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<SOH> SUMMARY OF THE INVENTION <EOH>The present invention features 8-mer peptides that are suitable substrates for β-secretase, particularly human β-secretase. In vitro assays for measuring β-secretase activity employing such substrates are also disclosed. The peptide substrates are characterized as comprising at least one β-secretase cleavage site. In one embodiment, the peptide substrates of the invention, when presented as an immunogen, elicit the production of a antibodies which specifically bind to a region of native APP having an amino acid sequence that is substantially homologous to that of any of the disclosed invention peptides. In another embodiment, the antibodies specifically bind the peptide substrates of the invention but do not specifically bind to native APP. In another aspect of the present invention, antibodies are provided which are specific for an amino terminal fragment or carboxy terminal fragment (a/k/a cleavage product) of one of the peptides listed in Table 1 and/or Table 2 that results from cleavage of the substrates by β-secretase. The antibodies may be polyclonal or monoclonal. In another aspect, the invention provides antibodies that recognize the synthetic β-secretase cleavage site of any of the 8-mer peptides listed in Table 1 and/or Table 2. In particular, antibodies that do not recognize the β-secretase cleavage site of native APP are provided. An aspect of the present invention describes a nucleic acid comprising a nucleotide base sequence encoding any of the herein disclosed β-secretase substrates. Preferably, the nucleic acid is contained in an expression vector. Another aspect of the present invention describes a recombinant cell comprising a nucleic acid encoding any of the disclosed β-secretase substrates or fragments thereof. Another aspect of the present invention describes a method for assaying β-secretase activity. β-Secretase activity can be obtained from cells producing β-secretase in a solubilized form or in a membrane-bound form. The method can be performed by measuring cleavage product formation resulting from β-secretase substrate cleavage. Measuring can be performed by qualitative or quantitative techniques. Thus an aspect of the present invention describes a method for measuring the ability of a compound to affect β-secretase activity comprising the steps of: (a) combining together a β-secretase substrate, a test compound, and a preparation comprising β-secretase activity, under reaction conditions allowing for β-secretase activity, and (b) measuring β-secretase activity. An exemplary method utilizes a reaction system including β-secretase and a peptide substrate of the invention present in initial amounts. The reaction system is maintained under conditions which permit the β-secretase to cleave the peptide substrate into cleavage products. The β-secretase cleavage reaction is monitored by detecting the amount of at least one of the β-secretase cleavage products, where the amount of cleavage product(s) will increase over time as the reaction progresses. Such methods are particularly useful for screening test compounds for the ability to inhibit β-secretase activity. Test compounds are introduced to the reaction system, and the ability of the test compound to inhibit the β-secretase activity is determined based on the ability to decrease the amount of cleavage product produced, usually in comparison to a control where β-secretase mediated cleavage in the reaction system is observed and measured in the absence of test compound(s). The methods of the present invention allow identification of test substances which inhibit proteolytic cleavage of to disclosed peptide substrates by β-secretase. The methods comprise exposing a peptide of the invention comprising a β-secretase cleavage site to β-secretase in the presence of the test substance under conditions such that the β-secretase would be expected to cleave the peptide substrate into an amino-terminal fragment and a carboxy-terminal fragment in the absence of the test substance. Test substances which inhibit such cleavage are thus identified as having β-secretase inhibition activity. Usually, generation of the amino-terminal fragment and/or the carboxy-terminal fragment is detected by an antibody specific for the carboxy end of the amino-terminal fragment or the amino end of the carboxy-terminal fragment. Alternative methods of detecting the amino-terminal and/or carboxyl-terminal fragments include liquid chromatography/mass spectrometry (LC/MS). The present invention further comprises methods for inhibiting the cleavage of β-amyloid substrate in cells. Such methods comprise administering to the cells an amount of a compound effective to at least partially inhibit β-secretase activity. Usually, such compounds will be selected by the screening methods described above. Such compounds, will also find use in inhibiting binding of native or recombinant β-secretase to APP in vivo. The present invention further provides methods for inhibiting the cleavage of β-amyloid precursor protein in mammalian hosts. Such methods comprise administering to the host an amount of a compound effective to inhibit β-secretase activity in cells of the host, usually in brain cells of the host. Such compounds will usually be selected by the screening assays described in this application. Such methods will be useful for treating conditions related to Aβ peptide deposition such as Alzheimer's disease, Down's syndrome, and the like. In another aspect of the present invention, Aβ production inhibitors identified by the methods described herein may be studied in transgenic animal models. The animals are exposed to test compound(s) and those compounds which affect (usually by diminishing) the production of any of the cleavage products of APP are considered candidates for further testing as drugs for the treatment of Aβ-related conditions. Methods and compositions are provided for detecting and monitoring an amino-terminal fragment resulting from β-secretase cleavage of any of the herein included peptide substrates. The resulting fragment, referred to as a an amino terminal cleavage product, βATF-PS (amino terminal fragment of the peptide substrates) may be detected in biological samples and is useful for monitoring the processing of the disclosed substrates in animal models. In particular, the present invention provides for monitoring in vivo processing of any of the disclosed peptide substrates where the presence of the βATF-PS is detected in a specimen from an animal transformed to express the substrates and where the βATF-PS has been cleaved from the substrate between amino acids 596 and 597, based on the numbering of Kang et al., 1987, Nature 325:733-736 in the 695 amino acid isoform. It has been found that cells expressing the gene encoding any of the herein disclosed peptide substrates are particularly prolific producers of the cleavage products of said peptide substrate that are the amino- and the carboxy-terminal fragments thereof. That is, such cells are able to cleave the substrates of the invention at a greater frequency than cleavage of either the endogenous APP, the wild type human APP, or the Swedish mutant APP. It is further believed that intracellular processing of the substrates of the invention results in greater production of the βATF-PS than is produced by other human mutations of the APP gene. Thus, transgenic animal systems, such as transgenic mice, expressing any of the herein disclosed peptide substrates are particularly suitable as models for monitoring intracellular processing of the disclosed substrates as well as for screening test compounds for the ability to inhibit or modulate cleavage of APP as a result of β-secretase activity, the apparently pathogenic form of APP processing. Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention. |
Corticotropin releasing factor antagonists |
The present invention provides compounds of formula (I) including stereoisomers, prodrugs and pharmaceutically acceptable salts or solvates thereof wherein R is aryl or heteroaryl and each of the above groups R may be substituted by 1 to 4 groups selected from: halogen, C1-C6 alkyl, C1-C6 alkoxy, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo C1-C6 alkoxy, —COR4, nitro, —NR3R4 cyano, or a group R5; R1 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo C1-C6 alkyl, halo C1-C6 alkoxy, halogen, NR3R4 or cyano; R2 corresponds to a group CHR6R7; R3 is hydrogen, C1-C6 alkyl; R4 independently from R3, has the same meanings; R5 is C3-C7 cycloalkyl, which may contain one or more double bonds; aryl; or a 5-6 membered heterocycle; wherein each of the above groups R5 may be substituted by one or more groups selected from: halogen, C1-C6 alkyl, C1-C6 alkoxy, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo C1-C6 alkoxy, C1-C6 dialkylamino, nitro or cyano; R6 is hydrogen, C2-C6 alkenyl or C1-C6 alkyl, wherein each of the above groups R6 may be substituted by one or more groups selected from: C1-C6 alkoxy and hydroxy; R7 independently from R6, has the same meanings; X is carbon or nitrogen; to processes for their preparation, to pharmaceutical compositions containing them and to their use in the treatment of conditions mediated by corticotropin-releasing factor (CRF). |
1. Compounds of formula (I) including stereoisomers, prodrugs and pharmaceutically acceptable salts or solvates thereof wherein R is aryl or heteroaryl and each of the above groups R may be substituted by 1 to 4 groups selected from: halogen, C1-C6 alkyl, C1-C6 alkoxy, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo C1-C6 alkoxy, —COR4, nitro, —NR3R4 cyano, or a group R5; R1 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo C1-C6 alkyl, halo C1-C6 alkoxy, halogen, NR3R4 or cyano; R2 corresponds to a group CHR6R7; R3 is hydrogen, C1-C6 alkyl; R4 independently from R3, has the same meanings; R5 is C3-C7 cycloalkyl, which may contain one or more double bonds; aryl; or a 5-6 membered heterocycle; wherein each of the above groups R5 may be substituted by one or more groups selected from: halogen, C1-C6 alkyl, C1-C6 alkoxy, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, halo C1-C6 alkoxy, C1-C6 dialkylamino, nitro or cyano; R6 is hydrogen, C2-C6 alkenyl or C1-C6 alkyl, wherein each of the above groups R6 may be substituted by one or more groups selected from: C1-C6 alkoxy and hydroxy; R7 independently from R6, has the same meanings; X is carbon or nitrogen. 2. Compounds, according to claim 1, of general formula (Ia) in which R, R1, and R2 are defined as in claim 1. 3. Compounds, according to claim 1, of general formula (Ib) in which R, R1, and R2 are defined as in claim 1. 4. Compounds, according to claim 1, wherein R1 is C1-C3 alkyl group or halo C1-C3 alkyl group. 5. Compounds, according to claim 1, wherein R is an aryl group selected from: 2,4-dichlorophenyl, 2-chloro-4-methylphenyl, 2-chloro-4-trifluoromethyl, 2-chloro-4-methoxyphenyl, 2,4-dimethylphenyl, 2-methyl-4-methoxyphenyl, 2-methyl-4-chlorophenyl, 2-methyl-4-trifluoromethyl, 2,4-dimethoxyphenyl, 2-methoxy-4-trifluoromethylphenyl, 2-methoxy-4-chlorophenyl, 3-methoxy-4-chlorophenyl, 2,5-dimethoxy-4-chlorophenyl, 2-methoxy-4-isopropylphenyl, 2-methoxy-4-trifluoromethylphenyl, 2-methoxy-4-isopropylphenyl, 2-methoxy-4-methylphenyl, 2-trifluoromethyl-4-chlorophenyl, 2,4-trifluoromethylphenyl, 2-trifluoromethyl-4-methylphenyl, 2-trifluoromethyl-4-methoxyphenyl, 2-bromo-4-isopropylphenyl, 4-methyl-6-dimethylaminopyridin-3-yl, 3,5-dichloro-pyridin-2-yl, 2,6-bismethoxy-pyridin-3-yl and 3-chloro-5-tricloromethyl-pyridin-2-yl. 6. A compound, according to claim 1, selected in a group consisting from: 7. A process for the preparation of a compound of formula (I) as claimed in claim 1, which comprises the reaction of a compound of formula (II), wherein L is a leaving group, with the alcohol compound (III) HOCHR2aR3a wherein R2a and R3a have the meanings defined in claim 1 for R2 and R3 or are a group convertible thereto. 8-14. (Cancelled). 15. A pharmaceutical composition comprising a compound according to claim 1, in admixture with one or more physiologically acceptable carriers or excipients. 16. A diagnostic formulation comprising a radiolabelled compound according to claim 1, in admixture with one or more physiologically acceptable carriers or excipients. 17. A method for the treatment of a mammal, including man, in particular in the treatment of conditions mediated by CRF (corticotropin-releasing factor), comprising administration of an effective amount of a compound according to claim 1. 18. A method, according to claim 17, in the treatment of depression and anxiety, comprising administration of an effective amount of a compound according to claim 1. 19. A method, according to claim 17, in the treatment of IBS (irritable bowel disease) and IBD (inflammatory bowel disease), comprising administration of an effective amount of a compound according to claim 1. 20. A method for the diagnosis of conditions mediated by CRF (corticotropin-releasing factor) in an animal, including man, comprising administration of an effective amount of a radiolabelled compound according to claim 1. |
Oxazolidines containing a sulfonimid group as antibiotics |
Compounds of the formula (I), or a pharmaceutically-acceptable salt, or an in-vivo-hydrolysable ester thereof, wherein, for example, HET is an N-linked 5-membered, fully or partially unsaturated heterocyclic ring, or an N-linked 6-membered di-hydro-heteroaryl ring; and Q is, for example, Q1 or Q2: wherein R2 and R3 are independently hydrogen or fluoro; T is selected, for example, from a group of the formula (TA1) or (TA2): wherein, for example, X1m is O═ and X2m is R2s—(E)ms—N—; wherein E is an electron withdrawing group, for example, —SO2— or —CO—; and, for example, R2s is hydrogen or (1-6C)alkyl; are useful as pharmaceutical agents; and processes for their manufacture and pharmaceutical compositions containing them are described. |
1. A compound of the formula (I), or a pharmaceutically-acceptable salt, or an in-vivo-hydrolysable ester thereof, wherein (i) HET is an N-linked 5-membered, fully or partially unsaturated heterocyclic ring, containing either (i) 1 to 3 further nitrogen heteroatoms or (ii) a further heteroatom selected from O and S together with an optional further nitrogen heteroatom; which ring is optionally substituted on a C atom, other than a C atom adjacent to the linking N atom, by an oxo or thioxo group; and/or which ring is optionally substituted on any available C atom, other than a C atom adjacent to the linking N atom, by a substituent selected from (1-4C)alkyl, (2-4C)alkenyl, (3-6C)cycloalkyl, amino, (1-4C)alkylamino, di-(1-4C)alkylamino, (1-4C)alkylthio, (1-4C)alkoxy, (1-4C)alkoxycarbonyl, halogen, cyano and trifluoromethyl and/or on an available nitrogen atom (provided that the ring is not thereby quatermised) by (1-4C)alkyl; or HET is an N-linked 6-membered di-hydro-heteroaryl ring containing up to three nitrogen heteroatoms in total (including the linking heteroatom), which ring is substituted on a suitable C atom, other than a C atom adjacent to the linking N atom, by oxo or thioxo and/or which ring is optionally substituted on any available C atom, other than a C atom adjacent to the linking N atom, by one or two substituents independently selected from (1-4C)alkyl, (2-4C)alkenyl, (3-6C)cycloalkyl, amino, (1-4C)alkylamino, di-(1-4C)alkylamino, (1-4C)alkylthio, (1-4C)alkoxy, (1-4C)alkoxycarbonyl, halogen, cyano and trifluoromethyl and/or on an available nitrogen atom (provided that the ring is not thereby quatermised) by (1-4C)alkyl; and wherein at each occurrence of alkyl, alkenyl and cycloalkyl HET substituents, each is optionally substituted with one or more F, Cl or CN; or (ii) HET is selected from the structures (Za) to (Zf) below: wherein u and v are independently 0 or 1; RT is selected from a substituent from the group (RTa) wherein RT is hydrogen, halogen, (1-4C)alkoxy, (2-4C)alkenyloxy, (2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, amino, (1-4C)alkylamino, di-(1-4C)alkylamino, (2-4C)alkenylamino, (1-4C)alkylcarbonylamino, (1-4C)alkylthiocarbonylamino, (1-4C)alkyl-OCO—NH—, (1-4C)alkyl-NH—CO—NH—, (1-4C)alkyl-NH—CS—NH—, (1-4C)alkyl-SO2—NH— or (1-4C)alkyl-S(O)q— (wherein q is 0, 1 or 2); or RT is selected from the group (RTb) wherein RT is a (1-4C)alkyl group which is optionally substituted by one substituent selected from hydroxy, (1-4C)alkoxy, amino, cyano, azido, (2-4C)alkenyloxy, (1-4C)alkylcarbonyl, (1-4C)alkoxycarbonyl, (1-4C)alkylamino, (2-4C)alkenylamino, (1-4C)alkyl-SO2—NH—, (1-4C)alkylcarbonylamino, (1-4C)alkylthiocarbonylamino, (1-4C)alkyl-OCO—NH—, (1-4C)alkyl-NH—CO—NH—, (1-4C)alkyl-NH—CS—NH—, (1-4C)alkyl-SO2—NH—, (1-4C)alkyl-S(O)q—(wherein q is 0, 1 or 2), (3-6C)cycloalkyl, (3-6C)cycloalkenyl or an N-linked 5-membered heteroaryl ring, which ring contains either (i) 1 to 3 further nitrogen heteroatoms or (ii) a further heteroatom selected from O and S together with an optional further nitrogen heteroatom; which ring is optionally substituted on a carbon atom by an oxo or thioxo group; and/or the ring is optionally substituted on a carbon atom by 1 or 2 (1-4C)alkyl groups; and/or on an available nitrogen atom (provided that the ring is not thereby quatermised) by (1-4C)alkyl; or RT is selected from a group of formula (RTc1) to (RTc3): (RTc1) a fully saturated 4-membered monocyclic ring containing 1 or 2 heteroatoms independently selected from O, N and S (optionally oxidised), and linked via a ring nitrogen or carbon atom; or (RTc2) a saturated or unsaturated 5-membered monocyclic ring containing 1 heteroatom selected from O, N and S (optionally oxidised), and linked via a ring nitrogen atom if the ring is not thereby quatermised, or a ring carbon atom; or (RTc3) a saturated or unsaturated 6- to 8-membered monocyclic ring containing 1 or 2 heteroatoms independently selected from O, N and S (optionally oxidised), and linked via a ring nitrogen atom if the ring is not thereby quatermised, or a ring carbon atom; wherein said rings in (RTc1) to (RTc3) are optionally substituted on an available carbon atom by 1 or 2 substituents independently selected from hydroxy, (1-4C)alkoxy, amino, cyano, azido, (2-4C)alkenyloxy, (1-4C)alkylcarbonyl, (1-4C)alkoxycarbonyl, (1-4C)alkylamino, (2-4C)alkenylamino, (1-4C)alkyl-SO2—NH—, (1-4C)alkylcarbonylamino, (1-4C)alkylthiocarbonylamino, (1-4C)alkyl-OCO—NH—, (1-4C)alkyl-NH—CO—NH—, (1-4C)alkyl-NH—CS—NH—, (1-4C)alkyl-SO2—NH—, (1-4C)alkyl-S(O)q— (wherein q is 0, 1 or 2), (3-6C)cycloalkyl or (3-6C)cycloalkenyl; or RT is selected from the group (RTd) cyano, nitro, azido, formyl, (1-4C)alkylcarbonyl or (1-4C)alkoxycarbonyl; and wherein at each occurrence of an RT substituent containing an alkyl, alkenyl, alkynyl, cycloalkyl or cycloalkenyl moiety in (RTa), (RTb) or (RTc1) to (RTc3) each such moiety is optionally further substituted on an available carbon atom with one or more substituents independently selected from F and Cl and/or by one cyano group; Q is selected from Q1 to Q10: wherein R2 and R3 are independently hydrogen or fluoro; wherein A1 is carbon or nitrogen; B1 is O or S (or, in Q9 only, NH); Xq is O, S or N—R1 (wherein R1 is hydrogen, (1-4C)alkyl or hydroxy-(1-4C)alkyl); and wherein in Q7 each A1 is independently selected from carbon or nitrogen, with a maximum of 2 nitrogen heteroatoms in the 6-membered ring, and Q7 is linked to T via any of the A1 atoms (when A1 is carbon), and linked in the 5-membered ring via the specified carbon atom, or via A1 when A1 is carbon; Q8 and Q10 are linked to T via either of the specified carbon atoms in the 5-membered ring, and linked in the benzo-ring via either of the two specified carbon atoms on either side of the linking bond shown; and Q9 is linked via either of the two specified carbon atoms on either side of the linking bond shown; wherein T is selected from the groups in (TA) & (TB) below (wherein AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a and CY are defined herein); (TA) T is selected from the following groups (TA1) and (TA2): wherein: in (TA1), ( )o1 is 0 or 1 and represents a chain of carbon atoms (optionally substituted as defined for AR1) of length o1 and M is a bond joining the adjacent carbon atoms, or M represents one or two carbon atoms, and defines a 4- to 7-membered monocyclic ring, which ring may optionally have one of (i) one double bond between any two ring carbon atoms; or (ii) a C1-C3 bridge connecting any two appropriate, non-adjacent ring carbon atoms, which bridge may optionally contain one heteroatom selected from oxygen or >NRc; or (iii) a C2-C5 cyclic moiety including a ring carbon atom to define a spiro C2-C5 ring system, which ring may optionally contain one heteroatom selected from oxygen or >NRc; or (iv) a C1-C4 bridge connecting adjacent carbon atoms to define a fused ring, wherein a C2-C4 bridge may optionally contain one heteroatom selected from oxygen or >NRc; wherein Rc is as defined hereinafter; wherein in (TA2), ( )n1 and ( )o1 are independently 0, 1 or 2 and represent chains of carbon atoms (optionally substituted as defined for AR1) of length n1 and o1 respectively, and define a 4- to 8-membered monocyclic ring, which ring may optionally have one of (i) a C1-C3 bridge connecting any two appropriate, non-adjacent ring carbon atoms, which bridge contains one heteroatom selected from oxygen or >NRc; or (ii) a C2-C5 cyclic moiety including a ring carbon atom to define a spiro C2-C5 ring system, which ring may optionally contain one heteroatom selected from oxygen or >NRc; or (iii) a C1-C4 bridge connecting adjacent carbon atoms to define a fused ring, wherein a C2-C4 bridge may optionally contain one heteroatom selected from oxygen or >NRc; wherein Rc is as defined hereinafter; and wherein in (TA1) and (TA2), X1m and X2m taken together represent R2s—(E)ms—N═; or X1m is O═ and X2m is R2s—(E)ms—N—, and vice versa; wherein E is an electron withdrawing group selected from —SO2—, —CO—, —O—CO—, —CO—O—, —CS—, —CON(Rs)—, —SO2N(Rs)—, or E may represent a group of the formula R3s—C(═N—O—R3s)—C(═O)—, wherein R3s is H or as defined in R2s at (i) below; or, when E is —CON(Rs)— or —SO2N(Rs)—, R2s and Rs may link together to form a carbon chain which defines a 5- or 6-membered saturated, unsaturated or partially unsaturated ring linked via the N atom in E, which ring is optionally further substituted by an oxo substituent, and which ring may be optionally fused with a phenyl group to form a benzo-fused system, wherein the phenyl group is optionally substituted by up to three substituents independently selected from halo, cyano, (1-4C)alkyl and (1-4C)alkoxy; ms is 0 or 1; R2s and Rs are independently selected from: (i) hydrogen (except where E is —SO2— or —O—CO—), or (1-6C)alkyl {optionally substituted by one or more (1-4C)alkanoyl groups (including geminal disubstitution) and/or optionally monosubstituted by cyano, cyano-imino, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as defined for AR1 herein), optionally substituted heteroaryl group of the formula AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein, (1-4C)alkylS(O)q— (q is 0, 1 or 2); and/or (with the proviso that where R2s is —SO2 or —O—CO— not on the first carbon atom of the (1-6C) alkyl chain) optionally substituted by one or more groups (including geminal disubstitution) each independently selected from hydroxy and fluoro, and/or optionally further substituted, by no more than one of each of, oxo, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N-(-4C)alkyl-N-(1-6C)alkanoylamino, (1-4C)alkylS(O)pNH— or (1-4C)alkylS(O)p-((1-4C)alkyl)N— (p is 1 or 2)}; or (ii) an optionally substituted aryl or optionally substituted heteroaryl group of the formula AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein; or (where ms is 0 only); (iii) cyano, —CO—NRvRw, —CO—NRv Rw′, —SO2—NRvRw, —SO2—NRv Rw′ [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl; Rw′ is phenyl (optionally substituted as defined for AR1 herein), or a heteroaryl group selected from AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a (optionally substituted as defined herein)], (1-4C)alkoxycarbonyl, trifluoromethyl, ethenyl, 2-(1-4C)alkylethenyl, 2-cyanoethenyl, 2-cyano-2-((1-4C)alkyl)ethenyl, 2-nitroethenyl, 2-nitro-2-((1-4C)alkyl)ethenyl, 2-((1-4C)alkylaminocarbonyl)ethenyl, 2-((1-4C)alkoxycarbonyl)ethenyl, 2-(AR1)ethenyl, 2-(AR2)ethenyl, or 2-(AR2a)ethenyl; or (TB) T is selected from the following groups (TB1) to (TB3): wherein: X1m and X2m taken together represent R2s—(E)ms—N═; or X1m is O═ and X2m is R2s—(E)ms—N—, and vice versa; wherein E is an electron withdrawing group selected from —SO2—, —CO—, —O—CO—, —CO—O—, —CS—, —CON(Rs)—, —SO2N(Rs)—, or E may represent a group of the formula R3s—C(═N—O—R3s)—C(═O)—, wherein R3s is H or as defined in R2s at (i) below; or, when E is —CON(Rs)— or —SO2N(Rs)—, R2s and Rs may link together to form a carbon chain which defines a 5- or 6-membered saturated, unsaturated or partially unsaturated ring linked via the N atom in E, which ring is optionally further substituted by an oxo substituent, and which ring may be optionally fused with a phenyl group to form a benzo-fused system, wherein the phenyl group is optionally substituted by up to three substituents independently selected from halo, cyano, (1-4C)alkyl and (1-4C)alkoxy; ms is 0 or 1; R2s and Rs are independently selected from: (i) hydrogen (except where E is —SO2— or —O—CO—), or (1-6C)alkyl {optionally substituted by one or more (1-4C)alkanoyl groups (including geminal disubstitution) and/or optionally monosubstituted by cyano, cyano-imino, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as for AR1), optionally substituted heteroaryl group of the formula AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined hereinafter, (1-4C)alkylS(O)q— (q is 0, 1 or 2); and/or (with the proviso that where R2s is —SO2 or —O—CO— not on the first carbon atom of the (1-6C) alkyl chain) optionally substituted by one or more groups (including geminal disubstitution) each independently selected from hydroxy and fluoro, and/or optionally further substituted, by no more than one of each of, oxo, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N-(1-4C)alkyl-N-(1-6C)alkanoylamino, (1-4C)alkylS(O)pNH— or (1-4C)alkylS(O)p-((1-4C)alkyl)N— (p is 1 or 2)}; or (ii) an optionally substituted aryl or optionally substituted heteroaryl group of the formula AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined herein; or (where ms is 0 only); (iii) cyano, —CO—NRvRw, —CO—NRv Rw′, —SO2—NRvRw, —SO2—NRv Rw′ [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl; Rw′ is phenyl (optionally substituted as for AR1), or a heteroaryl group selected from AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a (as defined herein)], (1-4C)alkoxycarbonyl, trifluoromethyl, ethenyl, 2-(1-4C)alkylethenyl, 2-cyanoethenyl, 2-cyano-2-((1-4C)alkyl)ethenyl, 2-nitroethenyl, 2-nitro-2-((1-4C)alkyl)ethenyl, 2-((1-4C)alkylaminocarbonyl)ethenyl, 2-((1-4C)alkoxycarbonyl)ethenyl, 2-(AR1)ethenyl, 2-(AR2)ethenyl, or 2-(AR2a)ethenyl; and wherein ( )n1, ( )o1, ( )n1′, ( )O1′, ( )p1 and ( )p1′ represent chains of carbon atoms (optionally substituted as for AR1) of length( )n1, ( )o1, ( )n1′, ( )O1′, ( )p1 and ( )p1′ respectively, and are independently 0-2, with the proviso that in (TB1) and (TB2) the sum of n1, o1, n1′ and o1′ does not exceed 8 (giving a maximum ring size of 14 in (TB1) and 11 in (TB2)), and in (TB3) the sum of n1, o1, n1′, o1′, p1 and p1′ does not exceed 6 (giving a maximum ring size of 12); wherein Rc is selected from groups (Rc1) to (Rc5): (Rc1) optionally substituted (1-6C)alkyl; (Rc2) R13CO—, R13SO2— or R13CS— wherein R13 is selected from (Rc2a) to (Rc2e): (Rc2a) AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a, CY; (Rc2b) hydrogen, (1-4C)alkoxycarbonyl, trifluoromethyl, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], ethenyl, 2-(1-4C)alkylethenyl, 2-cyanoethenyl 2-cyano-2-((1-4C)alkyl)ethenyl, 2-nitroethenyl, 2-nitro-2-((1-4C)alkyl)ethenyl, 2-((1-4C)alkylaminocarbonyl)ethenyl, 2-((1-4C)alkoxycarbonyl)ethenyl, 2-(AR1)ethenyl, 2-(AR2)ethenyl, 2-(AR2a)ethenyl; (Rc2c) optionally substituted (1-10C)alkyl; (Rc2d) R14C(O)O(1-6C)alkyl wherein R14 is AR1, AR2, (1-4C)alkylamino (the (1-4C)alkyl group being optionally substituted by (1-4C)alkoxycarbonyl or by carboxy), benzyloxy-(1-4C)alkyl or (1-10C)alkyl {optionally substituted as defined for (Rc2c)}; (Rc2e) R15O— wherein R15 is benzyl, (1-6C)alkyl {optionally substituted as defined for (Rc2c)}, CY, or AR2b; (Rc3) hydrogen, cyano, 2-cyanoethenyl, 2-cyano-2-((1-4C)alkyl)ethenyl, 2-((1-4C)alkylaminocarbonyl)ethenyl, 2-((1-4C)alkoxycarbonyl)ethenyl, 2-nitroethenyl, 2-nitro-2-((1-4C)alkyl)ethenyl, 2-(AR1)ethenyl, 2-(AR2)ethenyl, or of the formula (Rc3a) wherein X00 is —OR17, —SR17, —NHR17and —N(R17)2; wherein R17 is hydrogen (when X00 is —NHR17and —N(R17)2), and R17 is (1-4C)alkyl, phenyl or AR2 (when X00 is —OR17, —SR17 and —NHR17); and R16 is cyano, nitro, (1-4C)alkylsulfonyl, (4-7C)cycloalkylsulfonyl, phenylsulfonyl, (1-4C)alkanoyl and (1-4C)alkoxycarbonyl; (Rc4) trityl, AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b; (Rc5) RdOC(Re)═CH(C═O)—, RfC(═O)C(═O)—, RgN═C(Rh)C(═O)— or RiNHC(Rj)=CHC(═O)— wherein Rd is (1-6C)alkyl; Re is hydrogen or (1-6C)alkyl, or Rd and Re together form a (3-4C)alkylene chain; Rf is hydrogen, (1-6C)alkyl, hydroxy(1-6C)alkyl, (1-6C)alkoxy(1-6C)alkyl, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkoxy, (1-6C)alkoxy(1-6C)alkoxy, hydroxy(2-6C)alkoxy, (1-4C)alkylamino(2-6C)alkoxy, di-(1-4C)alkylamino(2-6C)alkoxy; Rg is (1-6C)alkyl, hydroxy or (1-6C)alkoxy; Rh is hydrogen or (1-6C)alkyl; Ri is hydrogen, (1-6C)alkyl, AR1, AR2, AR2a, AR2b and Rj is hydrogen or (1-6C)alkyl; wherein CY is an optionally substituted cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl or cyclohexenyl ring. 2. A compound as claimed in claim 1, or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof, wherein Q is selected from Q1, Q2, Q4, Q6 and Q9. 3. A compound as claimed in claim 1 or claim 2, or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof, wherein T is TA1. 4. A compound as claimed in claim 1 or claim 2, or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof, wherein T is TA2 or TB. 5. A compound as claimed in claim 1 or claim 2 of the formula (IA), or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof wherein HET is 1,2,3-triazole, 1,2,4-triazole or tetrazole; or HET is a di-hydro version of pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine and pyridine; R2 and R3 are independently hydrogen or fluoro; and T is selected from (TA1), (TA2) and (TB1) to (TB3), wherein (TA1), (TA2) and (TB1) to (TB3) are as hereinbefore defined. 6. A compound as claimed in claim 5, or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof, wherein HET is 1,2,3-triazole, 1,2,4-triazole or tetrazole; R2 and R3 are independently hydrogen or fluoro; and T is selected from (TA1a & b), (TA2a) and (TB1a & b); or in-vivo hydrolysable esters or pharmaceutically-acceptable salts thereof. 7. A compound as claimed in claim 6, or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof, wherein RT is selected from (RTa) or (RTb) as hereinbefore defined. 8. A compound as claimed in claim 7, or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof, wherein RT is optionally substituted methyl. 9. A compound as claimed in claim 5, or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof, wherein HET is unsubstituted. 10. A compound as claimed claim 1 or 2 or a pharmaceutically-acceptable salt or in-vivo hydrolysable ester thereof, wherein X1m is O═ and X2m is R2s—(E)ms—N—, and vice versa; and when ms is 0, R2s is selected from (i) hydrogen, a (1-6C) alkyl group {optionally monosubstituted by (1-4C)alkanoyl group, cyano, cyano-imino, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as for AR1 defined herein), optionally substituted heteroaryl group of the formula AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein, (1-4C)alkylS(O)q— (q is 0, 1 or 2); or optionally substituted by one or more fluoro groups (including geminal disubstitution); or optionally substituted by one or more hydroxy groups (excluding geminal disubstitution), and/or optionally further substituted, by no more than one of each of, oxo, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N-(1-4C)alkyl-N-(1-6C)alkanoylamino, (1-4C)alkylS(O)pNH— or (1-4C)alkylS(O)p-((1-4C)alkyl)N— (p is 1 or 2)}; or (ii) an optionally substituted aryl or optionally substituted heteroaryl group of the formula AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein; or (iii) cyano, —CO—NRvRw, —CO—NRv Rw′, —SO2—NRvRw, —SO2—NRv Rw′ [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl; Rw′ is phenyl (optionally substituted as for AR1 defined herein), or a heteroaryl group selected from AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a (optionally substituted as defined herein)], (1-4C)alkoxycarbonyl, trifluoromethyl; and when ms is 1, E is —CO— or —SO2— and R2s is selected from: (i) (1-6C)alkyl {optionally monosubstituted by cyano, cyano-imino, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as for AR1 defined herein), optionally substituted heteroaryl group of the formula AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein, (1-4C)alkylS(O)q—(q is 0, 1 or 2); and/or (with the proviso that where R2s is —SO2— or —O—CO— not on the first carbon atom of the (1-6C) alkyl chain) optionally substituted by one or more groups (including geminal disubstitution) each independently selected from hydroxy and fluoro, and/or optionally monosubstituted by —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N-(1-4C)alkyl-N-(1-6C)alkanoylamino, (1-4C)alkylS(O)pNH— or (1-4C)alkylS(O)p-((1-4C)alkyl)N— (p is 1 or 2)}; or (ii) an optionally substituted aryl or heteroaryl group of the formula AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein. 11. A compound as claimed in claim 1 or 2 or a pharmaceutically-acceptable salt or in-vivo hydrolysable ester thereof, wherein X1m is O═ and X2m is R2s—(E)ms—N—, and vice versa; and when ms is 0, R2s is selected from (i) hydrogen, (1-6C)alkyl {optionally monosubstituted by (1-4C)alkoxy, trifluoromethyl, (1-4C)alkylS(O)q— (q is 0, 1 or 2); or optionally substituted by one or more fluoro-groups (including geminal disubstitution); or optionally substituted by one or more hydroxy groups (excluding geminal disubstitution)}; or (iii) —CO—NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], —CO—NRv Rw′ [wherein Rv is hydrogen or (1-4C)alkyl; Rw′ is phenyl (optionally substituted as for AR1 defined herein)], (1-4C)alkoxycarbonyl; and when ms is 1, E is —CO— or —SO2— and R2s is selected from: (i) (1-6C)alkyl {optionally monosubstituted by (1-4C)alkoxy, trifluoromethyl, (1-4C)alkylS(O)q— (q is 0, 1 or 2); or optionally substituted by one or more fluoro groups (including geminal disubstitution); or optionally substituted by one or more hydroxy groups (excluding geminal disubstitution)}, (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino. 12. A compound as claimed in claim 1 or 2 or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof wherein R2 and R3 are independently selected from hydrogen and fluorine. 13. A compound as claimed in claim 1 or 2 or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof wherein Rc is R13CO— and R13 is (1-4C)alkoxycarbonyl, hydroxy(1-4C)alkyl, (1-4C)alkyl (optionally substituted by one or two hydroxy groups, or by an (1-4C)alkanoyl group), (1-4C)alkylamino, dimethylamino(1-14C)alkyl, (1-4C)alkoxymethyl, (1-4C)alkanoylmethyl, (1-4C)alkanoyloxy(1-4C)alkyl, (1-5C)alkoxy or 2-cyanoethyl. 14. A compound as claimed in claim 1 or 2 or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof wherein Rc is R13CO— and R13 is 1,2-dihydroxyethyl, 1,3-dihydroxyprop-2-yl, 1,2,3-trihydroxyprop-1-yl, methoxycarbonyl, hydroxymethyl, methyl, methylamino, dimethylaminomethyl, methoxymethyl, acetoxymethyl, methoxy, methylthio, naphthyl, tert-butoxy or 2-cyanoethyl. 16. A method for producing an antibacterial effect in a warm blooded animal, comprising combining a compound as claimed in claim 1 or 2, or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof with a pharmaceutically acceptable diluent or carrier. 17. A pharmaceutical composition which comprises a compound as claimed in claim 1 or 2, or a pharmaceutically-acceptable salt or an in-vivo hydrolysable ester thereof, and a pharmaceutically-acceptable diluent or carrier. 18. A method of manufacture of a compound as claimed in claim 1 and pharmaceutically-acceptable salts and in vivo hydrolysable esters thereof, according to a process (a) to (h) as follows (wherein the variables are as defined above unless otherwise stated): (a) by modifying a substituent in or introducing a substituent into another compound of formula (I); or (b) by reaction of a compound of formula (II): wherein LG is a displaceable group, with a compound of the formula (III): HET (III) wherein HET is HET-H free-base form or HET-anion formed from the free base form; or (c) by reaction of a compound of the formula (IV): T—Q—LG1 (IV) wherein LG1 is an isocyanate, amine or urethane group with an epoxide of the formula (V); or with a related compound of formula (VA) where the hydroxy group at the internal C-atom is conventionally protected and where the leaving group Y at the terminal C-atom is a conventional leaving group; or (d) by oxidation (i) with an aminating agent of a lower valent sulfur compound (VI), or an analogue thereof, which is suitable to give a T substituent as defined by (TA2), or a bi-, or tri-cyclic ring analogue of (VI) which is suitable to give a T substituent as defined by (TB); or (ii) with an oxygenating agent of a lower valent sulfur compound (VII), or an analogue thereof, which is suitable to give a T substituent as defined by (TA2), or a bi-, or tri-cyclic ring analogue of (VII) which is suitable to give a T substituent as defined by (TB); where n=0 or 1 and ( )x and ( )x′ are chains of length x and x′; or (e) (i) by coupling of a compound of formula (VIII): wherein LG2 is a group HET as hereinbefore defined and LG3 is a replaceable substituent, with a compound of the formula (IX), or an analogue thereof, which is suitable to give a T substituent as defined by (TA1), in which the link is via an sp2 carbon atom, or (TA2), or a bi-, or tri-cyclic ring analogue of (IX) which is suitable to give a T substituent as defined by (TB); where n=0 or 1 and ( )x and ( )x′ are chains of length x and x′; D is NH or CH═C—Lg4 where Lg4 is a leaving group; or (e) (ii) by coupling, of a compound of formula (X): wherein LG2 is a group HET as hereinbefore defined, with a compound [Aryl]-LG4, where LG4 is a replaceable substituent; or (f) for HET as 1,2,3-triazole by cycloaddition via the azide (wherein e.g. LG in (II) is azide); or (g) Where HET is 4-substituted 1,2,3-triazole by reaction of a compound of formula (II) where LG═NH2 (primary amine) with a compound of formula (XI), namely the arenesulfonylhydrazone of a methyl ketone that is further geminally substituted on the methyl group by two substituents (Y′ and Y″) capable of being eliminated from this initial, and the intermediate, substituted hydrazones as HY′ and HY″ (or as conjugate bases thereof); (h) by reduction of a compound formed by process (e) (i) in which the T substituent (as defined by (TA1)) is linked via an sp2 carbon atom, to form the saturated analogue; and thereafter if necessary: (i) removing any protecting groups; (ii) forming a pharmaceutically-acceptable salt; (iii) forming an in-vivo hydrolysable ester. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The international microbiological community continues to express serious concern that the evolution of antibiotic resistance could result in strains against which currently available antibacterial agents will be ineffective. In general, bacterial pathogens may be classified as either Gram-positive or Gram-negative pathogens. Antibiotic compounds with effective activity against both Gram-positive and Gram-negative pathogens are generally regarded as having a broad spectrum of activity. The compounds of the present invention are regarded as effective against both Gram-positive and certain Gram-negative pathogens. Gram-positive pathogens, for example Staphylococci, Enterococci , and Streptococci are particularly important because of the development of resistant strains which are both difficult to treat and difficult to eradicate from the hospital environment once established. Examples of such strains are methicillin resistant staphylococcus (MRSA), methicillin resistant coagulase negative staphylococci (MRCNS), penicillin resistant Streptococcus pneumoniae and multiply resistant Enterococcus faecium. The major clinically effective antibiotic for treatment of such resistant Gram-positive pathogens is vancomycin. Vancomycin is a glycopeptide and is associated with nephrotoxicity and ototoxicity. Furthermore, and most importantly, antibacterial resistance to vancomycin and other glycopeptides is also appearing. This resistance is increasing at a steady rate rendering these agents less and less effective in the treatment of Gram-positive pathogens. There is also now increasing resistance appearing towards agents such as lactams, quinolones and macrolides used for the treatment of upper respiratory tract infections, also caused by certain Gram negative strains including H.influenzae and M.catarrhalis. Certain antibacterial compounds containing an oxazolidinone ring have been described in the art (for example, Walter A. Gregory et al in J. Med. Chem. 1990, 33, 2569-2578 and Chung-Ho Park et al in J. Med. Chem. 1992, 35, 1156-1165). Such antibacterial oxazolidinone compounds with a 5-acetamidomethyl sidechain may be subject to mammalian peptidase metabolism. Furthermore, bacterial resistance to known antibacterial agents may develop, for example, by (i) the evolution of active binding sites in the bacteria rendering a previously active pharmacophore less effective or redundant, (ii) the evolution of means to chemically deactivate a given pharmacophore and/or (iii) the development and/or up-regulation of efflux mechanisms. Therefore, there remains an ongoing need to find new antibacterial agents with a favourable pharmacological profile, in particular for compounds containing new pharmacophores. We have discovered a new class of antibiotic compounds containing an aryl substituted oxazolidinone ring in which the aryl ring is itself substituted by certain novel sulfilimine and sulfoximine-containing rings. These compounds have useful activity against Gram-positive pathogens including MRSA and MRCNS and, in particular, against various strains exhibiting resistance to vancomycin and against E. faecium strains resistant to both aminoglycosides and clinically used β-lactams, but also to fastidious Gram negative strains such as H.influenzae, M.catarrbalis, mycoplasma spp. and chlamydial strains. Accordingly the present invention provides a compound of the formula (I), or a pharmaceutically-acceptable salt, or an in-vivo-hydrolysable ester thereof, wherein i) HET is an N-linked 5-membered, fully or partially unsaturated heterocyclic ring, containing either (i) 1 to 3 further nitrogen heteroatoms or (ii) a further heteroatom selected from O and S together with an optional further nitrogen heteroatom; which ring is optionally substituted on a C atom, other than a C atom adjacent to the linking N atom, by an oxo or thioxo group; and/or which ring is optionally substituted on any available C atom, other than a C atom adjacent to the linking N atom, by a substituent selected from (1-4C)alkyl, (2-4C)alkenyl, (3-6C)cycloalkyl, amino, (1-4C)alkylamino, di-(1-4C)alkylamino, (1-4C)alkylthio, (1-4C)alkoxy, (1-4C)alkoxycarbonyl, halogen, cyano and trifluoromethyl and/or on an available nitrogen atom (provided that the ring is not thereby quatermised) by (1-4C)alkyl; or HET (which may also be described as —N-HET herein) is an N-linked 6-membered di-hydro-heteroaryl ring containing up to three nitrogen heteroatoms in total (including the linking heteroatom), which ring is substituted on a suitable C atom, other than a C atom adjacent to the linking N atom, by oxo or thioxo and/or which ring is optionally substituted on any available C atom, other than a C atom adjacent to the linking N atom, by one or two substituents independently selected from (1-4C)alkyl, (2-4C)alkenyl, (3-6C)cycloalkyl, amino, (1-4C)alkylamino, di-(1-4C)alkylamino, (1-4C)alkylthio, (1-4C)alkoxy, (1-4C)alkoxycarbonyl, halogen, cyano and trifluoromethyl and/or on an available nitrogen atom (provided that the ring is not thereby quatermised) by (1-4C)alkyl; and wherein at each occurrence of alkyl, alkenyl and cycloalkyl HET substituents, each is optionally substituted with one or more F, Cl or CN; or ii) HET is selected from the structures (Za) to (Zf) below: wherein u and v are independently 0 or 1; RT is selected from a substituent from the group (RTa) wherein RT is hydrogen, halogen, (1-4C)alkoxy, (2-4C)alkenyloxy, (2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, amino, (1-4C)alkylamino, di-(1-4C)alkylamino, (2-4C)alkenylamino, (1-4C)alkylcarbonylamino, (1-4C)alkylthiocarbonylamino, (1-4C)alkyl-OCO—NH—, (1-4C)alkyl-NH—CO—NH—, (1-4C)alkyl-NH—CS—NH—, (1-4C)alkyl-SO 2 —NH— or (1-4C)alkyl-S(O)q- (wherein q is 0, 1 or 2); or RT is selected from the group (RTb) wherein RT is a (1-4C)alkyl group which is optionally substituted by one substituent selected from hydroxy, (1-4C)alkoxy, amino, cyano, azido, (2-4C)alkenyloxy, (1-4C)alkylcarbonyl, (1-4C)alkoxycarbonyl, (1-4C)alkylamino, (2-4C)alkenylamino, (1-4C)alkyl-SO 2 —NH—, (1-4C)alkylcarbonylamino, (1-4C)alkylthiocarbonylamino, (1-4C)alkyl-OCO—NH—, (1-4C)alkyl-NH—CO—NH—, (1-4C)alkyl-NH—CS—NH—, (1-4C)alkyl-SO 2 —NH—, (1-4C)alkyl-S(O)q- (wherein q is 0, 1 or 2), (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or an N-linked 5-membered heteroaryl ring, which ring contains either (i) 1 to 3 further nitrogen heteroatoms or (ii) a further heteroatom selected from O and S together with an optional further nitrogen heteroatom; which ring is optionally substituted on a carbon atom by an oxo or thioxo group; and/or the ring is optionally substituted on a carbon atom by 1 or 2 (1-4C)alkyl groups; and/or on an available nitrogen atom (provided that the ring is not thereby quatermised) by (1-4C)alkyl; or RT is selected from a group of formula (RTc1) to (RTc3): (RTc1) a fully saturated 4-membered monocyclic ring containing 1 or 2 heteroatoms independently selected from O, N and S (optionally oxidised), and linked via a ring nitrogen or carbon atom; or (RTc2) a saturated or unsaturated 5-membered monocyclic ring containing 1 heteroatom selected from O, N and S (optionally oxidised), and linked via a ring nitrogen atom if the ring is not thereby quatermised, or a ring carbon atom; or (RTc3) a saturated or unsaturated 6- to 8-membered monocyclic ring containing 1 or 2 heteroatoms independently selected from O, N and S (optionally oxidised), and linked via a ring nitrogen atom if the ring is not thereby quatermised, or a ring carbon atom; wherein said rings in (RTc1) to (RTc3) are optionally substituted on an available carbon atom by 1 or 2 substituents independently selected from hydroxy, (1-4C)alkoxy, amino, cyano, azido, (2-4C)alkenyloxy, (1-4C)alkylcarbonyl, (1-4C)alkoxycarbonyl, (1-4C)alkylamino, (2-4C)alkenylamino, (1-4C)alkyl-SO 2 —NH—, (1-4C)alkylcarbonylamino, (1-4C)alkylthiocarbonylamino, (1-4C)alkyl-OCO—NH—, (1-4C)alkyl-NH—CO—NH—, (1-4C)alkyl-NH—CS—NH—, (1-4C)alkyl-SO 2 —NH—, (1-4C)alkyl-S(O)q- (wherein q is 0, 1 or 2), (3-6C)cycloalkyl or (3-6C)cycloalkenyl; or RT is selected from the group (RTd) cyano, nitro, azido, formyl, (1-4C)alkylcarbonyl or (1-4C)alkoxycarbonyl; and wherein at each occurrence of an RT substituent containing an alkyl, alkenyl, alkynyl, cycloalkyl or cycloalkenyl moiety in (RTa), (RTb) or (RTc1) to (RTc3) each such moiety is optionally further substituted on an available carbon atom with one or more substituents independently selected from F and Cl and/or by one cyano group; Q is selected from Q1 to Q10: wherein R 2 and R 3 are independently hydrogen or fluoro; wherein A 1 is carbon or nitrogen; B 1 is O or S (or, in Q9 only, NH); X q is O, S or N—R 1 (wherein R 1 is hydrogen, (1-4C)alkyl or hydroxy-(1-4C)alkyl); and wherein in Q7 each A 1 is independently selected from carbon or nitrogen, with a maximum of 2 nitrogen heteroatoms in the 6-membered ring, and Q7 is linked to T via any of the A 1 atoms (when A 1 is carbon), and linked in the 5-membered ring via the specified carbon atom, or via A 1 when A 1 is carbon; Q8 and Q10 are linked to T via either of the specified carbon atoms in the 5-membered ring, and linked in the benzo-ring via either of the two specified carbon atoms on either side of the linking bond shown; and Q9 is linked via either of the two specified carbon atoms on either side of the linking bond shown; wherein T is selected from the groups in (TA) & (TB) below (wherein AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a and CY are defined hereinbelow); (TA) T is selected from the following groups (TA1) and (TA2): wherein: in (TA1), ( )o 1 is 0 or 1 and represents a chain of carbon atoms (optionally substituted as defined for AR1) of length o 1 and M is a bond joining the adjacent carbon atoms, or M represents one or two carbon atoms, and defines a 4- to 7-membered monocyclic ring, which ring may optionally have one of (i) one double bond between any two ring carbon atoms; or (ii) a C1-C3 bridge connecting any two appropriate, non-adjacent ring carbon atoms, which bridge may optionally contain one heteroatom selected from oxygen or >NRc; or (iii) a C2-C5 cyclic moiety including a ring carbon atom to define a spiro C2-C5 ring system, which ring may optionally contain one heteroatom selected from oxygen or >NRc; or (iv) a C1-C4 bridge connecting adjacent carbon atoms to define a fused ring, wherein a C2-C4 bridge may optionally contain one heteroatom selected from oxygen or >NRc; wherein Rc is as defined hereinafter; wherein in (TA2), ( )n 1 and ( )o 1 are independently 0, 1 or 2 and represent chains of carbon atoms (optionally substituted as defined for ARl) of length n 1 and o 1 respectively, and define a 4- to 8-membered monocyclic ring, which ring may optionally have one of (i) a C1-C3 bridge connecting any two appropriate, non-adjacent ring carbon atoms, which bridge contains one heteroatom selected from oxygen or >NRc; or (ii) a C2-C5 cyclic moiety including a ring carbon atom to define a spiro C2-C5 ring system, which ring may optionally contain one heteroatom selected from oxygen or >NRc; or (iii) a C1-C4 bridge connecting adjacent carbon atoms to define a fused ring, wherein a C2-C4 bridge may optionally contain one heteroatom selected from oxygen or >NRc; wherein Rc is as defined hereinafter; and wherein in (TA1) and (TA2), X 1m and X 2m taken together represent R 2s —(E) ms —N═; or X 1m is O═ and X 2m is R 2s —(E) ms —N—, and vice versa; wherein E is an electron withdrawing group selected from —SO 2 —, —CO—, —O—CO—, —CO—O—, —CS—, —CON(R s )—, —SO 2 N(R s )—, or E may represent a group of the formula R 3s —C(═N—O—R 3 s)—C(═O)—, wherein R 3s is H or as defined in R 2s at (i) below; or, when E is —CON(R s )— or —SO 2 N(R s )—, R 2s and R s may link together to form a carbon chain which defines a 5- or 6-membered saturated, unsaturated or partially unsaturated ring linked via the N atom in E, which ring is optionally further substituted by an oxo substituent, and which ring may be optionally fused with a phenyl group to form a benzo-fused system, wherein the phenyl group is optionally substituted by up to three substituents independently selected from halo, cyano, (1-4C)alkyl and (1-4C)alkoxy; ms is 0 or 1; R 2s and R s are independently selected from: (i) hydrogen (except where E is —SO 2 — or —O—CO—), or (1-6C)alkyl {optionally substituted by one or more (1-4C)alkanoyl groups (including geminal disubstitution) and/or optionally monosubstituted by cyano, cyano-imino, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as defined for AR1 herein), optionally substituted heteroaryl group of the formula AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein, (1-4C)alkylS(O)q- (q is 0, 1 or 2); and/or (with the proviso that where R 2s is —SO 2 or —O—CO— not on the first carbon atom of the (1-6C) alkyl chain) optionally substituted by one or more groups (including geminal disubstitution) each independently selected from hydroxy and fluoro, and/or optionally further substituted, by no more than one of each of, oxo, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N -(1-4C)alkyl- N -(1-6C)alkanoylamino, (1-4C)alkylS(O) p NH— or (1-4C)alkylS(O) p -((1-4C)alkyl)N— (p is 1 or 2)}; or (ii) an optionally substituted aryl or optionally substituted heteroaryl group of the formula AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein; or (where ms is 0 only); (iii) cyano, —CO—NRvRw, —CO—NRv Rw′, —SO 2 —NRvRw, —SO 2 —NRv Rw′ [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl; Rw′ is phenyl (optionally substituted as defined for AR1 herein), or a heteroaryl group selected from AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a (optionally substituted as defined herein)], (1-4C)alkoxycarbonyl, trifluoromethyl, ethenyl, 2-(1-4C)alkylethenyl, 2-cyanoethenyl, 2-cyano-2-((1-4C)alkyl)ethenyl, 2-nitroethenyl, 2-nitro-2-((1-4C)alkyl)ethenyl, 2-((1-4C)alkylaminocarbonyl)ethenyl, 2-((1-4C)alkoxycarbonyl)ethenyl, 2-(AR1)ethenyl, 2-(AR2)ethenyl, or 2-(AR2a)ethenyl; or (TB) T is selected from the following groups (TB1) to (TB3): wherein: X 1m and X 2m taken together represent R 2s —(E) ms —N═; or X 1m is O═ and X 2m is R 2s —(E) ms —N—, and vice versa; wherein E is an electron withdrawing group selected from —SO 2 —, —CO—, —O—CO—, —CO—O—, —CS—, —CON(R s )—, —SO 2 N(R s )—, or E may represent a group of the formula R 3s —C(═N—O—R 3s )—C(═O)—, wherein R 3s is H or as defined in R 2s at (i) below; or, when E is —CON(R s )— or —SO 2 N(R s )—, R 2s and R s may link together to form a carbon chain which defines a 5- or 6-membered saturated, unsaturated or partially unsaturated ring linked via the N atom in E, which ring is optionally further substituted by an oxo substituent, and which ring may be optionally fused with a phenyl group to form a benzo-fused system, wherein the phenyl group is optionally substituted by up to three substituents independently selected from halo, cyano, (1-4C)alkyl and (1-4C)alkoxy; ms is 0 or 1; R 2s and R s are independently selected from: (i) hydrogen (except where E is —SO 2 — or —O—CO—), or (1-6C)alkyl {optionally substituted by one or more (1-4C)alkanoyl groups (including geminal disubstitution) and/or optionally monosubstituted by cyano, cyano-imino, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as defined for AR1 hereinafter), optionally substituted heteroaryl group of the formula AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) hereinafter, (1-4C)alkylS(O) q — (q is 0, 1 or 2); and/or (with the proviso that where R 2s is —SO 2 or —O—CO— not on the first carbon atom of the (1-6C) alkyl chain) optionally substituted by one or more groups (including geminal disubstitution) each independently selected from hydroxy and fluoro, and/or optionally further substituted, by no more than one of each of, oxo, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N-(1-4C)alkyl-N-(1-6C)alkanoylamino, (1-4C)alkylS(O) p NH— or (1-4C)alkylS(O) p -((1-4C)alkyl)N— (p is 1 or 2)}; or (ii) an optionally substituted aryl or optionally substituted heteroaryl group of the formula AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) hereinafter; or (where ms is 0 only); (iii) cyano, —CO—NRvRw, —CO—NRv Rw′, —SO 2 —NRvRw, —SO 2 —NRv Rw′ [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl; Rw′ is phenyl (optionally substituted as defined for AR1 hereinafter), or a heteroaryl group selected from AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a (optionally substituted as defined hereinafter)], (1-4C)alkoxycarbonyl, trifluoromethyl, ethenyl, 2-(1-4C)alkylethenyl, 2-cyanoethenyl, 2-cyano-2-((1-4C)alkyl)ethenyl, 2-nitroethenyl, 2-nitro-2-((1-4C)alkyl)ethenyl, 2-((1-4C)alkylaminocarbonyl)ethenyl, 2-((1-4C)alkoxycarbonyl)ethenyl, 2-(AR1)ethenyl, 2-(AR2)ethenyl, or 2-(AR2a)ethenyl; and wherein ( )n 1 , ( )o 1 , ( )n 1 , ( )o 1 , ( )p 1 and ( )p 1′ , represent chains of carbon atoms (optionally substituted as defined for AR1 hereinafter) of length n 1 , o 1 , n 1′ , o 1′ , p 1 and p 1′ respectively, and are independently 0-2, with the proviso that in (TB1) and (TB2) the sum of n 1 , o 1 , n 1′ and o 1′ does not exceed 8 (giving a maximum ring size of 14 in (TB1) and 11 in (TB2)), and in (TB3) the sum of n 1 , o 1 , n 1′ , o 1′ , p 1 and p 1′ does not exceed 6 (giving a maximum ring size of 12); wherein Rc is selected from groups (Rc1) to (Rc5): (Rc1) (1-6C)alkyl {optionally substituted by one or more (1-4C)alkanoyl groups (including geminal disubstitution) and/or optionally monosubstituted by cyano, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as for AR1 defined hereinafter), (1-4C)alkylS(O) q — (q is 0, 1 or 2); or, on any but the first carbon atom of the (1-6C)alkyl chain, optionally substituted by one or more groups (including geminal disubstitution) each independently selected from hydroxy and fluoro, and/or optionally monosubstituted by oxo, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N -(1-4C)alkyl- N -(1-6C)alkanoylamino, (1-4C)alkylS(O) p NH— or (1-4C)alkylS(O) p -((1-4C)alkyl)N— (p is 1 or 2)}; (Rc2) R 13 CO—, R 13 SO 2 — or R 13 CS— wherein R 13 is selected from (Rc2a) to (Rc2e): (Rc2a) AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a, CY; (Rc2b) hydrogen, (1-4C)alkoxycarbonyl, trifluoromethyl, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], ethenyl, 2-(1-4C)alkylethenyl, 2-cyanoethenyl, 2-cyano-2-((1-4C)alkyl)ethenyl, 2-nitroethenyl, 2-nitro-2-((1-4C)alkyl)ethenyl, 2-((1-4C)alkylaminocarbonyl)ethenyl, 2-((1-4C)alkoxycarbonyl)ethenyl, 2-(AR1)ethenyl, 2-(AR2)ethenyl, 2-(AR2a)ethenyl; (Rc2c) (1-10C)alkyl {optionally substituted by one or more groups (including geminal disubstitution) each independently selected from hydroxy, (1-10C)alkoxy, (1-4C)alkoxy-(1-4C)alkoxy, (1-4C)alkoxy-(1-4C)alkoxy-(1-4C)alkoxy, (1-4C)alkanoyl, carboxy, phosphoryl [—O—P(O)(OH) 2 , and mono- and di-(1-4C)alkoxy derivatives thereof], phosphiryl [—O—P(OH) 2 and mono- and di-(1-4C)alkoxy derivatives thereof], and amino; and/or optionally substituted by one group selected from phosphonate [phosphono, —P(O)(OH) 2 , and mono- and di-(1-4C)alkoxy derivatives thereof], phosphinate [—P(OH) 2 and mono- and di-(1-4C)alkoxy derivatives thereof], cyano, halo, trifluoromethyl, (1-4C)alkoxycarbonyl, (1-4C)alkoxy-(1-4C)alkoxycarbonyl, (1-4C)alkoxy-(1-4C)alkoxy-(1-4C)alkoxycarbonyl, (1-4C)alkylamino, di((1-4C)alkyl)amino, (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N-(1-4C)alkyl-N-(1-6C)alkanoylamino, (1-4C)alkylaminocarbonyl, di((1-4C)alkyl)aminocarbonyl, (1-4C)alkylS(O) p NH—, (1-4C)alkylS(O) p -((1-4C)alkyl)N—, fluoro(1-4C)alkylS(O) p NH—, fluoro(1-4C)alkylS(O) p ((1-4C)alkyl)N—, (1-4C)alkylS(O) q — [the (1-4C)alkyl group of (1-4C)alkylS(O) q — being optionally substituted by one substituent selected from hydroxy, (1-4C)alkoxy, (1-4C)alkanoyl, phosphoryl [—O—P(O)(OH) 2 , and mono- and di-(1-4C)alkoxy derivatives thereof], phosphiryl [—O—P(OH) 2 and mono- and di-(1-4C)alkoxy derivatives thereof], amino, cyano, halo, trifluoromethyl, (1-4C)alkoxycarbonyl, (1-4C)alkoxy-(1-4C)alkoxycarbonyl, (1-4C)alkoxy-(1-4C)alkoxy-(1-4C)alkoxycarbonyl, carboxy, (1-4C)alkylamino, di((1-4C)alkyl)amino, (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N-(1-4C)alkyl-N-(1-6C)alkanoylamino, (1-4C)alkylaminocarbonyl, di((1-4C)alkyl)aminocarbonyl, (1-4C)alkylS(O) p NH—, (1-4C)alkylS(O) p -((1-4C)alkyl)N—, (1-4C)alkylS(O) q —, AR1-S(O) q —, AR2-S(O) q —, AR 3 -S(O) q — and also AR2a, AR2b, AR3a and AR3b versions of AR2 and AR3 containing groups], CY, AR1, AR2, AR3, AR1-O—, AR2-O—, AR3-O—, AR1-S(O) q —, AR2-S(O) q —, AR3-S(O) q —, AR1-NH—, AR2-NH—, AR3—NH— (p is 1 or 2 and q is 0, 1 or 2), and also AR2a, AR2b, AR3a and AR3b versions of AR2 and AR3 containing groups}; (Rc2d) R 14 C(O)O(1-6C)alkyl wherein R 14 is AR1, AR2, (1-4C)alkylamino (the (1-4C)alkyl group being optionally substituted by (1-4C)alkoxycarbonyl or by carboxy), benzyloxy-(1-4C)alkyl or (1-10C)alkyl {optionally substituted as defined for (Rc2c)}; (Rc2e) R 15 O— wherein R 15 is benzyl, (1-6C)alkyl {optionally substituted as defined for (Rc2c)}, CY, or AR2b; (Rc3) hydrogen, cyano, 2-cyanoethenyl, 2-cyano-2-((1-4C)alkyl)ethenyl, 2-((1-4C)alkylaminocarbonyl)ethenyl, 2-((1-4C)alkoxycarbonyl)ethenyl, 2-nitroethenyl, 2-nitro-2-((1-4C)alkyl)ethenyl, 2-(AR1)ethenyl, 2-(AR2)ethenyl, or of the formula (Rc3a) wherein X 00 is —OR 17 , —SR 17 , —NHR 17 and —N(R 17 ) 2 ; wherein R 17 is hydrogen (when X 00 is —NHR 17 and —N(R 17 ) 2 ), and R 17 is (1-4C)alkyl, phenyl or AR2 (when X 00 is —OR 17 , —SR 17 and —NHR 17 ); and R 16 is cyano, nitro, (1-4C)alkylsulfonyl, (4-7C)cycloalkylsulfonyl, phenylsulfonyl, (1-4C)alkanoyl and (1-4C)alkoxycarbonyl; (Rc4) trityl, AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b; (Rc5) RdOC(Re)═CH(C═O)—, RfC(═O)C(═O)—, RgN═C(Rh)C(═O)— or RiNHC(Rj)=CHC(═O)— wherein Rd is (1-6C)alkyl; Re is hydrogen or (1-6C)alkyl, or Rd and Re together form a (3-4C)alkylene chain; Rf is hydrogen, (1-6C)alkyl, hydroxy(1-6C)alkyl, (1-6C)alkoxy(1-6C)alkyl, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkoxy, (1-6C)alkoxy(1-6C)alkoxy, hydroxy(2-6C)alkoxy, (1-4C)alkylamino(2-6C)alkoxy, di-(1-4C)alkylamino(2-6C)alkoxy; Rg is (1-6C)alkyl, hydroxy or (1-6C)alkoxy; Rh is hydrogen or (1-6C)alkyl; Ri is hydrogen, (1-6C)alkyl, AR1, AR2, AR2a, AR2b and Rj is hydrogen or (1-6C)alkyl; wherein AR1 is an optionally substituted phenyl or optionally substituted naphthyl; AR2 is an optionally substituted 5- or 6-membered, fully unsaturated (i.e with the maximum degree of unsaturation) monocyclic heteroaryl ring containing up to four heteroatoms independently selected from O, N and S (but not containing any O—O, O—S or S—S bonds), and linked via a ring carbon atom, or a ring nitrogen atom if the ring is not thereby quatermised; AR2a is a partially hydrogenated version of AR2 (i.e. AR2 systems retaining some, but not the full, degree of unsaturation), linked via a ring carbon atom or linked via a ring nitrogen atom if the ring is not thereby quatermised; AR2b is a fully hydrogenated version of AR2 (i.e. AR2 systems having no unsaturation), linked via a ring carbon atom or linked via a ring nitrogen atom; AR3 is an optionally substituted 8-, 9- or 10-membered, fully unsaturated (i.e with the maximum degree of unsaturation) bicyclic heteroaryl ring containing up to four heteroatoms independently selected from O, N and S (but not containing any O—O, O—S or S—S bonds), and linked via a ring carbon atom in either of the rings comprising the bicyclic system; AR3a is a partially hydrogenated version of AR3 (i.e. AR3 systems retaining some, but not the full, degree of unsaturation), linked via a ring carbon atom, or linked via a ring nitrogen atom if the ring is not thereby quatermised, in either of the rings comprising the bicyclic system; AR3b is a fully hydrogenated version of AR3 (i.e. AR3 systems having no unsaturation), linked via a ring carbon atom, or linked via a ring nitrogen atom, in either of the rings comprising the bicyclic system; AR4 is an optionally substituted 13- or 14-membered, fully unsaturated (i.e with the maximum degree of unsaturation) tricyclic heteroaryl ring containing up to four heteroatoms independently selected from O, N and S (but not containing any O—O, O—S or S—S bonds), and linked via a ring carbon atom in any of the rings comprising the tricyclic system; AR4a is a partially hydrogenated version of AR4 (i.e. AR4 systems retaining some, but not the full, degree of unsaturation), linked via a ring carbon atom, or linked via a ring nitrogen atom if the ring is not thereby quatermised, in any of the rings comprising the tricyclic system; CY is an optionally substituted cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl or cyclohexenyl ring. For the avoidance of doubt in the definition of (TA1) & (TA2) and (TB), it is to be understood that when R 2s and R s are independently selected from (ii) (1-6C)alkyl {optionally substituted, for example, by no more than one of each of oxo and —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl]}, to avoid duplication with the substituent —CO—NRvRw provided in section (iii) of the definition for R 2s and R s , then oxo and —NRvRw are not to be both selected together when (1-6C)alkyl is methyl. For the avoidance of doubt, in the above definitions of TA1, TA2 and TB, ( )n 1 , ( )o 1 , ( )n 1 ′, ( )o 1 ′, ( )p 1 and ( )p 1 indicate (—CH 2 —)n 1 , (—CH2-)o 1 , (—CH2-)n 1 ′, (—CH2-)o 1 ′, (—CH2-)p 1 and (—CH2-)p 1 ′ respectively. In this specification, HET as an N-linked 5-membered ring, as defined in definition (i) above, may be a fully or partially unsaturated heterocyclic ring, provided there is some degree of unsaturation in the ring. Particular examples of N-linked 5-membered heteroaryl rings containing 2 to 4 heteroatoms independently selected from N, O and S (with no O—O, O—S or S—S bonds) are preferably rings containing 2 to 4 N atoms, in particular pyrazole, imidazole, 1,2,3-triazole (preferably 1,2,3-triazol-1-yl), 1,2,4-triazole (preferably 1,2,4-triazol-1-yl) and tetrazole (preferably tetrazol-2-yl). Particular examples of N-linked 6-membered di-hydro-heteroaryl rings containing up to three nitrogen heteroatoms in total (including the linking heteroatom) include di-hydro versions of pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine and pyridine. In this specification, where it is stated that a ring may be linked via an sp 2 carbon atom, which ring is fully saturated other than (where appropriate) at a linking sp 2 carbon atom, it is to be understood that the ring is linked via one of the carbon atoms in a C═C double bond. In this specification the term ‘alkyl’ includes straight chained and branched structures. For example, (1-6C)alkyl includes propyl, isopropyl and tert-butyl. However, references to individual alkyl groups such as “propyl” are specific for the straight chained version only, and references to individual branched chain alkyl groups such as “isopropyl” are specific for the branched chain version only. A similar convention applies to other radicals, for example halo(1-4C)alkyl includes 1-bromoethyl and 2-bromoethyl. In general “halogen” when present as an aromatic ring substituent is selected from any one of bromine, chlorine or fluorine, as an aliphatic substituent from chlorine or fluorine. There follow particular and suitable values for certain substituents and groups referred to in this specification. These values may be used where appropriate with any of the definitions and embodiments disclosed hereinbefore, or hereinafter. For the avoidance of doubt each stated species represents a particular and independent aspect of this invention. Examples of (1-4C)alkyl and (1-5C)alkyl include methyl, ethyl, propyl, isopropyl and t-butyl; examples of (1-6C)alkyl include methyl, ethyl, propyl, isopropyl, t-butyl, pentyl and hexyl; examples of (1-10C)alkyl include methyl, ethyl, propyl, isopropyl, pentyl, hexyl, heptyl, octyl and nonyl; examples of (1-4C)alkanoylamino-(1-4C)alkyl include formamidomethyl, acetamidomethyl and acetamidoethyl; examples of hydroxy(1-4C)alkyl and hydroxy(1-6C)alkyl include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl and 3-hydroxypropyl; examples of (1-4C)alkoxycarbonyl include methoxycarbonyl, ethoxycarbonyl and propoxycarbonyl; examples of 2-((1-4C)alkoxycarbonyl)ethenyl include 2-(methoxycarbonyl)ethenyl and 2-(ethoxycarbonyl)ethenyl; examples of 2-cyano-2-((1-4C)alkyl)ethenyl include 2-cyano-2-methylethenyl and 2-cyano-2-ethylethenyl; examples of 2-nitro-2-((1-4C)alkyl)ethenyl include 2-nitro-2-methylethenyl and 2-nitro-2-ethylethenyl; examples of 2-((1-4C)alkylaminocarbonyl)ethenyl include 2-(methylaminocarbonyl)ethenyl and 2-(ethylaminocarbonyl)ethenyl; examples of (2-4C)alkenyl include allyl and vinyl; examples of (2-4C)alkynyl include ethynyl and 2-propynyl; examples of (1-4C)alkanoyl include formyl, acetyl and propionyl; examples of (1-4C)alkoxy include methoxy, ethoxy and propoxy; examples of (1-6C)alkoxy and (1-10C)alkoxy include methoxy, ethoxy, propoxy and pentoxy; examples of (1-4C)alkylthio include methylthio and ethylthio; examples of (1-4C)alkylamino include methylamino, ethylamino and propylamino; examples of di-((1-4C)alkyl)amino include dimethylamino, N-ethyl-N-methylamino, diethylamino, N-methyl-N-propylamino and dipropylamino; examples of halo groups include fluoro, chloro and bromo; examples of (1-4C)alkylsulfonyl include methylsulfonyl and ethylsulfonyl; examples of (1-4C)alkoxy-(1-4C)alkoxy and (1-6C)alkoxy-(1-6C)alkoxy include methoxymethoxy, 2-methoxyethoxy, 2-ethoxyethoxy and 3-methoxypropoxy; examples of (1-4C)alkoxy-(1-4C)alkoxy-(1-4C)alkoxy include 2-(methoxymethoxy)ethoxy, 2-(2-methoxyethoxy)ethoxy; 3-(2-methoxyethoxy)propoxy and 2-(2-ethoxyethoxy)ethoxy; examples of (1-4C)alkylS(O) 2 amino include methylsulfonylamino and ethylsulfonylamino; examples of (1-4C)alkanoylamino and (1-6C)alkanoylamino include formamido, acetamido and propionylamino; examples of (1-4C)alkoxycarbonylamino include methoxycarbonylamino and ethoxycarbonylamino; examples of N-(1-4C)alkyl-N-(1-6C)alkanoylamino include N-methylacetamido, N-ethylacetamido and N-methylpropionamido; examples of (1-4C)alkylS(O) p NH— wherein p is 1 or 2 include methylsulfinylamino, methylsulfonylamino, ethylsulfinylamino and ethylsulfonylamino; examples of (1-4C)alkylS(O) p ((1-4C)alkyl)N— wherein p is 1 or 2 include methylsulfinylmethylamino, methylsulfonylmethylamino, 2-(ethylsulfinyl)ethylamino and 2-(ethylsulfonyl)ethylamino; examples of fluoro(1-4C)alkylS(O) p NH— wherein p is 1 or 2 include trifluoromethylsulfinylamino and trifluoromethylsulfonylamino; examples of fluoro(1-4C)alkylS(O) p ((1-4C)alkyl)NH— wherein p is 1 or 2 include trifluoromethylsulfinylmethylamino and trifluoromethylsulfonylmethylamino examples of (1-4C)alkoxy(hydroxy)phosphoryl include methoxy(hydroxy)phosphoryl and ethoxy(hydroxy)phosphoryl; examples of di-(1-4C)alkoxyphosphoryl include di-methoxyphosphoryl, di-ethoxyphosphoryl and ethoxy(methoxy)phosphoryl; examples of (1-4C)alkylS(O) q — wherein q is 0, 1 or 2 include methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, methylsulfonyl and ethylsulfonyl; examples of phenylS(O) q and naphthylS(O) q — wherein q is 0, 1 or 2 are phenylthio, phenylsulfinyl, phenylsulfonyl and naphthylthio, naphthylsulfinyl and naphthylsulfonyl respectively; examples of benzyloxy-(1-4C)alkyl include benzyloxymethyl and benzyloxyethyl; examples of a (3-4C)alkylene chain are trimethylene or tetramethylene; examples of (1-6C)alkoxy-(1-6C)alkyl include methoxymethyl, ethoxymethyl and 2-methoxyethyl; examples of hydroxy-(2-6C)alkoxy include 2-hydroxyethoxy and 3-hydroxypropoxy; examples of (1-4C)alkylamino-(2-6C)alkoxy include 2-methylaminoethoxy and 2-ethylaminoethoxy; examples of di-(1-4C)alkylamino-(2-6C)alkoxy include 2-dimethylaminoethoxy and 2-diethylaminoethoxy; examples of phenyl(1-4C)alkyl include benzyl and phenethyl; examples of (1-4C)alkylcarbamoyl include methylcarbamoyl and ethylcarbamoyl; examples of di((1-4C)alkyl)carbamoyl include di(methyl)carbamoyl and di(ethyl)carbamoyl; examples of hydroxyimino(1-4C)alkyl include hydroxyiminomethyl, 2-(hydroxyimino)ethyl and 1-(hydroxyimino)ethyl; examples of (1-4C)alkoxyimino-(1-4C)alkyl include methoxyiminomethyl, ethoxyiminomethyl, 1-(methoxyimino)ethyl and 2-(methoxyimino)ethyl; examples of halo(1-4C)alkyl include, halomethyl, 1-haloethyl, 2-haloethyl, and 3-halopropyl; examples of nitro(1-4C)alkyl include nitromethyl, 1-nitroethyl, 2-nitroethyl and 3-nitropropyl; examples of amino(1-4C)alkyl include aminomethyl, 1-aminoethyl, 2-aminoethyl and 3-aminopropyl; examples of cyano(1-4C)alkyl include cyanomethyl, 1-cyanoethyl, 2-cyanoethyl and 3-cyanopropyl; examples of (1-4C)alkanesulfonamido include methanesulfonamido and ethanesulfonamido; examples of (1-4C)alkylaminosulfonyl include methylaminosulfonyl and ethylaminosulfonyl; examples of di-(1-4C)alkylaminosulfonyl include dimethylaminosulfonyl, diethylaminosulfonyl and N-methyl-N-ethylaminosulfonyl; examples of (1-4C)alkanesulfonyloxy include methylsulfonyloxy, ethylsulfonyloxy and propylsulfonyloxy; examples of (1-4C)alkanoyloxy include acetoxy; examples of (1-4C)alkylaminocarbonyl include methylaminocarbonyl and ethylaminocarbonyl; examples of di((1-4C)alkyl)aminocarbonyl include dimethylaminocarbonyl and diethylaminocarbonyl; examples of (3-8C)cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; examples of (4-7C)cycloalkyl include cyclobutyl, cyclopentyl and cyclohexyl; examples of di(N-(1-4C)alkyl)aminomethylimino include dimethylaminomethylimino and diethylaminomethylimino. Particular values for AR2 include, for example, for those AR2 containing one heteroatom, furan, pyrrole, thiophene; for those AR2 containing one to four N atoms, pyrazole, imidazole, pyridine, pyrimidine, pyrazine, pyridazine, 1,2,3- & 1,2,4-triazole and tetrazole; for those AR2 containing one N and one O atom, oxazole, isoxazole and oxazine; for those AR2 containing one N and one S atom, thiazole and isothiazole; for those AR2 containing two N atoms and one S atom, 1,2,4- and 1,3,4-thiadiazole. Particular examples of AR2a include, for example, dihydropyrrole (especially 2,5-dihydropyrrol-4-yl) and tetrahydropyridine (especially 1,2,5,6-tetrahydropyrid-4-yl). Particular examples of AR2b include, for example, tetrahydrofuran, pyrrolidine, morpholine (preferably morpholino), thiomorpholine (preferably thiomorpholino), piperazine (preferably piperazino), imidazoline and piperidine, 1,3-dioxolan-4-yl, 1,3-dioxan-4-yl, 1,3-dioxan-5-yl and 1,4-dioxan-2-yl. Particular values for AR3 include, for example, bicyclic benzo-fused systems containing a 5- or 6-membered heteroaryl ring containing one nitrogen atom and optionally 1-3 further heteroatoms chosen from oxygen, sulfur and nitrogen. Specific examples of such ring systems include, for example, indole, benzofuran, benzothiophene, benzimidazole, benzothiazole, benzisothiazole, benzoxazole, benzisoxazole, quinoline, quinoxaline, quinazoline, phthalazine and cinnoline. Other particular examples of AR3 include 5/5-, 5/6 and 6/6 bicyclic ring systems containing heteroatoms in both of the rings. Specific examples of such ring systems include, for example, purine and naphthyridine. Further particular examples of AR3 include bicyclic heteroaryl ring systems with at least one bridgehead nitrogen and optionally a further 1-3 heteroatoms chosen from oxygen, sulfur and nitrogen. Specific examples of such ring systems include, for example, 3H-pyrrolo[1,2-a]pyrrole, pyrrolo[2, 1-b]thiazole, 1H-imidazo[1,2-a]pyrrole, 1H-imidazo[1,2-a]imidazole, 1H,3H-pyrrolo[1,2-c]oxazole, 1H-imidazo[1,5-a]pyrrole, pyrrolo[1,2-b]isoxazole, imidazo[5,1-b]thiazole, imidazo[2,1-b]thiazole, indolizine, imidazo[1,2-a]pyridine, imidazo[1,5-a]pyridine, pyrazolo[1,5-a]pyridine, pyrrolo[1,2-b]pyridazine, pyrrolo[1,2-c]pyrimidine, pyrrolo[1,2-a]pyrazine, pyrrolo[1,2-a]pyrimidine, pyrido[2,1-c]-s-triazole, s-triazole[1,5-a]pyridine, imidazo[1,2-c]pyrimidine, imidazo[1,2-a]pyrazine, imidazo[1,2-a]pyrimidine, imidazo[1,5-a]pyrazine, imidazo[1,5-a]pyrimidine, imidazo[1,2-b]-pyridazine, s-triazolo[4,3-a]pyrimidine, imidazo[5,1-b]oxazole and imidazo[2,1-b]oxazole. Other specific examples of such ring systems include, for example, [1H]-pyrrolo[2, 1-c]oxazine, [3H]-oxazolo[3,4-a]pyridine, [6H]-pyrrolo[2,1-c]oxazine and pyrido[2,1-c][1,4]oxazine. Other specific examples of 5/5-bicyclic ring systems are imidazooxazole or imidazothiazole, in particular imidazo[5,1-b]thiazole, imidazo[2,1-b]thiazole, imidazo[5,1-b]oxazole or imidazo[2,1-b]oxazole. Particular examples of AR3a and AR3b include, for example, indoline, 1,3,4,6,9,9a-hexahydropyrido[2,1c][1,4]oxazin-8-yl, 1,2,3,5,8,8a-hexahydroimidazo[1,5a]pyridin-7-yl, 1,5,8,8a-tetrahydrooxazolo[3,4a]pyridin-7-yl, 1,5,6,7,8,8a-hexahydrooxazolo[3,4a]pyridin-7-yl, (7aS)[3H,5H]-1,7a-dihydropyrrolo[1,2c]oxazol-6-yl, (7aS)[5H]-1,2,3,7a-tetrabydropyrrolo[1,2c]imidazol-6-yl, (7aR)[3H,5H]-1,7a-dihydropyrrolo[1,2c]oxazol-6-yl, [3H,5H]-pyrrolo[1,2-c]oxazol-6-yl, [5H]-2,3-dihydropyrrolo[1,2-c]imidazol-6-yl, [3H,5H]-pyrrolo[1,2-c]thiazol-6-yl, [3H,5H]-1,7a-dihydropyrrolo[1,2-c]thiazol-6-yl, [5H]-pyrrolo[1,2-c]imidazol-6-yl, [1H]-3,4,8,8a-tetrahydropyrrolo[2,1-c]oxazin-7-yl, [3H]-1,5,8,8a-tetrahydrooxazolo[3,4-a]pyrid-7-yl, [3H]-5,8-dihydroxazolo[3,4-a]pyrid-7-yl and 5,8-dihydroimidazo[1,5-a]pyrid-7-yl. Particular values for AR4 include, for example, pyrrolo[a]quinoline, 2,3-pyrroloisoquinoline, pyrrolo[a]isoquinoline,1H-pyrrolo[1,2-a]benzimidazole, 9H-imidazo[1,2-a]indole, 5H-imidazo[2,1-a]isoindole,1H-imidazo[3,4-a]indole, imidazo[1,2-a]quinoline, imidazo[2,1-a]isoquinoline, imidazo[1,5-a]quinoline and imidazo[5,1-a]isoquinoline. The nomenclature used is that found in, for example, “Heterocyclic Compounds (Systems with bridgehead nitrogen), W. L. Mosby (Intercsience Publishers Inc., New York), 1961, Parts 1 and 2. Where optional substituents are listed such substitution is preferably not geminal disubstitution unless stated otherwise. If not stated elsewhere suitable optional substituents for a particular group are those as stated for similar groups herein. Suitable substituents on AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a and CY are (on an available carbon atom) up to three substituents independently selected from (1-4C)alkyl {optionally substituted by (preferably one) substituents selected independently from hydroxy, trifluoromethyl, (1-4C)alkyl S(O) q — (q is 0, 1 or 2) (this last substituent preferably on AR1 only), (1-4C)alkoxy, (1-4C)alkoxycarbonyl, cyano, nitro, (1-4C)alkanoylamino, —CONRvRw or —NRvRw}, trifluoromethyl, hydroxy, halo, nitro, cyano, thiol, (1-4C)alkoxy, (1-4C)alkanoyloxy, dimethylaminomethyleneaminocarbonyl, di(N-(1-4C)alkyl)aminomethylimino, carboxy, (1-4C)alkoxycarbonyl, (1-4C)alkanoyl, (1-4C)alkylSO 2 amino, (2-4C)alkenyl {optionally substituted by carboxy or (1-4C)alkoxycarbonyl}, (2-4C)alkynyl, (1-4C)alkanoylamino, oxo (═O), thioxo (═S), (1-4C)alkanoylamino {the (1-4C)alkanoyl group being optionally substituted by hydroxy}, (1-4C)alkyl S(O) q — (q is 0, 1 or 2) {the (1-4C)alkyl group being optionally substituted by one or more groups independently selected from cyano, hydroxy and (1-4C)alkoxy}, —CONRvRw or —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl]. Further suitable substituents on AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a, and CY (on an available carbon atom), and also on alkyl groups (unless indicated otherwise) are up to three substituents independently selected from trifluoromethoxy, benzoylamino, benzoyl, phenyl {optionally substituted by up to three substituents independently selected from halo, (1-4C)alkoxy or cyano}, furan, pyrrole, pyrazole, imidazole, triazole, pyrimidine, pyridazine, pyridine, isoxazole, oxazole, isothiazole, thiazole, thiophene, hydroxyimino(1-4C)alkyl, (1-4C)alkoxyimino(1-4C)alkyl, halo-(1-4C)alkyl, (1-4C)alkanesulfonamido, —SO 2 NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl]. Preferable optional substituents on Ar2b as 1,3-dioxolan-4-yl, 1,3-dioxan-4-yl, 1,3-dioxan-5-yl or 1,4-dioxan-2-yl are mono- or disubstitution by substituents independently selected from (1-4C)alkyl (including geminal disubstitution), (1-4C)alkoxy, (1-4C)alkylthio, acetamido, (1-4C)alkanoyl, cyano, trifluoromethyl and phenyl]. Preferable optional substituents on CY are mono- or disubstitution by substituents independently selected from (1-4C)alkyl (including geminal disubstitution), hydroxy, (1-4C)alkoxy, (1-4C)alkylthio, acetamido, (1-4C)alkanoyl, cyano, and trifluoromethyl. Suitable substituents on AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4 and AR4a are (on an available nitrogen atom, where such substitution does not result in quaternization) (1-4C)alkyl, (1-4C)alkanoyl {wherein the (1-4C)alkyl and (1-4C)alkanoyl groups are optionally substituted by (preferably one) substituents independently selected from cyano, hydroxy, nitro, trifluoromethyl, (1-4C)alkyl S(O) q — (q is 0, 1 or 2), (1-4C)alkoxy, (1-4C)alkoxycarbonyl, (1-4C)alkanoylamino, —CONRvRw or —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl]}, (2-4C)alkenyl, (2-4C)alkynyl, (1-4C)alkoxycarbonyl or oxo (to form an N-oxide). Suitable pharmaceutically-acceptable salts include acid addition salts such as methanesulfonate, fumarate, hydrochloride, citrate, maleate, tartrate and (less preferably) hydrobromide. Also suitable are salts formed with phosphoric and sulfuric acid. In another aspect suitable salts are base salts such as an alkali metal salt for example sodium, an alkaline earth metal salt for example calcium or magnesium, an organic amine salt for example triethylamine, morpholine, N -methylpiperidine, N -ethylpiperidine, procaine, dibenzylamine, N , N -dibenzylethylamine, tris-(2-hydroxyethyl)amine, N-methyl d-glucamine and amino acids such as lysine. There may be more than one cation or anion depending on the number of charged functions and the valency of the cations or anions. A preferred pharmaceutically-acceptable salt is the sodium salt. In addition certain salts of the sulfoximine NH residue are envisaged, by way of non-limiting example sulphonic acid derivatives, methane sulfonate, hydrochloride and hydrobromide salts. However, to facilitate isolation of the salt during preparation, salts which are less soluble in the chosen solvent may be preferred whether pharmaceutically-acceptable or not. As stated before, we have discovered a range of compounds that have good activity against a broad range of Gram-positive pathogens including organisms known to be resistant to most commonly used antibiotics, together with activity against fastidious Gram negative pathogens such as H.influenzae, M.catarrhalis, Mycoplasma and Chlamydia strains. They have good physical and/or pharmacokinetic properties in general, and favourable toxicological profiles. Particularly preferred compounds of the invention comprise a compound of formula (I), or a pharmaceutically-acceptable salt or an in-vivo hydrolysable ester thereof, wherein the substituents Q, HET, T and other substituents mentioned above have values disclosed hereinbefore, or any of the following values (which may be used where appropriate with any of the definitions and embodiments disclosed hereinbefore or hereinafter): In one embodiment of the invention are provided compounds of formula (I), in an alternative embodiment are provided pharmaceutically-acceptable salts of compounds of formula (I), and in a further alternative embodiment are provided in-vivo hydrolysable esters of compounds of formula (I). In one embodiment is provided a compound of formula (I) or a pharmaceutically-acceptable salt or in-vivo hydrolysable ester thereof, as defined herein wherein Q is selected from Q1 to Q9. In another embodiment is provided a compound of formula (I) or a pharmaceutically-acceptable salt or in-vivo hydrolysable ester thereof, as defined herein wherein Q is Q10. Preferably Q is selected from Q1, Q2, Q4, Q6 and Q9; especially Q1, Q2 and Q9; more particularly Q1 and Q2; and most preferably Q is Q1. In another embodiment of the invention are provided compounds of formula (I), or a pharmaceutically-acceptable salt or in-vivo hydrolysable ester thereof, in which Q, T and other substituents mentioned above have the values disclosed hereinbefore, HET is selected from structures Za to Zf as hereinbefore defined (ie HET is as defined in definition (ii) for HET, as hereinbefore defined) and RT is selected from the group RTb. In one embodiment RT has values (RTa) to (RTc1-3). Preferable RT groups are those of (RTa) and (RTb). Even more preferable RT group is (RTb). In (RTb), in one aspect, the (1-4C)alkyl group is preferably substituted, and