of the AGONIST or the ANTAGONIST of claim 3. 6. A method of preventing or treating an obesity-related disease or disorder comprising providing or administering to an individual in need of such treatment the composition of claim 5. 7. A method of screening of a candidate substance for interaction with a polypeptide comprising XOBESIN extracellular domain, said method comprising the following steps: a) providing said polypeptide comprising XOBESIN extracellular domain; b) obtaining a candidate substance; c) bringing into contact said polypeptide with said candidate substance; d) detecting the complexes formed between said polypeptide and said candidate substance. 8. The method according to claim 7, wherein said candidate substance is an AGONIST or ANTAGONIST of XOBESIN activity. 9. The method according to claim 7, wherein said detecting step comprises assaying the activity or expression of the XOBESIN polypeptide. 10. The method according to claim 9, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, insulin-like activity, free fatty acid oxidation, and weight reduction. 11. The method according to claim 8, wherein said candidate substance is an AGONIST. 12. The method according to claim 11, wherein said AGONIST is a composition consisting essentially of self-assembling homotrimers comprising gAPM1, gC2P, gZADJ2 or gZADJ-7 fragments. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention. Obesity is a public health problem that is serious, widespread, and increasing. In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634). Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643). Even modest weight loss ameliorates these associated conditions. Recently it was shown that particular carboxyl-terminal fragments of the full-length ACRP30 (mouse) and APM1 (human) polypeptides have unexpected effects in vitro and in vivo, including utility for weight reduction, prevention of weight gain, and control of blood glucose levels (Fruebis et al (2001) Proc Natl Acad Sci USA 98:2005-10). The effects of ACRP30 fragment administration in mammals also include reduction of elevated free fatty acid levels including elevated free fatty acid levels caused by administration of epinephrine, iv. injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, and weight reduction in mammals consuming a normal or high fat/high sucrose diet. Throughout this application, various publications, patents and published patent applications are cited. The disclosures of these publications, patents and published patent specification referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains. |
<SOH> SUMMARY OF THE INVENTION <EOH>APM1 belongs to an expanding family of related secreted polypeptides that includes among others C2P, ZADJ-2 and ZADJ-7. These polypeptides have in common the structure: signal peptide, N-terminally disposed unique region, collagen-like region, and globular C-terminal C1q homology domain. APM1, C2P, ZADJ-2 and ZADJ-7 further share an NGLXXD amino acid motif C-terminally disposed within the globular domain within a loop implicated in receptor binding, wherein said receptor is XOBESIN. Fragments of APM1, C2P, ZADJ-2 and ZADJ-7 polypeptide comprising the globular domain are herein referred to as gAPM1, gC2P, gZADJ-2 and gZADJ-7. It is further taken to be understood herein that LIGAND refers to a composition consisting essentially of or consisting of in vitro or in vivo self-assembling homotrimer comprised of gAPM1, gC2P, gZADJ-2, or gZADJ-7 polypeptide fragment. XOBESIN is a member of the Tumor Necrosis Factor Receptor Super Family (TNFRSF) and is a Type I transmembrane protein. The instant invention is based on XOBESIN as receptor for LIGAND that mediates effects, including utility for weight reduction, maintenance of weight loss, prevention of weight gain, increased insulin sensitivity, and control of blood glucose levels in humans and other mammals. These effects in mammals of XOBESIN engagement by LIGAND also include reduction of elevated free fatty acid levels including elevated free fatty acid levels including elevated free fatty acid levels caused by administration of epinephrine, i.v. injection of “intralipid”, or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, and weight reduction in mammals consuming a normal or high fat/high sucrose diet. More specifically, the present invention is directed to XOBESIN to which LIGAND binds and through which LIGAND mediates said effects. In particular, the invention provides for methods of identifying and using AGONISTS and ANTAGONISTS of XOBESIN activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity, as well as to pharmaceutical and physiologically acceptable compositions comprising said XOBESIN AGONISTS or ANTAGONISTS and methods of administering said pharmaceutical and physiologically acceptable compositions in order to increase or reduce body weight, maintain weight loss, or to treat obesity-related diseases and disorders. Assays for identifying AGONISTS and ANTAGONISTS of obesity-related activity are also part of the invention. Preferably said XOBESIN AGONIST or ANTAGONIST is a compound selected from the group consisting of polypeptide, polypeptide fragment, peptide, protein, antibody, carbohydrate, lipid, small molecular weight organic compound and small molecular weight inorganic compound. Preferably said XOBESIN AGONIST or ANTAGONIST is a compound that selectively binds to the extracellular domain of XOBESIN. In other embodiment, said XOBESIN AGONIST or ANTAGONIST is a compound that selectively binds to the intracellular domain of a polypeptide comprising the extracellular domain of XOBESIN. The present invention also provides a method of assaying test compounds to identify a test compound that binds to XOBESIN polypeptide. The method comprises contacting XOBESIN polypeptide with a test compound and to determine the extent of binding of the test compound to said XOBESIN polypeptide. The method further comprises determining whether such test compounds are AGONISTS or ANTAGONISTS of XOBESIN polypeptide. The present invention further provides a method of testing the impact of molecules on the expression of XOBESIN polypeptide or on the activity of XOBESIN polypeptide. The present invention also relates to diagnostic methods of identifying individuals or non-human animals having elevated or reduced levels of XOBESIN products, which individuals are likely to benefit from therapies to suppress or enhance XOBESIN expression, respectively, and to methods of identifying individuals or non-human animals at increased risk for developing, or present state of having, certain diseases/disorders associated with XOBESIN abnormal expression or biological activity. The present invention provides for methods of identifying AGONISTS of XOBESIN polypeptide biological activity comprising contacting a small molecule compound with XOBESIN polypeptides and measuring XOBESIN polypeptide biological activity in the presence and absence of these small molecules. The present invention further provides for methods of identifying ANTAGONISTS of XOBESIN polypeptide biological activity comprising contacting a small molecule compound with XOBESIN polypeptides and measuring XOBESIN polypeptide biological activity in the presence and absence of these small molecules. These small molecules can be a naturally occurring medicinal compound or derived from combinatorial chemical libraries. The present invention also relates to pharmaceutical or physiologically acceptable compositions comprising, an active agent, including AGONIST or ANTAGONIST of the present invention. In a first aspect, the invention is directed to XOBESIN AGONISTS, wherein said AGONIST is an antibody that specifically binds XOBESIN, a compound excluding said XOBESIN antibody (e.g., small organic or inorganic compound, protein, peptide, carbohydrate, lipid), or a LIGAND polypeptide or fragment thereof. In a further preferred embodiment, the invention is directed to a XOBESIN AGONIST, wherein said AGONIST is an antibody that specifically binds XOBESIN. More preferably the invention is directed to said XOBESIN antibody, wherein said XOBESIN antibody binds XOBESIN and manifests LIGAND activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein. In a further preferred embodiment, the invention is directed to a XOBESIN AGONIST, wherein said AGONIST is a compound excluding said XOBESIN antibody. More preferably the invention is directed to said compound, wherein said compound binds XOBESIN and manifests LIGAND activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein. Further more preferably the invention is directed to said compound, wherein said compound manifests LIGAND activity exclusive of binding to XOBESIN, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein. Further more preferably the invention is directed to said compound, wherein said compound increases XOBESIN expression. In a further preferred embodiment, the invention is directed to a XOBESIN AGONIST that selectively binds to a polypeptide comprising the extracellular domain of XOBESIN. In a further preferred embodiment, the invention is directed to a XOBESIN AGONIST, wherein said AGONIST is LIGAND, and wherein it is understood that LIGAND refers to a composition consisting essentially of or consisting of in vitro or in vivo self-assembling homotrimer comprised of gAPM1, gC2P, gZADJ-2, or gZADJ-7 polypeptide fragment. More preferably the invention is directed to said LIGAND, wherein said LIGAND binds XOBESIN and elicits biological activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein. More preferably the invention is directed to said LIGAND, wherein said LIGAND induces, enhances, or potentiates said biological activity exclusive of binding to XOBESIN. In preferred embodiment, said homotrimer is comprised of preferred gAPM1, gC2P, gZADJ-2 or gZADJ-7 polypeptide fragment APM1. Preferred gAPM1 polypeptide fragment is selected from amino acids 18-244, 34-244, 49-244, 56-244, 59-244, 66-244, 69-244, 78-244, 85-244, 93-244, 101-244, 102-244, 103-244, 104-244, 107-244, 110-244 or 113-244, wherein said numbering of said amino acids within APM1 amino acid sequence is understood to be taken from said APM1 amino acid sequence presented in Table 2. Less preferred gAPM1 fragments are indicated in bold. C2P. Preferred gC2P polypeptide fragment is selected from amino acids 20-333, 25-333, 43-333, 45-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333, 81-333, 82-333, 86-333, 89-333, 95-333, 100-333, 104-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, or 202-333, wherein said numbering of said amino acids within C2P amino acid sequence is understood to be taken from said C2P amino acid sequence presented in Table 2. Less preferred gC2P fragments are indicated in bold. ZADJ-2. Preferred gZADJ-2 polypeptide fragment is selected from amino acids 16-285, 25-285, 26285, 29-285, 30-285, 91-285, 93-285, 97-285, 98-285, 99-285, 105-285, 109-285, 112-285, 120-285, 126-285, 127-285, 130-285, 132-285, 133-285, 134-285, or 150-285, wherein said numbering of said amino acids within ZADJ-2 amino acid sequence is understood to be taken from said ZADJ-2 amino acid sequence presented in Table 2. Less preferred gZADJ-2 fragments are indicated in bold. ZADJ-7. Preferred gZADJ-7 polypeptide fragment is selected from amino acids 31-303, 39-303, 78-303, 81-303, 84-303, 85-303, 88-303, 91-303, 97-303, 99-303, 109-303, 117-303, 118-303, 127-303, 139-303, 142-303, 155-303, or 162-303, wherein said numbering of said amino acids within ZADJ-7 amino acid sequence is understood to be taken from said ZADJ-7 amino acid sequence presented in Table 2. Less preferred gZADJ-7 fragments are indicated in bold. More preferred LIGAND is APM1. In a further preferred embodiment, said AGONIST is able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides. Further preferred AGONISTS are those that significantly stimulate muscle lipid or free fatty acid oxidation as compared to untreated cells. Further preferred AGONISTS are those that cause C2C12 cells differentiated in the presence of said AGONISTS to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells. Further preferred AGONISTS are those that increase by at least 10%, 20%, 30%, 35%, or 40% leptin uptake in a liver cell line [preferably BPRCL mouse liver cells (ATCC CRL-2217)] as compared to untreated cells. Further preferred AGONISTS are those that significantly reduce the postprandial increase in plasma free fatty acids or triglycerides, particularly following a high fat meal. Further preferred AGONISTS are those that significantly reduce or eliminate ketone body production, particularly following a high fat meal. Further preferred AGONISTS are those that increase glucose uptake in skeletal muscle cells. Further preferred AGONISTS are those that increase glucose uptake in adipose cells. Further preferred AGONISTS are those that increase glucose uptake in neuronal cells. Further preferred AGONISTS are those that increase glucose uptake in red blood cells. Further preferred AGONISTS are those that increase glucose uptake in the brain. Further preferred AGONISTS are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal. Further preferred AGONISTS are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal. Further preferred AGONISTS are those that improve insulin sensitivity. Further preferred said AGONISTS are those that decrease body mass, wherein said decrease in body mass is comprised of a change in mass of the subcutaneous adipose tissue. Further preferred said AGONISTS are those that decrease body mass, wherein said decrease in body mass is comprised of a change in mass of the visceral (omental) adipose tissue. In a second aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said AGONIST described in the first aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent. In a third aspect, the invention features a method of reducing body mass comprising providing or administering to individuals in need of reducing body mass said pharmaceutical or physiologically acceptable composition described in the second aspect. In a fourth aspect, the invention features a method of preventing or treating an obesity-related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the second aspect. Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by said XOBESIN AGONIST of the invention include hyperlipidemia and hyperuricemia. In preferred embodiments, said individual is a mammal, preferably a human. In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising AGONIST, wherein said biological response is selected from the group consisting of: (a) lowering circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids; (b) lowering circulating (either blood, serum or plasma) levels (concentration) of glucose; (c) lowering circulating (either blood, serum or plasma) levels (concentration) of triglycerides; (d) stimulating muscle lipid or free fatty acid oxidation; (c) increasing leptin uptake in the liver or liver cells; (e) reducing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; (f) reducing or eliminating ketone body production, particularly following a high fat meal; (g) increasing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain, and further wherein said biological response is significantly greater than, or at least 10%, 20%, 30%, 35%, or 40% greater than that observed in the absence of treatment; or alternatively wherein said biological response is greater than a transient response; or alternatively wherein said biological response is sustained. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy. In a further preferred embodiment, the present invention may be used in complementary therapy of NIDDM patients to improve their weight or glucose control in combination with an insulin secretagogue or an insulin sensitising agent. Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide. Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone. The present invention further provides a method of improving the body weight or glucose control of NIDDM patients alone, without an insulin secretagogue or an insulin sensitising agent. In a further preferred embodiment, the present invention may be used in complementary therapy of IDDM patients to improve their weight or glucose control in combination with an insulin secretagogue or an insulin sensitising agent. Preferably, the insulin secretagogue is 1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide. Preferably, the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone. The present invention further provides a method of improving the body weight or glucose control of IDDM patients alone, without an insulin secretagogue or an insulin sensitising agent. In a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent). Accordingly, the present invention further provides for a composition of pharmaceutical or physiologically acceptable composition and an insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose control in NIDDM or IDDM patients. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitiser. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) without insulin therapy. In a fifth aspect, the invention features a use of AGONIST described in the first aspect for treatment of obesity-related diseases and disorders and/or reducing body mass. Preferably, said obesity-related diseases and disorders are selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by said AGONIST of the invention include hyperlipidemia and hyperuricemia. In a sixth aspect, the invention features a use of AGONIST described in the first aspect for the preparation of a medicament for the treatment of obesity-related diseases and disorders and/or for reducing body mass. Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. In referred embodiments, said individual is a mammal, preferably a human. In a seventh aspect, the invention provides AGONIST of the first aspect of the invention, or a composition of the second aspect of the invention, for use in a method of treatment of the human or animal body. In an eighth aspect, the invention features methods of reducing body weight comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the second aspect, or AGONIST described in the first aspect. Where the reduction of body weight is practiced for cosmetic purposes, the individual has a BMI of at least 20 and no more than 25. In embodiments for the treatment of obesity, the individual may have a BMI of at least 20. One embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 25. Another embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 30. Yet another embodiment provides for the treatment of individuals with BMI values of at least 40. In further embodiment, the invention features methods of maintaining weight loss comprising providing to an individual said pharmaceutical or physiologically acceptable composition. In a ninth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the second aspect for reducing body mass and/or for treatment or prevention of obesity-related diseases or disorders. Preferably, said obesity-related disease or disorder is selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes). Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure. Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia. In preferred embodiments, said individual is a mammal, preferably a human. In preferred embodiments, the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping LIGAND single nucleotide polymorphisms (SNPs) or measuring LIGAND polypeptide or mRNA levels in clinical samples from said individuals. Preferably, said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva. In a tenth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the second aspect for reducing body weight for cosmetic reasons. In an eleventh aspect, AGONIST of the invention is used in methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the second aspect, or AGONIST described in the first aspect. In a preferred aspect of the methods above and disclosed herein, the amount of AGONIST administered to an individual is sufficient to bring levels of XOBESIN activation to their normal levels (levels in individuals without obesity-related disease or disorder). “Normal levels” of XOBESIN activation may be followed using surrogate markers including circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides. In a twelfth aspect, the invention is directed to a XOBESIN ANTAGONIST, wherein said ANTAGONIST is a soluble fragment of XOBESIN polypeptide, an antibody that specifically binds XOBESIN, a compound excluding said soluble fragment of XOBESIN polypeptide and said XOBESIN antibody (e.g., small molecular weight organic or inorganic compound, protein, peptide, carbohydrate, lipid), or a variant or fragment of LIGAND polypeptide. In a further preferred embodiment, the invention is directed to a XOBESIN ANTAGONIST, wherein said ANTAGONIST is a soluble fragment of XOBESIN polypeptide. More preferably the invention is directed to purified, isolated, or recombinant soluble fragments of XOBESIN polypeptide. More preferably the invention is directed to said soluble fragment of XOBESIN polypeptide, wherein said soluble fragment binds LIGAND and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said soluble fragment of XOBESIN polypeptide does not activate XOBESIN. Preferably said soluble fragment of XOBESIN polypeptide blocks or inhibits LIGAND binding to XOBESIN. In preferred embodiments, said soluble fragment of XOBESIN polypeptide comprises, consists essentially of, or consists of, at least 6 and not more than 246 consecutive amino acids of SEQ ID NO:2, more preferably of amino acids comprising the extracellular domain of XOBESIN. Preferred said soluble fragment of XOBESIN comprises the extracellular domain of mature XOBESIN polypeptide. Particularly preferred soluble fragment of XOBESIN comprises amino acids 30-197, 30-199, 30-207, 30-212, 30-224, 30-231, 30-245 or 30-262 of SEQ ID NO:2, where it is understood that amino acid 30 is predicted to be the N-terminal amino acid of the mature XOBESIN polypeptide absent the putative signal peptide. In other preferred embodiments, said soluble fragment of XOBESIN polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of SEQ ID NO:2. Further preferred embodiments include heterologous polypeptides comprising a XOBESIN polypeptide of the invention. In further preferred embodiment, a XOBESIN polypeptide of the invention is conjugated at its N- or C-terminus to an antibody Fc region or portion thereof. In a further preferred embodiment, the invention is directed to a XOBESIN ANTAGONIST, wherein said ANTAGONIST is an antibody that specifically binds XOBESIN. More preferably the invention is directed to said XOBESIN antibody, wherein said XOBESIN antibody binds XOBESIN and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said XOBESIN antibody does not activate XOBESIN. Preferably said XOBESIN antibody blocks or inhibits LIGAND binding to XOBESIN. In a further preferred embodiment, the invention is directed to a XOBESIN ANTAGONIST, wherein said ANTAGONIST is a compound excluding said soluble fragment of XOBESIN polypeptide and said XOBESIN antibody (e.g., small organic molecule, protein, peptide). More preferably the invention is directed to said compound, wherein said compound binds to XOBESIN and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said compound does not activate XOBESIN. Preferably said compound that binds to XOBESIN blocks or inhibits LIGAND binding to XOBESIN. Further more preferably the invention is directed to said compound, wherein said compound blocks or inhibits LIGAND activity exclusive of binding to XOBESIN, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said compound does not activate XOBESIN. Further more preferably the invention is directed to said compound, wherein said compound blocks or inhibits XOBESIN expression and wherein said compound does not have LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said compound does not activate XOBESIN. In a further preferred embodiment, the invention is directed to a XOBESIN ANTAGONIST, wherein said ANTAGONIST is a variant or fragment of LIGAND polypeptide. More preferably the invention is directed to said variant of fragment of LIGAND polypeptide, wherein said variant or fragment of LIGAND polypeptide binds XOBESIN and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said variant or fragment of LIGAND polypeptide does not activate XOBESIN. Preferably said variant or fragment of LIGAND polypeptide blocks or inhibits LIGAND binding to XOBESIN. More preferably the invention is directed to said variant or fragment of LIGAND polypeptide, wherein said variant or fragment of LIGAND polypeptide inhibits the induction, enhancement, or potentiation of said biological activity exclusive of binding to XOBESIN. In a further preferred embodiment, the invention is directed to a XOBESIN ANTAGONIST that selectively binds to a polypeptide comprising the extracellular domain of XOBESIN. In a further preferred embodiment, said ANTAGONIST is able to raise circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides. Further preferred said ANTAGONISTS are those that significantly inhibit muscle lipid or free fatty acid oxidation stimulated by its LIGAND. Further preferred said ANTAGONISTS are those that cause C2C12 cells differentiated in the presence of LIGAND to undergo at least 10%, 20%, 30%, 35%, or 40% less oleate oxidation as compared to untreated cells. Further preferred said ANTAGONISTS are those that inhibit by at least 10%, 20%, 30%, 35%, or 40% the increase in leptin uptake stimulated by LIGAND polypeptide in a liver cell line [preferably BPRCL mouse liver cells (ATCC CRL-2217)] as compared to untreated cells. Further preferred said ANTAGONISTS are those that significantly increase the postprandial increase in plasma free fatty acids, particularly following a high fat meal. Further preferred said ANTAGONISTS are those that significantly increase ketone body production, particularly following a high fat meal. Further preferred said ANTAGONISTS are those that decrease glucose uptake in skeletal muscle cells stimulated by LIGAND. Further preferred said ANTAGONISTS are those that decrease glucose uptake in adipose cells stimulated by LIGAND. Further preferred said ANTAGONISTS are those that decrease glucose uptake in neuronal cells stimulated by LIGAND. Further preferred said ANTAGONISTS are those that decrease glucose uptake in red blood cells stimulated by LIGAND. Further preferred said ANTAGONISTS are those that decrease glucose uptake in the brain stimulated by LIGAND. Further preferred said ANTAGONISTS are those that significantly increase the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal. Further preferred said ANTAGONISTS are those that significantly facilitate the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal. Further preferred said ANTAGONISTS are those that reduce the insulin sensitivity stimulated by LIGAND. Further preferred said ANTAGONISTS are those that increase body mass, wherein said increase in body mass is comprised of a change in mass of the subcutaneous adipose tissue. Further preferred said ANTAGONISTS are those that increase body mass, wherein said increase in body mass is comprised of a change in mass of the visceral (omental) adipose tissue. In a thirteenth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said ANTAGONIST described in the twelfth aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent. In a fourteenth aspect, the invention features a method of increasing body mass comprising providing or administering to individuals in need of increasing body mass said pharmaceutical or physiologically acceptable composition described in the thirteenth aspect. In a fifteenth aspect, the invention features a method of preventing or treating disorders associated with excessive weight loss comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the thirteenth aspect. Preferably said disorder is selected from the group consisting of cachexia, wasting, cancer-related weight loss, AIDS-related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia. Said disorders associated with excessive weight loss are comprised of those mediated by tumor necrosis factor (TNFalpha) alone, those mediated by TNFalpha plus one or more additional factors, and those mediated only by one or more factors exclusive of TNFalpha. Said factors include, but are not restricted to, macrophage migration inhibitory factor, interleukin 1, and interleukin 6. In preferred embodiments, said individual is a mammal, preferably a human. In related aspects, embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising ANTAGONIST, wherein said biological response is selected from the group consisting of: (a) raising circulating (either blood, serum, or plasma) levels (concentration) of free fatty acids (FFA) or triglycerides (TG); (b) raising circulating (either blood, serum or plasma) levels (concentration) of glucose; (c) raising circulating (either blood, serum or plasma) levels (concentration) of triglycerides; (d) inhibiting muscle lipid or free fatty acid oxidation; (c) inhibiting leptin uptake in the liver or liver cells; (e) increasing the postprandial increase in plasma free fatty acids, particularly following a high fat meal; and, (f) increasing or eliminating ketone body production, particularly following a high fit meal; (g) reducing tissue sensitivity to insulin, particularly muscle, adipose, liver or brain, and further wherein said biological response is greater than a transient response; or alternatively herein said biological response is sustained. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method of increasing body mass in some persons with cachexia, wasting, cancer-related weight loss, AIDS-related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia. In further preferred embodiments, the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin de-sensitiser, wherein the sensitivity of a cell or tissue to insulin is reduced. In a sixteenth aspect, the invention features a method of making the XOBESIN polypeptide described in the twelfth aspect, wherein said method is selected from the group consisting of proteolytic cleavage, recombinant methodology and artificial synthesis. In a preferred embodiment, proteolytic cleavage is carried out using trypsin, plasmin, or collagenase. In a seventeenth aspect, the invention features a use of ANTAGONIST described in the twelfth aspect for the preparation of a medicament for the treatment of disorders associated with excessive weight loss and/or for increasing body mass. Preferably, said disorder is selected from the group consisting of cachexia, wasting, cancer-related weight loss, AIDS-related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia. In preferred embodiments, said individual is a mammal, preferably a human. In an eighteenth aspect, the invention provides ANTAGONIST of the twelfth aspect of the invention, or a composition of the thirteenth aspect of the invention, for use in a method of treatment of the human or animal body. In a nineteenth aspect, the invention features methods of increasing body weight comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the thirteenth aspect, or ANTAGONIST described in the twelfth aspect. Where the increase of body weight is practiced for cosmetic purposes, the individual has a BMI of no greater than 25 and at least 20. In embodiments for the treatment of disorders associated with excessive weight loss, the individual may have a BMI no greater than 20. One embodiment for the treatment of disorders associated with excessive weight loss provides for the treatment of individuals with BMI values of no greater than 15. Alternatively, for increasing the body weight of an individual, the BMI value should be at least 15 and no more than 20. In a twentieth aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the thirteenth aspect for increasing body mass and/or for treatment of disorders associated with excessive weight loss. Preferably, said disorder is selected from the group consisting of cachexia, wasting, cancer-related weight loss, AIDS-related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia. In preferred embodiments, said individual is a mammal, preferably a human. In a twenty-first aspect, the invention features the pharmaceutical or physiologically acceptable composition described in the thirteenth aspect for increasing body weight for cosmetic reasons. In a preferred aspect of the methods above and disclosed herein, the amount of ANTAGONIST administered to an individual is sufficient to bring levels of XOBESIN activation to their normal levels (levels in healthy individuals). “Normal levels” of XOBESIN activation may be followed using surrogate markers including circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides. |
Hook for attaching a portable tool to a carrying device |
Engine powered tools that are carned in a harness on he operator must be easy to release from the harness if the rool is not working properly. The tool is hanging in a hook (14) that is secured to a hip pad (10) on the harness. The hook (14) is releasable from the hip pad (10) by a clip (13) that is opend in case of tool problems. When the clasp (13) is opened the tool will drop to the ground by the gravity. |
1. A device for attaching a hook (14), that is supporting a portable tool, to a hip pad (10) that is hanging down from a harness, characterised in that the hook (14) is attached to the hip pad (10) via a clasp (13) that could be opened so that the hook (14) is released from the hip pad (14). 2. A device according to claim 1, characterised in that the hook (14) is provided with a strap (17) that is fastened in the clasp (13) secured to the hip pad (10). 3. A device according to claim 2, characteiised in that the strap (17) is pressed between different parts of the clasp (13). 4. A device according to claim 3, characterised in that clasp (13) comprises a component (18) that is rotated around an axle (19) and a plate (20) secured in the hip pad (10) 5. A device according to claim 4, characterised in that the section of the component (18) that surrounds the axle (I9) is provided with a protruding flange (22) in the same direction as the axial direction of the axle (19). 6. A device according to claimr 5, characterised in that the strap (17) is pressed between the protruding flange (22) on the component (18) and the plate (20) so that the strap (17) is secured in the clasp (13). 7. A device according to claim 4, 5 or 6, characterised in that the hook (14) is released from the hip pad (10) by rotating the component (18) outwards in relation to the hip pad (10) around the axle (19) so that the pressure on the strap (17) disappear and the strap is released. 8. A device according to claim 4, 5, 6 or 7, characterised in that the end of the component (18) that not is connected to the axle (19) is provided witb a small hook (24) that connects to a protruding part (23) in the end of the plate (20) so that the clasp (13) not is opened unintentionally. |
Carrier device for an engine powered tool |
A harness is used for carrying portable tools like trimmer and clearing saws. The harness comprises at least two straps (11) that pass over the shoulders of the operator and a waist belt (17) secured to a plate (12) placed on the back of the operator. A hip pad (15) hanging in a string or a rope (16) running between the plate (12) and a device (13) placed on the chest of the operator. In order to make the operator carry more of the tool weight by the waist belt is the hip pad (15) secured to the plate (12) on the back of the operator by a third strap (19). |
1. A carrier device for a portable tool comprising at least two straps (11) that pass over the shoulders of the operator and a waist belt (17) secured to a plate (12) placed on the back of the operator and a hip pad (15) hanging in a string or a rope (16) running between the plate (12) and a device (13) placed on the chest of the operator, characterised in that the hip pad (15) is secured to the plate (12) on the back of the operator by a third strap (19). 2. A carrier device according to claim 1, characterised in that the strap (19) is running from the hip pad (15) and behind the back of the operator. 3. A carrier device according to claim 1 or 2, characterised in that the strap (19) is secured to the hip pad (15) and/or the plate (12) in such a way that the length of the strap (19) can be adjusted. 4. A carrier device according to any of the previous claims, characterised in that the strap (19) is secured in the top section of the hip pad (15). 5. A carrier device according to any of the previous claims, characterised in that at least one section of the strap (19) is made of an elastic material. 6. A carrier device according to any of the previous claims, characterised in that the plate (12) placed on the back of the operator is made of a stiff material. |
Hinge construction and hinge actuator, in particular for a wing mirror of a motor vehicle |
A hinge construction comprising two hinge members provided with a guide track. The guide tracks of each of the hinge members comprise at least three substantially flat, circular segment-shaped track parts, which each extend between first and second track ends and which are distributed at uniform intermediate distances over a corresponding circular circumference, so that the track parts of the hinge members pairwise form a guide for at least one guide element. The hinge members are pivotable relative to each other between a first angular position in which the guide elements are received in the guides so as to be displaceable along the track parts, and a second angular position in which the guide elements are received in the guides so as to be clamped between first and second track ends of the track parts. |
1. A hinge construction comprising two hinge members provided with a guide track, which hinge members are arranged so as to be pivotable relative to a common rotation axis, so that the guide tracks together form a guide for guide elements displaceably received between the hinge members, which guide elements, distributed at fixed intermediate distances along the circumference of a circle located around the rotation axis, are included on a common carrier, the guide tracks of each of the hinge members comprising at least three substantially flat, circular segment-shaped track parts, which each extend between first and second track ends and which are distributed at uniform intermediate distances over a corresponding circular circumference, so that the track parts of the hinge members pairwise form a guide for at least one guide element, such that the hinge members are pivotable relative to each other between a first angular position in which the guide elements are received in the guides so as to be displaceable along the track parts, and a second angular position in which the guide elements are received in the guides so as to be clamped between first and second track ends of the track parts. 2. A hinge construction according to claim 1, wherein the track parts per hinge member are located in one flat plane. 3. A hinge construction according to claim 1 or 2, wherein the hinge members are provided with auxiliary guide track parts linking up with the track parts, which auxiliary guide track parts pairwise form an auxiliary guide, such that the hinge members are pivotable further from the second angular position into a third angular position in which the guide elements are received in the auxiliary guides. 4. A hinge construction according to claim 3, wherein the auxiliary guide track parts per hinge member are located in one flat plane. 5. A hinge construction according to claim 3 or 4, wherein the guide tracks and the auxiliary guide track parts per hinge member link up via the track ends to form a stepped annular guide track. 6. A hinge construction according to claim 5, wherein the stepped annular track is formed from sheet material. 7. A hinge construction according to any one of the preceding claims, wherein the guide elements are rolling elements, and wherein the common carrier is a cage in which the rolling elements are bearing-mounted. 8. A hinge construction according to any one of the preceding claims, wherein the common carrier carries three guide elements. 9. A hinge construction according to any one of the preceding claims, wherein the common carrier, with the aid of a catch device, cooperates with at least one of the hinge members. 10. A hinge construction according to claim 9, wherein the catch device comprises a cam placed on the common carrier, which cooperates with a stop arranged on a hinge member. 11. A hinge construction according to any one of the preceding claims 3-10, wherein the hinge members are provided with a limiter which limits pivotal motion of the hinge members relative to each other when the hinge members are in an intermediate angular position in which at least one guide element cooperates with a guide track part of a first hinge member on the one hand and with an auxiliary guide track part of another hinge member on the other hand. 12. A hinge construction according to claim 11, wherein the limiter comprises a stop, which extends between the hinge members to a length less than the sum of the distance between the planes in which the track parts and the auxiliary guide track parts are located in the two hinge members. 13. A hinge construction according to any one of the preceding claims, wherein the guide tracks of the hinge members cooperate in axial direction relative to the rotation axis. 14. A hinge construction according to claim 13, wherein the hinge members cooperate in axial direction under spring action. 15. A hinge actuator, comprising a mirror base which carries a base shaft and a frame part for a mirror housing, which frame part is connected with said base shaft so as to be pivotable relative to the base shaft by means of a hinge construction according to any one of the preceding claims. 16. A hinge actuator according to claim 15, wherein the first hinge member is integrated with the mirror base and/or the second hinge member is integrated with the frame part for the mirror housing. 17. A wing mirror unit for a motor vehicle, provided with a hinge construction according to any one of the preceding claims. |
Anti-coronavirus vaccine |
The invention relates to a vaccine against coronavirus infections and, in particular, against feline infectious peritonitis (FIP). The inventive vaccine comprises immunogenic peptides included in the S protein of feline coronaviruses (FcoV), which do not result from immunologic enhancement. The invention also relates to the use of at least one peptide for the preparation of a vaccine that induces protection against coronavirus infections, said peptide being selected from the group comprising fragments of an S protein of coronavirus of at least 12 amino acids, included in the SEQ ID NO: 5, or nucleic acid fragments of at least 36 nucleotides, included in the SEQ ID NO: 10 and coding for one of said peptides. |
1-23. (Cancelled). 24. A peptide, which is selected from the group consisting of SEQ ID NO: 3, from 12 to 20 amino acids of SEQ ID NO: 3, SEQ ID NO: 4, from 12 to 20 amino acids of SEQ ID NO: 4, the peptide SEQ ID NO: 5 and from 12 to 20 amino acids of SEQ ID NO: 5. 25. A modified peptide, which corresponds to the peptide as claimed in claim 24, into which there have been introduced artificial mutations, deletions, insertions, variations or combinations of these events, provided that the peptides thus modified do not induce enhancement phenomena. 26. The peptide as claimed in claim 24, whichs is in the form of a synthetic peptide, a repeated peptide or a protein fused at the N- or C-terminal end with a protein of another feline pathogenic agent. 27. A method of preventing or protecting a cat against feline infections peritonitis (FIP), wherein the method comprises administering to the cat an amount of a peptide according to claim 24 sufficient for inducing an exclusively neutralizing protection against feline infectious peritonitis. 28. A vaccine for protecting cats against feline infectious peritonitis (FIP), which comprises at least one peptide as claimed in claim 24, in combination with at least one of a carrier substance, an adjuvant, and at least one pharmaceutically acceptable vehicle. 29. The vaccine as claimed in claim 28, comprising an adjuvant; wherein the adjuvant is selected from the group consisting of an oily emulsion, saponin, an inorganic substance, a bacterial extract, aluminum hydroxide, squalene, and mixtures thereof. 30. The vaccine as claimed in claim 28, comprising a carrier substance; wherein the carrier substance is selected from the group consisting of a unilamellar liposome, a multi lamellar liposome, a saponin micelle, a solid micro sphere of a saccharide, a solid microsphere of auriferous nature, and mixtures thereof. 31. The vaccine as claimed in claim 28, which further comprises other viral proteins or peptides. 32. A nucleic acid molecule encoding a peptide as claimed in claim 24. 33. The nucleic acid molecule as claimed in claim 32, which is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, from 36 to 60 nucleotides of SEQ ID NO: 8 and from 36 to 60 nucleotides of SEQ ID NO: 9. 34. The use of at least one nucleic acid molecule as claimed in claim 32 for the construction of recombinant vectors, which are useful as vaccines. 35. A method of preventing or protecting a cat against feline infectious peritonitis (FIP), wherein the method comprises administering to the cat an amount of a nucleic acid according to claim 32 sufficient for inducing an exclusively neutralizing protection against feline infectious peritonitis. 36. A recombinant vector, wherein said vector comprises a nucleic acid molecule as claimed in claim 32. 37. The vector as claimed in claim 36, wherein said vector is a viral vector selected from the group consisting of poxviruses, adenoviruses, retroviruses, and herpes viruses; a bacterial vector selected from the group consisting of mycobacteria, enterobacteria, and lactobacilli; or a plasmid comprising a sequence encoding at least one of peptide which is selected from the group consisting of SEQ ID NO: 3, from 12 to 20 amino acids of SEQ ID NO: 3, SEQ ID NO: 4, from 12 to 20 amino acids of SEQ ID NO: 4, the peptide SEQ ID NO: 5 and from 12 to 20 amino acids of SEQ ID NO: 5. 38. A vaccine for protecting cats against feline infectious peritonitis (FIP), wherein said vaccine comprises a recombinant vector as claimed in claim 36 or at least one nucleic acid molecule encoding a peptide that is selected from the group consisting of the SEQ ID NO: 3, from 12 to 20 amino acids of SEQ ID NO: 3, SEQ ID NO: 4, from 12 to 20 amino acids of SEQ ID NO: 4, SEQ ID NO: 5 and from 12 to 20 amino acids of SEQ ID NO: 5. 39. A vaccine for protecting cats against feline infectious peritonitis (FIP), wherein said vaccine comprises a recombinant vector as claimed in claim 37 and at least one nucleic acid molecule wherein at least one nucleic acid molecule is selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, from 36 to 60 nucleotides of SEQ ID NO: 8 and from 36 to 60 nucleotides of SEQ ID NO: 9. 40. A method for selecting immunogenic peptides corresponding to a fragment of a coronavirus S protein, and not inducing enhancement phenomena, which comprises: constructing a random peptide library corresponding to a coronavirus S protein fragment, from at least one FIPV viral genome, bringing said peptides into contact with at least four different spontaneously regressing (SR) cat sera, after infection with FIPV, at a dilution of at least 1/1000, and immunoselecting the peptides interacting with said spontaneously regressing cat sera, but scarcely or not even interacting at all with sera from cats exhibiting clinical symptoms (CS) or sera from cats exhibiting subclinical signs of chronic infection (CI) with FIPV. |