more preferably is a substituted methyl group. In another aspect the (1-4C) alkyl group is prefeably unsubstituted, and more preferably is a methyl group. In (RTb), when the (1-4C)alkyl group is substituted by a N-linked 5-membered heteroaryl ring it will be appreciated that the ring is aromatic and that when the ring is optionally substituted on an available carbon atom by oxo or thioxo then, when HET contains 1 to 3 further nitrogen heteroatoms, one of the further nitrogen heteroatoms is present as NH or as N-(1-4C)alkyl. Similarly, when the ring is optionally substituted on an available nitrogen atom by (1-4C)alkyl then the ring is substituted on an available carbon atom by oxo or thioxo. Preferred values for the N-linked 5-membered heteroaryl ring as a substituent in (RTb) are the following rings (HET-P1 to HET-P5): In (RTc1) to (RTc3), particular rings are morpholino, tetrahydropyridyl and dihydropyrrolyl. Preferable (RT) groups provided by optional F and/or Cl and/or one cyano further substituents in (RTa) and (RTb) are, for example, RT as trifluoromethyl, —CHF 2 , —CH 2 F, —CH 2 CN, —CF 2 NH(1-4C)alkyl, —CF 2 CH 2 OH, —CH 2 OCF 3 , —CH 2 OCHF 2 , —CH 2 OCH 2 F, —NHCF 2 CH 3 . In one embodiment is provided a compound of formula (I) or a pharmaceutically-acceptable salt or in-vivo hydrolysable ester thereof, as defined herein wherein T is selected from (TA2) and (TB). In another embodiment is provided a compound of formula (I) as defined herein wherein T is (TA1). In (TA1), when the ring has an optional double bond between any two ring carbon atoms, the ring is preferably linked via an sp 2 carbon atom of the double bond. Preferably (TA1) is (TA1a) or (TA1b), and preferably (TA2) is (TA2a): wherein X 1m and X 2m are as defined above, and hereinafter. In (TB1) to (TB3), preferably n 1 =o 1 & n 1′ =o 1′ (most preferably all are 1); p 1 =p 1′ (most preferably both are 0); and further preferred values for the groups defined in (TB) are defined by formulae (TB1a, b), (TB2a) and (TB3a): wherein X 1m and X 2m are as defined above, and hereinafter. Preferably X 1m is O═ and X 2m is R 2s —(E) ms —N—, and vice versa. When ms is 0, R 2s is preferably selected from: (i) hydrogen, a (1-6C)alkyl group {optionally monosubstituted by (1-4C)alkanoyl group, cyano, cyano-imino, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as for AR1 defined herein), optionally substituted heteroaryl group of the formula AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein, (1-4C)alkylS(O) q — (q is 0, 1 or 2); or optionally substituted by one or more fluoro groups (including geminal disubstitution); or optionally substituted by one or more hydroxy groups (excluding geminal disubstitution), and/or optionally further substituted, by no more than one of each of, oxo, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N-(1-4C)alkyl-N-(1-6C)alkanoylamino, (1-4C)alkylS(O) p NH— or (1-4C)alkylS(O) p -((1-4C)alkyl)N— (p is 1 or 2)}; or (ii) an optionally substituted aryl or optionally substituted heteroaryl group of the formula AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein; or (iii) cyano, —CO—NRvRw, —CO—NRv Rw′, —SO 2 —NRvRw, —SO 2 —NRv Rw′ [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl; Rw′ is phenyl (optionally substituted as for AR1 defined herein), or a heteroaryl group selected from AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a (optionally substituted as defined herein)], (1-4C)alkoxycarbonyl, trifluoromethyl. When ms is 0, R 2s is most preferably selected from (i) hydrogen, (1-6C)alkyl {optionally monosubstituted by (1-4C)alkoxy, trifluoromethyl, (1-4C)alkylS(O) q — (q is 0, 1 or 2); or optionally substituted by one or more fluoro-groups (including geminal disubstitution); or optionally substituted by one or more hydroxy groups (excluding geminal disubstitution)}; or (iii) —CO—NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], —CO—NRv Rw′ [wherein Rv is hydrogen or (1-4C)alkyl; Rw′ is phenyl (optionally substituted as for AR1 defined herein)], (1-4C)alkoxycarbonyl. When ms is 1, E is preferably —CO— or —SO 2 — and R 2s is preferably selected from: (i) (1-6C)alkyl {optionally monosubstituted by cyano, cyano-imino, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as for AR1 defined herein), optionally substituted heteroaryl group of the formula AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein, (1-4C)alkylS(O) q — (q is 0, 1 or 2); and/or (with the proviso that where R 2 , is —SO 2 — or —O—CO— not on the first carbon atom of the (1-6C) alkyl chain) optionally substituted by one or more groups (including geminal disubstitution) each independently selected from hydroxy and fluoro, and/or optionally monosubstituted by —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N -(1-4C)alkyl- N -(1-6C)alkanoylamino, (1-4C)alkylS(O) p NH— or (1-4C)alkylS(O) p -((1-4C)alkyl)N— (p is 1 or 2)}; or (ii) an optionally substituted aryl or heteroaryl group of the formula AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein. When ms is 1, E is preferably —CO— or —SO 2 — and R 2s is most preferably selected from: (i) (1-6C)alkyl {optionally monosubstituted by (1-4C)alkoxy, trifluoromethyl, (1-4C)alkylS(O) q — (q is 0, 1 or 2); or optionally substituted by one or more fluoro groups (including geminal disubstitution); or optionally substituted by one or more hydroxy groups (excluding geminal disubstitution)}, (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino. In (TB) and (TA2), where ( )n 1 , ( )o 1 , ( )n 1 ′, ( )o 1 ′, ( )p 1 and ( )p 1 ′ represent chains of carbon atoms optionally substituted as defined for AR1 herein, preferable optional substituents are selected from (preferably one of) hydroxy, trifluoromethyl, (1-4C)alkyl S(O) q — (q is 0, 1 or 2), (1-4C)alkoxy, (1-4C)alkoxycarbonyl, cyano, nitro, (1-4C)alkanoylamino, —CONRvRw or —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl]. Most preferably, ( )n 1 , ( )o 1 , ( )n 1 ′, ( )o 1 ′, ( )p 1 and ( )p 1 ′ represent unsubstituted chains of carbon atoms. The above preferred values of (TAa) to (TAc) and (TB) are particularly preferred when present in Q1 or Q2, especially Q1. Preferably T is selected from (TA1a & b), (TA2a) and (TB1a & b). Especially preferred is each of these values of T when present in Q1 and Q2, particularly in Q1. Preferable values for other substituents (which may be used where appropriate with any of the definitions and embodiments disclosed hereinbefore or hereinafter) are: (a) In one embodiment HET is a 6-membered heteroaryl as defined herein, and in another embodiment HET is a 5-membered heteroaryl as defined hereinbefore in definition (i) for HET. (b) When HET is a 5-membered heteroaryl as defined hereinbefore in definition (i) for HET, preferably HET is 1,2,3-triazole (especially 1,2,3-triazol-1-yl), 1,2,4-triazole (especially 1,2,4-triazol-1-yl) and tetrazole (preferably tetrazol-2-yl). (c) When HET is a 6-membered heteroaryl as defined herein, preferably HET is a di-hydro version of pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine and pyridine. (d) Preferably HET is unsubstituted. (e) In another embodiment, HET is preferably of formula (Zc), (Zd) or (Zf). (f) In one aspect preferably one of R 2 and R 3 is hydrogen and the other fluoro. In another aspect both R 2 and R 3 are fluoro. (g) Preferably Rc is R 13 CO— and preferably R 13 is (1-4C)alkoxycarbonyl, hydroxy(1-4C)alkyl, (1-4C)alkyl (optionally substituted by one or two hydroxy groups, or by an (1-4C)alkanoyl group), (1-4C)alkylamino, dimethylamino(1-4C)alkyl, (1-4C)alkoxymethyl, (1-4C)alkanoylmethyl, (1-4C)alkanoyloxy(1-4C)alkyl, (1-5C)alkoxy or 2-cyanoethyl. (h) More preferably R 13 is 1,2-dihydroxyethyl, 1,3-dihydroxyprop-2-yl, 1,2,3-trihydroxyprop-1-yl, methoxycarbonyl, hydroxymethyl, methyl, methylamino, dimethylaminomethyl, methoxymethyl, acetoxymethyl, methoxy, methylthio, naphthyl, tert-butoxy or 2-cyanoethyl. (i) Particularly preferred as R 13 is 1,2-dihydroxyethyl, 1,3-dihydroxyprop-2-yl or 1,2,3-trihydroxyprop-1-yl. (j) In another aspect preferably R 13 is hydrogen, (1-10C)alkyl [optionally substituted by one or more hydroxy] or R 14 C(O)O(1-6C)alkyl. For compounds of formula (I) preferred values for Rc are those in group (Rc2) when present in any of the definitions herein containing Rc. In the definition of (Rc2c) the AR2a, AR2b, AR3a and AR3b versions of AR2 and AR3 containing groups are preferably excluded. Especially preferred compounds of the present invention are of the formula (IA): wherein HET is 1,2,3-triazole (especially 1,2,3-triazol-1-yl), 1,2,4-triazole (especially 1,2,4-triazol-1-yl) and tetrazole (preferably tetrazol-2-yl) or HET is a di-hydro version of pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine and pyridine; R 2 and R 3 are independently hydrogen or fluoro; and T is selected from (TA1), (TA2) and (TB1) to (TB3); or in-vivo hydrolysable esters or pharmaceutically-acceptable salts thereof. Further especially preferred compounds of the present invention are of the formula (1A) defined above, wherein HET is selected from structures Za to Zf (as hereinbefore defined) and is 1,2,3-triazole (especially 1,2,3-triazol-1-yl), 1,2,4-triazole (especially 1,2,4-triazol-1-yl) and tetrazole (preferably tetrazol-2-yl); RT is selected from (RTa) or (RTb); R 2 and R 3 are independently hydrogen or fluoro; and T is selected from (TA1), (TA2) and (TB1) to (TB3); or in-vivo hydrolysable esters or pharmaceutically-acceptable salts thereof. Further particularly preferred compounds of the present invention are of the formula (1A) defined above wherein RT is a methyl group from (RTb), substituted with any of those substituents defined herein in (RTb) other than an N-linked 5-membered heteroaryl ring; or in-vivo hydrolysable esters or pharmaceutically-acceptable salts thereof. Further especially preferred compounds of the invention are of the formula (IA) wherein HET is 1,2,3-triazole (especially 1,2,3-triazol-1-yl), 1,2,4-triazole (especially 1,2,4-triazol-1-yl) or tetrazole (preferably tetrazol-2-yl; R 2 and R 3 are independently hydrogen or fluoro; T is selected from (TA1a & b), (TA2a) and (TB1a & b); or in-vivo hydrolysable esters or pharmaceutically-acceptable salts thereof. In the above aspects and preferred compounds of formula (IA), in (TA1), (TA2) and (TB1) to (TB3); and especially in (TA1a & b), (TA2a) and (TB1a & b); preferably X 1m is O═ and X 2m is R 2s —(E) ms —N—, and vice versa; and when ms is 0, R 2s is preferably selected from (i) hydrogen, a (1-6C)alkyl group {optionally monosubstituted by (1-4C)alkanoyl group, cyano, cyano-imino, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as for AR1 defined herein), optionally substituted heteroaryl group of the formula AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein, (1-4C)alkylS(O) q — (q is 0, 1 or 2); or optionally substituted by one or more fluoro groups (including geminal disubstitution); or optionally substituted by one or more hydroxy groups (excluding geminal disubstitution), and/or optionally further substituted, by no more than one of each of, oxo, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N -(1-4C)alkyl- N -(1-6C)alkanoylamino, (1-4C)alkylS(O) p NH— or (1-4C)alkylS(O) p -((1-4C)alkyl)N— (p is 1 or 2)}; or (ii) an optionally substituted aryl or optionally substituted heteroaryl group of the formula AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein; or (where ms is 0 only), (iii) cyano, —CO—NRvRw, —CO—NRv Rw′, —SO 2 —NRvRw, —SO 2 —NRv Rw′ [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl; Rw′ is phenyl (optionally substituted as for AR1 defined herein), or a heteroaryl group selected from AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a (optionally substituted as defined herein)], (1-4C)alkoxycarbonyl, trifluoromethyl; and when ms is 1, E is preferably —CO— or —SO 2 — and R 2s is preferably selected from: (i) (1-6C)alkyl {optionally monosubstituted by cyano, cyano-imino, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as for AR1 defined herein), optionally substituted heteroaryl group of the formula AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein, (1-4C)alkylS(O) q — (q is 0, 1 or 2); and/or (with the proviso that where R 2s is —SO 2 — or —O—CO— not on the first carbon atom of the (1-6C) alkyl chain) optionally substituted by one or more groups (including geminal disubstitution) each independently selected from hydroxy and fluoro, and/or optionally monosubstituted by —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N -(1-4C)alkyl- N -(1-6C)alkanoylamino, (1-4C)alkylS(O) p NH— or (1-4C)alkylS(O) p -((1-4C)alkyl)N— (p is 1 or 2)}; or (ii) an optionally substituted aryl or heteroaryl group of the formula AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a or CY all as defined (and optionally substituted as defined) herein. In a further aspect of the present invention is provided a compound of the formula (IB), or a pharmaceutically-acceptable salt, or an in-vivo-hydrolysable ester thereof, wherein: X1 and X2 taken together represent R2 F —(E)m-N═, wherein E is an electron withdrawing group selected from SO2-, CO—, O—CO—, CO—O—, CS—, CON(R F )—, SO2N(R F )—, or E may represent a group of the formula R3 F —C(═N—O—R3 F )—C(═O)—, wherein R3 F is H or as defined in R2 F (i) below; or X1 is O═ and X2 is R2 F —(E)m-N—, and vice versa; and R2 F and R F may be linked as a 5- or 6-membered unsaturated or partially unsaturated ring; m 0 or 1; R2 F and R F are independently selected from: (i) hydrogen (except where E is SO2 or O—CO—), a (1-6C)alkyl group {optionally substituted by one or more (1-4C)alkanoyl groups (including geminal disubstitution) and/or optionally monosubstituted by cyano, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as for AR defined herein after, heteroaryl(optionally substituted and defined as below),(1-4C)alkylS(O) q — (q is 0, 1 or 2); or (with the proviso that where R2 is SO2 or O—CO— not on the first carbon atom of the (1-6C) alkyl chain) optionally substituted by one or more groups (including geminal disubstitution) each independently selected from hydroxy and fluoro, and/or optionally monosubstituted by oxo, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N -(1-4C)alkyl- N -(1-6C)alkanoylamino, (1-4C)alkylS(O) p NH— or (1-4C)alkylS(O) p -((1-4C)alkyl)N— (p is 1 or 2)}; or (ii) an optionally substituted aryl or heteroaryl group of the formula AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a, or CY all as hereinbefore defined, or where m=0 only, (iii) cyano (1-4C)alkoxycarbonyl, trifluoromethyl, ethenyl, 2-(1-4C)alkylethenyl, 2-cyanoethenyl, 2-cyano-2-((1-4C)alkyl)ethenyl, 2-nitroethenyl, 2-nitro-2-((1-4C)alkyl)ethenyl, 2-((1-4C)alkylaminocarbonyl)ethenyl, 2-((1-4C)alkoxycarbonyl)ethenyl, 2-(AR1)ethenyl, 2-(AR2)ethenyl, or 2-(AR2a)ethenyl; W is a bond joining the adjacent carbon atoms or represents one or two carbon atoms (each —CH2- or —CH—), the heterocyclic ring comprising W therefore has 5-7 ring atoms and may optionally have one or more of (i) one double bond between ring carbon atoms, (ii) a C1-C3 bridge connecting two ring carbon atoms and optionally containing a heteroatom selected from oxygen or nitrogen, and (iii) a C2-C5 cyclic moiety around a ring carbon atom; (HET)AR is a 5-6 membered aromatic or heteroaromatic ring, (i) when a 5-membered ring this may be a thiophene ring, comprising a single sulphur atom sited ortho to the nitrogen atom on the adjacent oxazolidinone ring, such a ring may have a single optional substituent R1 F as hereinafter defined sited ortho to the carbon atom on the adjacent sulfilimine/sulfoximine ring, (ii) when a 6-membered ring this may be a phenyl ring or comprise a single nitrogen atom sited ortho to the nitrogen atom on the adjacent oxazolidinone ring, such ring may be optionally substituted at one or both positions ortho to the carbon atom on the adjacent sulfilimine/sulfoximine ring by R1 F , where each R1 F is independently selected from hydrogen, halogen, methyl, methoxy, ethyl and ethoxy; Y and Z taken together represent (a) an N-linked 5-membered heteroaryl ring, containing either (i) 1 to 3 further nitrogen heteroatoms or (ii) a further heteroatom selected from O and S together with an optional further nitrogen heteroatom; which ring is optionally substituted on a C atom by an oxo or thioxo group; and/or the ring is optionally substituted on a C atom by 1 or 2 (1-4C)alkyl groups; and/or on an available nitrogen atom (provided that the ring is not thereby quatermised) by (1-4C)alkyl; or (b) an N-linked 6-membered heteroaryl ring containing up to three nitrogen heteroatoms in total (including the linking heteroatom), which ring is substituted on a suitable C atom by oxo or thioxo and optionally substituted on any available C atom by 1 or 2 (1-4C)alkyl substituents; For compounds of the formula (IB) the term “a C5-C6 heteroaromatic ring” means a 5- or 6-membered aryl ring wherein (unless stated otherwise) 1, 2 or 3 of the ring atoms are selected from nitrogen, oxygen and sulfur. Unless stated otherwise, such rings are fully aromatic. Particular examples of 5- or 6-membered heteroaryl ring systems are furan, pyrrole, pyrazole, imidazole, triazole, pyrimidine, pyridazine, pyridine, isoxazole, oxazole, isothiazole, thiazole and thiophene. For compounds of the formula (IB), particular optional substituents for alkyl, phenyl (and phenyl containing moieties) and naphthyl groups and ring carbon atoms in heteroaryl (mono or bicyclic) rings (such as set out hereinbefore in groups AR1 to AR4a and CY inclusive) include halo, (1-4C)alkyl , hydroxy, nitro, carbamoyl, (1-4C)alkylcarbamoyl, di-((1-4C)alkyl)carbamoyl, cyano, trifluoromethyl, trifluoromethoxy, amino, (1-4C)alkylamino, di((1-4C)alkyl)amino, (1-4C)alkyl S(O) q — (q is 0, 1 or 2), carboxy, (1-4C)alkoxycarbonyl, (2-4C)alkenyl, (2-4C)alkynyl, (1-4C)alkanoyl, (1-4C)alkoxy, (1-4C)alkylS(O) 2 amino, (1-4C)alkanoylamino, benzoylamino, benzoyl, phenyl (optionally substituted by up to three substituents selected from halo, (1-4C)alkoxy or cyano), furan, pyrrole, pyrazole, imidazole, triazole, pyrimidine, pyridazine, pyridine, isoxazole, oxazole, isothiazole, thiazole, thiophene, hydroxyimino(1-4C)alkyl, (1-4C)alkoxyimino(1-4C)alkyl, hydroxy-(1-4C)alkyl, halo-(1-4C)alkyl, nitro(1-4C)alkyl, amino(1-4C)alkyl, cyano(1-4C)alkyl, (1-4C)alkanesulfonamido, aminosulfonyl, (1-4C)alkylaminosulfonyl and di-((1-4C)alkyl)aminosulfonyl. The phenyl and naphthyl groups and heteroaryl (mono- or bicyclic) rings may be mono- or di-substituted on ring carbon atoms with substituents independently selected from the above list of particular optional substituents, or on ring nitrogen atoms provided the ring is not thereby quatermised. For compounds of the formula (IB), particular examples of 5-membered heteroaryl rings containing 2 or 3 heteroatoms independently selected from N, O and S (with the proviso that there are no O—O, O—S or S—S bonds) are pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole; and also in an alternative embodiment, isothiazole, 1,2,5-thiadiazole, 1,2,4-thiadiazole or 1,2,3-thiadiazole. AR1, AR2, AR2a, AR2b, AR3, AR3a, AR3b, AR4, AR4a and CY are understood to be as hereinbefore defined for formula I. Particular values for X1 and X2 are as follows: (i) X1 is O═ and X2 is R2-(E)m-N—, wherein m=0 and vice versa, (ii) X1 is O═ and X2 is R2-(E)m-N—, wherein m is 1 and vice versa (iii) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —SO2— and m is 0 (iv) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —SO2— and m is 1 (v) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —CO— and m is 0 (vi) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —CO— and m is 1 (vii) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —O—CO— and m is 0 (viii) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —O—CO— and m is 1 (ix) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —CO—O— and m is 0 (x) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —COO— and m is 1 (xi) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —CS— and m is 0 (xii) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —CS— and m is 1 (xiii) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —CON(R)— and m is 0 (xiv) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —CON(R)— and m is 1 (xv) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —SO2N(R)— and m is 0 (xvi) X1 and X2 taken together represent R2-(E)m-N═, wherein E is —SO2N(R)— and m is 1 R1 is hydrogen or halogen; R2 and R are independently hydrogen (except where E is SO2 or O—CO—), a (1-6C)alkyl group {optionally substituted by one or more (1-4C)alkanoyl groups (including geminal disubstitution) and/or optionally monosubstituted by cyano, (1-4C)alkoxy, trifluoromethyl, (1-4C)alkoxycarbonyl, phenyl (optionally substituted as for AR defined hereinafter, heteroaryl(optionally substituted and defined as below),(1-4C)alkylS(O) q — (q is 0, 1 or 2); or, optionally substituted by one or more groups (including geminal disubstitution) each independently selected from hydroxy and fluoro, and/or optionally monosubstituted by oxo, —NRvRw [wherein Rv is hydrogen or (1-4C)alkyl; Rw is hydrogen or (1-4C)alkyl], (1-6C)alkanoylamino, (1-4C)alkoxycarbonylamino, N-(1-4C)alkyl-N-(1-6C)alkanoylamino, (1-4C)alkylS(O) p NH— or (1-4C)alkylS(O) p -((1-4C)alkyl)N— (p is 1 or 2)}; m is 1; Y and Z together are an N-linked triazole or tetrazole ring. More particular values are as follows: E is absent or is SO2-; R1 is halogen; R2 and R are independently hydrogen (except where E is SO2 or O—CO—), an alkyl, cycloalkyl, alkenyl or alkynyl group [especially cyclopropyl, or cyclobutyl, ethyl or methyl], all being optionally substituted by one or more of hydroxy, O-alkyl, alkanoyl (including geminal disubstitution), CN, SO2CH3, fluorine, chlorine, trifluoromethyl, COOH, COO-alkyl, CONH2, CONH-alkyl, or CON-dialkyl; and wherein any group has up to 6, such as up to 4 carbon atoms, the O-alkyl and alkanoyl groups may be further substituted by any convenient substituent such as for example trifluoromethyl; Y and Z together are 1,2,3-triazol-1-yl. In all of the above aspects and preferred compounds of formula (IA) and (IB), in-vivo hydrolysable esters are preferred where appropriate, especially phosphoryl esters (as defined by formula (PD3) with npd as 1, or of formula (PS1)). In all of the above definitions the preferred compounds are as shown in formula (IC) as described hereinafter; i.e. the pharmaceutically active enantiomer. Particularly preferred compounds of the present invention include the compounds described in the following examples. Therefore the present invention also provides a compound described in any one of the following examples, or a pharmaceutically-acceptable salt or an in-vivo hydrolysable ester thereof (and in particular compounds and salts thereof); and their use as a medicament (as herein described). The compounds of the formula (I) may be administered in the form of a pro-drug which is broken down in the human or animal body to give a compound of the formula (I). A prodrug may be used to alter or improve the physical and/or pharmacokinetic profile of the parent compound and can be formed when the parent compound contains a suitable group or substituent which can be derivatised to form a prodrug. Examples of pro-drugs include in-vivo hydrolysable esters of a compound of the formula (I) or a pharmaceutically-acceptable salt thereof. Various forms of prodrugs are known in the art, for examples see: a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol. 42, p.309-396, edited by K. Widder, et al. (Academic Press, 1985); b) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 “Design and Application of Prodrugs”, by H. Bundgaard p. 113-191 (1991); c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); and e) N. Kakeya, et al., Chem Pharm Bull, 32, 692 (1984). An in-vivo hydrolysable ester of a compound of the formula (I) or a pharmaceutically-acceptable salt thereof containing carboxy or hydroxy group is, for example, a pharmaceutically-acceptable ester which is hydrolysed in the human or animal body to produce the parent acid or alcohol. Suitable pharmaceutically-acceptable esters for carboxy include (1-6C)alkoxymethyl esters for example methoxymethyl, (1-6C)alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, (3-8C)cycloalkoxycarbonyloxy(1-6C)alkyl esters for example 1-cyclohexylcarbonyloxyethyl; 1,3-dioxolan-2-onylmethyl esters for example 5-methyl-1,3-dioxolan-2-ylmethyl; and (1-6C)alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyloxyethyl and may be formed at any carboxy group in the compounds of this invention. An in-vivo hydrolysable ester of a compound of the formula (I) or a pharmaceutically-acceptable salt thereof containing a hydroxy group or groups includes inorganic esters such as phosphate esters (including phosphoramidic cyclic esters) and α-acyloxyalkyl ethers and related compounds which as a result of the in-vivo hydrolysis of the ester breakdown to give the parent hydroxy group/s. In addition the sulphoximine residue may be derivatised by a convenient biologically labile group to give a derivative suitable for use as a solubilising pro-drug. Examples of α-acyloxyalkyl ethers include acetoxymethoxy and 2,2-dimethylpropionyloxymethoxy. A selection of in-vivo hydrolysable ester forming groups for hydroxy include (1-10C)alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, (1-10C)alkoxycarbonyl (to give alkyl carbonate esters), di-(1-4C)alkylcarbamoyl and N-(di-(1-4C)alkylaminoethyl)-N-(1-4C)alkylcarbamoyl (to give carbamates), di-(1-4C)alkylaminoacetyl and carboxyacetyl. Examples of substituents on benzoyl and phenylacetyl include chloromethyl or aminomethyl, (1-4C)alkylaminomethyl and di-((1-4C)alkyl)aminomethyl, and morpholino or piperazino linked from a ring nitrogen atom via a methylene linking group to the 3- or 4-position of the benzoyl ring. In addition a sulphoximine residue may be derivatised by a convenient biologically labile group to give a derivative suitable for use as a solubilising pro-drug. Certain suitable in-vivo hydrolysable esters of a compound of the formula (I) are described within the definitions listed in this specification, for example esters described by the definition (Rc2d), and some groups within (Rc2c). Suitable in-vivo hydrolysable esters of a compound of the formula (I) are described as follows. For example, a 1,2-diol may be cyclised to form a cyclic ester of formula (PD1) or a pyrophosphate of formula (PD2): Particularly interesting are such cyclised pro-drugs when the 1,2-diol is on a (1-4C)alkyl chain linked to a carbonyl group in a substituent of formula Rc borne by a nitrogen atom in structures (TA1) or (TA2). Esters of compounds of formula (I) wherein the HO— function/s in (PD1) and(PD2) are protected by (1-4C)alkyl, phenyl or benzyl are useful intermediates for the preparation of such pro-drugs. Further in-vivo hydrolysable esters include phosphoramidic esters, and also compounds of formula (I) in which any free hydroxy group, or sulfoxime group, independently forms a phosphoryl (npd is 1) or phosphiryl (npd is 0) ester of the formula (PD3) or (PS1), wherein npd is independently 0 or 1 for each oxo group: For the avoidance of doubt, phosphono is —P(O)(OH) 2 ; (1-4C)alkoxy(hydroxy)-phosphoryl is a mono-(1-4C)alkoxy derivative of —O—P(O)(OH) 2 ; and di-(1-4C)alkoxyphosphoryl is a di-(1-4C)alkoxy derivative of —O—P(O)(OH) 2 . Useful intermediates for the preparation of such esters include compounds containing a group/s of formula (PD3) in which either or both of the —OH groups in (PD3) is independently protected by (1-4C)alkyl (such compounds also being interesting compounds in their own right), phenyl or phenyl-(1-4C)alkyl (such phenyl groups being optionally substituted by 1 or 2 groups independently selected from (1-4C)alkyl, nitro, halo and (1-4C)alkoxy). Thus, prodrugs containing groups such as (PD1), (PD2) and (PD3) may be prepared by reaction of a compound of formula (I) containing suitable hydroxy group/s with a suitably protected phosphorylating agent (for example, containing a chloro or dialkylamino leaving group), followed by oxidation (if necessary) and deprotection. Prodrugs containing a group such as (PS1) may be obtained by analagous chemistry. When a compound of formula (I) contains a number of free hydroxy group, those groups not being converted into a prodrug functionality may be protected (for example, using a t-butyl-dimethylsilyl group), and later deprotected. Also, enzymatic methods may be used to selectively phosphorylate or dephosphorylate alcohol functionalities. Other interesting in-vivo hydrolysable esters include, for example, those in which Rc is defined by, for example, R 14 C(O)O(1-6C)alkyl-CO— (wherein R 14 is for example, benzyloxy-(1-4C)alkyl, or phenyl). Suitable substituents on a phenyl group in such esters include, for example, 4-(1-4C)piperazino-(1-4C)alkyl, piperazino-(1-4C)alkyl and morpholino-(1-4C)alkyl. Where pharmaceutically-acceptable salts of an in-vivo hydrolysable ester may be formed this is achieved by conventional techniques. Thus, for example, compounds containing a group of formula (PD1), (PD2) and/or (PD3) may ionise (partially or fully) to form salts with an appropriate number of counter-ions. Thus, by way of example, if an in-vivo hydrolysable ester prodrug of a compound of formula (I) contains two (PD3) groups, there are four HO—P— functionalities present in the overall molecule, each of which may form an appropriate salt (i.e. the overall molecule may form, for example, a mono-, di-, tri- or tetra-sodium salt). The compounds of the present invention have a chiral centre at the C-5 position of the oxazolidinone ring. The pharmaceutically active enantiomer is of the formula (IC): The present invention includes the pure enantiomer depicted above or mixtures of the 5R and 5S enantiomers, for example a racemic mixture. If a mixture of enantiomers is used, a larger amount (depending upon the ratio of the enantiomers) will be required to achieve the same effect as the same weight of the pharmaceutically active enantiomer. For example, the enantiomer depicted above is the 5(R) isomer when HET is 1,2,3- or 1,2,4-triazole or tetrazole. Furthermore, some compounds of the formula (I) may have other chiral centres, for example, certain sulfoxime compounds may be chiral at the sulfur atom. It is to be understood that the invention encompasses all such optical and diastereo-isomers, and racemic mixtures, that possess antibacterial activity. It is well known in the art how to prepare optically-active forms (for example by resolution of the racemic form by recrystallisation techniques, by chiral synthesis, by enzymatic resolution, by biotransformation or by chromatographic separation) and how to determine antibacterial activity as described hereinafter. Furthermore, some compounds of the formula (I), for example certain sulfoxime compounds may exist as cis- and trans-isomers. It is to be understood that the invention encompasses all such isomers, and mixtures thereof, that possess antibacterial activity. The invention relates to all tautomeric forms of the compounds of the formula (I) that possess antibacterial activity. It is also to be understood that certain compounds of the formula (I) can exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms which possess antibacterial activity. It is also to be understood that certain compounds of the formula (I) may exhibit polymorphism, and that the invention encompasses all such forms which possess antibacterial activity. Process Section: In a further aspect the present invention provides a process for preparing a compound of formula (I) or a pharmaceutically-acceptable salt or an in-vivo hydrolysable ester thereof. It will be appreciated that during certain of the following processes certain substituents may require protection to prevent their undesired reaction. The skilled chemist will appreciate when such protection is required, and how such protecting groups may be put in place, and later removed. For examples of protecting groups see one of the many general texts on the subject, for example, ‘Protective Groups in Organic Synthesis’ by Theodora Green (publisher: John Wiley & Sons). Protecting groups may be removed by any convenient method as described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with minimum disturbance of groups elsewhere in the molecule. Thus, if reactants include, for example, groups such as amino, carboxy or hydroxy it may be desirable to protect the group in some of the reactions mentioned herein. A suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or t-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an acyl group such as a t-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulphuric or phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine. A suitable protecting group for a hydroxy group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an aroyl group, for example benzoyl, or an arylmethyl group, for example benzyl. The deprotection conditions for the above protecting groups will necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or an aroyl group may be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an arylmethyl group such as a benzyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon. A suitable protecting group for a carboxy group is, for example, an esterifying group, for example a methyl or an ethyl group which may be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a t-butyl group which may be removed, for example, by treatment with an acid, for example an organic acid such as trifluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon. Resins may also be used as a protecting group. The protecting groups may be removed at any convenient stage in the synthesis using conventional techniques well known in the chemical art. A compound of the formula (I), or a pharmaceutically-acceptable salt or an in vivo hydrolysable ester thereof, may be prepared by any process known to be applicable to the preparation of chemically-related compounds. Such processes, when used to prepare a compound of the formula (I), or a pharmaceutically-acceptable salt or an in vivo hydrolysable ester thereof, are provided as a further feature of the invention and are illustrated by the following representative examples. Necessary starting materials may be obtained by standard procedures of organic chemistry (see, for example, Advanced Organic Chemistry (Wiley-Interscience), Jerry March). The preparation of such starting materials is described within the accompanying non-limiting Examples (in which, for example, 3,5-difluorophenyl, 3-fluorophenyl and (des-fluoro)phenyl containing intermediates may all be prepared by analogous procedures. Alternatively, necessary starting materials are obtainable by analogous procedures to those illustrated which are within the ordinary skill of an organic chemist. Information on the preparation of necessary starting materials or related compounds (which may be adapted to form necessary starting materials) may also be found in the following Patent and Application Publications, the contents of the relevant process sections of which are hereby incorporated herein by reference: WO99/02525; WO98/54161; WO97/37980; WO97/30981 (& U.S. Pat. No. 5,736,545); WO97/21708 (& U.S. Pat. No. 5,719,154); WO97/10223; WO97/09328; WO96/35691; WO96/23788; WO96/15130; WO96/13502; WO95/25106 (& U.S. Pat. No. 5,668,286); WO95/14684 (& U.S. Pat. No. 5,652,238); WO95/07271 (& U.S. Pat. No. 5,688,792); WO94/13649; WO94/01 110; WO93/23384 (& U.S. Pat. Nos. 5,547,950 & 5,700,799); WO93/09103 (& U.S. Pat. Nos. 5,565,571, 5,654,428, 5,654,435, 5,756,732 & 5,801,246); 5,231,188; 5,247,090; 5,523,403; WO97/27188; WO97/30995; WO97/31917; WO98/01447; WO98/01446; WO99/10342; WO99/10343; WO99/11642; WO99/64416; WO99/64417 and GB99/03299; European Patent Application Nos. 0,359,418 and 0,609,905; 0,693,491 A1 (& U.S. Pat. No. 5,698,574); 0,694,543 A1 (& AU 24985/95); 0,694,544 A1 (& CA 2,154,024); 0,697,412 A1 (& U.S. Pat. No. 5,529,998); 0,738,726 A1 (& AU 50735/96); 0,785,201 A1 (& AU 10123/97); German Patent Application Nos. DE 195 14 313 A1 (& U.S. Pat. No. 5,529,998); DE 196 01 264 A1 (& AU 10098/97); DE 196 01 265 A1 (& AU 10097/97); DE 196 04 223 A1 (& AU 12516/97); DE 196 49 095 A1 (& AU 12517/97). The following Patent and Application Publications may also provide useful information and the contents of the relevant process sections are hereby incorporated herein by reference: FR 2458547; FR 2500450(& GB 2094299, GB 2141716 & U.S. Pat. No. 4,476,136); DE 2923295 (& GB 2028306, GB 2054575, U.S. Pat. Nos. 4,287,351, 4,348,393, 4,413,001, 4,435,415 & 4,526,786), DE 3017499 (& GB 2053196, U.S. Pat. Nos. 4,346,102 & 4,372,967); 4,705,799; European Patent Application Nos. 0,312,000; 0,127,902; 0,184,170; 0,352,781; 0,316,594. Information on the preparation of necessary starting materials or related compounds (which may be adapted to form necessary starting materials) may also be found in WO 01/46185. The skilled organic chemist will be able to use and adapt the information contained and referenced within the above references to obtain necessary starting materials. In particular we refer to our PCT patent applications WO-99/64417 and WO-00/21960 wherein detailed guidance is given on convenient methods for preparing oxazolidinone compounds. The present invention also provides that compounds of the formulae (I) and pharmaceutically-acceptable salts and in vivo hydrolysable esters thereof, can be prepared by a process (a) to (h) as follows (wherein a variable sulfoximine/sulfimine substituent is designated by R and the other variables are as defined above unless otherwise stated) (a) (i) by modifying a substituent in, or introducing a new substituent into, the substituent group RT of HET of another compound of formula (I)—for instance by (i) displacement of a functional group from a compound of formula (I) by another functional group, (ii) by oxidation or (iii) reduction of a compound of formula (I), by (iv) addition of a reagent to or (v) elimination of a reagent from a compound of formula (I), by (vi) metathesis of a compound of formula (I) into a modified compound of formula (I), or by (vii) rearrangement of a compound of formula (I) to an isomeric compound of formula (I); or (a) (ii) by modifying a substituent in, or introducing a new substituent into, the group Q of another compound of formula (I)—for instance by (i) displacement of a functional group from a compound of formula (I) by another functional group, (ii) by oxidation or (iii) reduction of a compound of formula (I), by (iv) addition of a reagent to or (v) elimination of a reagent from a compound of formula (I), by (vi) metathesis of a compound of formula (I) into a modified compound of formula (I), or by (vii) rearrangement of a compound of formula (I) to an isomeric compound of formula (I) (Scheme 1 shows examples drawn from the range of suitable methods); or (b) by reaction of a compound of formula (II): wherein LG is a displaceable group (which may be (i) generated in-situ, for example under Mitsunobu conditions, or (ii) preformed, such as chloro or mesylate) with a compound of the formula (III): in-line-formulae description="In-line Formulae" end="lead"? HET (III) in-line-formulae description="In-line Formulae" end="tail"? wherein HET is HET-H free-base form or HET-anion formed from the free base form (Scheme 2 shows examples drawn from the range of suitable methods); or (c) by reaction of a compound of the formula (IV): in-line-formulae description="In-line Formulae" end="lead"? T—Q—LG1 (IV) in-line-formulae description="In-line Formulae" end="tail"? wherein LG1 is an isocyanate, amine or urethane group with an epoxide of the formula (V) wherein Z is an isocyanate, amine or urethane group with an epoxide of the formula (V) wherein the epoxide group serves as a leaving group at the terminal C-atom and as a protected hydroxy group at the internal C-atom; or with a related compound of formula (VA) where the hydroxy group at the internal C-atom is conventionally protected e.g. with an acetyl group and where the leaving group Y at the terminal C-atom is a conventional leaving group e.g. a chloro- or mesyloxy-group (Scheme 3 shows examples drawn from a range of suitable methods); or (d) by oxidation (i) with an aminating agent of a lower valent sulfur compound (VI), or an analogue thereof, which is suitable to give a T substituent as defined by (TA2), or a bi-, or tri-cyclic ring analogue of (VI) which is suitable to give a T substituent as defined by (TB); or (ii) with an oxygenating agent of a lower valent sulfur compound (VII), or an analogue thereof, which is suitable to give a T substituent as defined by (TA2), or a bi-, or tri-cyclic ring analogue of (VII) which is suitable to give a T substituent as defined by (TB); where n=0 or 1 and ( )x and ( )x′ are chains of length x and x′. Suitable aminating agents include mesitylenesulfonyl hydroxylamine, sodium azide and polyphosphoric acid, and chloramine-T; suitable oxygenating agents include peracids and osmium tetroxide—amine N-oxide mixtures (Scheme 4 shows examples drawn from a range of suitable methods); or (e) (i) by coupling, using catalysis by transition metals such as palladium(0), of a compound of formula (VIII): wherein LG2 is a group HET as hereinbefore defined, LG3 is a replaceable substituent—such as chloride, bromide, iodide, or trifluoromethylsulfonyloxy, with a compound of the formula (IX), or an analogue thereof, which is suitable to give a T substituent as defined by (TA1), in which the link is via an sp 2 carbon atom, or (TA2), or a bi- or tri-cyclic ring analogue of (IX) which is suitable to give a T substituent as defined by (TB); where n=0 or 1 and ( )x and ( )x′ are chains of length x and x′; D is NH or CH═C—LG4 where LG4 is a replaceable substituent such as chloride, bromide, iodide, or trifluoromethylsulfonyloxy, or (for instance under conditions of the Heck reaction) also hydrogen (Scheme 5 shows examples drawn from the range of suitable methods); (e) (ii) by coupling, using catalysis by transition metals such as palladium(0), of a compound of formula (X): wherein LG2 is a group HET as hereinbefore defined, with a compound [Aryl]-LG4, where LG4 is a replaceable substituent such as chloride, bromide, iodide, or trifluoromethylsulfonyloxy, or an analogue thereof (Scheme 5 shows an example drawn from the range of suitable methods); or (f) Where HET is 1,2,3-triazole there is the additional possibility by cycloaddition via the azide (wherein LG in (II) is azide), with a substituted acetylene or a masked acetylene (such as a vinyl sulfone, a nitroloefin, or an enamine, or a substituted cyclohexa-1,4-diene derivative (Scheme 2 shows examples drawn from the range of suitable methods) (g) Where HET is 4-substituted 1,2,3-triazole there is the additional possibility of synthesis by reaction of a compound of formula (II) where LG═NH 2 (primary amine) with a compound of formula (XI), namely the arenesulfonylhydrazone of a methyl ketone that is further geminally substituted on the methyl group by two substituents (Y′ and Y″) capable of being eliminated from this initial, and the intermediate, substituted hydrazones as HY′ and HY″ (or as conjugate bases thereof) (Scheme 6 shows an example drawn from the range of suitable methods); (h) by reduction of the carbon-carbon double bond of an unsaturated compound formed for instance by process (e) (i) in which the T substituent (as defined by (TA1)) is linked via an sp 2 carbon atom, to form the saturated analogue (Scheme 7 shows examples drawn from a range of suitable methods); (i) and thereafter if necessary: (i) removing any protecting groups; (ii) forming a pharmaceutically-acceptable salt; (iii) forming an in-vivo hydrolysable ester. (a) Methods for converting substituents into other substituents are known in the art. For example an alkylthio group may be oxidised to an alkylsulfinyl or alkysulfonyl group, a cyano group reduced to an amino group, a nitro group reduced to an amino group, a hydroxy group alkylated to a methoxy group, a hydroxy group thiomethylated to an arylthiomethyl or a heteroarylthiomethyl group (see, for example, Tet.Lett., 585, 1972), a carbonyl group converted to a thiocarbonyl group (eg. using Lawsson's reagent) or a bromo group converted to an alkylthio group. (b)(i) Reaction (b)(i) (in which LG is initially hydroxy) is performed under Mitsunobu conditions, for example, in the presence of tri-n-butylphosphine and diethyl azodicarboxylate (DEAD) in an organic solvent such as THF, and in the temperature range 0° C.-60° C., but preferably at ambient temperature. Details of Mitsunobu reactions are contained in Tet. Letts., 31, 699, (1990); The Mitsunobu Reaction, D. L. Hughes, Organic Reactions, 1992, Vol.42, 335-656 and Progress in the Mitsunobu Reaction, D. L. Hughes, Organic Preparations and Procedures International, 1996, Vol.28, 127-164. The general method is illustrated in Scheme 2. (b)(ii) Reactions (b)(ii) are performed conveniently in the presence of a suitable base such as, for example, an alkali or alkaline earth metal carbonate, alkoxide or hydroxide, for example sodium carbonate or potassium carbonate, or, for example, an organic amine base such as, for example, pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, morpholine or diazabicyclo-[5.4.0]undec-7-ene, the reaction is also preferably carried out in a suitable inert solvent or diluent, for example methylene chloride, acetonitrile, tetrahydrofuran, 1,2-dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidin-2-one or dimethylsulfoxide at and at a temperature in the range 25-60° C. When LG is chloro, the compound of the formula (II) may be formed by reacting a compound of the formula (II) wherein LG is hydroxy (hydroxy compound) with a chlorinating agent. For example, by reacting the hydroxy compound with thionyl chloride, in a temperature range of ambient temperature to reflux, optionally in a chlorinated solvent such as dichloromethane or by reacting the hydroxy compound with carbon tetrachloride/triphenyl phosphine in dichloromethane, in a temperature range of 0° C. to ambient temperature. A compound of the formula (II) wherein LG is chloro or iodo may also be prepared from a compound of the formula (II) wherein LG is mesylate or tosylate, by reacting the latter compound with lithium chloride or lithium iodide and crown ether, in a suitable organic solvent such as THF, in a temperature range of ambient temperature to reflux When LG is (1-4C)alkanesulfonyloxy or tosylate the compound (II) may be prepared by reacting the hydroxy compound with (1-4C)alkanesulfonyl chloride or tosyl chloride in the presence of a mild base such as triethylamine or pyridine. When LG is a phosphoryl ester (such as PhO 2 —P(O)—O—) or Ph 2 —P(O)—O— the compound (II) may be prepared from the hydroxy compound under standard conditions. If not commercially available, compounds of the formula (III) may be prepared by procedures which are selected from standard chemical techniques, techniques which are analogous to the synthesis of known, structurally similar compounds, or techniques which are analogous to the procedures described in the Examples. For example, standard chemical techniques are as described in Houben Weyl. The general method is illustrated in Scheme 2. (c) by reaction of T—Q—LG1 (IV) wherein LG1 is an amine, urethane, or isocyanate with an N-epoxypropyl hetercycle (V). Epoxides of the formula (V) may be prepared from the corresponding N-allylheterocycle of formula (XII): Certain such epoxide and alkene intermediates are novel and are provided as a further feature of the invention. Asymmetric epoxidation may be used to give the desired optical isomer. Compounds of formula (VA) may be obtained from epoxides of formula (V); alternatively compounds of formula (VA) may be used as precursors for epoxides of formula (V) according to the relative ease of synthesis in each case. The skilled chemist will appreciate that the epoxides of formula (V) and the compounds of formula (VA) are structurally equivalent and the choice between them will be made on the grounds of availability, convenience, and cost. Furthermore, a similar reaction to reaction (c) may be performed in which Q—LG1 wherein LG1 is an amine group is reacted with the epoxide (V) (optionally in the presence of an organic base), and the product is reacted with, for example, phosgene to form the oxazolidinone ring. Alternatively, a precursor of the group HET may be incorporated in place of the group HET in the epoxide of formula (V). Such reactions and the preparation of starting materials in within the skill of the ordinary chemist with reference to the above-cited documents disclosing analogous reactions and preparations. Compounds of the formula (II) wherein LG is hydroxy may be obtained as described in the references cited herein, for example, by reacting a compound T—Q—LG1 (IV) where LG1 is an amine, an isocyanate, or a urethane, especially a compound of the formula (IV, LG1=NHCO 2 R 21 ) with a compound of formula (XIII): wherein R 21 is (1-6C)alkyl or benzyl and R 22 is (1-4C)alkyl or —S(O) n (1-4C)alkyl where n is 0, 1 or 2. Preferably R 22 is (1-4C)alkyl. Compounds of the formula (II), (IV), and (XIII) may be prepared by the skilled man, for example as described in International Patent Application Publication Nos. cited herein, the contents of which are hereby incorporated by reference, and by analogous processes. Compounds of the formula T—Q—LG1 wherein LG1 is a urethane may be prepared by the skilled chemist, for example by analogous processes to those described in International Patent Application Publication Nos. WO 97/30995 and WO 97/37980. Compounds of the formula Q—LG1 wherein LG1 is an isocyanate may be prepared by the skilled chemist, for example by analogous processes to those described in Walter A. Gregory et al in J. Med. Chem. 1990, 33, 2569-2578 and Chung-Ho Park et al in J. Med. Chem. 1992, 35, 1156-1165. The general method is illustrated in Scheme 3. Compounds of the formula T—Q—LG1 wherein LG1 is an amine may be prepared by arylating an amine of formula (XIV), ( )x and ( )x′ are chains of length x and x′, which is suitable to give a T substituent as defined by (TA2), or a bi-, or tricyclic ring analogue of (XII) which is suitable to give a T substituent as defined by (TB); with a nitroarylhalide, such as 3,4-difluoronitrobenzene, and reducing the nitro-compound so produced to the corresponding amine. The thioether may be oxidized to a sulfimine or sulfoximine at any convenient stage of the synthesis. Examples of the way that such reactions can be employed in the overall synthesis in different orders according to convenience are shown in Scheme 3A. Suitable amine thioethers of the type shown in formula (XIV) may be synthesized by combination of the methods well-known in the art for the separate synthesis of cyclic amines and cyclic thioethers. Cyclic thioethers are readily available by reaction of sulfide anion with bifunctional alkylating agents, such as dibromides or bis-mesylates derived from diols. Certain cyclic thioethers are also available by cycloadditions, such as 1,3-dipolarcycloadditions of thiocarbonyl-ylids to olefins to give tetrahydrothiophenes and 1,4-cycloaddition of thiocarbonyl compounds to 1,3-dienes to give dihydrothiopyrans. Cyclic amines are available by similar reactions of analogous nitrogen compounds. In addition, cyclic amines are available by reduction of a wide range of imides and lactams. It will be apparent to the skilled chemist that the similar functional groups used to prepare the cyclic thioether and cyclic amine functionality may need to be selectively protected by methods known in the art. (d) Convenient methods for aminating thioethers or sulfoxides are indicated in Michael Reggelin and Cornelia Zur in Synthesis, 2000, 1, 1-64. Further references include Reggelin et al, Tetrahedron Letters, 1992, 33 (46), 6959-6962; Reggelin et al, Tetrahedron Letters, 1992, 36 (33), 5885-5886; and Gage et al, Tetrahedron Letters, 2000, 41, 4301-4305. For substrates containing nucleophilic nitrogen atoms such as tertiary arylamines it is advantageous to use an acidic reaction mixture such as sodium azide in polyphosphoric acid to reduce the amount of amination on nitrogen. Sufoximines may be made either by oxidizing thioethers first to the corresponding sufoxides and then to the sulfoximines or by oxidizing thioethers first to the corresponding sulfilimines (sulfimines) and then to the sulfoximine. The general method for aminating thioethers or sulfoxides and for oxidizing sulfimines is illustrated in Scheme 4. Convenient methods for the preparation of functionalised sulfilimines and sulfoximines include those in which a sulfilimine or sulfoximine is (i) alkylated, for instance by reductive amination using aldehydes, (ii) acylated for instance using acid chlorides in pyridine, or (iii) arylated, for instance by palladium coupling with (hetero)aryl halides or by cyclisation and heteroaromatisation of an acyclic substituent on the sulfoximine N. The general method for refunctionalizing sulfimines or sulfoximines in the final step is also illustrated in Scheme 4. (e) (i) The transition metal catalysed coupling reaction to form a C—C or N—C bond from the corresponding aryl derivatives and the cyclic sulfoximines and sulfimines is performed under conventional conditions (see for instance J. K. Stille, Angew. Chem. Int. Ed. Eng., 1986, 25, 509-524; N. Miyaura and A. Suzuki, Chem. Rev., 1995, 95, 22457-2483; D. Baranano, G. Mann, and J. F. Hartwig, Current Org. Che., 1997, 1, 287-305; S. P. Stanforth, Tetrahedron, 1998, 54, 263-303). The cyclic sulfoxides and sulfimines used in reaction (e) (i) may be obtained by oxidation of the corresponding cyclic aminothioethers described for (c) according to the methods analogous to those of reaction (d). The general method is illustrated in Scheme 5. (e) (ii) The reaction e (ii) may be conveniently carried out under the conditions described Tetrahedron Letters (2001), 42(22), 3681-3684, or in the analogous conventional conditions described in the above mentioned literature. In such a procedure a preferred variation of LG4 may be bromine. (f) The cycloaddition-cycloreversion reaction to form 1,2,3 triazoles from the corresponding azide is performed under conventional Diels-Alder reaction conditions. The method is illustrated in Scheme 2. Compounds of the formula (II) wherein LG is azide may be obtained as described in the references cited herein (particularly in the section proceeding the discussion of protecting groups), for example from the corresponding compounds in which LG is hydroxy or mesylate. (g) The reaction of amines of formula (II, LG═NH2) with arenesulfonyl hydrazones to form 1,2,3 triazoles may be carried out as described in the literature (Sakai, Kunikazu; Hida, Nobuko; Kondo, Kiyosi. Reactions of α-polyhalo ketone tosylhydrazones with sulfide ion and primary amines. Cyclization to 1,2,3-thiadiazoles and 1,2,3-triazoles. Bull. Chem. Soc. Jpn. (1986), 59(1), 179-83; Sakai, Kunikazu; Tsunemoto, Daiei; Kobori, Takeo; Kondo, Kiyoshi; Hida, Nobuko. 1,2,3-Trihetero 5-membered heterocyclic compounds, EP 103840 A2 19840328). The leaving groups Y, Y′ may be chloro or any other group capable of being eliminated from the arenesulfonyl hydrazone during the reaction with the amine. The skilled chemist will also appreciate that a similar reaction may be used to produce other substituted triazoles suitable for incorporation into related processes such as reaction with compounds of formula (IV) in process (c). (h) The reduction of a compound formed by process (e) in which the T substituent (as defined by (TA1)) is linked via an sp 2 carbon atom, to form the saturated analogue, may be performed using methods from the standard range of hydrogenations. For example, a dihydrothiopyran may be reduced to produce the tetrahydrothiopyran analogue. The following Schemes illustrate process chemistry which allows preparation of compounds of the formula (I); wherein A and R are values suitable to provide the compounds of formula (I) defined herein. The Schemes may be genericised by the skilled man to apply to compounds within the present specification which are not specifically illustrated in the Schemes (for example to HET as a 6-membered ring as defined herein). The removal of any protecting groups, the formation of a pharmaceutically-acceptable salt and/or the formation of an in vivo hydrolysable ester are within the skill of an ordinary organic chemist using standard techniques. Furthermore, details on the these steps, for example the preparation of in-vivo hydrolysable ester prodrugs has been provided in the section above on such esters, and in certain of the following non-limiting Examples. Certain novel intermediates utilised in the above processes are provided as a further feature of the invention. Convenient methods for the preparation of compounds of the formula (IB) include those in which as a last step; (i) a sulfoxide is converted into a sulfoximine; (ii) a sulfilimine is oxidised to the corresponding sulfoximine (iii) an appropriate compound heterocycle —Y—Z is coupled to an appropriate corresponding oxazolidinone intermediate. (iv) a preformed sulfilimine or sulfoximine ring-containing intermediate is coupled to an aryloxazolidinone. Such methods are shown by way of non-limiting illustration below wherein LG6 represents a convenient leaving group: Convenient methods for functionalised sulfilimines and sulfoximines include those in which a sulfilimine or sulfoximine is (i) alkylated, (ii) acylated or (iii) arylated. A detailed review of sulfoximine chemistry is provided by Michael Reggelin and Cornelia Zur in Synthesis, 2000, 1, 1-64. Further references include Reggelin et al, Tetrahedron Letters, 1992, 33 (46), 6959-6962; Reggelin et al, Tetrahedron Letters, 1992, 36 (33), 5885-5886; and Gage et al, Tetrahedron Letters, 2000, 41, 4301-4305. General guidance on reaction conditions and reagents may be obtained in Advanced Organic Chemistry, 4 th Edition, Jerry March (publisher: J. Wiley & Sons), 1992. Necessary starting materials may be obtained by standard procedures of organic chemistry, such as described in this process section, in the Examples section or by analogous procedures within the ordinary skill of an organic chemist. Certain references are also provided (see above) which describe the preparation of certain suitable starting materials, for particular example see International Patent Application Publication No. WO 97/37980, the contents of which are incorporated here by reference. Processes analogous to those described in the references may also be used by the ordinary organic chemist to obtain necessary starting materials. Methods for converting substituents into other substituents are known in the art. For example an alkylthio group may be oxidised to an alkylsulfinyl or alkylsulfonyl group, a cyano group reduced to an amino group, a nitro group reduced to an amino group, a hydroxy group alkylated to a methoxy group, a hydroxy group converted to an arylthiomethyl or a heteroarylthiomethyl group (see, for example, Tet.Lett., 585, 1972), a carbonyl group converted to a thiocarbonyl group (eg. using Lawsson's reagent) or a bromo group converted to an alkylthio group. It is also possible to convert one R2 F group into another R2 F group as a final step in the preparation of a compound of the formula (IB). One compound of formula (IB) may be converted into another compound of formula (IB) by reacting a compound of formula (IB) in which a substituent is halo with a suitable compound to form another compound. Thus, for example, halo may be displaced by suitable vinyl, aromatic, tropolone and nitrogen-linked systems by reaction using known Pd(0) coupling techniques. Further examples of converting substituents into other substituents are contained in the accompanying non-limiting Examples. Certain compounds may be prepared by the skilled chemist, for example as described in International Patent Application Publication Nos. WO95/07271, WO97/27188, WO 97/30995, WO 98/01446 and WO 98/01447, the contents of which are hereby incorporated by reference, and by analogous processes. If not commercially available, compounds may be prepared by procedures which are selected from standard chemical techniques, techniques which are analogous to the synthesis of known, structurally similar compounds, or techniques which are analogous to the procedures described in the Examples. For example, standard chemical techniques are as described in Houben Weyl, Methoden der Organische Chemie, E8a, Pt.I (1993), 45-225, B. J. Wakefield (for isoxazoles) and E8c, Pt.I (1994), 409-525, U. Kraatz (for 1,2,4-oxadiazoles). Also, for example, 3-hydroxyisoxazole may be prepared by cyclisation of CH≡C—CO—NHOH (prepared from CH≡C—CO—O-(1-4C)alkyl) as described in Chem.Pharm.Bull.Japan, 14, 92, (1966). The removal of any protecting groups, the formation of a pharmaceutically-acceptable salt and/or the formation of an in vivo hydrolysable ester are within the skill of an ordinary organic chemist using standard techniques. Furthermore, details on the these steps, for example the preparation of in-vivo hydrolysable ester prodrugs has been provided in the section above on such esters, and in certain of the following non-limiting Examples. When an optically active form of a compound of the formula (I) is required, it may be obtained by carrying out one of the above procedures using an optically active starting material (formed, for example, by asymmetric induction of a suitable reaction step), or by resolution of a racemic form of the compound or intermediate using a standard procedure, or by chromatographic separation of diastereoisomers (when produced). Enzymatic techniques may also be useful for the preparation of optically active compounds and/or intermediates. Similarly, when a pure regioisomer of a compound of the formula (I) is required, it may be obtained by carrying out one of the above procedures using a pure regioisomer as a starting material, or by separation of a mixture of the regioisomers or intermediates using a standard procedure. According to a further feature of the invention there is provided a compound of the formula (I), or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester or amide thereof for use in a method of treatment of the human or animal body by therapy. According to a further feature of the present invention there is provided a method for producing an antibacterial effect in a warm blooded animal, such as man, in need of such treatment, which comprises administering to said animal an effective amount of a compound of the present invention, or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof. The invention also provides a compound of the formula (I), or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof, for use as a medicament, and for use as an antibacterial agent; and the use of a compound of the formula (I) of the present invention, or a pharmaceutically-acceptable salt, or in-vivo hydrolysable ester thereof, in the manufacture of a medicament for use in the production of an antibacterial effect in a warm blooded animal, such as man. In order to use a compound of the formula (I), an in-vivo hydrolysable ester or a pharmaceutically-acceptable salt thereof, including a pharmaceutically-acceptable salt of an in-vivo hydrolysable ester, (hereinafter in this section relating to pharmaceutical composition “a compound of this invention”) for the therapeutic (including prophylactic) treatment of mammals including humans, in particular in treating infection, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition. Therefore in another aspect the present invention provides a pharmaceutical composition which comprises a compound of the formula (I), an in-vivo hydrolysable ester or a pharmaceutically-acceptable salt thereof, including a pharmaceutically-acceptable salt of an in-vivo hydrolysable ester, and a pharmaceutically-acceptable diluent or carrier. The pharmaceutical compositions of this invention may be administered in standard manner for the disease condition that it is desired to treat, for example by oral, rectal, topical or parenteral administration. For these purposes the compounds of this invention may be formulated by means known in the art into the form of, for example, tablets, capsules, aqueous or oily solutions or suspensions, (lipid) emulsions, dispersible powders, suppositories, ointments, creams, aerosols (or sprays), drops and sterile injectable aqueous or oily solutions or suspensions. In addition to the compounds of the present invention the pharmaceutical composition of this invention may also contain or be co-administered (simultaneously, sequentially or separately) with one or more known drugs selected from other clinically useful antibacterial agents (for example, β-lactams or aminoglycosides) and/or other anti-infective agents (for example, an antifungal triazole or amphotericin). These may include carbapenems, for example meropenem or imipenem, to broaden the therapeutic effectiveness. Compounds of this invention may also contain or be co-administered with bactericidal/permeability-increasing protein (BPI) products or efflux pump inhibitors to improve activity against gram negative bacteria and bacteria resistant to antimicrobial agents. A suitable pharmaceutical composition of this invention is one suitable for oral administration in unit dosage form, for example a tablet or capsule which contains between 1 mg and 1 g of a compound of this invention, preferably between 100 mg and 1 g of a compound. Especially preferred is a tablet or capsule which contains between 50 mg and 800 mg of a compound of this invention, particularly in the range 100 mg to 500 mg. In another aspect a pharmaceutical composition of the invention is one suitable for intravenous, subcutaneous or intramuscular injection, for example an injection which contains between 0.1% w/v and 50% w/v (between 1 mg/ml and 500 mg/ml) of a compound of this invention. Each patient may receive, for example, a daily intravenous, subcutaneous or intramuscular dose of 0.5 mgkg- 1 to 20 mgkg- 1 of a compound of this invention, the composition being administered 1 to 4 times per day. In another embodiment a daily dose of 5 mgkg- 1 to 20 mgkg- 1 of a compound of this invention is administered. The intravenous, subcutaneous and intramuscular dose may be given by means of a bolus injection. Alternatively the intravenous dose may be given by continuous infusion over a period of time. Alternatively each patient may receive a daily oral dose which may be approximately equivalent to the daily parenteral dose, the composition being administered 1 to 4 times per day. A pharmaceutical composition to be dosed intravenously may contain advantageously (for example to enhance stability) a suitable bactericide, antioxidant or reducing agent, or a suitable sequestering agent. In the above other, pharmaceutical composition, process, method, use and medicament manufacture features, the alternative and preferred embodiments of the compounds of the invention described herein also apply. Antibacterial Activity: The pharmaceutically-acceptable compounds of the present invention are useful antibacterial agents having a good spectrum of activity in vitro against standard Gram-positive organisms, which are used to screen for activity against pathogenic bacteria. Notably, the pharmaceutically-acceptable compounds of the present invention show activity against enterococci, pneumococci , methicillin resistant strains of S.aureus and coagulase negative staphylococci, haemophilus and moraxella strains. The antibacterial spectrum and potency of a particular compound may be determined in a standard test system. The (antibacterial) properties of the compounds of the invention may also be demonstrated and assessed in-vivo in conventional tests, for example by oral and/or intravenous dosing of a compound to a warm-blooded mammal using standard techniques. The following results were obtained on a standard in-vitro test system. The activity is described in terms of the minimum inhibitory concentration (MIC) determined by the broth-dilution technique with an inoculum size of 5×10 4 CFU/spot. Typically, compounds are active in the range 0.01 to 256 μg/ml. Staphylococci were tested in broth using an inoculum of 5×10 4 CFU/spot and an incubation temperature of 37° C. for 16-24 hours. Streptococci were tested in Mueller-Hinton broth supplemented with 2.5% clarified lake horse blood with an innoculum of 10 4 CFU/well and an incubation temperature of 37° C. aerobically for 24 hours. Fastidious Gram negative organisms were tested in Mueller-Hinton broth supplemented with hemin and NAD, grown aerobically for 24 h at 37° C., and with an innoculum of 5×10 4 CFU/well. MIC (μg/ml) Organism Example 2 Staphylococcus aureus: MSQS 1 MRQR 8 Streptococcus pneumoniae 2 Streptococcus pyogenes 2 Haemophilus influenzae 8 Moraxella catarrhalis 8 MSQS=methicillin sensitive and quinolone sensitive MRQR=methincillin resistant and quinolone resistant Certain intermediates and/or Reference Examples described hereinafter within the scope of the invention may also possess useful activity, and are provided as a further feature of the invention. The invention is now illustrated but not limited by the following Examples in which unless otherwise stated: i) evaporations were carried out by rotary evaporation in vacuo and work-up procedures were carried out after removal of residual solids by filtration; (ii) operations were carried out at ambient temperature, that is typically in the range 18-26° C. and in air unless otherwise stated, or unless the skilled person would otherwise work under an inert atmosphere; where unspecified, temperatures are quoted in ° C.; (iii) column chromatography (by the flash procedure) was used to purify compounds and was performed on Merck Kieselgel silica (Art. 9385) unless otherwise stated; (iv) yields are given for illustration only and are not necessarily the maximum attainable; (v) the structure of the end-products of the invention were generally confirmed by NMR and mass spectral techniques [proton magnetic resonance spectra were generally determined in DMSO-D6 unless otherwise stated using a Varian Gemini 2000 spectrometer operating at a field strength of 300 MHz, or a Bruker AM250 spectrometer operating at a field strength of 250 MHz; chemical shifts are reported in parts per million downfield from tetramethysilane as an internal standard (6 scale) and peak multiplicities are shown thus: s, singlet; d, doublet; AB or dd, doublet of doublets; t, triplet, m, multiplet; fast-atom bombardment (FAB) mass spectral data were generally obtained using a Platform spectrometer (supplied by Micromass) run in electrospray and, where appropriate, either positive ion data or negative ion data were collected]; (vi) each intermediate was purified to the standard required for the subsequent stage and was characterised in sufficient detail to confirm that the assigned structure was correct; purity was in general assessed by HPLC, TLC, infra-red (IR), MS or NMR analysis; and identity was determined by IR, MS or NMR spectroscopy as appropriate; and (vii) in which the following abbreviations may be used: ® is a Trademark; DMF is N,N-dimethylformamide; DMA is N,N-dimethylacetamide; TLC is thin layer chromatography; HPLC is high pressure liquid chromatography; MPLC is medium pressure liquid chromatography; DMSO is dimethylsulfoxide; DMSO-d6 is deuterated DMSO; CDCl 3 is deuterated chloroform; MS is mass spectroscopy; ESP is electrospray; EI is electron impact; CI is chemical ionisation; APCI is atmospheric pressure chemical ionisation; THF is tetrahydrofuran; TFA is trifluoroacetic acid; NMP is N-methylpyrrolidone; HOBT is 1-hydroxy-benzotriazole; EtOAc is ethyl acetate; MeOH is methanol; phosphoryl is (HO) 2 —P(O)—O—; phosphiryl is (HO) 2 —P—O—; EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (hydrochloride); PTSA is para-toluenesulfonic acid. detailed-description description="Detailed Description" end="lead"? |
Intracellular signaling molecules |
Various embodiments of the invention provide human intracellular signaling molecules (INTSIG) and polynucleotides which identify and encode INTSIG. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of INTSIG. |