Cosmetic and dermatological preparations in the form of o/w-emulsions containing sterols and/or c12-c40 fatty acids |
A cosmetic or dermatological composition which comprises a sterol and/or a C12-C40-fatty acid, an ester of a C12-C40-fatty acid with glycerol and/or a glycol, an ester of a C12-C40-fatty acid and sorbitan, an ethoxylated C12-C40-fatty acid and a C12-C40-fatty alcohol. |
1-7. (canceled) 8. A cosmetic or dermatological composition, wherein the composition comprises: (I) from 1% to 5% by weight of at least one substance which is selected from sterols and C12-C40-fatty acids; (II) from 0.1% to 1.5% by weight of at least one ester of one or more C14-C40-fatty acids with one or more of glycerol, propylene glycol and glycol; (III) from 0.1% to 1.5% by weight of at least one ester of a C14-C40-fatty acid of sorbitan, which ester may be ethoxylated with a degree of ethoxylation of up to 100; (IV) from 0.1% to 1.5% by weight of at least one ethoxylated C12-C40-fatty acid having a degree of ethoxylation of up to 100; and (V) from 0.5% to 7% by weight of at least one C12-C40-fatty alcohol. 9. The composition of claim 8, wherein the composition comprises at least one sterol. 10. The composition of claim 9, wherein the at least one sterol comprises at least one of cholesterol and lanosterol. 11. The composition of claim 10, wherein the composition comprises cholesterol. 12. The composition of claim 8, wherein the composition comprises at least one C12-C40-fatty acid. 13. The composition of claim 12, wherein the at least one C12-C40-fatty acid comprises at least one of stearic acid, palmitic acid and lignoceric acid. 14. The composition of claim 13, wherein the composition comprises stearic acid. 15. The composition of claim 8, wherein the composition comprises at least one C12-C40-fatty acid and at least one sterol. 16. The composition of claim 15, wherein the composition comprises cholesterol and stearic acid. 17. The composition of claim 8, wherein the composition comprises at least one of glycerol monostearate, glycerol distearate, propylene glycol monostearate, glycerol isostearate, glycerol lanolate, glycerol myristate, glycerol laurate, glycerol oleate and glycerol stearate citrate. 18. The composition of claim 17, wherein the composition comprises at least one of glycerol monostearate and glycerol distearate. 19. The composition of claim 8, wherein the composition comprises glycerol monostearate. 20. The composition of claim 8, wherein the composition comprises at least one of sorbitan stearate, sorbitan distearate, sorbitan isostearate, sorbitan oleate, PEG-40 sorbitan peroleate, PEG-40 sorbitan perisostearate and sorbitan sesquioleate. 21. The composition of claim 20, wherein the composition comprises sorbitan stearate. 22. The composition of claim 8, wherein the at least one ester of a C14-C40-fatty acid of sorbitan comprises an ester which has a degree of ethoxylation of at least 10. 23. The composition of claim 8, wherein at least one ethoxylated C12-C40-fatty acid having a degree of ethoxylation of up to 100 comprises at least one ester which has a degree of ethoxylation of at least 5. 24. The composition of claim 20, wherein the composition comprises at least one of PEG-20 stearate, PEG-30 stearate, PEG-40 stearate, and PEG-100 stearate. 25. The composition of claim 19, wherein the composition comprises PEG-100 stearate. 26. The composition of claim 8, wherein the at least one C12-C40-fatty alcohol comprises at least one of myristyl alcohol, cetyl alcohol, isocetyl alcohol, cetylstearyl alcohol, stearyl alcohol, isostearyl alcohol and behenyl alcohol. 27. The composition of claim 8, wherein the at least one C12-C40-fatty alcohol comprises at least one wool wax alcohol. 28. The composition of claim 8, wherein a weight ratio (II): (III): (IV) is a b c and a, b and c independently are rational numbers of from 1 to 5. 29. The composition of claim 28, wherein a, b and c independently are rational numbers of from 1 to 3. 30. The composition of claim 28, wherein a weight ratio [(II)+(III)+(IV)]: (V) is from 5:1 to 1:5. 31. The composition of claim 30, wherein the composition has a pH of from 3.5 to 8. 32. The composition of claim 8, wherein the composition has a pH of from 4.5 to 6.5. 33. The composition of claim 8, wherein components (I) to (V) are present in a total amount of at least about 5.5% by weight. 34. The composition of claim 33, wherein components (I) to (V) are present in a total amount of not more than about 11.5% by weight. 35. The composition of claim 32, wherein the composition further comprises at least 2% by weight of liquid lipids. 36. The composition of claim 35, wherein the liquid lipids comprise at least one of a Guerbet alcohol, a saturated triglyceride, an ether of a medium-chain fatty alcohol, a nonpolar lipid, a silicone oil and a dialkyl carbonate. 37. A cosmetic or dermatological composition, wherein the composition comprises: (I) from 1% to 5% by weight of at least one substance which is selected from sterols and C12-C40-fatty acids, which substance comprises at least one of cholesterol, lanosterol, stearic acid, palmitic acid and lignoceric acid; (II) from 0.1% to 1.5% by weight of at least one ester of one or more C14-C40-fatty acids with one or more of glycerol, propylene glycol and glycol, which ester comprises at least one of glycerol monostearate, glycerol distearate, propylene glycol monostearate, glycerol isostearate, glycerol lanolate, glycerol myristate, glycerol laurate, glycerol oleate and glycerol stearate citrate; (III) from 0.1% to 1.5% by weight of at least one ester of a C14-C40-fatty acid of sorbitan, which ester may be ethoxylated with a degree of ethoxylation of up to 100 and comprises at least one of sorbitan stearate, sorbitan distearate, sorbitan isostearate, sorbitan oleate, PEG-40 sorbitan peroleate, PEG-40 sorbitan perisostearate and sorbitan sesquioleate; (IV) from 0.1% to 1.5% by weight of at least one ethoxylated C12-C40-fatty acid having a degree of ethoxylation of up to 100, which ethoxylated fatty acid comprises at least one of PEG-20 stearate, PEG-30 stearate, PEG-40 stearate and PEG-100 stearate; and (V) from 0.5% to 7% by weight of at least one C12-C40-fatty alcohol, which fatty alcohol comprises at least one of myristyl alcohol, cetyl alcohol, isocetyl alcohol, cetylstearyl alcohol, stearyl alcohol, isostearyl alcohol and behenyl alcohol myristyl alcohol, cetyl alcohol, isocetyl alcohol, cetylstearyl alcohol, stearyl alcohol, isostearyl alcohol and behenyl alcohol. 38. The composition of claim 37, wherein the composition comprises: (I) from 1% to 5% by weight of at least one of cholesterol, lanosterol, stearic acid, palmitic acid and lignoceric acid; (II) from 0.1% to 1.5% by weight of at least one of glycerol monostearate, glycerol distearate, propylene glycol monostearate, glycerol isostearate, glycerol lanolate, glycerol myristate, glycerol laurate, glycerol oleate and glycerol stearate citrate; (III) from 0.1% to 1.5% by weight of at least one of sorbitan stearate, sorbitan distearate, sorbitan isostearate, sorbitan oleate, PEG-40 sorbitan peroleate, PEG-40 sorbitan perisostearate and sorbitan sesquioleate; (IV) from 0.1% to 1.5% by weight of at least one of PEG-20 stearate, PEG-30 stearate, PEG-40 stearate and PEG-100 stearate; and (V) from 0.5% to 7% by weight of at least one of myristyl alcohol, cetyl alcohol, isocetyl alcohol, cetylstearyl alcohol, stearyl alcohol, isostearyl alcohol and behenyl alcohol. 39. A cosmetic or dermatological composition, wherein the composition comprises: (I) from 1% to 5% by weight of at least one substance which is selected from sterols and C12-C40-fatty acids; (II) from 0.1% to 1.5% by weight of at least one ester of one or more even-numbered C12-C40-fatty acids with one or more of glycerol and a glycol; (III) from 0.1% to 1.5% by weight of at least one ester of a C12-C40-fatty acid of a sorbitan, which ester may be ethoxylated with a degree of ethoxylation of from 10 to 100; (IV) from 0.1% to 1.5% by weight of at least one ethoxylated C12-C40-fatty acid having a degree of ethoxylation of from 5 to 100; and (V) from 0.5% to 7% by weight of at least one C12-C40-fatty alcohol. 40. A skin lotion which comprises the composition of claim 8. 41. A skin protection cream which comprises the composition of claim 8. 42. A cosmetic milk which comprises the composition of claim 37. 43. A sunscreen lotion which comprises the composition of claim 8. 44. The composition of claim 8, wherein the composition further comprises at least one UV absorbing substance. 45. The composition of claim 8, wherein the composition further comprises at least one antioxidant. 46. The composition of claim 8, wherein the composition further comprises at least one of a cosmetically active ingredient and a dermatologically active ingredient. 47. A method for the cosmetic or dermatological treatment of skin, wherein the method comprises applying onto at least parts of the skin the composition of claim 8. |
Structure for reducing internal circuit of fuel cell |
In a structure for reducing internal circuit of a fuel cell including adjacently stacked unit cells; a fuel side distributing means for connecting each fuel side inflow path of the unit cells and insulating them; and an air side distributing means for connecting each air side inflow path of the unit cells, electric connection among the stacked plural unit cells by fuel as an electrolyte solution and electric leakage by additional parts can be minimized. |
1. A structure for reducing internal circuit of a fuel cell, comprising: adjacently stacked unit cells; a fuel side distributing means for connecting each fuel side inflow path of the unit cells and insulating them electrically; and an air side distributing means for connecting each air side inflow path of the unit cells. 2. The structure of claim 1, wherein the fuel side distributing means is a fuel side distributing pipe for connecting fuel side inflow paths of the unit cells and forming an insulating space, and the fuel side distributing pipe is made of an insulating material. 3. The structure of claim 1, wherein the fuel side inflow paths and the air side inflow paths are arranged so as to be opposite to each other. 4. The structure of claim 1, wherein a pump for supplying fuel is installed as the fuel side distributing means. 5. The structure of claim 1, wherein outflow pipes are respectively connected with fuel side outflow paths of the unit cells. 6. The structure of claim 1, wherein a pump for supplying air is installed as the air side distributing means. 7. A structure for reducing internal circuit of a fuel cell, comprising: a stack consisting of adjacently stacked unit cells; a first and a second manifolds respectively arranged on both sides of the stack so as to have fuel side connection paths for connecting fuel side paths of the unit cells and air side connection paths for connecting air side paths of the unit cells; a first insulating member combined between the stack and the first manifold so as to have fuel side through holes for connecting the fuel side paths of the unit cell with the fuel side connection path of the first manifold and air side through holes for connecting the air side paths of the unit cell with the air side connection path of the first manifold; and a second insulating member combined between the stack and the second manifold so as to have fuel side through holes for connecting the fuel side paths of the unit cell with the fuel side connection path of the second manifold and air side through holes for connecting the air side paths of the unit cell with the air side connection path of the second manifold. 8. The structure of claim 7, wherein the first and second insulating members respectively have a certain thickness so as to make internal through holes thereof have an insulating space. 9. The structure of claim 7, wherein the first and second manifolds are made of an insulating material. 10. The structure of claim 7, wherein the fuel side connection path of the first manifold is formed so as to connect fuel side inflow paths of adjacent two unit cells with each other, and the air side connection path of the first manifold is formed so as to connect air side outflow paths of the two unit cells with each other. 11. The structure of claim 7, wherein the first manifold is divided into a part including the fuel side connection path and a part including the air side connection path. 12. The structure of claim 7, wherein the fuel side connection path of the second manifold is formed so as to connect fuel side outflow paths of adjacent two unit cells with each other, and the air side connection path of the second manifold is formed so as to connect air side inflow paths of the two unit cells with each other. 13. The structure of claim 7, wherein the second manifold is divided into a part including the fuel side connection path and a part including the air side connection path. |
<SOH> BACKGROUND ART <EOH>Fuel cell has been presented as a substitute for fossil fuel, and it converts chemical energy generated by oxidation of fuel such as hydrogen into electric energy directly. FIG. 1 illustrates an example of a fuel cell. As depicted in FIG. 1 , in the fuel cell, when hydrogen-included fuel and air as a oxidant are supplied to a fuel electrode (anode) 11 and an air electrode (cathode) 12 arranged on both sides of an electrolyte layer 10 respectively, electrochemical oxidation reaction occurs on the fuel electrode 11 , hydrogen ions and electrons are emitted, the hydrogen ions are moved to the air electrode 12 through the electrolyte layer 10 , and the electrons are moved to the air electrode 12 through a load 20 connecting the fuel electrode 11 to the air electrode 12 . Simultaneously electrochemical reduction reaction occurs on the air electrode 12 , and heat and by-products are generated while the hydrogen ions are combined with oxygen. Herein, current is generated while the electrons emitted from the fuel electrode 11 are moved to the air electrode 12 . One unit fuel cell is constructed with the structure. Herein, in order to generate greater electric energy, a fuel cell can be constructed by combining plural unit cells. In addition, fuel cells can be classified into various kinds according to kinds of fuel, operational temperature and catalyzers, etc. When fuel of hydrogen group such as NaBH4, KBH4, LiA1H4, KH, NaH, etc. is dissolved in an alkali aqueous solution, the fuel becomes an electrolyte solution, electrons generated with hydrogen ions are moved through the electrolyte solution (fuel). FIG. 2 is a sectional view illustrating an example of a fuel cell using the electrolyte solution as fuel in accordance with the conventional art, FIG. 3 is a plane view illustrating a stack of the fuel cell, FIG. 4 is a plane view illustrating a first manifold of the fuel cell, and FIG. 5 is a plane view illustrating a second manifold of the fuel cell. As depicted in FIGS. 2 ˜ 5 , in the fuel cell, monopolar plates 110 , 120 are respectively arranged on both sides of one bipolar plate 100 , two M.E.As (membrane electrode assembly) 130 are respectively inserted between the bipolar plate 100 and the monopolar plate 110 , 120 , and an end plate 140 is respectively arranged on both sides of the monopolar plates 110 , 120 . The bipolar plate 100 , the monopolar plate 110 , 120 , the M.E.A 130 and the end plate 140 are fixedly combined by fastening means 150 , and accordingly a stack is constructed. In the bipolar plate 100 , fluid flowing channels 102 , 103 are respectively formed on both sides of a plate 101 having a certain thickness and area; and inflow paths 104 , 105 and outflow paths 106 , 107 in which fuel and air flow respectively are formed so as to be connected with the channels 102 , 103 . In the monopolar plates 110 , 120 , fluid flowing channels 112 , 122 are formed on a side of plates 111 , 121 having a certain thickness and area; and inflow paths 113 , 123 and outflow paths 114 , 124 connected to the channels 112 , 122 are formed on the plates 111 , 121 so as to receive and discharge a fluid. The fuel side inflow paths 104 , 123 of the bipolar plate 100 and the monopolar plates 110 , 120 are arranged on the same line, and the air side inflow paths 105 , 113 are arranged on the same line with the fuel side inflow paths 104 , 123 so as to have a certain interval. In the M.E.A 130 , a fuel side electrode 132 contacted to fuel is formed on a side of the electrolyte layer 131 having a certain area, and an air side electrode 133 contacted to air is formed on the other side of the electrolyte layer 131 . In the M.E.As 130 , the same electrode is arranged on the same position. A first manifold 160 for distributing fuel and air so as to make them flow into the fuel side inflow paths 104 , 123 and the air side inflow paths 105 , 113 respectively is arranged on a side of the stack, a second manifold 170 for gathering fuel and air to be respectively discharged to the fuel side outflow paths 106 , 124 and the air side outflow paths 107 , 114 is arranged on the other side of the stack, and the first and second manifolds 160 , 170 are fixedly combined by additional fastening means 180 . In the first manifold 160 , a fuel side space 162 and an air side space 163 are respectively formed in a body unit 162 having a certain thickness and rectangular area, through holes 164 connected with the fuel side inflow paths 104 , 123 are formed on the bottom of the fuel side space 162 , and through holes 165 connected with the air side inflow paths 105 , 113 are formed on the bottom of the air side space 163 . And, in the second manifold 170 , a fuel side space 172 and an air side space 173 are respectively formed in a body unit 171 having a certain thickness and rectangular area, through holes 174 connected with the fuel side outflow paths 106 , 124 are formed on the bottom of the fuel side space 172 , and through holes 175 connected with the air side outflow paths 107 , 114 are formed on the bottom of the air side space 173 . The fuel side space 162 of the first manifold is connected to a fuel tank (not shown) and a pump (not shown) by a pipe (not shown), and the fuel side space 172 of the second manifold is connected to the fuel tank by an additional reproducing means (not shown). In the above-described fuel cell, when fuel in the fuel tank flows into the fuel side space 162 of the first manifold, simultaneously air flows into the air side space 163 of the first manifold. The fuel in the fuel side space 162 flows into the bipolar plate 100 and the inflow paths 104 , 123 of the monopolar plate 120 of the stack through the through holes 164 . When the fuel flows in the channels 102 , 122 , electrochemical oxidation occurs on the fuel side electrode 132 of the M.E.A 130 , hydrogen ions and electrons are generated, the hydrogen ions are moved to the air side electrode 133 through the electrolyte layer 131 of the M.E.A, and the electrons are moved to the air side electrode 133 through the bipolar plate 100 or the monopolar plates 110 , 120 . Simultaneously, when the air in the air side space 163 of the first manifold flows into the channels 103 , 112 through the through holes 165 in the air side space, each bipolar plate 100 and the inflow paths 105 , 113 of the monopolar plate 110 of the stack, electrochemical reduction reaction occurs with the hydrogen ions on the air side electrode 133 of the M.E.A. In the meantime, the fuel discharged into the fuel side space 172 of the second manifold flows into the fuel tank through the reproducing means and is supplied again to the stack. And, when a load is connected between the monopolar plates 110 , 120 , electric energy is generated while current flows through the load by electric potential difference. However, in the conventional structure, because the electrolyte solution is used as fuel, the fuel connects the stacked unit cells electrically so as to construct an internal circuit, electric leakage may occur, and accordingly electrical loss may occur. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention. In the drawings: FIG. 1 is a sectional view illustrating a general fuel cell; FIG. 2 is a sectional view illustrating an example of the conventional fuel cell; FIG. 3 is a plane view illustrating a stack of a fuel cell in accordance with the conventional art; FIGS. 4 and 5 are plane views respectively illustrating partial-exploded first and second manifolds of the fuel cell in accordance with the conventional art; FIG. 6 is a sectional view illustrating a fuel cell having a structure for reducing internal circuit of a fuel cell in accordance with a first embodiment of the present invention; FIG. 7 is a plane view illustrating the fuel cell in FIG. 6 ; FIG. 8 is a sectional view illustrating a fuel cell having an internal circuit reducing structure in accordance with a second embodiment of the present invention; FIG. 9 is a sectional view illustrating the fuel cell taken along a line A-B in FIG. 8 ; FIG. 10 is a sectional view illustrating the fuel cell taken along a line C-D in FIG. 8 ; and FIG. 11 is a graph showing comparison results of unit cells in accordance with the first and second embodiments of the present invention. detailed-description description="Detailed Description" end="lead"? |
Data processing circuit, display device, and mobile terminal |
When a data processing circuit is formed on an insulating substrate by using TFTs, it is difficult to process a data signal having a high data rate, such as digital display data, at a high speed. In a data processing circuit formed on an insulating substrate by using TFTs, a data signal having a small voltage amplitude input in series is increased in level to a data signal having a large voltage amplitude by a level shift circuit (11), the serial data signal having the large voltage amplitude is converted to parallel data signals by a serial-parallel conversion circuit (12), and then, the parallel data signals are reduced in level to data signals having a small voltage amplitude by level shift circuits (13A and 13B). Therefore, high-speed processing can be applied to digital data signals at a low power consumption. |
1. A data processing circuit characterized by comprising: first level-conversion means for level-converting a data signal having a first voltage amplitude input in series to a data signal having a second voltage amplitude larger than the first voltage amplitude; serial-parallel conversion means for converting the data signal level-converted by the first level-conversion means to parallel data signals; and second level-conversion means for level-converting the parallel data signals to data signals having a third voltage amplitude smaller than the second voltage amplitude, and characterized in that the data processing circuit is formed on an insulating substrate by using thin-film transistors. 2. A data processing circuit according to claim 1, characterized in that the first level-conversion means and the serial-parallel conversion means comprise a plurality of sample-and-latch level conversion circuits to which the data signal having the first voltage amplitude is input in common, and sampling is performed in the plurality of sample-and-latch level conversion circuits by a plurality of sampling signals having different timing. 3. A display apparatus characterized by comprising: a display section formed of pixels disposed in a matrix manner on a transparent, insulating substrate; a plurality of horizontal driving circuits mounted on the transparent, insulating substrate together with the display section, for writing display data into the pixels in the display section; and a data processing circuit mounted on the transparent, insulating substrate together with the display section, for processing a display data signal having a first voltage amplitude input in series from the outside of the substrate and for sending to the plurality of horizontal driving circuits, and characterized in that the data processing circuit is formed of thin-film transistors, and the data processing circuit comprises first level-conversion means for level-converting the display data signal having the first voltage amplitude to a display data signal having a second voltage amplitude larger than the first voltage amplitude; serial-parallel conversion means for converting the display data signal level-converted by the first level-conversion means to parallel display data signals; and second level-conversion means for level-converting the parallel display data signals to display data signals having a third voltage amplitude smaller than the second voltage amplitude and for sending the display data signals to the plurality of horizontal driving circuits. 4. A display apparatus according to claim 3, characterized in that the plurality of horizontal driving circuits is operated at the third voltage of a power supply, and comprises a data latch circuit group for latching the display data signals sent from the data processing circuit. 5. A display apparatus according to claim 4, characterized in that the plurality of horizontal driving circuits is operated at the third voltage of a power supply, and comprises a second latch circuit group for latching display data signals collectively sent from the data latch circuit group, and a level shift circuit group for level-converting the display data signals latched by the second latch circuit group to display data signals having a fourth voltage amplitude larger than the third voltage amplitude. 6. A display apparatus according to claim 5, characterized in that the fourth voltage amplitude is set to a voltage amplitude required for processing in a DA conversion circuit group for converting the display data signals output from the level shift circuit group, to analog display signals. 7. A portable terminal characterized by having mounted thereon a display apparatus as a screen display section, the display apparatus comprising: a display section formed of pixels disposed in a matrix manner on a transparent, insulating substrate; a plurality of horizontal driving circuits mounted on the transparent, insulating substrate together with the display section, for writing display data into the pixels in the display section; and a data processing circuit mounted on the transparent, insulating substrate together with the display section, for processing a display data signal having a first voltage amplitude input in series from the outside of the substrate and for sending to the plurality of horizontal driving circuits, wherein the data processing circuit is formed of thin-film transistors, and the data processing circuit comprises first level-conversion means for level-converting the display data signal having the first voltage amplitude to a display data signal having a second voltage amplitude larger than the first voltage amplitude; serial-parallel conversion means for converting the display data signal level-converted by the first level-conversion means to parallel display data signals; and second level-conversion means for level-converting the parallel display data signals to display data signals having a third voltage amplitude smaller than the second voltage amplitude and for sending the display data signals to the plurality of horizontal driving circuits. |
<SOH> BACKGROUND ART <EOH>In the field of flat-panel-type display apparatuses, typical of which are liquid-crystal display apparatuses and EL (electroluminescence) display apparatuses, so-called driving-circuit-united-type display apparatuses have been developed in order to make the frames of the panels smaller and make the panels thinner. In the driving-circuit-united-type display apparatuses, a display section in which pixels are arranged in a matrix manner and peripheral driving circuits for driving the display section are mounted on a transparent, insulating substrate as a unit. In liquid-crystal display apparatuses and EL display apparatuses, since thin-film transistors (TFT) are used as pixel transistors, the peripheral driving circuits are also formed by using TFTs when the peripheral driving circuits are mounted on a transparent, insulating substrate. The peripheral driving circuits of the display apparatuses include a vertical driving circuit for selecting pixels in the display section in units of lines and a horizontal driving circuit for writing display data into each pixel in the selected line. In addition, a data processing circuit for applying various processes to display data to be sent to the horizontal driving circuit needs to be included. It is assumed here that the data processing circuit is formed by using TFTs on a transparent, insulating substrate, such as a glass substrate, in a display apparatus. TFTs have much variance in element characteristics and the absolute values of their thresholds Vth are large. When TFTs are formed on an insulating substrate, such as a glass substrate, it is known that their element characteristics become worse than when TFTs are formed on a silicon substrate. Therefore, when a data processing circuit is formed on an insulating substrate by using TFTs, where the absolute values of the thresholds Vth are large, it is difficult to process at a high speed, data signals having high data rates, such as digital display data signals. Even when the absolute values of the thresholds Vth are large, if the power-supply voltage of the circuit is set high and the data signals are handled as large-amplitude signals, it is possible to handle digital data signals having high data rates at a high speed. When the power-supply voltage of the data processing circuit is set high, however, the power consumption of the data processing circuit increases very much. Therefore, it is disadvantageous when the display apparatus has a driving-circuit-united-type structure to reduce its power consumption. The present invention has been made in consideration of the above issues. An object of the present invention is to provide a data processing circuit capable of processing digital data signals at a high speed with a low power consumption even if the data processing circuit is formed on an insulating substrate by using TFTs, a display apparatus which uses the data processing circuit as one of peripheral driving circuits for a display section, and a portable terminal in which the display apparatus is mounted as an image display section. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a block diagram showing an example structure of a data processing circuit according to an embodiment of the present invention. FIG. 2 is a block diagram showing an example specific structure of a level shift circuit and a serial-parallel conversion circuit. FIG. 3 is a circuit diagram showing a specific circuit example of a sample-and-latch level shift circuit which also functions as a serial-parallel conversion circuit. FIG. 4 is a block diagram showing an example structure of a driving-circuit-united-type liquid-crystal display apparatus according to the present invention. FIG. 5 is a circuit diagram showing an example structure of a pixel in a display section. FIG. 6 is a circuit diagram showing an example specific circuit of a data sample-and-latch section, a second latch section, and a level shifter in a horizontal driver. FIG. 7 is an appearance view showing an outlined structure of a PDA according to the present invention. detailed-description description="Detailed Description" end="lead"? |
Graft polymer martrices |
A three-dimensional, non-crosslinked, linear or branched graft polymer matrix suitable for microassays comprises one or more active chemical moieties having inherent specificity for binding to a chemical, biochemical, or biological probe or target, the moieties being permanently attached to and distributed throughout the graft polymer matrix, optionally including one or more probes. |
1. A three-dimensional, non-crosslinked, linear or branched graft polymer matrix comprising one or more active chemical moieties having inherent specificity for binding to a chemical, biochemical, or biological probe, wherein said moieties are permanently attached to and distributed throughout the graft polymer matrix. 2. The matrix of claim 1 additionally comprising at least one probe having selective affinity for a target. 3. The matrix of claim 1 wherein the matrix is comprised of linear or branched graft polymer molecules of controlled length and density. 4. The matrix of claim 1 wherein the active chemical moieties are attached as side chains to the backbone of the graft polymer chain. 5. The matrix of claim 1 wherein the probe is a nucleic acid or protein. 6. The matrix of claim 1 wherein the probe is a toxin, pathogen or pharmaceutical agent. 7. The matrix of one of claim 2, wherein the target is selected from the group consisting of viruses, bacteria, fungi, parasites, and molecules or molecular fragments of DNA, RNA, proteins, carbohydrates and lipids. 8. The matrix of claim 1 additionally comprising at least one structural modifier. 9. The matrix of claim 1 wherein the active chemical moieties are selected from the group consisting of amines, carboxylic acids, epoxides, aldehydes, sulfhydryls, haloacetamides and carboxylic acid succinimidyl esters. 10. The matrix of claim 9 wherein the active chemical moiety is a N-hydroxysuccinimidyl ester of a carboxylic acid. 11. The matrix of claim 1 that contains a plurality of different chemical moieties. 12. The matrix of claim 1 wherein the chemical moieties are further modified to alter their reactivity. 13. The matrix of claim 12 wherein the chemical moieties are side-chain chemical moieties, and are further modified with spacer groups to further enhance their reactivity. 14. An article comprising the matrix of claim 1. 15. (canceled) 16. (canceled) 17. (canceled) 18. (canceled) 19. An article having on its surface a coating comprising a three-dimensional, non-crosslinked, linear or branched graft polymer matrix, bonded to the article, comprising one or more chemical moieties that are permanently attached to and distributed throughout the graft polymer matrix wherein said chemical moieties impart specific functional utility to the article. 20. (canceled) 21. (canceled) 22. (canceled) 23. (canceled) 24. (canceled) 25. (canceled) 26. (canceled) 27. (canceled) 28. (canceled) 29. (canceled) 30. (canceled) 31. (canceled) 32. (canceled) 33. (canceled) 34. (canceled) 35. (canceled) 36. (canceled) 37. An article having on its surface a multilayer coating comprising: a) an adhesive layer comprising a polymer or polymer mixture, bound to the article surface; and b) a three-dimensional, non-crosslinked, linear or branched graft polymer matrix, bonded to the polymer layer, comprising one or more chemical moieties that are permanently attached to and distributed throughout the graft polymer matrix, wherein said chemical moieties impart specific functional utility to the article. 38. (canceled) 39. (canceled) 40. (canceled) 41. (canceled) 42. (canceled) 43. (canceled) 44. (canceled) 45. (canceled) 46. (canceled) 47. (canceled) 48. (canceled) 49. (canceled) 50. (canceled) 51. (canceled) 52. (canceled) 53. (canceled) 54. (canceled) 55. (canceled) 56. A method for applying a three-dimensional, linear or branched graft polymer matrix to an article with a controlled graft polymer surface density, said method comprising the steps of: a) applying to said surface a solution comprising: i) an organic solvent or mixture of organic solvents, i) a polymer or mixture of polymers which is soluble in said solvent or solvent mixture and insoluble in water, and iii) a radical initiator that is soluble in said solvent or solvent mixture, said initiator being capable of generating reactive radical sites on the polymer-coated surface and initiating a graft polymerization reaction on said surface by generating reactive radical sites thereon; b) removing the solvents to leave upon the polymer-coated surface a coating of initiator dissolved in polymer; c) immersing the polymer coated surface in a medium comprising one or more monomers in solution that are capable of reacting with the reactive sites created by surface bound initiator to form a polymer chain grafted onto said polymer-coated surface; d) initiating a graft polymerization reaction on said polymer-coated surface by generating reactive radical sites thereon; e) graft polymerizing onto the polymer-coated surface the reactive monomers from the medium by forming covalent bonds between monomer molecules and the polymer-coated surface at reactive radical sites on the polymer-coated surface; to obtain a coated article. 57. (canceled). 58. (canceled) 59. (canceled) 60. (canceled) 61. (canceled) 62. (canceled) 63. (canceled) 64. (canceled) 65. (canceled) 66. (canceled) 67. (canceled) 68. (canceled) 69. (canceled) 70. (canceled) 71. (canceled) 72. (canceled) 73. (canceled) 74. (canceled) 75. (canceled) 76. (canceled) 77. (canceled) 78. (canceled) 79. A method for applying a three-dimensional, linear or branched graft polymer matrix with a controlled graft polymer surface density to an article, said method comprising the steps of: a) applying to said surface a solution comprising: i) an organic solvent or mixture of organic solvents and ii) a polymer or mixture of polymers which is soluble in said solvent or solvent mixture and insoluble in water; b) removing the solvents to obtain a polymer-coated surface; c) exposing said polymer-coated surface to an initiator capable of generating reactive radical sites on the polymer coated surface and initiating a graft polymerization reaction on said surface by generating reactive radical sites thereon; d) immersing the polymer-coated surface in a medium comprising one or more monomers in solution that are capable of reacting with the reactive sites created by surface bound initiator to form a polymer chain grafted onto said surface; e) initiating a graft polymerization reaction on said coated surface by generating reactive radical sites thereon; f) graft polymerizing onto the coated surface the reactive monomers from the medium by forming covalent bonds between monomer molecules and the surface at reactive radical sites on the polymer coated surface; to obtain an article with a coated surface having a controlled graft polymer surface density. 80. (canceled) 81. (canceled) 82. (canceled) 83. (canceled) 84. (canceled) 85. (canceled) 86. (canceled) 87. (canceled) 88. (canceled) 89. (canceled) 90. (canceled) 91. (canceled) 92. (canceled) 93. (canceled) 94. (canceled) 95. (canceled) 96. (canceled) 97. (canceled) 98. (canceled) 99. (canceled) 100. (canceled) 101. (canceled) 102. A method for applying a three-dimensional, non-crosslinked, linear or branched graft polymer matrix with a controlled graft polymer surface density to an article, said method comprising the steps of: a) applying to said surface a solution comprising: i) an organic solvent or mixture of organic solvents with water, and ii) a silane monomer or mixture of silane monomers or nonreactive silanes which is soluble in said solvent or solvent water mixture; b) removing the solvent to obtain a coated surface with silane attached monomers or other silane attached non-reactive groups upon the surface; c) exposing said coated surface to an initiator capable of generating reactive radicals on the surface and initiating a graft polymerization reaction on said surface by generating reactive radical sites thereon; d) immersing the coated surface in a medium comprising one or more monomers in solution that are capable of reacting with the reactive radical sites created by surface bound initiator to form a polymer chain grafted onto said surface; and e) initiating a graft polymerization reaction on said coated surface by generating reactive radical sites thereon; f) graft polymerizing onto the coated surface the reactive monomers from the medium by forming covalent bonds between monomer molecules and the surface at reactive radical sites on the coated surface to obtain an article with a three-dimensional, non-crosslinked, linear or lightly branched graft polymer matrix with a controlled graft polymer surface density. 103. (canceled) 104. (canceled) 105. (canceled) 106. (canceled) 107. (canceled) 108. (canceled) 109. (canceled) 110. (canceled) 111. (canceled). 112. (canceled) 113. (canceled) 114. (canceled) 115. (canceled) 116. (canceled) 117. (canceled) 118. (canceled) 119. (canceled) 120. (canceled) 121. (canceled) 122. (canceled) 123. A method for applying a three-dimensional, non-crosslinked, linear or lightly branched graft polymer matrix with a controlled graft polymer surface density to an article, said method comprising the steps of: a) applying to said surface a solution comprising: i) an organic solvent or mixture of organic solvents with water and ii) a silane with an attached initiator a or mixture of nonreactive silanes with attached initiators or silanes which is soluble in said solvent or solvent water mixture; b) removing the solvents to leave a coated surface with silane attached initiators or other silane attached non-reactive groups upon the surface, c) immersing the coated surface in a medium comprising one or more monomers in solution that are capable of reacting with the reactive sites created by surface bound initiator to form a polymer chain grafted onto said surface; d) initiating a graft polymerization reaction on said coated surface by generating reactive radical sites thereon; and e) graft polymerizing onto the coated surface the reactive monomers from the medium by forming covalent bonds between monomer molecules and the surface at reactive radical sites on the coated surface; to obtain an article having a three-dimensional, non-crosslinked, linear or lightly branched graft polymer matrix with a controlled graft polymer surface density. 124. (canceled) 125. (canceled) 126. (canceled) 127. (canceled) 128. (canceled) 129. (canceled) 130. (canceled) 131. (canceled) 132. (canceled) 133. (canceled) 134. (canceled) 135. (canceled) 136. (canceled) 137. (canceled) 138. (canceled) 139. (canceled) 140. (canceled) 141. (canceled) 142. (canceled) 143. (canceled) 144. A method of making a microarray comprising the steps of a) applying the matrix of claim 1 to a surface, wherein said matrix contains chemical moieties that react with, or can be modified to react with a biomolecule; b) providing a solution of a probe biomolecule; c) applying said solution to the matrix under conditions such that said probe biomolecules become bound to said chemical moieties. 145. (canceled) 146. (canceled) 147. A microarray made by the method of claim 144. 148. A biochip comprising the microarray of claim 147. 149. A method of detecting a target biomolecule, comprising contacting the biochip of claim 148 with a test sample in which the target biomolecule may be present. |
<SOH> BACKGROUND OF THE INVENTION <EOH>An important development in medicine has been the ability to quickly and reliably screen individuals for diseases and more recently genetic markers which may pre-dispose individuals to develop illnesses during their lifetime. The development of clinical diagnostic methods based on gene expression profiles is growing rapidly due to genetic information gathered by the Human Genome Project, the generation of animal models to study human disease and many other genomic and proteomic approaches being applied to decipher the molecular pathogenesis of disease over the last decade. A consequence of these efforts has been to strive for the development of high speed, high throughput, and highly reproducible DNA and protein microarray technologies. The basic concepts of these technologies are the same as those for enzyme based immunoassay and RNA-based northern blotting techniques that have been used for years to detect protein antigens and gene expression levels. Highly specific probes generated to query a large number of molecules (e.g. antigens or nucleic acid sequences) are attached to the surface of a sample slide and exposed to target molecules generated from a wide variety of biological materials. If target molecules are present in the hybridization cocktail, they will bind with high affinity and specificity to substrate-bound probe molecules immobilized on the slide. The slide is processed and imaged in a way that only areas containing bound target/probe substrate are detectable and fully quantifiable through radiolabeling, enzymatic colorimetry, fluorescence spectroscopy, mass spectroscopy or other techniques known to those skilled in the art. An important difference between gene expression approaches using microarrays and more standard molecular biological approaches is their relative size scales. An example of this is the comparison of a microtiter plate based approach where the well of a typical 96-well microtiter plate is about 1 cm in diameter and can contain up to several hundred microliters of probe-containing solution. In contrast, a DNA microarray spot is only about 25-100 μm in diameter and can be printed with just 200-500 pl of probe-containing solution. Thus, a substrate the size of a microscope slide can contain an array of tens of thousands of spots, with each spot containing a different molecular probe or probe concentration. With high resolution and high speed automated printing processes that are now available, microarrays can be manufactured reliably and on a large scale. One common microarray surface structure comprises a linker molecule connecting the surface of the substrate (typically glass) with one end of the probe molecule. Typical linker molecules for DNA microarrays are amino-terminated silanes. They bind covalently to the glass surface through the silane end and are photo-crosslinked through the amine end to a DNA probe. Alternatively, aldhehyde-terminated linker molecules can be used. In this scheme, the aldehyde functionalities form Schiff base adducts with amine groups conjugated to the thymidine residues of the DNA oligomer. Protein microarray substrates containing binding proteins such as monoclonal antibodies, single chain antibodies and/or peptides, for example, may also be prepared in this manner. A significant limitation to this approach is the inability to achieve more than one layer of probes on the slide surface. This two-dimensionality imposes an important spatial constraint given the surface area of a microarray spot. The inability to stack probes in three dimensions directly impacts the maximum dynamic range and sensitivity that may be achieved with this substrate structure. A simple calculation underscores the significance of this limitation. The surface density of oligonucleotides on aminated glass surfaces has been estimated at 0.1 pmol/mm 2 (˜1 molecule per 1600 Å 2 ), meaning that there are ˜1.2×10 8 sites available for probe binding on a 50 μm diameter spot. A 260 pl drop of a DNA oligomer solution containing 500 ng/μl DNA will contain ˜4×10 11 oligomer units (assuming a 20 unit oligomer and average MW of 200/nucleic acid residue). Thus, such a droplet will have