1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, SEQ ID NO:5-9, and SEQ ID NO: 13-17, c) a polypeptide comprising a naturally occurring amino acid sequence at least 91% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 12, d) a polypeptide comprising a naturally occurring amino acid sequence at least 97% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 18-19, e) a polypeptide comprising a naturally occurring amino acid sequence at least 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO: 11, f) a polypeptide consisting essentially of a naturally occurring amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:20, g) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and h) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. 3. An isolated polynucleotide encoding a polypeptide of claim 1. 4. An isolated polynucleotide encoding a polypeptide of claim 2. 5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3. 7. A cell transformed with a recombinant polynucleotide of claim 6. 8. A transgenic organism comprising a recombinant polynucleotide of claim 6. 9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed. 10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. 11. An isolated antibody which specifically binds to a polypeptide of claim 1. 12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-23, and SEQ ID NO:25-40, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 98% identical to the amino acid sequence of SEQ ID NO:24, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), f) a polynucleotide complementary to a polynucleotide of c), and g) an RNA equivalent of a)-f). 13. (canceled) 14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof. 15. (canceled) 16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. 19. (canceled) 20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample. 21. (canceled) 22. (canceled) 23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample. 24. (canceled) 25. (canceled) 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. 27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1. 28. (canceled) 29. A method of assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. 30-95. (canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>Cell-cell communication is essential for the growth, development, and survival of multicellular organisms. Cells communicate by sending and receiving molecular signals. An example of a molecular signal is a growth factor, which binds and activates a specific transmembrane receptor on the surface of a target cell. The activated receptor transduces the signal intracellularly, thus initiating a cascade of biochemical reactions that ultimately affect gene transcription and cell cycle progression in the target cell. Intracellular signaling is the process by which cells respond to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.) through a cascade of biochemical reactions that begins with the binding of a signaling molecule to a cell membrane receptor and ends with the activation of an intracellular target molecule. Intermediate steps in the process involve the activation of various cytoplasmic proteins by phosphorylation via protein inases, and their deactivation by protein phosphatases, and the eventual translocation of some of these activated proteins to the cell nucleus where the transcription of specific genes is triggered. The intracellular signaling process regulates all types of cell functions including cell proliferation, cell differentiation, and gene transcription, and involves a diversity of molecules including protein kinases and phosphatases, and second messenger molecules such as cyclic nucleotides, calcium-calmodulin, inositol, and various mitogens that regulate protein phosphorylation. A distinctive class of signal transduction molecules are involved in odorant detection. The process of odorant detection involves specific recognition by odorant receptors. The olfactory mucosa also appears to possess an additional group of odorant-binding proteins which recognize and bind separate classes of odorants. For example, cDNA clones from rat have been isolated which correspond to mRNAs highly expressed in olfactory mucosa but not detected in other tissues. The proteins encoded by these clones are homologous to proteins that bind lipopolysaccharides or polychlorinated biphenyls, and the different proteins appear to be expressed in specific areas of the mucosal tissue. These proteins are believed to interact with odorants before or after specific recognition by odorant receptors, perhaps acting as selective signal filters (Dear, T. N. et al. (1991) EMBO J. 10:2813-2819; Vogt, R. G. et al. (1991) J. Neurobiol. 22:74-84). Cells also respond to changing conditions by switching off signals. Many signal transduction proteins are short-lived and rapidly targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Cells also maintain mechanisms to monitor changes in the concentration of denatured or unfolded proteins in membrane-bound extracytoplasmic compartments, including a transmembrane receptor that monitors the concentration of available chaperone molecules in the endoplasmic reticulum and transmits a signal to the cytosol to activate the transcription of nuclear genes encoding chaperones in the endoplasmic reticulum. Certain proteins in intracellular signaling pathways serve to link or cluster other proteins involved in the signaling cascade. These proteins are referred to as scaffold, anchoring, or adaptor proteins. (For review, see Pawson, T. and J. D. Scott (1997) Science 278:2075-2080.) As many intracellular signaling proteins such as protein kinases and phosphatases have relatively broad substrate specificities, the adaptors help to organize the component signaling proteins into specific biochemical pathways. Many of the above signaling molecules are characterized by the presence of particular domains that promote protein-protein interactions. A sampling of these domains is discussed below, along with other important intracellular messengers. Intracellular Signaling Second Messenger Molecules Protein Phosphorylation Protein kinases and phosphatases play a key role in the intracellular signaling process by controlling the phosphorylation and activation of various signaling proteins. The high energy phosphate for this reaction is generally transferred from the adenosine triphosphate molecule (ATP) to a particular protein by a protein kinase and removed from that protein by a protein phosphatase. Protein kinases are roughly divided into two groups: those that phosphorylate serine or threonine residues (serine/threonine kinases, STK) and those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK). A few protein kinases have dual specificity for serine/threonine and tyrosine residues. Almost all kinases contain a conserved 250-300 amino acid catalytic domain containing specific residues and sequence motifs characteristic of the kinase family (Hardie, G. and S. Hanks (1995) The Protein Kinase Facts Books, Vol 1:7-20, Academic Press, San Diego, Calif.). STKs include the second messenger dependent protein kinases such as the cyclic-AMP dependent protein kinases (PKA), involved in mediating hormone-induced cellular responses; calcium-calmodulin (CaM) dependent protein kinases, involved in regulation of smooth muscle contraction, glycogen breakdown, and neurotransmission; and the mitogen-activated protein kinases (MAP kinases) which mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison's Pinci les of Internal Medicine, McGraw-Hill, New York, N.Y., pp. 416-431, 1887). PTKs are divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembrane PTKs are receptors for most growth factors. Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors. Receptors that function through non-receptor PTKs include those for cytokines and hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes. Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells in which their activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs, and it is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Charbonneau H and N. K. Tonks (1992) Annu. Rev. Cell Biol. 8:463-493). An additional family of protein kinases previously thought to exist only in prokaryotes is the histidine protein kinase family (HPK). HPKs bear little homology with mammalian STKs or PTKs but have distinctive sequence motifs of their own (Davie, J. R. et al. (1995) J. Biol. Chem. 270:19861-19867). A histidine residue in the N-terminal half of the molecule (region I) is an autophosphorylation site. Three additional motifs located in the C-terminal half of the molecule include an invariant asparagine residue in region II and two glycine-rich loops characteristic of nucleotide binding domains in regions III and IV. Recently a branched chain alpha-ketoacid dehydrogenase kinase has been found with characteristics of HPK in rat (Davie et al., supra). Protein phosphatases regulate the effects of protein kinases by removing phosphate groups from molecules previously activated by kinases. The two principal categories of protein phosphatases are the protein (serine/threonine) phosphatases (PPs) and the protein tyrosine phosphatases (PTPs). PPs dephosphorylate phosphoserine/threonine residues and are important regulators of many cAMP-mediated hormone responses (Cohen, P. (1989) Annu. Rev. Biochem. 58:453-508). PIPs reverse the effects of protein tyrosine kinases and play a significant role in cell cycle and cell signaling processes (Charbonneau and Tonks, supra). As previously noted, many PTKs are encoded by oncogenes, and oncogenesis is often accompanied by increased tyrosine phosphorylation activity. It is therefore possible that PTPs may prevent or reverse cell transformation and the growth of various cancers by controlling the levels of tyrosine phosphorylation in cells. This hypothesis is supported by studies showing that overexpression of PTPs can suppress transformation in cells, and that specific inhibition of PTPs can enhance cell transformation (Charbonneau and Totks, supra). Phospholipid and Inositol-Phosphate Signaling Inositol phospholipids (phosphoinositides) are involved in an intracellular signaling pathway that begins with binding of a signaling molecule to a G-protein linked receptor in the plasma membrane. This leads to the phosphorylation of phosphatidylinositol (PI) residues on the inner side of the plasma membrane to the biphosphate state (PIP 2 ) by inositol kinases. Simultaneously, the G-protein linked receptor binding stimulates a trimeric G-protein which in turn activates a phosphoinositide-specific phospholipase C-β. Phospholipase C-β then cleaves PIP 2 into two products, inositol triphosphate (IP 3 ) and diacylglycerol. These two products act as mediators for separate signaling events. IP 3 diffuses through the plasma membrane to induce calcium release from the endoplasmnic reticulum (ER), while diacylglycerol remains in the membrane and helps activate protein kinase C, a serine-threonine linase that phosphorylates selected proteins in the target cell. The calcium response initiated by IP 3 is terminated by the dephosphorylation of IP 3 by specific inositol phosphatases. Cellular responses that are mediated by this pathway are glycogen breakdown in the liver in response to vasopressin, smooth muscle contraction in response to acetylcholine, and thrombin-induced platelet aggregation. Inositol-phosphate signaling controls tubby, a membrane bound transcriptional regulator that serves as an intracellular messenger of Ge-coupled receptors (Santagata et al. (2001) Science 292:2041-2050). Members of the tubby family contain a C-terminal tubby domain of about 260 amino acids that binds to double-stranded DNA and an N-terminal transcriptional activation domain. Tabby binds to phosphatidylinositol 4,5-bisphosphate, which localizes tubby to the plasma membrane. Activation of the G-protein t leads to activation of phospholipase C-β and hydrolysis of phosphoinositide. Loss of phosphatidylinositol 4,5-bisphosphate causes tubby to dissociate from the plasma membrane and to translocate to the nucleus where tubby regulates transcription of its target genes. Defects in the tubby gene are associated with obesity, retinal degeneration, and hearing loss (Boggon, T. J. et al. (1999) Science 286:2119-2125). Cyclic Nucleotide Signaling Cyclic nucleotides (cAMP and cGMP) function as intracellular second messengers to transduce a variety of extracellular signals including hormones, light, and neurotransmitters. In particular, cyclic-AMP dependent protein kinases (PKA) are thought to account for all of the effects of cAMP in most mammalian cells, including various hormone-induced cellular responses. Visual excitation and the phototransmission of light signals in the eye is controlled by cyclic-GMP regulated, Ca 2 +-specific channels. Because of the importance of cellular levels of cyclic nucleotides in mediating these various responses, regulating the synthesis and breakdown of cyclic nucleotides is an important matter. Thus adenylyl cyclase, which synthesizes cAMP from AMP, is activated to increase cAMP levels in muscle by binding of adrenaline to β-adrenergic receptors, while activation of guanylat cyclase and increased cGMP levels in photoreceptors leads to reopening of the Ca 2+ -specific channels and recovery of the dark state in the eye. There are nine known transmembrane isoforms of mammalian adenylyl cyclase, as well as a soluble form preferentially expressed in testis. Soluble adenylyl cyclase contains a P-loop, or nucleotide binding domain, and may be involved in male fertility (Buck, J. et al. (1999) Proc. Natl. Acad. Sci. USA 96:79-84). In contrast, hydrolysis of cyclic nucleotides by cAMP and cGMP-specific phosphodiesterases (PDEs) produces the opposite of these and other effects mediated by increased cyclic nucleotide levels. PDEs appear to be particularly important in the regulation of cyclic nucleotides, considering the diversity found in this family of proteins. At least seven families of mammalian PDEs (PDE1-7) have been identified based on substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitory drugs (Beavo, J. A. (1995) Physiol. Rev. 75:725-748). PDE inhibitors have been found to be particularly useful in treating various clinical disorders. Rolipram, a specific inhibitor of PD134, has been used in the treatment of depression, and similar inhibitors are undergoing evaluation as anti-inflammatory agents. Theophylline is a nonspecific PDE inhibitor used in the treatment of bronchial asthma and other respiratory diseases (Banner, K. H. and C. P. Page (1995) Eur. Respir. J. 8:996-1000). Calcium Signaling Molecules Ca 2+ is another second messenger molecule that is even more widely used as an intracellular mediator than cAMP. Ca 2+ can enter the cytosol by two pathways, in response to extracellular signals. One pathway acts primarily in nerve signal transduction where Ca 2+ enters a nerve terminal through a voltage-gated Ca 2+ channel. The second is a more ubiquitous pathway in which Ca 2+ is released from the ER into the cytosol in response to binding of an extracellular signaling molecule to a receptor. Ca 2+ directly activates regulatory enzymes, such as protein kinase C, which trigger signal transduction pathways. Ca 2+ also binds to specific Ca 2+ -binding proteins (CBPs) such as calmodulin (CaM) which then activate multiple target proteins in the cell including enzymes, membrane transport pumps, and ion channels. CaM interactions are involved in a multitude of cellular processes including, but not limited to, gene regulation, DNA synthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signal transduction, ion homeostasis, exocytosis, and metabolic regulation (Celio, M. R. et al. (1996) Guidebook to Calcium - binding Proteins, Oxford University Press, Oxford, UK, pp. 15-20). Some Ca 2+ binding proteins are characterized by the presence of one or more EF-hand Ca 2+ binding motifs, which are comprised of 12 amino acids flanked by α-helices (Celio, supra). The regulation of CBPs has implications for the control of a variety of disorders. Calcineurin, a CaM-regulated protein phosphatase, is a target for inhibition by the immunosuppressive agents cyclosporin and FK506. This indicates the importance of calcineurin and CaM in the immune response and immune disorders (Schwaninger M. et al. (1993) J. Biol Chem. 268:23111-23115). The level of CaM is increased several-fold in tumors and tumor-derived cell lines for various types of cancer (Rasmussen, C. D. and A. R. Means (1989) Trends Neurosci. 12:433-438). The annexins are a family of calcium-binding proteins that associate with the cell membrane (Towle, C. A. and B. V. Treadwell (1992) J. Biol. Chem. 267:5416-5423). Annexins reversibly bind to negatively charged phospholipids (phosphatidylcholine and phosphatidylserine) in a calcium dependent manner. Annexins participate in various processes pertaining to signal transduction at the plasma membrane, including membrane-cytoskeleton interactions, phospholipase inhibition, anticoagulation, and membrane fusion. Annexins contain four to eight repeated segments of about 60 residues. Each repeat folds-into five alpha helices wound into a right-handed superhelix. G-Protein Signaling Guanine nucleotide binding proteins (G-proteins) are critical mediators of signal transduction between a particular class of extracellular receptors, the G-protein coupled receptors (GPCRs), and intracellular second messengers such as cAMP and Ca 2+ . G-proteins are linked to the cytosolic side of a GPCR such that activation of the GPCR by ligand binding stimulates binding of the G-protein to GTP, inducing an “active” state in the G-protein. In the active state, the G-protein acts as a signal to trigger other events in the cell such as the increase of cAMP levels or the release of Ca 2+ into the cytosol from the ER, which, in turn, regulate phosphorylation and activation of other intracellular proteins. Recycling of the G-protein to the inactive state involves hydrolysis of the bound GTP to GDP by a GTPase activity in the G-protein. (See Alberts, B. et al. (1994) Molecular Biology of the Cell Garland Publishing, Inc. New York, N.Y., pp.734-759.) The superfamily of G-proteins consists of several families which may be grouped as translational factors, heterotrimeric G-proteins involved in transmembrane signaling processes, and low molecular weight (LMW) G-proteins including the proto-oncogene Ras proteins and products of rab, rap, rho, rac, smg21, smg25, YPT, SBC4, and ARF genes, and tubulins (Kaziro, Y. et al (1991) Annu. Rev. Biochem. 60:349400). In all cases, the GTPase activity is regulated through interactions with other proteins. Heterotrimeric G-proteins are composed of 3 subunits, α, β, and γ, which in their inactive conformation associate as a trimer at the inner face of the plasma membrane. Gabinds GDP or GTP and contains the GTPase activity. The βγ complex enhances binding of Gα to a receptor. Gγ is necessary for the folding and activity of Gβ (Neer, E. J. et al. (1994) Nature 371:297-300). Multiple homologs of each subunit have been identified in mammalian tissues, and different combinations of subunits have specific functions and tissue specificities (Spiegel, A. M. (1997) J. Inher. Metab. Dis. 20:113-121). The alpha subunits of heterotrimeric G-proteins can be divided into four distinct classes. The α-s class is sensitive to ADP-ribosylation by pertussis toxin which uncouples the receptor:G-protein interaction. This uncoupling blocks signal transduction to receptors that decrease cAMP levels which normally regulate ion channels and activate phospholipases. The inhibitory α-I class is also susceptible to modification by pertussis toxin which prevents α-I from lowering cAMP levels. Two novel classes of α subunits refractory to pertussis toxin modification are a-q, which activates phospholipase C, and α-12, which has sequence homology with the Drosophila gene concertina and may contribute to the regulation of embryonic development (Simon, M. L. (1991) Science 252:802-808). The mammalian Gβ and Gγ subunits, each about 340 amino acids long, share more than 80% homology. The Gβ subunit (also called transducin) contains seven repeating units, each about 43 amino acids long. The activity of both subunits may be regulated by other proteins such as calmodulin and phosducin or the neural protein GAP 43 (Clapham, D. and E. Neer (1993) Nature 365:403-406). The, β and γ subunits are tightly associated. The β subunit sequences are highly conserved between species, implying that they perform a fundamentally important role in the organization and function of G-protein linked systems (Van der Voorn, L. (1992) FEBS Lett. 307:131-134). They contain seven tandem repeats of the WD-repeat sequence motif, a motif found in many proteins with regulatory functions. WD-repeat proteins contain from four to eight copies of a loosely conserved repeat of approximately 40 amino acids which participates in protein-protein interactions. Mutations and variant expression of β transducin proteins are linked with various disorders. Mutations in LIS1, a subunit of the human platelet activating factor acetylhydrolase, cause Miller-Dieker lissencephaly. RACK1 binds activated protein kinase C, and RbAp48 binds retinoblastoma protein. CstF is required for polyadenylation of mammalian pre-mRNA in vitro and associates with subunits of cleavage-stimulating factor. Defects in the regulation of β-catenin contribute to the neoplastic transformation of human cells. The WD40 repeats of the human F-box protein bTrCP mediate binding to β-catenin, thus regulating the targeted degradation of β-catenin by ubiquitin ligase (Neer et al., supra; Hart, M. et al. (1999) Curr. Biol. 9:207-210). The γ subunit primary structures are more variable than those of the β subunits. They are often post-translationally modified by isoprenylation and carboxyl-methylation of a cysteine residue four amino acids from the C-terminus; this appears to be necessary for the interaction of the βγ subunit with the membrane and with other G-proteins. The βγ subunit has been shown to modulate the activity of isoforms of adenylyl cyclase, phospholipase C, and some ion channels. It is involved in receptor phosphorylation via specific kinases, and has been implicated in the p21ras-dependent activation of the MAP kinase cascade and the recognition of specific receptors by G-proteins (Clapham and Neer, supra). G-proteins interact with a variety of effectors including adenylyl cyclase (Clapham and Neer, supra). The signaling pathway mediated by cAMP is mitogenic in hormone-dependent endocrine tissues such as adrenal cortex, thyroid, ovary, pituitary, and testes. Cancers in these tissues have been related to a mutationally activated form of a Gα, known as the gsp (Gs protein) oncogene (Dhanasekaran, N. et al. (1998) Oncogene 17:1383-1394). Another effector is phosducin, a retinal phosphoprotein, which forms a specific complex with retinal Gβ and Gγ (Gβγ) and modulates the ability of Gβγ to interact with retinal Gα (Clapham and Neer, supra). Irregularities in the G-protein signaling cascade may result in abnormal activation of leukocytes and lymphocytes, leading to the tissue damage and destruction seen in many inflammatory and autoimmune diseases such as rheumatoid arthritis, biliary cirrhosis, hemolytic anemia, lupus erythematosus, and thyroiditis. Abnormal cell proliferation, including cyclic AMP stimulation of brain, thyroid, adrenal, and gonadal tissue proliferation is regulated by G proteins. Mutations in Gα subunits have been found in growth-hormone-secreting pituitary somatotroph tumors, hyperfunctioning thyroid adenomas, and ovarian and adrenal neoplasms (Meij, J. T. A. (1996) Mol. Cell Biochem 157:31-38; Aussel, C. et al. (1988) J. Immunol 140:215-220). LMW G-proteins are GTPases which regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. They consist of single polypeptides which, like the alpha subunit of the heterotrimeric G-proteins, are able to bind to and hydrolyze GTP, thus cycling between an inactive and an active state. LMW G-proteins respond to extracellular signals from receptors and activating proteins by transducing mitogenic signals involved in various cell functions. The binding and hydrolysis of GTP regulates the response of LMW G-proteins and acts as an energy source during this process (Bokoch, G. M. and C. J. Der (1993) FASEB J. 7:750-759). At least sixty members of the LMW G-protein superfamily have been identified and are currently grouped into the ras, rho, arf, sar1, ran, and rab subfamilies. Activated ras genes were initially found in human cancers, and subsequent studies confirmed that ras function is critical in determining whether cells continue to grow or become differentiated. Ras1 and Ras2 proteins stimulate adenylate cyclase (Kaziro et al., supra), affecting abroad array of cellular processes. Stimulation of cell surface receptors activates Ras which, in turn, activates cytoplasmic kinases. These kinases translocate to the nucleus and activate key transcription factors that control gene expression and protein synthesis (Barbacid, M. (1987) Annu. Rev. Biochem. 56:779-827; Treisman, R. (1994) Curr. Opin. Genet. Dev. 4:96-98). Other members of the LMW G-protein superfamily have roles in signal transduction that vary with the function of the activated genes and the locations of the G-proteins that initiate the activity. Rho G-proteins control signal transduction pathways that link growth factor receptors to actin polymerization, which is necessary for normal cellular growth and division. The rab, arf, and sar1 families of proteins control the translocation of vesicles to and from membranes for protein processing, localization, and secretion. Vesicle- and target-specific identifiers (v-SNAREs and t-SNAREs) bind to each other and dock the vesicle to the acceptor membrane. The budding process is regulated by the closely related ADP ribosylation factors (ARFs) and SAR proteins, while rab proteins allow assembly of SNARE complexes and may play a role in removal of defective complexes (Rothman, J. and F. Wieland (1996) Science 272:227-234). Ran G-proteins are located in the nucleus of cells and have a key role in nuclear protein import, the control of DNA synthesis, and cell-cycle progression (Hall, A. (1990) Science 249:635-640; Barbacid, supra; Ktistakis, N. (1998) BioEssays 20:495-504; and Sasaki, T. and Y. Takai (1998) Biochem. Biophys. Res. Commun. 245:641-645). Rab proteins have a highly variable amino terminus containing membrane-specific signal information and a prenylated carboxy terminus which determines the target membrane to which the Rab proteins anchor. More than 30 Rab proteins have been identified in a variety of species, and each has a characteristic intracellular location and distinct transport function. In particular, Rab1 and Rab2 are important in ER-to-Golgi transport; Rab3 transports secretory vesicles to the extracellular membrane; Rab5 is localized to endosomes and regulates the fusion of early endosomes into late endosomes; Rab6 is specific to the Golgi apparatus and regulates intra-Golgi transport events; Rab7 and Rab9 stimulate the fusion of late endosomes and Golgi vesicles with lysosomes, respectively; and Rab10 mediates vesicle fusion from the medial Golgi to the trans Golgi. Mutant forms of Rab proteins are able to block protein transport along a given pathway or alter the sizes of entire organelles. Therefore, Rabs play key regulatory roles in membrane trafficking (Schimmöller, I. S. and S. R. Pfeffer (1998) J. Biol Chem. 243:22161-22164). The function of Rab proteins in vesicular transport requires the cooperation of many other proteins. Specifically, the membrane-targeting process is assisted by a series of escort proteins (Khosravi-Far, R. et al. (1991) Proc. Natl. Acad. Sci. USA 88:6264-6268). In the medial Golgi, it has been shown that GTP-bound Rab proteins initiate the binding of VAMP-like proteins of the transport vesicle to syntaxin-like proteins on the acceptor membrane, which subsequently triggers a cascade of protein-binding and membrane-fusion events. After transport, GTPase-activating proteins (GAPs) in the target membrane are responsible for converting the GTP-bound Rab proteins to their GDP-bound state. And finally, guanine-nucleotide dissociation inhibitor (GDI) recruits the GDP-bound proteins to their membrane of origin. The cycling of LMW G-proteins between the GTP-bound active form and the GDP-bound inactive form is regulated by a variety of proteins. Guanosine nucleotide exchange factors (GEFs) increase the rate of nucleotide dissociation by several orders of magnitude, thus facilitating release of GDP and loading with GTP. The best characterized is the mammalian homolog of the Drosophila Son-of-Sevenless protein. Certain Ras-family proteins are also regulated by guanine nucleotide dissociation inhibitors (GDIs), which inhibit GDP dissociation. The intrinsic rate of GTP hydrolysis of the LMW G-proteins is typically very slow, but it can be stimulated by several orders of magnitude by GAPs (Geyer, M. and A. Wittinghofer (1997) Curr. Opin. Struct. Biol. 7:786-792). Both GEF and GAP activity may be controlled in response to extracellular stimuli and modulated by accessory proteins such as RalBP1 and POB1. Mutant Ras-family proteins, which bind but cannot hydrolyze GTPP; are permanently activated, and cause cell proliferation or cancer, as do GEPs that inappropriately activate LMW G-proteins, such as the human oncogene NET1, a Rho-GEF (Drivas, G. T. et al (1990) Mol. Cell Biol. 10:1793-1798; Alberts, A. S. and R. Treisman (1998) EMBO J. 14:4075-4085). A member of the ARF family of G-proteins is centaurin beta 1A, a regulator of membrane traffic and the actin cytoskeleton. The centaurin β family of GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors contain pleckstrin homology (PE) domains which are activated by phosphoinositides. PH domains bind phosphoinositides, implicating PH domains in signaling processes. Phosphoinositides have a role in converting Arf-GTP to Arf-GDP via the centaurin β family and a role in Arf activation (Kam, J. L. et al (2000) J. Biol. Chem. 275:9653-9663). The rho GAP family is also implicated in the regulation of actin polymerization at the plasma membrane and in several cellular processes. The gene ARHGAP6 encodes GTPase-activating protein 6 isoform 4. Mutations in ARHGAP6, seen as a deletion of a 500 kb critical region in Xp22.3, causes the syndrome microphthalmia with linear skin defects (MIS). MS is an X-linked dominant, male-lethal syndrome (Prakash, S. K. et al. (2000) Hum. Mol. Genet. 9:477-488). A member of the Rho family of G-proteins is CDC42, a regulator of cytoskeletal rearrangements required for cell division. CDC42 is inactivated by a specific GAP (CDC42GAP) that strongly stimulates the GTPase activity of CDC42 while having a much lesser effect on other Rho family members. CDC42GAP also contains an SH3-binding domain that interacts with the SH3 domains of cell signaling proteins such as p85 alpha and c-Src, suggesting that CDC42GAP may serve as a link between CDC42 and other cell signaling pathways (Barfod, E. T. et al. (1993) J. Biol. Chem. 268:26059-26062). The Dbl proteins are a family of GEFs for the Rho and Ras G-proteins (Whitehead, I. P. et al. (1997) Biochim. Biophys. Acta 1332:F1-F23). All Dbl family members contain a Dbl homology (DH) domain of approximately 180 amino acids, as well as a pleckstrin homology (PH) domain located immediately C-terminal to the DH domain Most Dbl proteins have oncogenic activity, as demonstrated by the ability to transform various cell lines, consistent with roles as regulators of Rho-mediated oncogenic signaling pathways. The kalrin proteins are neuron-specific members of the Dbl family, which are located to distinct subcellular regions of cultured neurons (Johnson, R. C. (2000) J. Cell Biol. 275:19324-19333). Other regulators of G-protein signaling (RGS) also exist that act primarily by negatively regulating the G-protein pathway by an unknown mechanism (Druey, K. M. et al. (1996) Nature 379:742-746). Some 15 members of the RGS family have been identified. RGS family members are related structurally through similarities in an approximately 120 amino acid region termed the RGS domain and functionally by their ability to inhibit the interleukin (cytokine) induction of MAP kinase in cultured mammalian 293T cells (Druey et al., supra). The Immuno-associated nucleotide (LAN) family of proteins has GTP-binding activity as indicated by the conserved ATP/GTP-binding site P-loop motif. The IAN family includes IAN-1, IAN-4, IAP38, and IAG-1. IAN-1 is expressed in the immune system, specifically in T cells and thymocytes. Its expression is induced during thymic events (Poirier, G. M. C. et al. (1999) J. Immunol. 163:4960-4969). IAP38 is expressed in B cells and macrophages and its expression is induced in splenocytes by pathogens. IAG-1, which is a plant molecule, is induced upon bacterial infection (Krucken, J. et al. (1997) Biochem. Biophys. Res. Commun. 230:167-170). IAN-4 is a mitochondrial membrane protein which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the bcr/abl oncogene. The bcr/abl oncogene is known to be associated with chronic myelogenous leukemia, a clonal myelo-proliferative disorder, which is due to the translocation between the bcr gene on chromosome 22 and the abl gene on chromosome 9. Bcr is the breakpoint cluster region gene and abl is the cellular homolog of the transforming gene of the Abelson murine leukemia virus. Therefore, the IAN family of proteins appears to play a role in cell survival in immune responses and cellular transformation (Daheron, L. et al. (2001) Nucleic Acids Res. 29:1308-1316). Formin-related genes (FRL) comprise a large family of morphoregulatory genes and have been shown to play important roles in morphogenesis, embryogenesis, cell polarity, cell migration, and cytokinesis through their interaction with Rho family small GTPases. Formin was first identified in mouse limb deformitity (ld) mutants where the distal bones and digits of all limbs are fused and reduced in size. FRL contains formin homology domains FH1, FH2, and FH3. The FH1 domain has been shown to bind the Src homology 3 (SH3) domain, WWP/WW domains, and profilin. The FH2 domain is conserved and was shown to be essential for formin function as disruption at the FH2 domain results in the characteristic ld phenotype. The FH3 domain is located at the N-terminus of FRL, and is required for associating with Rac, a Rho family GTPase (Yayoshi-Yamamoto, S. et al. (2000) Mol. Cell. Biol 20:6872-6881). Signaling Complex Protein Domains PDZ domains were named for three proteins in which this domain was initially discovered. These proteins include PSD-95 (postsynaptic density 95), Dlg ( Drosophila lethal(1)discs large-1), and ZO-1 (zonula occludens-1). These proteins play important roles in neuronal synaptic transmission, tumor suppression, and cell junction formation, respectively. Since the discovery of these proteins, over sixty additional PDZ-containing proteins have been identified in diverse prokaryotic and eukaryotic organisms. This domain has been implicated in receptor and ion channel clustering and in the targeting of multiprotein signaling complexes to specialized functional regions of the cytosolic face of the plasma membrane. (For a review of PDZ domain-containing proteins, see Ponting, C. P. et al. (1997) Bioessays 19:469-479.) A large proportion of PDZ domains are found in the eukaryotic MAGUK (membrane-associated guanylate kinase) protein family, members of which bind to the intracellular domains of receptors and channels. However, PDZ domains are also found in diverse membrane-localized proteins such as protein tyrosine phosphatases, serine/threonine kinases, G-protein cofactors, and synapse-associated proteins such as syntrophins and neuronal nitric oxide synthase (nNOS). Generally, about one to three PDZ domains are found in a given protein, although up to nine PDZ domains have been identified in a single protein. The glutamate receptor interacting protein (GRIP) contains seven PDZ domains. GRIP is an adaptor that links certain glutamate receptors to other proteins and may be responsible for the clustering of these receptors at excitatory synapses in the brain (Dong, H. et al. (1997) Nature 386:279-284). The Drosophila scribble (SCRIB) protein contains both multiple PDZ domains and leucine-rich repeats. SCRIB is located at the epithelial septate junction, which is analogous to the vertebrate tight junction, at the boundary of the apical and basolateral cell surface. SCRIB is involved in the distribution of apical proteins and correct placement of adherens junctions to the basolateral cell surface (Bilder, D. and N. Perrimon (2000) Nature 403:676-680). The PX domain is an example of a domain specialized for promoting protein-protein interactions. The PX domain is found in sorting nexins and in a variety of other proteins, including the PhoX components of NADPH oxidase and the Cpk class of phosphatidylinositol 3-kinase. Most PX domains contain a polyproline motif which is characteristic of SH3 domain-binding proteins (Ponting, C. P. (1996) Protein Sci. 5:2353-2357). SH3 domain-mediated interactions involving the PhoX components of NADPH oxidase play a role in the formation of the NADPH oxidase multi-protein complex (Leto, T. L. et al. (1994) Proc. Natl. Acad. Sci. USA 91:10650-10654; Wilson, L. et al. (1997) Inflamm. Res. 46:265-271). The SH3 domain is defined by homology to a region of the proto-oncogene c-Src, a cytoplasmic protein tyrosine kinase. SH3 is a small domain of 50 to 60 amino acids that interacts with proline-rich ligands. SH3 domains are found in a variety of eukaryotic proteins involved in signal transduction, cell polarization, and membrane-cytoskeleton interactions. In some cases, SH3 domain-containing proteins interact directly with receptor tyrosine kinases. For example, the SLAP-130 protein is a substrate of the T-cell receptor (TCR) stimulated protein kinase. SLAP-130 interacts via its SH-3 domain with the protein SLP-76 to affect the TCR-induced expression of interleukin-2 (Musci, M. A. et al. (1997) J. Biol. Chem. 272:11674-11677). Another recently identified S53 domain protein is macrophage actin-associated tyrosine-phosphorylated protein (MAYP) which is phosphorylated during the response of macrophages to colony stimulating factor-1 (CSF-1) and is likely to play a role in regulating the CSF-1-induced reorganization of the actin cytoskeleton (Yeung, Y.-G. et al (1998) J. Biol. Chem. 273:30638-30642). The structure of the SH3 domain is characterized by two antiparallel beta sheets packed against each other at right angles. This packing forms a hydrophobic pocket lined with residues that are highly conserved between different SH3 domains. This pocket makes critical hydrophobic contacts with proline residues in the ligand (Feng, S. et al. (1994) Science 266:1241-1247). A novel domain, called the WW domain, resembles the SH3 domain in its ability to bind proline-rich ligands. This domain was originally discovered in dystrophin, a cytoskeletal protein with direct involvement in Duchenne muscular dystrophy (Bork, P. and M. Sudol (1994) Trends Biochem. Sci. 19:531-533). WW domains have since been discovered in a variety of intracellular signaling molecules involved in development, cell differentiation, and cell proliferation. The structure of the WW domain is composed of beta strands grouped around four conserved aromatic residues, generally tryptophan. Like SH3, the SH2 domain is defined by homology to a region of c-Src. SH2 domains interact directly with phospho-tyrosine residues, thus providing an immediate mechanism for the regulation and transduction of receptor tyrosine kinase-mediated signaling pathways. For example, as many as ten distinct SH2 domains are capable of binding to phosphorylated tyrosine residues in the activated PDGF receptor, thereby providing a highly coordinated and finely tuned response to ligand-mediated receptor activation. (Reviewed in Schaffhausen, B. (1995) Biochim. Biophys. Acta. 1242:61-75.) The BLNK protein is a linker protein involved in B cell activation, that bridges B cell receptor-associated kinases with SH2 domain effectors that link to various signaling pathways (Fu, C. et al. (1998) Immunity 9:93-103). The pleckstrin homology (PH) domain was originally identified in pleckstrin, the predominant substrate for protein kinase C in platelets. Since its discovery, this domain has been identified in over 90 proteins involved in intracellular signaling or cytoskeletal organization. Proteins containing the pleckstrin homology domain include a variety of kinases, phospholipase-C isoforms, guanine nucleotide release factors, and GTPase activating proteins. For example, members of the FGD1 family contain both Rho-guanine nucleotide exchange factor (GEF) and PH domains, as well as a FYVE zinc finger domain. FGD1 is the gene responsible for faciogenital dysplasia, an inherited skeletal dysplasia (Pasteris, N. G. and J. L. Gorski (1999) Genomics 60:57-66). Many PH domain proteins function in association with the plasma membrane, and this association appears to be mediated by the PH domain itself. PH domains share a common structure composed of two antiparallel beta sheets flanked by an amphipathic alpha helix Variable loops connecting the component beta strands generally occur within a positively charged environment and may function as ligand binding sites (Lemmon, M. A. et al. (1996) Cell 85:621-624). Ankyrin (ANK) repeats mediate protein-protein interactions associated with diverse intracellular signaling functions. For example, ANK repeats are found in proteins involved in cell proliferation such as kinases, kinase inhibitors, tumor suppressors, and cell cycle control proteins. (See, for example, Kalus, W. et al. (1997) FEBS Lett. 401:127-132; Ferrante, A. W. et al (1995) Proc. Natl. Acad. Sci. USA 92:1911-1915.) These proteins generally contain multiple ANK repeats, each composed of about 33 amino acids. Myotrophin is an ANK repeat protein that plays a key role in the development of cardiac hypertrophy, a contributing factor to many heart diseases. Structural studies show that the myotrophin ANK repeats, like other ANK repeats, each form a helix-turn-helix core preceded by a protruding “tip.” These tips are of variable sequence and may play a role in protein-protein interactions. The helix-turn-helix region of the ANK repeats stack on top of one another and are stabilized by hydrophobic interactions (Yang, Y. et al. (1998) Structure 6:619-626). Members of the ASB protein family contain a suppressor of cytokine signaling (SOCS) domain as well as multiple ankyrin repeats (Hilton, D. J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:114-119). The tetratricopeptide repeat (TPR) is a 34 amino acid repeated motif found in organisms from bacteria to humans. TPRs are predicted to form ampipathic helices, and appear to mediate protein-protein interactions. TPR domains are found in CDC16, CDC23, and CDC27, members of the anaphase promoting complex which targets proteins for degradation at the onset of anaphase. Other processes involving TPR proteins include cell cycle control, transcription repression, stress response, and protein kinase inhibition (Lamb, J. R. et al (1995) Trends Biochem. Sci. 20:257-259). The armadillo/beta-catenin repeat is a 42 amino acid motif which forms a superhelix of alpha helices when tandemly repeated. The structure of the armadillo repeat region from beta-catenin revealed a shallow groove of positive charge on one face of the superhelix, which is a potential binding surface. The armadillo repeats of beta-catenin, plakoglobin, and p120 cas bind the cytoplasmnic domains of cadherins. Beta-cateninicadherin complexes are targets of regulatory signals that govern cell adhesion and mobility (Huber, A. R et al (1997) Cell 90:871-882). Eight tandem repeats of about 40 residues (WD-40 repeats), each containing a central Trp-Asp motif, make up beta-transducin (G-beta), which is one of the three subunits (alpha, beta, and gamma) of the guanine nucleotide-binding proteins (G proteins). In higher eukaryotes G-beta exists as a small multigene family of highly conserved proteins of about 340 amino acid residues. COP1 (constitutive photomorphogenic protein) from plants and PML (promyelocytic leukemia protein) from mammals both contain RING-fingers and have similarities in cellular distribution, dynamics, and structure. They possibly function in regulating the targeting of nuclear proteins to specific nuclear compartments for degradation through the ubiquitin-proteasome pathway keyes, J. C. (2001) Trends Biochem. Sci. 26:18-20). More specifically, in the dark, COP1 accumulates in the plant nucleus where it functions in the degradation of the HY5 protein, a positive regulator of photomorphogenesis. In the light, COP1 is excluded from the nucleus allowing the constitutively nuclear HY5 protein to accumulate (Schwechheimer, C. and Deng, X. W. (2000) Semin. Cell Dev. Biol. 11:495-503). The Gab2 protein is a scaffolding protein attaching to inositol lipids at the cytoplasmic face of the plasma membrane through its PH domain. Gab2 contains a pleckstrin homology domain, and potential binding sites for proteins containing SH2- and SH3-domains as well as for 14-3-3 proteins. Gab2, like DOS (daughter of sevenless) in Drosophila, controls the development of cells. Gab2 acts downstream of a broad range of cytokine, growth factor receptors, and the T and B antigen receptors, linking these receptors to MAP kinase by somehow switching between the MAP kinase pathway and the GAB2 mediated pathway (See http://www.fhcrc.org/labs/rohrschneider/GabPagel.html; Liu, Y. et al. (2001) Mol. Cell Biol. 21:3047-3056; and Crouin, C. et al. (2001) FEBS Let. 495:148-153). The epidermal growth factor (EGF) superfamily is a diverse group of proteins that function as secreted signaling molecules, growth factors, and components of the extracellular matrix, which are involved in vertebrate development. The Scube1 (signal peptide-CUB domain-EGF-related 1) gene is a novel mammalian gene encoding an EGF-related protein with a CUB (C1s-like) domain that defines a new mammalian gene family. The Scube1 gene is on chromosome 15 and is expressed in developing gonad, nervous system, somites, surface ectoderm, and limb buds. It is similar to a human gene in the syntenic region of chromosome 22q13 (Grimmond, S. (2000) Genomics 70:74-81). Intracellular Trafficking Proteins Eukaryotic cells are bound by a lipid bilayer membrane and subdivided into functionally distinct, membrane-bound compartments. The membranes maintain the essential differences between the cytosol, the extracellular environment, and the lumenal space of each intracellular organelle. Eukaryotic proteins including integral membrane proteins, secreted proteins, and proteins destined for the lumen of organelles are synthesized within the endoplasmic reticulum (ER), delivered to the Golgi complex for post-translational processing and sorting, and then transported to specific intracellular and extracellular destinations. Material is internalized from the extracellular environment by endocytosis, a process essential for transmission of neuronal, metabolic, and proliferative signals; uptake of many essential nutrients; and defense against invading organisms. This intracellular and extracellular movement of protein molecules is termed vesicle trafficking. Trafficking is accomplished by the packaging of protein molecules into specialized vesicles which bud from the donor organelle membrane and fuse to the target membrane (Rothman, J. E and Wieland, F. T. (1996) Science 272:227-234). Several steps in the transit of material along the secretory and endocytic pathways require the formation of transport vesicles. Specifically, vesicles form at the transitional endoplasmic reticulum (tER), the rim of Golgi cisternae, the face of the Trans-Golgi Network (TGN), the plasma membrane (PM), and tubular extensions of the endosomes. Vesicle formation occurs when a region of membrane buds off from the donor organelle. The membrane-bound vesicle contains proteins to be transported and is surrounded by a proteinaceous coat, the components of which are recruited from the cytosol The initial budding and coating processes are controlled by a cytosolic ras-like GTP-binding protein, ADP-ribosylating factor (Arf), and adapter proteins (AP). Cytosolic GTP-bound Arf is also incorporated into the vesicle as it forms. Different isoforms of both Arf and AP are involved at different sites of budding. For example, Arfs 1, 3, and 5 are required for Golgi budding, Arf4 for endosomal budding, and Arf6 for plasma membrane budding. Two different classes of coat protein have also been identified. Clathrin coats form on vesicles derived from the TGN and PM, whereas coatomer (COP) coats form on vesicles derived from the ER and Golgi (Mellman, I. (1996) Annu. Rev. Cell Dev. Biol. 12:575-625). In clathrin-based vesicle formation, APs bring vesicle cargo and coat proteins together at the surface of the budding membrane. APs are heterotetrameric complexes composed of two large chains: one chain comprised of an α, γ, δ, or ε chain with a β chain, a medium chain (μ), and a small chain (σ). Clathrin binds to APs via the carboxy-terminal appendage domain of the β-adaptin subunit (Le Bourgne, R. and Hoflack, B. (1998) Curr. Opin. Cell. Biol 10:499-503). AP-1 functions in protein sorting from the TGN and endosomes to compartments of the endosomal/lysosomal system. AP-2 functions in clathrin-mediated endocytosis at the plasma membrane, while AP-3 is associated with endosomes and/or the TGN and recruit& integral membrane proteins for transport to lysosomes and lysosome-related organelles. The recently isolated AP-4 complex localizes to the TGN or a neighboring compartment and may play a role in sorting events thought to take place in post-Golgi compartments (Dell'Angelica, E. C. et al. (1999) J. Biol. Chem. 274:7278-7285). Cytosolic GTP-bound Arf is also incorporated into the vesicle as it forms. Another GTP-binding protein, dynamin, forms a ring complex around the neck of the forming vesicle and provides the mechanochemical force required to release the vesicle from the donor membrane. The coated vesicle complex is then transported through the cytosol. During the transport process, Arf-bound GTP is hydrolyzed to GDP and the coat dissociates from the transport vesicle (West, M. A. et al (1997) J. Cell Biol. 138:1239-1254). Coatomer (COP) coats, a second class of coat proteins, form on vesicles derived from the ER and Golgi. COP coats can further be classified as COPI, involved in retrograde traffic through the Golgi and from the Golgi to the ER, and COPII, involved in anterograde traffic from the ER to the Golgi (Mellman, supra). The COP coat consists of two major components, a GTP-binding protein (Arf or Sar) and coat protomer (coatomer). Coatomer is an equimolar complex of seven proteins, termed α-, β-, βα-, γ-, Δ-, ε- and Z-COP. The coatomer complex binds to dilysine motifs contained on the cytoplasmic tails of integral membrane proteins. These include the dilysine-containing retrieval motif of membrane proteins of the ER and dibasic/diphenylamine motifs of members of the p24 family. The p24 family of type I membrane proteins represents the major membrane proteins of COPI vesicles. (Harter, C. and Wieland, F. T. (1998) Proc. Natl. Acad. Sci. USA 95:11649-11654.) Vesicles can undergo homotypic, fusing with a same type vesicle, or heterotypic, fusing with a different type vesicle, fusion. Molecules required for appropriate targeting and fusion of vesicles include proteins in the vesicle membrane, the target membrane, and proteins recruited from the cytosol. During budding of the vesicle from the donor compartment, an integral membrane protein, VAMP (vesicle-associated membrane protein) is incorporated into the vesicle. Soon after the vesicle uncoats, a cytosolic prenylated GTP-binding protein, Rab, is inserted into the vesicle membrane. The amino acid sequence of Rab proteins reveals conserved GTP-binding domains characteristic of Ras superfamily members. In the vesicle membrane, GTP-bound Rab interacts with VAMP. Once the vesicle reaches the target membrane, a GTPase activating protein (GAP) in the target membrane converts the Rab protein to the GDP-bound form. A cytosolic protein, guanine-nucleotide dissociation inhibitor (GDI) then removes GDP-bound Rab from the vesicle membrane. Several Rab isoforms have been identified and appear to associate with specific compartments within the cell. For example, Rabs, 4, 5, and 11 are associated with the early endosome, whereas Rabs 7 and 9 associate with the late endosome. These differences may provide selectivity in the association between vesicles and their target membranes. (Novick, P., and Zerial, M. (1997) Cur. Opin. Cell Biol. 9:496-504.) Docking of the transport vesicle with the target membrane involves the formation of a complex between the vesicle SNAP receptor (v-SNARE), target membrane (t-) SNAREs, and certain other-membrane and cytosolic proteins. Many of these other proteins have been identified although their exact functions in the docling complex remain uncertain (Tellam, J. T. et al. (1995) J. Biol. Chem. 270:5857-5863; Hata, Y. and Sudhof, T. C. (1995) J. Biol. Chem. 270:13022-13028). N-ethylmaleimide sensitive factor (NSF) and soluble NSF-attachment protein (α-SNAP and β-SNAP) are two such proteins that are conserved from yeast to man and function in most intracellular membrane fusion reactions. Sec1 represents a family of yeast proteins that function at many different stages in the secretory pathway including membrane fusion. Recently, mammalian homologs of Sec1, called Munc-18 proteins, have been identified (Katagiri, X et al. (1995) J. Biol Chem. 270:4963-4966; Hata et al. sutra). The SNARE complex involves three SNARE molecules, one in the vesicular membrane and two in the target membrane. Together they form a rod-shaped complex of four α-helical coiled-coils. The membrane anchoring domains of all three SNAREs project from one end of the rod. This complex is similar to the rod-like structures formed by fusion proteins characteristic of the enveloped viruses, such as myxovirus, influenza, filovirus (Ebola), and the HIV and SIV retroviruses (Skehel, J. J., and Wiley, D. C. (1998) Cell 95:871-874). It has been proposed that the SNARE complex is sufficient for membrane fusion, suggesting that the proteins which associate with the complex provide regulation over the fusion event (Weber, T. et al. (1998) Cell 92:759-772). For example, in neurons, which exhibit regulated exocytosis, docked vesicles do not fuse with the presynaptic membrane until depolarization, which leads to an influx of calcium (Bennett, M. K., and Scheller, R. H. (1994) Annu. Rev. Biochem. 63:63-100). Synaptotagmin, an integral membrane protein in the synaptic vesicle, associates with the t-SNARE syntaxin in the docking complex. Synaptotagmin binds calcium in a complex with negatively charged phospholipids, which allows the cytosolic SNAP protein to displace synaptotagmin from syntaxin and fusion to occur. Thus, synaptotagmin is a negative regulator of fusion in the neuron. (Littleton, J. T. et al. (1993) Cell 74:1125-1134.) In many cases the tSNARE exists as a complex of syntaxin with a member of the syntaptosome-associated protein-25 (SNAP-25) family of palmitoylated proteins. In neurons and neuroendocrine cells, the tSNAREs consist of syntaxin and SNAP-25, while SNAP-23 replaces SNAP-25 in nonneuronal tissues (Ravichindran, V. et al (1996) J. Biol. Chem. 271:13300-13303). The human SNAP-23 gene was recently mapped to human chromosome region 15q15-21. Several neurological syndromes have been mapped to this region, including some forms of schizophrenia, autism, epilepsy, and a variant of late infantile neuronal ceroid lipofuccinosis in which there is an accumulation of large intracellular vesicles. Alterations of membrane fusion proteins is highly likely to result in distinct clinical syndromes, as in the case of Williams syndrome, a neurological defect resulting from hemizygous deletions of the syntaxin 1A gene. Therefore SNAP-23 is considered to be a candidate gene for any of the neurological syndromes that map to its region of human chromosome 15 (Lazo, P. A. et al. (2001) Hum. Genet. 108:211-215). The etiology of numerous other human diseases and disorders can be attributed to defects in the trafficking of proteins to organelles or the cell surface. Defects in the trafficking of membrane-bound receptors and ion channels are associated with cystic fibrosis (cystic fibrosis transmembrane conductance regulator; CFTR), glucose-galactose malabsorption syndrome (Na+/glucose cotransporter), hypercholesterolemia (low-density lipoprotein (LDL) receptor), and forms of diabetes mellitus (insulin receptor). Abnormal hormonal secretion is linked to disorders including diabetes insipidus (vasopressin), hyper- and hypoglycemia (insulin, glucagon), Grave's disease and goiter (thyroid hormone), and Cushing's and Addison's diseases (adrenocorticotropic hormone; ACTH). Cancer cells secrete excessive amounts of hormones or other biologically active peptides. Disorders related to excessive secretion of biologically active peptides by tumor cells include: fasting hypoglycemia due to increased insulin secretion from insulinoma-islet cell tumors; hypertension due to increased epinephrine and norepinephrine secreted from pheochromocytomas of the adrenal medulla and sympathetic paraganglia; and carcinoid syndrome, which includes abdominal cramps, diarrhea, and valvular heart disease, caused by excessive amounts of vasoactive substances (serotonin, bradykinin, histamine, prostaglandins, and polypeptide hormones) secreted from intestinal tumors. Ectopic synthesis and secretion of biologically active peptides (peptides not expected from a tumor) includes s ACTH and vasopressin in lung and pancreatic cancers; parathyroid hormone in lung and bladder cancers; calcitonin in lung and breast cancers; and thyroid-stimulating hormone in medullary thyroid carcinoma. Various human pathogens alter host cell protein trafficking pathways to their own advantage. For example, the HIV protein Nef down-regulates cell surface expression of CD4 molecules by accelerating their endocytosis through clathrin coated pits. This function of Nef is important for the spread of H[V from the infected cell (Harris, M. (1999) Curr. Biol. 9:R449-R461). A recently identified human protein, Nef-associated factor 1 (Naf1), a protein with four extended coiled-coil domains, has been found to associate with Nef. Overexpression of Naf1 increased cell surface expression of CD4, an effect which could be suppressed by Nef (Fukushi, M. et al. (1999) FEBS Lett. 442:83-88). PACS-1 (phosphofurin acidic cluster sorting protein-1) controls the endosome to Golgi trafficking of integral membrane proteins that contain acidic cluster sorting motifs, including furin, Nef, and herpes virus envelope glycoproteins, by connecting the acidic cluster domains of these proteins with AP-1. Nef downregulates the surface expression of major histocompatibility complex class I (MHC-1) proteins, thereby promoting immune evasion by HN-1. This process is dependent upon binding of Nef to PACS-1 (Piguet, V. et al. (2000) Nature Cell Biol. 2:163-167). A PACS-1 mutant altered in the adaptor binding site was able to disrupt the Nef-dependent redistribution of MHC-1, suggesting the possibility of controlling IRV immune evasion through inlubtion of PACS-1 (Crump, C. M. et al. (2001) EMBO J. 20:2191-2201). Expression Profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. The potential application of gene expression profiling is particularly relevant to improving the diagnosis, prognosis, and treatment of cancers, such as lung cancer. Lung Cancer Lung cancer is the leading cause of cancer death in the United States, affecting more than 100,000 men and 50,000 women each year. Nearly 90% of the patients diagnosed with lung cancer are cigarette smokers. Tobacco smoke contains thousands of noxious substances that induce carcinogen metabolizing enzymes and covalent DNA adduct formation in the exposed bronchial epithelium. In nearly 80% of patients diagnosed with lung cancer, metastasis has already occurred. Most commonly lung cancers metastasize to pleura, brain, bone, pericardium, and liver. The decision to treat with surgery, radiation therapy, or chemotherapy is made on the basis of tumor histology, response to growth factors or hormones, and sensitivity to inhibitors or drugs. With current treatments, most patients die within one year of diagnosis. Earlier diagnosis and a systematic approach to identification, staging, and treatment of lung cancer could positively affect patient outcome. Lung cancers progress through a series of morphologically distinct stages from hyperplasia to invasive carcinoma. Malignant lung cancers are divided into two groups comprising four histopathological classes. The Non Small Cell Lung Carcinoma (NSCLC) group includes squamous cell carcinomas, adenocarcinomas, and large cell carcinomas and accounts for about 70% of all lung cancer cases. Adenocarcinomas typically arise in the peripheral airways and often form mucin secreting glands. Squamous cell carcinomas typically arise in proximal airways. The histogenesis of squamous cell carcinomas maybe related to chronic inflammation and injury to the bronchial epithelium, leading to squamous metaplasia. The Small Cell Lung Carcinoma (SCLC) group accounts for about 20% of lung cancer cases. SCLCs typically arise in proximal airways and exbibit a number of paraneoplastic syndromes including inappropriate production of adrenocorticotropin and anti-diuretic hormone. Lung cancer cells accumulate numerous genetic lesions, many of which are associated with cytologically visible chromosomal aberrations. The high frequency of chromosomal deletions associated with lung cancer may reflect the role of multiple tumor suppressor loci in the etiology of this disease. Deletion of the short arm of chromosome 3 is found in over 90% of cases and represents one of the earliest genetic lesions leading to lung cancer. Deletions at chromosome arms 9p and 17p are also common. Other frequently observed genetic lesions include overexpression of telomerase, activation of oncogenes such as K-ras and c-myc, and inactivation of tumor suppressor genes such as RB, p53 and CDKN2. Genes differentially regulated in lung cancer have been identified by a variety of methods. Using mRNA differential display technology, Manda et al. (1999; Genomics 51:5-14) identified five genes differentially expressed in hug cancer cell lines compared to normal bronchial epithelial cells. Among the known genes, pulmonary surfactant apoprotein A and alpha 2 macroglobulin were down regulated whereas nm23H1 was upregulated. Petersen et al. (2000; Int J. Cancer, 86:512-517) used suppression subtractive hybridization to identify 552 clones differentially expressed in lung tumor derived cell lines, 205 of which represented known genes. Among the known genes, thrombospondin-1, fibronectin, intercellular adhesion molecule 1, and cytokeratins 6 and 18 were previously observed to be differentially expressed in lung cancers. Wang et al. (2000; Oncogene 19:1519-1528) used a combination of microarray analysis and subtractive hybridization to identify 17 genes differentially overexpresssed in squamous cell carcinoma compared with normal lung epithelium. Among the known genes they identified were keratin isoform 6, KOC, SPRC, IGFb2, connexin 26, plakofillin 1 and cytokeratin 13. Alzheimer's Disease The potential application of gene expression profiling is also particularly relevant to improving diagnosis, prognosis, and treatment of neurological disorders, such as Alzheimer's disease (AD). Characterization of region-specific gene expression in the human brain provides a context and background for molecular neurobiology on a variety of neurological disorders. For example, AD is a progressive, neurodestructive process of the human neocortex, characterized by the deterioration of memory and higher cognitive function. A progressive and irreversible brain disorder, AD is characterized by three major pathogenic episodes involving (a) an aberrant processing and deposition of beta-amyloid precursor protein (betaAPP) to form neurotoxic beta-amyloid (betaA) peptides and an aggregated insoluble polymer of betaA that forms the senile plaque, (b) the establishment of intraneuronal neuritic tau pathology yielding widespread deposits of agyrophilic neurofibrillary tangles (NFT) and (c) the initiation and proliferation of a brain-specific inflammatory response. These three seemingly disperse attributes of AD etiopathogenesis are linked by the fact that proinflammatory microglia, reactive astrocytes and their associated cytokines and chemokines are associated with the biology of the microtubule associated protein tan, betaA speciation and aggregation. Missense mutations in the presenilin genes PS1 and PS2, implicated in early onset familial AD, cause abnormal betaAPP processing with resultant overproduction of betaA42 and related neurotoxic peptides. Specific betaA fragments such as betaA42 can further potentiate proinflammatory mechanisms. Expression of the inducible oxidoreductase cyclooxygenase-2 and cytosolic phospholipase A2 (cPLA2) are strongly activated during cerebral ischemia and trauma, epilepsy and AD, indicating the induction of proinflammatory gene pathways as a response to brain injury. Neurotoxic metals such as aluminum and zinc, both implicated in AD etiopathogenesis, and arachidonic acid, a major metabolite of brain cPLA2 activity, each polymerize hyperphosphorylated tan to form NFT-like bundles. Studies have identified a reduced risk for AD in patients aged over 70 years who were previously treated with non-steroidal anti-inflammatory drugs for non-CNS afflictions that include arthritis. (For a review of the interrelationships between the mechanisms of PS1, PS2 and betaAPP gene expression, tau and betaA deposition and the induction, regulation and proliferation in AD of the neuroinflammatory response, see Lukiw W. J, and Bazan N. G.(2000) Neurochem. Res. 2000 25:1173-1184). One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of cell proliferative, autoimmune/inflammatory, neurological, gastrointestinal, reproductive, developmental, and vesicle trafficking disorders. |
<SOH> SUMMARY OF THE INVENTION <EOH>Various embodiments of the invention provide purified polypeptides, intracellular signaling molecules, referred to collectively as “INTSIG” and individually as “INTSIG-1,” “INTSIG-2,” ΘINTSIG-3,” “INTSIG-4,” “INTSIG-5,” “INTSIG-6,” “INTSIG-7,” “INTSIG-8,” “INTSIG-9,” “INTSIG-10,” “INTSIG-11,” “INTSIG-12,” “INTSIG-13,” “INTSIG-14,” “INTSIG-15,” “INTSIG-16,” “INTSIG-17,” “INTSIG-18,” “INTSIG-19,” and ”INTSIG-20,” and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified intracellular signaling molecules and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified intracellular signaling molecules and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions. An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-20. Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-20. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:21-40. Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ D) NO:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20. Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed. Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20. Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof. Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and a pharmaceutically acceptable excipient In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional INTSIG, comprising administering to a patient in need of such treatment the composition. Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) abiologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional INTSIG, comprising administering to a patient in need of such treatment the composition. Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional INTSIG, comprising administering to a patient in need of such treatment the composition. Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide. Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide. Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of in), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, iin) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. |
Methods of treating alzheimer's disease |
Disclosed are methods for treating Alzheimer's disease, and other diseases, and/or inhibiting beta-secretase enzyme, and/or inhibiting deposition of A beta peptide in a mammal, by use of hydrazine compounds of formula (I) wherein the variables R1-R9 are defined herein. |
1. A method of treating or preventing Alzheimer's disease in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof: wherein R1 and R9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl, alkenyl or alkynyl; heterocyclyl; sulfo; sulfonyl substituted by unsubstituted or substituted alkyl, aryl, heterocyclyl, alkoxy, which is unsubstituted or substituted, or by aryloxy; sulfamoyl that is unsubstituted or substituted at the nitrogen atom; or phosphoryl substituted by one or two radicals, which may be identical or different, selected from unsubstituted or substituted alkyl, from unsubstituted or substituted cycloalkyl, from aryl, from hydroxy, from unsubstituted or substituted alkoxy, from cycloalkoxy and from aryloxy; with the proviso that not more than one of the radicals R1 and R9 is hydrogen; R2 and R8 are each independently of the other hydrogen or one of the radicals mentioned above for R1 and R9; or the pairs of substituents R1 and R2, and R8 and R9, each independently of the other, may form together with the nitrogen atom to which they are bonded a heterocyclic ring consisting of the bonding nitrogen atom together with a radical selected from ethylene, trimethylene, tetramethylene and pentamethylene in which a carbon atom may have been replaced by nitrogen, oxygen, sulfur or by sulfur mono- or di-substituted by oxygen and which may be unsaturated, or one of those radicals with an oxo substituent at each of the two carbon atoms linked to the bonding carbon atom and with or without a fused-on benzene or naphthalene ring; R3 and R4 are each independently of the other hydrogen, unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; or R3 and R4 together form unsubstituted or substituted alkylene, alkylidene or benzo-fused alkylene; R5 is hydroxy; R6 is hydrogen; or R5 and R6 together are oxo; and R7 is unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl. 2. A method of treating Alzheimer's disease in a patient in need of such treatment comprising administering to the patient a compound disclosed in claim 1, or a pharmaceutically acceptable salt thereof. 3. A method of treating Alzheimer's disease by modulating the activity of beta amyloid converting enzyme, comprising administering to a patient in need of such treatment a compound disclosed in claim 1, or a pharmaceutically acceptable salt thereof. 4. The method according to claim 1, further comprising the administration of a P-gp inhibitor, or a pharmaceutically acceptable salt thereof. 5. A method of treating a patient who has, or in preventing a patient from getting, a disease or condition selected from the group consisting of Alzheimer's disease, for helping prevent or delay the onset of Alzheimer's disease, for treating patients with mild cognitive impairment (MCI) and preventing or delaying the onset of Alzheimer's disease in those who would progress from MCI to AD, for treating Down's syndrome, for treating humans who have Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type, for treating cerebral amyloid angiopathy and preventing its potential consequences, i.e. single and recurrent lobar hemorrhages, for treating other degenerative dementias, including dementias of mixed vascular and degenerative origin, dementia associated with Parkinson's disease, dementia associated with progressive supranuclear palsy, dementia associated with cortical basal degeneration, or diffuse Lewy body type of Alzheimer's disease and who is in need of such treatment which includes administration of a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof: wherein R1 and R9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl, alkenyl or alkynyl; heterocyclyl; sulfo; sulfonyl substituted by unsubstituted or substituted alkyl, aryl, heterocyclyl, alkoxy, which is unsubstituted or substituted, or by aryloxy; sulfamoyl that is unsubstituted or substituted at the nitrogen atom; or phosphoryl substituted by one or two radicals, which may be identical or different, selected from unsubstituted or substituted alkyl, from unsubstituted or substituted cycloalkyl, from aryl, from hydroxy, from unsubstituted or substituted alkoxy, from cycloalkoxy and from aryloxy; with the proviso that not more than one of the radicals R1 and R9 is hydrogen; R2 and R8 are each independently of the other hydrogen or one of the radicals mentioned above for R1 and R9; or the pairs of substituents R1 and R2, and R8 and R9, each independently of the other, may form together with the nitrogen atom to which they are bonded a heterocyclic ring consisting of the bonding nitrogen atom together with a radical selected from ethylene, trimethylene, tetramethylene and pentamethylene in which a carbon atom may have been replaced by nitrogen, oxygen, sulfur or by sulfur mono- or di-substituted by oxygen and which may be unsaturated, or one of those radicals with an oxo substituent at each of the two carbon atoms linked to the bonding carbon atom and with or without a fused-on benzene or naphthalene ring; R3 and R4 are each independently of the other hydrogen, unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; or R3 and R4 together form unsubstituted or substituted alkylene, alkylidene or benzo-fused alkylene; R5 is hydroxy; R6 is hydrogen; or R5 and R6 together are oxo; and R7 is unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl. 6. The method according to claim 5 wherein the compound of formula (I) is selected from the group consisting of: 1-[2(S)-(2-pyridylcarbonyl)oxy-3(S)-(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino4-phenylbutyl-1-[phenylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-butyryloxy-3(S)-(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenylbutyl-1-[phenylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(2-pyridylcarbonyl)oxy-3(S)-(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino4-phenylbutyl-1-[4-methoxyphenylmethyl]-2-[N(benzyloxycarbonyl)-(L) valyl]hydrazine; 1-[2(S)-(methoxy-acetyl)oxy-3(S)-(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenylbutyl-1-[4-methoxyphenylmethyl]-2-[N(benzyloxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-propionyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-butyryloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-pentanoyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-(2(S)-octanoyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-decanoyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-dodecanoyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-pivaloyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(2-furylcarbonyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(4-imidazolylcarbonyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(4-imidazolylacetyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(3-(4-imidazolyl)-propionyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-benzoyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(2-pyridylacetyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(3-(pyridin-2-yl)-propionyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenylbutyl)]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(quinolin-2-ylcarbonyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(aminoacetyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(N-methylaminoacetyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(N,N-dimethylaminoacetyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(N-benzyloxycarbonyl-N-methyl-aminoacetyl)oxy-3(S)-(N-(methoxycarbonyl)(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl) (L)-valyl]hydrazine; 1-[2(S)-prolyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(4-morpholinomethylbenzoyl)oxy-3 (S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(4-chloromethylbenzoyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(3-carboxypropionyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(benzyloxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[tert-butoxy-carbonyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[3,3-dimethylbutyryl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[tert-butylamino-carbonyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[benzylamino-carbonyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[benzyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[benzyl]-2-[N-ethoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[benzyl]-2-[N-benzyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[benzyl]-2-[N-allyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-.sup.2 carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4-methoxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L) valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4-methoxyphenylmethyl]-2-[N-benzyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4-methoxyphenylmethyl]-2-[N-allyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-(4-methoxyphenyl)-butyl]-1-[benzyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-(4-methoxyphenyl)-butyl]-1-[benzyl]-2-[N-benzyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-(4-benzyloxyphenyl)-butyl]-1-[benzyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-(4-benzyloxyphenyl)-butyl]-1-[benzyl]-2-[N-allyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4methoxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4-methoxyphenylmethyl]-2-[N-allyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4-benzyloxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[2(S)-hydroxy-3(S)—(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenylbutyl-1-[phenylmethyl]-2-[N-(2-(2-methoxyethoxy)ethoxycarbonyl)-(L)-valyl]hydrazine 1-[2(S)-hydroxy-3(S)-(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenylbutyl-1-[4-methoxyphenylmethyl]-2-[tert-butoxy-carbonyl]hydrazine; and 1-[2(S)-hydroxy-3(S)-(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenylbutyl-1-[4-methoxyphenylmethyl]-2-[N-(ethoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(2-methoxyethoxy)acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-(2-methoxyethoxy)acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(2-(2-methoxyethoxy)ethoxy)carbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-(2-(2-methoxyethoxy)ethoxy)carbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-ylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-yl-methyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-yl-methyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-(2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-ylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[thien-2-ylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-ylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-ylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[2, 3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6tetrahydropyran-4-ylmethyl]-2-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl)-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)-aminocarbonyl)(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3 (S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]-hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3 (S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine: 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylmethyl]-2-N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine and 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-trifluoroacetyl-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-trifluoroacetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(methoxy-carbonyl)-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[cyclohexylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(n-propoxy-carbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(n-propyl)oxy-carbonyl-(L)-valyl]hydrazine; 1-[2(R)-hydroxy-3(S)-(N-(methoxy-carbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1-[2(R)-hydroxy-3(R)—(N-(methoxy-carbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(benzyloxy-carbonyl-amino)-4-phenyl-butyl]-1-[phenylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(methoxy-carbonyl)-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[phenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[4-methoxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[4-isobutoxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[4-ethoxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[4-benzyloxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[2-pyridylcarbonyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(benzyloxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[tert-butoxy-carbonyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[3,3-dimethylbutyryl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[tert-butylamino-carbonyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[benzylamino-carbonyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[benzyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[benzyl]-2-[N-ethoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[benzyl]-2-[N-benzyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[benzyl]-2-[N-allyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-.sup.2 carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4-methoxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4-methoxyphenylmethyl]-2-[N-benzyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4-methoxyphenylmethyl]-2-[N-allyloxy-carbonyl)-(L)-valyl]hydrazine 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-(4-methoxyphenyl)-butyl]-1-[benzyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-(4-methoxyphenyl)-butyl]-1-[benzyl]-2-[N-benzyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-(4-benzyloxyphenyl)-butyl]-1-[benzyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-(4-benzyloxyphenyl)-butyl]-1-[benzyl]-2-[N-allyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4-methoxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4-methoxyphenylmethyl]-2-[N-allyloxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[4-benzyloxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenyl-butyl]-1-[2(S)-hydroxy-3(S)—(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenylbutyl-1-[phenylmethyl]-2-[N-(2-(2-methoxyethoxy)ethoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenylbutyl-1-[4-methoxyphenylmethyl]-2-[tert-butoxy-carbonyl]hydrazine; and 1-[2(S)-hydroxy-3(S)-(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenylbutyl-1-[4-methoxyphenylmethyl]-2-[N-(ethoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-allyloxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-allyloxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(2-(2-methoxyethoxy)ethoxy)carbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-(2-(2-methoxyethoxy)ethoxy)carbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(2-methoxyethoxy)acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-(2-methoxyethoxy)acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-ylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-yl-methyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-yl-methyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-ylmethyl]-2-[N—(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3 (S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[thien-2-ylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-ylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[thien-2-ylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran4-ylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-(N,N-dimethylaminocarbo nyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; and 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[2,3,5,6-tetrahydropyran-4-ylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3 (S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-hydroxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3 (S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-methoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl)-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)-aminocarbonyl)(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-benzyloxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3 (S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(3,4-dimethoxybenzyl)oxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]-hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-isobutoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-(2-methoxyethoxy)phenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3 (S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[methylene-3,4-dioxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[3, 4-dimethoxyphenylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine: 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3 (S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[3,4-dimethoxyphenylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)-N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-acetyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-acetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylmethyl]-2-N-methoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N,N-dimethylaminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-(N,N-dimethylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3 (S)-(N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-(N-(2-methoxyethyl)aminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)aminocarbonyl)-(L)-valyl]hydrazine and 1-[2(S)-hydroxy-3(S)-(N-(N-(2-(morpholin-4-yl)ethyl)-N-methyl-aminocarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[4-biphenylylmethyl]-2-[N-(N-(2-(morpholin-4-yl)ethyl)N-methylaminocarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-ethoxycarbonyl-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-ethoxycarbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-trifluoroacetyl-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-trifluoroacetyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(methoxy-carbonyl)-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[cyclohexylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(n-propoxy-carbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(n-propyl)oxy-carbonyl-(L)-valyl]hydrazine; 1-[2(R)-hydroxy-3(S)-(N-(methoxy-carbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1-[2(R)-hydroxy-3(R)-(N-(methoxy-carbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(benzyloxy-carbonyl-amino)-4-phenyl-butyl]-1-[phenylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-(methoxy-carbonyl)-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[phenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[4-methoxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[4-isobutoxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[4-ethoxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; 1-[2(S)-hydroxy-3(S)-(N-methoxycarbonyl-(L)-valyl)amino-4-cyclohexyl-butyl]-1-[4-benzyloxyphenylmethyl]-2-[N-methoxy-carbonyl)-(L)-valyl]hydrazine; N-[(1S,2R)-3-(2-benzoyl-1-ethylhydrazino)-1-benzyl-2-hydroxypropyl]-2-[(methylsulfonyl)amino]-1,3-thiazole-4-carboxamide; N-{(1S,2R)-1-benzyl-3-[1-ethyl-2-(4-methylpentanoyl)hydrazino]-2-hydroxypropyl}-2-[(methylsulfonyl)amino]-1,3-thiazole-4-carboxamide; N-{(1S,2R)-1-benzyl-3-[1-ethyl-2-(4-methylpentanoyl)hydrazino]-2-hydroxypropyl}-4-methylpentanamide; N1-{(1S,2S)-1-benzyl-3-[1-ethyl-2-(4-methylpentanoyl)hydrazino]-2-hydroxypropyl}-5-methyl-N3,N3-dipropylisophthalamide; N1-{(1S,2S)-1-(3,5-difluorobenzyl)-3-[1-ethyl-2-(4-methylpentanoyl)hydrazino]-2-hydroxypropyl}-5-methyl-N3,N3-dipropylisophthalamide; N1-{(1S,2S)-1-(3,5-difluorobenzyl)-3-[1-ethyl-2-(4-methylbutanoyl)hydrazino]-2-hydroxypropyl}-5-methyl-N3,N3-dipropylisophthalamide; or pharmaceutically acceptable salts thereof. 7. A method of treating or preventing Alzheimer's disease in a patient in need of such treatment comprising administering a therapeutically effective amount of a composition comprising one or more pharmaceutically acceptable carriers and a compound of Formula (I) or a pharmaceutically acceptable salt thereof: wherein R1 and R9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl, alkenyl or alkynyl; heterocyclyl; sulfo; sulfonyl substituted by unsubstituted or substituted alkyl, aryl, heterocyclyl, alkoxy, which is unsubstituted or substituted, or by aryloxy; sulfamoyl that is unsubstituted or substituted at the nitrogen atom; or phosphoryl substituted by one or two radicals, which may be identical or different, selected from unsubstituted or substituted alkyl, from unsubstituted or substituted cycloalkyl, from aryl, from hydroxy, from unsubstituted or substituted alkoxy, from cycloalkoxy and from aryloxy; with the proviso that not more than one of the radicals R1 and R9 is hydrogen; R2 and R8 are each independently of the other hydrogen or one of the radicals mentioned above for R1 and R9; or the pairs of substituents R1 and R2, and R8 and R9, each independently of the other, may form together with the nitrogen atom to which they are bonded a heterocyclic ring consisting of the bonding nitrogen atom together with a radical selected from ethylene, trimethylene, tetramethylene and pentamethylene in which a carbon atom may have been replaced by nitrogen, oxygen, sulfur or by sulfur mono- or di-substituted by oxygen and which may be unsaturated, or one of those radicals with an oxo substituent at each of the two carbon atoms linked to the bonding carbon atom and with or without a fused-on benzene or naphthalene ring; R3 and R4 are each independently of the other hydrogen, unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; or R3 and R4 together form unsubstituted or substituted alkylene, alkylidene or benzo-fused alkylene; R5 is hydroxy; R6 is hydrogen; or R5 and R6 together are oxo; and R7 is unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl. 8. (canceled) 9. A method for inhibiting beta-secretase activity, comprising contacting an effective amount for inhibition of a compound of formula (I): wherein R1 and R9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl, alkenyl or alkynyl; heterocyclyl; sulfo; sulfonyl substituted by unsubstituted or substituted alkyl, aryl, heterocyclyl, alkoxy, which is unsubstituted or substituted, or by aryloxy; sulfamoyl that is unsubstituted or substituted at the nitrogen atom; or phosphoryl substituted by one or two radicals, which may be identical or different, selected from unsubstituted or substituted alkyl, from unsubstituted or substituted cycloalkyl, from aryl, from hydroxy, from unsubstituted or substituted alkoxy, from cycloalkoxy and from aryloxy; with the proviso that not more than one of the radicals R1 and R9 is hydrogen; R2 and R8 are each independently of the other hydrogen or one of the radicals mentioned above for R1 and R9; or the pairs of substituents R1 and R2, and R8 and R9, each independently of the other, may form together with the nitrogen atom to which they are bonded a heterocyclic ring consisting of the bonding nitrogen atom together with a radical selected from ethylene, trimethylene, tetramethylene and pentamethylene in which a carbon atom may have been replaced by nitrogen, oxygen, sulfur or by sulfur mono- or di-substituted by oxygen and which may be unsaturated, or one of those radicals with an oxo substituent at each of the two carbon atoms linked to the bonding carbon atom and with or without a fused-on benzene or naphthalene ring; R3 and R4 are each independently of the other hydrogen, unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; or R3 and R4 together form unsubstituted or substituted alkylene, alkylidene or benzo-fused alkylene; R5 is hydroxy; R6 is hydrogen; or R5 and R6 together are oxo; and R7 is unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl. 10. A method for inhibiting cleavage of an amyloid precursor protein (APP) isotype at a site in the APP isotype that is susceptible to cleavage, comprising contacting said APP isotype with an effective cleavage inhibitory amount of a compound of formula (I): wherein R1 and R9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl, alkenyl or alkynyl; heterocyclyl; sulfo; sulfonyl substituted by unsubstituted or substituted alkyl, aryl, heterocyclyl, alkoxy, which is unsubstituted or substituted, or by aryloxy; sulfamoyl that is unsubstituted or substituted at the nitrogen atom; or phosphoryl substituted by one or two radicals, which may be identical or different, selected from unsubstituted or substituted alkyl, from unsubstituted or substituted cycloalkyl, from aryl, from hydroxy, from unsubstituted or substituted alkoxy, from cycloalkoxy and from aryloxy; with the proviso that not more than one of the radicals R1 and R9 is hydrogen; R2 and R8 are each independently of the other hydrogen or one of the radicals mentioned above for R1 and R9; or the pairs of substituents R1 and R2, and R8 and R9, each independently of the other, may form together with the nitrogen atom to which they are bonded a heterocyclic ring consisting of the bonding nitrogen atom together with a radical selected from ethylene, trimethylene, tetramethylene and pentamethylene in which a carbon atom may have been replaced by nitrogen, oxygen, sulfur or by sulfur mono- or di-substituted by oxygen and which may be unsaturated, or one of those radicals with an oxo substituent at each of the two carbon atoms linked to the bonding carbon atom and with or without a fused-on benzene or naphthalene ring; R3 and R4 are each independently of the other hydrogen, unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; or R3 and R4 together form unsubstituted or substituted alkylene, alkylidene or benzo-fused alkylene; R5 is hydroxy; R6 is hydrogen; or R5 and R6 together are oxo; and R7 is unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl. 11. A method for inhibiting production of amyloid beta peptide (A beta) in a cell, comprising administering to said cell an effective inhibitory amount of a compound of formula (I): wherein R1 and R9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl, alkenyl or alkynyl; heterocyclyl; sulfo; sulfonyl substituted by unsubstituted or substituted alkyl, aryl, heterocyclyl, alkoxy, which is unsubstituted or substituted, or by aryloxy; sulfamoyl that is unsubstituted or substituted at the nitrogen atom; or phosphoryl substituted by one or two radicals, which may be identical or different, selected from unsubstituted or substituted alkyl, from unsubstituted or substituted cycloalkyl, from aryl, from hydroxy, from unsubstituted or substituted alkoxy, from cycloalkoxy and from aryloxy; with the proviso that not more than one of the radicals R1 and R9 is hydrogen; R2 and R8 are each independently of the other hydrogen or one of the radicals mentioned above for R1 and R9; or the pairs of substituents R1 and R2, and R8 and R9, each independently of the other, may form together with the nitrogen atom to which they are bonded a heterocyclic ring consisting of the bonding nitrogen atom together with a radical selected from ethylene, trimethylene, tetramethylene and pentamethylene in which a carbon atom may have been replaced by nitrogen, oxygen, sulfur or by sulfur mono- or di-substituted by oxygen and which may be unsaturated, or one of those radicals with an oxo substituent at each of the two carbon atoms linked to the bonding carbon atom and with or without a fused-on benzene or naphthalene ring; R3 and R4 are each independently of the other hydrogen, unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; or R3 and R4 together form unsubstituted or substituted alkylene, alkylidene or benzo-fused alkylene; R5 is hydroxy; R6 is hydrogen; or R5 and R6 together are oxo; and R7 is unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl. 12. The method of claim 11, wherein the cell is an animal cell. 13. The method of claim 12, wherein the animal cell is a mammalian cell. 14. The method of claim 13, wherein the mammalian cell is human. 15. A composition comprising beta-secretase complexed with a compound of formula (I), wherein R1 and R9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl, alkenyl or alkynyl; heterocyclyl; sulfo; sulfonyl substituted by unsubstituted or substituted alkyl, aryl, heterocyclyl, alkoxy, which is unsubstituted or substituted, or by aryloxy; sulfamoyl that is unsubstituted or substituted at the nitrogen atom; or phosphoryl substituted by one or two radicals, which may be identical or different, selected from unsubstituted or substituted alkyl, from unsubstituted or substituted cycloalkyl, from aryl, from hydroxy, from unsubstituted or substituted alkoxy, from cycloalkoxy and from aryloxy; with the proviso that not more than one of the radicals R1 and R9 is hydrogen; R2 and R8 are each independently of the other hydrogen or one of the radicals mentioned above for R1 and R9; or the pairs of substituents R1 and R2, and R8 and R9, each independently of the other, may form together with the nitrogen atom to which they are bonded a heterocyclic ring consisting of the bonding nitrogen atom together with a radical selected from ethylene, trimethylene, tetramethylene and pentamethylene in which a carbon atom may have been replaced by nitrogen, oxygen, sulfur or by sulfur mono- or di-substituted by oxygen and which may be unsaturated, or one of those radicals with an oxo substituent at each of the two carbon atoms linked to the bonding carbon atom and with or without a fused-on benzene or naphthalene ring; R3 and R4 are each independently of the other hydrogen, unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; or R3 and R4 together form unsubstituted or substituted alkylene, alkylidene or benzo-fused alkylene; R5 is hydroxy; R6 is hydrogen; or R5 and R6 together are oxo; and R7 is unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl. 16. A method for producing a beta-secretase complex comprising the composition of claim 15. 17. A method for inhibiting the production of beta-amyloid plaque in an animal, comprising administering to said animal an effective inhibiting amount of a compound of formula (I): wherein R1 and R9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl, alkenyl or alkynyl; heterocyclyl; sulfo; sulfonyl substituted by unsubstituted or substituted alkyl, aryl, heterocyclyl, alkoxy, which is unsubstituted or substituted, or by aryloxy; sulfamoyl that is unsubstituted or substituted at the nitrogen atom; or phosphoryl substituted by one or two radicals, which may be identical or different, selected from unsubstituted or substituted alkyl, from unsubstituted or substituted cycloalkyl, from aryl, from hydroxy, from unsubstituted or substituted alkoxy, from cycloalkoxy and from aryloxy; with the proviso that not more than one of the radicals R1 and R9 is hydrogen; R2 and R8 are each independently of the other hydrogen or one of the radicals mentioned above for R1 and R9; or the pairs of substituents R1 and R2, and R8 and R9, each independently of the other, may form together with the nitrogen atom to which they are bonded a heterocyclic ring consisting of the bonding nitrogen atom together with a radical selected from ethylene, trimethylene, tetramethylene and pentamethylene in which a carbon atom may have been replaced by nitrogen, oxygen, sulfur or by sulfur mono- or di-substituted by oxygen and which may be unsaturated, or one of those radicals with an oxo substituent at each of the two carbon atoms linked to the bonding carbon atom and with or without a fused-on benzene or naphthalene ring; R3 and R4 are each independently of the other hydrogen, unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; or R3 and R4 together form unsubstituted or substituted alkylene, alkylidene or benzo-fused alkylene; R5 is hydroxy; R6 is hydrogen; or R5 and R6 together are oxo; and R7 is unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl. 18. The method of claim 17, wherein said animal is a human. 19. A method for treating or preventing a disease characterized by beta-amyloid deposits on or in the brain, comprising administering to a patient in need of such treatment or prevention an effective therapeutic amount of a compound of formula (I): wherein R1 and R9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl, alkenyl or alkynyl; heterocyclyl; sulfo; sulfonyl substituted by unsubstituted or substituted alkyl, aryl, heterocyclyl, alkoxy, which is unsubstituted or substituted, or by aryloxy; sulfamoyl that is unsubstituted or substituted at the nitrogen atom; or phosphoryl substituted by one or two radicals, which may be identical or different, selected from unsubstituted or substituted alkyl, from unsubstituted or substituted cycloalkyl, from aryl, from hydroxy, from unsubstituted or substituted alkoxy, from cycloalkoxy and from aryloxy; with the proviso that not more than one of the radicals R1 and R9 is hydrogen; R2 and R8 are each independently of the other hydrogen or one of the radicals mentioned above for R1 and R9; or the pairs of substituents R1 and R2, and R8 and R9, each independently of the other, may form together with the nitrogen atom to which they are bonded a heterocyclic ring consisting of the bonding nitrogen atom together with a radical selected from ethylene, trimethylene, tetramethylene and pentamethylene in which a carbon atom may have been replaced by nitrogen, oxygen, sulfur or by sulfur mono- or di-substituted by oxygen and which may be unsaturated, or one of those radicals with an oxo substituent at each of the two carbon atoms linked to the bonding carbon atom and with or without a fused-on benzene or naphthalene ring; R3 and R4 are each independently of the other hydrogen, unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; or R3 and R4 together form unsubstituted or substituted alkylene, alkylidene or benzo-fused alkylene; R5 is hydroxy; R6 is hydrogen; or R5 and R6 together are oxo; and R7 is unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl. 20. A method of treatment according to claim 5, further comprising administration of one or more therapeutic agents selected from the group consisting of an antioxidant, an anti-inflammatory, a gamma secretase inhibitor, a neurotrophic agent, an acetyl cholinesterase inhibitor, a statin, an A beta peptide, and an anti-A beta peptide. 21. (canceled) 22. A method of treating or preventing Alzheimer's disease in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound of Formula (I-A) or a pharmaceutically acceptable salt thereof: wherein R1 and R9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl; sulfo; or sulfonyl substituted by unsubstituted or substituted alkyl, aryl or by heterocyclyl, with the proviso that not more than one of the radicals R1 and R9 is hydrogen; R2 and R8 are each independently of the other hydrogen or unsubstituted or substituted alkyl; R3 and R4 are each independently of the other hydrogen, unsubstituted or substituted alkyl, unsubstituted or substituted cycloalkyl or aryl; R5 is acyloxy; R6 is hydrogen; and R7 is unsubstituted or substituted alkyl, unsubstituted or substituted cycloalkyl or aryl; and salts of the mentioned compounds where salt-forming groups are present, with the exception of the compound wherein R1 and R9 are each acetyl, R2, R3, R4, R6 and R8 are each hydrogen, R5 is acetoxy and R7 is 2,2-[N-ethoxycarbonylmethyl)-N-methyl]hydrazin-1-ylcarbonylmethyl. 23. A method of treating a patient who has, or in preventing a patient from getting, a disease or condition selected from the group consisting of Alzheimer's disease, for helping prevent or delay the onset of Alzheimer's disease, for treating patients with mild cognitive impairment (MCI) and preventing or delaying the onset of Alzheimer's disease in those who would progress from MCI to AD, for treating Down's syndrome, for treating humans who have Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type, for treating cerebral amyloid angiopathy and preventing its potential consequences, i.e. single and recurrent lobar hemorrhages, for treating other degenerative dementias, including dementias of mixed vascular and degenerative origin, dementia associated with Parkinson's disease, dementia associated with progressive supranuclear palsy, dementia associated with cortical basal degeneration, or diffuse Lewy body type of Alzheimer's disease and who is in need of such treatment which includes administration of a therapeutically effective amount of a compound of formula (I-A), or a pharmaceutically acceptable salt thereof: wherein R1 and R9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl; sulfo; or sulfonyl substituted by unsubstituted or substituted alkyl, aryl or by heterocyclyl, with the proviso that not more than one of the radicals R1 and R9 is hydrogen; R2 and R8 are each independently of the other hydrogen or unsubstituted or substituted alkyl; R3 and R4 are each independently of the other hydrogen, unsubstituted or substituted alkyl, unsubstituted or substituted cycloalkyl or aryl; R5 is acyloxy; R6 is hydrogen; and R7 is unsubstituted or substituted alkyl, unsubstituted or substituted cycloalkyl or aryl; and salts of the mentioned compounds where salt-forming groups are present, with the exception of the compound wherein R1 and R9 are each acetyl, R2, R3, R4, R6 and R8 are each hydrogen, R5 is acetoxy and R7 is 2,2-[N-ethoxycarbonylmethyl)-N-methyl]hydrazin-1-ylcarbonylmethyl. 24. A method according to claim 23, wherein the compound is selected from the group consisting of: 1-[2(S)-palmitoyloxy-3(S)-(N-(methoxy-carbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1[-2(S)-(methoxy-acetoxy)-3(S)-(N-(methoxy-carbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-methoxy-carbonyl-(L)-valyl]hydrazine; 1-[2(S)-(2-pyridyl-carbonyl)oxy-3(S)-(tert-butoxy-carbonyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[tert-butoxy-carbonyl]hydrazine; 1-[2(S)-(2-pyridylcarbonyl)oxy-3(S)-(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino4-phenylbutyl-1-[phenylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-butyryloxy-3(S)-((N-quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenylbutyl-1-[phenylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(2-pyridylcarbonyl)oxy-3(S)-(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino4-phenylbutyl-1-[4-methoxyphenylmethyl]-2-[N-(benzyloxycarbonyl)-(L) valyl]hydrazine; 1-[2(S)-(methoxy-acetyl)oxy-3(S)-(N-quinoline-2-carbonyl)-(L)-asparaginyl)amino-4-phenylbutyl-1-[4-methoxyphenylmethyl]-2-[N-(benzyloxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-propionyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-butyryloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-pentanoyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-octanoyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-decanoyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-dodecanoyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-pivaloyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(2-furylcarbonyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(4-imidazolylcarbonyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(4-imidazolylacetyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(3-(4-imidazolyl)-propionyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-benzoyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(2-pyridylacetyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(3-(pyridin-2-yl)-propionyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenylbutyl)]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(quinolin-2-ylcarbonyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(aminoacetyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(N-methylaminoacetyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(N,N-dimethylaminoacetyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(N-benzyloxycarbonyl-N-methyl-aminoacetyl)oxy-3(S)-(N-(methoxycarbonyl)(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)(L)-valyl]hydrazine; 1-[2(S)-prolyloxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(4-morpholinomethylbenzoyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; 1-[2(S)-(4-chloromethylbenzoyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenyl-butyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; and 1-[2(S)-(3-carboxypropionyl)oxy-3(S)-(N-(methoxycarbonyl)-(L)-valyl)amino-4-phenylbutyl]-1-[cyclohexylmethyl]-2-[N-(methoxycarbonyl)-(L)-valyl]hydrazine; or a pharmaceutically acceptable salt thereof. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Alzheimer's disease (AD) is a progressive degenerative disease of the brain primarily associated with aging. Clinical presentation of AD is characterized by loss of memory, cognition, reasoning, judgment, and orientation. As the disease progresses, motor, sensory, and linguistic abilities are also affected until there is global impairment of multiple cognitive functions. These cognitive losses occur gradually, but typically lead to severe impairment and eventual death in the range of four to twelve years. Alzheimer's disease is characterized by two major pathologic observations in the brain: neurofibrillary tangles and beta amyloid (or neuritic) plaques, comprised predominantly of an aggregate of a peptide fragment know as A beta. Individuals with AD exhibit characteristic beta-amyloid deposits in the brain (beta amyloid plaques) and in cerebral blood vessels (beta amyloid angiopathy) as well as neurofibrillary tangles. Neurofibrillary tangles occur not only in Alzheimer's disease but also in other dementia-inducing disorders. On autopsy, large numbers of these lesions are generally found in areas of the human brain important for memory and cognition. Smaller numbers of these lesions in a more restricted anatomical distribution are found in the brains of most aged humans who do not have clinical AD. Amyloidogenic plaques and vascular amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down's Syndrome), Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D), and other neurodegenerative disorders. Beta-amyloid is a defining feature of AD, now believed to be a causative precursor or factor in the development of disease. Deposition of A beta in areas of the brain responsible for cognitive activities is a major factor in the development of AD. Beta-amyloid plaques are predominantly composed of amyloid beta peptide (A beta, also sometimes designated betaA4). A beta peptide is derived by proteolysis of the amyloid precursor protein (APP) and is comprised of 39-42 amino acids. Several proteases called secretases are involved in the processing of APP. Cleavage of APP at the N-terminus of the A beta peptide by beta-secretase and at the C-terminus by one or more gamma-secretases constitutes the beta-amyloidogenic pathway, i.e. the pathway by which A beta is formed. Cleavage of APP by alpha-secretase produces alpha-sAPP, a secreted form of APP that does not result in beta-amyloid plaque formation. This alternate pathway precludes the formation of A beta peptide. A description of the proteolytic processing fragments of APP is found, for example, in U.S. Pat. Nos. 5,441,870; 5,721,130; and 5,942,400. An aspartyl protease has been identified as the enzyme responsible for processing of APP at the beta-secretase cleavage site. The beta-secretase enzyme has been disclosed using varied nomenclature, including BACE, Asp, and Memapsin. See, for example, Sindha et al., 1999 , Nature 402:537-554 (p501) and published PCT application WO00/17369. Several lines of evidence indicate that progressive cerebral deposition of beta-amyloid peptide (A beta) plays a seminal role in the pathogenesis of AD and can precede cognitive symptoms by years or decades. See, for example, Selkoe, 1991 , Neuron 6:487. Release of A beta from neuronal cells grown in culture and the presence of A beta in cerebrospinal fluid (CSF) of both normal individuals and AD patients has been demonstrated. See, for example, Seubert et al., 1992 , Nature 359:325-327. It has been proposed that A beta peptide accumulates as a result of APP processing by beta-secretase, thus inhibition of this enzyme's activity is desirable for the treatment of AD. In vivo processing of APP at the beta-secretase cleavage site is thought to be a rate-limiting step in A beta production, and is thus a therapeutic target for the treatment of AD. See for example, Sabbagh, M., et al., 1997 , Alz. Dis. Rev. 3, 1-19. BACE1 knockout mice fail to produce A beta, and present a normal phenotype. When crossed with transgenic mice that over express APP, the progeny show reduced amounts of A beta in brain extracts as compared with control animals (Luo et al., 2001 Nature Neuroscience 4:231-232). This evidence further supports the proposal that inhibition of beta-secretase activity and reduction of A beta in the brain provides a therapeutic method for the treatment of AD and other beta amyloid disorders. At present there are no effective treatments for halting, preventing, or reversing the progression of Alzheimer's disease. Therefore, there is an urgent need for pharmaceutical agents capable of slowing the progression of Alzheimer's disease and/or preventing it in the first place. Compounds that are effective inhibitors of beta-secretase, that inhibit beta-secretase-mediated cleavage of APP, that are effective inhibitors of A beta production, and/or are effective to reduce amyloid beta deposits or plaques, are needed for the treatment and prevention of disease characterized by amyloid beta deposits or plaques, such as AD. U.S. Pat. No. 5,753,652 discloses hydrazine compounds of the formula wherein R 1 and R 9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl, alkenyl or alkynyl; heterocyclyl; sulfo; sulfonyl substituted by unsubstituted or substituted alkyl, aryl, heterocyclyl, alkoxy, which is unsubstituted or substituted, or by aryloxy; sulfamoyl that is unsubstituted or substituted at the nitrogen atom; or phosphoryl substituted by one or two radicals, which may be identical or different, selected from unsubstituted or substituted alkyl, from unsubstituted or substituted cycloalkyl, from aryl, from hydroxy, from unsubstituted or substituted alkoxy, from cycloalkoxy and from aryloxy; with the proviso that not more than one of the radicals R 1 and R 9 is hydrogen; R 2 and R 8 are each independently of the other hydrogen or one of the radicals mentioned above for R 1 and R 9 ; or the pairs of substituents R 1 and R 2 , and R 8 and R 9 , each independently of the other, may form together with the nitrogen atom to which they are bonded a heterocyclic ring consisting of the bonding nitrogen atom together with a radical selected from ethylene, trimethylene, tetramethylene and pentamethylene in which a carbon atom may have been replaced by nitrogen, oxygen, sulfur or by sulfur mono- or di-substituted by oxygen and which may be unsaturated, or one of those radicals with an oxo substituent at each of the two carbon atoms linked to the bonding carbon atom and with or without a fused-on benzene or naphthalene ring; R 3 and R 4 are each independently of the other hydrogen, unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; or R 3 and R 4 together form unsubstituted or substituted alkylene, alkylidene or benzo-fused alkylene; R 5 is hydroxy; R 6 is hydrogen, or R 5 and R 6 together are oxo; and R 7 is unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; and salts of the mentioned compounds where salt-forming groups are present. U.S. Pat. No. 5,753,652 discloses how to make the above compounds and how to use them in treating HIV and AIDS; the disclosure of U.S. Pat. No. 5,753,652 is incorporated herein by reference in its entirety. |
<SOH> SUMMARY OF INVENTION <EOH>The present invention relates to methods of treating a patient who has, or in preventing a patient from developing, a disease or condition selected from the group consisting of Alzheimer's disease, for helping prevent or delay the onset of Alzheimer's disease, for helping to slow the progression of Alzheimer's disease, for treating patients with mild cognitive impairment (MCI) and preventing or delaying the onset of Alzheimer's disease in those who would progress from MCI to AD, for treating Down's syndrome, for treating humans who have Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type, for treating cerebral amyloid angiopathy and preventing its potential consequences, i.e. single and recurrent lobar hemorrhages, for treating other degenerative dementias, including dementias of mixed vascular and degenerative origin, dementia associated with Parkinson's disease, dementia associated with progressive supranuclear palsy, dementia associated with cortical basal degeneration, or diffuse Lewy body type of Alzheimer's disease and who is in need of such treatment which comprises administration of a therapeutically effective amount of a compound of formula (I): wherein R 1 and R 9 are each independently of the other hydrogen; acyl; unsubstituted or substituted alkyl, alkenyl or alkynyl; heterocyclyl; sulfo; sulfonyl substituted by unsubstituted or substituted alkyl, aryl, heterocyclyl, alkoxy, which is unsubstituted or substituted, or by aryloxy; sulfamoyl that is unsubstituted or substituted at the nitrogen atom; or phosphoryl substituted by one or two radicals, which may be identical or different, selected from unsubstituted or substituted alkyl, from unsubstituted or substituted cycloalkyl, from aryl, from hydroxy, from unsubstituted or substituted alkoxy, from cycloalkoxy and from aryloxy; with the proviso that not more than one of the radicals R 1 and R 9 is hydrogen; R 2 and R 8 are each independently of the other hydrogen or one of the radicals mentioned above for R 1 and R 9 ; or the pairs of substituents R 1 and R 2 , and R 8 and R 9 , each independently of the other, may form together with the nitrogen atom to which they are bonded a heterocyclic ring consisting of the bonding nitrogen atom together with a radical selected from ethylene, trimethylene, tetramethylene and pentamethylene in which a carbon atom may have been replaced by nitrogen, oxygen, sulfur or by sulfur mono- or di-substituted by oxygen and which may be unsaturated, or one of those radicals with an oxo substituent at each of the two carbon atoms linked to the bonding carbon atom and with or without a fused-on benzene or naphthalene ring; R 3 and R 4 are each independently of the other hydrogen, unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; or R 3 and R 4 together form unsubstituted or substituted alkylene, alkylidene or benzo-fused alkylene; R 5 is hydroxy; R 6 is hydrogen, or R 5 and R 6 together are oxo; and R 7 is unsubstituted or substituted alkyl or cycloalkyl; aryl; heterocyclyl; or unsubstituted or substituted alkenyl; and salts of the mentioned compounds where salt-forming groups are present. detailed-description description="Detailed Description" end="lead"? |
Inspection state check system |
A system for verifying a check status includes a display 41, a mobile terminal 30 to which a mechanic inputs a status information indicative of a check status, and a server 10 which makes a display indicate the check status on in real time, in response to the status information sent from the mobile terminal. Accordingly, a user can verify the check status of a vehicle. |
1. A system for verifying a check status, comprising: a display; an information terminal to which a mechanic inputs a status information indicative of a check status; and a processor which makes said display indicate said check status thereon in real time, in response to said status information sent from said information terminal. 2. The system for verifying a check status, according to claim 1, wherein said information terminal comprises a bar code reader which reads a bar code put on a repair card on which a check item is noted, and information read by said bar code reader is sent as said status information to said processor. 3. The system for verifying a check status, according to claim 1, wherein said processor comprises a transmit unit which sends data indicative of said check status to said information terminal, in response to a request from said information terminal, and said information terminal comprises a display unit which indicates said data indicative of said check status sent by said transmit unit of said processor. 4. The system for verifying a check status, according to claim 1, wherein said information terminal further comprises a check status obtaining unit which obtains said status information of an apparatus that is being checked by said mechanic, and wherein said check status obtaining unit sends said status information to said processor. 5. The system for verifying a check status, according to claim 1, wherein said display is included in a terminal device, said terminal device further includes a input unit to which a user of an apparatus that is being checked by said mechanic inputs a indication request information, and said processor transmits said check status to said terminal device such that said display indicates said check status, in response to said indication request information sent from said terminal device. 6. The system for verifying a check status, according to claim 5, further comprising: a memory device which stores said check status and an ID number of said apparatus, wherein said check status is related to said ID number, wherein said processor transmits said check status to said terminal device, in response to said indication request information which includes said ID number sent from said terminal device. 7. The system for verifying a check status, according to claim 6, wherein said apparatus includes a vehicle. 8. The system for verifying a check status, according to claim 7, wherein said terminal device is installed in a waiting room for said user who is waiting for completion of said check by said mechanic. 9. The system for verifying a check status, according to claims 8, wherein said check status includes check items. 10. A method of verifying a check status, comprising: receiving a status information indicative of a check status of an apparatus that is being checked by a mechanic; and indicating said check status on a display in real time, in response to said status information, wherein said display is installed in a place where at least one of a user of said apparatus and said mechanic are in. 11. The method of verifying a check status, according to claim 10, further comprising the steps of: storing said status information in a memory device; receiving an indication request information including an ID number of said apparatus, wherein said indication request information shows a request for indicating said status information on said display; obtaining said status information from said memory device, based on said indication request information; and indicating said check status on said display, in response to said indication request information. 12. The method of verifying a check status, according to claim 11, wherein said display is installed in a waiting room for said user who is waiting for completion of said check by said mechanic. 13. The method of verifying a check status, according to claim 12, wherein said apparatus includes a vehicle. 14. The method of verifying a check status, according to claims 13, wherein said check status includes check items. 15. The system for verifying a check status, according to claim 1, wherein said information terminal is a mobile information terminal. 16. The system for verifying a check status, according to claim 1, wherein said terminal device is installed in a waiting room for said user who is waiting for completion of said check by said mechanic. 17. The method of verifying a check status, according to claim 10, wherein the mechanic inputs said status information via a mobile information terminal. 18. The method of verifying a check status, according to claim 17, wherein said mobile information terminal comprises a bar code reader which reads a bar code placed on a repair card on which a check item is noted, and said status information is read by said bar code reader. |
<SOH> BACKGROUND ART <EOH>Conventionally, a check is carried out such that a vehicle, which a user brings into a maintenance shop, is checked and then delivered to the user after the completion of the check. Actually, when the vehicle is brought into the maintenance shop, a reception is firstly carried out by a service adviser. Next, after a current status of the vehicle is verified by a work controller, a diagnosis is carried out. In this diagnosis, after questioning of the condition, symptom reproduction verification and the like are carried out, a reason for a trouble is supposed. Next, the estimation of a cost and a time required to remove the supposed reason for the trouble is carried out, and presented to the user. Here, if the user admits the presented estimation, a mechanic starts carrying out the check. The user waits for the completion of the check in a waiting room. In the conventional check as mentioned above, the user only waits for the elapse of the estimated check time in the waiting room. However, there may be a case that the estimated time is postponed, for example, if the presumption of the reason for the trouble is erroneous. Even in such a case, that the user can do is only to wait for. Even if the user asks the service adviser about the check status, since the service adviser himself can not know the check status, he can not explain the check status. Therefore, there is a desire from the user, such as a request to know the current check status. The present invention is proposed in order to cope with the above-mentioned request. The object of the present invention is to provide a system for verifying a check status, in which a user can verify a check status of a vehicle. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a block diagram showing a configuration of a system for verifying a check status, according to a best mode for carrying out the present invention. FIG. 2 is a flowchart describing an operation of the system for verifying a check status, according to a best mode for carrying out the present invention. detailed-description description="Detailed Description" end="lead"? |
Chimaeric phages |
The invention relates to the field of generating helper phages and phage display libraries for the identification of binding molecules. The invention provide chimaeric phages having a coat comprising a protein mixture. The protein mixture comprises a fusion protein having a proteinaceous molecule fused to a functional form of a phage coat protein and a mutant form of the phage coat protein, wherein the mutant form is impaired in binding to a host cell receptor. The invention further provides new phage collections, novel helper phages and methods and means for producing chimaeric phages, infectious phages and helper phages. |
1. A chimaeric phage having a coat comprising a mixture of proteins, said mixture of proteins comprising: a fusion protein comprising a proteinaceous molecule is fused to a functional form of a phage coat protein; and a mutant form of said phage coat protein, wherein said mutant form is characterized in that a phage comprising no wild type phage coat protein from which said mutant form originates and has a coat comprising said mutant form and no copies of said functional form, is less infectious than a phage comprising no wild type phage coat protein from which said mutant form originates and has a coat comprising said mutant form and at least one copy of said functional form. 2. A chimaeric phage having a coat comprising a mixture of proteins, said mixture of proteins comprising: a fusion protein comprising a proteinaceous molecule is fused to a functional form of a phage coat protein; and a mutant form of said phage coat protein, wherein said mutant form is impaired in binding to a host cell receptor. 3. The chimaeric phage to of claim 1, wherein said phage coat protein is the g3 protein. 4. The chimaeric phage to of claim 3, wherein said mutant form comprises a mutation in the D1 region, the D2 region or the D1 region and the D2 region of said g3 protein. 5. The chimaeric phage of claim 4, wherein said mutation comprises a deletion of substantially all of said D1 and said D2 region of said g3 protein. 6. The chimaeric phage of claim 1, further comprising a nucleic acid encoding said fusion protein. 7. The chimaeric phage of claim 1, wherein said chimaeric phage is of a M13, M13K07, VCSM13 or R408 phage origin. 8. The chimaeric phage of claim 1, wherein said proteinaceous molecule comprises a peptide or a protein. 9. The chimaeric phage of claim 1, wherein said proteinaceous molecule is selected from the group consisting of an antibody, a Fab fragment, a single chain Fv fragment, a variable region, a CDR region, an immunoglobulin and any combination thereof. 10. A chimaeric phage having a coat comprising a mixture of proteins, said mixture of proteins comprising. a fusion protein comprising a proteinaceous molecule is fused to a phage coat protein, or to a fragment or derivative thereof; wherein said fusion protein is functional so as to render the chimaeric phage infectious; and a mutant form of said phage coat protein, wherein said mutant form is characterized in that a phage comprising no wild type phage coat protein from which said mutant form is originates and carrying said mutant form and no copies of said fusion protein, is less infectious than a phage comprising no wild type phage coat protein from which said mutant form originates and carrying in addition to said mutant form at least one copy of said fusion protein. 11. A chimaeric phage having a coat comprising a mixture of proteins, said mixture of proteins comprising. a fusion protein comprising a proteinaceous molecule is fused to a phage coat protein, or to a fragment or derivative thereof; wherein said fusion protein is functional so as to render the chimaeric phage infectious; and a mutant form of said phage coat protein, said mutant form being impaired in binding to a host cell receptor. 12. The chimaeric phage of claim 10, wherein said mutant form is characterized in that a phage comprising no wild type phage coat protein from which said mutant form originates and carrying said mutant form and no copies of said fusion protein is non-infectious. 13. The chimaeric phage of claim 1, wherein said mutant form is further characterized in that a phage having a coat comprising said mutant form in the presence or absence of copies of said functional form, is stable. 14. An infectious phage comprising: at least one copy of a mutant form of a phage coat protein, wherein said mutant form has lost the ability to mediate infection of a natural host by said infectious phage. 15. A phage collection comprising a the chimaeric phage of claim 1. 16. The phage collection of claim 15, wherein said phage collection is a phage display library. 17. A phage collection consisting essentially of the chimaeric phage of claim 1. 18. A process for producing a phage particle, said process comprising: providing a host cell with a first nucleic acid encoding a fusion protein, said fusion protein comprising a proteinaceous molecule fused to a functional form of a phage coat protein; providing said host cell with a second nucleic acid encoding a mutant form of said phage coat protein, said mutant form being characterized in that a phage comprising no wild type phage coat protein from which said mutant form originates and having a coat comprising said mutant form and no copies of said functional form, is less infectious than a phage comprising no wild type phage coat protein from which said mutant form originates and having a coat comprising at least one copy of said functional form; wherein said host cell comprises an additional nucleic acid encoding at least all other proteins, or functional equivalents thereof, that are essential for the assembly of said phage particle in said host cell; and culturing said host cell to allow assembly of said phage particle. 19. A process for producing a phage particle, said process comprising: providing a host cell with a first nucleic acid encoding a fusion protein, said fusion protein comprising a proteinaceous molecule fused to a functional form of a phage coat protein; providing said host cell with a second nucleic acid encoding a mutant form of said phage coat protein, said mutant form being impaired in binding to a host cell receptor; and culturing said host cell to allow assembly of said phage particle. 20. The process according to claim 18, wherein expression of said fusion protein, said mutant form or a combination thereof is regulatable by altering the culturing conditions of said host cell. 21. The process according to claim 18, wherein expression of said fusion protein, said mutant form or a combination thereof is under the control of a regulatable promoter. 22. The process according to claim 21, wherein said regulatable promoter comprises the AraC/BAD promoter, the psp promoter, the lac promoter, or a functional equivalent of any thereof. 23. The process according to claim 18, wherein said additional nucleic acid sequence is provided by a helper phage to said host cell. 24. The process according to claim 23, wherein said helper phage comprises said second nucleic acid. 25. The process according to claim 18, wherein said fusion protein and said mutant form are encoded by separate nucleic acids and each unique selection marker. 26. The process according to claim 25, wherein said separate nucleic acids each comprises a unique origin of replication. 27. The process according to claim 25, wherein said separate nucleic acids each comprises codons that essentially do not render a homologous recombination event between said separate nucleic acids. 28. A chimaeric phase produced by the process according to claim 18. 29. A helper phage comprising: nucleic acid encoding phage proteins or functional equivalents thereof that are essential for the assembly of said helper phage; wherein said nucleic acid further encodes a mutant form of a phage coat protein, said mutant form characterized in that a phage comprising no wild type phage coat protein from which said mutant form originates and having a coat comprising said mutant form and no copies of a functional form of said phage coat protein, is less infectious than a phage comprising no wild type phage coat protein from which said mutant form originates and having a coat comprising at least one copy of said functional form; wherein said functional form is characterized in that it renders a phage particle carrying said functional form in its coat infectious; wherein said helper phage does not comprise a nucleic acid encoding said functional form. 30. A helper phage comprising: nucleic acid encoding phage proteins or functional equivalents thereof that are essential for the assembly of said helper phage; wherein said nucleic acid further encodes a mutant form of a phage coat protein, said mutant form being impaired in binding to a host cell receptor; wherein said helper phage does not comprise a nucleic acid encoding a functional form of said phage coat protein. 31. The helper phage of claim 29, wherein said phage coat protein is the g3 protein. 32. The helper phage of claim 31, wherein said mutant form comprises a mutation in the D1 region, the D2 region, or the D1 region and the D2 region of said g3 protein. 33. The helper phage of claim 32, wherein said mutation comprises a deletion of substantially all of said D1 and said D2 region of said g3 protein. 34. The helper phage of claim 29, wherein said mutant form is further characterized in that a phage having a coat comprising said mutant form in the presence or absence of a copy of said functional form, is stable. 35. A process for producing a helper phage the process comprising: providing a host cell with a first nucleic acid encoding a functional form of a phage coat protein; providing said host cell with a second nucleic acid encoding a mutant form of said phage coat protein, wherein said mutant form is characterized in that a phage comprising no wild type phage coat protein from which said mutant form is originates and having a coat comprising said mutant form and no copies of said functional form, is less infectious than a phage comprising no wild type phage coat protein from which said mutant form originates and having a coat comprising at least one copy of said functional form; wherein said host cell comprises an additional nucleic acid sequence encoding at least all other proteins or functional equivalents thereof that are essential for the assembly of said helper phage in said host cell; and culturing said host cell to allow assembly of said helper phage. 36. A process for producing a helper phage, the process comprising: providing a host cell with a first nucleic acid encoding a functional form of a phage coat protein; providing said host cell with a second nucleic acid encoding a mutant form of a phage coat protein, said mutant form being impaired in binding to a host cell receptor; wherein said host cell comprises an additional nucleic acid sequence encoding at least all other proteins or functional equivalents thereof that are essential for the assembly of said helper phage in said host cell; and culturing said host cell to allow assembly of said helper phage. 37. The process according to claim 35, wherein said all other proteins or functional equivalents thereof that are essential for the assembly of said helper phage in said host cell are encoded by said second nucleic acid. 38. The process according to claim 35, wherein expression of said functional form, said mutant form or a combination thereof is regulatable by altering the culturing conditions of said host cell. 39. The process according to claim 35, wherein expression of said functional form, said mutant form or a combination thereof is under the control of a regulatable promoter. 40. The process according to claim 39, wherein said regulatable promoter comprises the AraC/BAD promoter, the psp promoter, the lac promoter, or a functional equivalent of any thereof. 41. The process according to claim 35, wherein said phage coat protein is the g3 protein. 42. The process according to claim 41, wherein said mutant form comprises a mutation in the D1 region, the D2 region or the D1 region and the D2 region of said g3 protein. 43. The process according to claim 42, wherein said mutation comprises a deletion of substantially all of said D1 and said D2 region of said g3 protein. 44. The process according to claim 35, wherein said first nucleic acid and said second nucleic acid each comprises a unique selection marker. 45. The process according to claim 35, wherein said first nucleic acid and said second nucleic acid each comprises a unique origin of replication. 46. The process according to claim 35, wherein said first nucleic acid and said second nucleic acid comprise codons that essentially do not render a homologous recombination event between said first nucleic acid and said second nucleic acid. 47. A helper phage produced by the process according to claim 35, wherein said helper phage does not comprise nucleic acid encoding said functional form. 48. A process for the enrichment of a first binding pair member in a repertoire of first binding pair members selected from the group consisting of: an antibody, an antibody fragment, a single chain Fv fragment, a Fab fragment, a variable region, a CDR region, an immunoglobulin and a functional part of any thereof, wherein said first binding pair member is specific for a second binding pair member, the process comprising: contacting a the phage collection of claim 15 with material comprising said second binding pair member under conditions allowing specific binding; removing non-specific binders; and recovering specific binders, said specific binders comprising said first binding pair member. 49. The process according to claim 48, further comprising: recovering a DNA sequence encoding said first specific binding pair member from a phase; subcloning said DNA sequence in an expression vector; expressing said DNA sequence in a host; and culturing said host under conditions, wherein said first specific binding pair member is produced. 50. A chimaeric phage having a coat, the coat comprising: a fusion protein comprising a phage coat protein fused to a proteinaceous molecule; and a means for rendering the chimaeric phage less infectious than a wild-type phage from which the chimaeric phage originates. 51. The chimaeric phage of claim 50, wherein the phage coat protein is g3 protein. 52. The chimaeric phage of claim 50, wherein the proteinaceous molecule is selected from the group consisting of an antibody, a Fab fragment, a single chain Fv fragment, a variable region, a CDR region, an immunoglobulin and any combination thereof. 53. The chimaeric phage of claim 50, wherein the means for rendering the chimaeric phage less infectious comprises a mutated g3 protein. 54. The chimaeric phage of claim 53, wherein the mutated g3 protein comprises a mutation in a D1 region, a D2 region, or the D1 region and the D2 region. |
<SOH> BACKGROUND <EOH>An individual needs to have a dynamic immune system that is able to adapt rapidly and respond adequately to potentially harmful microorganisms, and to respond to the exposure of a highly diverse and continuously changing environment. Higher organisms have evolved specialized molecular mechanisms to ensure the implementation of clonally-distributed, highly diverse repertoires of antigen-receptor molecules expressed by cells of the immune system: immunoglobulin (Ig) molecules on B lymphocytes and T cell receptors on T lymphocytes. A primary repertoire of (generally low affinity) Ig receptors is established during B cell differentiation in the bone marrow as a result of rearrangement of germ line-encoded gene segments. Further refinement of Ig receptor specificity and affinity occurs in peripheral lymphoid organs where antigen-stimulated B lymphocytes activate a somatic hypermutation machinery that specifically targets the immunoglobulin variable (V) regions. During this process, B cell clones with mutant Ig receptors of higher affinity for the inciting antigen are stimulated into clonal proliferation and maturation into antibody-secreting plasma cells (reviewed in Berek and Milstein. 1987). Recombinant DNA technology has been used to mimic many aspects of the processes that govern the generation and selection of natural human antibody repertoires (reviewed in Winter and Milstein. 1991; Vaughan et al. 1998). The construction of large repertoires of antibody fragments (such as Fab fragments or single chain Fv fragments, scFv's) expressed on the surface of filamentous phage particles and the selection of such phages by “panning” on antigens has been developed as a versatile and rapid method to obtain antibodies of desired specificities (reviewed in Burton and Barbas. 1994). A subsequent optimization of the affinity of individual phage antibodies was achieved by creating mutant antibody repertoires of the selected phages and sampled for higher affinity descendents by selection for binding to antigen under more stringent conditions (reviewed in Hoogenboom. 1994). M13 and M13-derived phages (sometimes also called viruses) are filamentous phages that can selectively infect F-pili bearing (F + ) Escherichia coli ( E.coli ) cells. The phage genome encodes 11 proteins, while the phage coat itself consists of 5 of these proteins: gene3, -6, -7, -8 and -9 (g3, g6, g7, g8 and g9) proteins that are bound to and that protect the (circular) single stranded DNA (ssDNA) of the viral genome. The life cycle of the virus can be subdivided into different phases. The g3 protein (g3p) of M13 phages and M13-derivatives comprises three functional domains: D1, D2 and D3, linked by two glycine-rich linkers. An alternative nomenclature for g3p domains has also been generally accepted, in which D1, D2 and D3 are named “N1,” “N2” and “CT,” respectively. The N-terminal D1/D2 regions interact with the C-terminal D3 region as has been found by Chatellier et al. (1999) using several deletion mutants of g3p. Considering that functionality of a D3 domain of the protein is required for assembly of stable phages, a less-, or non-infectious mutant of the phage coat protein preferably comprises a D3 region of the g3p, or comprises a functional part, derivative and/or analogue of the D3 region. The D3 domain is thought to bind to DNA inside the viral particle. Loss of the D3 domain functionally results in rare phage-like particles that are very long and very fragile (Pratt et al. 1969; Crissman and Smith. 1984; Rakonjac and Model. 1998). The D1 and D2 domain are thought to interact with each other until the phage binds to the bacteria, while D1/D2 also interact with D3 at certain stages (Chatellier et al. 1999). The linkers present in g3p between D1, D2 and D3 apparently also play a role in infectivity of the phage particle (Nilsson et al. 2000). Studies in which a protease cleavage site was introduced between D1 and D2 showed that after cleavage, the phage particle became non-infectious (Kristensen and Winter. 1998). Functional analysis of g3p showed that of the g3p N-terminal regions, the D1 domain is essential for infection. Loss of this domain results in phages that cannot infect bacteria (Lubkowski et al. 1998; Nelson et al. 1981; Deng et al. 1999; Riechmann and Holliger. 1997; Holliger and Riechmann. 1997). It has been shown that the D2 domain interacts with the D1 domain of g3p on the phage ( FIG. 1 ). Due to competition of proteins located on the F-pilus (on F + bacteria) that have higher affinity for D2 than for D1, the D1 and D2 domain of the g3p dissociate from each other. The binding of D2 to the F-pilus results in a process that leads to retraction of the F-pilus towards the E.coli cell membrane. Due to this process, the phage particle comes in close contact with the bacterial membrane. The dissociated D1 domain can interact with bacterial proteins such as the TolA receptor, leading to the introduction of the phage DNA into the E. coli cell (Lubkowski et al. 1999). The fact that removal of the D2 domain does not prevent infection, but enables phages to infect E.coli lacking F-pili (Riechmann and Holliger. 1997; Deng et al. 1999) shows that the presence of the D2 domain increases specificity and that D2 has an important role in preventing F-pili independent infections. The binding of D1 to the specific receptors on the surface of the E.coli cell (a feature that is not F + -specific) is represented in FIG. 2 . This process triggers the injection of the viral genome into the bacterium (as depicted in FIG. 3 ). Although loss of the D2 domain results in the formation of phage particles that can infect E.coli in a somewhat reduced specific manner, it appears that the level of infections from such a population of phages is significantly reduced. After infection of an E.coli by a phage particle, the ssDNA of the virus becomes double stranded due to the action of a number of bacterial enzymes. The double stranded phage genome serves as a template for the transcription and translation of all 11 genes located on the phage genome. Besides these protein-encoding regions, the phage genome contains an intergenic region: the F1-origin of replication initiation (F1-ORI). The DNA sequence of this F1-ORI can be divided in 2 separate subregions. One subregion is responsible for the initiation and termination of the synthesis of ssDNA via the so-called ‘rolling circle mechanism’ and the other subregion is responsible for the packaging initiation of the formed circular ssDNA leading to the formation and release of new virus particles. It has been shown that polypeptides, such as stretches of amino acids, protein parts or even entire proteins can be added by means of molecular genetics to the terminal ends of a number of particle coat proteins, without disturbing the functionality of these proteins in the phage life cycle (Smith. 1985; Cwirla et al. 1990; Devlin et al. 1990; Bass et al. 1990; Felici et al. 1993; Luzzago et al. 1993). This feature enables investigators to display peptides or proteins on phages, resulting in the generation of peptide- or protein expression phage display libraries. One of the proteins that has been used to fuse with polypeptides for phage display purposes, is the g3 protein (g3p), which is a coat protein that is required for an efficient and effective infectivity and subsequent entry of the viral genome into the E. coli cell. For the production of phages that display polypeptides fused to the g3p coat protein, investigators introduced a plasmid together with the phage genome in E.coli cells. This plasmid contains an active promoter upstream of an in-frame fusion between the g3 encoding gene and a gene of interest (X) encoding, for instance, polypeptides such as proteins such as antibodies or fragments such as Fab fragments or scFv's. The introduction of this plasmid together with the genome of the helper phage in an E.coli cell results in the generation of phages that contain on their coat either the wild type g3p from the viral genome, the fusion product g3p-X from the plasmid or a mixture of the two, since one phage particle carries five g3p's on its surface. The process of g3p or g3p-X incorporation is generally random. The presence of an F1-ORI sequence in the g3p-X expression vector (plasmid) misleads the phage synthesis machinery in such a way that two types of circular ssDNA are formed: one is derived from the genome of the phage and the other is derived from the expression vector. During the synthesis of new phages, the machinery is unable to distinguish the difference between these two forms of ssDNA resulting in the synthesis of a mixed population of phages, one part containing the phage genome and one part harboring the vector DNA. Due to these processes, the mixture contains at least some phages in which the phenotypic information on the outside (the g3p-X fusion protein) is conserved within the genotypic information inside the particle (the g3p-X expression vector). An infectious wild type phage and a phage carrying a fusion protein attached to g3p are depicted in FIG. 4 . The art teaches that there are several problems that concern the use of these basic set-ups. The high level of genotypic wild type phages in phage populations grown in bacteria that contain both the phage genome and the expression vector compelled investigators to design mutant F1-ORI sequences in M13 genomes. Such mutant M13-strains are less effective in incorporating their genome in phage particles during phage assembly, resulting in an increased percentage of phages containing vector sequences when co-expressed. These mutant phages, such as the commercially available strains R408, VCSM13 and M13KO7, are called “helper phages.” The genome of these helper phages may contain genes required to assemble new (helper-) phages in E.coli and to subsequently infect new F-pili expressing E.coli . Both VCSM13 and M13K07 were provided with an origin of replication initiation (ORI) of the P15A type that results in the multiplication of the viral genome in E.coli . Moreover, the ORI introduction ensures that after cell division the old and newly formed E.coli contains at least one copy of the viral genome. It was suggested and finally proven by several investigators that the introduction of plasmids containing a g3p-scFv fusion product together with the genome of the helper phages in E.coli cells results in approximately 99% of newly formed phages that harbor the g3p-scFv fusion protein expression plasmid, but nevertheless lack the g3p-scFv fusion on its surface (Beekwilder et al. 1999). The absence of g3p-X is a significant disadvantage in the use of display libraries for the identification of specific proteins or peptides such as scFv's that bind to a target of interest (such as tumor antigens). It implies that in the case of phage display libraries, at least a 100-fold excess of produced phages must be used in an experiment in order to perform a selection with all possible fusion proteins present. The art teaches that this overload of relatively useless phages in an experiment leads to (too) many false positives. For instance, at least 10 12 phages should be added to a panning experiment in order to have 1 copy of each possible fusion present in the experiment, since such a library contains approximately 10 10 different g3p-scFv fusions (1%). The phages in this approximate 1% express generally only 1 g3p-scFv fusion on their coat together with four normal g3p's (no fusions), while the rest of the helper phages (approximately 99%) express five g3p's and no g3p-scFv fusions. To ensure, theoretically, the presence of 100 copies of each separate fusion protein in a panning experiment, one needs to use approximately 10 14 phages in such an experiment. Persons skilled in the art generally attempt to use an excess of at least 100-fold of each single unique fusion protein, to ensure the presence of sufficient numbers of each separate fusion and not to lose relevant binders too quickly in first panning rounds. That number of phages (10 14 ) is more or less the maximum of phage particles that a milliliter (ml) can hold. The viscosity of such a solution is extremely high and therefore relatively useless. Especially when ELISA panning strategies are used (in which the volume of one well is only 200 μl) such libraries cannot be used. In addition to these problems, it is assumed that, generally, depending on the antigen and the stringency of washing procedures, an average of 1 in every 10 7 phages will bind to the antigen due to a-specific binding. Thus, for the application of 10 12 scFv expressing input phages (1%) to a panning procedure, one has to add approximately 10 14 phages (99% of which do not express a scFv fragment). It is generally assumed that from these 10 12 phages, approximately 10 4 particles might be putatively interesting phages. However, depending on the washing conditions, the number of calculated background phages that are normally found by using libraries present in the art after one round of panning, was approximately 10 6 -10 7 while only a few of these phages appear to be relevant binders. This is one of the most significant problems recognized in the art: too many background phages show up as initial binders in the phage mix after the first round of panning, while only a few significant and interesting binders are present in this mix. Thus, the absolute number of isolated phages after one round of panning is clearly too high (10 6 -10 7 ). Moreover, in subsequent rounds of panning, non-specific background phages remain present. In libraries used in the art, most of these non-specific binders will amplify on bacteria that, upon amplification, continue to in a second round of panning. Therefore, the art teaches that the background level of a-specifically binding phages and the total number of phages per ml in these types of libraries is unacceptably high and remains high during subsequent rounds of panning. A possibility that was suggested by investigators in the art as a solution to the problem of obtaining too many background phages that lack a g3p-X fusion was to remove the g3p-encoding gene entirely from the helper phage genome. In principle, this system ensures that during phage synthesis in an E.coli cell (that received the g3-less phage genome and a g3p-X fusion protein expression vector), only g3p-X proteins are incorporated in the newly formed phage coat. By doing so, each synthesized phage will express five copies of the g3p-X fusion product and hardly any phages are synthesized that express the g3p alone or that express less than five g3p-X fusions. R408-d3 and M13MDΔD3 are two examples of g3-minus helper phages (Dueñias and Borrebaeck. 1995; Rakonjac et al. 1997). Because the genome of these phages is not capable of supporting g3p synthesis, phage particles that carry less than five g3p-X fusion proteins can hardly be formed; or, if formed, are found to be non-infectious due to instability, since the art teaches that five g3p's are necessary to ensure a stable phage particle. To produce helper phages that do not contain the g3 gene, but that are nevertheless infectious and that can be used to generate libraries of phages that carry five g3p-X fusion proteins, and that lack phages with less than five g3p-X fusions, it was recognized in the art that an external source for g3p was required. Such a source can be a vector without F1-ORI but that nevertheless contains an active promoter upstream of the full open reading frame (ORF) of g3. One major problem that is recognized by persons skilled in the art is that after the generation step of producing newly formed helper phages lacking a g3 gene, the yield is dramatically low. In fact, the yield of all described systems is below 10 10 phages per liter, meaning that for a library of 10 10 individual clones, at least 100 liters of helper phage culture are necessary (NB: the helper phages need to be purified) in order to grow the library once. The art, thus, teaches that phage libraries generated with such low titers of helper phages are not useful for phage display purposes and that these libraries cannot be used for panning experiments. One method for complementation of g3p deletion phages was presented recently, in which wild-type g3p was provided by a nucleic acid encoding the wild-type g3p, wherein the nucleic acid was stably incorporated into the host cell genome (Rondot et al. 2001). Phages that express deleted g3p's fused to heterologous proteins have been generated. For the construction of most conventional Fab libraries and some scFv phage display libraries, the D1 domain and parts of the D2 domain were removed to ensure a shorter fusion protein, which was considered in the art as a product that could be translated easier than a full length g3p linked to a full length Fab fragment. The shorter g3p part would not prevent the generation of a viable and useful helper phage. Of course, such phages still depend on full length g3p's that are present on their surface next to the deleted g3p fusion with the Fab fragment for functional infectivity of E.coli cells. Also, phages that express deleted g3p's fused to ligand-binding proteins have been generated that depend on their infectious abilities on antigens that were fused to the parts of g3p that were missing from the non-infectious phage particle (Krebber et al. 1997; Spada et al. 1997). These particles depend for their infectivity on an interaction between the ligand-binding protein, such as an antibody or a fragment thereof and their respective ligand (or antigen). However, this interaction-dependency reduces the efficiency of infection, due to elimination of a direct linkage between the g3p domains, and a general inhibitory effect of the soluble N-terminal part of g3p coupled to the antigen. The g3-minus helper phages R408-d3 and M13MDΔD3 mentioned above lack a bacterial ORI and a selection marker in their genome. The absence of a selection marker in the g3-minus genomes has a significant effect on the production scale of helper phages, because it results in an overgrowth of bacteria that do not contain the helper phage genome. It is known that bacteria grow slower when infected with the helper phage or virus. Therefore, bacteria that lack the phage genome quickly overgrow the other bacteria that do contain the genome. Another effect of the lack of an ORI or a selection marker is that g3-minus phage genomes cannot be kept in dividing bacteria during the production and expansion of phage display libraries. This is a very important negative feature because overgrowth of bacteria that lost the phage genome or that never received one, appear to have a growth advantage over bacteria that do contain the phage genome. In addition, such ‘empty’ bacteria are not capable of producing any phage and as a result, the phage display vectors including fusion protein fragments in such helper phages lacking-bacteria are lost permanently. As mentioned, the g3p's are thought to be essential for the assembly of stable M13-like phages and because of their crucial role in infection, g3p's should be provided otherwise when g3-minus helper phages are to be generated. There is a prejudice in the art against making phage display libraries that lack g3p's because phages lacking g3p's are not stable. Rakonjac et al. (1997) constructed a VCSM13 g3-minus helper phage in parallel to a R408 g3-minus helper phage and used helper plasmids with either the psp or the lac promoter upstream of a full length g3 sequence to substitute g3 during helper phage synthesis (Model et al. 1997). However, the art teaches that the lac promoter has the disadvantage that it cannot be shut off completely, even not in the presence of high concentrations of glucose (3-5%) in the medium (Rakonjac and Model. 1998). An additional problem that is well known in the art is that even very low levels of g3p in E.coli can block infection of M13-like phages. Moreover, it has been shown that co-encapsidation of plasmids together with the phage genome can occur (Russel and Model. 1989; Krebber et al. 1995; Rakonjac et al. 1997). If co-encapsidation occurs with the lac driven helper plasmid, it will compete with the lac driven vectors used in the phage display resulting in the efficient production of infectious phage particles that will not contain the g3p-X fusion product. Together, the art thus teaches that the lac promoter is not the best candidate promoter in the helper plasmid system. The psp promoter has the advantage to be relatively silent in E.coli until infection (Rakonjac et al. 1997). Upon M13-class phage infection, the psp promoter becomes activated and the helper plasmid will produce g3 proteins. However, the disadvantage of this promoter is that the level of RNA production cannot be regulated with external factors, but has to be regulated by either mutating (and change the activity of) the promotor, changing the ribosomal binding site (RBS) or other elements that influence the promotor activity. To figure out the ideal level of promotor activity in a specific E.coli strain can be time consuming and needs to be optimized for each E.coli strain separately. The art also teaches that the psp promotor system is not very attractive for large-scale helper phage production due to the inflexibility of E.coli strains, the time consuming optimization and the significant low level of helper phage production. A significant problematic feature helper phage systems described is the occurrence of unwanted recombination events between the helper genome and the (helper-) plasmids. The problem that confronts investigators in the art is the fact that the g3 DNA sequences in the helper phages are homologous to the g3 sequences in the phage display vector and/or the helper phage plasmid. This results, in many cases, in recombination between the two DNA strains and therefore loss of functionality of the library as a whole. |
<SOH> SUMMARY OF THE INVENTION <EOH>The current invention provides chimaeric phages, novel helper phages, libraries comprising the chimaeric phages and methods and means to produce the chimaeric phages and the helper phages. In one embodiment, the invention provides a chimaeric phage having a coat comprising a mixture of proteins, the mixture comprising a fusion protein, wherein a proteinaceous molecule is fused to a functional form of a phage coat protein. The mixture further comprises a mutant form of the phage coat protein, the mutant form being impaired in binding to a host cell receptor. The invention also provides a chimaeric phage having a coat comprising a mixture of proteins, the mixture comprising a fusion protein, wherein a proteinaceous molecule is fused to a phage coat protein, or to a fragment or derivative thereof The fusion protein is functional so as to render the chimaeric phage infectious, the mixture further comprises a mutant form of the phage coat protein, the mutant form being impaired in binding to a host cell receptor. In another embodiment, the invention provides a helper phage comprising a nucleic acid encoding phage proteins or functional equivalents thereof that are essential for the assembly of the helper phage. The nucleic acid further encodes a mutant form of a phage coat protein, the mutant form being impaired in binding to a host cell receptor, and wherein the helper phage does not comprise nucleic acid encoding a functional form of the phage coat protein. In yet another embodiment, the invention further provides methods and means for producing phage particles, chimaeric phages, infectious phages and helper phages. The invention also provides phage collections, such as phage display libraries comprising chimaeric phages and/or infectious phages. |
Protein modification and maintenance molecules |
Various embodiments of the invention provide human proteinmodification and maintenance molecules (PMOD) and polynucleotides which identify and encode PMOD. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, andantagonists. Other embodiments provide methods for diagnosing, eating, or preventing disorders associated with aberrant expression of PMOD. |
1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3-7, SEQ ID NO:9-19, SEQ I) NO:21-26, and SEQ ID NO:28, c) a polypeptide comprising a naturally occurring amino acid sequence at least 94% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:20, d) a polypeptide comprising a naturally occurring amino acid sequence at least 96% identical to an amino acid sequence of SEQ ID NO:8, e) a polypeptide comprising a naturally occurring amino acid sequence at least 97% identical to an amino acid sequence of SEQ ID NO:27, f) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and g) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ I) NO:1-28. 3. An isolated polynucleotide encoding a polypeptide of claim 1. 4. An isolated polynucleotide encoding a polypeptide of claim 2. 5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3. 7. A cell transformed with a recombinant polynucleotide of claim 6. 8. (CANCELED). 9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed. 10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. 11. An isolated antibody which specifically binds to a polypeptide of claim 1. 12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-53 and SEQ ID NO:55-56, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 91% identical to a polynucleotide sequence of SEQ ID NO:54, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), f) a polynucleotide complementary to a polynucleotide of c), and g) an RNA equivalent of a)-f). 13. (CANCELED). 14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof. 15. (CANCELED). 16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. 19. (CANCELED). 20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample. 21. (CANCELED). 22. (CANCELED). 23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample. 24. (CANCELED). 25. (CANCELED). 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. 27. (CANCELED). 28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. 29. A method of assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. 30-111. (CANCELED). |