over a 1000-fold more genetic material than the surface is capable of accepting. It is clear that the limiting factor in increasing microarray dynamic range is not the concentration of the target in solution but the rather the number of sites on the microarray to which the target can bind. As microarray features become smaller and smaller with the continued interest in miniaturization, the space available for those binding sites will continue to decrease. Construction of microarray surfaces that are capable of accepting probe molecules in three dimensions will allow a substantial increase in the density of probes per unit area on the microarray surface. Three-dimensional surfaces also make it possible for probe or target molecules to attach to the array without steric hindrance or surface interference. This sort of steric hindrance could most likely be associated with the use of large proteins or nucleic acid sequences. It is for this reason that we have developed technology that will allow the three-dimensional placement of probes on microarray surfaces. One solution to these problems is the development of three-dimensional matrices. Such three-dimensional matrices should be rapidly porous to liquids so that probes may be attached throughout the depth of the matrix, thus making it possible to achieve higher spatial densities of probe molecules than has been achieved previously. Increased matrix porosity will improve the efficiency and degree of the target/probe hybridization process. It is well known that protein interactions are adversely affected due to the inherent instability and denaturation of proteins adhered on solid surfaces. This limits the use of specific binding proteins, for example as found with antibody/antigen and receptor/ligand interactions, on two-dimensional surfaces. Therefore, a three-dimensional matrix will allow for a pseudo-solution phase where polymer-bound and target proteins are removed from the solid surface and evenly dispersed throughout the matrix increase binding efficiency. A number of approaches have been taken to develop three-dimensional surfaces capable of binding probe molecules to them. For example, hydrogel layers in various configurations are often cumbersome and expensive and/or unreliable to manufacture in large volume. Potential problems with some of these approaches arise from the possible limited access to the inner part of the matrix of probes and targets as a result of the crosslinked matrix. Duran et al. disclose in U.S. Pat. No. 5,858,653 reagent compositions for covalent attachment of target molecules, such as nucleic acids, onto the surface of a substrate. The reagent compositions include groups capable of attracting the target molecule as well as groups capable of covalently binding to the target molecule, once attracted. Optionally, the compositions can contain photoreactive groups for use in attaching the reagent composition to a surface. This method has several limitations and disadvantages. First, it can be used to bond onto polymeric surfaces only. In addition, initiator contained in the aqueous copolymer medium may lead to non-grafted polymer and block copolymer, which requires crosslinking to be permanently retained in the layer. Finally, the patent does not disclose methods and compositions for use on glass surfaces, and therefore does not enable one skilled in the art to make coatings on glass without undue experimentation. Hahn, et al. disclose in U.S. Pat. No. 6,174,683, methods for preparing a biochip wherein the biomolecular probe to be used with the biochip is alternately bound to a hydrogel prepolymer prior to or simultaneously with polymerization of the prepolymer. In particularly preferred embodiments, a polyurethane-based hydrogel prepolymer is derivitized with an organic solvent soluble biomolecule, such as a peptide nucleic acid probe in aprotic, organic solvent. Following derivitization of the prepolymer, an aqueous solution, for example sodium bicarbonate, preferably buffered to a pH of about 7.2 to about 9.5, is added to the derivatized prepolymer solution to initiate polymerization of the hydrogel. Alternatively, a water soluble biomolecule, such as DNA or other oligonucleotide, is prepared in an aqueous solution and added to the polyurethane-based hydrogel prepolymer such that derivitization and polymerization occur, essentially, simultaneously. While the hydrogel is polymerizing, it is microspotted onto a solid substrate, preferably a silinated glass substrate, to which the hydrogel microdroplet allegedly becomes covalently bound. Most preferably the hydrogel microdroplets are at least about 30 μm thick, for example about 50 μm to about 100 μm thick. This process is complicated compared to the more usual technique of hybridizing directly onto a hydrogel surface. In addition, it uses organic solvents which may denature nucleotides, the hydrogel microdroplets may not adhere well to glass surfaces, the hydrogel droplets may not be printed in as dense a format as conventional oligo solutions, and the technology is expensive to accomplish because of its complexity. Chenchik, et al. disclose in U.S. Pat. No. 6,087,102 arrays of polymeric targets stably associated with the surface of a rigid solid support. The polymeric targets are arranged according size via electrophoresis. The polymeric targets are generally biopolymeric compounds, e.g. nucleic acids and proteins, where ribonucleic acids and proteins are the preferred polymeric targets. The technology uses multiple silanization, rinse, and polish steps to first prepare the surface for coating with a hydrogel. The plates must be rinsed and polished between and following each silanization step, a cumbersome and expensive process. The gel polymerization is conducted between plates that are clamped together. This gel is also crosslinked following the polymerization step, which can slow diffusion of aqueous fluids into it. Lockhart, et al. disclose in U.S. Pat. No. 6,040,138 methods of monitoring the expression levels of a multiplicity of genes. The methods involve hybridizing a nucleic acid sample to a high density array of oligonucleotide probes where the high density array contains complimentary subsequences to target nucleic acids in the nucleic acid sample. In one embodiment, the method involves providing a pool of target nucleic acids comprising RNA transcripts of one or more target genes, or nucleic acids derived from the RNA transcripts, hybridizing said pool of nucleic acids to an array of oligonucleotide probes immobilized on the surface, where the array comprises more than 100 different oligonucleotides and each different oligonucleotide is localized in a predetermined region of the surface, the density of the different oligonucleotides is greater than about 60 different oligonucleotides per cm 2 , and the oligonucleotide probes are complimentary to the RNA transcripts or nucleic acids derived from the RNA transcripts; and quantifying the hybridized nucleic acids in the array. This technology is complicated to manufacture. Although it discloses certain probes and probe arrangements, it does not disclose new substrate technology. Pirrung et al. disclose in U.S. Pat. No. 6,225,625 a method and apparatus for preparation of a substrate containing a plurality of sequences. Photoremovable groups are attached to a surface of a substrate. Selected regions of the substrate are exposed to light so as to activate the selected areas. A monomer, also containing a photoremovable group, is provided to the substrate to bind at the selected areas. The process is repeated using a variety of monomers such as amino acids until sequences of a desired length are obtained. Detection methods and apparatus are also disclosed. This process is designed to facilitate efficient synthesis of polynucleotides or other biopolymers. However, the process is limited to a two-dimensional configuration. Felder et al. disclose in U.S. Pat. No. 6,232,066 compositions, apparatus and methods for concurrently performing multiple, high throughput, biological or chemical assays, using repeated arrays of probes. A combination of the invention comprises a surface, which comprises a plurality of test regions, several of which are substantially identical, wherein each of the test regions comprises an array of generic anchor molecules. The anchors are associated with bi-functional linker molecules, each containing a portion which is specific for at least one of the anchors and a portion of which is a probe specific for a target of interest. This technology produces a two-dimensional surface which necessarily limits the amount of probe that can be arrayed in a given area. Anders et al. disclose in U.S. Pat. No. 6,096,369 a process for making the surface of polymeric substrates hydrophilic, which includes coating the surface of a polymeric substrate with a solution of a macroinitiator, wherein the macroinitiator includes a polymer framework and side chains attached to the polymer framework, and wherein at least one of the side chains includes at least one free-radical-forming group. Optionally, a hydrophilic vinyl monomer or monomers may then be free-radical polymerized or graft polymerized onto the macroinitiator-coated substrate. A crosslinking vinyl monomer may optionally be used together with the macroinitiator or the hydrophilic vinyl monomer. This process requires a polymeric substrate. The hydrophilic polymers are not designed to react with biomaterial probes. Turner et al. disclose in U.S. Pat. No. 5,948,62 a stamp for transferring molecules and molecular patterns to a substrate face which includes a backing and a polymeric gel bound to the backing and loaded with the molecular species. Where the molecule to be patterned is a biomolecule, such as a protein or nucleic acid, the polymeric gel is typically a hydrogel, such as sugar-based polyacrylates and polyacrylamides. The process includes preparation of silanized glass plates and formation thereon of hydrogel layers via polymerization of 6-acryloyl-B-O-methylgalactoside (2% crosslinking) and N,N′-methylenebisacrylamide. A relief image-wise pattern is created on the hydrogel surface, which is used to transfer monoclonal antibodies or other biomolecules onto a substrate. This process uses a crosslinked hydrogel like a relief printing plate, and is unlikely to achieve the same level of pattern sharpness that is achieved by modem printing methods. In addition, the process is cumbersome to accomplish, and may be expensive. Jannsen, et al. disclose in U.S. Pat. No. 4,978,481 a process for encapsulating a preformed polymeric substrate by forming peroxide or hydroperoxide sites on the substrate surface using ozone and then carrying out a crosslinked graft polymerization of selected ethyleneically unsaturated monomers both on and surrounding (encapsulating) said substrate. This method relies on the use of ozone which is undesirable. The control of the density of graft polymer links is limited, and no provisions are incorporated for probe linkages. Clapper, et al. disclose in U.S. Pat. No. 6,121,027 a polyfunctional reagent having a polymeric crosslinked backbone, one or more pendant photoreactive moieties, and two or more pendant bioactive groups. The reagent can be activated to form a coating on a polymeric surface. The pendant bioactive groups function by promoting the attachment of specific molecules or cells to the coated surface. This method is cumbersome and requires crosslinking to entrain non-grafted polymer to sustain layer integrity in aqueous media. Matsuda et al. disclose in U.S. Pat. No. 5,128,170 a medical device having a biocompatible surface wherein a hydrophilic polymer is bonded onto a surface of the medical device covalently through a nitrogen atom, and a method for manufacturing such a medical device is provided. The process includes the steps of applying a hydrophilic polymer having an azido group and/or a composition comprising a compound having at least two azido groups and a hydrophilic polymer onto the surface of the medical device, and irradiating the biocompatible material with light so that the hydrophilic polymer is bonded to the medical device surface. This process is likely to produce non-grafted polymer or copolymer which must be dealt with in a washing step to remove non-grafted polymer or copolymer from the layer, or crosslinking so that the non-grafted polymer or copolymer is retained permanently in the coated layer. No provision is made to link probes in/on the coated surface. Surface matrices must have uniform thickness, high diffusivity of unbound target throughout the matrix, be suitable for patterning of probe-rich and probe-poor areas and exhibit negligible non-specific binding. Attachment of graft polymers is a highly useful method of surface modification. The functional utility of a graft polymer-modified surface is in certain cases dependent upon the surface density of grafts. Graft polymer surface coatings are low density and porous by nature when compared to most other polymer coatings, this allows for the defusing and binding of larger molecules throughout the coating. This porosity can be further enhanced if the graft polymer chain density and length can be controlled. Furthermore, all surfaces are not inherently susceptible to the formation of permanent graft polymer attachment. Thus, there is a need for a three-dimensional non-crosslinked linear or branched graft polymer matrix comprising one or more active chemical moieties having inherent specificity for binding to chemical or biochemical, or biological probes or targets, wherein said moieties are permanently attached to and distributed throughout the graft polymer matrix, with a controlled graft polymer chain density and length. There also is a need for a method for graft polymer surface modification that allows for control of the surface density and chain length of the resulting graft polymer matrix, and also makes it possible to graft polymers onto surfaces on which graft polymerization was not heretofore possible. There also is a need to spatially separate proteins from the proximity of a surface to allow efficient protein binding interactions and to avoid denaturation, inactivation and steric hindrance. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides for a novel three-dimensional, non-crosslinked graft polymer matrix containing one or more chemical, biochemical or biological moieties attached to the graft polymer chain, said moieties having been selected to have reactivity with specific probe or target molecular species. The graft polymer matrix differs significantly from those generated from other emerging three dimensional coating technologies in that its advantages are achieved without the need for covalent crosslinks. The invention calls for individual polymer chains grafted to a surface, as depicted schematically in FIG. 8 . Probe reactive groups (active chemical moieties) can be incorporated into the graft polymer matrix that bind to either DNA or protein based probes. The system is not confined to any particular type of linking or reactive group chemistry. The term “non-crosslinked” is intended to refer to a polymer matrix in which the benefits of a porous, coherent material are achieved with individual polymer chains bound to the substrate without the requirement of extensive crosslinking, and the term “non-crosslinked” is intended to distinguish known crosslinked polymer matrices as in prior publications and products described here and otherwise known. The system is not limited to, but is well suited for microarray assay and nanotechnology. The graft polymer matrix also can include a spacer arm between the probe reactive groups and graft polymer backbone to reduce steric hindrance from the graft polymer backbone. Moreover, for protein assays the printed probe, e.g. a monoclonal antibody or enzyme, can exist in a pseudo solution phase. It is well know that proteins denature or change conformation after binding to a solid surface (e.g. polystryrene microtiter plates). Using the graft polymer matrix the proteins may be attached by a single endpoint, and are spatially removed from the solid interface and can exist in a semi-soluble state. This may be particularly relevant to discovery research where the goal is to develop therapeutic reagents, such as peptides of monoclonal antibodies, for in vivo targets. In these situations the selection of target-specific agents is only relevant if the target (e.g. antigen) is present in its native in vivo state during screening. The graft polymer matrix may also contain long pendant side-chains (structural modifiers) that may provide structural integrity to the coatings without the rigidity imposed by covalent crosslinks. The degree of hydrophilicity can be controlled by the structural modifiers or the monomeric groups incorporated into graft polymer matrix. This will be important for fine-tuning the matrix, for example, if one wishes to control the spot diffusion during microarray printing. To increase diffusion into the matrix one may wish to increase the hydrophilic nature of the matrix. This may also increase the performance of assays that require hydrophilic conditions, such as found with typical protein assays. This greater flexibility and the controlled graft polymer chain surface density allows for better probe and target diffusion throughout the matrix compared to other three dimensional systems. In addition, the open structure of graft polymer matrix surfaces will allow easier and more efficient washing, thus reducing nonspecific binding due to entrapment in known densely packed coated or crosslinked polymer chains. By controlling the length of the graft polymer chains and the distribution of probe reactive groups and structural modifiers within each grafted chain one can tailor the assay milieu to meet specific assay or biological performance needs. The structural modifiers may also serve as buffers, or to increase or decrease ionic binding, by the incorporation of appropriate charged groups. A limited amount of crosslinking is also contemplated within the scope of the invention so long as the polymer chains are directly bound to the surface, and the crosslinking does not interfere with the functionality of the matrix. By combining these features, the invention substantially increases the sensitivity and dynamic range of microarrays for both genomics and proteomics. In one aspect, the invention provides a method for producing a three-dimensional, non-crosslinked graft polymer matrix having chemical moieties permanently attached to it, said moieties being distributed throughout the matrix in a known and controlled manner. In yet another aspect, the invention provides for an article having at its surface a permanently attached three-dimensional, non-crosslinked graft polymer matrix containing probe molecules, said probe molecules having inherent specificity for binding to target chemical or biochemical species for which information on its presence or concentration in a sample is of interest. The probe molecules may be permanently attached to the graft polymer matrix and their distribution throughout the matrix may be chosen in such a way as to provide optimal sensitivity and dynamic range to the detection of said target molecules. In a preferred embodiment of this aspect of the invention, the probes are attached as side-chains to the backbone of the graft polymer. In still another aspect, the invention provides a method for permanently attaching a multiplicity of graft polymer chains to a surface wherein the density of graft chains per unit area can be controlled to allow for large probe and target binding throughout the graft polymer matrix leading to increased sensitivity and dynamic range. In still another aspect, the invention provides a method for permanently attaching a multiplicity of graft polymer chains to a surface wherein the length of graft chains can be controlled to allow for increased sensitivity and dynamic range. One object of the invention is to provide a three-dimensional, non-crosslinked, linear or branched graft polymer matrix containing one or more active chemical moieties having inherent specificity for binding to chemical or biochemical, or biological probes or targets, wherein said moieties are permanently attached to and distributed throughout the graft polymer matrix. In a preferred embodiment of this aspect of the invention the biological targets are selected from the group consisting of viruses, fungi, parasites, and bacteria. In another preferred embodiment, the chemical or biochemical functional targets are selected from the group consisting of molecules or molecular fragments of DNA, RNA, protein, carbohydrates and lipids. In still other preferred embodiments, the probe or target may be a toxin. In still another preferred embodiment, the active chemical moiety is selected from the group consisting of amines, carboxylic acids, epoxides, aldehydes, sulfhydryls, thioesters, haloacetamides and carboxylic acid succinimidyl esters. In an especially preferred embodiment of this aspect of the invention, the active chemical moiety is the N-hydroxysuccinimidyl ester of a carboxylic acid. It is another object of the invention to provide a three-dimensional, non-crosslinked, linear or branched graft polymer matrix containing one or more active chemical moieties imparting specific functional utility to the coating matrix, wherein said moieties are permanently attached to and distributed throughout the graft polymer matrix, with a controlled graft polymer chain density and length. In one preferred embodiment of this aspect of the invention, the functional utility is an enhanced ability to be imaged by an imaging method selected from the group consisting of magnetic resonance imaging, computer tomography, x-ray radiology, fluorescence, and ultrasonography. In another preferred embodiment, the functional utility is an enhanced resistance to infection and thrombosis. It is another object of the invention to provide an article having on its surface a multilayered coating comprising: a) An adhesive polymer layer bound to the surface of the article b) A three-dimensional, non-crosslinked, linear or branched graft polymer matrix containing one or more chemical moieties that are permanently attached to and distributed throughout the graft polymer matrix wherein said chemical moieties impart specific functional utility to the article. In one preferred embodiment of this aspect of the invention, the chemical moieties are attached with side-chain spacer arms to the backbone of the graft polymer. Monomers having functional groups that remain intact during the graft polymerization process appear as attached side chains on the graft polymer backbone. In one preferred embodiment of this aspect of the invention, the thickness of the matrix coating upon the article can be varied, for example, by varying the time of the graft polymerization reaction. Such variation may be desirable depending upon the number of different probes and targets, to be used, sample size, and other factors dependent upon the type of measurements to be made. In one preferred embodiment of this aspect of the invention, the article is a microarray. The article according to this aspect of the invention may contain microchannels, one purpose of which is to facilitate the diffusion of large probe and target species to reactive sites within the matrix. The size of these microchannels being determined by the open space between the graft polymer chains or their surface density. Other preferred embodiments of this aspect of the invention include devices whose performance and utility could be enhanced through the application of this invention. Examples of such devices include multi-welled