<SOH> BACKGROUND OF THE INVENTION <EOH>The cellular processes regulating modification and maintenance of protein molecules coordinate their function, conformation, stabilization, and degradation. Each of these processes is mediated by key enzymes or proteins such as kinases, phosphatases, proteases, protease inhibitors, isomerases, transferases, and molecular chaperones. Kinases Kinases catalyze the transfer of high-energy phosphate groups from adenosine triphosphate (ATP) to target proteins on the hydroxyamino acid residues serine, threonine, or tyrosine. Addition of a phosphate group alters the local charge on the acceptor molecule, causing internal conformational changes and potentially influencing intermolecular contacts. Reversible protein phosphorylation is the ubiquitous strategy used to control many of the intracellular events in eukaryotic cells. It is estimated that more than ten percent of proteins active in a typical mammalian cell are phosphorylated. Extracellular signals including hormones, neurotransmitters, and growth and differentiation factor can activate kinases, which can occur as cell surface receptors or as the activator of the final effector protein, but can also occur along the signal transduction pathway. Kinases are involved in all aspects of a cell's function, from basic metabolic processes, such as glycolysis, to cell-cycle regulation, differentiation, and communication with the extracellular environment through signal transduction cascades. Inappropriate phosphorylation of proteins in cells has been linked to changes in cell cycle progression and cell differentiation. Changes in the cell cycle have been linked to induction of apoptosis or cancer. Changes in cell differentiation have been linked to diseases and disorders of the reproductive system, immune system, and skeletal muscle. There are two classes of protein kinases. One class, protein tyrosine kinases (PTKs), phosphorylates tyrosine residues, and the other class, protein serine/threonine kinases (STKs), phosphorylates serine and threonine residues. Some PTKs and STKs possess structural characteristics of both families and have dual specificity for both tyrosine and serine/threonine residues. Almost all kinases contain a conserved 250-300 amino acid catalytic domain containing specific residues and sequence motifs characteristic of the kinase family. (Reviewed in Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Book. Vol I p.p. 17-20 Academic Press, San Diego, Calif.). Phosphatases Phosphatases hydrolytically remove phosphate groups from proteins. Phosphatases are essential in determining the extent of phosphorylation in the cell and, together with kinases, regulate key cellular processes such as metabolic enzyme activity, proliferation, cell growth and differentiation, cell adhesion, and cell cycle progression. Protein phosphatases are characterized as either serine/threonine- or tyrosine-specific based on their preferred phospho-amino acid substrate. Some phosphatases (DSPs, for dual specificity phosphatases) can act on phosphorylated tyrosine, serine, or threonine residues. The protein serine/threonine phosphatases (PSPs) are important regulators of many cAMP-mediated hormone responses in cells. Protein tyrosine phosphatases (PTPs) play a significant role in cell cycle and cell signaling processes. Proteases Proteases cleave proteins and peptides at the peptide bond that forms the backbone of the protein or peptide chain. Proteolysis is one of the most important and frequent enzymatic reactions that occurs both within and outside of cells. Proteolysis is responsible for the activation and maturation of nascent polypeptides, the degradation of misfolded and damaged proteins, and the controlled turnover of peptides within the cell. Proteases participate in digestion, endocrine function, and tissue remodeling during embryonic development, wound healing, and normal growth. Proteases can play a role in regulatory processes by affecting the half life of regulatory proteins. Proteases are involved in the etiology or progression of disease states such as inflammation, angiogenesis, tumor dispersion and metastasis, cardiovascular disease, neurological disease, and bacterial, parasitic, and viral infections. Proteases can be categorized on the basis of where they cleave their substrates. Exopeptidases, which include aminopeptidases, dipeptidyl peptidases, tripeptidases, carboxypeptidases, peptidyl-di-peptidases, dipeptidases, and omega peptidases, cleave residues at the termini of their substrates. Endopeptidases, including serine proteases, cysteine proteases, and metalloproteases, cleave at residues within the peptide. Four principal categories of mammalian proteases have been identified based on active site structure, mechanism of action, and overall three-dimensional structure. (See Beynon, R. J. and J. S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York, N.Y., pp. 1-5.) Serine Proteases The serine proteases (SPs) are a large, widespread family of proteolytic enzymes that include the digestive enzymes trypsin and chymotrypsin, components of the complement and blood-clotting cascades, and enzymes that control the degradation and turnover of macromolecules within the cell and in the extracellular matrix. Most of the more than 20 subfamilies can be grouped into six clans, each with a common ancestor. These six clans are hypothesized to have descended from at least four evolutionarily distinct ancestors. SPs are named for the presence of a serine residue found in the active catalytic site of most families. The active site is defined by the catalytic triad, a set of conserved asparagine, histidine, and serine residues critical for catalysis. These residues form a charge relay network that facilitates substrate binding. Other residues outside the active site form an oxyanion hole that stabilizes the tetrahedral transition intermediate formed during catalysis. SPs have a wide range of substrates and can be subdivided into subfamilies on the basis of their substrate specificity. The main subfamilies are named for the residue(s) after which they cleave: trypases (after arginine or lysine), aspases (after aspartate), chymases (after phenylalanine or leucine), metases (methionine), and serases (after serine) (Rawlings, N. D. and A. J. Barrett (1994) Meth. Enz. 244:19-61). Most mammalian serine proteases are synthesized as zymogens, inactive precursors that are activated by proteolysis. For example, trypsinogen is converted to its active form, trypsin, by enteropeptidase. Enteropeptidase is an intestinal protease that removes an N-terminal fragment from trypsinogen. The remaining active fragment is trypsin, which in turn activates the precursors of the other pancreatic enzymes. Likewise, proteolysis of prothrombin, the precursor of thrombin, generates three separate polypeptide fragments. The N-terminal fragment is released while the other two fragments, which comprise active thrombin, remain associated through disulfide bonds. The two largest SP subfamilies are the chymotrypsin (S1) and subtilisin (S8) families. Some members of the chymotrypsin family contain two structural domains unique to this family. Kringle domains are triple-looped, disulfide cross-linked domains found in varying copy number. Kringles are thought to play a role in binding mediators such as membranes, other proteins or phospholipids, and in the regulation of proteolytic activity (PROSITE PDOC00020). Apple domains are 90 amino-acid repeated domains, each containing six conserved cysteines. Three disulfide bonds link the first and sixth, second and fifth, and third and fourth cysteines (PROSITE PDOC00376). Apple domains are involved in protein-protein interactions. S1 family members include trypsin, chymotrypsin, coagulation factors IX-XII, complement factors B, C, and D, granzymes, kallikrein, and tissue- and urokinase-plasminogen activators. The subtilisin family has members found in the eubacteria, archaebacteria, eukaryotes, and viruses. Subtilisins include the proprotein-processing endopeptidases kexin and furin and the pituitary prohormone convertases PC1, PC2, PC3, PC6, and PACE4 (Rawlings and Barrett, supra). SPs have functions in many normal processes and some have been implicated in the etiology or treatment of disease. Enterokinase, the initiator of intestinal digestion, is found in the intestinal brush border, where it cleaves the acidic propeptide from trypsinogen to yield active trypsin (Kitamoto, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91: 7588-7592). Prolylcarboxypeptidase, a lysosomal serine peptidase that cleaves peptides such as angiotensin II and III and [des-Arg9] bradykinin, shares sequence homology with members of both the serine carboxypeptidase and prolylendopeptidase families (Tan, F. et al. (1993) J. Biol. Chem. 268:16631-16638). The protease neuropsin may influence synapse formation and neuronal connectivity in the hippocampus in response to neural signaling (Chen, Z.-L. et al. (1995) J Neurosci 15:5088-5097). Tissue plasminogen activator is useful for acute management of stroke (Zivin, J. A. (1999) Neurology 53:14-9) and myocardial infarction (Ross, A. M. (1999) Clin Cardiol 22:165-71). Some receptors (PAR, for proteinase-activated receptor), highly expressed throughout the digestive tract, are activated by proteolytic cleavage of an extracellular domain. The major agonists for PARs, thrombin, trypsin, and mast cell tryptase, are released in allergy and inflammatory conditions. Control of PAR activation by proteases has been suggested as a promising therapeutic target (Vergnolle, N. (2000) Aliment. Pharmacol. Ther. 14:257-266; Rice, K. D. et al. (1998) Curr. Pharm. Des. 4:381-396). Prostate-specific antigen (PSA) is a kallikrein-like serine protease synthesized and secreted exclusively by epithelial cells in the prostate gland. Serum PSA is elevated in prostate cancer and is the most sensitive physiological marker for monitoring cancer progression and response to therapy. PSA can also identify the prostate as the origin of a metastatic tumor. (Brawer, M. K. and Lange, P. H. (1989) Urology 33:11-16). The kallikreins are a subfamily of serine proteases. KLK14 is a kallikrein gene located within the human kallikrein locus at 19q13.4. KLK14 is approximately 5.4 kb in length and transcribes two alternative transcripts present only in prostate and skeletal muscle. In prostate, KLK14 is expressed by both benign and malignant glandular epithelial cells, thus exhibiting an expression pattern similar to that of two other prostatic kallikreins, KLK2 and KLK3, which encode K2 and prostate-specific antigen, respectively (Hooper, J. D. et al. (2001) Genomics 73:117-122). The signal peptidase is a specialized class of SP found in all prokaryotic and eukaryotic cell types that serves in the processing of signal peptides from certain proteins. Signal peptides are amino-terminal domains of a protein which direct the protein from its ribosomal assembly site to a particular cellular or extracellular location. Once the protein has been exported, removal of the signal sequence by a signal peptidase and posttranslational processing, e.g., glycosylation or phosphorylation, activate the protein. Signal peptidases exist as multi-subunit complexes in both yeast and mammals. The canine signal peptidase complex is composed of five subunits, all associated with the microsomal membrane and containing hydrophobic regions that span the membrane one or more times (Shelness, G. S. and G. Blobel (1990) J. Biol. Chem. 265:9512-9519). Some of these subunits serve to fix the complex in its proper position on the membrane while others contain the actual catalytic activity. Another family of proteases which have a serine in their active site are dependent on the hydrolysis of ATP for their activity. These proteases contain proteolytic core domains and regulatory ATPase domains which can be identified by the presence of the P-loop, an ATP/GTP-binding motif (PROSITE PDOC00803). Members of this family include the eukaryotic mitochondrial matrix proteases, Clp protease and the proteasome. Clp protease was originally found in plant chloroplasts but is believed to be widespread in both prokaryotic and eukaryotic cells. The gene for early-onset torsion dystonia encodes a protein related to Clp protease (Ozelius, L. J. et al. (1998) Adv. Neurol. 78:93-105). The proteasome is an intracellular protease complex found in some bacteria and in all eukaryotic cells, and plays an important role in cellular physiology. Proteasomes are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins of all types, including proteins that function to activate or repress cellular processes such as transcription and cell cycle progression (Ciechanover, A. (1994) Cell 79:13-21). In the UCS pathway, proteins targeted for degradation are conjugated to ubiquitin, a small heat stable protein. The ubiquitinated protein is then recognized and degraded by the proteasome. The resultant ubiquitin-peptide complex is hydrolyzed by a ubiquitin carboxyl terminal hydrolase, and free ubiquitin is released for reutilization by the UCS. Ubiquitin-proteasome systems are implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes (p53), cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, supra). This pathway has been implicated in a number of diseases, including cystic fibrosis, Angelman's syndrome, and Liddle syndrome (reviewed in Schwartz, A. L. and A. Ciechanover (1999) Ann. Rev. Med. 50:57-74). A murine proto-oncogene, Unp, encodes a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation of NIH3T3 cells. The human homologue of this gene is consistently elevated in small cell tumors and adenocarcinomas of the lung (Gray, D. A. (1995) Oncogene 10:2179-2183). Ubiquitin carboxyl terminal hydrolase is involved in the differentiation of a lymphoblastic leukemia cell line to a non-dividing mature state (Maki, A. et al. (1996) Differentiation 60:59-66). In neurons, ubiquitin carboxyl terminal hydrolase (PGP 9.5) expression is strong in the abnormal structures that occur in human neurodegenerative diseases (Lowe, J. et al. (1990) J. Pathol. 161:153-160). The proteasome is a large (˜2000 kDa) multisubunit complex composed of a central catalytic core containing a variety of proteases arranged in four seven-membered rings with the active sites facing inwards into the central cavity, and terminal ATPase subunits covering the outer port of the cavity and regulating substrate entry (for review, see Schmidt, M. et al. (1999) Curr. Op. Chem. Biol. 3:584-591). Cysteine Proteases Cysteine proteases (CPs) are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. Nearly half of the CPs known are present only in viruses. CPs have a cysteine as the major catalytic residue at the active site where catalysis proceeds via a thioester intermediate and is facilitated by nearby histidine and asparagine residues. A glutamine residue is also important, as it helps to form an oxyanion hole. Two important CP families include the papain-like enzymes (C1) and the calpains (C2). Papain-like family members are generally lysosomal or secreted and therefore are synthesized with signal peptides as well as propeptides. Most members bear a conserved motif in the propeptide that may have structural significance (Karrer, K. M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3063-3067). Three-dimensional structures of papain family members show a bilobed molecule with the catalytic site located between the two lobes. Papains include cathepsins B, C, H, L, and S, certain plant allergens and dipeptidyl peptidase (for a review, see Rawlings, N. D. and A. J. Barrett (1994) Meth. Enz. 244:461-486). Some CPs are expressed ubiquitously, while others are produced only by cells of the immune system. Of particular note, CPs are produced by monocytes, macrophages and other cells which migrate to sites of inflammation and secrete molecules involved in tissue repair. Overabundance of these repair molecules plays a role in certain disorders. In autoimmune diseases such as rheumatoid arthritis, secretion of the cysteine peptidase cathepsin C degrades collagen, laminin, elastin and other structural proteins found in the extracellular matrix of bones. Bone weakened by such degradation is also more susceptible to tumor invasion and metastasis. Cathepsin L expression may also contribute to the influx of mononuclear cells which exacerbates the destruction of the rheumatoid synovium (Keyszer, G. M. (1995) Arthritis Rheum. 38:976-984). Calpains are calcium-dependent cytosolic endopeptidases which contain both an N-terminal catalytic domain and a C-terminal calcium-binding domain. Calpain is expressed as a proenzyme heterodimer consisting of a catalytic subunit unique to each isoform and a regulatory subunit common to different isoforms. Each subunit bears a calcium-binding EF-hand domain. The regulatory subunit also contains a hydrophobic glycine-rich domain that allows the enzyme to associate with cell membranes. Calpains are activated by increased intracellular calcium concentration, which induces a change in conformation and limited autolysis. The resultant active molecule requires a lower calcium concentration for its activity (Chan S. L. and Mattson M. P. (1999) J. Neurosci. Res. 58:167-190). Calpain expression is predominantly neuronal, although it is present in other tissues. Several chronic neurodegenerative disorders, including ALS, Parkinson's disease and Alzheimer's disease are associated with increased calpain expression (Chan and Mattson, supra). Calpain-mediated breakdown of the cytoskeleton has been proposed to contribute to brain damage resulting from head injury (McCracken E. et al. (1999) J. Neurotrauma 16:749-61). Calpain-3 is predominantly expressed in skeletal muscle, and is responsible for limb-girdle muscular dystrophy type 2A (Minami, N. et al. (1999) J. Neurol. Sci. 171:31-37). Another family of thiol proteases is the caspases, which are involved in the initiation and execution phases of apoptosis. A pro-apoptotic signal can activate initiator caspases that trigger a proteolytic caspase cascade, leading to the hydrolysis of target proteins and the classic apoptotic death of the cell. Two active site residues, a cysteine and a histidine, have been implicated in the catalytic mechanism. Caspases are among the most specific endopeptidases, cleaving after aspartate residues. Caspases are synthesized as inactive zymogens consisting of one large (p20) and one small (p10) subunit separated by a small spacer region, and a variable N-terminal prodomain. This prodomain interacts with cofactors that can positively or negatively affect apoptosis. An activating signal causes autoproteolytic cleavage of a specific aspartate residue (D297 in the caspase-1 numbering convention) and removal of the spacer and prodomain, leaving a p10/p20 heterodimer. Two of these heterodimers interact via their small subunits to form the catalytically active tetramer. The long prodomains of some caspase family members have been shown to promote dimerization and auto-processing of procaspases. Some caspases contain a “death effector domain” in their prodomain by which they can be recruited into self-activating complexes with other caspases and FADD protein associated death receptors or the TNF receptor complex. In addition, two dimers from different caspase family members can associate, changing the substrate specificity of the resultant tetramer. Endogenous caspase inhibitors (inhibitor of apoptosis proteins, or IAPs) also exist. All these interactions have clear effects on the control of apoptosis (reviewed in Chan and Mattson, supra; Salveson, G. S. and V. M. Dixit (1999) Proc. Nat. Acad. Sci. USA 96:10964-10967). Caspases have been implicated in a number of diseases. Mice lacking some caspases have severe nervous system defects due to failed apoptosis in the neuroepithelium and suffer early lethality. Others show severe defects in the inflammatory response, as caspases are responsible for processing IL-1b and possibly other inflammatory cytokines (Chan and Mattson, supra). Cowpox virus and baculoviruses target caspases to avoid the death of their host cell and promote successful infection. In addition, increases in inappropriate apoptosis have been reported in AIDS, neurodegenerative diseases and ischemic injury, while a decrease in cell death is associated with cancer (Salveson and Dixit, supra; Thompson, C. B. (1995) Science 267:1456-1462). Aspartyl Proteases Aspartyl proteases (APs) include the lysosomal proteases cathepsins D and E, as well as chymosin, renin, and the gastric pepsins. Most retroviruses encode an AP, usually as part of the pol polyprotein. APs, also called acid proteases, are monomeric enzymes consisting of two domains, each domain containing one half of the active site with its own catalytic aspartic acid residue. APs are most active in the range of pH 2-3, at which one of the aspartate residues is ionized and the other neutral. The pepsin family of APs contains many secreted enzymes, and all are likely to be synthesized with signal peptides and propeptides. Most family members have three disulfide loops, the first 5 residue loop following the first aspartate, the second 5-6 residue loop preceding the second aspartate, and the third and largest loop occurring toward the C terminus. Retropepsins, on the other hand, are analogous to a single domain of pepsin, and become active as homodimers with each retropepsin monomer contributing one half of the active site. Retropepsins are required for processing the viral polyproteins. APs have roles in various tissues, and some have been associated with disease. Renin mediates the first step in processing the hormone angiotensin, which is responsible for regulating electrolyte balance and blood pressure (reviewed in Crews, D. E. and S. R. Williams (1999) Hum. Biol. 71:475-503). Abnormal regulation and expression of cathepsins are evident in various inflammatory disease states. Expression of cathepsin D is elevated in synovial tissues from patients with rheumatoid arthritis and osteoarthritis. The increased expression and differential regulation of the cathepsins are linked to the metastatic potential of a variety of cancers (Chambers, A. F. et al. (1993) Crit. Rev. Oncol. 4:95-114). Metalloproteases Metalloproteases require a metal ion for activity, usually manganese or zinc. Most zinc-dependent metalloproteases share a common sequence in the zinc-binding domain. The active site is made up of two histidines which act as zinc ligands and a catalytic glutamic acid C-terminal to the first histidine. Proteins containing this signature sequence are known as the metzincins and include aminopeptidase N, angiotensin-converting enzyme, neurolysin, the matrix metalloproteases and the adamalysins (ADAMS). An alternate sequence is found in the zinc carboxypeptidases, in which all three conserved residues—two histidines and a glutamic acid—are involved in zinc binding. A number of the neutral metalloendopeptidases, including angiotensin converting enzyme and the aminopeptidases, are involved in the metabolism of peptide hormones. High aminopeptidase B activity, for example, is found in the adrenal glands and neurohypophyses of hypertensive rats (Prieto, I. Et al. (1998) Horm. Metab. Res. 30:246-248). Oligopeptidase M/neurolysin can hydrolyze bradykinin as well as neurotensin (Serizawa, A. et al. (1995) J. Biol. Chem 270:2092-2098). Neurotensin is a vasoactive peptide that can act as a neurotransmitter in the brain, where it has been implicated in limiting food intake (Tritos, N. A. et al. (1999) Neuropeptides 33:339-349). The matrix metalloproteases (MMPs) are a family of at least 23 enzymes that can degrade components of the extracellular matrix (ECM). They are Zn +2 endopeptidases with an N-terminal catalytic domain. Nearly all members of the family have a hinge peptide and C-terminal domain which can bind to substrate molecules in the ECM or to inhibitors produced by the tissue (TIMPs, for tissue inhibitor of metalloprotease; Campbell, I. L. et al. (1999) Trends Neurosci. 22:285). The presence of fibronectin-like repeats, transmembrane domains, or C-terminal hemopexinase-like domains can be used to separate MMPs into collagenase, gelatinase, stromelysin and membrane-type MMP subfamilies. In the inactive form, the Zn +2 ion in the active site interacts with a cysteine in the pro-sequence. Activating factors disrupt the Zn +2 -cysteine interaction, or “cysteine switch,” exposing the active site. This partially activates the enzyme, which then cleaves off its propeptide and becomes fully active. MMPs are often activated by the serine proteases plasmin and furin. MMPs are often regulated by stoichiometric, noncovalent interactions with inhibitors; the balance of protease to inhibitor, then, is very important in tissue homeostasis (reviewed in Yong, V. W. et al. (1998) Trends Neurosci. 21:75). MMPs are implicated in a number of diseases including osteoarthritis (Mitchell, P. et al. (1996) J. Clin. Inv. 97:761), atherosclerotic plaque rupture (Sukhova, G. K. et al. (1999) Circulation 99:2503), aortic aneurysm (Schneiderman, J. et al. (1998) Am. J. Path. 152:703), non-healing wounds (Saarialho-Kere, U.K. et al. (1994) J. Clin. Inv. 94:79), bone resorption (Blavier, L. and J. M. Delaisse (1995) J. Cell Sci. 108:3649), age-related macular degeneration (Steen, B. et al. (1998) Invest. Ophthalmol. Vis. Sci. 39:2194), emphysema (Finlay, G. A. et al. (1997) Thorax 52:502), myocardial infarction (Rohde, L. E. et al. (1999) Circulation 99:3063) and dilated cardiomyopathy (Thomas, C. V. et al. (1998) Circulation 97:1708). MMP inhibitors prevent metastasis of mammary carcinoma and experimental tumors in rat, and Lewis lung carcinoma, hemangioma, and human ovarian carcinoma xenografts in mice (Eccles S. A. et al. (1996) Cancer Res. 56:2815; Anderson et al. (1996) Cancer Res. 56:715-718; Volpert, O. V. et al. (1996) J. Clin. Invest. 98:671; Taraboletti, G. et al. (1995) JNCI 87:293; Davies, B. et al. (1993) Cancer Res. 53:2087). MMPs may be active in Alzheimer's disease. A number of MMPs are implicated in multiple sclerosis, and administration of MMP inhibitors can relieve some of its symptoms (reviewed in Yong, supra). Another family of metalloproteases is the ADAMs, for A Disintegrin and Metalloprotease Domain, which they share with their close relatives the adamalysins, snake venom metalloproteases (SVMPs). ADAMs combine features of both cell surface adhesion molecules and proteases, containing a prodomain, a protease domain, a disintegrin domain, a cysteine rich domain, an epidermal growth factor repeat, a transmembrane domain, and a cytoplasmic tail. The first three domains listed above are also found in the SVMPs. The ADAMs possess four potential functions: proteolysis, adhesion, signaling and fusion. The ADAMs share the metzincin zinc binding sequence and are inhibited by some MMP antagonists such as TIMP-1. ADAMs are implicated in such processes as sperm-egg binding and fusion, myoblast fusion, and protein-ectodomain processing or shedding of cytokines, cytokine receptors, adhesion proteins and other extracellular protein domains (Schlöndorff, J. and C. P. Blobel (1999) J. Cell. Sci. 112:3603-3617). The Kuzbanian protein cleaves a substrate in the NOTCH pathway (possibly NOTCH itself), activating the program for lateral inhibition in Drosophila neural development. Two ADAMs, TACE (ADAM 17) and ADAM 10, are proposed to have analogous roles in the processing of amyloid precursor protein in the brain (Schlöndorff and Blobel, supra). TACE has also been identified as the TNF activating enzyme (Black, R. A. et al. (1997) Nature 385:729). TNF is a pleiotropic cytokine that is important in mobilizing host defenses in response to infection or trauma, but can cause severe damage in excess and is often overproduced in autoimmune disease. TACE cleaves membrane-bound pro-TNF to release a soluble form. Other ADAMs may be involved in a similar type of processing of other membrane-bound molecules. The ADAMTS sub-family has all of the features of ADAM family metalloproteases and contain an additional thrombospondin domain (TS). The prototypic ADAMTS was identified in mouse, found to be expressed in heart and kidney and upregulated by proinflammatory stimuli (Kuno, K. et al. (1997) J. Biol. Chem. 272:556). To date eleven members are recognized by the Human Genome Organization (HUGO; http://www.gene.ucl.ac.uk/users/hester/adamts.html#Approved). Members of this family have the ability to degrade aggrecan, a high molecular weight proteoglycan which provides cartilage with important mechanical properties including compressibility, and which is lost during the development of arthritis. Enzymes which degrade aggrecan are thus considered attractive targets to prevent and slow the degradation of articular cartilage (See, e.g., Tortorella, M. D. (1999) Science 284:1664; Abbaszade, I. (1999) J. Biol. Chem. 274:23443). Other members are reported to have antiangiogenic potential (Kuno et al., supra) and/or procollagen processing (Colige, A. et al. (1997) Proc.Natl. Acad. Sci. USA 94:2374). All members of the MDC family of integral membrane proteins contain a metalloproteinase-like domain, a disintegrin-like domain and a cysteine-rich domain. They have been identified in a wide range of mammalian tissues and many are abundantly expressed in the male reproductive tract. A number of MDC proteins (fertilin alpha, fertilin beta, tMDC I, tMDC II and tMDC III) are localized to spermatogenic cells and processed as spermatozoa pass through the epididymis, yielding proteins that retain their disintegrin domain on mature spermatozoa. Fertilin beta and tMDC I have been implicated in egg recognition, mediated by a disintegrin-integrin interaction (Frayne, J. et al. (1998) J. Reprod. Fertil. Suppl. 53:149-155). Examples of manganese metalloenzymes include aminopeptidase P and human proline dipeptidase (PEPD). Aminopeptidase P can degrade bradykinin, a nonapeptide activated in a variety of inflammatory responses. Aminopeptidase P has been implicated in coronary ischemia/reperfusion injury. Administration of aminopeptidase P inhibitors has been shown to have a cardioprotective effect in rats (Ersahin, C. et al (1999) J. Cardiovasc. Pharmacol. 34:604-611). Protease Inhibitors Protease inhibitors and other regulators of protease activity control the activity and effects of proteases. Protease inhibitors have been shown to control pathogenesis in animal models of proteolytic disorders (Murphy, G. (1991) Agents Actions Suppl. 35:69-76). In patients with HIV disease protease inhibitors have been shown to be effective in preventing disease progression and reducing mortality (Barry, M. et al. (1997) Clin. Pharmacokinet. 32:194-209). Low levels of the cystatins, low molecular weight inhibitors of the cysteine proteases, correlate with malignant progression of tumors. (Calkins, C. et al. (1995) Biol. Biochem. Hoppe Seyler 376:71-80). The cystatin superfamily of protease inhibitors is characterized by a particular pattern of linearly arranged and tandemly repeated disulfide loops (Kellermann, J. et al. (1989) J. Biol. Chem. 264:14121-14128). An example of a representative of a structural prototype of a novel family among the cystatin superfamily is human alpha 2-HS glycoprotein (AHSG), a plasma protein synthesized in liver and selectively concentrated in bone matrix, dentine, and other mineralized tissues (Triffitt, J. T. (1976) Calcif. Tissue Res. 22:27-33), which is also classified as belonging to the fetuin family. Fetuins are characterized by the presence of 2 N-terminally located cystatin-like repeats and a unique C-terminal domain which is not present in other proteins of the cystatin superfamily (PROSITE PDOC00966). AHSG has been reported to be involved in bone formation and resorption as well as immune responses (Yang, F. et al. (1992) 1130:149-156; Lee, C. C. et al. (1987) PNAS USA 84:4403-4407; Nakamura, O. et al. (1999) Biosci. Biotechnol. Biochem. 63:1383-1391). Additionally, AHSG has been implicated in infertility associated with endometriosis (Mathur, S. P. (2000) Am. J. Reprod. Immunol. 44:89-95; Mathur, S. P. et al. (1999) Autoimmunity 29:121-127) and inhibition of osteogenesis (Binkert, C. et al, (1999) J. Biol Chem. 274:28514-28520). Decreased serum levels of AHSG have been detected in patients with acute leukemias, chronic granulocyte and myelomonocyte leukemias, lymphomas, myelofibrosis, multiple myeloma, metastatizing solid tumors, systemic lupus erythematosus, rheumatoid arthritis, acute alcoholic hepatitis, fatty liver, chronic active hepatitis, liver cirrhosis, acute and chronic pancreatitis, and Crohn's disease (Kalabay, L. et al. (1992) Orv. Hetil. 133:1553-1554; 1559-1560). Serpins are inhibitors of mammalian plasma serine proteases. Many serpins serve to regulate the blood clotting cascade and/or the complement cascade in mammals. Sp32 is a positive regulator of the mammalian acrosomal protease, acrosin, that binds the proenzyme, proacrosin, and thereby aides in packaging the enzyme into the acrosomal matrix (T. Baba et al. (1994) J. Biol. Chem. 269:10133-10140). The Kunitz family of serine protease inhibitors are characterized by one or more “Kunitz domains” containing a series of cysteine residues that are regularly spaced over approximately 50 amino acid residues and form three intrachain disulfide bonds. Members of this family include aprotinin, tissue factor pathway inhibitor (TFPI-1 and TFPI-2), inter-α-trypsin inhibitor (ITI), and bikunin. (Marlor, C. W. et al. (1997) J. Biol. Chem. 272:12202-12208.) Members of this family are potent inhibitors (in the nanomolar range) against serine proteases such as kallikrein and plasmin. has clinical utility in reduction of perioperative blood loss. ITI has been found to inactivate human trypsin, chymotrypsin, neutrophil elastase and cathepsin G (Morii, M. et al. (1985) Biol. Chem. Hoppe Seyler 366:19-21); and is suspected of playing a key role in the biology of the extracellular matrix and in the pathophysiology of chronic bronchopulmonary diseases or lung cancer progression (Cuvelier, A. et al. (2000) Rev. Mal. Respir. 17:437-446). Eppin (Epididymal protease inhibitor) is a family of protease inhibitors expressed in the epididymis and testis. Two eppin isoforms contain both Kunitz-type and WAP-type four disulfide core protease inhibitor consensus sequences. Eppin-1 is expressed only in the testis and epididymis; Eppin-2 is expressed only in the epididymis and Eppin-3 only in the testis (Richardson, R. T. et al. (2001) Gene 270:93-102). Human cystatin C is a potent inihibitor of cysteine proteases. Further, it has amyloidogenic properties. It refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The structure suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe ‘conformational disease’ is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage, and death in young adults (Janowski, R. et al. (2001) Nat. Struct. Biol. 8(4):316-20). A major portion of all proteins synthesized in eukaryotic cells are synthesized on the cytosolic surface of the endoplasmic reticulum (ER). Before these immature proteins are distributed to other organelles in the cell or are secreted, they must be transported into the interior lumen of the ER where post-translational modifications are performed. These modifications include protein folding and the formation of disulfide bonds, and N-linked glycosylations. Protein Isomerases Protein folding in the ER is aided by two principal types of protein isomerases, protein disulfide isomerase (PDI), and peptidyl-prolyl isomerase (PPI). PDI catalyzes the oxidation of free sulfhydryl groups in cysteine residues to form intramolecular disulfide bonds in proteins. PPI, an enzyme that catalyzes the isomerization of certain proline imidic bonds in oligopeptides and proteins, is considered to govern one of the rate limiting steps in the folding of many proteins to their final functional conformation. The cyclophilins represent a major class of PPI that was originally identified as the major receptor for the immunosuppressive drug cyclosporin A (Handschumacher, R. E. et al. (1984) Science 226: 544-547). Protein Glycosylation The glycosylation of most soluble secreted and membrane-bound proteins by oligosaccharides linked to asparagine residues in proteins is also performed in the ER. This reaction is catalyzed by a membrane-bound enzyme, oligosaccharyl transferase. Although the exact purpose of this “N-linked” glycosylation is unknown, the presence of oligosaccharides tends to make a glycoprotein resistant to protease digestion. In addition, oligosaccharides attached to cell-surface proteins called selectins are known to function in cell-cell adhesion processes (Alberts, B. et al. (1994) Molecular Biology of the Cell Garland Publishing Co., New York, N.Y. p.608). “O-linked” glycosylation of proteins also occurs in the ER by the addition of N-acetylgalactosamine to the hydroxyl group of a serine or threonine residue followed by the sequential addition of other sugar residues to the first. This process is catalyzed by a series of glycosyltransferases each specific for a particular donor sugar nucleotide and acceptor molecule (Lodish, H. et al. (1995) Molecular Cell Biology, W. H. Freeman and Co., New York, N.Y. pp.700-708). In many cases, both − and O-linked oligosaccharides appear to be required for the secretion of proteins or the movement of plasma membrane glycoproteins to the cell surface. For example, one of the glycosyltransferases in the dolichol pathway, dolichol phosphate mannose synthase, is required in N:-glycosylation, O-mannosylation, and glycosylphosphatidylinositol membrane anchoring of protein (Tomita, S. et al. (1998) J. Biol. Chem. 9249-9254). Thus, in many cases, both N- and O-linked oligosaccharides appear to be required for the secretion of proteins or the movement of plasma membrane glycoproteins to the cell surface. An additional glycosylation mechanism operates in the ER specifically to target lysosomal enzymes to lysosomes and prevent their secretion. Lysosomal enzymes in the ER receive an N-linked oligosaccharide, like plasma membrane and secreted proteins, but are then phosphorylated on one or two mannose residues. The phosphorylation of mannose residues occurs in two steps, the first step being the addition of an N-acetylglucosamine phosphate residue by N-acetylglucosamine phosphotransferase, and the second the removal of the N-acetylglucosamine group by phosphodiesterase. The phosphorylated mannose residue then targets the lysosomal enzyme to a mannose 6-phosphate receptor which transports it to a lysosome vesicle (Lodish et al. supra, pp. 708-711). Chaperones Molecular chaperones are proteins that aid in the proper folding of immature proteins and refolding of improperly folded ones, the assembly of protein subunits, and in the transport of unfolded proteins across membranes. Chaperones are also called heat-shock proteins (hsp) because of their tendency to be expressed in dramatically increased amounts following brief exposure of cells to elevated temperatures. This latter property most likely reflects their need in the refolding of proteins that have become denatured by the high temperatures. Chaperones may be divided into several classes according to their location, function, and molecular weight, and include hsp60, TCP1, hsp70, hsp40 (also called DnaJ), and hsp90. For example, hsp90 binds to steroid hormone receptors, represses transcription in the absence of the ligand, and provides proper folding of the ligand-binding domain of the receptor in the presence of the hormone (Burston, S. G. and A. R. Clarke (1995) Essays Biochem. 29:125-136). Hsp60 and hsp70 chaperones aid in the transport and folding of newly synthesized proteins. Hsp70 acts early in protein folding, binding a newly synthesized protein before it leaves the ribosome and transporting the protein to the mitochondria or ER before releasing the folded protein. Hsp60, along with hsp10, binds misfolded proteins and gives them the opportunity to refold correctly. All chaperones share an affinity for hydrophobic patches on incompletely folded proteins and the ability to hydrolyze ATP. The energy of ATP hydrolysis is used to release the hsp-bound protein in its properly folded state (Alberts, B. et al. supra, pp 214, 571-572). Dipeptidyl-peptidase I, a lysosomal cysteine proteinase, is important in intracellular degradation of proteins and appears to be a central coordinator for activation of many serine proteinases in immune/inflammatory cells. The gene has been mapped to chromosomal region 11q14.1-q14.3. Dipeptidyl-peptidase I is expressed at high levels in lung, kidney, and placenta, and also at high levels in polymorphonuclear leukocytes and alveolar macrophages and their precursor cells (Rao, N. V. et al. (1997) J. Biol. Chem. 272:10260-10265). IAP is a protein family that has baculovirus IAP repeat (BIR) domains and inhibits apoptosis. A human IAP family gene, Apollon, encodes a 530 kDa protein that contains a single BIR domain and a ubiquitin-conjugating enzyme domain. Apollon has been observed to protect cells from undergoing apoptosis and implicated in tumorigenesis and drug resistance (Chen, Z. et al. (1999) Biochem. Biophys. Res. Commun. 264:847-854). The RTVL-H family is a medium repetitive family of endogenous retrovirus-like sequences found in the genomes of humans and other primates. Different subfamilies of RTVL-H elements are designated Type I, Type Ia, and Type II (Goodchild, N. L. (1993) Virology 196:778-788). Lysyl Hydroxylases Lysyl hydroxylase is an enzyme involved in collagen biosynthesis. Collagens are a family of fibrous structural proteins that are found in essentially all tissues. Collagens are the most abundant proteins in mammals, and are essential for the formation of connective tissue such as skin, bone, tendon, cartilage, blood vessels and teeth. Members of the collagen family can be distinguished from one another by the degree of cross-linking between collagen fibers and by the number of carbohydrate units (e.g., galactose or glucosylgalactose) attached to the collagen fibers. Hydroxylated lysine residues (hydroxylysine) are essential for stability of cross-linking and as attachment points for carbohydrate units. The enzyme lysyl hydroxylase catalyzes the hydroxylation of lysine residues to form hydroxylysine. Lysyl hydroxylase targets the lysine residue of the sequence, X-lys-gly (lys=lysine, gly=glycine, and X=any amino acid residue). Three isoforms of lysyl hydroxylase have been characterized, termed LH1 (or PLOD; procollagen-lysine, 2-oxoglutarate 5-dioxygenase), LH2 (or PLOD2), and LH3. The three enzymes share 60% sequence identity overall, with even higher similarity in the C-terminal region. In addition, there are regions in the middle of the molecule that have an identity of more than 80% (Valtavaara, M. et al. (1998) J. Biol. Chem. 273:12881-12886). Diminished lysyl hydroxylase activity is involved in certain connective tissue disorders. In particular mutations, including a truncation and duplications within the coding region of the gene for PLOD, have been described in patients with type VI Ehlers-Danos syndrome (Hyland, J. et al. (1992) Nature Genet. 2:228-31; Hautala, T. et al. (1993) Genomics 15:399-404). Ubiquitin-Associated Proteins The ubiquitin conjugation system (UCS), is a major pathway for the degradation of cellular proteins of all types, including proteins that function to activate or repress cellular processes such as transcription, cell cycle progression, and immune recognition (Ciechanover, A. (1994) Cell 79:13-21). The process of ubiquitin conjugation and protein degradation involves several steps (Jentsch, S. (1992) Annu. Rev. Genet. 26:179-207). First ubiquitin (Ub), a small, heat stable protein is activated by a ubiquitin-activating enzyme (E1) in an ATP dependent reaction which binds the C-terminus of Ub to the thiol group of an internal cysteine residue in E1. Activated Ub is then transferred to one of several Ub-conjugating enzymes (E2). Different ubiquitin-dependent proteolytic pathways employ structurally similar, but distinct ubiquitin-conjugating enzymes that are associated with recognition subunits which direct them to proteins carrying a particular degradation signal. E2 then transfers the Ub molecule through its C-terminal glycine to a member of the ubiquitin-protein ligase family, E3. Next, E3 transfers the Ub molecule to the target protein. Additional Ub molecules may be added to the target protein forming a multi-Ub chain structure. The ubiquitinated protein is then recognized and degraded by the proteasome, an intracellular protease complex found in some bacteria and in all eukaryotic cells. The resultant ubiquitin-peptide complex is hydrolyzed by a ubiquitin carboxyl terminal hydrolase, and free ubiquitin is released for reutilization by the UCS. Ubiquitin-proteasome systems are implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes (p53), cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, supra). This pathway has been implicated in a number of diseases, including cystic fibrosis, Angelman's syndrome, and Liddle syndrome (reviewed in Schwartz, A. L. and A. Ciechanover (1999) Annu. Rev. Med. 50:57-74). A murine proto-oncogene, Unp, encodes a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation of NIH3T3 cells. The human homologue of this gene is consistently elevated in small cell tumors and adenocarcinomas of the lung (Gray, D. A. (1995) Oncogene 10:2179-2183). Ubiquitin carboxyl terminal hydrolase is involved in the differentiation of a lymphoblastic leukemia cell line to a non-dividing mature state (Maki, A. et al. (1996) Differentiation 60:59-66). In neurons, ubiquitin carboxyl terminal hydrolase (PGP 9.5) expression is strong in the abnormal structures that occur in human neurodegenerative diseases (Lowe, J. et al. (1990) J. Pathol. 161:153-160). Additional ubiquitin-like proteins which also possess the ability to covalently modify other cellular proteins have been identified in recent years. (For review, see Yeh, E. T. H. et al. (2000) Gene 248:1-14; and Jentsch, S. and Pyrowolakis, G. (2000) Trends Cell Biol. 10:335-342.) These ubiquitin-like protein modifiers include the sentrins (also known as SUMO proteins), NEDD8, and Apg12. The conjugation pathways for these proteins closely resemble that for ubiquitin. For example, conjugation of sentrin requires the E1 heterodimer AOS1/UBA2, and a single E2 enzyme, UBC9. The recently discovered protein S3 may function as a sentrin ligase. The yeast protein Ulp1 is a sentrin hydrolase. Inactivation of Ulp1 in yeast results in severe cell cycle defects. In humans, seven sentrin specific proteases (SENP) have been identified, which range in size from 238 to 1112 amino acid residues (Yeh, supra). All human SENPs share a conserved C-terminal domain. The N-terminal regions may regulate cellular location and substrate specificity. Sentrinization does not promote protein degradation as does ubiquitin. In some cases sentrinization appears to be important for stable localization of target proteins in nuclear bodies. Substrates for sentrinization include PML, a RING finger protein with tumor suppressor activity, HIPK2, a co-repressor for homeodomain transcription factors, and the tumor suppressor p53. IκBα, a cytosolic inhibitor of NFκB, a transcription factor involved in induction of inflammation associated proteins, is also a substrate for sentrinization. Sentrinized IκBα cannot be ubiquitinated and is resistant to proteasomal degradation, suggesting links between the ubiquitin and sentrin pathways. Jentsch, supra). Expression Profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. Steroids Affecting Protein Modification Steroids are a class of lipid-soluble molecules, including cholesterol, bile acids, vitamin D, and hormones, that share a common four-ring structure based on cyclopentanoperhydrophenanthrene and that carrry out a wide variety of functions. Cholesterol, for example, is a component of cell membranes that controls membrane fluidity. It is also a precursor for bile acids which solubilize lipids and facilitate absorption in the small intestine during digestion. Vitamin D regulates the absorption of calcium in the small intestine and controls the concentration of calcium in plasma. Steroid hormones, produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens. They control various biological processes by binding to intracellular receptors that regulate transcription of specific genes in the nucleus. Glucocorticoids, for example, increase blood glucose concentrations by regulation of gluconeogenesis in the liver, increase blood concentrations of fatty acids by promoting lipolysis in adipose tissues, modulate sensitivity to catcholamines in the central nervous system, and reduce inflammation. The principal mineralocorticoid, aldosterone, is produced by the adrenal cortex and acts on cells of the distal tubules of the kidney to enhance sodium ion reabsorption. Androgens, produced by the interstitial cells of Leydig in the testis, include the male sex hormone testosterone, which triggers changes at puberty, the production of sperm and maintenance of secondary sexual characteristics. Female sex hormones, estrogen and progesterone, are produced by the ovaries and also by the placenta and adrenal cortex of the fetus during pregnancy. Estrogen regulates female reproductive processes and secondary sexual characteristics. Progesterone regulates changes in the endometrium during the menstrual cycle and pregnancy. Steroid hormones are widely used for fertility control and in anti-inflammatory treatments for physical injuries and diseases such as arthritis, asthma, and auto-immune disorders. Progesterone, a naturally occurring progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding, or as a contraceptive. Endogenous progesterone is responsible for inducing secretory activity in the endometrium of the estrogen-primed uterus in preparation for the implantation of a fertilized egg and for the maintenance of pregnancy. It is secreted from the corpus luteum in response to luteinizing hormone (LH). The primary contraceptive effect of exogenous progestins involves the suppression of the midcycle surge of LH. At the cellular level, progestins diffuse freely into target cells and bind to the progesterone receptor. Target cells include the female reproductive tract, the mammary gland, the hypothalamus, and the pituitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone from the hypothalamus and blunt the pre-ovulatory LH surge, thereby preventing follicular maturation and ovulation. Progesterone has minimal estrogenic and androgenic activity. Progesterone is metabolized hepatically to pregnanediol and conjugated with glucuronic acid. Medroxyprogesterone (MAH), also known as 6α-methyl-17-hydroxyprogesterone, is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone. MAH is used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance. MAH has a stimulatory effect on respiratory centers and has been used in cases of low blood oxygenation caused by sleep apnea, chronic obstructive pulmonary disease, or hypercapnia. Mifepristone, also known as RU-486, is an antiprogesterone drug that blocks receptors of progesterone. It counteracts the effects of progesterone, which is needed to sustain pregnancy. Mifepristone induces spontaneous abortion when administered in early pregnancy followed by treatment with the prostaglandin, misoprostol. Further, studies show that mifepristone at a substantially lower dose can be highly effective as a postcoital contraceptive when administered within five days after unprotected intercourse, thus providing women with a “morning-after pill” in case of contraceptive failure or sexual assault. Mifepristone also has potential uses in the treatment of breast and ovarian cancers in cases in which tumors are progesterone-dependent. It interferes with steroid-dependent growth of brain meningiomas, and may be useful in treatment of endometriosis where it blocks the estrogen-dependent growth of endometrial tissues. It may also be useful in treatment of uterine fibroid tumors and Cushing's Syndrome. Mifepristone binds to glucocorticoid receptors and interferes with cortisol binding. Mifepristone also may act as an anti-glucocorticoid and be effective for treating conditions where cortisol levels are elevated such as AIDS, anorexia nervosa, ulcers, diabetes, Parkinson's disease, multiple sclerosis, and Alzheimer's disease. Danazol is a synthetic steroid derived from ethinyl testosterone. Danazol indirectly reduces estrogen production by lowering pituitary synthesis of follicle-stimulating hormone and LH. Danazol also binds to sex hormone receptors in target tissues, thereby exhibiting anabolic, antiestrognic, and weakly androgenic activity. Danazol does not possess any progestogenic activity, and does not suppress normal pituitary release of corticotropin or release of cortisol by the adrenal glands. Danazol is used in the treatment of endometriosis to relieve pain and inhibit endometrial cell growth. It is also used to treat fibrocystic breast disease and hereditary angioedema. Corticosteroids are used to relieve inflammation and to suppress the immune response. They inhibit eosinophil, basophil, and airway epithelial cell function by regulation of cytokines that mediate the inflammatory response. They inhibit leukocyte infiltration at the site of inflammation, interfere in the function of mediators of the inflammatory response, and suppress the humoral immune response. Corticosteroids are used to treat allergies, asthma, arthritis, and skin conditions. Beclomethasone is a synthetic glucocorticoid that is used to treat steroid-dependent asthma, to relieve symptoms associated with allergic or nonallergic (vasomotor) rhinitis, or to prevent recurrent nasal polyps following surgical removal. The anti-inflammatory and vasoconstrictive effects of intranasal beclomethasone are 5000 times greater than those produced by hydrocortisone. Budesonide is a corticosteroid used to control symptoms associated with allergic rhinitis or asthma. Budesonide has high topical anti-inflammatory activity but low systemic activity. Dexamethasone is a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive compositions. It is also used in inhalants to prevent symptoms of asthma. Due to its greater ability to reach the central nervous system, dexamethasone is usually the treatment of choice to control cerebral edema. Dexamethasone is approximately 20-30 times more potent than hydrocortisone and 5-7 times more potent than prednisone. Prednisone is metabolized in the liver to its active form, prednisolone, a glucocorticoid with anti-inflammatory properties. Prednisone is approximately 4 times more potent than hydrocortisone and the duration of action of prednisone is intermediate between hydrocortisone and dexamethasone. Prednisone is used to treat allograft rejection, asthma, systemic lupus erythematosus, arthritis, ulcerative colitis, and other inflammatory conditions. Betamethasone is a synthetic glucocorticoid with antiinflammatory and immunosuppressive activity and is used to treat psoriasis and fungal infections, such as athlete's foot and ringworm. The anti-inflammatory actions of corticosteroids are thought to involve phospholipase A 2 inhibitory proteins, collectively called lipocortins. Lipocortins, in turn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic acid. Proposed mechanisms of action include decreased IgE synthesis, increased number of β-adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism. During an immediate allergic reaction, such as in chronic bronchial asthma, allergens bridge the IgE antibodies on the surface of mast cells, which triggers these cells to release chemotactic substances. Mast cell influx and activation, therefore, is partially responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of corticosteroids. Toxicology Testing: Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for examining which genes are tissue specific, carry out housekeeping functions, are parts of a signaling cascade, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of disease. For example, both the levels and sequences expressed in tissues from subjects with hyperlipidemia may be compared with the levels and sequences expressed in normal tissue. Toxicity testing is a mandatory and time-consuming part of drug development programs in the pharmaceutical industry. A more rapid screen to determine the effects upon metabolism and to detect toxicity of lead drug candidates may be the use of gene expression microarrays. For example, microarrays of various kinds may be produced using full length genes or gene fragments. These arrays can then be used to test samples treated with the drug candidates to elucidate the gene expression pattern associated with drug treatment. This gene pattern can be compared with gene expression patterns associated with compounds which produce known metabolic and toxicological responses. The human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth. The use of a clonal population enhances the reproducibility of the cells. C3A cells have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insulin-like growth factor II receptor; ii) secretion of a high ratio of serum albumin compared with α-fetoprotein iii) convertion of ammonia to urea and glutamine; iv) metabolism of aromatic amino acids; and v) ability to proliferate in glucose-free and insulin-free medium. The C3A cell line is now well established as an in vitro model of the mature human liver (Mickelson et al. (1995) Hepatology 22:866-875; Nagendra et al. (1997) Am. J. Physiol. 272:G408-416). Clofibrate is an hypolidemic drug which lowers elevated levels of serum triglycerides. In rodents, chronic treatment produces hepatomegaly and an increase in hepatic peroxisomes (peroxisome proliferation). Peroxisome proliferators (PPs) are a class of drugs which activate the PP-activated receptor in rodent liver, leading to enzyme induction, stimulation of S-phase, and a suppression of apoptosis (Hasmall and Roberts (1999) Pharmacol. Ther. 82:63-70). PPs include the fibrate class of hypolidemic drugs, phenobarbitone, thiazolidinediones, certain non-steroidal anti-inflammatory drugs, and naturally-occuring fatty acid-derived molecules (Gelman et al. (1999) Cell. Mol. Life Sci. 55:932-943). Clofibrate has been shown to increase levels of cytochrome P450 4A. It is also involved in transcription of β-oxidation genes as well as induction of PP-activated receptors (Kawashima et al. (1997) Arch. Biochem. Biophys. 347:148-154). Peroxisome proliferation that is induced by both clofibrate and the chemically-related compound fenofibrate is mediated by a common inhibitory effect on mitochondrial membrane depolarization (Zhou and Wallace (1999) Toxicol. Sci. 48:82-89). Dexamethasone and its derivatives, dexamethasone sodium phosphate and dexamethasone acetate, are synthetic glucocorticoids used as anti-inflammatory or immunosuppressive agents. Dexamethasone has little to no mineralocorticoid activity and is usually selected for management of cerebral edema because of its superior ability to penetrate the central nervous sytem. Glucocorticoids are naturally occurring hormones that prevent or suppress inflammation and immune responses when administered at pharmacological doses. Responses can include inhibition of leukocyte infiltration at the site of inflammation, interference in the function of mediators of inflammatory response, and suppression of humoral immune responses. The anti-inflammatory actions of corticosteroids are thought to involve phospholipase A 2 inhibitory proteins, collectively called lipocortins. The numerous adverse effects related to corticosteroid use usually depend on the dose administered and the duration of therapy. Proposed mechanisms of action include decreased IgE synthesis, increased number of β-adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism. During an immediate allergic reaction, such as in chronic bronchial asthma, allergens bridge the IgE antibodies on the surface of mast cells, which triggers these cells to release chemotactic substances. Mast cell influx and activation, therefore, is partially responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of adrenocorticoids. As with other corticosteroids, the effects upon liver metabolism and hormone clearance mechanisms are important to understand the pharmacodynamics of a drug. Cancer Prostate cancer develops through a multistage progression ultimately resulting in an aggressive tumor phenotype. The initial step in tumor progression involves the hyperproliferation of normal luminal and/or basal epithelial cells. Androgen responsive cells become hyperplastic and evolve into early-stage tumors. Although early-stage tumors are often androgen sensitive and respond to androgen ablation, a population of androgen independent cells evolve from the hyperplastic population. These cells represent a more advanced form of prostate tumor that may become invasive and potentially become metastatic to the bone, brain, or lung. Breast cancer develops through a multi-step process in which pre-malignant mammary epithelial cells undergo a relatively defined sequence of events leading to tumor formation. An early event in tumor development is ductal hyperplasia. Cells undergoing rapid neoplastic growth gradually progress to invasive carcinoma and become metastatic to the lung, bone, and potentially other organs. Several variables that may influence the process of tumor progression and malignant transformation include genetic factors, environmental factors, growth factors, and hormones. Based on the complexity of this process, it is critical to study a population of human mammary epithelial cells undergoing the process of malignant transformation, and to associate specific stages of progression with phenotypic and molecular characteristics. Immune Response Proteins Interleukin 12 (IL-12) is a pleiotropic cytokine produced by macrophages and B lymphocytes that can have multiple effects on T cells and natural killer (NK) cells. Effects include inducing production of IFN-γ and TNF by resting and activated T and NK cells; enhancing the cytotoxic activity of resting NK and T cells, inducing and synergizing with IL-2 in the generation of lymphokine-activated killer (LAK) cells; acting as a comitogen to stimulate proliferation of resting T cells; and inducing proliferation of activated T and NK cells. Current evidence indicates that IL-12, produced by macrophages in response to infectious agents, is a central mediator of the cell-mediated immune response by its actions on the development, proliferation, and activities of TH1 cells. As the initiator of cell-mediated immunity, IL-12 may stimulate cell-mediated immune responses to microbial pathogens, metastatic cancers, and viral infections such as AIDS. There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders. |
<SOH> SUMMARY OF THE INVENTION <EOH>Various embodiments of the invention provide purified polypeptides, protein modification and maintenance molecules, referred to collectively as “PMOD” and individually as “PMOD-1,” “PMOD-2,” “PMOD-3,” “PMOD-4,” “PMOD-5,” “PMOD-6,” “PMOD-7,” “PMOD-8,” “PMOD-9,” “PMOD-10,” “PMOD-11,” “PMOD-12,” “PMOD-13,” “PMOD-14,” “PMOD-15,” “PMOD-16,” “PMOD-17,” “PMOD-18,” “PMOD-19,” “PMOD-20,” “PMOD-21,” “PMOD-22,” “PMOD-23,” “PMOD-24,” “PMOD-25,” “PMOD-26,” “PMOD-27,” and “PMOD-28,” and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified protein modification and maintenance molecules and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified protein modification and maintenance molecules and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions. An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-28. Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-28. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:29-56. Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed. Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof. Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional PMOD, comprising administering to a patient in need of such treatment the composition. Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional PMOD, comprising administering to a patient in need of such treatment the composition. Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional PMOD, comprising administering to a patient in need of such treatment the composition. Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide. Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide. Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:29-56, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. |
Memory for producing a memory component |
The invention relates to a method for producing a memory component comprising a memory location (104) having memory cells and first control electrode strips (162) for controlling the individual memory cells, and a peripheral area (106) having peripheral elements and second control electrode strips (164) for controlling said peripheral elements. The inventive method enables the expansion of the second control electrode strips (164) in the peripheral area (106) to be approximately randomly adjusted to minimum line widths, without influencing or changing the expansion of the first control electrode strips (162) in the memory location (104). |
1. A method for producing a memory component comprising a memory cell region having memory cells and first control electrode tracks for driving the individual memory cells and a peripheral region having peripheral elements and second control electrode tracks for driving the peripheral elements, comprising: applying, in a first direction, to a provision of a substrate having memory cell structures, an insulation layer, a layer stack and a patterning layer; applying a first mask layer on the patterning layer in the first direction; patterning of the first mask layer in the first direction, the first mask layer comprising, in the memory cell region 44 and the peripheral region, closed regions, in which the first mask layer is not removed and which are assigned to the first and second control electrode tracks, and open regions, in which the first mask layer is removed, in at least one second direction perpendicular to the first direction; transferring the mask structure of the first mask layer to the patterning layer in the first direction, such that the structure of the patterning layer corresponds to the mask structure; removing the first mask layer; filling open regions of the patterning layer in at least the memory cell region with a protective material, such that the open regions are filled with the protective material in the second direction in a manner essentially flush with the patterning layer; selectively setting the expansion of closed regions of the patterning layer in the peripheral region in at least the second direction, such that the expansion of closed regions bounded by at least one open region of the patterning layer, the open region having no protective material, is set; removing the protective material in the memory cell region and in the peripheral region selectively with respect to the patterning layer and the layer stack; transferring the structures of the patterning layer in the first direction to the layer stack in order to produce the first and the second control electrode tracks. 2. The method as claimed in claim 1, in which the fillings comprises: applying the protective material in the memory cell region and the peripheral region. 3. The method as claimed in claim 2, in which the filling further comprises: direction-selective removing the protective material in the first direction, such that the protective material is removed in the first direction from the upper ends of the closed regions of the patterning layer, and the open regions are filled with the protective material in at least the second direction in a manner essentially flush with the upper ends of the closed regions of the patterning layers. 4. The method as claimed in claim 3, in which the filling further comprises: applying a second mask layer at least on the memory cell region. 5. The method as claimed in claim 4, in which the filling further comprises: removing the protective material in the regions of the peripheral region which are not covered by the second mask layer, selectively with respect to the patterning layer and the layer stack. 6. The method as claimed in claim 5, in which the filling further comprises: removing the second mask layer. 7. The method as claimed in claim 1, in which the selectively setting further comprises: partially removing the patterning layer in the memory cell region and in the peripheral region selectively with respect to the protective material. 8. The method as claimed in claim 1, in which the patterning of the first mask layer comprises patterning by means of photolithography. 9. The method as claimed in claim 1, in which the step of transfer of the mask structure of the first mask layer to the patterning layer comprises selectively etching the patterning layer. 10. The method as claimed in claim 1, in which the transfer of the structures of the patterning layer to the layer stack comprises selectively etching the layer stack with respect to the insulation layer. 11. The method as claimed in claim 1, in which the layer stack has a control electrode layer and a conductivity increasing layer. 12. The method as claimed in claim 1, in which the provision comprises: a provision of the insulation layer having insulators which are embedded in the substrates and which isolate the memory cell regions from the peripheral regions. 13. The method as claimed in claim 1, in which the memory component comprises a dynamic random access memory. 14. The method as claimed in claim 1, in which the first and second control electrode tracks comprise gate stacks of MOS field-effect transistors. 15. The method as claimed in claim 1, in which the substrate comprises silicon. 16. The method as claimed in claim 1, in which the insulation layer comprises silicon oxide. 17. The method as claimed in claim 11, in which the control electrode layer comprises polysilicon. 18. The method as claimed in claim 11, in which the conductivity increasing layer comprises tungsten silicide. 19. The method as claimed in claim 11, in which the patterning layer comprises silicon nitride. 20. The method as claimed in claim 11, in which the first and/or the second mask layer comprise a resist layer. 21. The method as claimed in claim 1, in which the protective layer comprises an oxide. 22. The method as claimed in claim 21, in which the oxide is formed by means of a subatmospheric chemical vapor deposition or a low pressure chemical vapor deposition. 23. The method as claimed in claim 5, in which removing the protective material in the regions of the peripheral region which are not covered by the second mask layer comprises removing the protective material using hydrofluoric acid. 24. The method as claimed claim 7, in which partially removing the patterning layer comprises removing the patterning layer using a mixture of hydrofluoric acid and ethylene glycol. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The speed or performance of an integrated circuit is greatly dependent on the smallest control electrode length or gate length of an insulated transistor that can be reliably realized. The magnitude of the control electrode length may be subject to technological boundary conditions which limit said length. In a dynamic random access memory (DRAM), both a memory cell region or cell array and a peripheral region have to be produced in a process sequence. The memory cell region comprises control electrode tracks or gate conductor tracks for field-effect selection transistors which are assigned to memory cells, and gaps between the control electrode tracks having a specific distance (on pitch). By contrast, the peripheral region comprises the driving logic and clock generation, etc. for the memory cells in the memory cell region and/or another logic and usually likewise field-effect transistors with control electrode tracks and gaps between the control electrode tracks. Since it is necessary to effect optimization to the memory cell region in particular with regard to the control electrode lithography, however, the minimum insulated line width of a control electrode track of a transistor in the peripheral region cannot be chosen freely. This has the effect that a dynamic random access memory or an embedded dynamic random access memory which comprises both a memory cell region and a peripheral region is at a disadvantage with regard to the performance of the peripheral region compared with a pure logic circuit in which the entire lithography can be concentrated on the smallest insulated control electrode track. However, since the demands with regard to the performance of memory components, such as e.g. dynamic random access memories (DRAMs), are also increasing, improvements which are suitable for production and improve the performance of the transistors in the peripheral region of memory components are desirable. In the figures, reference symbols which differ only in respect of the first numeral designate identical or functionally identical constituent parts. FIG. 2 shows a known method for producing a memory component, and in particular the method for patterning the control electrode plane for a DRAM. FIG. 2A shows a substrate 200 , in which there are already situated parts, such as e.g. wells, etc., of the later memory components and insulations 202 which divide the substrate 200 into a memory cell region 204 and a peripheral region 206 . A control electrode oxide layer 208 or a gate oxide layer is applied on the substrate 200 . A layer stack comprising a polysilicon layer 210 , which is usually n-doped, and a tungsten silicide (WSi x ) layer 212 for increasing the conductivity is applied on the control electrode oxide layer 208 . A patterning layer 214 or a cap layer made of silicon nitride (SiN) is applied on the layer stack. The patterning layer 214 is very important for patterning in the memory cell region 204 , and there in particular for the production of the bit line contacts, which are not discussed in any further detail. In contrast to a logic circuit which is not divided into memory cell region and peripheral region, however, attention shall be drawn explicitly to the need for said patterning layer, even if the latter is rather disturbing in the peripheral region of a memory component. A resist mask 216 applied on the patterning layer 214 is patterned by means of photolithography, in such a way that it has open regions 216 a and closed regions 216 b . As already mentioned above, in the memory cell region 204 , optimization is effected to the dimension of the line width 218 of a control electrode track in the memory cell region 204 . The minimum line width 220 of a closed region 216 b of the resist mask 216 which is assigned to an insulated control electrode track in the peripheral region 206 is then defined by the illumination conditions and the material parameters of the resist mask 216 . FIG. 2B shows that the patterning layer 214 is etched selectively with respect to the tungsten silicide layer 212 , and the resist mask 216 is removed. The patterning layer 214 has open regions 214 a and closed regions 214 b equivalent to the resist mask 216 . The etching changes the line width 218 of the closed regions 214 b in the memory cell region 204 , which are assigned to the control electrode tracks in the memory cell region 204 , to a line width 222 and the line width 220 of the closed regions 214 b in the peripheral region 206 , which are assigned to control electrode tracks in the peripheral region 206 , to a line width 224 , which is referred to as the etching deviation or the etching bias of the mask opening step for opening the mask in the patterning layer 214 . FIG. 2C shows control electrode tracks 226 or control electrode stacks (gate stacks) for driving individual memory cells in the memory cell region 204 and control electrode tracks 228 for driving peripheral elements in the peripheral region 206 after the structures of the patterning layer 214 have been transferred to the layer stack of the polysilicon layer 210 and the tungsten silicide layer 212 . The patterned patterning layer 214 was used as a hard mask for patterning the polysilicon layer 210 and the tungsten silicide layer 212 . This control electrode etching step is designed in such a way that it stops on the control electrode oxide layer 208 . During this method, once again the line width 222 of a closed region 214 b assigned to a control electrode track in the memory cell region 204 changes to an actual line width 230 of the control electrode track 226 for driving the individual memory cells in the memory cell region 204 , and the line width 224 of a closed region 214 b assigned to a control electrode track in the peripheral region 206 changes to an actual line width 232 of the control electrode track 228 for driving the peripheral elements. This change in the line width corresponds to the etching deviation of the control electrode etching step. The change in the line width from FIG. 2B to 2 C is small, however, during this step. The thickness of the patterning layer 214 additionally changes during the transfer of the structures, said patterning layer being reduced to a thickness 234 in this case. This change in the thickness is identical for both the memory cell region 204 and the peripheral region 206 after the control electrode track etching, within the bounds of small fluctuations. FIG. 3A shows the production of typical control electrode tracks of a pure logic circuit which does not comprise different regions, such as e.g. a memory cell region and a peripheral region. These control electrode tracks differ in several points from the control electrode tracks of a memory component, such as e.g. a DRAM. The layer structure of the control electrode tracks comprises, similarly to FIG. 1 , a substrate 300 , a control electrode oxide layer 308 applied on the substrate 308 , and a polysilicon layer 310 applied on the control electrode oxide layer 308 . The polysilicon of polysilicon layer 310 is undoped at this point in time in the method, in order later to be able to realize transistors having n- and p-doped control electrodes or gates. In comparison with the structure of a memory component as shown in FIG. 2 , the layer structure shown in FIG. 3 does not have a tungsten silicide layer, since the low resistance of the control electrode tracks can later be achieved by means of saliciding. This is possible in particular because no patterning layer or cap layer made of silicon nitride is used, rather an oxide layer 336 is instead deposited on the polysilicon layer 310 , which is later consumed during the method. In the logic circuit shown in FIG. 3 , there is no memory cell region in which the smallest insulated track of a control electrode track determines the process window, and the resist and the exposure conditions can be optimized thereto. A resist layer 316 is applied on the oxide layer 336 , which resist layer is already patterned and has open regions 316 a and closed regions 316 b , the closed regions 316 b having line widths 338 and 340 assigned to control electrode tracks. FIG. 3B shows the layer structure after the transfer of the structure of the resist layer 316 to the oxide layer 336 and after the removal of the resist layer 316 . During this transfer, open regions 336 a and closed regions 336 b are produced in the control electrode oxide layer 336 , the closed regions 336 b having line widths 342 and 344 assigned to control electrode tracks. Finally, FIG. 3C shows the layer structure after the transfer of the structure of the oxide layer 336 to the polysilicon layer 310 . The line widths 342 , 344 of closed regions 336 b of the oxide layer 336 are transferred into actual line widths 346 , 348 of the control electrode tracks 350 or control stacks. The remaining oxide layer 336 is thinned compared with the original oxide layer shown in FIG. 3A and is removed in later method steps before the saliciding. FIG. 4 shows a method for reducing the line width and/or the line length of a control electrode track or a control stack of individual transistors in logic circuits additionally below the lithographically governed minima. The structure shown in FIG. 4A once again has a substrate 400 , on which a control electrode oxide layer 408 and a polysilicon layer 410 are applied. The logic circuit is divided into a first region 404 and a second region 406 by insulators 402 . There is applied on the polysilicon layer 410 a patterned oxide layer 436 having open regions 436 a and closed regions 436 b , the structure of which corresponds to the structure shown in FIG. 3B . The closed regions 436 b produced in the structure of the oxide layer 436 , which are assigned to control electrode tracks, have line widths 442 and 444 . In FIG. 4A , a resist mask 452 is applied on a part of the logic circuit. In order to reduce the line width 444 of a closed region 436 b assigned to a control electrode track in FIG. 4A , an isotropic etching is carried out, e.g. in hydrofluoric acid (HF), as a result of which the patterned closed regions 436 b of the oxide layer 436 which are not covered by the resist layer 452 are reduced laterally to a line width 445 and vertically to a thickness 447 . This step is generally called pull-back. The resist mask 452 is stripped or removed in a next step, e.g. by incineration, and an oxide layer 436 having different local thicknesses remains, which is shown in FIG. 4B . The oxide layer 436 therefore does not form a uniform plane, which may lead to problems e.g. in later polishing methods. Such problems must be avoided in particular in the case of memory components, such as e.g. DRAM memory components. In the case of logic circuits, in contrast to memory components, this is unimportant, however, since the oxide layer 436 has already fulfilled its function and can be removed. In the case of logic circuits, the isotropic etching step may, of course, also be carried out without a resist layer 452 , and closed regions 436 b assigned to control electrode tracks may simultaneously be diminished. Finally, FIG. 4C shows the transfer of the structure of the oxide layer 436 to the polysilicon layer 410 in order to form control electrode tracks 450 having actual line widths 446 and 448 . A further possibility for realizing cell regions and very narrow insulated control electrode tracks in the control electrode conductor plane consists in a double exposure. This can be applied in principle to memory components, but has the disadvantages of a high outlay and of overlay problems during the exposure of subsequent planes. Therefore, one disadvantage in the prior art is that, during the production of control electrode tracks for memory components, although the line width of control electrode tracks assigned to memory cells in a memory cell region of a memory component can be optimized optically and in terms of magnitude, at the same time it is possible as a result only to effect a limited reduction of the extent, such as e.g. reduction of the line width, of the control electrode tracks assigned to peripheral elements in a peripheral region of memory components. This problem is due to the fact that peripheral regions of memory components are typically provided with logic circuits, such as e.g. a driving logic or a clock generation, which do not have periodic structures but rather structures that are far away from one another, such as e.g. control electrode tracks, which do not afford any optical support during the exposure of the structures whereby the resolution could be improved and the line width minimized. A further disadvantage in the prior art is that in alternative methods for setting the extent, such as e.g. the line width of control electrode tracks, in different regions of an integrated circuit, the known methods have the effect that the thickness of a patterning layer, such as e.g. a silicon nitride layer, varies, which leads to problems during later required polishing of the structure of the memory component. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention discloses a method for producing a memory component which makes it possible to reduce the extent of control electrode tracks in a peripheral region of a memory component without impairing the yield in the production of the memory component. The invention has the advantage over the known solutions, in particular the known method of FIG. 2 , that it is possible to form narrow insulated control electrode tracks or narrow control stacks in a peripheral region of a memory component by using a small number of additional steps after the step of patterning of a patterning layer or after a mask opening etching for the memory component, such as e.g. a dynamic random access memory (DRAM). In this case, the lithography remains untouched and continues to be optimized to the cell region of the memory component. Additionally used layers are removed again in the course of the method according to the invention, so that the final structure achieved is identical to a typical memory component structure, merely with the difference that a reduced line width of the control electrode tracks is achieved in the peripheral region, and that a more greatly reduced but uniform thickness of a patterning layer, such as e.g. a silicon nitride layer, occurs, it being possible for this greater reduction of the thickness to be readily corrected by means of a larger deposition thickness of the patterning layer. Therefore, a further advantage of the invention is that, during later polishing methods for the memory component, the patterning layer has a uniform thickness, and this therefore cannot lead to damage to the memory component and, therefore, also cannot lead to a reduced yield in the production of the memory component. A further advantage of the the invention over the known solutions is that the method steps of the invention are known in the production of memory components or from other production methods and the main steps of a production method remain unchanged, which ensures a simplified implementation in existing production methods. In accordance with one preferred embodiment of the invention, the filling comprises application of the protective material in the memory cell region and the peripheral region. In accordance with a further preferred embodiment of the invention, the filling comprises the direction-selective removal of the protective material in the first direction, in such a way that the protective material is removed in the first direction from the upper ends of the closed regions of the patterning layers, and the open regions are filled with the protective material in at least the second direction in a manner essentially flush with the upper ends of the closed regions of the patterning layer. In accordance with a further preferred embodiment of the invention, the filling comprises the application of a second mask layer at least on the memory cell region. In accordance with a further preferred embodiment of the invention, the filling comprises the removal of the protective material in those regions of the peripheral region which are not covered by the second mask layer, selectively with respect to the patterning layer and the layer stack. In accordance with a further preferred embodiment of the invention, the filling furthermore comprises removal of the second mask layer. In accordance with a further preferred embodiment of the invention, the selective setting furthermore comprises partial removal of the patterning layer in the memory cell region and in the peripheral region selectively with respect to the protective material. In accordance with a further preferred embodiment of the invention, the patterning of the first mask layer comprises patterning by means of photolithography. In accordance with a further preferred embodiment of the invention, the transfer of the mask structure of the first mask layer to the patterning layer comprises selective etching of the patterning layer. In accordance with a further preferred embodiment of the invention, the transfer of the structures of the patterning layer to the layer stack comprises selective etching of the layer stack with respect to the insulation layer. In accordance with a further preferred embodiment of the invention, the layer stack has a control electrode layer and a conductivity increasing layer. In accordance with a further preferred embodiment of the invention, the provision comprises provision of the insulation layer having insulators which are embedded in the substrate and which isolate the memory cell regions from the peripheral regions. In accordance with a further preferred embodiment of the invention, the memory component comprises a dynamic random access memory (DRAM). In accordance with a further preferred embodiment of the invention, the first and second control electrode tracks are gate stacks of MOS field-effect transistors (MOSFETs). In accordance with a further preferred embodiment of the invention, the substrate comprises silicon. In accordance with a further preferred embodiment of the invention, the insulation layer comprises silicon oxide. In accordance with a further preferred embodiment of the invention, the control electrode layer comprises polysilicon. In accordance with a further preferred embodiment of the invention, the conductivity increasing layer comprises tungsten silicide (WSi x ). In accordance with a further preferred embodiment of the invention, the patterning layer comprises SiNx. In accordance with a further preferred embodiment of the invention, the first and/or the second mask layer comprise a resist layer. In accordance with a further preferred embodiment of the invention, the protective layer comprises an oxide. In accordance with a further preferred embodiment of the invention, the oxide is formed by means of a subatmospheric chemical vapor deposition (SACVD) or a low pressure chemical vapor deposition (LPCVD). In accordance with a further preferred embodiment of the invention, the removal of the protective material in that region of the peripheral region which are not covered by the second mask layer comprises removal of the protective material using hydrofluoric acid (HF). In accordance with a further preferred embodiment of the invention, the partial removal of the patterning layer comprises removal of the patterning layer using a mixture of hydrofluoric acid (HF) and ethylene glycol (EG). |
Culture medium for detecting and/or discriminating enterococcus and method therefor |
The invention concerns a culture medium for isolating enterococcus comprising violet crystal, and preferably gram-negative bacteria inhibitors and chromogens. The invention also concerns a method for detecting enterococcus using said medium. |
1. A culture medium for detecting and/or distinguishing enterococci, characterized in that it contains, in a culture medium for enterococci, Crystal Violet at a concentration allowing the growth of enterococci and the inhibition of the growth of most Gram-positive bacteria, said concentration being between 0.1 and 1.5 mg/l. 2. The culture medium as claimed in claim 1, characterized in that it additionally comprises at least one chromogenic agent, a substrate for an enzyme for sugar fermentation. 3. The medium as claimed in claim 2, characterized in that said enzyme is a glucosidase, in particular β-glucosidase or a galactosidase, in particular β-galactosidase. 4. The culture medium as claimed in claim 2 or 3, characterized in that said chromogenic agent releases, by hydrolysis, a precipitable chromophore chosen from indoxyl, haloindoxyl (bromoindoxyl, chloroindoxyl, fluoroindoxyl, iodoindoxyl, dichloroindoxyl, chlorobromoindoxyl, trichloroindoxyl), methylindoxyl or hydroxyquinoline derivatives, in particular the following derivatives: 6-chloroindoxyl, 5-bromoindoxyl, 3-bromoindoxyl, 6-fluoroindoxyl, 5-iodoindoxyl, 4,6-dichloroindoxyl, 6-7-dichloroindoxyl, 5-bromo-4-chloroindoxyl, 5-bromo-6-chloroindoxyl, 4,6,7-trichloroindoxyl, N-methylindoxyl or 8-hydroxyquinoline. 5. The culture medium as claimed in claim 3 or 4, characterized in that said β-glucosidase substrate is an indoxylglucoside, and/or said β-galactosidase substrate is an indoxyl-galactoside. 6. The culture medium as claimed in claim 5, characterized in that the β-glucosidase substrate is 5-bromo-4-chloro-3-indoxyl-β-glucoside and/or the β-galactosidase substrate is 5-bromo-6-chloro-3-indoxyl-β-galactoside. 7. The culture medium as claimed in one of claims 1 to 6, characterized in that it also contains growth inhibitors for Gram-negative bacteria. 8. The culture medium as claimed in one of claims 1 to 7, characterized in that it comprises (for one liter): Agar 15 g Yeast extract and peptones 9 g NaCl 5 g Nalidixic acid 50 mg Colistin 5 mg Crystal Violet 0.5 mg 5-bromo-4-chloro-3-indoxyl-β-glucoside 50 mg 9. The culture medium as claimed in one of claims 1 to 8, additionally containing antibiotics. 10. The culture medium as claimed in one of claims 1 to 9, characterized in that it does not contain sodium azide. 11. The use of a culture medium as defined in one of claims 1 to 10 for detecting and/or distinguishing enterococci. 12. A method for detecting and/or distinguishing enterococci in a sample, characterized in that it comprises the steps consisting in: a. inoculating a culture medium as defined in one of claims 1 to 10 with said sample of an inoculum derived from the sample, b. detecting the presence of enterococci on said culture medium. 13. A culture medium for the detection of Gram-positive or Gram-negative bacteria comprising, in addition to growth factors for said Gram-positive or Gram-negative bacteria, a chromogenic agent and Crystal Violet, the Crystal Violet being present at a concentration allowing the growth of said bacteria which it is sought to detect and the differential inhibition of the growth of Gram-positive bacteria, said concentration being between 0.1 and 1.5 mg/l. 14. The use of Crystal Violet as a growth-selective inhibitor for the preparation of a culture medium for the detection of Gram-positive or Gram-negative bacteria, additionally containing a chromogenic agent. |
Biofunctional fibers |
The present invention is directed to surface functionalization of polymeric fibers. Surface biofunctionalization is achieved by covalent conjugation of biofunctional igands and/or cell growth factors that are crucial for cell attachment, proliferation and functions. Biofunctional fibers could be fabricated into three-dimensional scaffolds. Polymer fibers described here comprise of biocompatible polymers that are either biodegradable ornon-biodegradable. This patent also describes a series of new biodegradable polyphosphoramidates for the processing of biodegradable fibers. Scaffolds made of non-biodegradable functional fibers could be used for in vitro cell culture (for example, ex vivo cell expansion), while biodegradable functional fibers could be fabricated into tissue engineering scaffolds. |