plates, drug release devices that bind drugs or other therapeutic compounds for in vivo release; devices which sequester and thus remove target compounds from solution, such as in dialysis, or the like or other applications that could be envisioned by a person of skill in the art who would understand how to make the necessary adaptations. In another preferred embodiment of this aspect of the invention, the article is a chemical sensor. The article according to this aspect of the invention may contain chemical moieties that are permanently attached to the graft polymer matrix. Said chemical moieties may have reactivity towards specific nonbiological chemical species or classes of species. Representative examples of such classes include, but are not limited to, monovalent metal ions, divalent metal ions, transition metal ions, inorganic halides, carbonates, sulfates, phosphates, borates, arsenates, zero valent heavy metals, and the like. Upon attachment of one or more of the chemical species to the graft polymer, one or more physical properties of said coating will be altered in a way that is detectable by an appropriate detection device. Representative examples of physical properties that could be altered are, but are not limited to, electrical conductivity, capacitance or impedence, paramagnetism or diamagnetism, optical clarity, optical transmittance over a narrow or wide range of wavelengths, or the like. In another preferred embodiment, the article has an enhanced ability to be imaged by an imaging method selected from the list magnetic resonance imaging, computer tomography, x-ray radiology, and ultrasonography relative to analogous articles that are not coated as described herein. In one particularly preferred embodiment, the functional utility is an enhanced resistance to infection. Coatings of this type are particularly useful on medical devices. Medical devices that may be coated according to the methods of the invention include catheters (including, for example, arterial, short term central venous, long term tunneled central venous, peripheral venous, peripherally insertable central venous, pulmonary artery Swan-Ganz, PTCA or PTA, and vascular port), dialysis devices, introducers, needles (including, for example, amniocentesis, biopsy, introducer), obdurators, pacemaker leads, penile prosthesis, shunts (including, for example, arteriovenous and hydrocephalus shunts), small or temporary joint replacements, stents (e.g. biliary, coronary, neurological, urological, and vascular), syringes, tubes (e.g. drain, endotracheal, gastroenteric, nasogastric), urinary devices (e.g. long term and tissue bonding), urinary dilators, urinary sphincters, urethral inserts, and wound drains. Other devices that may be advantageously coated will be familiar to those of skill in the art. The surface of an article to be coated according to methods of the invention may be comprised of glass, metal, or polymeric material. The initiator can generally be applied directly to an article having a polymeric surface without using a primer, whereas in the case of a glass or metal surface, a primer may be necessary or desirable to ensure optimal adhesion of the initiator and the graft polymer matrix to the surface. It is therefore a further object of the invention to provide a primer and a method of applying a three dimensional matrix according to the invention that includes a primer, for use on surfaces where the use of a primer may be necessary or desirable. The primer may comprise a solution of a polymer or mixture of polymers in an organic solvent or mixture of organic solvents. The primer solution is applied to the surface of the article either prior to, or simultaneously with, the initiator. Suitable primer solutions can be prepared, for example, using organic solvents such as tetrahydrofuran, toluene, methylethylketone combined with a suitable polymer or polymer mixture. It has been found that in some instances the initiator may be combined with the primer solution to produce superior results. It is therefore one object of the invention to provide a method for applying a three-dimensional, non-crosslinked, linear or branched graft polymer matrix with a controlled graft polymer chain surface density to an uncoated surface, said method comprising the steps of: a) applying to said surface a solution comprising i) an organic solvent or mixture of organic solvents, ii) a polymer or mixture of polymers which is soluble in said solvent or solvent mixture and iii) a radical initiator which is soluble in said solvent or solvent mixture, said initiator being capable of generating reactive radical sites on one or more of the polymers present on the-coated substrate surface and initiating a graft polymerization reaction on said polymer-coated surface by generating reactive radical sites thereon. b) removing the solvents to leave upon the surface of the substrate a coating of initiator dissolved in polymeric primer c) immersing the polymer-coated surface in a medium comprising one or more monomers in solution that are capable of reacting with the reactive sites created by surface bound initiator to form a polymer chain grafted onto said polymer-coated surface; d) initiating a graft polymerization reaction on said polymer-coated surface by generating reactive radical sites thereon, e) graft polymerizing onto the polymer-coated surface the reactive monomers from the medium by forming covalent bonds between monomer molecules and the polymeric surface at reactive radical sites on the polymer-coated surface. The graft polymer surface density is controlled by mixing together different ratios of reactive polymers and unreactive polymers and/or by using different concentrations of initiator. In a preferred embodiment of this aspect of the invention, the monomer solution used comprises at least one monomer that, when incorporated into the three-dimensional non-crosslinked graft polymer matrix, provides the resulting graft polymer with side-chain chemical moieties that are permanently attached to and distributed throughout the graft polymer matrix, said side-chain chemical moieties having inherent specificity for binding to chemical, biochemical, or biological probes or targets. The side-chain chemical moieties may be modified to alter their reactivity. The side chain moieties may have inherent specificity for binding to chemical, biochemical, or biological probes or targets, or have other features that impart specific functional utility to the article. It is another object of the invention to provide a method for applying a three-dimensional, non-crosslinked, linear or branched graft polymer matrix with a controlled graft polymer chain density to an uncoated surface, said method comprising the steps of: a) applying to said surface a solution comprising: i) an organic solvent or mixture of organic solvents and ii) a polymer or mixture of polymers which is soluble in said solvent or solvent mixture b) removing the solvents to leave a coating of polymeric primer upon the substrate surface, c) exposing said polymer-coated surface to an initiator capable of generating reactive radical sites on one or more of the polymers present on the coated surface of the substrate and initiating a graft polymerization reaction on said surface by generating reactive radical sites thereon d) immersing the polymer-coated surface in a medium comprising: i) one or more monomers in solution that are capable of reacting with the reactive sites created by surface bound initiator to form a polymer chain grafted onto said surface; and ii) a concentration of solute in sufficient concentration to induce a ‘salting-out effect’ wherein reactive monomers preferentially localize at the surface of the substrate, wherein the initiator used is insoluble or poorly soluble; e) initiating a graft polymerization reaction on said coated surface by generating reactive radical sites thereon, f) graft polymerizing onto the coated surface the reactive monomers from the medium by forming covalent bonds between monomer molecules and the surface at reactive radical sites on the polymer coated surface. The graft polymer surface density is controlled by mixing together different ratios of reactive polymers and unreactive polymers and/or by changing the amount of initiator the polymer coated surface is exposed to. It is another object of the invention to provide a method for applying a three-dimensional, non-crosslinked, linear or branched graft polymer matrix with a controlled graft polymer chain density to an uncoated glass surface, said method comprising the steps of: a) applying to a glass surface a solution comprising: i) an organic solvent or mixture of organic solvents with water and ii) a silane monomer or mixture of silane monomers and unreactive silanes which is soluble in said solvent or solvent water mixture b) removing the solvents to leave a coating of silane attached monomers or a mixture of silane attached monomers and silane attached unreactive groups. c) exposing said silane coated surface to an initiator capable of generating reactive radical sites on one or more of the silane attached groups present on the coated surface of the substrate and initiating a graft polymerization reaction on said surface by generating reactive radical sites thereon d) immersing the silane-coated surface in a medium having one or more monomers in solution that are capable of reacting with the reactive sites created by surface bound initiator to form a polymer chain grafted onto said surface; e) initiating a graft polymerization reaction on said coated surface by generating reactive radical sites thereon, f) graft polymerizing onto the coated surface the reactive monomers from the medium by forming covalent bonds between monomer molecules and the surface at reactive radical sites on the silane coated surface. The graft polymer surface density is controlled by mixing together different ratios of reactive silane attached monomers and unreactive silane attached groups on the glass surface and/or by changing the amount of initiator the silane coated surface is exposed to. It is another object of the invention to provide a method for applying a three-dimensional, non-crosslinked, linear or branched graft polymer matrix with a controlled graft polymer chain density to an uncoated glass surface, said method comprising the steps of: a) applying to a glass surface a solution comprising: i) an organic solvent or mixture of organic solvents with water and ii) a silane with an attached initiator or mixture of silane initiators and unreactive silane groups which is soluble in said solvent or solvent water mixture b) removing the solvents to leave a coating of silane attached initiator or a mixture of silane attached initiators and silane attached unreactive groups. c) immersing the silane-coated surface in a medium comprising one or more monomers in solution that are capable of reacting with the reactive sites created by surface bound initiator to form a polymer chain grafted onto said surface; d) initiating a graft polymerization reaction on said coated surface by generating reactive radical sites thereon, e) graft polymerizing onto the coated surface the reactive monomers from the medium by forming covalent bonds between monomer molecules and the surface at reactive radical sites on the silane coated surface. The graft polymer surface density is controlled by mixing together different ratios of reactive silane attached initiators and unreactive silane attached groups on the glass surface. In a preferred embodiment of this aspect of the invention, the monomer solution used comprises at least one monomer that, when incorporated into the three-dimensional non-crosslinked graft polymer matrix, provides the resulting graft polymer with chemical moieties that are permanently attached to and distributed throughout the graft polymer matrix, said chemical moieties having inherent specificity for binding to chemical, biochemical, or biological probes or targets. The chemical moieties may be modified to alter their reactivity. The moieties have inherent specificity for binding to chemical, biochemical, or biological probes or targets, or have other characteristics that impart specific functional utility to the article. In an especially preferred embodiment of this aspect of the invention, the chemical moieties are attached as side-chains to the backbone of the graft polymer matrix and are separated from the graft polymer backbone by a side chain spacer arm that can enhance probe and target binding efficiency. In a particularly preferred embodiment of the invention, graft polymer matrix is applied to the article surface in a pattern. This facilitates the placement of different types of reactive probes, and the reading of results once the matrix has been allowed to react with a test sample. The graft polymer matrices of the present invention may be constructed and used so as to provide an increased capacity and efficiency for a single type of reactive group or probe, or may be constructed and used for mixtures of or multiple layers of probes/functional groups in a single location. Matrices prepared according to the present invention have a surface density that can be controlled as well as controlled chain lengths to provide a uniform surface for printing and hybridization. It will be appreciated that the individual features and aspects of the invention are intended to be combined in all combinations that will be operable and practical, and that all of such combinations are intended to be included within the invention. |
Gas treating device and gas treating method |
A gas processing apparatus 1 includes a processing container 2 for applying a processing to a wafer W while using a processing gas, a mount table 5 arranged in the processing container 2 to mount the wafer W, a shower head 22 arranged corresponding to the wafer W on the mount table 5 to discharge the processing gas into the processing container 2 and exhausting means 132 for exhausting the interior of the processing container 2. The shower head 22 has first gas discharging holes 46 arranged corresponding to the wafer W mounted on the mount table 5 and second gas discharging holes 47 arranged around the first gas discharging holes 46 independently to discharge the processing gas to the peripheral part of the wafer W. Thus, with a uniform gas supply to a substrate, it is possible to perform a uniform gas processing. |
1-66. (Canceled) 67. A gas processing apparatus comprising: a processing container for housing a substrate to be processed; a mount table arranged in the processing container to mount the substrate to be processed thereon; a processing-gas discharging mechanism arranged in a position opposing the substrate to be processed mounted on the mount table to discharge a processing gas into the processing container; and exhausting means for exhausting an interior of the processing container, wherein the processing-gas discharging mechanism includes a first gas discharging part provided corresponding to the substrate to be processed mounted in the mount table; and a second gas discharging part arranged around the first gas discharging part independently to discharge the processing gas into the periphery of the substrate to be processed mounted on the mount table. 68. A gas processing apparatus for applying a gas processing to a substrate to be processed while using a gas containing a first processing gas of a relatively high diffusion velocity and a second processing gas of a relatively low diffusion velocity, the gas processing apparatus comprising: a processing container for housing a substrate to be processed; a mount table arranged in the processing container to mount the substrate to be processed thereon; a processing-gas discharging mechanism arranged in a position opposing the substrate to be processed mounted on the mount table to discharge a gas containing the first processing gas and the second processing gas into the processing container; and exhausting means for exhausting an interior of the processing container, wherein the processing-gas discharging mechanism includes a first gas discharging part provided corresponding to the substrate to be processed mounted in the mount table to discharge the gas containing the first processing gas and the second processing gas; and a second gas discharging part arranged around the first gas discharging part independently, to discharge the first processing gas into the periphery of the substrate to be processed mounted on the mount table. 69. A gas processing apparatus as claimed in claim 67 or claim 68, wherein the processing-gas discharging mechanism has a heater. 70. A gas processing apparatus as claimed in claim 67, wherein the processing-gas discharging mechanism includes a gas discharging plate having the first gas discharging part and the second gas discharging part, and the first gas discharging part and the second discharging part each have a plurality of gas discharging holes formed in the gas discharging plate. 71. A gas processing apparatus as claimed in claim 67 or claim 68, wherein the processing-gas discharging mechanism further includes a base part for supporting the gas discharging plate and a gap layer between the gas discharging plate and the base part. 72. A gas processing apparatus as claimed in claim 67 or claim 68, wherein the processing-gas discharging mechanism includes cooling means for cooling the gas discharging plate, the cooling means having a coolant supply path arranged in the outer peripheral part of the processing-gas discharging mechanism to introduce a coolant, a coolant discharging path arranged in the outer peripheral part of the processing-gas discharging mechanism to discharge the coolant and a coolant passage communicating the coolant supply path with the coolant discharging path. 73. A gas processing apparatus as claimed in claim 72, wherein the coolant passage is arranged in an area of the gas discharging plate where the gas discharging holes are formed. 74. A gas processing apparatus as claimed in claim 73, wherein the coolant passage is formed so as to correspond to the shape of a gas discharging plate's part interposed among the plural gas discharging holes in the gas discharging plate's area where the gas discharging holes are formed. 75. A gas processing apparatus as claimed in claim 73, wherein the coolant passage is formed concentrically. 76. A gas processing apparatus as claimed in claim 67 or claim 68, further comprising: a coolant flow piping arranged both in upstream of the coolant passage arranged in the processing-gas discharging mechanism and in the downstream of the coolant passage; a bypass piping connected, both in upstream of the processing-gas discharging mechanism and in the downstream, to the coolant flow piping while bypassing the processing-gas discharging mechanism; a pressure relief valve arranged on the downstream side of the coolant passage in the coolant flow piping; a group of valves defining a flowing pathway of the coolant; control means for controlling the group of valves; and a heater for heating the processing-gas discharging mechanism, wherein when cooling the processing-gas discharging mechanism, the control means controls the group of valves so as to allow the coolant to flow into the coolant passage, when heating the processing-gas discharging mechanism, the control means operates the heater and further controls the group of valves so as to stop the inflow of the coolant into the coolant passage and allow the coolant to flow into the bypass piping, and when lowering a temperature of the processing-gas discharging mechanism in its elevated condition in temperature, the control means controls the valves so as to allow the coolant to flow into both of the coolant passage and the bypass piping. 77. A gas processing apparatus as claimed in claim 70, wherein the plural gas discharging holes included in the second gas discharging part are arranged outside the periphery of the substrate to be processed on the mount table. 78. A gas processing apparatus as claimed in claim 77, wherein the plural gas discharging holes included in the second gas discharging part are arranged perpendicularly to the substrate to be processed on the mount table. 79. A gas processing apparatus as claimed in claim 77, wherein the plural gas discharging holes included in the second gas discharging part are arranged in the periphery of the first gas discharging part, in one or more lines. 80. A gas processing apparatus as claimed in claim 77, providing that the plural gas discharging holes included in the second gas discharging part are arranged in the periphery of the first gas discharging part in two or more lines, wherein the plural gas discharging holes are arranged so as to alternate with each other. 81. A gas processing apparatus as claimed in claim 67, wherein the exhausting means includes a baffle plate for exhausting from the peripheral side of the substrate to be processed on the mount table, an annular exhaust space arranged below the baffle plate and an exhaust hole in communication with the exhaust space, which is arranged in a diagonal position of the processing container. 82. A gas processing apparatus as claimed in claim 81, wherein a bottom partition wall is arranged in the exhaust space adjacent to the exhaust hole. 83. A gas processing method for applying a gas processing to a substrate to be processed in a processing container while supplying a processing gas to the substrate, the gas processing method comprising the steps of: discharging the processing gas through a first gas discharging part provided so as to oppose the substrate to be processed; and discharging the processing gas to the circumference of the substrate to be processed through a second gas discharging part provided around the first gas discharging part independently, thereby performing the gas processing. 84. A gas processing method as claimed in claim 83, wherein gas containing the processing gas of a relatively low diffusion velocity is discharged from the first gas discharging part provided so as to oppose the substrate to be processed, and the processing gas of a relatively high diffusion velocity is discharged to the circumference of the substrate to be processed from the second gas discharging part provided around the first gas discharging part independently, thereby performing the gas processing. 85. A gas processing method as claimed in claim 84, wherein the processing gas containing WF6-gas is discharging from the first gas discharging part, while the processing gas containing H2-gas is discharging from the second gas discharging part, thereby forming a film on the substrate to be processed. 86. A gas processing method as claimed in claim 85, wherein the processing gas discharged from the first gas discharging part contains H2-gas, and the flow rate of H2-gas from the second gas discharging part is 50% or more percent of the flow rate of H2-gas from the first gas discharging part. 87. A gas processing apparatus comprising: a processing container for housing a substrate to be processed; a mount table arranged in the processing container to mount the substrate to be processed thereon; a processing-gas discharging mechanism arranged in a position opposing the substrate to be processed mounted on the mount table to discharge a processing gas into the processing container; and exhausting means for exhausting an interior of the processing container, wherein the processing-gas discharging mechanism includes a gas discharging plate having a discharging hole for discharging the gas; a base part supporting the gas discharging part; a heater provided in the gas discharging part; and a gap layer defined between the gas discharging part and the base part. 88. A gas processing apparatus as claimed in claim 87, wherein the gap layer has a fastening mechanism for fastening the gas discharging plate to the base part so as to allow a relative displacement therebetween. 89. A gas processing apparatus as claimed in claim 88, wherein the fastening mechanism includes a holding part for fixing the gas discharging plate to the base part and a moving part arranged on the opposite side of the holding part to allow a relative displacement between the gas discharging plate and the base part. 90. A gas processing apparatus as claimed in claim 87, wherein the processing-gas discharging mechanism has a coolant passage. 91. A gas processing apparatus as claimed in claim 90, further comprising: a coolant flow piping arranged both in upstream of the coolant passage and in the downstream; a bypass piping connected, both in upstream of the processing-gas discharging mechanism and in the downstream, to the coolant flow piping while bypassing the processing-gas discharging mechanism; a pressure relief valve arranged on the downstream side of the coolant passage in the coolant flow piping; a group of valves defining a flowing pathway of the coolant; control means for controlling the group of valves; and a heater for heating the processing-gas discharging mechanism, wherein when cooling the processing-gas discharging mechanism, the control means controls the group of valves so as to allow the coolant to flow into the coolant passage, when heating the processing-gas discharging mechanism, the control means operates the heater and further controls the group of valves so as to stop the inflow of the coolant into the coolant passage and allow the coolant to flow into the bypass piping, and when lowering a temperature of the processing-gas discharging mechanism in its elevated condition in temperature, the control means controls the group of valves so as to allow the coolant to flow into both of the coolant passage and the bypass piping. 92. A gas processing apparatus as claimed in claim 87, wherein a spacer ring is arranged on the outer peripheral side of the gas discharging plate to fill up a space between the gas discharging plate and a peripheral wall in the processing container. 93. A gas processing apparatus as claimed in claim 87, wherein the heater is embedded in the outer peripheral part of a lower part of the gas discharging plate. 94. A gas processing apparatus as claimed in claim 87, wherein a seal member is arranged in an inner peripheral part between the gas discharging plate and the base part. 95. A gas processing apparatus as claimed in claim 88, wherein a member of fluorocarbon resin is arranged between the fastening mechanism and the gas discharging plate in a manner that when the member is expanded thermally, the relative displacement between the fastening mechanism and the gas discharging plate can be absorbed by slipping of the member. 96. A gas processing apparatus as claimed in claim 87, wherein the exhausting means includes a baffle plate for exhausting from the peripheral side of the substrate to be processed on the mount table, an annular exhaust space arranged below the baffle plate and an exhaust hole in communication with the exhaust space, which is arranged in a diagonal position of the processing container. 97. A gas processing apparatus as claimed in claim 96, wherein a bottom partition wall is arranged in the exhaust space proximity to the exhaust hole. |
<SOH> BACKGROUND OF ART <EOH>In the semiconductor manufacturing process, metal, for example, W (tungsten), WSi (tungsten silicide), Ti (titanium), TiN (titanium nitride), TiSi (titanium silicide), etc. or metallic compound thereof is deposited to form a film in order to fill up contact holes formed on a semiconductor wafer as an object to be processed (referred “wafer” hereinafter) or wiring holes for connecting wires to each other. As the film deposition for these elements, physical vapor deposition (PVD) technique has been employed conventionally. Recently, however, both of miniaturization and high integration of a device have been particularly required and therefore, its design rule becomes severe in particular. Correspondingly, as both device's line-width and diameter of holes become smaller with the progress of high aspect ratio, a “PVD” film has been getting incapacitated. Therefore, it has been recently carried out to form a film of such a metal or metal compounds by chemical vapor deposition (CVD) technique promising an ability of forming a film of better quality. For example, by use of WF 6 (tungsten hexafluoride) gas as the processing gas and H 2 -gas as the reduction gas, a W-film is produced due to a reaction on a wafer represented by the formula of “WF 6 +H 2 →W+6HF”. The CVD film deposition process like this is carried out by mounting a wafer on a mount table in a processing container and further supplying the container with WF 6 -gas and H 2 -gas discharged from a shower head as being a gas discharging mechanism arranged in a position opposing the wafer while exhausting the interior of the processing container, thereby forming a designated “processing-gas” atmosphere in the processing container. Under the process like this, however, as a reduction gas having a high diffusion velocity, e.g. H 1 -gas, quickly diffuses in the processing container throughout and is discharged therefrom, the concentration of the reduction gas is easy to drop around the peripheral part of a wafer. Particularly, since the film deposition apparatus has been large-sized corresponding to a recent large-sized wafer from 200 mm to 300 mm in size, the above reduction in the concentration of the reduction gas in the periphery of the wafer becomes remarkable to cause a film deposition rate to be lowered in the same area. Consequently, the uniformity in film thickness is lowered remarkably. Meanwhile, when forming a W-film on SiO 2 or Si, it is performed in advance of the deposition of W-film to cover the SiO 2 or Si with thin and uniform Ti-film, TiN-film or their lamination film as the barrier layer in view of improvement in adhesive property between a W-film and the SiO 2 or Si, restriction of a reaction of W with Si etc. In connection, when filling in recesses or the like, hydrogen gas exhibiting reduction property less than that of silane gas (Si n H 2m+n , SiH n Cl 4−n ) is mainly used in order to make its embedding property excellent. Then, there is a possibility that the “under” barrier layer is attacked by non-reacted WF 6 -gas, so that the barrier layer reacts with fluorine to expand its volume thereby producing a projecting defect called “volcano” and further, there is an occasion that voids occur in holes to be embedded. In order to prevent the occurrence of such defects, it is attempted to firstly form a nucleate W-film (nucleation film) by a minimal thickness in the order from 30 to 50 nm with by the use of silane gas having more intensive reduction power in place of hydrogen gas and subsequently, to form a main W-film with the nucleation film as the starting point by the use of H 2 -gas and WF 6 -gas. However, in spite of the adoption of such a method, the step coverage of a nucleation film is deteriorated due to contamination etc. on the surface of a barrier layer as the under layer, so that the fill-in property of the main W-film gets worse. This tendency becomes remarkable with the progress of miniaturization in semiconductor devices. In order to solve such a problem, it is also attempted, in advance of the formation of the nucleation film, to perform an initiation process to allow the under barrier layer to absorb SiH X (X<4) with the supply of only silane gas for a predetermined period and subsequently, to make a growth of the nucleation film with the so-absorbed barrier layer as the starting point. However, this measure is believed to be insufficient. Therefore, we and applicant previously proposed a technique to form an initial W-film on the surface of a substrate to be processed (Japanese Patent Application No. 2001-246089). According to the technique, there are repeatedly performed a reduction-gas supply process of supplying the reduction gas and a W-gas supply process of supplying a W-content gas with the interposition of a purging process of evacuating while supplying an inert gas between the above processes. With this technique, it is possible to form a uniform nucleation film in even a minute hole, with high step coverage, whereby the above problem can be solved. Nevertheless, if the above technique is applied to a normal W-film deposition apparatus, then WF 6 -gas reacts to silane gas in a shower head as a gas discharging mechanism, so that a W-film is formed in the shower head, thereby decreasing the reproducibility among the surfaces of wafers. In order to avoid an occurrence of such a problem, it is necessary to lower a temperature of a gas discharging part of the shower head less than 30° C. However, since the shower head is generally cooled down from its lateral surface, it is difficult to attain the temperature of a central part of the shower head less than 30° C. by means of generally cooling water. In the present circumstances where the shower head is also large-sized because of large-sized wafers, the requirement of attaining the temperature of the central part of the shower head less than 30° C. would require an ultra cold chiller to cause a great increase in the installation cost of a system due to countermeasures of dew condensation etc. In the CVD film deposition apparatus of this kind, meanwhile, if forming a W-film on a substrate having an exposed TiN-film, then a compound “TiN” is etched by fluorine during the film depositing operation, so that reaction by-product materials, such as titanium fluoride (TiF x ), stick to the shower head and the inner wall of the chamber and thereafter, the by-product materials are peeled off to be the origin of particles. Therefore, after completing a designated film deposition, it is carried out to introduce ClF 3 -gas (as a cleaning gas) into a chamber through a shower head thereby cleaning the apparatus. Regarding this cleaning, since the cleaning efficiency is increased with elevated temperature, there is performed a “flashing” process to introduce ClF 3 -gas into the chamber while heating the shower head at predetermined intervals by a heater embedded in the shower head. However, due to the shower head being large-sized for large wafers that requires for the heater to have a high-power output, heat from the shower head to a container lid is also heat transferred, so that the heater is required to have more power to compensate such a dissipative heat. The requirement makes it difficult to elevate the temperature of the shower head up to a predetermined temperature. Additionally, with an apparatus being large-sized, if heating the shower head by the heater, then the shower head has a thermal expansion of the order of 1 mm, so that a problem of heat distortion about the shower head arises. Under such a situation, an object of the present invention is to provide a gas processing apparatus and a gas processing method by which it is possible to avoid defects about a gas discharging mechanism, the defects being accompanied with the apparatus being large-sized. More in detail, an object of the invention is to provide a gas processing apparatus and a gas processing method that can perform a uniform gas processing by supplying a substrate with gas uniformly. Additionally, an object of the invention is to provide a gas processing apparatus that allows a gas discharging mechanism to be heated with high efficiency. Further, an object of the invention is to provide a gas processing apparatus that can reduce an influence of thermal expansion when the gas discharging mechanism is heated. Still further, in case of an apparatus that alternately supplies two processing gases required to keep a temperature of the gas discharging mechanism low, an object of the invention is to provide the gas processing apparatus that can cool the whole gas discharging mechanism to a desired temperature without using any special installation, such as ultra cold chiller, despite that the gas discharging mechanism is large-sized. Further, in case of supplying two processing gases alternately to form a film, an object of the invention is to provide a gas processing apparatus and a gas processing method that can prevent formation of an unnecessary film in the gas discharging mechanism without cooling specially. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1A is a front view of a CVD film deposition apparatus in accordance with the first embodiment of the present invention. FIG. 1B is a side view of the CVD film deposition apparatus in accordance with the first embodiment of the present invention. FIG. 2 is a schematic sectional view showing a main body of the CVD film deposition apparatus of FIGS. 1A and 1B . FIG. 3 is a sectional view taken along a line A-A of the apparatus of FIG. 2 . FIG. 4 is a sectional view taken along a line B-B of the apparatus of FIG. 2 . FIG. 5 is a sectional view showing a joint part between a shower plate and a shower base in the CVD film deposition apparatus in accordance with the first embodiment of the present invention, in enlargement. FIG. 6 is a view showing a top surface of the shower plate 35 in the CVD film deposition apparatus in accordance with the first embodiment of the present invention. FIG. 7 is a sectional view showing the peripheral part of a lower part of the shower head in the apparatus of FIG. 2 , in enlargement. FIG. 8 is a sectional view showing the vicinity of the peripheral part of the lower part of the shower head in enlargement, in case of arranging the second gas discharging holes doubly. FIG. 9A is a view showing one example of the arrangement of the second gas discharging holes in enlargement, in case of arranging the second gas discharging holes doubly. FIG. 9B is a view showing another example of the arrangement of the second gas discharging holes in enlargement, in case of arranging the second gas discharging holes doubly. FIG. 10 is a sectional view showing the vicinity of the peripheral part of the lower part of the shower head in enlargement, in case of arranging the second gas discharging holes obliquely. FIG. 11 is a sectional view showing the vicinity of the peripheral part of the lower part of the shower head in enlargement, in case of arranging the second gas discharging holes inside the outer periphery of a wafer W obliquely. FIG. 12 is a sectional plan view showing the other structure of the shower head. FIG. 13 is a perspective view showing an interior structure of a casing of a gas introducing part of FIG. 2 , in its exploded state. FIG. 14 is a sectional view taken along a line C-C of the apparatus of FIG. 3 . FIG. 15 is a sectional view taken along a line D-D of the apparatus of FIG. 3 . FIG. 16 is a back view showing the opening-and-closing conditions of a lid body in the CVD film deposition apparatus shown in FIGS. 1A and 1B . FIG. 17 is a circuit diagram for explanation of a cooling control system used in the CVD film deposition apparatus in accordance with the first embodiment. FIG. 18 is a graph where its horizontal axis represents the flow rate of H 2 -gas, while the vertical axis represents the uniformity of W-film. FIG. 19 is a graph showing the distribution of film thickness, which is obtained by measuring the thickness of W-film at respective measuring points 1 to 161 established along the diameter of a wafer W on film deposition as a result of changing the supply rate of H 2 -gas to peripheral H 2 -gas discharging holes variously and of which horizontal axis represents the measuring points, while the vertical axis represents the thickness of W-film at the respective measuring points. FIG. 20 is a view in cooling a shower head by using the conventional coolant passage, showing the relationship between the diametric position of a shower plate and its temperature at respective temperatures of cooling water. FIG. 21 is a vertical sectional view showing a shower head part of the main body of a CVD apparatus in accordance with the second embodiment of the present invention. FIG. 22 is a horizontal sectional view taken along a line E-E of FIG. 21 , showing the shower head part of the main body of the CVD apparatus in accordance with the second embodiment of the present invention. FIG. 23A is a sectional view showing the structure of a first circular passage in the shower head of FIG. 21 . FIG. 23B is a sectional view showing the structure of a third circular passage in the shower head of FIG. 21 . FIG. 24 is a sectional view showing the structure of a semiconductor wafer on which a W-film is formed by the apparatus in accordance with the second embodiment of the present invention. FIG. 25 is a view for explanatory of an example of W-film formation flow carried out by the apparatus in accordance with the second embodiment of the present invention. FIG. 26 is a sectional view showing a condition where an initial W-film is formed on a under barrier layer of the semiconductor wafer of FIG. 24 . FIG. 27 is a view showing a calculation example of the cooling condition of a shower plate of the apparatus in accordance with the second embodiment of the present invention. FIG. 28 is a sectional view showing a condition where a main W-film is formed on the initial W-film on the under barrier layer of the semiconductor wafer of FIG. 26 . FIG. 29 is a sectional view showing a condition where a reactive intermediate represented by SiH x is formed by the application of an initiation processing on the under barrier layer of the semiconductor wafer of FIG. 26 . FIG. 30 is a sectional view showing a condition where a passivation W-film is formed on the first W-film of FIG. 26 . FIG. 31 is a sectional view showing another example of the coolant passage applied to the second embodiment of the present invention. FIG. 32 is a sectional view showing a CVD apparatus in accordance with the third embodiment of the present invention. FIG. 33A is a pattern diagram for explanation of the gas-flow in a SiH 4 -gas supply process when forming a first W-film by using the apparatus of the third embodiment of the present invention. FIG. 33B is a pattern diagram for explanation of the gas-flow in a WF 6 -gas supply process when forming a first W-film by using the apparatus of the third embodiment of the present invention. FIG. 34 is a schematic sectional view showing another example of the shower head of the third embodiment of the present invention. FIG. 35 is a horizontal sectional view taken along a line F-F of FIG. 34 . detailed-description description="Detailed Description" end="lead"? |
Working device and method for working a stack of plate-shaped elements |
A working station for working vertical edges of a stack (9) of plate-shaped elements (10). The working station comprises a support (11) for supporting the stack (9) and a number of working devices (1, 2, 3, 4) that are each configured for being able to slide towards and away from the stack (9). The support (11) is configured for being able to rotate the stack (9) about a vertical axis (13), and the working devices (1, 2, 3, 4) are configured for being, in a fixed path, able to slide between an outermost position away from the stack (9) and an innermost position towards or in proximity away from the stack (9) and an innermost position towards or in proximity of the vertical edges of the stack (9), irrespective of the angulation of the stack (9). Furthermore, a method is disclosed for working vertical edges of a stack (9) of plate-shaped elements (10) by means of such working station. |
1. A working station for working vertical edges of a stack (9) of plate-shaped elements (10), which working station comprises a support (11) for supporting the stack (9) and a plurality of working devices (1, 2, 3, 4; 15, 17) that are each configured to be able to slide towards and away from the stack (9), which support (11) is configured for being able to rotate the stack about a vertical axis (13); wherein the working devices (1, 2, 3, 4; 15, 17) are configured for being, in a fixed path, able to slide between an outermost position away from the stack (9) and an innermost position towards or in proximity of the vertical edges of the stack (9), irrespective of the angulation of the stack (9), wherein each of the working devices (1, 2, 3, 4; 15, 17) is mounted on a carriage (5) that can be caused to slide radially towards and away from the stack (9) along a bracket (6); wherein one of the working devices (1) comprises measuring means provided for detecting the peripheral geometry of the stack (9); and wherein control means are provided for automatically moving the other working devices (2, 3, 4; 15, 17) towards and away from the stack (9) in response to the detected geometry and the rotation of the stack (9). 2. A working station according to claim 1, wherein the working devices comprise at least one grinding cylinder (2, 3) that is configured to be turnable about a vertical axis. 3. A working station according to claim 2, wherein the grinding cylinder (2, 3) is provided with a flexible grinding material. 4. A working station according to claim 2, wherein precisely two grinding cylinders (2, 3) are provided that are configured for rotation in opposite directions. 5. A working station to the measuring means comprise a measuring wheel (1) mounted on a carriage (5) that can be caused to slide radially towards and away from the stack (9). 6. A working station according to claim 5, further comprising means for removing grinding dust from the vertical edges of the stack (9). 7. A working station according to claim 6, wherein the means for removing grinding dust comprise a blower device (4) mounted on a carriage (5) that can be caused to slide radially towards and away from the stack (9). 8. A working station according to claim 6, wherein the means for removing grinding dust comprise a brush that rotates about a vertical axis and is mounted on a carriage that can be caused to slide radially towards and away from the stack. 9. A working station according to claim 1, wherein the working devices comprise a paint or varnish applicator device (15). 10. A working station according to claim 9, further comprising a hardening device (17) provided with hardening means for the paint or the varnish. 11. A working station according to claim 10, wherein the hardening means are UV lamps (18). 12. A method of working vertical edges of a stack (9) of plate-shaped elements (10) by means of plurality of working devices (1, 2, 3, 4; 15, 17) that are caused to successively move towards the vertical edges of the stack (9) and to work same along their entire circumference, in which method the stack (9) is rotated about a vertical axis (13), and the working devices (1, 2, 3, 4; 15, 17) are caused to slide in a fixed path for working of the vertical edges of the stack (9), wherein the working devices (1, 2, 3, 4; 15, 17) are displaced radially towards and away from the stack (9), and that as a first step a measuring wheel (1) is displaced radially for abutment on the periphery of the stack (9), the stack (9) is rotated one full revolution while simultaneously its peripheral geometry is registered, and the measuring wheel (1) is displaced radially away from the stack (9). 13. A method according to claim 12, wherein the vertical edges of the stack (9) are subjected to grinding in the following steps following the first step: a first grinding cylinder (2) that rotates in a first direction about a vertical axis is displaced radially for abutment on the vertical edges of the stack (9), while the stack (9) rotates one full revolution, the first grinding cylinder (2) is displaced radially away from the stack (9); a second grinding cylinder (3) that rotates in a second direction about a vertical axis is displaced radially for abutment on the vertical edges of the stack (9), while the stack (9) rotates one full revolution, the second grinding cylinder (3) is caused to slide radially away from the stack (9); a blower device (4) or a rotating brush is caused to slide radially towards the vertical edges of the stack (9), while the stack (9) rotates one full revolution, the blower device (4) or the brush is caused to slide radially away from the stack (9). |