1. A biofunctional fiber comprising a biological molecule conjugated to a polymer, wherein the polymer comprises one or more reactive groups for attaching the biological molecule. 2. The biofunctional fiber of claim 1, comprising two or more distinct biological molecules conjugated to the polymer. 3. The biofunctional fiber of claim 1, wherein the polymer is biodegradable or non-biodegradable. 4. The biofunctional fiber of claim 1, wherein the polymer comprises polyphosphoester, polyester, polyethylene, polymethacrylic, polyacrylic, polysulfone, polyurethane or nylon. 5. The biofunctional fiber of claim 1, wherein the polymer comprises a plurality of polyphosphoramidates. 6. The biofunctional fiber of claim 1, wherein the reactive group is selected from carboxyl, hydroxyl, amino and polyacrylic acid groups. 7. The biofunctional fiber of claim 1, wherein the biological molecule and polymer are conjugated through a covalent bond. 8. The biofunctional fiber of claim 1, wherein the biological molecule and polymer are separated by a spacer. 9. The biofunctional fiber of claim 8, wherein the space is between about 2 and 500 angstroms in length. 10. The biofunctional fiber of claim 1, wherein the biological molecule comprises an amino acid sequence, nucleic acid, sugar, oligosaccharide, carbohydrate, lipid, fatty acid or a combination thereof. 11. The biofunctional fiber of claim 1, wherein the biological molecule modulates cell or tissue growth survival, apoptosis, proliferation, adhesion, differentiation, chemotaxis, signaling or gene expression. 12. The biofunctional fiber of claim 1, wherein the biological molecule comprises a receptor, ligand, growth factor, survival factor, proliferation factor, adhesion molecule, differentiation factor, chemotactic factor, or a molecule modulating signaling or gene expression. 13. The biofunctional fiber of claim 1, wherein the biological molecule is selected from collagen, fibronectin, extracellular matrix molecule, galactose, galactosamine, cluster ligands specific for hepatocytes, SCF, Flt-3 Ligand, TPO, G-CSF, GM-CSF, IL-3, IL-6 and Epo. 14. A method for producing a biofunctional fiber with a bioactivity comprising conjugating a biological molecule to a polymer, wherein the polymer comprises one or more reactive groups for attaching the biological molecule. 15. The method of claim 14, wherein the polymer is biodegradable or non-biodegradable. 16. The method of claim 14, wherein the polymer comprises polyphosphoester, polyester, polyethylene, polymethacrylic, polyacrylic, polysulfone, polyurethane or nylon. 17. The method of claim 14, wherein the polymer comprises a plurality of polyphosphoramidates. 18. The method of claim 14, wherein the reactive group is selected from carboxyl, hydroxyl, amino and polyacrylic acid groups. 19. The method of claim 14, wherein the biological molecule and polymer are conjugated through a covalent bond. 20. The method of claim 14, wherein the biological molecule and polymer are separated by a spacer. 21. The method of claim 20, wherein the spacer is between about 2 and 500 angstroms in length. 22. The method of claim 14, wherein the biological molecule comprises two or more distinct biological molecules. 23. The method of claim 14, wherein the biological molecule comprises an amino acid sequence, nucleic acid, sugar, oligosaccharide, carbohydrate, lipid, fatty acid or a combination thereof. 24. The method of claim 14, wherein the biological molecule modulates cell or tissue growth survival, apoptosis, proliferation, adhesion, differentiation, chemotaxis, signaling or gene expression. 25. The method of claim 14, wherein the biological molecule comprises a receptor, ligand, growth factor, survival factor, proliferation factor, adhesion molecule, differentiation factor, chemotactic factor, or a molecule modulating signaling or gene expression. 26. The method of claim 12, wherein the biological molecule is selected from collagen, fibronectin, extracellular matrix molecule, galactose, galactosamine, cluster ligands specific for hepatocytes, SCF, Flt-3 Ligand, TPO, G-CSF, GM-CSF, 113, IL-6 and Epo. 27. A two- or three-dimensional structure of biofunctional fibers comprising two or more biofunctional fibers of claim 1 knitted or weaved together. 28. The three-dimensional structure of claim 27, wherein at least one fiber has a biological molecule distinct from another fiber. 29. The three-dimensional structure of claim 27, wherein the structure comprises a scaffold, a tube or a chamber. 30. The three-dimensional structure of claim 27, wherein the structure comprises two or more tubes or chambers. 31. The three-dimensional structure of claim 27, wherein the structure mimics a body part or organ. 32. The three-dimensional structure of claim 27, wherein the structure is substantially impermeable to a biomolecule or a cell. 33. The three dimensional structure of claim 27, wherein the structure is porous to a biomolecule or a cell. 34. The three dimensional structure of claim 33, wherein the porosity of the structure is uniform or non-uniform. 35. The three-dimensional structure of claim 33, wherein the porosity of the structure is dictated by the knit or weave pattern or a biological property of the biofunctional fibers. 36. The three-dimensional structure of claim 33, wherein the structure is configured to allow permeation of a biomolecule or cell from within the structure to the outside of the structure. 37. The three-dimensional structure of claim 33, wherein the structure is configured to allow permeation of a biomolecule or cell from outside the structure to inside of the structure. 38. The three-dimensional structure of claims 36 or 37, wherein the biomolecule comprises a drug, amino acid sequence, nucleic acid, sugar, oligosaccharide, carbohydrate, lipid, fatty acid or combination thereof. 39. The three-dimensional structure of claim 33, wherein the biomolecule comprises a slow release formulation. 40. The three-dimensional structure of claim 33, wherein the slow release formulation comprises a colloidal dispersion system. 41. The three-dimensional structure of claim 33, wherein the slow release formulation comprises a microsphere. 42. A method for producing a two- or three-dimensional structure of biofunctional fibers comprising knitting or weaving together two or more biofunctional fibers of claim 1. 43. The method of claim 42, wherein the structure comprises a scaffold, a tube or a chamber. 44. The method of claim 42, wherein the structure comprises two or more tubes or chambers. 45. The method of claim 42, wherein the structure is substantially impermeable to a biomolecule or a cell. 46. The method of claim 42, wherein the structure is porous to a biomolecule or a cell. 47. The method of claim 46, wherein the porosity of the structure is uniform or non-uniform. 48. The method of claim 46, wherein the porosity of the structure is dictated by the knit or weave pattern or a biological property of the biofunctional fibers. 49. A bioreactor for cell proliferation or differentiation comprising the three-dimensional structure of claim 27. 50. The bioreactor of claim 49, wherein the biological molecule is presented in a paracrine fashion. 51. The bioreactor of claim 49, wherein the biofunctional fibers are knitted or woven together to mimic a thee-dimensional in vivo microenvironment that promotes cell proliferation or differentiation. 52. A method for introducing a biomolecule into a subject for controlled release comprising implanting the three-dimensional structure of claim 27 having the biomolecule into the subject under conditions allowing controlled release of the biomolecule into the subject. |
<SOH> BACKGROUND AND PRIOR ARTS <EOH>Effective scaffolding is crucial to the success of all tissue-engineering applications and ex vivo cell expansion applications. The design of effective scaffolds has recently been focused on incorporation of specific matrix chemistry, substrate surface configuration and three-dimensional macrostructure design. Polymer scaffolds must possess several key characteristics, including high porosity and surface area, structural strength, and specific three-dimensional shapes, to be useful for tissue engineering applications. Developing polymeric scaffolds with high porosity, i.e. high surface to volume ratio to provide a large amount of surface for cell attachment has been one of the most active research topics. Several techniques have been established for processing polymers into a porous structure. Most of these methods are based on a class of biodegradable polymers, poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and their polymers (PLGA). Particulate leaching is the first method that has been employed for the fabrication of biodegradable porous foams. This method, however, has less control of the microarchitecture of the pore structure and uniform porosity. An obvious limitation is the difficulties of scaling up of this fabrication technique (Mikos, et al. 1993; Ma, et al. 1998). Recently, textile technologies are used to fabricate biodegradable woven or nonwoven fabrics as tissue engineering scaffolds (Ma, et al. 1995). Fibers provide a large surface area to volume ratio and therefore are desirable as scaffold materials. The first studied fabric scaffold is a nonwoven mesh made of PGA sutures. Nonwoven PGA fibrous matrix is prepared by entangling fibers or filaments to form an isotropic 3-D matrix structure, leaving a space with a high void volume and a typical porosity in the range of 80-90%. These fibrous matrix lacks of structural stability necessary for the cell culture use. Therefore, several fiber-bonding techniques have been developed to prepare the interconnected fiber networks with different shapes as tissue engineering scaffolds (Thomson, et al. 2000). Nonwoven fabrics design, compared with biodegradable foams formed by particulate leaching, offers a better control over the scaffold porosity and the fabrication process is more reproducible. These nonwoven mesh scaffolds have achieved good success in several tissue engineering applications, including urinary bladder (Oberpenning, et al. 1999), vascular graft (Niklason, et al. 1999), Trileaflet Heart Valves (Hoerstrup, et al. 2000), cardiac graft (Li, et al. 2000), skeletal muscle (Saxena, et al. 1999), cartilage (Naumann, et al. 1998), etc. Nevertheless, the current available scaffold designs using polymer fibers (mostly non-woven mesh) still pose several limitations. Firstly, the surface of the fibers used to fabricate scaffolds or matrixes lacks of functional ligands required for cell attachment, proliferation and function. PGA fiber surfaces are not the natural substrate for cell attachment and growth. In almost all the studies mentioned-above, the non-woven meshes have been coated by another biodegradable polymer as a binder (e.g. poly-4-hydrobutyrate, PHB) or treated by partial alkali hydrolysis to modify the adsorption of serum proteins onto the surface-hydrolyzed fibers to improve cell attachment and seeding density (Gao, et al. 1998). This process would affect the degradation kinetics of the biodegradable fibers, and is also much less controllable. Moreover, the modified surface adsorbed with serum proteins has no specificity to cell types. Similar approach is taken for non-degradable fibrous matrix. Polyethylene terephtahlate (PET) fibers are partially hydrolyzed and to create enough functionalities on fiber surface to enhance the attachment of the extracellular proteins and therefore improve cell adhesion (Ma, et al. 1999). This patent provides methods to conjugate bioactive signal proteins to the surface of biodegradable fibers and non-degradable fibers. Secondly, polymer materials used to process biodegradable fibrous scaffolds have been limited to PGA although different bonding materials have been used to stabilize the scaffolds, mostly PLA or PHB. The degradation products of PLA, PGA and PLGA are glycolic acid and lactic acid. They would create an acidic microenvironment at the cell-scaffold interface. Low pH microenvironment is known to be detrimental to maturation of many types of cells and tissue development. Shum-Tim et al. have engineered an ovine pulmonary valve leaflet and the pulmonary arteries from autologous cells using nonwoven PGA mesh (Shum-Tim, et al. 1999). Use of this cell-polymer construct in the systemic circulation resulted in aneurysm formation. This is possibly due to the acidic degradation products or lacking the structural integrity throughout the remodeling process. New biodegradable materials suitable for fiber processing are in great demand to overcome this limitation. This patent also provides a serious of new biodegradable materials that could be processed into fibers and amendable to surface conjugation. Lastly, nonwoven fabric designs lack of the control of scaffold microarchitecture. Obtaining a uniform porosity is not possible. In addition, nonwoven fabric scaffolds generally have weak mechanical structures. Certain bonding or backing materials are needed to ensure the structural stability. Examples of structural re-enforcing techniques include polypropylene fiber backing for PET meshes (Wang, et al. 1992), solution coating or spray coating of a PLA or PLGA layer (Mikos et al. 1993; Mooney, et al. 1996), sewing with Dexon suture (Niklason et al. 1999), and polyglactin suture (Oberpenning et al. 1999) for PGA meshes. This patent provides methods using textile technologies to provide scaffolds with coherent and ordered structures. Polymer fibers are woven or knitted to form three-dimensional scaffolds with different designed pattern to obtain various degrees of porosity (Wintermantel, et al. 1996), microtopology of the cell culture environment and microdistribution of the functional ligands using surface modified fibers. This patent describes methods of preparing biofunctional fibers based on non-degradable fibers and biodegradable fibers, describes a serious of new biodegradable materials that could be processed into fibers and amendable to surface conjugation, describes methods of preparing fibrous scaffolds by surface biofunctionalization or using biofunctionalized fibers. These technologies will find wide applications in tissue-engineering and bioprocessing fields. Two specific examples are illustrated below to demonstrate the advantages of this scaffolding technology—stern cell expansion for nondegradable fibrous scaffolds, and vascular graft engineering for the biodegradable scaffolds. 1. Current Stem Cell Expansion Methodologies A technology for efficient and practical ex vivo expansion of hematopoietic stem cells and progenitor cells would find wide applications in stem cell transplantation and somatic gene therapy. For detailed clinical applications of the expanded haemopoietic progenitor cells, see reference (Alcorn, et al. 1996). Current methodologies for ex vivo stem cell expansion are still far from optimal in achieving high expansion rate and maintaining pluripotency. The goal of ex vivo expansion is to induce cell division and proliferation of stem cells while maintaining their primary functional phenotypes, namely, their ability to engraft and sustain long-term hematopoiesis. Over the past few years, techniques have become available that allow the extensive proliferation of haemopoietic progenitor cells in ex vivo culture systems. One method of stem cell expansion utilizes an adherent monolayer of stromal cell, which supports the viability of stem cells and early progenitor cells (Dexter, et al. 1977). Briefly, in the first few weeks of culture, a complex adherent layer of stromal cells is laid down. This stromal layer comprises fibroblasts, macrophages, adipocytes, endothelial cells and reticular cells. Hematopoesis can be maintained for months in a long-term bone marrow culture and it is thought that direct adhesive interactions between the hematopoietic cells and various elements of the stroma are crucial to the regulation of primitive hematopoietic cells. This suggests that the complex stromal layer can, to some extent, successfully mimic the unique microenvironment present in the bone marrow. The major advantage of these stromal-based culture systems is their ability to expand the numbers of primitive hematopoietic cells. Although stromal layer may provide a suitable substrate for hematopoietic cell immobilization and culture, it has a number of limitations. The stromal layer is fragile. Therefore, it requires a rigid substrate on which the layers of stromal cells should be grown in order lo maintain the integrity of the stroma. Moreover, cells grown on stroma only have a limited culturing lifetime of about six to eight weeks due to death of the stromal cells. More importantly, the use of stroma for a clinical ex vivo application poses a considerable logistic problem. In most cases, the stromal cells are obtained from the patient lo avoid the immuno-rejection. The need lo first collect and then grow a layer of the patient's stromal cells before they can be used lo culture the hematopoielic cells adds to the time, cost, and complexity of the production of the autologous HPC cells. Moreover the stromal layers are much less defined. It introduces an additional highly variable factor into the culture system. This renders the controlled culturing difficult if reproducible stromal cultures of predictable characteristics are to be obtained. Allogeneic source of stroma, although feasible, is unreliable. The fact that a primary allogeneic stroma has to be irradiated suffers, as any donor-derived tissues would, the potential risks of infection. The quantity to which primary stromal cells can be expanded is limited. Immortalized human stromal cell lines are potentially unlimited in quantity (Roecklein, et al. 1995). However, no allogeneic stromal support is currently available that is suitable for clinical use yet (von Kalle, et al. 1998). For these reasons, ex vivo culture of HSCs in suspension without stroma a has been actively pursued in recent years. The most widely used method for ex vivo expansion has been a relatively simple liquid suspension culture system supplemented with a combination of a range of cytokines (Hoffman, et al. 1995). The development of HSC in vivo is thought to be regulated, at least in part, by interactions of cytokine receptor signals. Various combinations of cytokines have therefore been. studied to obtain the optimal culture conditions for HSC expansion. In particular, stem cell factor (SCF) and Flk-2/Flt-3 ligand (FL) have been used as key cytokines for HSC expansion, because c-Kit and Flk-2/Flt-3, tyrosine kinase receptors for SCF and FL, respectively, have been shown to transduce signals crucial for HSC development. Thrombopoietin (TPO), a ligand for c-Mpl, originally identified as a primary regulator for megakaryopoiesis, has also been shown to stimulate the expansion of primitive hematopoietic cells. A recent study showed that a combination of SCF, FL, TPO, and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), was able to induce a significant ex vivo expansion of human hematopoietic stem cells for 7 days. The expanded cells were capable of repopulating in NOD/SCID mice, leading to successful bone marrow engraftment in the recipient animals as measured by considerable numbers of human CD45 + cells 10-12 weeks after transplantation (Ueda, et al. 2000). Simplicity is a major advantage of the cytokine-supplemented suspension culture. In a typical process, CD34 + cells are suspended in culture medium and incubated in an appropriate vessel (tissue culture flasks (Brugger, et al. 1995) or gas-permeable culture bags (Alcorn, et al. 1996; Mellado-Damas, et al. 1999)) for between eight to twelve days. The culture cells can then be harvested with ease and used as required. The medium is preferably serum-free, although a number of studies have used serum-supplemented medium. Serum-free culture allows the researcher to develop a chemically defined medium with known amount of cytokines, therefore the cell expansion process is more controlled and reproducible, and easy to scale up. More importantly, the use of serum free conditions is highly recommended for cell therapy protocols such as employing HPC-derived dendritic cells (DC) and T cells, whose exposure to exogenous antigens can be limited to a minimal level. While the general protocols for suspension culture are similar, there are a variety of different cytokine recipes developed by various groups. The cytokines most commonly used include a combination of SCF, Flt-3 Ligand, TPO. G-CSF, GM-CSF, IL-3, IL-6, and erythropoietin (Epo). Several recent studies have suggested that SCF, Flt-3 ligand, TPO, and IL-3 might play key roles in the early human hematopoiesis. The combination of these cytokines (especially Flt-3 ligand and TPO) significantly enhanced the amplification of primitive HSCs (Petzer, et al. 1996; Petzer, et al. 1996; Piacibello, et al. 1997; Yagi, et al. 1999). The degree of ex vivo expansion is normally assessed by calculating the fold-increase in total numbers of cells, committed progenitors, CD34 + cells, and LTBMC-IC with respect to the input cells. Routinely, extensive expansion of cell numbers is obtained. Depending on the duration of culture, this can vary from a 30-fold increase in cell numbers from an eight-day culture, up to over 1000-fold increases with longer periods of 14 to 21 days. Similarly, numbers of committed progenitor cells also increase, for example, 41-fold following an eight-day culture, up to 190-fold from a 14-day culture. By repeated feeding of cultures, cell numbers can continue to increase for up to 21 days. Generally speaking, no stromal influence is incorporated into the suspension culture system, although various combinations of cytokines are utilized to provide the proliferation and differentiation signals that stroma is thought to deliver. The addition of cytokines is thought to compensate for the absence of stroma-associated support. This represents a major disadvantage when one considers that, in vivo, blood cell production is regulated at a local level by interactions of hematopoietic stem cells with a variety of cell-bound and secreted factors produced by adjacent bone marrow stromal cells. It is unlikely that the cytokine combination currently in use will be adequate substitutes for stroma. Another limitation of the serum-free suspension culture is the low expansion of the true stem cells, which is measured by long-term-culture-initiating cell (LTC-IC) assay. There is little evidence of significant LTC-IC proliferation, with, at best, maintenance of LTC-IC numbers over the culture period under these conditions. This is probably related to the fact that the current system lacks the unique regulatory microenvironment of bone marrow stroma. Nevertheless, a recent study showed that using a much higher concentration (30-fold higher) of cytokines than for maximal amplification of colony-forming cells, a 60-fold expansion of LTC-ICs from primitive cells has been achieved (Zandstra, et al. 1997). However, other studies have suggested the induction of differentiation of murine stem cells and thus loss of their repopulating ability when high concentration of IL-1, IL-3 and IL-6 are used for the ex vivo expansion (Jonsson, et al. 1997). Down regulation of surface IL-3 receptor in response to the high concentration of soluble IL-3 may have played a role. Immobilized HGFs may alleviate this problem by only providing high concentration of growth factors at the “reaction site”. Recent insights into hematopoietic stem cell biology have demonstrated that the three-dimensional architecture of the culture environment may influence the maintenance of stem cell pluripotency in vitro. Several studies employing three-dimensional devices made of synthetic polymers support the hypothesis that physical topography of bone marrow microenvironments plays an important role in maintaining hematopoietic stem cell viability and pluripotency (Naughton, et al. 1989; Naughton, et al. 1990). These studies show that a 3-D microenvironment supports HPC survival, proliferation and multilineage differentiation. Naughton and Naughton have developed a three-dimensional cell culture apparatus for HSC expansion, in which a stromal support matrix is pre-estabilished and grown on the polymeric mesh surface (Naughton, et al. 1992). An interesting study by Rosenzweig et al. indicates that culturing hematopoietic progenitor cells (HPCs) in a three-dimensional tantalium-coated porous biomaterial structure enhances HPC survival, and preserves primitive CD34 + CD38 31 cells, even without using hematopoietic growth factors as compared with standard culture techniques. This culture technique improves retroviral transduction of CD34 + cells and LTC-ICs without loss of multipotency (Rosenzweig, et al. 1997). In summary, other than defining the source of HSCs and developing methods to obtain a purer CD34 + cell source, optimizing the ex vivo culture methodology represents the major challenge for HSC expansion. Considering the various aspects of ex vivo culture of HSCs, we hypothesize that a successful new generation of HSC culture system should include the following key features: (1) a three-dimensional culture device that mimic the microenvironment in the bone marrow stroma, (2) matrix-bound cytokines (including SCF, Flt-3 ligand, TPO, etc.) that mimic the in vivo configuration where these crucial cytokines interact with HSCs in vivo in early hematopolesis, (3) a bioreactor system that is easy to scale up to obtain a clinically acceptable expanded stem cell population. 2 Tissue Engineering of Small Diameter Vascular Grafts Surgical treatment of vascular disease is now a common medical procedure. However, to date, the use of synthetic polymeric materials is limited to grafts larger than 5-6 mm due to the frequency of occlusion observed with synthetic vessels of smaller diameters. Consequently, significant efforts in the past 15 years have been focused on the development of a small-diameter blood vessel equivalent using tissue-engineering approach. The seeding of synthetic grafts with endothelial cells has been investigated as a means to increase patency, but has been limited by the challenges associated with maintaining effective surface coverage. As an alternative to the use of synthetic materials, two approaches have been taken to create a blood vessel using cell and matrix components. One approach is to create an acellular graft constructed of a material, such as collagen, that would provide the required mechanical properties on implant but would also facilitate remodeling and infiltration of host cells into a cellular vessel (Sullivan, et al. 2000). In this approach, the acellular matrix allografts or xenografts often times require a crosslinking process to provide the requisite mechanical characteristics, and the potential inflammatory response to the acellular grafts still persists. Another approach has gain great attention recently, uses techniques to create a cellular vessel through culture of smooth muscle cells within a biodegradable fibrous matrix and lining the lumen with endothelial cells (Niklason, et al. 1997; Shinoka, et al. 1998; Zund, et al. 1998; Niklason et al. 1999; Shum-Tim et al. 1999). Weinberg C B and Bell E have first demonstrated in vitro development of a model blood vessel in a porous collagen scaffold in 1986. The remodeled blood vessel has three layers corresponding to an intima, media, and adventitia (Weinberg, et al. 1986). A confluent layer of endothelial cells was grown in culture onto the lumen of a tubular collagen construct consisting of an outer layer of fibroblasts and a middle layer of smooth muscle cells. An external Dacron mesh was used to provide additional mechanical support. However, elastin, the principal arterial-tissue-matrix protein besides collagen, was not present in the model. Matsuda T and Miwa H also created a hybrid construct using a polyureathane scaffold seeded with smooth muscle and endothelial cells (Matsuda, et al. 1995). This construct was shown to remodel in vivo successsfully in a canine model for up to 1 year. It is worth noting that in both of these two designs, a nondegradable polymer support was used to reinforce the strength of the cellular layers. The state-of-art scaffolding technology in tissue engineering of blood vessel is to employ synthetic nonwoven biodegradable fibrous meshes. Using a partially hydrolyzed PGA nonwoven fabric scaffold, Niklason L E et al. have cultured bovine vessels under pulsatile media flow conditions (Niklason et al. 1999). In this study, vascular biopsy derived aortic smooth muscle cells have been seeded in the scaffold and cultured for 8 weeks, before seeding the endothelial cells in the luminal surface. Pulsatile radical stress is applied to the vessels at 165 beats per minute and 5% radical distention. The remodeled vessels have rupture strengths greater than 2000 mmHg and suture retention strengths of up to 90 grams, and exhibit the beginnings of vascular contractile responses. These engineered arteries have been implanted in miniature swine, and remain patent: for up to 3 weeks postimplantation. However, these engineered vessels are also notably lacking in elastin content. In another in vivo blood vessel engineering model, Shum-Tim D et al. have reported a tissue engineered ovine pulmonary artery from autologous cells cultured in a PGA fibrous scaffold (nonwoven mesh) (Shum-Tim et al. 1999). Polyhydroxyalkanoate (PHA) layers have been used to provide the temporary mechanical characteristics of the tubular scaffold as the cells lay down their own extracellular matrix on the PGA surface, which ultimately takes over the structural integrity and biomechanical profile of the engineered tissue. Ovine carotid arteries are harvested, expanded in vitro, and seeded onto 7-mm diameter PHA-PGA tubular scaffolds. The autologous cell-polymer vascular constructs have been used to replace 3-4 cm abdominal aortic segments in lambs. All tissue-engineered grafts remain patent for up to 5 months, and no aneurysms developed by the time of sacrifice. The mechanical strain-stress curve of the TE aorta approaches that of the native vessel. In both studies, scaffolds have been used without any cell adhesive molecules on the surface. A bioadhesive surface would obviously increase the cell seeding efficiency and shorten the time needed for in vitro modeling. This has been difficult to achieve using the current available polymeric materials. Another key challenge in developing a tissue-engineered blood vessel is to create a construct with the required mechanical properties. Several studies have demonstrated that optimizing the in vitro culture conditions would increase the burst strength of the engineered blood vessel. A few factors that would significantly affect the mechanical characteristics of the remodeled blood vessels include media flow (Ziegler, et al. 1995), ascorbic acid supplement (L'Heureux, et al. 1998)), glycation of the media equivalents (Girton, et al. 1999; Girton, et al. 2000), and particularly, applying pulsatile mechanical stimulus to the cellularized constructs (Niklason et al. 1999). This requires a scaffold with good mechanical strength, which nonwoven-mesh scaffold lacks. As an alternative, additional biodegradable suture, coating or silicon tubing has been used to provide structural integrity and mechanical properties for these non-woven mesh scaffolds (Niklason et al. 1999; Oberpenning et al. 1999; Shum-Tim et al. 1999). This patent provides biodegradable polymers with functional side chains for the conjugation of adhesion molecules, provides methods of preparing fibrous scaffolds based on biofunctional fibers derived from these polymers. |
<SOH> SUMMARY OF THE INVENTION <EOH>1. Biofunctional Fibers—Nonbiodegradable Fiborus Scaffolds for Cell Expansion Wale propose a new cell culture system composed of three-dimensional fibrous scaffolds surface-engineered with essential cytokines for hematopoietic stem cells growth and differentiation. The key features include: (1) The surface of polymer fibers (non-biodegradable) is conjugated with several different growth factors (SCF, Flt-3 Ligand, TPO, CSFs, etc.) with appropriate spacer and 2-D pattern conducive to the cell attachment and function. Cell adhesion molecules (e.g. RGD sequence) may also be conjugated to the fiber surface to facilitate the binding of HSC, and provide the synergy for the Interaction between HSC and surface-bound hematopoietic growth factors. (2) The surface engineered fibers are woven/knitted into a three-dimensional scaffold with various textures (different mesh sizes and patterns) to accommodate cells and facilitate cell-cell interaction. (3) A bioreactor system can be designed based on this fibrous scaffold. The system can potentially be operated under a continuous condition. The expanded cells are “leached out” from the fibrous scaffolds, and are harvested at any time from the suspension simply by centrifugation. 2 Biofunctional Fibers for Biodegradable Fibrous Scaffolds This patent provides a new type of biodegradable polymeric fibers processed from polyphosphoramidates (Formula I, see Detailed Description for the structure parameters), which are biodegradable and have good mechanical properties. The side chains of these polymers are conjugated with cell adhesion peptides. The polyphosphoramidates described in this patent are biodegradable. The degradation rate could be adjusted by varying the structure parameters. The present patent also provides the methods for preparation of these biodegradable polymers. Biofunctional fibers from these polymers can be obtained by conjugating biofunctional ligands to the side chains of the polymers or by surface modification of the polyphosphoramidate fibers, in later case, polyphosphoramidates carry reactive side chains to allow the further conjugation of biofunctional proteins, peptides or oligosccharides. These biofunctional polymeric fibers could be fabricated into a three-dimensional scaffold by woven/knitting methods. These scaffolds provide optimal supports for cell attachment, proliferation and functions, and allows cells to grow in three dimensions. Potential Advantages: 1. Nonbiodegradable Biofunctional Fibers for Cell Expansion This biofunctional fiber design for configuring and constructing cell culture devices provides an optimal microenvironment for hematopoietic stem cell expansion. It also allows various designs of extra-cellular matrices with a reasonable porosity for other applications. The proposed matrix structure allows for a higher immobilized cell density than can normally be achieved by traditional cell culture techniques (flasks or plastic bags). When surface immobilization and microencapsulation of hematopoietic growth factors and adhesion molecules were incorporated in the three-dimensional culture device, higher expansion rate and better LTC-IC maintenance are expected. This is due to increased contact with HGF immobilized matrix and co-stimulation or synergy of different growth factors/cytokines at a local level, while costs are lowered through controlled release of growth factors. Compare to the conventional culture devices, this newly proposed scaffold has a higher surface area and a higher cell density can be achieved. It also has a low pressure drop across the fibrous structure due to the high porosity, and allows for high mass-transfer of nutrients and oxygen at high cell densities. The potential applications of this proposed three-dimensional fibrous device are beyond the expansion of hematopoietic stem cells. This biofunctional fibrous scaffold can easily be adapted to the expansion of other growth factor dependent cells, e.g. T-cell expansion and dendritic-cell expansion for adoptive cellular immunotherapy. It is also a useful tool for in vitro studies, such as biochemical signals for growth, differentiation, migration and various extracellular matrix components. These studies are useful in understanding cell-cell interaction: behavior, communication, control, and morphogenesis, and studying the effect of surface properties on cell functions and spatial control of cell micro-organization. 2. Biofunctional Fibers for Biodegradable Fibrous Scaffolds This patent provides a new type of biodegradable polymeric fibers processed from polyphosphoramidates, which are biodegradable and have good mechanical properties. The side chains of these polymers are conjugated with cell adhesion peptides. These biofunctional polymeric fibers could be fabricated into a three-dimensional scaffold by woven/knitting methods. These scaffolds provide optimal supports for cell attachment, proliferation and functions, and allows cells to grow in three dimensions. The salient and attractive features are: (1) The scaffold fibers have surface conjugated bioadhesion ligands, which are not available on the PGA/PLA/PLGA fibers. The polyphosphoesters we proposed have available side chains for conjugation of bioadhesive ligands. These ligands could be conjugated through a flexible spacer on the fiber surface. As an alternative, ligands could also be linked to the side chains of the polymer before being processed into fiber. In later case, bioadhesion ligands are distributed throughout the bulk of polymer fiber. (2) This fibrous scaffold design offers good control of the 3-D porous microarchitecture. The surface engineered fibers or fibers made of bioadhesive polymers are arranged into 3-D scaffolds using nonwoven or woven/knitting techniques. The microporous structures are defined to accommodate cell attachment, facilitate cell differentiation, and guide cell growth and tissue regeneration in three dimensions. This design offers a wide range of suprastructures by chancing fiber diameter, orientation, porosity, and woven and knitting characteristics; (3) Biofunctional oradient scaffolds can be fabricated through the 3-D arrangement of functional fibers. Biofunctional gradient scaffolds have a single or multiple ligands arranged with a spatial gradient change of their surface concentration. This type of scaffolds is particularly useful in directing tissue growth (e.g. for nerve tissue engineering) or coculture of multiple cell types (e.g. for vascular graft engineering). (4) The scaffolds have good biocompatibility, mechanical properties, and more steady degradation profile. Polymer fibers are fabricated from new biodegradable polyphosphoesters, tailored to be biocompatible and with no acidic degradation products. |
Boron-based wood preservatives and treatment of wood with boron-based preservatives |
A process for treating wood comprising applying to the surface of the wood a boron based preservative which reacts with moisture within the wood to form a boron compound and alcohol and subjecting the wood with the applied preservative to a substantially moisture-free and enclosed environment for a period sufficient for the applied preservative to be absorbed into the wood and to produce the boron compound on reaction with the moisture in the wood and for the alcohol by-product of the reaction to be adsorbed within the wood structure. |
1. A process for treating wood comprising applying to the surface of the wood a boron based preservative which reacts with moisture within the wood to form a boron compound and alcohol and subjecting the wood with the applied preservative to a substantially moisture-free and enclosed environment for a period sufficient for the applied preservative to be absorbed into the wood and to produce the boron compound on reaction with the moisture in the wood and for the alcohol by-product of the reaction to be adsorbed within the wood structure. 2. A process according to claim 1, wherein, prior to the application of the boron based preservative to the wood, the wood is dried to reduce the moisture content of the wood. 3. A process according to claim 1, wherein the substantially moisture-free and enclosed environment to which the wood is subjected following application of the boron based preservative is such as to prevent the ingress of moisture into the treated timber, as may be provided from humidity in the atmosphere, and to substantially prevent the evaporation of the applied preservative from the wood into the atmosphere. 4. A process according to claim 3, wherein the wood with the applied preservative is introduced to a container or other preformed envelope, such as of steel or plastics, which is then sealed to provide the substantially moisture free and enclosed environment, or is wrapped to exclude atmosphere and thereby provide the substantially moisture free and enclosed environment. 5. A process according to claim 4, wherein the wood is wrapped in a plastics material selected from polyethylene film, polyester film, preferably polyethylene terephthalate (PET) film. 6. A process according to claim 5, wherein the wood is wrapped in a heat sealable co-extruded PET film, preferably having a thickness in the range of 15 to 30 μm. 7. A process according to claim 1, wherein the period of retention in the substantially moisture free and enclosed environment at ambient temperature and pressure is less than 3 days, preferably less than about 24 hours. 8. A process according to claim 1, wherein the moisture content of the wood is about 6% by weight or less of the oven dry weight of the wood. 9. A process according to claim 1, wherein the boron based preservative is TMB or a combination of TMB and methanol at or about the azeotropic composition thereof, or is triethyl borate. 10. A process according to claim 1, wherein the boron based preservative includes one or more additives selected from additives to enhance fire-proofing attributes, such as a compatible compound of zinc, additives to enhance activity, such as waxes, resins, oils and oil-based pigments which improve the water repellency of timber surfaces and may improve the colour and aesthetic appeal of the treated timber, and dimension stabilising chemicals such as acetic anhydride. 11. A process according to claim 1, wherein the boron based preservative is applied to the surface of the wood by pressure impregnation, vacuum/pressure impregnation, dipping, insizing and dipping, soaking, spraying/atomizing/fogging, electrostatic spraying, vaporising, evacuation and vapour or gaseous application, brushing, rolling and compression rolling. 12. A process according to claim 11, wherein the boron based preservative is applied to the wood by dipping for a period of about 2 minute or less, preferably about 1 minute or less, more preferably about 30 seconds or less and most preferably about 15 seconds or less. 13. A process according to claim 1, wherein following treatment of the wood the wood is surface treated, for example, with a resin, to immobilize the boron. 14. A process according to claim 1, wherein the boron based preservative is a light organic solvent wood preservative comprising a trialkyl borate and a non-polar carrier. 15. A process according to claim 14, wherein the trialkyl borates is one having C1-20 alkyl, preferably C1-9 alkyl, more preferably C1-6 alkyl groups and most preferably TMB or triethyl borate. 16. A process according to claim 14, wherein the non-polar carrier is a non-polar solvent selected from aliphatic or aromatic hydrocarbons and heterocycles or derivatives thereof, preferably kerosene, petroleum or turpentine; an oil; or mixtures thereof. 17. A process according to claim 14, wherein the light organic solvent wood preservative further includes one of more additives selected from water repellents, such as waxes, resins or polymers, for example, polyethylene glycol; dimensional stabilisers, such as acetic anhydride; fire retardants, such as zinc compounds; mildewicides/fungicides; insecticides, such as, pyrethroids or triazoles; mouldicides; dyes and pigments. 18. A process according to claim 14, wherein the wood to be treated has a moisture content of from 10-14%. 19. A process according to claim 1, wherein the boron based preservative is a boroxine and/or a polyborate. 20. A process according to claim 19, wherein the boroxine has a general formula (I): wherein R1, R2, and R3 may be the same or different and are selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted aryl or optionally substituted heterocycyl. 21. A process according to claim 20, wherein R1, R2, and R3 are C1-10 alkyl or phenol. 22. A process according to claim 19, wherein the polyborate has the general formula (II): wherein R1 and R2 may be the same or different and are selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted aryl or optionally substituted heterocycyl. 23. A process according to claim 22, wherein R1 and R2 are C1-10 alkyl or phenol. 24. A process according to claim 19, wherein the boron based preservative is applied to the wood alone, in the form of an emulsion, or in combination with a suitable carrier which may be polar or non-polar and which is selected from water, alcohols, aromatic or aliphatic solvents or oils. 25. A process according to claim 19, wherein the boron based preservative includes one or more additives selected from water repellants, such as waxes, resins or polymers, such as polyethylene glycol; dimensional stabilisers, such as acetic anhydride; fire retardants, such as zinc compounds; mildewicides; fungicides/insecticides; such as pyrethroids or triazoles; mouldicides; dyes and pigments. 26. A light organic solvent wood preservative comprising a trialkyl borate and a non-polar carrier. 27. (cancelled) 28. (cancelled) 29. (cancelled) 30. (cancelled) 31. (cancelled) 32. A boron based preservative comprising a boroxine having the general formula (I): wherein R1, R2, and R3 may be the same or different and are selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted aryl or optionally substituted heterocycyl. 33. A boron based preservative according to claim 32, wherein R1, R2, and R3 are C1-10 alkyl or phenol. 34. A boron based preservative comprising a polyborate having the general formula (II): wherein R1 and R2 may be the same or different and are selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted aryl or optionally substituted heterocycyl. 35. A boron based preservative according to claim 34, wherein R1 and R2 are C1-10 alkyl or phenol. |
<SOH> BACKGROUND ART <EOH>Compounds of boron have been used as preservatives for wood for many years. Since about 1955 the most common method of application of the boron compounds in many countries has been by dipping the wood into an aqueous solution of the compound and allowing the boron compound to diffuse into the wood. For example, the wood may be dipped in 16-18% boric acid solution for a period of about two minutes to give surface application of the preservative and then wrapped to prevent moisture loss for about 6 to 8 weeks while the boric acid preservative diffuses through the wood. New Zealand patent specification 115464 dated 2 Dec. 1955 proposed an alternative surface application using organic compounds of boron, which it is said may or may not hydrolyse within the wood during or after treatment. This specification proposes the use of a vast number of organic boron compounds and application methods, preferably to wood which is in a dry state either following special drying operations or in equilibrium with its climatic environment. While no methods are exemplified, one proposal is to apply the organic boron compound in the gaseous state with the wood being enclosed in a suitable vessel or envelope, such as of plastic film, from which air is excluded. However there is no discussion of any post-treatment of the wood following application of the preservative. Furthermore, momentary immersion, for periods of about two minutes, of the wood in the boron preservative has remained the standard technique of application. One boron compound mentioned in NZ-A-115464 as capable of being applied to wood in a gaseous treatment is trimethyl borate. Trimethyl borate (TMB) and some other boron compounds hydrolyse with the wood moisture to release the boron, as the well known preservative boric acid, and alcohol. For example, TMB reacts according to the reaction: in-line-formulae description="In-line Formulae" end="lead"? B(OCH 3 ) 3 +3H 2 O→H 3 BO 3 +3CH 3 OH. in-line-formulae description="In-line Formulae" end="tail"? One problem of applying a wood preservative to the surface of the wood is ensuring that it penetrates sufficiently into the wood for the treatment to be effective. In the case of TMB, if the wood moisture content is too high, the TMB may react to form boric acid before it has diffused into the wood so that the boric acid only appears at and adjacent the surface, rendering the treatment ineffective. This problem is resolved in Australian patent specification 18324/88 by drying the wood to a reduced moisture content in a treatment vessel, evacuating the treatment vessel, introducing gaseous TMB to the vessel for a period of time before evacuating or venting the vessel to atmosphere, and steaming the treated wood. Steaming is considered necessary in order to restore the moisture content of the wood and to relieve any stresses in the wood caused by the drying, but also has the advantage of rendering inert any remaining TMB on the wood so as to render the wood safe to handle. However, the TMB is applied in excess and is substantially recovered along with moisture and any solvent such as alcohol, as well as alcohol by-product of the TMB reaction with moisture, prior to steaming by evacuating the timber. Excess TMB is recovered because it represents both a health hazard and a flammability hazard on release from the treatment vessel. The condensate from the steam treatment (effectively a mixture of boric acid, alcohol and wood moisture) is a waste product which has a disposal cost. Drying of wood and its subsequent steam reconditioning are very well known procedures which have been used for many years. Another way of resolving the problem of ensuring that the conversion of TMB to boric acid is not only at the surface of the wood is proposed in Australian Patent Specification 40465/89 in which the need to pre-dry the wood is said to be avoided by exposing the wood to a vapour of a TMB-methanol azeotrope at a temperature below the boiling point of the methanol by-product of the TMB reaction with moisture in the wood. This is said to reduce the vapour pressure of the methanol by-product allowing improved boron preservative vaporisation and surprisingly improved boric acid deposition. However, the process requires careful temperature control since the boiling point of the azeotrope and of the methanol by-product may be close. The process further requires the recovery of residual vapours since although alcohol is said to be condensed in the wood structure, the alcohol is free to evaporate after the preservative treatment. Preservative formulations involving boric acid esters dissolved in organic solvents have been described in, for example, NZ Patent No. 115,464 referred to above, U.S. Pat. No. 4,970,201 and International Publication Nos. WO93/02557 and WO94/00988. The choice of organic solvent is important for this type of treatment. Organic solvents used in the wood industry can be classified by polarity. Light organic solvent processes (hereinafter referred to as “LOSP”) involve the use of non-polar solvents, such as, kerosene or white spirits which do not interact with the cell wall. The advantages include non-swelling of the wood, low uptakes and treatment of the wood in its final form. The other types of solvents proposed for boric acid ester formulations are polar solvents which interact with the cell wall. The swelling effect of this interaction requires a drying step after treatment and possible recovery of the solvent. Treatment of dry wood with polar solvents such as methanol results in substantially higher uptakes of the preservative solution as a result of swelling of the cell wall. The dilution of TMB or a TMB-methanol azeotrope with methanol or other polar solvents also poses the following problems: (a) methanol is a Class A solvent which means that it is very flammable and requires special equipment designed for its handling; (b) the preservative solution is susceptible to hydrolysis and requires careful handling; (c) the TMB-methanol azeotrope has a lower boiling point than TMB and is volatile requiring careful handling procedures both before and after treatment; and (d) the reaction as shown in equation (1) above produces methanol which has very similar swelling properties to water. The problem with the use of polar solvents in boron-based preservative solutions such as TMB or the TMB-methanol azeotrope is that they interact with the cell walls and result in swelling of the wood. It is difficult when using these preservative solutions to obtain a high concentration of boric acid in the wood as may be required to impart fire resistant properties. Different concentrations of boric acid are required in wood to achieve biocidal protection and fire retardant properties. Typically, the boric acid equivalents (hereinafter referred to as “BAE”) required for various applications are as follows: % wt/wt BAE Insect protection 0.25 Fungicidal protection 0.75 Fire retardant properties 7.00 The high volumes of TMB required to effect fire retardant properties result in unacceptable swelling of the wood with strength loss. On the other hand, the application of TMB for biocidal protection will usually require dilution of TMB. This may be achieved either by using the TMB-methanol azeotrope or by dilution of TMB or the azeotrope with an alcohol. This poses a similar problem of swelling of the wood as a result of the interaction between the alcohol and the cell wall. |
<SOH> SUMMARY OF THE INVENTION <EOH>According to a first aspect of the present invention there is provided a process for treating wood comprising applying to the surface of the wood, preferably having a reduced moisture content, a boron based preservative which reacts with moisture within the wood to form a boron compound and alcohol and subjecting the wood with the applied preservative to a substantially moisture-free and enclosed environment for a period sufficient for the applied preservative to be absorbed into the wood and to produce the boron compound on reaction with the moisture in the wood and for the alcohol by-product of the reaction to be adsorbed within the wood structure. Also according to the present invention there is provided wood, whether as timber or wood based products, which has been treated by the process described in the immediately preceding paragraph. By this aspect of the present invention it has been found that no treatment of the wood is necessary after a limited surface application of the boron based preservative except for subjecting the treated wood to a period in a substantially moisture-free and substantially enclosed environment. During this period, it has been found that the boron based preservative applied to the wood surface may substantially all diffuse into the wood to react with the wood moisture to form the effective boron compound, for example boric acid, and that at least substantially all the alcohol by-product is adsorbed into the wood structure and advantageously fixed in the cell walls. The adsorption process occurs over a prolonged period with the alcohol diffusing in either its condensed state or its vapour state through the wood cross-section, generally mainly in the vapour state. Molecules of the alcohol will eventually diffuse into the microstructure of the cell walls (the so-called transient capillaries) and form an adsorbed monolayer which is hydrogen bonded to the cellulose, hemi-cellulose and lignin in the wood structure. This means that no recovery of the alcohol by-product is necessary and that the wood is safe to handle following the treatment. Indications are that the formation of the monolayers in the wood structure is a permanent reaction whereby wood can adsorb or fix from 1 to 2% of its weight of alcohol which cannot be recovered even by prolonged evacuation, for example, up to a week. This fixed amount is generally in excess of the alcohol needed to be dissipated following the preservative treatment. However, it is considered likely that the alcohol may be leached out to some extent in water. It has also most advantageously been found that the alcohol retained in the structure of the wood may remove the need for any reconditioning of the wood by relieving at least some of the residual stresses which may be present in the wood and rendering the wood closer to its equilibrium moisture content. Preferably, however, the reduced moisture content wood to be treated in accordance with the invention is at least substantially stress-free such as, for example, kiln-dried “off-the-shelf” timber. The substantially moisture free and enclosed environment to which the wood is subjected following application of the boron based preservative is such as to prevent the ingress of moisture into the treated timber, as may be provided from humidity in the atmosphere, and to substantially prevent the evaporation of the applied preservative from the wood into the atmosphere. Various possible environments are envisaged for this post-treatment. For example, the wood with the applied preservative may be introduced to a container or other preformed envelope, such as of steel or plastics, which is then sealed. However, the wood preferably occupies at least a substantial part of the internal volume of the envelope, which may not be possible when the envelope is preformed. Thus, in a preferred embodiment, the treated wood is wrapped to exclude atmosphere and thereby provide the substantially moisture free and enclosed environment. Most advantageously, the wrapping is of plastics material such as polyethylene or, preferably, polyester. With boron based compounds such as TMB, plastics sheeting which is advantageously used to form the enclosed environment must be carefully selected if the enclosed environment is to be maintained over more than about 24 hours. TMB has very good properties as a solvent for different materials such as waxes, oils, resins, glues and plastics. It also has very low surface tension and low boiling point, properties which produce a high vapour pressure at normal temperatures and which increase the risk of loss of chemicals if the impervious nature of the plastics sheeting is damaged. Polyethylene has been found to be breached by TMB over a period of at least 24 hours, and extensive testing has shown that polyester films provide the optimum properties for forming the enclosed environment, for example polyethylene terephthalate (PET) films. Multilayer films, anti-static films and metallised oxygen barrier films such as 2100, 2100E and 2110E films marketed by 3M as well as metallised multi-layer films incorporating LDPE such as are used in wine bags made by Camvac (Europe) Limited are also appropriate. One possible difficulty with some of the above plastics sheeting, unless it is to be taped or glued to provide a seal, is that they may not be heat sealable. This difficulty is generally applicable to polyester films, but it has been found that a particularly advantageous film which can be heat sealed is a co-extruded PET film marketed under the Trade Mark MELINEX by ICI. The Melinex film may have a thickness selected as appropriate, for example in the range 15 to 30 μm. The period of retention in the substantially moisture free and enclosed environment is dependent on factors such as the wood structure, temperature, pressure and the like. Experiments at ambient temperature and pressure indicate only small amounts of unreacted preservative and by-product alcohol vapour after about 24 hours. However, under similar conditions a substantial reduction of both unreacted preservative and alcohol vapour was noted about 6 hours after enclosing the wood in the substantially moisture-free environment. Furthermore, the process involved in hydrolyzing the preservative to alcohol and water and the diffusion of the preservative and alcohol through the wood and uptake into wood structure are processes which can be accelerated by the application of heat. Thus, the period of retention may have to be determined on a trial basis according to the conditions. Generally, the boron based preservative will diffuse through the wood and hydrolyze within a few hours at most followed by complete adsorption of the alcohol by-product. Accordingly, no or negligible odour of the alcohol by-product vapour when the substantially enclosed environment is opened will indicate at least substantial completion of the post treatment. The wood may conveniently be dispatched for use immediately it is enclosed in the substantially moisture-free environment, minimising the holding time. This means, for example, that a bulk order for treated wood can be supplied in its plastic wrapping for the post-treatment, with the post-treatment continuing to completion of the hydrolysis of the preservative and adsorption of the alcohol by-product during the delivery of the wood and possibly subsequent storage. The reaction and adsorption would normally be expected to be complete within two to three days at most. The moisture content of the wood is preferably reduced prior to application of a boron based preservative to improve diffusion of the preservative into the wood, particularly to alleviate hydrolysis on the wood surface. Most preferably the moisture content is of the order of about 6% by weight or less of the oven dry weight of the wood. Somewhat higher moisture contents may be appropriate for some wood-based boards or composite products but with solid wood may lead to less efficient use of the preservative, although some additives may make it possible to treat wood with a higher moisture content as described hereafter. Drying can be achieved by an original drying operation of the wood, preferably entirely separate from the preservative treatment, or may be carried out subsequent to an original drying operation but prior to the preservative treatment from any previous wood moisture content. The application of the preservative and post treatment can be performed with the wood hot, for example out of the kiln or other drying apparatus, or cold. The boron based preservative which is applied to the surface of the wood may be any boron compound which hydrolyses with the wood moisture to form a preservative-effective boron compound and alcohol including any such organic compound listed in the aforementioned NZ 115464. The boron compound applied to the wood surface may be pure or substantially pure or an azeotrope or other mixture with, for example, alcohol or other solvents. A preferred boron based preservative is TMB or a combination of TMB and methanol at or about the azeotropic composition thereof. An alternative is tri-ethyl borate which hydrolyzes to form ethanol as a by-product and boric acid. Additives may be included in the boron based preservative including, for example, additives to enhance fire-proofing attributes, such as a compatible compound of zinc. Other additives may be included in the boron based preservative to enhance its activity, including a variety of waxes, resins, oils and oil-based pigments which improve the water repellency of timber surfaces and may improve the colour and aesthetic appeal of the treated timber. Dimension stabilising chemicals can be applied in conjunction with the boron based preservative. For example, one method for the dimensional stabilisation of wood involves the application of acetic anhydride, either in the vapour phase or as a liquid, and heating the wood to 130° C. until an acetylation reactions occur. A major problem with this technique is the corrosive nature of by-products of the reaction, requiring the use of a stainless steel reaction vessel. This problem can be alleviated by applying the acetic anhydride so that the chemical reactions proceed in the enclosed environment. Polyester based films are ideal for this purpose because they are acid resistant and heat resistant. Acetic anhydride is totally miscible with, for example, trimethyl borate and can therefore be applied in the liquid phase or vapour phase by any of the chemical application techniques mentioned hereinafter which can provide the necessary loading of chemical on the surfaces of the wood. The treated wood samples are then placed in the enclosed environment to allow extended diffusion, chemical reaction and chemical dissipation to take place. Trimethyl borate is more volatile than acetic anhydride and diffuses more quickly into the wood. The rate of reaction between chemical and wood moisture is more rapid at higher temperatures and therefore the dissipation reactions can be accelerated by applying heat. Higher temperatures are required to effect acetylation—typically 130° C. The by-products of acetylation (acetic acid) tend to undergo dissipation but the extent of this dissipation has yet to be determined. The level of protection provided by a treatment in accordance with this aspect of the present invention may be dependent upon the amount of the effective boron compound deposited into the wood. For example, boric acid produced by the hydrolysis of TMB is a broad spectrum preservative. At low retention levels, it provides timber with protection from borer ( Anobium punctatum ) and Lyctus attack. At higher retention levels it provides protection from termite attack and fungal decay, e.g. dry rot. At higher loadings again, it provides flame/fire-proofing for the wood. Most proposed applications of TMB for wood treatment involve the use of a lower-boiling azeotrope or mixture of TMB in alcohol. The alcohol (methanol) is a polar solvent which can be absorbed into the wood and, because of the relatively large volume involved, can result in the swelling of the wood. This can be most disadvantageous for some products which are to be treated with preservative, for example panel products such as medium density fibreboard, particle board and so forth, where swelling is an undesirable side effect. The swelling is particularly noticeable where large volumes of preservative are applied to achieve fire and flame resistance of the wood. Not all of the alcohol solvent may be adsorbed into the wood, because of the relatively large volume involved, in which case some recovery of the excess alcohol will be required following application of the preservative in alcohol for flame and fire proofing. This may be direct from the substantially moisture-free and enclosed environment, for example using heat pump technology, preferably vapour recompression. Because of this possible need to recover excess alcohol solvent, there is a preference for applying pure or substantially pure TMB in the process of the invention but TMB is itself a solvent for boric acid or boric oxide and the boron content of TMB can therefore be enhanced simply by refluxing boric oxide and TMB together to produce a boron rich TMB azeotrope which may have advantageous use in the process of this aspect of the invention for fire-proofing wood. The boron based preservative may be applied to the surface of the wood in any of many known methods, for example pressure impregnation, vacuum/pressure impregnation, dipping, insizing and dipping, soaking, spraying/atomizing/fogging, electrostatic spraying, vaporising, evacuation and vapour or gaseous application, brushing, rolling and compression rolling. The application may be hot or cold. For commercial use, the feasibility of any of these options depends on within-charge retention variability (i.e. variation in the amount of boron based preservative applied to different pieces of wood in the same charge), between-charge retention variability (i.e. the reproducibility of results between different charges given the same treatment schedule) and cost. In addition, it is desirable in accordance with the present invention to avoid excess application of the boron based preservative since there is no recovery of excess materials except, possibly, carrier solvents such as alcohol and kerosene. The application of boron based preservatives by dipping has been characterised by high retention variability since different amounts of the preservative may be deposited onto different portions of the wood. A typical packet of 100×50 mm radiata pine comprises 24 layers of block-stacked machined timber with fillets placed at the sixth and eighteenth layers, with the packet usually being strapped, and variability in the deposited preservative, both within and between charges, is encountered because of the variation in accessibility to the wood surfaces within the packet. Commonly, dipping is performed for about 2 minutes or more in an attempt to even up the application of the preservative. However, surprisingly, it has been found that variability in application of the preservative can be substantially reduced by reducing the dipping time to about 1 minute or less, preferably about 30 seconds or less and most preferably about 15 seconds or less. In experiments, it has been found that adequate application of preservative, in the form of substantially pure TMB, was achieved with minimum variability by reducing the dipping time of the charge to approximately 2 seconds. In practice, it is accepted that there may be commercial difficulties in restricting the dipping time of a substantial charge to approximately 2 seconds all over, but it will be appreciated that the proposed reduced dipping times, particularly 15 seconds or less will substantially reduce the uptake of chemical into the coarse capillary structure of the wood and limit uptake or retention of chemical to the surface of the wood, and thereby enhance the overall process of the invention. The preservative used for dipping or other non-vapour or gaseous application may be volatile at ambient temperature and advantageously the vapour pressure of the preservative is kept low by refrigerating the bath of preservative. Additionally, the bath may be sealed to prevent the escape of any vapours and, in a preferred embodiment, the wood with the preservative applied is introduced to the substantially moisture free and enclosed environment within the sealed atmosphere of the bath. Following treatment of the wood in accordance with this aspect of the present invention, the wood may be surface treated, for example, with a resin, to immobilize the boron, that is to render the boron leach resistant. It has been found that the use of a light organic solvent wood preservative containing a boron compound may allow wood to be treated at normal moisture contents i.e., 10 to 14%. That is the amount of solvent used is reduced thereby minimising flammability hazards and costs. Thus, according to a second aspect of the invention there is provided a light organic solvent wood preservative comprising a trialkyl borate and a non-polar carrier. This LOSP is preferably used in the process of the first aspect of the invention. Suitable trialkyl borates include those having C 1-20 alkyl, preferably C 1-9 alkyl and more preferably C 1-6 alkyl groups. A particularly preferred trialkyl borate is TMB which reacts with moisture present in the wood according to equation (1) above to form boric acid and methanol. An alternative trialkyl borate is triethyl borate which hydrolyses to form boric acid and ethanol. The non-polar carrier may include a non-polar solvent, such as, aliphatic or aromatic hydrocarbons and heterocycles or derivatives thereof, for example, kerosene, petroleum and turpentine; an oil; or mixtures thereof. While oil is slightly more expensive than other non-polar carriers, there are a number of advantages in its use. Oil has low volatility and odour and therefore requires no recovery. The efficacy of the preservative is also enhanced by the use of oil in a synergistic manner by reducing water ingress into the wood thereby delaying hydrolysis of the trialkyl borate. Oil also improves the aesthetic appearance of wood and reduces surface checking. It will be appreciated that the selection of the non-polar carrier may provide the wood with enhanced properties and reduce the amount of non-polar carrier needed to achieve total treatment of the wood. Thus, in another embodiment of this aspect of the invention the light organic solvent wood preservative comprises a trialkyl borate, a non-polar solvent and an oil. Additives may also be included in the wood preservative of this aspect of the present invention. Suitable additives are selected from water repellents, such as, waxes, resins or polymers, for example, polyethylene glycol; dimensional stabilisers, such as, acetic anhydride; fire retardants, such as, zinc compounds; mildewicides/fungicides; insecticides, such as, pyrethroids or triazoles; mouldicides; dyes and pigments. The wood, generally having a higher moisture content of from 10-14% may be any timber or wood based product, such as, refractory timber, softwoods or hardwoods. The softwood may include spruces, firs, cypresses or pine species, such as, P. Radiata , for example, heartwood or sapwood. Heartwood is the most difficult part of P. Radiata to treat with preservatives. The hardwoods may include eucalypts, oak, beech, poplar, maples, willows, elms or ashes. As discussed above, the wood may be treated at moisture contents above 6% which includes the moisture content of 10 to 14% which is regarded as optimum in the wood industry for drying and using wood in construction applications. Alternatively, as previously discussed the moisture content of the wood may be reduced prior to application of the preservative to about 6% or less to improve diffusion of the preservative into the wood, particularly to alleviate hydrolysis on the wood surface. Somewhat higher moisture contents may be appropriate for some wood-based boards or composite products, but with solid wood may lead to less efficient use of the preservative. Drying can be achieved as discussed above. The preservative treatment according to this aspect of invention can be performed with the wood hot, for example out of the kiln or other drying apparatus or cold because the presence of the non-polar carrier in the preservative means that there is no swelling of the wood because the non-polar carrier does not interact with the cell walls. This in turn reduces the rate of hydrolysis of the trialkyl borate so that the formation of boric acid and alcohol is retarded and will still penetrate into the wood. The preservative may be applied to the surface of the wood by any suitable known method as already discussed. In addition, it is desirable in accordance with this aspect of the present invention to avoid excess application of the preservative since there is no recovery of excess materials except, possibly, the non-polar carrier. By this aspect of the present invention, it has been found that no treatment of the wood is necessary after application of the preservative except for allowing sufficient time for the preservative to diffuse into the wood, preferably in a substantially moisture free and enclosed environment as discussed for the first aspect of the invention. The non-polar carrier which is substantially immiscible and repellent to water protects the trialkyl borate from contact with water contained in the wood cell wall. This enables the trialkyl borate to become dispersed throughout the wood before it reacts with the residual wood moisture to form boric acid and alcohol which is adsorbed into the wood structure and fixed in the cell walls. The adsorption process again generally occurs over a prolonged period with the alcohol diffusing in either its condensed state or its vapour state through the wood cross-section, generally mainly in the vapour state. Although no recovery of the alcohol is necessary and that the wood is safe to handle following the treatment, if desired the non-polar carrier may be recovered. The use of non-polar carriers in the preservative of this aspect of the present invention typically results in an uptake of carrier of 30 l/m 3 because there is substantially no interaction between the carrier and the wood (i.e. there is no swelling). This may be compared with 150 l/m 3 if polar solvents, such as, methanol are used. It has been found that there is a synergy in using non-polar carriers in applying TMB by vacuum/pressure impregnation. Normally, very low moisture contents are required for the application of TMB whether by liquid or vapour phase treatment, typically less than 6% moisture content, to achieve total preservative penetration. The application of TMB in non-polar carriers facilitates treatment of wood having a moisture content of greater than 6%. Thus, there are no special drying requirements for the wood to effect total TMB penetration. Furthermore, as the treatment may be conducted at higher wood moisture contents and there is total TMB penetration, the TMB is hydrolysed during treatment so that no recovery of the non-polar carrier is required after treatment. Other advantages of this aspect of the present invention include: (a) the ability to include other additives as described above to effect synergy and further reduce the quantity of trialkyl borate required to effect fire retardant properties and biocidal protection to the wood; (b) the use of Class B solvents which have reduced flammability hazards and enable treatment to be conducted in conventional LOSP treatment plants; and (c) the ability to treat wood in its final shape and form. The level of protection provided by a treatment in accordance with the present invention may be dependent upon the amount of boric acid deposited into the wood. For example, as already discussed, boric acid produced by the hydrolysis of TMB is a broad spectrum preservative. At low retention levels, it provides wood with protection from borer ( Anobium punctatum ) and Lyctus attack. At higher retention levels it provides protection from termite attack and fungal decay, e.g., dry rot. At higher loadings again, it provides flame/fire-proofing for the wood. Following treatment of the wood in accordance with this aspect of the present invention, the wood may be surface treated, for example, with a resin, to immobilise the boron, that is to render the boron leach resistant. According to a third aspect of the invention there is provided a boron-based wood preservative which will enable high or low concentrations of boric acid to be incorporated into the wood, but which avoids swelling of the wood so that drying and/or recovery steps are not required after the treatment. That is there is provided a wood preservative which is prepared by reacting a boron-based preservative with a boric oxide. Suitable boron-based preservatives include those disclosed in “The Chemistry of Wood Preservation” (1991), Ed. R. Thompson., Pub. The Royal Society of Chemistry Cambridge, such as, boron esters, for example, trisubstituted borates. Examples of trisubstituted borates include TMB, triethyl borate, tri-n-propyl borate, triisopropyl borate, tri-n-butyl borate, tri(hexylene glycol)borate, triphenyl borate, triisobutyl borate, tri-n-amyl borate, tri-(octelycene glycol)biborate, tri-sec-butyl borate, tri-n-octyl borate, tri-n-dodecyl borate, tri-tert-butyl borate, tri-3-pentyl borate, tri-3-heptyl borate, trialkyl amine borate, trialkanolamine borate and triphenyl borate. A preferred boron-based preservative is TMB. The wood preservative is advantageously prepared by reacting boric oxide with the boron-based preservative and refluxing the mixture until it dissolves. The exact identity of the product formed has not yet been identified, but is predicted to be a boroxine or a polyborate or mixtures thereof. The possible products formed by the reaction will now be described using TMB as the boron-based preservative. The reaction of boric oxide and TMB in appropriate proportions results in the production of trimethoxy boroxine as shown in equation (2): in-line-formulae description="In-line Formulae" end="lead"? (CH 3 O) 3 B+B 2 O 3 →(CH 3 OBO) 3 (2) in-line-formulae description="In-line Formulae" end="tail"? Trimethoxy boroxine has the following structure: One of the most important properties of the low molecular weight boroxines is their solubility in non-polar solvents. In addition, the amount of boric acid they deliver following hydrolysis makes these compounds useful as a wood preservative for a wide range of applications. The hydrolysis of trimethoxy boroxine is shown in equation (3): in-line-formulae description="In-line Formulae" end="lead"? (CH 3 O 3 )B 3 +9H 2 O→3H 3 BO 3 +3CH 3 OH (3) in-line-formulae description="In-line Formulae" end="tail"? The hydrolysis reaction indicates that more water is needed to hydrolyse one molecule of boroxine than is needed to hydrolyse TMB. It also shows that the amount of boric acid produced is 112% the initial weight of the boroxine. The methanol produced in comparison to the hydrolysis of TMB is also substantially lower. The hydrolysis is instantaneous and is therefore similar to TMB. Furthermore, the boiling point of trimethoxy boroxine is 130° C. which makes this compound easy to handle during preservative treatment. Thus, a wood preservative which comprises a boroxine is also provided as is a process for wood preservation which comprises treating the wood with a boroxine. Preferably, the boroxine has a general formula (I): wherein R 1 , R 2 , and R 3 may be the same or different and are selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted aryl or optionally substituted heterocycyl. The term “alkyl” used either alone or in compound words such as “optionally substituted alkyl” or “optionally substituted cycloalkyl” denotes straight chain, branched or mono- or poly-cyclic alkyl, preferably C 1-30 alkyl or cycloalkyl. Examples of straight chain and branched alkyl include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl, isoamyl, sec-amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2,-trimethylpropyl, 1,1,2-trimethylpropyl, heptyl, 5-methylhexyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl, 1-methylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-methyloctyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-, 2- or 3-propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and 8-methylnonyl, 1-, 2-, 3-, 4-, 5- or 6-ethyloctyl, 1-, 2-, 3- or 4-propylheptyl, undecyl 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-methyldecyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-ethylonyl, 1-, 2-, 3-, 4- or 5-propyloctyl, 1-, 2- or 3-butylheptyl, 1-pentylhexyl, dodecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-ethyldecyl, 1-, 2-, 3-, 4-, 5- or 6-propylnonyl, 1-, 2-, 3- or 4-butyloctyl, 1-2 pentylheptyl and the like. The term “alkenyl” used either alone or in compound words such as “optionally substituted alkenyl” or optionally substituted cycloalkenyl” denotes groups formed from straight chain, branched or mono- or poly-cyclic alkenes including ethylenically mono- or poly-unsaturated alkyl or cycloalkyl groups as defined above, preferably C 2-30 alkenyl. Examples of alkenyl include vinyl, allyl, 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl, 1-4,pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, 1,3,5,7-cyclooctatetraenyl and the like. The term “alkynyl” used either alone or in compound words, such as, “optionally substituted alkynyl” and “optionally substituted cycloalkynyl” denotes groups formed from straight chain, branched, or mono- or poly-cyclic alkynes. Examples of alkynyl include ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 10-undecynyl, 4-ethyl-1-octyn-3-yl, 7-dodecynyl, 9-dodecynyl, 10-dodecynyl, 3-methyl-1-dodecyn-3-yl, 2-tridecynyl, 11-tridecynyl, 3-tetradecynyl, 7-hexadecynyl, 3-octadecynyl and the like. The term “aryl” used either alone or in compound words such as “optionally substituted aryl” denotes single, polynuclear, conjugated and fused residues of aromatic hydrocarbons. Examples of aryl include phenyl, biphenyl, terphenyl, quarterphenyl, phenoxyphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl and the like. The term “heterocyclyl” used either alone or in compound words such as “optionally substituted heterocyclyl” denotes mono- or poly-cyclic heterocyclyl groups containing at least one heteroatom atom selected from nitrogen, sulphur and oxygen. Suitable heterocyclyl groups include N-containing heterocyclic groups, such as, unsaturated 3 to 6 membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl or tetrazolyl; saturated 3 to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, such as, pyrrolidinyl, imidazolidinyl, piperidino or piperazinyl; unsaturated condensed heterocyclic groups containing 1 to 5 nitrogen atoms, such as, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl or tetrazolopyridazinyl; unsaturated 3 to 6-membered heteromonocyclic group containing an oxygen atom, such as, pyranyl or furyl; unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms, such as, thienyl; unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl or oxadiazolyl; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, morpholinyl; unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, benzoxazolyl or benzoxadiazolyl; unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolyl or thiadiazolyl; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolidinyl; and unsaturated condensed heterocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, benzothiazolyl or benzothiadiazolyl. In this specification “optionally substituted” means that a group which may or may not be further substituted with one or more groups selected from alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, alkynyloxy, aryloxy, carboxy, benzyloxy haloalkoxy, haloalkenyloxy, haloalkynyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, azido, amino, alkylamino, alkenylamino, alkynylamino, arylamino, benzylamino, acyl, alkenylacyl, alkynylacyl, arylacyl, acylamino, acyloxy, aldehydo, alkylsulphonyl, arylsulphonyl, alkylsulphonylamino, arylsulphonylamino, alkylsulphonyloxy, arylsulphonyloxy, heterocyclyl, heterocycloxy, heterocyclylamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl, carboalkoxy, carboaryloxy, mercapto, alkylthio, arylthio, acylthio and the like. A particularly preferred boroxine for use in the present invention has the formula (I) as defined above wherein R 1 , R 2 and R 3 are C 1-10 alkyl or phenol. It will be appreciated that other methods may be used to prepare the boroxine, such as, for example, the methods disclosed in Steinberg, H., (1964), “Organoboron chemistry”, (First Edition ed.)., Interscience Publishers, pp 950. Polyborates formed from boroxines have the general formula (II): wherein R 1 and R 2 may be the same or different and are as defined in formula (I) above. Polyborates may be formed when boric oxide is reacted with trimethyl borate or trimethoxy boroxine in appropriate proportions. As the ratio of boron/alkyl groups increases, the polyborate starts to form complexes and becomes more viscous. In essence an alkoxy group becomes buried in a boron oxide type matrix. The advantages of polyborates include their higher boron content and slower hydrolysis. Thus, when used as a wood preservative in non-polar or polar solvents, good penetration can be achieved. Accordingly, there is further provided a wood preservative which comprises a polyborate and a process for wood preservation which comprises treating the wood with a polyborate. Preferably, the polyborate has the general formula (II) defined above, more preferably the polyborate compound has the general formula (II) wherein R 1 and R 2 are C 1-10 alkyl or phenol. The present invention still further provides a wood preservative comprising a boroxine and a polyborate. The present invention still further extends to a process for wood preservation which comprises treating the wood with a boroxine and a polyborate. The wood preservative may be applied to the wood alone, in the form of an emulsion or in combination with a suitable carrier which may be polar or non-polar and selected from water, alcohols, aromatic or aliphatic solvents or oils. A preferred polar carrier is methanol or TMB. Preferred non-polar carriers include kerosene, petroleum, turpentine, oil or mixtures thereof. In the case of polyborates, the dilution of the wood preservative provides lower viscosity solutions which are capable of vacuum pressure impregnation. Alternatively, the wood preservative may be used as a solid preservative, for example, in the form of a rod which may be inserted into the wood or a paste which may be applied to the surface of the wood so as to impart the appropriate fire retardant properties or biocidal protection. In particular, solid polyborates have been found to be very suitable for the treatment of wood which may be infected with decay fungi. The solid preservative may be shaped and applied into pre-drilled holes or can be melted at relatively low temperatures and injected into cavities. The particular advantages of the solid polyborates compared to other solid boron compounds include their stability, relatively low manufacturing costs and fast rates of dissolution and diffusion under high wood moisture leading to conditions normally suitable for decay. Additives may also be included in the wood preservative of the present invention. Suitable additives are selected from water repellants, such as, waxes, resins or polymers, such as polyethylene glycol; dimensional stabilisers, such as, acetic anhydride; fire retardants, such as, zinc compounds; mildewicides; fungicides/insecticides; such as pyrethroids or triazoles; mouldicides; dyes and pigments. The wood may be any timber or wood based product, such as, refractory timber, softwoods or hardwoods. The softwood may include spruces, firs, cypresses or pine species, such as, P. Radiata , for example, heartwood or sapwood. Heartwood is the most difficult part of P. Radiata to treat with preservatives. The hardwoods may include eucalypts, oak, beech, poplar, maples, willows, elms or ashes. The wood may be treated at moisture contents above 6% which includes the normal moisture content of 10 to 14% which is regarded as optimum in the wood industry for drying and using wood in construction applications. Alternatively, the moisture content of the wood may be reduced prior to application of the preservative to about 6% or less to improve diffusion of the preservative into the wood, particularly to alleviate hydrolysis on the wood surface. Somewhat higher moisture contents may be appropriate for some wood-based boards or composite products, but with solid wood may lead to less efficient use of the preservative. Drying can be achieved by an original drying operation of the wood, preferably entirely separate from the preservative treatment, or may be carried out subsequent to an original drying operation but prior to the preservative treatment from any previous wood moisture content. The preservative and treatment can be performed with the wood hot, for example out of the kiln or other drying apparatus or cold. The preservative may be applied to the surface of the wood in any suitable known method as previously described, for example pressure impregnation, vacuum/pressure impregnation, dipping, insizing and dipping, soaking, spraying/atomising/fogging, electrostatic spraying, vaporising, evacuation and vapour or gaseous application, brushing, rolling and compression rolling. The application may be hot or cold. For commercial use, the feasibility of any of these options depends on the within-charge retention variability (i.e. variation in the amount of preservative applied to different pieces of wood in the same charge), between-charge retention variability (i.e. the reproducibility of results between different charges given the same treatment schedule) and cost. In addition, it is desirable in accordance with the present invention to avoid excess application of the preservative since there is no recovery of excess materials except, possibly, the carrier solvents such as alcohol and kerosene. The low amount of alcohol present in the preservative enables it to become dispersed throughout the wood before it reacts with the residual wood moisture to form boric acid and alcohol which is adsorbed into the wood structure and fixed in the cell walls. The adsorption process occurs over a prolonged period with the alcohol diffusing in either its condensed state or its vapour state through the wood cross-section, generally mainly in the vapour state. Molecules of the alcohol will eventually diffuse into the microstructure of the cell walls (the so-called transient capillaries) and form an adsorbed monolayer which is hydrogen bonded to the cellulose, hemi-cellulose and lignin in the wood structure. This means that no recovery of the alcohol is necessary and that the wood is safe to handle following the treatment. The main advantages of the wood preservatives of this aspect of the present invention arise from their low alcohol content, which facilitates treatment of wood without swelling and strength loss of the product. Further advantages of the preservatives of the present invention relate to their lower cost compared to other boron compounds, their high boiling points and lower vapour pressures which reduce handling difficulties. The level of protection provided by a treatment in accordance with the present invention may be dependent upon the amount of boric acid deposited into the wood. For example, boric acid produced by the hydrolysis of TMB is a broad spectrum preservative. At low retention levels, it provides wood with protection from borer ( Anobium punctatum ) and Lyctus attack. At higher retention levels it provides protection from termite attack and fungal decay, e.g., dry rot. At higher loadings again, it provides flame/fire-proofing for the wood. |