Novel serine protease |
The invention relates to a enzyme predicted to be a serine protease, which is specifically expressed in association with embryo implantation and placentation in pregnant uterus. The enzyme of the invention is useful in the evaluation of fertility and monitoring of early pregnancy, placental development and function, fetal development, parturition, and conditions such as pre-eclampsia, intrauterine growth restriction, early abortion, abnormal uterine bleeding, endometriosis, and cancers, and may provide a potential target for contraception. It may also be important in diseases of the heart, testis or ovary, and may play a role in muscle function, including cardiac muscle, skeletal muscle, lung and the diaphragm. In addition the enzyme of the invention is useful in the screening of candidate drugs for fertility control or for treatment of fertility-related disorders. |
1-29. (canceled) 30. An isolated nucleic acid molecule which is expressed in endometrium and placenta, is upregulated in pregnant uterus, is highly expressed during placental development, and encodes a protein which has serine protease enzymatic activity and an insulin-like growth factor (IGF)-binding motif, which nucleic acid molecule has one or more of the following properties: (i) comprises the sequence SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:38; (ii) is able to hybridize under at least moderately stringent conditions to a nucleic acid molecule with the sequence SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:38; and (iii) has at least 75% sequence identity to SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:38; with the proviso that the nucleic acid molecule does not encode HtrA1. 31. A nucleic acid molecule according to claim 30, which comprises the serine protease active site sequence GNSGGPL (SEQ ID NO:29). 32. A nucleic acid molecule according to claim 30, which comprises the sequence TNAHV (SEQ ID NO:30) in the vicinity of the serine protease active site. 33. A nucleic acid molecule according to claim 31, which further comprises the sequence TNAHV (SEQ ID NO:30) in the vicinity of the serine protease active site. 34. A nucleic acid molecule according to claim 30, which is a cDNA molecule. 35. A nucleic acid molecule according to claim 30, which is able to hybridize under stringent conditions to a DNA molecule having the sequence SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:38. 36. A nucleic acid molecule according to claim 30, which has at least 80% sequence identity to a DNA molecule having the sequence SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:38. 37. A pregnancy-related serum protease protein (PRSP) having serine protease enzymatic activity and an IGF-binding motif, which is encoded by the nucleic acid molecule of claim 30. 38. A protein according to claim 37, which has a sequence SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:33, or SEQ ID NO:34, or which protein is a functionally active variant of a protein having said sequence. 39. A protein according to claim 38, which has the sequence SEQ ID NO:33 or SEQ ID NO:34. 40. The functionally active variant of claim 38, which has at least about 75% sequence identity with one or more of the amino acid sequences SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:33, or SEQ ID NO:34, and has one or more of the following properties: (a) serine protease enzymatic activity, (b) binding to IGF, and (c) immunological cross-reactivity with an antibody specific for an epitope of a protein that has the amino acid sequence SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:33 or SEQ ID NO:34. 41. A composition comprising a nucleic acid molecule according to claim 30, and a pharmaceutically acceptable carrier. 42. A composition comprising a protein according to claim 37 and a pharmaceutically acceptable carrier. 43. A nucleic acid probe for detection of a polynucleotide that encodes the PRSP protein of claim 37, which probe comprises at least 15 consecutive nucleotides of a reference nucleic acid molecule that is expressed in endometrium and placenta, is upregulated in pregnant uterus, is highly expressed during placental development, and encodes a protein with serine protease activity and an IGF-binding motif, and which reference nucleic acid molecule has one or more of the following three properties: (a) comprises the sequence SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:38; (b) is able to hybridize under at least moderately stringent conditions to a nucleic acid molecule with the sequence SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:38; and (c) has at least 75% sequence identity to SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:38, and which reference nucleic acid molecule optionally has one or more of the following properties: (i) is able to hybridize under stringent conditions to a DNA molecule having the sequence SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:38; (ii) has at least 80% sequence identity to a DNA molecule having the sequence SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:38; (iii) encodes a protein comprising the serine protease active site sequence SEQ ID NO:29; and (iv) encodes a protein comprising the sequence SEQ ID NO:30 in the vicinity of the serine protease active site. 44. A nucleic acid probe according to claim 43, which includes at least part of (a) SEQ ID NO:40; or (b) the first 1243 nucleotides of SEQ ID NO:31. 45. A diagnostic reagent comprising the probe of claim 43 and a diagnostically acceptable carrier. 46. A method of detecting, diagnosing, or monitoring a condition characterized by altered PRSP expression in a mammal suspected of having said condition, comprising performing a nucleic acid hybridization assay on a biological sample from said mammal using as a hybridization probe the nucleic acid molecule of claim 30, or a fragment having at least about 15 consecutive nucleotides therefrom. 47. A method of detecting, diagnosing, or monitoring a condition characterized by altered PRSP expression in a mammal suspected of having said condition, comprising performing a nucleic acid hybridization assay on a biological sample from said mammal using as a hybridization probe the probe of claim 43. 48. The method of claim 46, in which a mammal with said condition suffers from infertility caused by (i) an inability to achieve or sustain embryo implantation or (ii) an inability to sustain a pregnancy. 49. The method of claim 46 wherein the sample is placental or uterine tissue from said mammal, and the method comprises assaying total RNA in the sample for the presence of PRSP messenger RNA, wherein a decrease in the amount of PRSP messenger RNA is indicative of impaired fertility or of impending miscarriage. 50. A method of identifying a genetic polymorphism in a mammal which is indicative of predisposition or susceptibility to a condition of altered PS RP expression, comprising performing a nucleic acid hybridization assay on a biological sample from said mammal using as a hybridization probe the nucleic acid molecule of claim 30, or a fragment having at least about 15 nucleotides therefrom. 51. The method of claim 50, in which the mammal suffers from pre-eclampsia, early abortion, intrauterine growth restriction, abnormal uterine bleeding, endometriosis, cancer, or a disease of the heart, testis or ovaries. 52. An antibody specific for the protein of claim 37. 53. The antibody of claim 52 that is specific for an epitope formed by, or comprising, the amino acid sequence PSGLHQLTSPC (SEQ ID NO:5 1), A LQVSGT PVRQC (SEQ ID NO:52) or GPLVNLD GEVIGC (SEQ ID NO:53). 54. The antibody of claim 52 that is specific for an epitope within the common region of two isoforms of mouse or human PRSP. 55. The antibody of claim 52 that inhibits the serine protease activity and/or the IGF-binding activity of PRSP. 56. A method of detecting a PRSP protein comprising the step of reacting a biological sample with the antibody of claim 52, wherein reaction of the antibody with the sample is indicative of the presence of said protein. 57. A method of detecting, diagnosing, or monitoring a condition characterized by altered PRSP expression in a mammal which has, or is at risk for, said condition, comprising the step of measuring the amount or activity of PRSP in a biological sample from said mammal. 58. The method of claim 57, in which the mammal with said condition suffers from infertility caused by (i) an inability to achieve or sustain embryo implantation or (ii) an inability to sustain a pregnancy. 59. The method of claim 50 in which the biological sample is a biological fluid, a uterine or bladder washing, a tissue, cells, or a tissue or cell extract. 60. The method of claim 56 in which the biological sample is a biological fluid, a uterine or bladder washing, a tissue, cells, or a tissue or cell extract. 61. A method of screening a sample or a collection of compounds for a compound that has the ability to modulate the activity of the protein of claim 37, comprising the step of assessing the ability of the sample or a candidate compound of said collection to increase or decrease (a) the serine protease enzymatic activity of the protein, and/or (b) the IGF-binding activity of the protein. |
<SOH> BACKGROUND OF THE INVENTION <EOH>All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country. Embryo implantation, the process by which the blastocyst attaches and implants in the uterus, leads to the establishment of an intimate relationship between the embryo and the endometrium. Implantation is one of the most important limiting factors in establishing a successful pregnancy. It is a complex process involving active interactions between the blastocyst and the uterus. The uterus must undergo dramatic morphological and physiological changes to transform itself from a non-receptive to a receptive state. This differentiation process is largely mediated by the coordinated effects of the ovarian hormones, which act through their intracellular receptors to regulate gene expression, and hence to influence cellular proliferation and differentiation. It is also regulated by the blastocyst. While the details of the exact molecular events occurring in the uterus during this differentiation process towards receptivity are still unknown, in principle it can be predicted that a unique set of genes is up- or down-regulated in a temporally and spatially specific manner. Indeed, induction of specific genes in the uterus during the peri-implantation period, including those encoding some growth factors and cytokines, has been reported (Huet-Hudson et al., 1990; Stewart et al., 1992; Robb et al., 1998; Zhu et al., 1998; Das et al., 1999). However, given the complexity and the as-yet imprecisely defined molecular mechanism of the process, many other molecules critical for implantation are still unidentified. We have used the mouse as a model in a search for hitherto unrecognised molecules which are important in the early stage of implantation. In the mouse on day 4.5 of pregnancy (vaginal plug=day 0), the uterus undergoes dramatic morphological changes in association with cell proliferation and differentiation, leading to the acquisition of a receptive state (Abrahamsohn and Zorn, 1993). This uterine remodelling is associated with an increase in vascular permeability at implantation sites (Psychoyos, 1973). We hypothesised that the proliferation and differentiation of endometrial cells at this time is associated with up- or down-regulation of a number of genes, many of which are still unknown (Nie et al., 1997). To identify uterine genes which are potentially critical for uterine receptivity, we used the technique of RNA differential display (DDPCR) (Liang and Pardee, 1992; Liang and Pardee, 1993) and compared the mRNA expression patterns of implantation and interimplantation sites on day 4.5 of pregnancy (Nie et al., 2000a; Nie et al., 2000b). One of the mRNA molecules identified as being differently regulated between the two sites was found to encode a novel protein molecule, with a predicted serine protease motif (Zumbrunn & Traub, 1996). We isolated the cDNA encoding this protein, and examined its uterine expression during early pregnancy in the mouse; the protein is up-regulated in the pregnant mouse uterus from day 4.5 and further increased in the implantation site (including the maternal deciduum and the fetus and the placenta) from day 8.5 onwards. The observed expression pattern indicated a role for this protein in implantation, placentation and early pregnancy. We have also identified and isolated the cDNA encoding the corresponding human enzyme, and found that this encodes a protein with a predicted serine protease motif, which is expressed in endometrium, decidua and placenta, and also in ovary, heart, and certain other tissues. |
<SOH> SUMMARY OF THE INVENTION <EOH>In a first aspect the invention provides an isolated nucleic acid molecule which (a) is expressed in endometrium and placenta; (b) is up-regulated in pregnant uterus and highly expressed during placental development; and (c) encodes a protein which comprises a serine protease site and has an insulin-like growth factor (IGF)-binding motif. Preferably the protein comprises the serine protease active site sequence GNSGGPL (SEQ ID NO:29); more preferably the protein also comprises the sequence TNAHV (SEQ ID NO:30) in the vicinity of the serine protease active site. It will be appreciated that although the nucleic acid molecule of the invention encodes a protein which has serine protease activity and the ability to bind IGF, it may also have other activities which are significant for biological functions. The nucleic acid molecule may be a cDNA, a genomic DNA, or an RNA, and may be in the sense or the anti-sense orientation. Preferably the nucleic acid molecule is a cDNA. Preferably the nucleic acid molecule has a sequence selected from the group consisting of (a) a cDNA molecule having the sequence set out in FIG. 2 (SEQ ID NO:26), FIG. 3A (SEQ ID NO:31), FIG. 3B (SEQ ID NO:32), or FIG. 6A (SEQ ID NO:38); (b) a nucleic acid molecule which is able to hybridize under at least moderately stringent conditions to the molecule of (a); and (c) a nucleic acid molecule which has at least 75% sequence identity to the molecule of (a). More preferably in (b) the nucleic acid molecule is able to hybridize under stringent conditions to the molecule of (a). More preferably in (c) the nucleic acid molecule has at least 80%, even more preferably at least 90% sequence identity to the molecule of (a). In a second aspect the invention provides a protein having serine protease enzymic activity and an IGF-binding motif, which is encoded by the nucleic acid molecule of the invention. This protein is referred to herein as pregnancy-related serine protease (PRSP). It will be clearly understood that all isoforms of PRSP are within the scope of the invention. Preferably the protein has a sequence selected from the group consisting of the sequences set out in FIG. 2 (SEQ ID NO:27), FIG. 6B (SEQ ID NO:39), FIG. 4A (SEQ ID NO:33), or FIG. 4B (SEQ ID NO:34); more preferably the sequence is the one set out in FIG. 4A (SEQ ID NO:33) or FIG. 4B (SEQ ID NO:34). PRSP amino acid sequence variants are included within the scope of the invention, provided that they are functionally active. As used herein, the terms “functionally active” and “functional activity” in reference to PRSP mean that the PRSP is able to act as a serine protease and/or to bind IGF, and/or that the PRSP is immunologically cross-reactive with an antibody directed against an epitope of a naturally-occurring PRSP of the invention. It will be appreciated that PRSP may also have other biological functions in addition to those specifically mentioned herein. Therefore PRSP amino acid sequence variants will generally share at least about 75%, preferably greater than 80%, and more preferably greater than 90% sequence identity with one or more of the deduced amino acid sequences set out in in FIG. 2 (SEQ ID NO:27), FIG. 6B (SEQ ID NO:39), FIG. 4A (SEQ ID NO:33), or FIG. 4B (SEQ ID NO:34), after aligning the sequences to provide for maximum homology, for example as determined by the version described by Fitch et al., (1983), of the algorithm described by Needleman et al., (1970). In a third aspect the invention provides a composition comprising a nucleic acid molecule according to the invention, together with a pharmaceutically acceptable carrier. In a fourth aspect the invention provides a composition comprising a protein according to the invention, together with a pharmaceutically acceptable carrier. In a fifth aspect the invention provides a probe for detection of nucleic acid encoding PRSP, comprising at least 15, preferably at least 20, more preferably at least 30 consecutive nucleotides from the nucleic acid molecule of the invention. In a particularly preferred embodiment the probe encompasses at least part of the common region of the two isoforms disclosed herein for mouse PRSP (SEQ ID NO:40), or human PRSP (nucleotides 1-1243 of the long form sequence shown in SEQ ID NO:31). Thus the invention provides a method of detecting, diagnosing, or monitoring a condition which involves a change in PRSP expression, comprising the step of using a nucleic acid molecule according to the invention, or a fragment thereof comprising at least about 15 nucleotides, as a probe in a hybridization assay performed on a biological sample from a mammal suspected to be suffering from such a condition. The sample may be a sample of a biological fluid such as plasma, serum, uterine or bladder washings, or amniotic fluid, or may be a tissue or cell sample or an extract thereof. Such conditions include infertility caused by inability to achieve or sustain embryo implantation or to sustain pregnancy, in which the assay is performed on a sample. In one embodiment of the invention, total RNA in a sample of placental or uterine tissue from the mammal is assayed for the presence of PRSP messenger RNA, wherein an alteration in the amount of PRSP messenger RNA is indicative of impaired fertility or of impending miscarriage. It will be appreciated that probes according to this aspect of the invention may be used to identify genetic polymorphisms which are indicative of predisposition or susceptibility to PSRP-related conditions. Such conditions include but are not limited to pre-eclampsia, intrauterine growth restriction (IUGR), early abortion, abnormal uterine bleeding, endometriosis, cancers, and diseases of the heart, testis or ovaries. In a sixth aspect the invention provides an antibody directed against PRSP. The antibody may be polyclonal or monoclonal, and is preferably monoclonal. The antibody may suitably be directed against one of the following segments of the mouse protease: 1. Amino acids 133-142; sequence PSGLHQLTSPC (SEQ ID NO:51). 2. Amino acids 116-126; sequence ALQVSGTPVRQC (SEQ ID NO:52). 3. A sequence common to both isoforms, represented by amino acids 133-142 of SEQ ID NO:26; sequence GPLVNLDGEVIGC (SEQ ID NO:53). These mouse sequences are highly homologous to corresponding regions of the human protein. More preferably the antibody is directed to an epitope within the common region of the two isoforms disclosed herein for mouse or human PRSP. In one particularly preferred embodiment the antibody has the ability to inhibit the serine protease activity and/or the IGF-binding activity of the PRSP. The antibody may also be used to detect the PRSP in biological fluids, washings from hollow viscera such as the uterus or bladder, or in tissues, cells or extracts thereof. In a seventh aspect the invention provides a method of screening for compounds which have the ability to modulate the activity of PRSP, comprising the step of assessing the ability of a candidate compound to increase or decrease (a) the serine protease activity and/or (b) the IGF-binding activity of PRSP. It will be appreciated that modulation of PRSP activity may be detected inter alia by monitoring the effects of the candidate compound on levels of a substrate for the enzyme, or on a cellular activity of PRSP. The substrate assay may utilise synthetic substrates, and suitable substrates are well known in the art. Assays for cellular activity may utilise cell lines which have been transfected with nucleic acid encoding PSRP so as to over express this protein; such transformed cell lines are particularly useful for phenotypic assays of biological function. Thus the invention provides a method of identifying agonists and antagonists of PRSP. In view of the crucial role of PRSP in implantation and in formation of the placenta indicated by the results reported herein, it is contemplated that antagonists of PRSP will be useful as contraceptives, and that agonists of PRSP will be useful as agents for promoting fertility or for supporting at least the early phases of pregnancy. It is further contemplated that antagonists of PRSP include, but are not limited to, antibodies and anti-sense nucleic acids. In an eighth aspect, the invention provides a method of detecting, diagnosing, or monitoring conditions which involve changes in PRSP expression, such as infertility caused by inability to achieve or sustain embryo implantation or to sustain pregnancy, or insufficiency of placentation (such as may occur in pre-eclampsia or IUGR), comprising the step of measuring the amount or activity of PRSP in a biological sample from a mammal suffering from or at risk of such a condition. Any suitable biological sample may be used, for example a tissue or cell sample or extract, or a sample of a biological fluid, such as plasma, serum or amniotic fluid, or uterine or bladder washings. For example, the probes of the invention may be used to diagnose impaired fertility or impending miscarriage, as described above. The antibodies of the invention are expected to be particularly useful for detecting PRSP in biological fluids such as plasma, serum or amniotic fluid, or in uterine or bladder washings. The mammal may be a human, or may be a domestic or companion animal. While it is particularly contemplated that the compounds of the invention are suitable for use in medical treatment of humans, they are also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, zoo animals such as non-human primates, felids, canids, bovids, and ungulates, or for the control of pest or feral species such as rabbits, rats and mice. Methods and pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins, Pennsylvania, USA. The compounds and compositions of the invention may be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the condition to be treated, the age and general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered. The carrier or diluent, and other excipients, will depend on the route of administration, and again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case. For the purposes of this specification it will be clearly understood that the word “comprising” means “including but not limited to”, and that the word “comprises” has a corresponding meaning. |
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