Image coding method and image decoding method |
A picture encoding method of the present invention is a picture encoding method of predictively encoding an input picture with reference to pictures stored in a picture buffer, decoding the encoded input picture, judging whether or not the decoded picture is a picture for reference and whether or not the decoded picture is a picture for output which needs to be stored until its display time, and storing, in the picture buffer, the picture for reference and the picture for output based on the determination result. |
1-30. (canceled) 31. A decoding method comprising: decoding a coded picture signal to obtain a decoded picture; judging, if the decoded picture is a non-reference picture, whether or not the non-reference picture can be displayed prior to each decoded picture stored in a buffer; and storing the non-reference picture in the buffer without outputting the non-reference picture prior to any decoded picture stored in the buffer to be outputted before the non-reference picture, if the non-reference picture cannot be displayed prior to each decoded picture stored in the buffer. 32. The decoding method according to claim 31, wherein said storing of the non-reference picture comprises, if the buffer does not have an empty space for the non-reference picture, removing a decoded picture stored in the buffer with an earliest display order and not to be used for reference after said decoding of the non-reference picture to obtain an empty space, and storing the non-reference picture in the empty space. 33. The decoding method according to claim 31, further comprising outputting for display (1) the non-reference picture, if the non-reference picture can be displayed prior to each decoded picture stored in the buffer, or (2) at least one other picture stored in the buffer, if the non-reference picture cannot be displayed prior to the at least one other decoded picture. 34. The decoding method according to claim 31, further comprising, prior to said judging, deciding whether the decoded picture is a reference picture or the non-reference picture, based on a coded picture signal. 35. The decoding method according to claim 31, further comprising: deciding whether the decoded picture is a reference picture or the non-reference picture prior to said judging, based on a coded picture signal; and storing the decoded picture in the buffer, if the decoded picture is the reference picture. 36. The decoding method according to claim 35, further comprising outputting at least one other decoded picture stored in the buffer, if the decoded picture is the reference picture. 37. The decoding method according to claim 31, wherein said decoding of the non-reference picture uses at least one reference picture stored in the buffer. 38. The decoding method according to claim 35, wherein said storing of the reference picture and said storing of the non-reference picture that cannot be displayed prior to each decoded picture stored in the buffer are capable of storing in a same area of the buffer. 39. The decoding method according to claim 35, wherein the buffer has a predetermined size, and said storing of the reference picture and said storing of the non-reference picture that cannot be displayed prior to each decoded picture stored in the buffer are capable of storing in a same area of the buffer. 40. The decoding method according to claim 35, wherein said storing of the decoded picture as the reference picture comprises storing the decoded picture as the reference picture in an area of the buffer storing a decoded non-reference picture that cannot be displayed prior to each decoded picture stored in the buffer, and said storing of the decoded non-reference picture that cannot be displayed prior to each decoded picture stored in the buffer comprises storing the decoded non-reference picture that cannot be displayed prior to each decoded picture stored in the buffer, in the area of the buffer storing a reference picture. 41. The decoding method according to claim 35, wherein the buffer has a predetermined size, said storing of the decoded picture as the reference picture comprises storing the decoded picture as the reference picture in an area of the buffer storing a decoded non-reference picture that cannot be displayed prior to each decoded picture stored in the buffer, and said storing of the decoded non-reference picture that cannot be displayed prior to each decoded picture stored in the buffer comprises storing the decoded non-reference picture that cannot be displayed prior to each decoded picture stored in the buffer, in the area of the buffer storing a reference picture. 42. The decoding method according to claim 41, wherein the area of the buffer has space for storing only one picture. 43. A decoding apparatus comprising: a decoding unit operable to decode a coded picture signal to obtain a decoded picture; a buffer operable to store the decoded picture; a judging unit operable to judge, if the decoded picture is a non-reference picture, whether or not the non-reference picture can be displayed prior to each decoded picture stored in said buffer; and a storing unit operable to store the non-reference picture in said buffer without outputting the non-reference picture prior to any decoded picture stored in said buffer to be outputted before the non-reference picture, if said judging unit judges that the non-reference picture cannot be displayed prior to each decoded picture stored in said buffer. 44. The decoding apparatus according to claim 43, wherein, if said buffer does not have an empty space for the non-reference picture, a decoded picture stored in said buffer with an earliest display order and not to be used for reference after said decoder decodes the non-reference picture is removed to obtain an empty space, and the non-reference picture is stored in the empty space of said buffer. 45. The decoding apparatus according to claim 43, further comprising an outputting unit operable to output for display (1) the non-reference picture, if the non-reference picture can be displayed prior to each decoded picture stored in said buffer, or (2) at least one other decoded picture stored in said buffer, if the non-reference picture cannot be displayed prior to the at least one other decoded picture. 46. The decoding apparatus according to claim 43, further comprising a deciding unit operable to decide whether the decoded picture is a reference picture or the non-reference picture, based on a coded picture signal. 47. The decoding apparatus according to claim 43, further comprising a deciding unit operable to decide whether the decoded picture is a reference picture or the non-reference picture prior to said judging unit performing the judging, based on a coded picture signal, wherein said storing unit is further operable to store the decoded picture in said buffer, if said deciding unit decides that the decoded picture is the reference picture. 48. The decoding apparatus according to claim 47, further comprising an outputting unit operable to output at least one other decoded picture stored in said buffer, if the decoded picture is the reference picture. 49. The decoding apparatus according to claim 43, wherein said decoder decodes the non-reference picture using at least one other reference picture stored in said buffer. 50. The decoding apparatus according to claim 47, wherein said storing unit is capable of storing the decoded picture in a same area of said buffer regardless of whether said deciding unit decides that the decoded picture is the reference picture or the non-reference picture that cannot be displayed prior to each decoded picture stored in said buffer. 51. The decoding apparatus according to claim 47, wherein said buffer has a predetermined size, and said storing unit is capable of storing the decoded picture in a same area of said buffer regardless of whether said deciding unit decides that the decoded picture is the reference picture or the non-reference picture that cannot be displayed prior to each decoded picture stored in said buffer. 52. The decoding apparatus according to claim 46, wherein said storing unit is operable to store the decoded picture as the reference picture in an area of said buffer storing a decoded non-reference picture that cannot be displayed prior to each decoded picture stored in said buffer, and is operable to store the decoded non-reference picture that cannot be displayed prior to each decoded picture stored in said buffer, in the area of said buffer storing a reference picture. 53. The decoding apparatus according to claim 46, wherein said buffer has a predetermined size, and said storing unit is operable to store the decoded picture as the reference picture in an area of said buffer storing a decoded non-reference picture that cannot be displayed prior to each decoded picture stored in said buffer, and is operable to store the decoded non-reference picture that cannot be displayed prior to each decoded picture stored in said buffer, in the area of said buffer storing a reference picture. 54. The decoding apparatus according to claim 53, wherein the area of said buffer has space for storing only one picture. 55. A memory management method comprising: making an indication in first information, if a reference picture stored in an area of a buffer is no longer referred to; making an indication in second information, if the reference picture has already been outputted for display; and making the area of the buffer storing the reference picture reusable for another decoded picture based on the first information and the second information. 56. The memory management method according to claim 55, wherein said making of the area of the buffer reusable comprises making the area of the buffer storing the reference picture reusable, if said making of the indication in the first information occurs before said making of the indication in the second information, after said making of the indication in the second information. 57. The memory management method according to claim 55, wherein said making of the area of the buffer reusable comprises making the area of the buffer storing the reference picture reusable, if said making of the indication in the first information occurs after said making of the indication in the second information, after said making of the indication in the first information. 58. A decoding apparatus comprising: a decoding unit operable to decode a coded picture signal to obtain a decoded picture; a buffer operable to store the decoded picture; a reference picture management unit operable to judge whether the decoded picture is to be stored in said buffer as a reference picture, and further operable to judge whether said buffer has a picture that is no longer used as a reference picture; and a display picture management unit operable to judge whether a non-reference picture is a picture to be displayed prior to at least one other decoded picture stored in said buffer or a picture to be stored in said buffer until a display time of the non-reference picture, and further operable to judge whether or not the at least one other decoded picture stored in said buffer was already outputted for display, wherein said reference picture management unit and said display picture management unit manage said buffer. |
<SOH> BACKGROUND ART <EOH>Recently, with an arrival of the age of multimedia which handles integrally audio, video and pixel values of others, existing information media, i.e., newspapers, journals, TVs, radios and telephones and other means through which information is conveyed to people, has come under the scope of multimedia. Generally speaking, multimedia refers to something that is represented by associating not only with characters but also with graphics, audio and especially pictures and the like together. However, in order to include the aforementioned existing information media in the scope of multimedia, it appears as a prerequisite to represent such information in digital form. However, when calculating the amount of information contained in each of the aforementioned information media as the amount of digital information, the information amount per character requires 1˜2 bytes whereas the audio requires more than 64 Kbits (telephone quality) per second and when it comes to the moving picture, it requires more than 100 Mbits (present television reception quality) per second. Therefore, it is not realistic to handle the vast information directly in the digital format via the information media mentioned above. For example, a videophone has already been put into practical use via Integrated Services Digital Network (ISDN) with a transmission rate of 64 Kbit/s˜1.5 Mbit/s, however, it is not practical to transmit video captured on the TV screen or shot by a TV camera. This therefore requires information compression techniques, and for instance, in the case of the videophone, video compression techniques compliant with H.261 and H.263 standards internationally standardized by ITU-T (International Telecommunication Union-Telecommunication Standardization Sector) are employed. According to information compression techniques compliant with the MPEG-1 standard, picture information as well as music information can be stored in an ordinary music CD (Compact Disc). Here, MPEG (Moving Picture Experts Group) is an international standard for compression of moving picture signals and MPEG-1 is a standard that compresses video signals down to 1.5 Mbit/s, that is, to compress information of TV signals approximately down to a hundredth. The transmission rate within the scope of the MPEG-1 standard is limited primarily to about 1.5 Mbit/s, therefore, MPEG-2 which was standardized with the view to meet the requirements of high-quality picture allows data transmission of moving picture signals at a rate of 2˜15 Mbit/s. In the present circumstances, a working group (ISO/IEC JTC1/SC29/WG11) in the charge of the standardization of the MPEG-1 and the MPEG-2 has achieved a compression rate which goes beyond what the MPEG-1 and the MPEG-2 have achieved, realized encoding/decoding operations on a per-object basis and standardized MPEG-4 in order to realize a new function required by the era of multi media. In the process of the standardization of the MPEG-4, the standardization of encoding method for a low bit rate was aimed, however, the aim is presently extended to a more versatile encoding of moving pictures at a high bit rate including interlaced pictures. Recently, a new picture encoding as a next generation encoding of the MPEG-4 called JVC is under the process of the standardization jointly worked by the ITU-T and the ISO/IEC. FIG. 24 is a diagram showing a prediction structure, a decoding order and a display order of pictures. “Picture” is a term indicating either a frame or a field and the term “picture” here is used in stead of frame or field in the present specification. The hatched pictures in FIG. 24 present the pictures to be stored in the memory for reference when other pictures are encoded/decoded. I 0 is an intra coded picture and P 3 , P 6 and P 9 are predictive coded pictures (P-picture). The predictive encoding in the scheme of the JVT standard differs from that of the conventional MPEG-1/2/4. An arbitrary picture is selected out of a plurality of encoded pictures as a reference picture and a predictive image can be generated from the reference picture. For example, a picture P 9 may select an arbitrary picture out of three pictures of I 0 , P 3 and P 6 and generate a predictive image using the selected picture. Consequently, it heightens a possibility to select the more applicable predictive image than the conventional case of applying MPEG-1/2/4 and thereby improves a compression rate. B 1 , B 2 , B 4 , B 5 , B 7 and B 8 are bi-directionally predictive coded pictures (B-picture), differing from inter-picture prediction, wherein a plurality of pictures (two pictures) are selected and a predictive image is generated using the selected pictures and then encoded. It is especially known that the accuracy of the predictive image can be greatly improved and so can be the compression rate by performing interpolation prediction using an average value of two pictures temporally previous and subsequent for generating a predictive image. The marks of “I” for an intra coded picture, “P” for a predictive coded picture and “B” for a bi-directionally predictive coded picture are used in order to differentiate encoding method of each picture. In order to refer to the temporally previous and subsequent pictures for the B-pictures, the temporally previous pictures shall be coded/decoded at first. This is called reordering of pictures and often takes place in the conventional MPEG-1/2/4. Therefore, in contrast with an encoding order (Stream Order), an order of displaying the pictures which are decoded (Display Order) is reordered as shown in FIG. 24 showing a prediction structure, a decoding order and a display order of pictures. B-pictures in the example of FIG. 24 are displayed at the moment when the stream is decoded, therefore, there is no need to store them when they are not referred to by other pictures. However, I-pictures and P-pictures have to be stored in a memory since they are displayed after being decoded when the decoding of the following B-picture is terminated. The terms and the meanings of the hatched pictures in the diagram showing the prediction structure, the decoding order and the display order of the pictures are the same as those used in FIG. 24 . FIG. 26 is a block diagram showing a picture encoding apparatus for realizing a conventional picture encoding method. The following illustrates an operation of the picture encoding apparatus for realizing the conventional picture encoding method in FIG. 26 . A picture structure determination unit PicStruct determines an encoding type (I-picture, P-picture and B-picture) for each picture, notifies a reference picture control unit RefPicCtrl of the encoding type and the pictures that can be referred to in the encoding and informs also a reordering unit ReOrder of the encoding order of the pictures. The reordering unit ReOrder reorders the order of an input picture PicIn into an encoding order and outputs the reordered pictures to a motion estimation unit ME and a subtraction unit Sub. The motion estimation unit ME refers to the reference pictures stored in a picture memory PicMem 1 , determines an applicable reference picture and detects a motion vector indicating a pixel position of the reference picture and sends them to a variable length coding unit VLC, the picture memory PicMem 1 and a motion compensation unit MC. The picture memory PicMem 1 outputs the pixels of the reference picture according to the motion vector MV to the motion compensation unit MC whereas the motion compensation unit MC generates a predictive image using the pixels in the reference picture gained from the picture memory PicMem 1 and the motion vector MV. The subtraction unit Sub calculates a difference between the picture reordered by the reordering unit ReOrder and the predictive image. The difference is converted to frequency coefficients by an orthogonal transformation unit T and then the frequency coefficients are quantized by the quantization unit Q and outputted as quantized values Coef. An inverse quantization unit IQ inverse quantizes the quantized values Coef and restores them as frequency coefficients. The inverse orthogonal transformation unit IT performs inverse frequency conversion for the frequency coefficients to be outputted as pixel differential values. An addition unit Add adds the predictive image to the pixel differential values and obtains a decoded picture. The reference picture control unit RefPicCtrl, according to the encoding type of the picture, judges whether or not the decoded picture is to be stored in the picture memory PicMem 1 to be referred to as a reference picture and whether or not the decoded picture is to be removed from the picture memory PicMem 1 (no longer referred to as a reference picture) and notifies of the operation using a memory control command MMCO. A switch SW is turned ON when the memory control command MMCO ordered a storage and thereby the decoded picture is stored in the picture memory PicMem 1 as a reference picture. The picture memory PicMem 1 releases the area where the decoded picture is stored so that other decoded pictures can be stored when the picture memory PicMem 1 instructs that the decoded picture shall be removed from the picture memory PicMem 1 . The variable length coding unit VLC encodes the quantized values Coef, the motion vector MV and the memory control command MMCO and outputs an encoded stream Str. The case in which the encoding includes the frequency conversion and the quantization is shown, however, the encoding may be the one without them such as DPCM, ADPCM, and linear predictive encoding. The encoding may be the one in which the frequency conversion and the quantization are integrated or the one that is not accompanied by the quantization after the frequency conversion as in a bit-plane encoding. FIG. 27 shows bit streams of the memory control command MMCO. The variable length coding unit VLC encodes “000” which means a release of a whole memory area so that the picture memory is initialized at the beginning of the encoding/decoding or in the head of the GOP (Group Of Picture). Also, the variable length coding unit VLC encodes “01”, when the decoded picture is stored in the picture memory. When a picture stored in the picture memory is released at the same time, the variable length coding unit VLC encodes a picture number following the “001” since the picture number to be released has to be indicated. When a plurality of pictures are released, the command to release a picture needs to be encoded for a plural number of times, therefore, a command to store a picture is encoded in addition to the command to release a picture. The variable length coding unit VLC encodes sequentially a plurality of memory control commands MMCO and encodes lastly “1” indicating that the memory control command MMCO is complete. In this way, the memory control command MMCO is encoded as an encoded stream Str. FIG. 28 is a block diagram showing a picture decoding apparatus for realizing a conventional picture decoding method. The same numbers are put for the devices that operate in the same manner as the picture encoding apparatus for realizing the conventional picture encoding method shown in FIG. 26 . A variable length decoding unit VLD decodes an encoded stream Str and outputs a memory control command MMCO, a motion vector MV and quantized values Coef. A picture time Time is inputted from outside and is a signal for specifying a picture to be displayed. When a picture to be displayed is a decoded picture, an output from the adding unit Add is selected at a selector Sel and sent out to a display unit Disp. When a picture to be displayed is a picture stored in the picture memory PicMem 1 , it is read out from the picture memory PicMem 1 , selected at the selector Sel and outputted to a display unit Disp. As described above, the picture memory PicMem 1 outputs, to the motion compensation unit MC, pixels according to the motion vector MV whereas the motion compensation unit MC generates a predictive image according to the pixels obtained from the picture memory PicMem 1 together with the motion vector MV. The inverse quantization unit IQ inverse quantizes the quantized values Coef and restores them as frequency coefficients. Furthermore, the inverse orthogonal transformation IT performs inverse frequency conversion for the frequency coefficients to be outputted as pixel differential values. The addition unit Add adds the predictive image to the pixel differential values to generate a decoded picture. The picture memory PicMem 1 releases the area in which the decoded picture is stored so that other decoded picture can be stored. The example of the decoding including the inverse frequency conversion and the inverse quantization is described above, however, the decoding may be the one without them such as DPCM, ADPCM and a linear predictive encoding. The decoding may be the one in which the inverse frequency conversion and the inverse quantization are integrated or the one that is not accompanied by the inverse quantization after the frequency conversion as in a bit-plane encoding. With the use of the picture decoding apparatus for realizing the conventional picture decoding method shown in FIG. 28 , it is obvious that the combination of the conventional picture encoding types shown in FIGS. 24 and 25 allows a correct decoding of the encoded stream Str encoded by the picture encoding apparatus for realizing the conventional picture encoding method shown in FIG. 26 . The more flexible combination is considered here as a picture encoding type. FIG. 1 is a diagram showing a prediction structure, a decoding order and a display order of the pictures, which do not exist in the related art. The prediction structure with respect to B-picture differs in the vicinity of Picture 4 in FIG. 1 . Namely, Picture 2 that is a B-picture is stored in the picture memory to be referred to as a predictive image of Picture 1 and Picture 3 . Consequently, the encoding order and the display order of each picture are as shown in FIG. 1 . Pictures B 5 and B 6 are B-pictures that are not stored since they are not referred to in a predictive coding. However, differing from FIG. 24 , the display time for the pictures B 5 and B 6 has not yet come at the time when they are decoded since it is the time for other picture to be displayed. That is, at the time of decoding the picture B 5 , the picture P 4 shall be displayed and at the time of decoding the picture B 6 , the picture B 5 shall be displayed. Since the pictures B 5 and B 6 are not stored, they cannot be taken out from the picture memory at the display time. Therefore, the pictures which are not referred to for predictive encoding are not stored in the picture memory, therefore, the pictures B 5 and B 6 cannot be displayed after being decoded with the use of the conventional encoding/decoding method. Namely in the case of not storing the pictures that are not referred to in predictive encoding as in the example shown in FIG. 24 , only Pictures 1 , 2 , 4 , and 7 can be displayed. Thus, considering the more flexible combination as a picture encoding type, it is a problem that the pictures which cannot be displayed after being decoded occur. It is conceivable to add another picture memory for display and store the pictures that are not stored in the picture memory PicMem 1 in this picture memory for display so that they can be displayed; however, the weak point is that this picture memory requires a huge memory in this case. Furthermore, there rises a new problem in the reproduction of a picture in the middle of the stream even if another picture memory for display is introduced. FIG. 2 is a diagram showing a prediction structure, a decoding order and a display order of pictures. The difference comparing with FIG. 25 is that the prediction structure in the vicinity of Picture 7 becomes completely independent. The pictures following a picture I 7 are not referred to when the pictures with display time preceding the picture I 7 are encoded/decoded. Therefore, the pictures following the picture I 7 can be encoded correctly if the decoding starts from the picture I 7 and the picture I 7 can be reproduced independently. In this way, the insertion of I picture while streaming often takes place. This system to reproduce a picture in the middle of the stream, which complies with MPEG-2, is called GOP (Group Of Picture). The correspondence of a reproduced picture of the picture decoding apparatus and that of the picture encoding apparatus in the case of reproducing the picture in the middle of the stream has to be assured, and the easy method is to initialize the whole area of the picture memory. However, Picture 6 is not yet displayed and stored in the picture memory when Picture 7 is decoded, Picture 6 therefore cannot be displayed from the picture memory at its display time if the entire picture memory is initialized before the display of Picture 6 takes place. The object of the present invention therefore is to allow the display of the pictures that cannot be displayed after being decoded by taking the memory amount necessary for encoding/decoding of the picture into consideration. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a diagram showing a prediction structure, a decoding order and a display order of pictures. FIG. 2 is a diagram showing a prediction structure, a decoding order and a display order of the pictures. FIG. 3 is a block diagram showing a picture encoding apparatus for realizing a picture encoding method of the present invention described in a first embodiment. FIG. 4 is a flowchart showing an operation of a reference picture control unit of the present invention described in the first embodiment. FIGS. 5A, 5B and 5 C are state diagrams showing a storage status of the pictures in the memory. FIG. 6 is a flowchart showing an operation of the picture encoding apparatus of the present invention described in a second embodiment. FIG. 7 is a flowchart showing an operation of the picture encoding apparatus of the present invention described in a third embodiment. FIG. 8 is a flowchart showing an operation of the picture encoding apparatus of the present invention described in a fourth embodiment. FIG. 9 is a block diagram showing a picture decoding apparatus for realizing a picture decoding method of the present invention described in a fifth embodiment. FIG. 10 is a flowchart showing an operation of the picture decoding apparatus of the present invention described in the fifth embodiment. FIG. 11 is a flowchart showing another operation of the picture decoding apparatus of the present invention described in the fifth embodiment. FIG. 12 is a flowchart showing yet another operation of the picture decking apparatus of the present invention described in the fifth embodiment. FIG. 13 is a flowchart showing another operation of the picture decoding apparatus of the present invention described in the fifth embodiment. FIG. 14 is a block diagram showing a usage of a virtual display delay buffer of a picture encoding apparatus. FIG. 15 is a block diagram showing a processing of post decoder buffer operation for encoding according to the present invention. FIG. 16 is a block diagram showing a processing of post decoder buffer operation for decoding according to the present invention. FIG. 17 is an example of using the virtual display delay buffer of the picture encoding apparatus for limiting the maximum number of the reference pictures. FIG. 18 is an example of using the virtual display delay buffer for deciding the time to display a first picture. FIG. 19 is an illustration of a storage medium in order to store a program for realizing the picture encoding method and the picture decoding method of each embodiment in a computing system, described in a seventh embodiment. FIG. 20 is a block diagram showing an overall structure of a content supply system described in a eighth embodiment. FIG. 21 is an outline view showing an example of a cell phone using the picture encoding/decoding method of the present invention described in the eighth embodiment. FIG. 22 is a block diagram of the cell phone. FIG. 23 is a block diagram showing an example of digital broadcasting system described in the eighth embodiment. FIG. 24 is a diagram showing a prediction structure, a decoding order and a display order of the pictures. FIG. 25 is a diagram showing a prediction structure, a decoding order and a display order of the pictures. FIG. 26 is a block diagram of the picture decoding apparatus for realizing the conventional picture encoding method. FIG. 27 is a mapping diagram showing examples of codes for a memory control commands MMCO. FIG. 28 is a block diagram of the picture decoding apparatus for realizing the conventional picture decoding method. detailed-description description="Detailed Description" end="lead"? |
Nitrogenous heterocyclic derivative, medicinal composition containing the same, medical use thereof, and intermediate therefor |
The present invention provides nitrogen-containing heterocyclic derivatives represented by the general formula: wherein X1 and X3 independently represent N or CH; X2 represents N or CR2; X4 represents N or CR3; and with the proviso that one or two of X1, X2, X3 and X4 represent N; R1 represents a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a lower alkylthio group, a lower alkoxy-substituted (lower alkyl) group, a lower alkoxy-substituted(lower alkoxy) group, a lower alkoxy(lower alkoxy) -substituted (lower alkyl) group, a cyclic lower alkyl group, a halo(lower alkyl) group or a group represented by the general formula: HO-A—wherein A represents a lower alkylene group, a lower alkyleneoxy group or a lower alkylenethio group; R2 represents a hydrogen atom, a halogen atom, a lower alkyl group, a cyclic lower alkyl group, a lower alkoxy group, an amino group, a (lower acyl)amino group, a mono(lower alkyl)amino group or a di(lower alkyl)amino group; and R3 represents a hydrogen atom or a lower alkyl group, or pharmaceutically acceptable salts thereof, or prodrugs thereof which are useful as agents for the prevention or treatment of a disease associated with hyperglycemia such as diabetes, diabetic complications or obesity, pharmaceutical compositions comprising the same, and pharmaceutical uses and production intermediates thereof. |
1. A nitrogen-containing heterocyclic derivative represented by the general formula: wherein X1 and X3 independently represent N or CH; X2 represents N or CR2; X4 represents N or CR3; and with the proviso that one or two of X1, X2, X3 and X4 represent N; R1 represents a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a lower alkylthio group, a lower alkoxy-substituted (lower alkyl) group, a lower alkoxy-substituted (lower alkoxy) group, a lower alkoxy(lower alkoxy)-substituted (lower alkyl) group, a cyclic lower alkyl group, a halo(lower alkyl) group or a group represented by the general formula: HO-A—wherein A represents a lower alkylene group, a lower alkyleneoxy group or a lower alkylenethio group; R2 represents a hydrogen atom, a halogen atom, a lower alkyl group, a cyclic lower alkyl group, a lower alkoxy group, an amino group, a (lower acyl)amino group, a mono(lower alkyl)amino group or a di(lower alkyl)amino group; and R3 represents a hydrogen atom or a lower alkyl group, or a pharmaceutically acceptable salt thereof, or a prodrug thereof. 2. A nitrogen-containing heterocyclic derivative represented by the general formula: wherein P represents a hydrogen atom or a group forming a prodrug; X1 and X3 independently represent N or CH; X2 represents N or CR2; X4 represents N or CR3; and with the proviso that one or two of X1, X2, X3 and X4 represent N; R2 represents a hydrogen atom, a halogen atom, a lower alkyl group, a cyclic lower alkyl group, a lower alkoxy group, an amino group, a (lower acyl)amino group, a mono(lower alkyl)amino group or a di(lower alkyl)amino group; R3 represents a hydrogen atom or a lower alkyl group; R11 represents a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a lower alkylthio group, a lower alkoxy-substituted (lower alkyl) group, a lower alkoxy-substituted (lower alkoxy) group, a lower alkoxy(lower alkoxy)-substituted (lower alkyl) group, a cyclic lower alkyl group, a halo(lower alkyl) group or a group represented by the general formula: P1-O-A—wherein P1 represents a hydrogen atom or a group forming a prodrug; and A represents a lower alkylene group, a lower alkyleneoxy group or a lower alkylenethio group, or a pharmaceutically acceptable salt thereof. 3. A nitrogen-containing heterocyclic derivative represented by the general formula: wherein X1 and X3 independently represent N or CH; X2 represents N or CR2; X4 represents N or CR3; and with the proviso that one or two of X1, X2, X3 and X4 represent N; R1 represents a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a lower alkylthio group, a lower alkoxy-substituted (lower alkyl) group, a lower alkoxy-substituted (lower alkoxy) group, a lower alkoxy(lower alkoxy)-substituted (lower alkyl) group, a cyclic lower alkyl group, a halo(lower alkyl) group or a group represented by the general formula: HO-A—wherein A represents a lower alkylene group, a lower alkyleneoxy group or a lower alkylenethio group; R2 represents a hydrogen atom, a halogen atom, a lower alkyl group, a cyclic lower alkyl group, a lower alkoxy group, an amino group, a (lower acyl)amino group, a mono(lower alkyl)amino group or a di(lower alkyl)amino group; and R3 represents a hydrogen atom or a lower alkyl group, or a pharmaceutically acceptable salt thereof. 4. A nitrogen-containing heterocyclic derivative as claimed in claim 2 wherein at least one of P or R11 has a group forming prodrug, or a pharmaceutically acceptable salt thereof. 5. A nitrogen-containing heterocyclic derivative as claimed in claim 4 wherein each group forming prodrug in P and P1 is a lower acyl group, a lower alkoxy-substituted (lower acyl) group, a lower alkoxycarbonyl-substituted (lower acyl) group, a lower alkoxycarbonyl group or a lower alkoxy-substituted (lower alokoxycarbonyl) group, or a pharmaceutically acceptable salt thereof. 6. A pharmaceutical composition comprising as an active ingredient a nitrogen-containing heterocyclic derivative as claimed in any one of claims 1-5, a pharmaceutically acceptable salt thereof or a prodrug thereof. 7. A pharmaceutical composition as claimed in claim 6 wherein the composition is a human SGLT2 inhibitor. 8. A pharmaceutical composition as claimed in claim 6 or 7 wherein the composition is a drug for the prevention or treatment of a disease associated with hyperglycemia. 9. A pharmaceutical composition as claimed in claim 8 wherein the disease associated with hyperglycemia is selected from the group consisting of diabetes, diabetic complications, obesity, hyperinsulinemia, glucose metabolism disorders, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, lipid metabolism disorders, atherosclerosis, hypertension, congestive heart failure, edema, hyperuricemia and gout. 10. A pharmaceutical composition as claimed in claim 9 wherein the disease associated with hyperglycemia is diabetes. 11. A pharmaceutical composition as claimed in claim 9 wherein the disease associated with hyperglycemia is diabetic complications. 12. A pharmaceutical composition as claimed in claim 9 wherein the disease associated with hyperglycemia is obesity. 13. A method for the prevention or treatment of a disease associated with hyperglycemia, which comprises administering an effective amount of a nitrogen-containing heterocyclic derivative as claimed in any one of claims 1-5, a pharmaceutically acceptable salt thereof or a prodrug thereof. 14. A use of a nitrogen-containing heterocyclic derivative as claimed in any one of claims 1-5, a pharmaceutically acceptable salt thereof or a prodrug thereof for the manufacture of a pharmaceutical composition for the prevention or treatment of a disease associated with hyperglycemia. 15. A pharmaceutical combination which comprises (A) a nitrogen-containing heterocyclic derivative claimed in any one of claims 1-5, a pharmaceutically acceptable salt thereof or a prodrug thereof, and (B) at least one member selected from the group consisting of an insulin sensitivity enhancer, a glucose absorption inhibitor, a biguanide, an insulin secretion enhancer, insulin or an insulin analogue, a glucagon receptor antagonist, an insulin receptor kinase stimulant, a tripeptidyl peptidase II inhibitor, a dipeptidyl peptidase IV inhibitor, a protein tyrosine phosphatase-1B inhibitor, a glycogen phosphorylase inhibitor, a glucose-6-phosphatase inhibitor, a fructose-bisphosphatase inhibitor, a pyruvate dehydrogenase inhibitor, a hepatic gluconeogenesis inhibitor, D-chiroinsitol, a glycogen synthasekinase-3 inhibitor, glucagon-likepeptide-1, a glucagon-like Peptide-1 analogue, a glucagon-like peptide-1 agonist, amylin, an amylin analogue, an amylin agonist, an aldose reductase inhibitor, an advanced glycation endproducts formation inhibitor, a protein kinase C inhibitor, a γ-aminobutyric acid receptor antagonist, a sodium channel antagonist, a transcript factor NF-κB inhibitor, a lipid peroxidase inhibitor, an N-acetylated-α-linked-acid-dipeptidase inhibitor, insulin-like growth factor-I, platelet-derived growth factor, a platelet-derived growth factor analogue, epidermal growth factor, nerve growth factor, a carnitine derivative, uridine, 5-hydroxy-1-methylhydantoin, EGB-761, bimoclomol, sulodexide, Y-128, a hydroxymethyl-glutaryl coenzyme A reductase inhibitor, a fibric acid derivative, a β3-adrenoceptor agonist, an acyl-coenzyme A: cholesterol acyltransferase inhibitor, probcol, a thyroid hormone receptor agonist, a cholesterol absorption inhibitor, a lipase inhibitor, a microsomal triglyceride transfer protein inhibitor, a lipoxygenase inhibitor, a carnitine palmitoyl:-transferase inhibitor, a squalene synthase inhibitor, a low-density lipoprotein receptor enhancer, a nicotinic acid derivative, a bile acid sequestrant, a sodium/bile acid cotransporter inhibitor, a cholesterol ester transfer protein inhibitor, an appetite suppressant, an angiotensin-converting enzyme inhibitor, a neutral endopeptidase inhibitor, an angiotensin II receptor antagonist, an endothelin-converting enzyme inhibitor, an endothelin receptor antagonist; a diuretic agent, a calcium antagonist, a vasodilating antihypertensive agent, a sympathetic blocking agent, a centrally acting antihypertensive agent, an α2-adrenoceptor agonist, an antiplatelets agent, a uric acid synthesis inhibitor, a uricosuric agent and a urinary alkalinizer. 16. A pharmaceutical combination claimed in claim 15 for the prevention or treatment of a disease associated with hyperglycemia. 17. A pharmaceutical combination claimed in claim 16 wherein a component (B) is at least one member selected from the group consisting of an insulin sensitivity enhancer, a glucose absorption inhibitor, a biguanide, an insulin secretion enhancer, insulin or an insulin analogue, a glucagon receptor antagonist, an insulin receptor kinase stimulant, a tripeptidyl peptidase II inhibitor, a dipeptidyl peptidase IV inhibitor, a protein tyrosine phosphatase-1B inhibitor, a glycogen phosphorylase inhibitor, a glucose-6-phosphatase inhibitor, a fructose-bisphosphatase inhibitor, a pyruvate dehydrogenase inhibitor, a hepatic gluconeogenesis inhibitor, D-chiroinsitol, a glycogen synthase kinase-3 inhibitor, glucagon-like peptide-1, a glucagon-like peptide-1 analogue, a glucagon-like peptide-1 agonist, amylin, an amylin analogue, an amylin agonist and an appetite suppressant, and the disease associated with hyperglycemia is diabetes. 18. A pharmaceutical combination claimed in claim 17 wherein a component (B) is at least one member selected from the group consisting of an insulin sensitivity enhancer, a glucose absorption inhibitor, a biguanide, an insulin secretion enhancer, insulin or an insulin analogue, a glucagon receptor antagonist, an insulin receptor kinase stimulant, a tripeptidyl peptidase II inhibitor, a dipeptidyl peptidase IV inhibitor, a protein tyrosine phosphatase-1B inhibitor, a glycogen phosphorylase inhibitor, a glucose-6-phosphatase inhibitor, a fructose-bisphosphatase inhibitor, a pyruvate dehydrogenase inhibitor, a hepatic gluconeogenesis inhibitor, D-chiroinsitol, a glycogen synthase kinase-3 inhibitor, glucagon-like peptide-1, a glucagon-like peptide-1 analogue, a glucagon-like peptide-1 agonist, amylin, an amylin analogue and an amylin agonist. 19. A pharmaceutical combination claimed in claim 18 wherein a component (B) is at least one member selected from the group consisting of an insulin sensitivity enhancer, a glucose absorption inhibitor, a biguanide, an insulin secretion enhancer and insulin or an insulin analogue. 20. A pharmaceutical combination claimed in claim 16 wherein a component (B) is at least one member selected from the group consisting of an insulin sensitivity enhancer, a glucose absorption inhibitor, a biguanide, an insulin secretion enhancer, insulin or an insulin analogue, a glucagon receptor antagonist, an insulin receptor kinase stimulant, a tripeptidyl peptidase II inhibitor, a dipeptidyl peptidase IV inhibitor, a protein tyrosine phosphatase-1B inhibitor, a glycogen phosphorylase inhibitor, a glucose-6-phosphatase inhibitor, a fructose-bisphosphatase inhibitor, a pyruvate dehydrogenase inhibitor, a hepatic gluconeogenesis inhibitor, D-chiroinsitol, glycogen synthase kinase-3 inhibitors, glucagon-like peptide-1, a glucagon-like peptide-1 analogue, a glucagon-like peptide-1 agonist, amylin, an amylinan alogue, an amylin agonist, an aldose reductase inhibitor, an advanced glycation endproducts formation inhibitor, a protein kinase C inhibitor, a γ-aminobutyric acid antagonist, a sodium channel antagonist, a transcript factor NF-κB inhibitor, a lipid peroxidase inhibitor, an N-acetylated-α-linked-acid-dipeptidase inhibitor, insulin-like growth factor-I, platelet-derived growth factor, a platelet derived growth factor analogue, epidermal growth factor, nerve growth factor, a carnitine derivative, uridine, 5-hydroxy-1-methylhydantoin, EGB-761, bimoclomol, sulodexide, Y-128, an angiotensin-converting enzyme inhibitor, a neutral endo-peptidase inhibitor, an angiotensin II receptor antagonist, an endothelin-converting enzyme inhibitor, an endothelin receptor antagonist and a diuretic agent, and the disease associated with hyperglycemia is diabetic complications. 21. A pharmaceutical combination claimed in claim 20 wherein a component (B) is at least one member selected from the group consisting of an aldose reductase inhibitor, an angiotensin-converting enzyme inhibitor, a neutral endopeptidase inhibitor and an angiotensin II receptor antagonist. 22. A pharmaceutical combination claimed in claim 16 wherein a component (B) is at least one member selected from the group consisting of an insulin sensitivity enhancer, a glucose absorption inhibitor, a biguanide, an insulin secretion enhancer, an insulin analogue, a glucagon receptor antagonist, an insulin receptor kinase stimulant, a tripeptidyl peptidase II inhibitor, a dipeptidyl peptidase IV inhibitor, a protein tyrosine phosphatase-1B inhibitor, a glycogen phosphorylase inhibitor, a glucose-6-phosphatase inhibitor, a fructose-bisphosphatase 10 inhibitor, a pyruvate dehydrogenase inhibitor, a hepatic gluconeogenesis inhibitor, D-chiroinsitol, a glycogen synthase kinase-3 inhibitor, glucagon-like peptide-1, a glucagon-like peptide-1 analogue, a glucagon-like peptide-1 agonist, amylin, an amylin analogue, an amylin agonist, a β3-adrenoceptor agonist and an appetite suppressant, and the disease associated with hyperglycemia is obesity. 23. A pharmaceutical combination claimed in claim 22 wherein a component. (B) is at least one member selected from the group consisting of a β3-adrenoceptor agonist and an appetite suppressant. 24. A pharmaceutical combination claimed in claim 23 wherein the appetite suppressant is a drug selected from the group consisting of a monoamine reuptake inhibitor, a serotonin reuptake inhibitor, a serotonin releasing stimulant, a serotonin agonist, a noradrenaline reuptake inhibitor, a noradrenaline releasing stimulant, an α1-adrenoceptor agonist, a β2-adrenoceptor agonist, a dopamine agonist, a cannabinoid receptor antagonist, a γ-aminobutyric acid receptor antagonist, a H3-histamine antagonist, L-histidine, leptin, a leptin analogue, a leptin receptor agonist, a melanocortin receptor agonist, α-melanocyte stimulating hormone, cocaine-and amphetamine-regulated transcript, mahogany protein, an enterostatin agonist, calcitonin, calcitonin-gene-related peptide, bombesin, a cholecystokinin agonist, corticotropin-releasing hormone, a corticotropin-releasing hormone analogue, a corticotropin-releasing hormone agonist, urocortin, somatostatin, a somatostatin analogue, a somatostatin receptor agonist, pituitary adenylate cyclase-activating peptide, brain-derived neurotrophic factor, ciliary neurotrophic factor, thyrotropin-releasing hormone, neurotensin, sauvagine, a neuropeptide Y antagonist, an opioid peptide antagonist, a galanin antagonist, a melanin-concentrating hormone receptor antagonist, an agouti-related protein inhibitor and an orexin receptor antagonist. 25. A method for the prevention or treatment of a disease associated with hyperglycemia, which comprises administering an effective amount of (A) a nitrogen-containing heterocyclic derivative claimed in any one of claims 1-5, a pharmaceutically acceptable salt thereof or a prodrug thereof, in combination with (B) at least one member selected from the group consisting of an insulin sensitivity enhancer, a glucose absorption inhibitor, a biguanide, an insulin secretion enhancer, insulin or an insulin analogue, a glucagon receptor antagonist, an insulin receptor kinase stimulant, a tripeptidyl peptidase II inhibitor, a dipeptidyl peptidase IV inhibitor, a protein tyrosine phosphatase-1B inhibitor, a glycogen phosphorylase inhibitor, a glucose-6-phosphatase inhibitor, a fructose-bisphosphatase inhibitor, a pyruvate dehydrogenase inhibitor, a hepatic gluconeogenesis inhibitor, D-chiroinsitol, a glycogen synthase kinase-3 inhibitor, glucagon-like peptide-1, a glucagon-like peptide-1 analogue, a glucagon-like peptide-1 agonist, amylin, an amylin analogue, an amylin agonist, an aldose reductase inhibitor, an advanced glycation endproducts formation inhibitor, a protein kinase C inhibitor, a γ-aminobutyric acid receptor antagonist, a sodium channel antagonist, a transcript factor NF-κB inhibitor, a lipid peroxidase inhibitor, an N-acetylated-a-linked-acid-dipeptidase inhibitor, insulin-like growth factor-I, platelet-derived growth factor, a platelet-derived growth factor analogue, epidermal growth factor, nerve growth factor, a carnitine derivative, uridine, 5-hydroxy-1-methylhydantoin, EGB-761, bimoclomol, sulodexide, Y-128, a hydroxymethyl-glutaryl coenzyme A reductase inhibitor, a fibric acid derivative, a β3-adrenoceptor agonist, an acyl-coenzyme A: cholesterol acyltransferase inhibitor, probcol, a thyroid hormone receptor agonist, a cholesterol absorption inhibitor, a lipase inhibitor, a microsomal triglyceride transfer protein inhibitor, a lipoxygenase inhibitor, a carnitine palmitoyl-transferase inhibitor, a squalene synthase inhibitor, a low-density lipoprotein receptor enhancer, a nicotinic acid derivative, a bile acid sequestrant, a sodium/bile acid cotransporter inhibitor, a cholesterol ester transfer protein inhibitor, an appetite suppressant, an angiotensin-converting enzyme inhibitor, a neutral endopeptidase inhibitor, an angiotensin II receptor antagonist, an endothelin-converting enzyme inhibitor, an endothelin receptor antagonist, a diuretic agent, a calcium antagonist, a vasodilating antihypertensive agent, a sympathetic blocking agent, a centrally acting antihypertensive agent, an α2-adrenoceptor agonist, an antiplatelets agent, a uric acid synthesis inhibitor, a uricosuric agent and a urinary alkalinizer. 26. A use of (A) anitrogen-containing heterocyclic derivative claimed in any one of claims 1-5, a pharmaceutically acceptable salt thereof or a prodrug thereof, and (B) at least one member selected from the group consisting of an insulin sensitivity enhancer, aglucoseabsorptioninhibitor, a biguanide, an insulin secretion enhancer, insulin or an insulin analogue, a glucagon receptor antagonist, an insulin receptor kinase stimulant, a tripeptidyl peptidase II inhibitor, a dipeptidyl peptidase IV inhibitor, a protein tyrosine phosphatase-1B inhibitor, a glycogen phosphorylase inhibitor, a glucose-6-phosphatase inhibitor, a fructose-bisphosphatase inhibitor, a pyruvate dehydrogenase inhibitor, a hepatic gluconeogenesis inhibitor, D-chiroinsitol, a glycogen synthase kinase-3 inhibitor, glucagon-like peptide-1, a glucagon-like peptide-1 analogue, a glucagon-like peptide-1 agonist, amylin, an amylin analogue, an amylin agonist, an aldose reductase inhibitor, an advanced glycation endproducts formation inhibitor, a protein kinase C inhibitor, a γ-aminobutyric acid receptor antagonist, a sodium channel antagonist, a transcript factor NF-κB inhibitor, a lipid peroxidase inhibitor, an N-acetylated-αlinked-acid-dipeptidase inhibitor, insulin-like growth factor-I, platelet-derived growth factor, a platelet-derived growth factor analogue, epidermal growth factor, nerve growth factor, a carnitine derivative, uridine, 5-hydroxy-1-methylhydantoin, EGB-761, bimoclomol, sulodexide, Y-128, a hydroxymethyl-glutaryl coenzyme A reductase inhibitor, a fibric acid derivative, a β3-adrenoceptor agonist, an acyl-coenzyme A: cholesterol acyltransferase inhibitor, probcol, a thyroid hormone receptor agonist, a cholesterol absorption inhibitor, a lipase inhibitor, a microsomal triglyceride transfer protein inhibitor, a lipoxygenase inhibitor, a carnitine palmitoyl-transferase inhibitor, a squalene synthase inhibitor, a low-density lipoprotein receptor enhancer, a nicotinic acid derivative, a bile acid sequestrant, a sodium/bile acid cotransporter inhibitor, a cholesterol ester transfer protein inhibitor, an appetite suppressant, an angiotensin-converting enzyme inhibitor, a neutral endopeptidase inhibitor, an angiotensin II receptor antagonist, an endothelin-converting enzyme inhibitor, an endothelin receptor antagonist, a diuretic agent, a calcium antagonist, a vasodilating antihypertensive agent, a sympathetic blocking agent, a centrally acting antihypertensive agent, an α2-adrenoceptor agonist, an antiplatelets agent, a uric acid synthesis inhibitor, a uricosuric agent and a urinary alkalinizer, for the manufacture of a pharmaceutical composition for the prevention or treatment of a disease associated with hyperglycemia. 27. A nitrogen-containing heterocyclic derivative represented by the general formula: wherein X1 and X3 independently represent N or CH; X4 represents N or CR3; X5 represents N or CR4; and with the proviso that one or two of X1, X3, X4 and X5 represent N; R0 represents a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a lower alkylthio group, or a lower alkoxy-substituted (lower alkyl) group, a lower alkoxy-substituted (lower alkoxy) group, a lower alkoxy(lower alkoxy)-substituted (lower alkyl) group, a cyclic lower alkyl group, a halo(lower alkyl) group or a group represented by the general formula: P10-O-A—wherein P10 represents a hydrogen atom or a hydroxy-protective group; and A represents a lower alkylene group, a lower alkyleneoxy group or a lower alkylenethio group; R3 represents a hydrogen atom or a lower alkyl group; and R4 represents a hydrogen atom, a halogen atom, a lower alkyl group, a cyclic lower alkyl group, a lower alkoxy group, an amino group which may have a protective group, a (lower acyl) amino group, a mono (lower alkyl) amino group which may have a protective group or a di (lower alkyl) amino group, or a salt thereof. 28. A nitrogen-containing heterocyclic derivative represented by the general formula: wherein X1 and X3 independently represent N or CH; X4represents N or CR3; X5 represents N or CR4; and with the proviso that one or two of X1, X3, X4 and X5 represent N; R0 represents a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a lower alkylthio group, or a lower alkoxy-substituted (lower alkyl) group, a lower alkoxy-substituted (lower alkoxy) group, a lower alkoxy(lower alkoxy)-substituted (lower alkyl) group, a cyclic lower alkyl group, a halo(lower alkyl) group or a group represented by the general formula: P10-O-A—wherein P10 represents a hydrogen atom or a hydroxy-protective group; and A represents a lower alkylene group, a lower alkyleneoxy group or a lower alkylenethio group; R3 represents a hydrogen atom or a lower alkyl group; and R4 represents a hydrogen atom, a halogen atom, a lower alkyl group, a cyclic lower alkyl group, a lower alkoxy group, an amino group which may have a protective group, a (lower acyl) amino group, a mono (lower alkyl) amino group which may have a protective group or a di (lower alkyl) amino group, or a salt thereof. |
<SOH> BACKGROUND ART <EOH>Diabetes is one of lifestyle-related diseases with the background of change of eating habit and lack of exercise. Hence, diet and exercise therapies are performed in patients with diabetes. Furthermore, when its sufficient control and continuous performance are difficult, drug treatment is simultaneously performed. Now, biguanides, sulfonylureas and insulin sensitivity enhancers have been employed as antidiabetic agents. However, biguanides and sulfonylureas show occasionally adverse effects such as lactic acidosis and hypoglycemia, respectively. Insulin sensitivity enhancers show occasionally adverse effects such as edema, and are concerned for advancing obesity. Therefore, in order to solve these problems, it has been desired to develop antidiabetic agents having a new mechanism. In recent years, research and development of new type antidiabetic agents have been progressing, which promote urinary glucose excretion and lower blood glucose level by preventing reabsorption of excess glucose at the kidney (J. Clin. Invest., Vol. 79, pp.1510-1515(1987)). In addition, it is reported that SGLT2 (Na + /glucose cotransporter 2) is present in the S1 segment of the kidney's proximal tubule and participates mainly in reabsorption of glucose filtrated through glomerular (J. Clin. Invest., Vol.93, pp.397-404 (1994)). Accordingly, inhibiting a human SGLT2 activity prevents reabsorption of excess glucose at the kidney, subsequently promotes excreting excess glucose though the urine, and normalizes blood glucose level. Therefore, fast development of antidiabetic agents which have a potent inhibitory activity in human SGLT2 and have a new mechanism has been desired. In addition, since such agents for promoting the excretion of urinary glucose excrete excess glucose though the urine and consequently the glucose accumulation in the body is decreased, they are also expected to have a preventing or alleviating effect on obesity and a diuretic effect. Furthermore, the agents are considered to be useful for various related diseases which occur accompanying the progress of diabetes or obesity due to hyperglycemia. |
Identification and characterization of plant genes |
The invention discloses a set of genes the expression products of which are up-regulated during the grain filling process in rice and active in different metabolic pathways involved in nutrient partitioning. The invention also discloses the use of said genes to modify the compositional and nutritional characteristics of the plant grain. |
1. A polynucleotide comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in or associated with the synthesis, metabolism or degradation of carbohydrates in the plant grain and the expression of which is up-regulated during grain filling, which nucleotide sequence is substantially similar to a sequence encoding a polypeptide as given in SEQ ID NOS: 70-210 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 2. The polynucleotide of claim 1 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 69-209 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NO: 69-209, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 3. A polynucleotide according to claim 1 comprising a nucleotide sequence encoding a polypeptide which is involved in associated with starch biosynthsis and up-regulated during grain filling, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 70-188 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 4. The polynucleotide of claim 3 comprising a nucleotide sequence a) as given in any one of the SEQ ID NOs of table 7 such as SEQ ID NOs: 69-187 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NOs: 69-187, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 5. The polynucleotide of claim 3 comprising a nucleotide sequence encoding a polypeptide with an activity of a small and large subunit ADPG pyrophosphorylase, respectively, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NOs: 136-142 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 6. The polynucleotide of claim 5 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 135-141 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides given in SEQ ID NO: 135-141, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 7. A polynucleotide according to claim 3 comprising a nucleotide sequence encoding a polypeptide involved in starch structure rearrangement, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 76-78 exhibiting isoamylase debranching enzyme activity; 70-74 exhibiting a branching enzyme activity, 80-92 exhibiting an α-amylase activity; 94-100 exhibiting an α-amylase inhibitor activity; 110 exhibiting a pullulanase activity; 102-108 exhibiting a O-amylase activity; 112-118 exhibiting a a-glucosidase activity, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 8. The polynucleotide of claim 7, comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 75-77 exhibiting isoamylase debranching enzyme activity; 69-73 exhibiting a branching enzyme activity, 79-91 exhibiting an α-amylase activity; 93-99 exhibiting an α-amylase inhibitor activity; 109 exhibiting a pullulanase activity; 101-107, exhibiting a β-amylase activity; 111-117 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NOs: 75-77 exhibiting isoamylase debranching enzyme activity; 69-73 exhibiting a branching enzyme activity, 79-91 exhibiting an α-amylase activity; 93-99 exhibiting an α-amylase inhibitor activity; 109 exhibiting a pullulanase activity; 101-107, exhibiting a β-amylase activity; 111-117; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 9. A polynucleotide according to claim 3 comprising a nucleotide sequence encoding a polypeptide exhibiting an amylase or an amylase inhibitor activity, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 80-92 exhibiting an α-amylase activity; and 94-100 exhibiting an α-amylase inhibitor activity, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 10. The polynucleotide of claim 9 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 79-91 exhibiting an α-amylase activity; and 93-99 exhibiting an α-amylase inhibitor activity or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ED NOs: 79-91 exhibiting an α-amylase activity; and 93-99 exhibiting an α-amylase inhibitor activity, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 11. A polynucleotide according to claim 3 comprising a nucleotide sequence encoding a polypeptide exhibiting a sucrose synthase activity, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 120-128 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 12. The polynucleotide of claim 11 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 119-127 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NOs: 119-127 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 13. A polynucleotide according to claim 3 comprising a nucleotide sequence encoding a polypeptide exhibiting a glucanase activity, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 192 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 14. The polynucleotide of claim 13 comprising a nucleotide sequence a) as given in SEQ ID NO: 191 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides given in SEQ ID NO: 191 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 15. A polynucleotide comprising a nucleotide sequence encoding a seed storage protein, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 212-250 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 16. The polynucleotide of claim 15 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 211-249 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: 211-249 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 17. The polynucleotide of claim 15 comprising a nucleotide sequence encoding a glutelin protein the expression of which is up-regulated during grain filling, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 224, 236, and 240 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 18. The polynucleotide of claim 17 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 223, 235, and 239 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: 223, 235, and 239, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 19. A polynucleotide according to claim 15 comprising a nucleotide sequence encoding a prolamin protein the expression of which is up-regulated during grain filling, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ HD NOs: 218, 220, 226 and 242 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 20. The polynucleotide of claim 19 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 217, 219, 225 and 241 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: 217, 219, 225 and 241, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 21. A polynucleotide according to claim 15 comprising a nucleotide sequence encoding a gliadin protein, the expression of which is up-regulated during grain filling, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NOs: 212, 219; 234, 248; and 250 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 22. The polynucleotide of claim 21 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 211,220; 233, 247; and 249 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: 135325; 135133; 10825,135101; and 135103, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 23. A polynucleotide the expression of which is up-regulated during grain filling comprising a nucleotide sequence encoding a polypeptide that is involved in or associated with fatty acid synthesis or lipid metabolism, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NOs: 252-280 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 24. The polynucleotide of claim 23 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 251-279 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides given in any one of SEQ ID NOs: 251-279 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 25. A polynucleotide according to claim 23 comprising a nucleotide sequence encoding an oleosin protein, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NOs: 258 and 260 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 26. The polynucleotide of claim 25 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 257 and 259 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: 257 and 259, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 27. A polynucleotide according to claim 23 comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in or associated with the dehydrogenation of phytoene and the expression of which is up-regulated during grain filling, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NO: 278 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 28. The polynucleotide of claim 27 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 277 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide-sequence given in any one of SEQ ID NOs: 277, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 29. A polynucleotide comprising a nucleotide sequence that encodes a polypeptide that acts as a transcription factor and the expression of which is up-regulates during grain filling, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NOs: 302-328 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 30. The polynucleotide of claim 29 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 301-327 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: 301-327, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 31. A polynucleotide comprising a nucleotide sequence encoding a polypeptide the activity of which is involved or associated with the metabolism of amino acids and the expression of which is up-regulated during grain filling, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NOs: 282-300 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide. 32. The polynucleotide of claim 31 comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 281-299 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: δ 281-299, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). 33. A polypeptide which has an amino acid sequence encoded by any one of the polynucleotides according to claim 1. 34. A polypeptide according to claim 33, which has an amino acid sequence encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 1 to 461, 501-511, and 513-641. 35. A polypeptide according to claim 33 wherein said polypeptide has at least 90% amino acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 2-462, 502-512, and 514-642. 36. An isolated nucleic acid molecule comprising a nucleotid sequence, which nucleotide sequence is obtained or obtainable from plant genomic DNA comprising a gene having an open reading frame (ORF) encoding a polypeptide which has at least between 70%, and 99% amino acid sequence identity to a polypeptide encoded by an Oryza, e.g., Oryza saliva, gene comprising a nucleotide sequence as given in SEQ ID NOs: 1 to 461, 501-511, and 513-641. 37. A recombinant vector comprising a polynucleotide of claim 1. 38. An expression cassette comprising as operably linked components, a promoter, a polynucleotide of claim 1 and a termination sequence. 39. A host cell comprising the expression cassette of claim 38. 40. The host cell of claim 39 wherein said host cell is a bacterial cell, a yeast cell, an animal cell or a plant cell. 41. The host cell of claim 40, wherein said plant cell is from a cereal plant. 42. A plant comprising a host cell of claim 39. 43. A plant according to claim 42, wherein said plant is selected from the group consisting of maize, soybean, barley, alfalfa, sunflower, tomato, banana, canola, cotton, peanut, sorghum, tobacco, sugarbeet, wheat, and rice. 44. A method of modulating carbohydrate composition of the plant grain, comprising functionally integrating an isolated nucleic acid molecule according to claim 1 comprising a nucleic acid sequence encoding a polypeptide, which is involved in or associated with the synthesis, metabolism or degradation of carbohydrates in the plant grain and the expression of which is up-regulated during grain filling, into a cell, group of cells, tissue or organ of a plant. 45. A method of modulating the protein content and composition of the plant grain, comprising functionally integrating an isolated nucleic acid molecule according to claim 15 comprising a nucleic acid sequence encoding a polypeptide, which is involved in or associated with the synthesis, metabolism or degradation of seed storage proteins in the plant grain and the expression of which is up-regulated during grain filling, into a cell, group of cells, tissue or organ of a plant. 46. A method of modulating the fatty acid and/or lipid content and composition of the plant grain, comprising functionally integrating an isolated nucleic acid molecule according to claim 23 comprising a nucleic acid sequence encoding a polypeptide, which is involved in or associated with fatty acid synthesis or lipid metabolism in the plant grain and the expression of which is up-regulated during grain filling, into a cell, group of cells, tissue or organ of a plant. 47. A method of modulating the grain filling process of the plant grain, comprising functionally integrating an isolated nucleic acid molecule according to claim 28 comprising a nucleic acid sequence encoding a transcription factor polypeptide, which is involved in or associated with the regulation and coordination of grain filling in plants and the expression of which is up-regulated during grain filling, into a cell, group of cells, tissue or organ of a plant. 48. A method of modulating the amino acid content and composition of the plant grain, comprising functionally integrating an isolated nucleic acid molecule according to claim 31 comprising a nucleic acid sequence encoding a polypeptide the activity of which is involved or associated with the metabolism of amino acids and the expression of which is up-regulated during grain filling, into a cell, group of cells, tissue or organ of a plant. 49. A method of modulating nutrient content and composition of the plant grain, comprising: a) functionally integrating i. an isolated nucleic acid molecule according to claim 1, or a portion thereof in an anti-sense orientation; or ii. an dsRNAi construct comprising an isolated nucleic acid molecule according to claim 1, or a portion thereof in both a sense and an anti-sense orientation, which, optionally, may be separated by a spacer region; under the transcriptional control of regulatory sequences required for expression in plants, into a cell, group of cells, tissue or organ of a plant; and b) expressing the constructs as provided in a) above in a cell, group of cells, a tissue or organ of a plant to produce a RNA transcript. 50. A method of identifying or isolating polynucleotide sequences that are orthologous to a nucleic acid molecule according to claim 1 comprising a nucleic acid fragment encoding a polypeptide that is up-regulated during grain filling, from the genome of another plant, wherein all or a portion of a particular nucleic acid sequence according to claim 1 is used as a probe that selectively hybridizes to gene sequences present in a population of cloned genomic DNA fragments or cDNA fragments from a chosen source organism. 51. A method to identify a nucleic acid molecule encoding a polypeptide the expression of which is up-regulated during grain filling a) contacting a plurality of isolated nucleic acid samples comprising all or a portion of a particular nucleic acid sequence according to claim 1 on a solid substrate with a probe comprising plant nucleic acid corresponding to RNA isolated from a specific plant tissue during grain filling so as to form a complex, wherein each sample comprises a plurality of oligonucleotides corresponding to at least a portion of one plant gene; and b) contacting a second plurality of isolated nucleic acid samples comprising all or a portion of a particular nucleic acid sequence according to claim 1 to on a solid substrate with a second probe comprising plant nucleic acid corresponding to RNA that is taken at a different development stage of the plant; c) comparing complex formation in a) with complex formation in b) so as to identify which samples correspond to genes that are expressed during grain filling. 52. A method for detecting the presence of a polynucleotide according to claim 1, or a fragment or a variant thereof, or a complementary sequence thereto in a sample, the method including the following steps of: a) bringing into contact a nucleotide probe or a plurality of nucleotide probes which can hybridize with a polynucleotide according to claim 1, or a fragment or a variant thereof, or a complementary sequence thereto and the sample to be assayed. b) detecting the hybrid complex formed between the probe and a nucleotide in the sample. 53. A kit for detecting the presence of a polynucleotide according to claim 1, or a fragment or a variant thereof, or a complementary sequence thereto in a sample, the kit including a nucleotide probe or a plurality of nucleotide probes which can hybridize with a nucleotide sequence comprised within a polynucleotide according to claim 1, or a fragment or a variant thereof, or a complementary sequence thereto and, optionally, the reagents necessary for performing the hybridization reaction. 54. A method of modifying the frequency of a grain filling gene in a plant population, comprising the steps of: a) screening a plurality of plants using an oligonucleotide as a marker to determine the presence or absence of a grain filling gene in an individual plant, the oligonucleotide consisting of not more than 300 bases of a nucleotide sequence selected from the group consisting of SEQ ID NOs 1 to SEQ ID NO: 461, b) selecting at least one individual plant for breeding based on the presence or absence of the grain filling gene; and c) breeding at least one plant thus selected to produce a population of plants having a modified frequency of the grain filling gene. 55. A method according to claim 54, wherein the oligonucleotide comprises a simple sequence repeat (SSR) sequence comprising at least two consecutive repeat units of an SSR, the start and end points of which are provided in Tables 2 and 3, and a flanking sequence of at least about 14 nucleic acids immediately adjacent to said at least two consecutive repeat units. 56. A method of plant breeding to select for or against a trait of interest which is associated with grain filling in plants, comprising the steps of: a. identifying the trait of interest; identifying at least one oligonucleotide that can be used as a marker for the trait, the oligonucleotide consisting of not more than 300 bases of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 461, b. screening at least one plant for the presence of the at least one oligonucleotide; c. selecting at least one plant based on presence or absence of the at least one oligonucleotide; d. breeding at least one plant thus selected to produce a population of plants having a modified frequency of the at least one oligonucleotide; and e. screening at least one plant of the population for the presence or absence of the grain filling trait. 57. A method according to claim 56, wherein the oligonucleotide comprises a simple sequence repeat (SSR) sequence comprising at least two consecutive repeat units of an SSR, the start and end points of which are provided in Tables 2 and 3, and a flanking sequence of at least about 14 nucleic acids immediately adjacent to said at least two consecutive repeat units. 58. A method of determining a varietal identity of a plant, comprising: a) obtaining a nucleic acid sample from a plant; b) identifying at least one oligonucleotide to obtain an oligonucleotide profile for the plant, wherein the oligonucleotide consists of not more than 300 bases of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 461, the oligonucleotide comprising a simple sequence repeat (SSR) sequence comprising at least two consecutive repeat units of an SSR, the start and end points of which are provided in Tables 2 and 3, and a flanking sequence of at least about 14 nucleic acids immediately adjacent to said at least two consecutive repeat units in the sample; and c) comparing the SSR profile to at least one known SSR profile corresponding to at least one known variety to determine the varietal identity of the plant. 59. An oligonucleotide primer consisting of between 8 and 150 bases which comprises at least 14 bases selected from the group of flanking sequences obtainable from a nucleotide sequence provided in SEQ ID NOs: 3435 to SEQ ID NO: 150133, which at least 14 bases are immediately adjacent to at least two consecutive repeat units of an SSR, the start and end points of which are provided in Tables 2 and 3. 60. A computer-readable medium having stored thereon a data structure comprising: a) Sequence information of a polynucleotide according to claim 1; 15-22; 23-28; 28-30 and 31 to 32 and/or; and a polynucleotide according to any one of claims . . . to . . . . b) a module receiving the nucleic acid molecule which compares the nucleic acid sequence of the molecule to at least one other nucleic acid sequence. |
<SOH> BRIEF DESCRIPTION OF THE SEQUENCE LISTING <EOH>In the following, a brief description of the sequences in the Sequence Listing is provided: Odd numbered SEQ ID NOs:1-461 are representing a first sub-group (sub-group I) of polynucleotides comprising nucleotide sequences which encode polypeptides that are up-regulated during grain filling and are described in Tables 1-11 below. Even numbered SEQ ID NOs:2-462 are protein sequences encoded by the immediately preceding nucleotide sequence, e.g., SEQ ID NO:2 is the protein encoded by the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4 is the protein encoded by the nucleotide sequence of SEQ ID NO:3, etc. Odd numbered SEQ ID NOs: 501-511 are representing a second sub-group (sub-group II) of polynucleotides comprising rice cDNA sequences. The correlation between the sequences in sub-groups I and II is illustrated in Table 13. Even numbered SEQ ID NOs:502-512 are protein sequences encoded by the immediately preceding nucleotide sequence. Odd numbered SEQ ID NOs: 513-641 are representing a third sub-group (sub-group III) of polynucleotides comprising nucleotide sequences that have homologies between 80% and 99.90% to the nucleotide sequences of sub-group I and possible variants or familiy members of rice sequences provided in SEQ ID NOs: 1-461. The correlation between the sequences in sub-groups I and III is illustrated in Table 12. Even numbered SEQ ID NOs:514-642 are protein sequences encoded by the immediately preceding nucleotide sequence. SEQ ID NOs: 643-883 are promoter sequences. SEQ ID NOs: 884-950 are banana sequences which show homology to rice “grain filling” genes. SEQ ID NOs: 951-1105 are wheat sequences which show homology to rice “grain filling” genes. SEQ ID NOs: 1106-1201 are maize sequences which show homology to rice “grain filling” genes. Definitions For clarity, certain terms used in the specification are defined and presented as follows: The term “gene” is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. For example, gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. The term “native” or “wild type” gene refers to a gene that is present in the genome of an untransformed cell, i.e., a cell not having a known mutation. A “marker gene” encodes a selectable or screenable trait. The term “chimeric gene” refers to any gene that contains 1) DNA sequences, including regulatory and coding sequences, that are not found together in nature, or 2) sequences encoding parts of proteins not naturally adjoined, or 3) parts of promoters that are not naturally adjoined. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature. A “transgene” refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed. Additionally, transgenes may comprise native genes inserted into a normative organism, or chimeric genes. The term “endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism but that is introduced by gene transfer. An “oligonucleotide” corresponding to a nucleotide sequence of the invention, e.g., for use in probing or amplification reactions, may be about 30 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21 or 24, or any number between 9 and 30). Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16 to 24 nucleotides in length may be preferred. Those skilled in the art are well versed in the design of primers for use processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 100's or even 1000's of nucleotides in length. The terms “protein,” “peptide” and “polypeptide” are used interchangeably herein. The nucleotide sequences of the invention can be introduced into any plant. The genes to be introduced can be conveniently used in expression cassettes for introduction and expression in any plant of interest. Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Preferred promoters include constitutive, tissue-specific, development-specific, inducible and/or viral promoters. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes. The cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants. The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens , such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al., 1991; Proudfoot, 1991; Sanfacon et al., 1991; Mogen et al., 1990; Munroe et al., 1990; Ballas et al., 1989; Joshi et al., 1987. “Coding sequence” refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an “uninterrupted coding sequence”, i.e., lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions. An “intron” is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein. The terms “open reading frame” and “ORF” refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. The terms “initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides (‘codon’) in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation). A “functional RNA” refers to an antisense RNA, ribozyme, or other RNA that is not translated. The term “RNA transcript” refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA” (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA. “Regulatory sequences” and “suitable regulatory sequences” each refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ noncoding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. As is noted above, the term “suitable regulatory sequences” is not limited to promoters. “5′ noncoding sequence” refers to a nucleotide sequence located 5′ (upstream) to the coding sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency (Turner et al., 1995). “3′ non-coding sequence” refers to nucleotide sequences located 3′ (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al., 1989. The term “translation leader sequence” refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5′) of the translation start codon. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. “Signal peptide” refers to the amino terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into the secretory pathway. The term “signal sequence” refers to a nucleotide sequence that encodes the signal peptide. “Promoter” refers to a nucleotide sequence, usually upstream (5′) to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. “Promoter” includes a minimal promoter that is a short DNA sequence comprised of a TATA box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. “Promoter” also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. The “initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position+1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e., further protein encoding sequences in the 3′ direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5′ direction) are denominated negative. Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as “minimal or core promoters.” In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator. “Constitutive expression” refers to expression using a constitutive or regulated promoter. “Conditional” and “regulated expression” refer to expression controlled by a regulated promoter. “Constitutive promoter” refers to a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant. Each of the transcription-activating elements do not exhibit an absolute tissue-specificity, but mediate transcriptional activation in most plant parts at a level of ≧1% of the level reached in the part of the plant in which transcription is most active. “Regulated promoter” refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and includes both tissue-specific and inducible promoters. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found in the compilation by Okamuro et al. (1989). Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol inducible systems, promoters derived from glucocorticoid-inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysome-inducible systems. “Tissue-specific promoter” refers to regulated promoters that are not expressed in all plant cells but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells). These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence. “Inducible promoter” refers to those regulated promoters that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, or a pathogen. “Operably-linked” refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. “Expression” refers to the transcription and/or translation of an endogenous gene, ORF or portion thereof, or a transgene in plants. For example, in the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. In addition, expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of protein. “Specific expression” is the expression of gene products which is limited to one or a few plant tissues (spatial limitation) and/or to one or a few plant developmental stages (temporal limitation). It is acknowledged that hardly a true specificity exists: promoters seem to be preferably switch on in some tissues, while in other tissues there can be no or only little activity. This phenomenon is known as leaky expression. However, with specific expression in this invention is meant preferable expression in one or a few plant tissues. The “expression pattern” of a promoter (with or without enhancer) is the pattern of expression levels which shows where in the plant and in what developmental stage transcription is initiated by said promoter. Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter. The level of expression of a promoter can be determined by measuring the ‘steady state’ concentration of a standard transcribed reporter mRNA. This measurement is indirect since the concentration of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded. Therefore, the steady state level is the product of synthesis rates and degradation rates. The rate of degradation can however be considered to proceed at a fixed rate when the transcribed sequences are identical, and thus this value can serve as a measure of synthesis rates. When promoters are compared in this way techniques available to those skilled in the art are hybridization S1-RNAse analysis, northern blots and competitive RT-PCR. This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA. The analysis of transcription start points in practically all promoters has revealed that there is usually no single base at which transcription starts, but rather a more or less clustered set of initiation sites, each of which accounts for some start points of the mRNA. Since this distribution varies from promoter to promoter the sequences of the reporter mRNA in each of the populations would differ from each other. Since each mRNA species is more or less prone to degradation, no single degradation rate can be expected for different reporter mRNAs. It has been shown for various eukaryotic promoter sequences that the sequence surrounding the initiation site (‘initiator’) plays an important role in determining the level of RNA expression directed by that specific promoter. This includes also part of the transcribed sequences. The direct fusion of promoter to reporter sequences would therefore lead to suboptimal levels of transcription. A commonly used procedure to analyze expression patterns and levels is through determination of the ‘steady state’ level of protein accumulation in a cell. Commonly used candidates for the reporter gene, known to those skilled in the art are β-glucuronidase (GUS), chloramphenicol acetyl transferase (CAT) and proteins with fluorescent properties, such as green fluorescent protein (GFP) from Aequora victoria . In principle, however, many more proteins are suitable for this purpose, provided the protein does not interfere with essential plant functions. For quantification and determination of localization a number of tools are suited. Detection systems can readily be created or are available which are based on, e.g., immunochemical, enzymatic, fluorescent detection and quantification. Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression. Generally, individual transformed lines with one chimeric promoter reporter construct will vary in their levels of expression of the reporter gene. Also frequently observed is the phenomenon that such transformants do not express any detectable product (RNA or protein). The variability in expression is commonly ascribed to ‘position effects’, although the molecular mechanisms underlying this inactivity are usually not clear. “Overexpression” refers to the level of expression in transgenic cells or organisms that exceeds levels of expression in normal or untransformed (nontransgenic) cells or organisms. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of protein from an endogenous gene or a transgene. “Gene silencing” refers to homology-dependent suppression of viral genes, transgenes, or endogenous nuclear genes. Gene silencing may be transcriptional, when the suppression is due to decreased transcription of the affected genes, or post-transcriptional, when the suppression is due to increased turnover (degradation) of RNA species homologous to the affected genes (English et al., 1996). Gene silencing includes virus-induced gene silencing (Ruiz et al. 1998). The terms “heterologous DNA sequence,” “exogenous DNA segment” or “heterologous nucleic acid,” as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include no-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A “homologous” DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced. “Homologous to” in the context of nucleotide sequence identity refers to the similarity between the nucleotide sequence of two nucleic acid molecules or between the amino acid sequences of two protein molecules. As used herein, “homology” and “homologous” refer to an evaluation of the similarity between two sequences based on measurements of sequence identity adjusted for variables including gaps, insertions, frame shifts, conservative substitutions, and sequencing errors, as described below. Two nucleotide sequences or polypeptides are the to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The term “complementary to” is used herein to mean that the sequence can form a Watson-Crick base pair with a reference polynucleotide sequence. Complementary sequences can include nucleotides, such as inosine, that neither disrupt Watson-Crick base pairing nor contribute to the pairing. A “reverse complement” of a sequence corresponds to the complementary sequence, but in the opposite orientation of bases from 5′ to 3′, or to the complement of the primary sequence, if the primary sequence is in a reverse orientation of bases from 5′ to 3′. Homology is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc Natl Acad Sci ( USA ) 85:2444 (1988); Altschul et al., J. Mol Biol 215:403 (1990)). In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Aligment Search Tool (“BLAST”) which is well known in the art (Karlin and Altschul, Proc Natl Acad Sci USA 87:2264 (1990); Altschul et al. (1990) supra, Altschul et al., Nucleic Acids Res 25:3389 (1997)). In particular, five specific BLAST programs are used to perform the following task: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (aligned) by means of a scoring matrix selected from the many scoring matrices known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science 256:1443 (1992); Henikoff and Henikoff, Proteins 17:49 (1993)). Likewise, the PAM or PAM250 matrices may also be used (Schwartz and Dayhoff, In Atlas of protein Sequence and Structure , Dayhoff, ed., Natl Biomed. Res. Found., pp. 353-358 (1978)). The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (Karlin and Altschul (1990) supra). “Percentage of sequence identity” can be determined from alignments performed using algorithms known in the art. Alignment of nucleotide or polypeptide sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman ( Add APL Math 2:482 (1981)), by the homology alignment algorithm of Needleman and Wunsch ( J. Mol Biol 48:443 (1970)), by the search for similarity method of Pearson and Lipman ( Proc Natl Acad Sci USA 85:2444 (1988)), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group), or by inspection. When two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. In a preferred embodiment, percenty identity is determined using the GAP program for global alignment using default parameters, using the version of GAP found in the GCG package (Wisconsin Package Version 10.1, Genetics Computer Group, 575 Science Dr., Madison, Wis.). “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window may include additions or deletions, including for example gaps or overhangs, as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotide base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. In a broad sense, the term “substantially similar”, when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure as the polypeptide encoded by the reference nucleotide sequence. Desirably, the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence. Preferably, “substantially similar” refers to nucleotide sequences having at least 50% sequence identity, preferably at least 60%, 70%, 80% or 85%, more preferably at least 90% or 95%, and even more preferably, at least 96%, 97% or 99% sequence identity compared to a reference sequence containing nucleotide sequences of Table 1, that encode a protein having at least 50% identity, more preferably at least 85% identity, yet still more preferably at least 90% identity to a region of sequence of a BIOPATH protein and/or an FPD, wherein the protein sequence comparisons are conducted using GAP analysis as described below. Also, “substantially similar” preferably also refers to nucleotide sequences having at least 50% identity, more preferably at least 80% identity, still more preferably 95% identity, yet still more preferably at least 99% identity, to a region of nucleotide sequence encoding a BIOPATH protein and/or an FPD, wherein the nucleotide sequence comparisons are conducted using GAP analysis as described below. The term “substantially similar” is specifically intended to include nucleotide sequences wherein the sequence has been modified to optimize expression in particular cells. A polynucleotide including a nucleotide sequence “substantially similar” to the reference nucleotide sequence preferably hybridizes to a polynucleotide including the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C. The term “substantially similar”, when used herein with respect to a protein or polypeptide, means a protein or polypeptide corresponding to a reference protein, wherein the protein has substantially the same structure and function as the reference protein, where only changes in amino acids sequence that do not materially affect the polypeptide function occur. When used for a protein or an amino acid sequence the percentage of identity between the substantially similar and the reference protein or amino acid sequence desirably is preferably at least 30%, more preferably at least 40%, 50%, 60%, 70%, 80%, 85%, or 90%, still more preferably at least 95%, still more preferably at least 99% with every individual number falling within this range of at least 30% to at least 99% also being part of the invention, using default GAP analysis parameters with the University of Wisconsin GCG (version 10), SEQWEB application of GAP, based on the algorithm of Needleman and Wunsch (1970), supra. As used herein the term “polypeptide of the present invention,” or any similar term refers to an amino acid sequence encoded by a DNA molecule including a nucleotide sequence substantially similar to an AC sequence. Homologs of BIOPATH protein and/or FPDs include amino acid sequences that are at least 30% identical to BIOPATH protein and/or FPD sequences found in searchable databases, as measured using the parameters described above. “Target gene” refers to a gene on the replicon that expresses the desired target coding sequence, functional RNA, or protein. The target gene is not essential for replicon replication. Additionally, target genes may comprise native non-viral genes inserted into a non-native organism, or chimeric genes, and will be under the control of suitable regulatory sequences. Thus, the regulatory sequences in the target gene may come from any source, including the virus. Target genes may include coding sequences that are either heterologous or homologous to the genes of a particular plant to be transformed. However, target genes do not include native viral genes. Typical target genes include, but are not limited to genes encoding a structural protein, a seed storage protein, a protein that conveys herbicide resistance, and a protein that conveys insect resistance. Proteins encoded by target genes are known as “foreign proteins”. The expression of a target gene in a plant will typically produce an altered plant trait. The term “altered plant trait” means any phenotypic or genotypic change in a transgenic plant relative to the wild-type or nor-transgenic plant host. “Chromosomally-integrated” refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes are not “chromosomally integrated” they may be “transiently expressed.” Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus. The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as “trausgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”. Examples of methods of transformation of plants and plant cells include Agrobacterium -mediated transformation (De Blaere et al., 1987) and particle bombardment technology (Klein et al. 1987; U.S. Pat. No. 4,945,050). Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan (see, for example, Fromm et al., 1990). “Transformed,” “transgenic,” and “recombinant” refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed in Sambrook et al., 1989. See also Innis et al., 1995 and Gelfand, 1995; and Innis and Gelfand, 1999. Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. For example, “transformed,” “transformant,” and “transgenic” plants or calli have been through the transformation process and contain a foreign gene integrated into their chromosome. The term “untransformed” refers to normal plants that have not been through the transformation process. “Transiently transformed” refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium -mediated transformation or biolistic bombardment), but not selected for stable maintenance. “Stably transformed” refers to cells that have been selected and regenerated on a selection media following transformation. “Transient expression” refers to expression in cells in which a virus or a transgene is introduced by viral infection or by such methods as Agrobacterium -mediated transformation, electroporation, or biolistic bombardment, but not selected for its stable maintenance. “Genetically stable” and “heritable” refer to chromosomally-integrated genetic elements that are stably maintained in the plant and stably inherited by progeny through successive generations. “Primary transformant” and “T0 generation” refer to transgenic plants that are of the same genetic generation as the tissue which was initially transformed (i.e., not having gone through meiosis and fertilization since transformation). “Secondary transformants” and the “T1, T2, T3, etc. generations” refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants. “Wild-type” refers to a virus or organism found in nature without any known mutation. “Genome” refers to the complete genetic material of an organism. The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991; Ohtsuka et al., 1985; Rossolini et al. 1994). A “nucleic acid fragment” is a fraction of a given nucleic acid molecule. In higher plants, deoxyribonucleic acid (DNA) is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term “nucleotide sequence” refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms “nucleic acid” or “nucleic acid sequence” may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene. The invention encompasses isolated or substantially purified nucleic acid or protein compositions. In the context of the present invention, an “isolated” or “purified” DNA molecule or an “isolated” or “purified” polypeptide is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non native environment such as, for example, a transgenic host cell. For example, an “isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an “isolated” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein of interest chemicals. The nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant (variant) forms. Such variants will continue to possess the desired activity, i.e., either promoter activity or the activity of the product encoded by the open reading frame of the non-variant nucleotide sequence. Thus, by “variants” is intended substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein. Generally, nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide sequence identity to the native (wild type or endogenous) nucleotide sequence. “Conservatively modified variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are “silent variations” which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each “silent variation” of a nucleic acid which encodes a polypeptide is implicit in each described sequence. The nucleic acid molecules of the invention can be “optimized” for enhanced expression in plants of interest. See, for example, EPA 035472; WO 91/16432; Perlak et al., 1991; and Murray et al., 1989. In this manner, the open reading frames in genes or gene fragments can be synthesized utilizing plant-preferred codons. See, for example, Campbell and Gowri, 1990 for a discussion of host-preferred codon usage. Thus, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used. Variant nucleotide sequences and proteins also encompass sequences and protein derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to create a new polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, 1994; Stemmer, 1994; Crameri et al., 1997; Moore et al., 1997; Zhang et al., 1997; Crameri et al., 1998; and U.S. Pat. Nos. 5,605,793 and 5,837,458. By “variant” polypeptide is intended a polypeptide derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art. Thus, the polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, 1985; Kunkel et al., 1987; U.S. Pat. No. 4,873,192; Walker and Gaastra, 1983 and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoffet al. (1978). Conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred. Individual substitutions deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are “conservatively modified variations,” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine I, Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q). See also, Creighton, 1984. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also “conservatively modified variations.” “Expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development. “Vector” is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication). Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast or fungal cells). Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell. The vector may be a bifunctional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell. “Cloning vectors” typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance. A “transgenic plant” is a plant having one or more plant cells that contain an expression vector. “Plant tissue” includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture such as single cells, protoplast, embryos, and callus tissue. The plant tissue may be in plants or in organ, tissue or cell culture. The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”. (a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA or gene sequence, or the complete cDNA or gene sequence. (b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches. Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Preferred, nonlimiting examples of such mathematical algorithms are the algorithm of Myers and Miller, 1988; the local homology algorithm of Smith et al. 1981; the homology alignment algorithm of Needleman and Wunsch 1970; the search for-similarity-method of Pearson and Lipman 1988; the algorithm of Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993. Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. 1988; Higgins et al. 1989; Corpet et al. 1988; Huang et al. 1992; and Pearson et al. 1994. The ALIGN program is based on the algorithm of Myers and Miller, supra. The BLAST programs of Altschul et al., 1990, are based on the algorithm of Karlin and Altschul supra. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. 1997. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al., supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g. BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, Nc=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection. For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences disclosed herein is preferably made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program. (c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.). (d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. (e)(i) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, more preferably at least 80%, 90%, and most preferably at least 95%. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions (see below). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C., depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. (e)(ii) The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. As noted above, another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s) substantially” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence. “Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence dependent, and are different under different environmental parameters. The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T m can be approximated from the equation of Meinkoth and Wahl, 1984; T m 81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T m is reduced by about 1° C. for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point I for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point I; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point I; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point 1. Using the equation, hybridization and wash compositions, and desired T, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, 1993. Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point T m for the specific sequence at a defined ionic strength and pH. An example of highly stringent wash conditions is 0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30° C. and at least about 60° C. for long robes (e.g., >50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. Very stringent conditions are selected to be equal to the T m for a particular probe. An example of stringent conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. The following are examples of sets of hybridization/wash conditions that may be used to clone orthologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C., more desirably in 7% A sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C. “DNA shuffling” is a method to introduce mutations or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly. The DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule. The shuffled DNA preferably encodes a variant polypeptide modified with respect to the polypeptide encoded by the template DNA, and may have an altered biological activity with respect to the polypeptide encoded by the template DNA. “Recombinant DNA molecule” is a combination of DNA sequences that are joined together using recombinant DNA technology and procedures used to join together DNA sequences as described, for example, in Sambrook et al., 1989. The word “plant” refers to any plant, particularly to seed plant, and “plant cell” is a structural and physiological unit of the plant, which comprises a cell wall but may also refer to a protoplast. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ. “Significant increase” is an increase that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater. “Significantly less” means that the decrease is larger than the margin of error inherent in the measurement technique, preferably a decrease by about 2-fold or greater. Within the scope of the present invention a set of nucleic acid molecules is provided which comprises polynucleotides relating to genes which are shown to be preferentially up-regulated and to share a similar expression pattern during the process of grain filling. The polynucleotides within this subgroup are useful tools for generating plants which produce grain with modified compositional characteristics leading to improved nutritional properties. In one embodiment, the present invention thus relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the expression of which is up-regulated during grain filling and the use of said molecule for modifying the nutritional composition and quality of the plant grain. The majority of the polynucleotides within this group encode protein products that are directly involved in or associated with three major pathways of nutrition partitioning: the synthesis and transport of (1) carbohydrates, (2) proteins, and (3) fatty acids. Carbohydrates are the most abundant organic molecules in nature and modulation of their synthesis, accumulation, and storage presents a vast template of possibilities for improving the quality and quantity of agricultural plants, food crops, consumer health products such as dietary supplements, and many industrial applications. In plants, carbohydrates occur as mono-, di, or polysaccharides and have the essential functions of providing the plant with chemical energy and structural stability. Although sugar uptake from external sources generally is not a relevant process, the redistribution of sugar (usually glucose) from photosynthesizing tissues to non-green cells is of major importance. Once translocated to terminal sink storage tissues, sugars are converted to starch and stored in the leucoplasts of seeds, fruits, tubers and roots, as well as actively growing photosynthetic tissues. These plant tissues provide the bulk of human dietary intake, and as such, the anabolic pathways of synthesis and assimilation (starch, fatty acids, and nitrogen) are of particular importance to agriculture and commercial industry. As major contributors to the global carbon cycle, plants and algae bind 100 billion metric tons of carbon into carbohydrates each year. Nucleotide sequences encoding at least one polypeptide involved in sugar and carbohydrate metabolism and their end products, as well as the polypeptides encoded thereby, or an antigene sequences thereof, are commercially useful materials that can be used to study these processes and to modify these processes to elicit desired modifications in the compositional and nutritional characteristics of the plant grain. In particular, the subset of nucleic acid molecules provided herein, which comprises polynucleotides relating to genes that are up-regulated during grain filling and involved in carbohydrate transport, synthesis, metabolism, or degradation is a valuable tool box from which an appropriate nucleic acid molecule can be chosen for modifying the quantity and quality of the carbohydrate and sugar content of the grain, respectively. This can be achieved by introducing and overexpressing at least one polynucleotide from the various subsets of nucleic acid molecules provided herein in the plant, but preferentially in the approproate tissues of the plant grain such as, for example, the plant endosperm or by reducing the expression level of the corresponding endogenous gene by methods known in the art including antisense and dsRNAi techniques. It is thus one of the major objectives of the present invention to identify and provide a subset of nucleic acid molecules comprising at least one polynucleotide which encodes a protein that is involved in the metabolism of carbohydrates during grain filling. By modifying the expression level of at least one of the polynucleotides from this subgroup in a plant, but preferably in the approproate tissues of the plant grain such as, for example, the plant endosperm, and even more preferably at an early stage in seed development, it is possible to modify the carbohydrate composition of the plant grain accordingly. In one embodiment, the invention thus relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in or associated with the synthesis, metabolism or degradation of carbohydrates in the plant grain and the expression of which is up-regulated during grain filling, which nucleotide sequence is substantially similar to a sequence encoding a polypeptide as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 70-210. In particular, the invention relates to polynucleotide comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in or associated with the synthesis, metabolism or degradation of carbohydrates in the plant grain and the expression of which is up-regulated during grain filling, and which is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide of SEQ ID NOs given in table 7 such as SEQ ID NOs: 70-210, with any individual number within this range of between 70% and 99% A also being part of the invention. The invention further relates to polynucleotide comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in or associated with the synthesis, metabolism or degradation of carbohydrates in the plant grain and the expression of which is up-regulated during grain filling, and which is immunologically reactive with antibodies raised against a polypeptide as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 70-210. More particularly, the invention relates to polynucleotide comprising a nucleotide sequence a) as given in any one of SEQ ID NOs of table 7 such as SEQ ID NOs: 69-209 or a part thereof which still encodes a partial length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NOs of table 7 such as SEQ ID NOs: 69-209 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). One of the defining questions in assimilate partitioning is understanding how plants regulate the allocation of photosynthate between competing sink organs. In addition to the number of competing organs, and the sink strength of each, exogenous factors such as abiotic stress or pathogen infection may also influence partitioning (Bush, Current Opinions in Plant Biology 2:187. (1999)). Within the present invention a subset of genes could be identified that are known to be involved in the plant's response to abiotic and/or biotic stresses and demonstrated to be up-regulated during grain filling. By providing these genes it is now possible to regulate the expression levels of the encoded protein products in the plant grain during the grain filling process by applying methods known in the art including overexpressing or down-regulating the nucleic acid molecule in a plant, or preferably a plant seed, thereby modifying the partitioning in the developing grain. In one aspect, the present invention relates to polynucleotide comprising a nucleotide sequence encoding a polypeptide the expression of which is up-regulated during grain filling and the activity of which is involved in or associated with the plant's response to abiotic and/or biotic stresses, which nucleotide sequence is substantially similar to a sequencen encoding a polypeptide as given in any one of the SEQ ID NOs of table 4 such as SEQ ID NOs: 2-18. In particular, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide the expression of which is up-regulated during grain filling and the activity of which is involved in or associated with the plant's response to abiotic and/or biotic stresses, and which is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide as given in any one of the SEQ ID NOs of table 4 such as SEQ ID NOs: 2-18, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide the expression of which is up-regulated during grain filling and the activity of which is involved in or associated with the plant's response to abiotic and/or biotic stresses, and which is immunologically reactive with antibodies raised against a polypeptide as given in any one of the SEQ ID NOs of table 4 such as SEQ ID NOs: 2-18. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in in any one of the SEQ ID NOs of table 4 such as SEQ ID NOs: 1-17 or a part thereof which still encodes a partial length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence as given in any one of the SEQ ID NOs of table 4 such as SEQ ID NOs 1-17 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). The regulation of source-sink pathways encompasses complex mechanisms that integrate the expression of enzymes involved in carbohydrate production in source tissue with those involved with utilization in sink tissue. The elucidation of the underlying signal transduction pathways of sink-source regulation is of critical importance to the genetic manipulation of source-sink relations in transgenic plants. Within the scope of the present invention a subset of genes was identified comprising genes that are up-regulated during grain filling and encode polypeptides with a kinase or phosphatase activity which are known to be involved in signal transduction pathways. In a specific embodiment, the present invention provides nucleic acid molecules such as those represented in SEQ ID NOs: 19-29 that encode enzymes which exhibit a kinase or phosphatase activity and/or are involved in a signalig pathway and are thus key to the ability of regulating utilization of carbon/sugar sources, and partitioning of assimilates between source and sink tissues. The invention thus relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which exhibits a kinase or phosphatase activity and/or are involved in a signal transduction pathway, the expression of which is up-regulated during grain filling, which nucleotide sequence is substantially similar to a sequence encoding a polypeptide as given in any one of the SEQ ID NOs of table 5 such as SEQ ID Nos: 20-30. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which exhibit a kinase or phosphatase activity and is up-regulated during grain filling and has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide as given in any one of the SEQ ID NOs of table 5 such as SEQ ID NOs: 20-30, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which exhibit a kinase or phosphatase activity and is up-regulated during gain filling and immunologically reactive with antibodies raised against a polypeptide as given in any one of the SEQ ID NOs of table 5 such as SEQ ID NOs: 20-30. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of the SEQ ID NOs of table 5 such as SEQ ID NOs: 19-29 or a part thereof which still encodes a partial length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof, d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence as given in any one of the SEQ ID NOs of table 5 such as SEQ ID NOs: 19-29 or the complement thereof, e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). Regulating the environment-induced carbon status in crop plants, particularly the partitioning in storage organs, provides industry with the ability to limit or expand growing seasons to better suit commercial markets, to enhance the quality and content of food products derived from storage organs or other tissue specific components of crop plants, and modulate many other metabolic pathways in plants (such as nitrogen assimilation, phosphorylation and the activation of regulatory proteins) that effect consumer end use. Another possibility for modifying the carbohydrate content of the grain is through regulation of the transport of sugars and carbohydrates during grain filling. Supplying carbohydrates to sink tissues via apoplastic mechanisms involves the release of sucrose into the apoplast by an exporter, cleavage by an extracellular invertase, and uptake of hexose monomers by monosaccharide transporters. In one specific embodiment the present invention thus relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide with an activity which is involved in or associated with sugar transport and up-regulated during grain filling, which nucleotide sequence is substantially similar to a sequence encoding a polypeptide as given in any one of the SEQ ID NOs of table 6 such as SEQ ID NOs: 36; 50, and 58. In particular, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide with an activity which is involved in or associated with sugar transport and up-regulated during grain filling and is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide as given in any one of the SEQ ID NOs of table 6 such as SEQ ID NOs: 36; 50, and 58, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide with an activity which is involved in or associated with sugar transport and up-regulated during grain filling and is immunologically reactive with antibodies raised against a polypeptide as given in any one of the SEQ ID NOs of table 6 such as SEQ ID NOs: 36; 50, and 58. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of the SEQ ID NOs of table 6 such as SEQ ID NOs: 35; 49, and 57 or a pail thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence as given in any one of the SEQ ID NOs of table 6 such as SEQ ID NOs: 35; 49, and 57 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). Transmembrane transport of sugars has been demonstrated by the presence of transporter genes for a few crop species (spinach, potato). For the uses and application of modifying sugar transport mechanisms with regard to controlling the timing and extent of grain fill durations, we incorporate all relevant sections of PCT Publication WO9953068 to Allen et al., and for uses and application of modifying cells or plastids involved in hexose carrier proteins we incorporate all relevant sections of PCT Publication WO9953082 to Allen et al. Glucosyl equivalents for starch biosynthesis are found within the scope of the present invention to be transported into the plastid (amyloplast) either as glucose-1-phosphate via a hexose-phosphate-Pi transporter (a representative example of which is given in SEQ ID NO: 35), as triose phosphates via a triose-phosphate-Pi translocator (a representative example of which are given in SEQ ID NO: 163), as phosphoenolpyruvate via a PEP-Pi translocator (SEQ ID NOs: 175), or as ADP-glucose via a Brittle-like adenylate translocator or via an oxoglutarate/malate transporter. One isoform of a triose-phosphate/phosphate translocator (SEQ ID NO: 163) is expressed to a slightly higher level during earlier stages of grain development. Pyruvate appears to play a more important role during early stages of grain development in that a gene encoding an isoform of a PEP-Pi translocator (SEQ ID NO: 175) is relatively more highly expressed at this stage. In maize endosperm, the majority of glucosyl moieties are transported to the amyloplast during the linear phase of starch accumulation as ADP-glucose (J. C. Shannon et al., Plant Physiol. 117, 1235 (1998)). For uses and application of modifying amyloplasts in the regulation of starch production via an ADP glucose transporter, we incorporate all relevant sections of PCT Publication WO9947681 to Emes et al. Further examples of genes encoding a sugar transporter are provided in SEQ ID NOs: 35; 49, and 57. By providing the nucleic acid molecules according to the invention encoding sugar transporters the expression of which is upregulated during grain filling such as those given in SEQ ID NOs: 36; 50, and 58; 36385; 53483; it is now possible to manipulate the translocation and storage of sugars and their carbohydrate end products in the plant grain. In still another embodiment the present invention provides further subset of nucleic acid molecules which are up-regulated during grain filling comprising a nucleotide sequence encoding a polypeptide that has a transmembrane domain and assists in the transport of amino acids and inorganic compounds including nitrate and various cations, which nucleotide sequence is substantially similar to a sequence encoding a polypeptide as given in SEQ ID NOs: 32; 38; 40; 42; 44; 46; 48; 52; 54; 56; 60; 62; 64, 66; and 68. In particular, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide, that has a transmembrane domain and assists in the transport of amino acids and inorganic compounds including nitrate and various cations and is up-regulated during grain filling and is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide of SEQ ID NOs: 32; 38; 40; 42; 44; 46; 48; 52; 54; 56; 60; 62; 64, 66; and 68, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide, that has a transmembrane domain and assists in the transport of amino acids and inorganic compounds including nitrate and various cations and is up-regulated during grain filling and is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 32; 38; 40; 42; 44; 46; 48; 52; 54; 56; 60; 62; 64, 66; and 68. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 31; 37; 39; 41; 43; 45; 47; 51; 53; 55; 59; 612; 63, 65; and 67 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NO: 31; 37; 39; 41; 43; 45; 47; 51; 53; 55; 59; 612; 63, 65; and 67, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). In particular, the invention provides a nucleic acid molecule which is up-regulated during grain filling and comprises a nucleotide sequence encoding a polypeptide that belongs to the POT or PTR family. Proteins of the POT family (also called the PTR (peptide transport) family) consists of proteins from animals, plants, yeast, archaea, and both Gram-negative and Gram-positive bacteria. Several of these organisms possess multiple POT family paralogues. The proteins are of about 450-600 amino acyl residues in length with the eukaryotic proteins in general being longer than the bacterial proteins. They exhibit 12 putative or established transmembrane ?-helical spanners. Some members of the POT family exhibit limited sequence similarity to protein members of the major facilitator superfamily (MFS; TC #2.A.1). (Comparison scores of up to 8 standard deviations for segments in excess of 60 residues in length.) Thus the POT family is probably a family within the MFS. While most members of the POT family catalyze peptide transport, one is a nitrate permease and one can transport histidine as well as peptides. Some of the peptide transporters can also transport antibiotics. They function by proton symport, but the substrate:H + stoichiometry is variable: the high affinity rat PepT2 carrier catalyzes uptake of 2 and 3H + with neutral and anionic dipeptides, respectively, while the low affinity PepT1 carrier catalyzes uptake of one H+ per neutral peptide. In eukaryotes, some of these transporters may be in organellar membranes such as the lysosomes. The generalized transport reaction catalyzed by the proteins of the POT family is: in-line-formulae description="In-line Formulae" end="lead"? substrate (out)+nH + (out)--->substrate (in)+nH + (in). in-line-formulae description="In-line Formulae" end="tail"? In a specific embodiment, the present invention relates to an isolated nucleic acid molecule which is up-regulated during grain filling and comprises a nucleotide sequence encoding a polypeptide that belongs to the POT or PTR family, which nucleotide sequence is substantially similar to a sequence encoding a polypeptide as given in SEQ ID NOs: 38; 52, and 68. In particular, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide, which belongs to the POT or PTR family and up-regulated during grain filling and is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide of SEQ ID NOs: 38; 52, and 68, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide, which belongs to the POT or PTR family and up-regulated during grain filling and is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 38; 52, and 68. More particularly, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 37; 51, and 67 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NO: 37; 51, and 67 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). One of the economically most important and valuable carbohydrate end products is starch, which is an essential component of many food, feed, and industrial products. It consists of two types of glucan polymers: relatively long chained polymers with few branches known as amylose, and shorter chained but highly branched molecules called amylopectin. Its biosynthesis depends on the complex interaction of multiple enzymes (Smith, A. et al., (1995) Plant Physio. 107:673-677; Preiss, J., (1988) Biochemistry of Plants 14:181-253). One of the key enzymes in starch biosynthesis is ADP-glucose pyrophosphorylase, which catalyzes the formation of ADP-glucose; a series of starch synthases which use ADP glucose as a substrate for polymer formation using alpha.-1-4 linkages; and several starch branching enzymes, which modify the polymer by transferring segments of polymer to other parts of the polymer using alpha.-1-6 linkages, creating branched structures. However, based on data from starch forming plants such as potato, and corn, it is becoming clear that other enzymes also play a role in the determination of the final structure of starch. In particular, debranching and disproportionating enzymes not only participate in starch degradation, but also in modification of starch structure during its biosynthesis. Different models for this action have been proposed, but all share the concept that such activities, or lack thereof, change the structure of the starch produced. In plants used typically for the production of starch, such as maize or potato, the synthesized starch consists of approximately 25% amylose-starch and of about 75% amylopectin-starch. With respect to the homogeneity of the basic component starch for its use in the industrial area, starch-producing plants are needed which contain, for example, only the component amylopectin or only the component amylose. For a number of other uses plants are needed that synthesize amylopectin types with different degrees of branchings. Such plants may for example be obtained by breeding or by means of mutagenesis techniques. It is known for various plant species, such as for maize, that by means of mutagenesis varieties may be produced in which only amylopectin is formed. Also in the case of potato a genotype was produced from a haploid line by means of chemical mutagenesis. Said genotype does not form amylose (Hovenkamp-Hermelink, Theor. Appl. Genet. 75 (1987), 217-221). Apart from conventional breeding and mutagenesis techniques, recombinant DNA techniques are now increasingly used in order to specifically interfere with the starch metabolism of starch storing plants. A prerequisite for this is that DNA sequences be provided which encode enzymes involved in the starch metabolism. The present invention now provides a subset of nucleic acid molecules that are involved in the starch biosynthesis pathway and were shown to be up-regulated during grain filling. Representative examples of those subset genes are provided in SEQ ID NOs: 69-187 of the Sequence Listing. In a particular embodiment, the present invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which is involved in associated with starch biosynthsis and up-regulated during grain filling, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in any one of the SEQ ID NOs of table 7 such as SEQ ID NOs: 70-188. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide, which is involved in or associated with starch biosynthesis and up-regulated during grain filling and is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide as given in any one of the SEQ ID NOs of table 7 such as SEQ ID NOs: 70-188, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide, which is involved in or associated with starch biosynthesis and up-regulated during grain filling and is immunologically reactive with antibodies raised against a polypeptide as given in any one of the SEQ ID NOs of table 7 such as SEQ ID NOs: 70-188. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of the SEQ ID NOs of table 7 such as SEQ ID NOs: 69-187 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the fill-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence as given in any one of the SEQ ID NOs of table 7 such as SEQ ID NOs: 69-187, or the complement thereof, e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). By providing a subset of genes encoding polypeptides that are involved in starch metabolism it is now possible to interfere with starch metabolism to produce starch with modified physico/chemical characteristics. A gene encoding the small subunit of ADPG pyrophosphorylase (SEQ ID NO: 138); is expressed at early stages of grain development in conjunction with a single gene encoding a large subunit (SEQ ID NO: 140). Three other large subunits (SEQ ID NOs: 136; 142); are up-regulated at a later stage in development from 4 days after anthesis, in conjunction with the up regulation of the starch synthase genes (SEQ ID NOs: 129; 131; and 133) and two genes for branching enzymes (SEQ ID NOs: 70; and 72) (involved in amylose and amylopectin biosynthesis, respectively). Only one (distinct from the two mentioned above) of the small subunit genes increases in this time period. The expression of different isoforms may be related to the shift to storage starch production and a postulated concomitant shift to cytoplasmic ADP-glucose production (Stark, D. M., et al., “Regulation of the Amount of Starch in Plant Tissues by ADP Glucose Pyrophosphorylase ”, Science, 258,287-291 (Oct. 9, 1992)). In one embodiment the present invention provides a nucleic acid molecule comprising a nucleotide sequence which encodes a small subunit of ADPG pyrophosphorylase. In another embodiment the invention provides a nucleic acid molecule comprising a nucleotide sequence which encodes a large subunit of ADPG pyrophosphorylase. In particular, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide with an activity of a small and large subunit ADPG pyrophosphorylase, respectively, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NOs: 136-142. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide with an activity of a small and large subunit ADPG pyrophosphorylase, respectively, which is up-regulated during grain filling and has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide of SEQ ID NOs: 136-142, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide with an activity of a small and large subunit ADPG pyrophosphorylase, respectively, which is up-regulated during grain and immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 136-142. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: SEQ ID NOs: 135-141 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides given in SEQ ID NO: SEQ ID NOs: 135-141, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). The nucleic acid molecules of the instant invention may be used to create transgenic plants in which the small and/or large subunits of ADPG pyrophosphorylase are present at higher or lower levels than normal or in cell types or developmental stages in which it is not normally found. This may have the effect of altering starch structure in those cells or tissues but especially in the developing grain. For a further targeted modification of the starch in plants, in particular of the degree of branching of starch synthesized in plants by means of recombinant DNA techniques, it is still necessary to identify DNA sequences that encode enzymes participating in the starch metabolism, particularly in the branching of starch molecules. In the case of potato, for example, DNA sequences have by now been described which encode a granule-bound starch synthase or a branching enzyme (Q enzyme), and they have been used in order to genetically modify plants. Apart from the Q enzymes that introduce branchings into starch molecules, enzymes occur in plants which are capable of dissolving branchings. These enzymes are called debranching enzymes. In the case of sugar beet, Li et al. (Plant Physiol. 98 (1992), 1277-1284) could only prove the occurrence of one debranching enzyme, apart from five endo- and two exoamylases. This enzyme having a size of approximately 100 kD and an optimum pH value of 5.5 is located within the chloroplasts. A debranching enzyme was also described for spinach. The debranching enzyme from spinach as well as that from sugar beet exhibit a fivefold lower activity in a reaction with amylopectin as substrate when compared to a reaction with pullulan as a substrate (Ludwig et al., Plant Physiol. 74 (1984), 856-861; Li et al., Plant Physiol. 98 (1992), 1277-1284). The isolation of a cDNA encoding a debranching enzyme was described for spinach (Renz et al., Plant Physiol. 108 (1995), 1342). The existence of a debranching enzyme for maize has been described in the prior art. The corresponding mutant was designated su (sugary). The gene of the sugary locus was cloned recently (see James et al., Plant Cell 7 (1995), 417-429). In the case of the agriculturally significant starch storing cultured plant potato, the activity of a debranching enzyme was examined by Hobson et al. (J. Chem. Soc., (1951), 1451). It was proven that the respective enzyme, contrary to the Q enzyme, does not exhibit any activities leading to an elongation of the polysaccharide chain, but merely hydrolyses .alpha.-1,6-glycosidic bonds. Within the scope of the present invention a subset of genes is provided that encode polypeptides the activity of which is associated with the structural shaping of the starch granule. In particular, the invention provides a subset of genes that encode polypeptides the activity of which is associated the branching/debranching (representative examples of wich are given in SEQ ID NOs: 69-73/75; 77 (isoamylase debranching enzyme)) and/or degradation of starch (a-amylase (SEQ ID NO: 79-91), pullulanase (SEQ ID NO: 109) [the last gene in the a-amylase series], a-amylase inhibitor (SEQ ID NOs: 93-99); β-amylase (SEQ ID NO101-107), a-glucosidase (SEQ ID NO: 111-117). By modulating the expression of the polypeptides according to the invention, the amylose amylopectin ratio can be changed in order to accommodate the varying quality standards for food and/or feed applications or specific processing requirements. For example, by over-expressing and inhibiting the expression of endogeneous branching and/or debranching enzyme genes in rice or any other cereal crop plant, respectively, a plant can be produced that exhibits increased or reduced amounts of branching/debranching enzyme activity for the purpose of modifying the degree of branching of the amylopectin starch. By inhibiting the expression of endogeneous branching and/or debranching enzyme genes, plants are produced that exhibit a reduced activity of these enzymes, which leads to the synthesis of a modified starch. Inhibition of branching/debranching gene expression can be achieved by applying method known in the art such as, for example, antisense or dsRNAi techniques. By applying these techniques it is possible to produce plants in which the expression of an endogeneous branching/debranching enzyme gene in rice or any other cereal crop plant is inhibited to different degrees within the range of 0.1% to 100%, which all individual numbers within this range also being part of the invention. This enables in particular the production of cereal plants synthesizing amylopectin starch with most various variations of the degree of branching. This constitutes an advantage with regard to conventional breeding and mutagenesis techniques in which a lot of time and costs are required in order to provide such a variety. Highly branched amylopectin has a particularly large surface and is therefore particularly suitable as a copolymer. A high degree of branching furthermore leads to an improvement of the amylopectin's solubility in water. This property is very advantageous for certain technical applications. Another way of modifying the branching characteristics of starch is by overexpressing the nucleic acid molecule according to the invention encoding a branching/debranching enzyme activity in rice in a transgenic plant, but especially a plant seed. The expression of a novel or additional branching/debranching enzyme activity from rice in the transgenic plant cells and plants of the invention influences the degree of branching of the amylopectin synthesized in the cells and plants. Therefore, a starch synthesized in these plants exhibits modified physical and/or chemical properties when compared to starch from wildtype plants. Genes encoding products involved in starch structure rearrangement (debranching enzyme is (SEQ ID NO: 75-77 (isoamylase debranching enzyme)); branching enzyme (SEQ ID NOs: 69-73)) and starch degradation (a-amylase (SEQ ID NOs 79-91), a-amylase inhibitor (SEQ ID NOs: 93-99); pullulanase (SEQ ID NOs 109) [the last gene in the a-amylase series], β-amylase (SEQ ID NOs 101-107), a-glucosidase (SEQ ID NOs 111-117)) are all strongly expressed towards the end of grain development, reflecting their involvement in the final stages of shaping the starch granule. Genes encoding isoforms of an a-amylase inhibitor (SEQ ID NOs: 93 and 95) are expressed most strongly in the aleurone and seed coat layers, and endosperm and not (or to a reduced extent) in the embryo. The embryo also shows a different expression of genes encoding starch synthase and branching enzymes, perhaps reflecting its status as an energy-requiring sink organ rather than as a storage tissue. Myers et al. discuss the interaction of starch synthases, branching enzymes, debranching enzymes and disproportionating enzymes in producing and trimming glucan molecules so that a final transition may take place to a crystalline form (A. M. Myers, M. K. Morell, M. G. James, S. G. Ball. Plant Physiol. 122, 989 (2000)). In a further embodiment, the present invention provides the ability to modulate the shape and the physico/chemical properties of the starch granule by modifying expression level and pattern of those genes that encode products involved in starch structure rearrangement such as, for example, SEQ ID NO: 75-77 (isoanylase debranching enzyme); branching enzyme (SEQ ID NOs: 69-73) and starch degradation (a-amylase (SEQ ID NOs 79-91)), a-amylase inhibitor (SEQ ID NOs: 93-99); pullulanase (SEQ ID NO: 109), β-amylase (SEQ ID NO: 101-107), and/or a-glucosidase (SEQ ID NO: 111-117). The invention thus also relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide involved in starch structure rearrangement, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 75-77 exhibiting isoamylase debranching enzyme activity, 69-73 exhibiting a branching enzyme activity, 80-92 exhibiting an a-amylase activity; 94-100 exhibiting an a-amylase inhibitor activity; 110 exhibiting a pullulanase activity; 102-108, exhibiting a β-amylase activity; 112-118, exhibiting a a-glucosidase activity. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which is involved in starch structure rearrangement and up-regulated during grain filling and has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 75-77 exhibiting isoamylase debranching enzyme activity, 69-73 exhibiting a branching enzyme activity, 80-92, 80-92 exhibiting an a-amylase activity; 94-100 exhibiting an a-amylase inhibitor activity; 110 exhibiting a pullulanase activity; 102-108, exhibiting a β-amylase activity; 112-118, exhibiting a a-glucosidase activity with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which is involved in starch structure rearrangement and up-regulated during grain filling and immunologically reactive with antibodies raised against a polypeptide as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 75-77 exhibiting isoamylase debranching enzyme activity, 69-73 exhibiting a branching enzyme activity, 80-92, 80-92 exhibiting an a-amylase activity; 94-100 exhibiting an a-amylase inhibitor activity; 110 exhibiting a pullulanase activity; 102-108, exhibiting a β-amylase activity; 112-118, exhibiting a a-glucosidase activity. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 75-77 exhibiting isoamylase debranching enzyme activity, 69-73 exhibiting a branching enzyme activity, 79-91 exhibiting an a-amylase activity; 93-99 exhibiting an a-amylase inhibitor activity; 109 exhibiting a pullulanase activity; 101-107, exhibiting a 6-amylase activity; 111-117, exhibiting a a-glucosidase activity or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the fill-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 75-77 exhibiting isoamylase debranching enzyme activity; 69-73 exhibiting a branching enzyme activity, 79-91 exhibiting an a-amylase activity; 93-99 exhibiting an a-amylase inhibitor activity; 109 exhibiting a pullulanase activity; 101-107, exhibiting a β-amylase activity; 111-117, exhibiting a a-glucosidase activity, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). The identification of a defined subset of genes that are involved in carbohydrate metabolism but especially in starch metabolism and the expression of which is coordinately up- or down-regulated during the grain filling process makes it now possible to improve grain quality by overexpressing and/or underexpressing or completely knocking out genes that are known to positively contribute to the nutritional or processing properties of grains such as, for example, genes encoding products involved in starch structure rearrangement and starch degradation as mentioned hereinbefore. The expression of a-amylase, which is central in the starch biosynthesis pathway, may further be modified to obtain plants producing a desirable content of reducing sugars. For, example, a high content of reducing sugar resulting from a high α-amylase activity is desirable when rice or other cereal plants are to be used for the production of alcohol. This can be achieved by modifying the expression of the plant endogenous genes encoding an α-amylase or α-amylase inhibitor activity, for example, by introducing and overexpressing in a target plant a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide the amino acid sequence of which is substantially similar to any one of those given in SEQ ID NOs: 80-92 exhibiting an a-amylase activity; and 94-100 exhibiting an a-amylase inhibitor activity. In the specific embodiment, the invention thus also relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide exhibiting an amylase or an amylase inhibitor activity, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 80-92 exhibiting an a-amylase activity; and 94-100 exhibiting an a-amylase inhibitor activity. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which has an activity of an amylase and is up-regulated during grain filling and has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 80-92 exhibiting an a-amylase activity; and 94-100 exhibiting an a-amylase inhibitor activity, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which which has an activity of an amylase and is up-regulated during grain filling and immunologically reactive with antibodies raised against a polypeptide as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 80-92 exhibiting an a-amylase activity; and 94-100 exhibiting an a-amylase inhibitor activity. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 79-91 exhibiting an a-amylase activity; and 93-99 exhibiting an a-amylase inhibitor activity or a part thereof which still encodes a partial length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence as given in the SEQ ID NOs of table 7 such as SEQ ID NOs: 79-91 exhibiting an a-amylase activity; and 93-99 exhibiting an a-amylase inhibitor activity or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). Different isoforms often show distinct spatial expression patterns. For example, three different sucrose synthase isoforms (SEQ ID NOs: 119-123) are expressed in developing grain tissue, two of which (SEQ ID NOs: 121 and 123) are expressed more highly at the start of grain development (0 days post anthesis) and one (SEQ ID NO: 119) which is up-regulated towards the end of grain development. The spatial distribution of each differs. Other isoforms (SEQ ID NOs: 125 and 127), showing low expression in the grain, are expressed strongly in stems or roots. The invention thus also relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide exhibiting a sucrose synthase activity, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 120-128. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which has an activity of an sucrose synthase and is up-regulated during grain filling and has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide of SEQ ID NOs: 120-128, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which which has an activity of a sucrose synthase and is up-regulated during grain filling and immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 120-128. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 119-127 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NOs: 119-127 or the complement thereof, e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). In a further embodiment, the present invention provides the ability to regulate glucanases (as represented by SEQ ID NO: 191). Glucanases can be used to minimize wet droppings in high wheat, or barley, poultry and swine diets by breaking down and reducing the viscosity of β-glucans and other non-starch polysaccharides and thus can provide benefit as a processing aid in animal feed. For uses and application of modifying crop plants by creating transgenic monocots and monocot seeds expressing rice β-glucanase enzymes and genes we incorporate all relevant section of PCT Publication WO9859046 to Rodriguez. The invention thus also relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide exhibiting a glucanase activity, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 192. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which has an activity of an glucanase and is up-regulated during grain filling and has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide of SEQ ID NOs: 192, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide which which has an activity of a glucanase and is up-regulated during grain filling and immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 192. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in SEQ ID NO: 191 or a part thereof which still encodes a partial length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof, d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides given in SEQ ID NO: 191 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). Thus, in an embodiment applicable to all of the above stated provisions, the present invention provides nucleotide sequences encoding at least one polypeptide involved in the synthesis, metabolism, transport or storage of carbohydrates, as well as any polypeptides encoded thereby, or any antigene sequences thereof, which have numerous applications using techniques that are known to those skilled in the art of molecular biology, biotechnology, biochemistry, genetics, physiology or pathology. These techniques include the use of nucleotide molecules as hybridization probes, for chromosome and gene mapping, in PCR technologies, in the production of sense or antisense nucleic acids, in screening for new therapeutic molecules, in production of plants and seeds having desirable, inheritable, commercially useful phenotypes, or in discovery of inhibitory compounds. In a further collective embodiment, the present invention provides the ability to modulate carbohydrates, sugars and their transporters in plant tissues, by over-expressing, under-expressing or knocking out one or more cell cycle genes or their gene products, in a plant cell, in vitro or in planta. Expression vectors comprising at least one nucleotide sequence involved in carbohydrate or sugar synthesis, metabolism, transport or storage, or any antigenes thereof, operably linked to at least one suitable promoter and/or regulatory sequence can be used to study the role of polypeptides encoded by said sequences, for example by transforming a host cell with said expression vector and measuring the effects of overexpression and underexpression of sequences. A host cell transformed with at least one expression vector comprising nucleotide sequences involved in carbohydrate modulation, operably linked to suitable promoters and/or regulatory sequences, can be useful to produce a dietary supplement comprising a polypeptide having a defined amino acid profile. In a further collective embodiment, the present invention provides a transformed plant host cell, or one obtained through breeding, capable of over-expressing, under-expressing, or having a knock out of said metabolic genes and/or their gene products. Such a plant cell, transformed with at least one expression vector comprising nucleotide sequences involved in carbohydrate synthesis, metabolism, transport or storage, operably linked to suitable promoters and/or regulatory sequences, can be used to regenerate plant tissue or an entire plant, or seed there from, in which the effects of expression, including overexpression or underexpression, of the introduced sequence or sequences can be measured in vitro or in planta. A further subset of genes provided herein comprises genes that encode polypeptides with an activity that is involved in or associated with the production of seed storage proteins. In seeds of higher plants, proteins are contained in an amount of 20-30% by weight in case of beans, and in an amount of about 10% by weight in case of cereals, based on dry weight. Among the proteins in seeds, 70-80% by weight are storage proteins. Particularly, in rice seeds, about 80% by weight of the seed storage proteins is glutelin which is only soluble in dilute acids and dilute alkalis. The remainders are prolamin (10-15% by weight) soluble in organic solvents and globulin (5-10% by weight) solubilized by salts. Seed storage proteins are important as a protein source in foods and feeds, so that they have been well studied from the view points of nutrition and protein chemistry. As a result, in cereals, storage protein genes of maize, wheat, barley and the like have been cloned, amino acid sequences of the proteins have been deduced from the nucleotide sequence, and regulatory regions of the genes have been analyzed. The present invention provides a subset of nucleic acid molecules that is up-regulated during grain filling and comprises a nucleotide sequence encoding a seed storage protein. Representative examples of these genes are given in SEQ ID NOs: 211-249. The invention thus also relates to a polynucleotide comprising a nucleotide sequence encoding a seed storage protein, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in any one of the SEQ ID NOs of table 8 such as SEQ ID NOs: 212-250. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a seed storage protein which is up-regulated during grain filling and has at least between to 70%, and 99% amino acid sequence identity to at least one polypeptide as given in any one of the SEQ ID NOs of table 8 such as SEQ ID NOs: 212-250, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a seed storage protein, which is up-regulated during grain filling and immunologically reactive with antibodies raised against a polypeptide as given in any one of the SEQ ID NOs of table 8 such as SEQ ID NOs: 212-250. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of the SEQ ID NOs of table 8 such as SEQ ID NOs: 211-249 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof, d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence as given in any one of the SEQ ID NOs of table 8 such as SEQ ID NOs: 211-249 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). By providing the above subset of genes, the protein content and composition in the plant grain can be modified by up- or down-regulating the expression of at least one nucleic acid molecule within this subgroup giving rise to altered levels or an altered composition of seed storage protein in the plant grain. For rice grains to be processed, it is advantageous that the protein content is small. In case of rice to be used for preparing fermented alcoholic beverage, this can be attained through well defined refinement measures, thereby removing the proteins in the peripheral portion of endosperm which contains large amounts of storage proteins. In producing rice starch, in order to promote the purity, proteins are removed by treatments with alkalis, surfactants and ultrasonication. The protein content in the rice grain also influences the taste of rice. Good tasting rice grains have usually low contents of proteins. Rice varieties with a low protein content have been developed by the conventional cross-breeding or by mutation-breeding. (U.S. Pat. No. 5,516,668; Maruta). U.S. Pat. No. 5,516,668 describes a method for decreasing the amount of glutelin in plant seeds, comprising introducing into a rice plant a gene which is a template for the transcription of an antisense RNA against rice glutelin; and transcribing said gene in seeds from said rice plant to inhibit translation of mRNA of glutelin, thereby decreasing the amount of glutelin in said seeds in comparison to the amount of glutelin contained in seeds from unmodified wild-type rice plants. The cDNA of glutelin which is a seed storage protein in rice has been cloned and complete primary structure of the protein has been determined by sequencing the cDNA. The gene of this protein has been isolated by using the cDNA as a probe (Japanese Laid-open Patent Application (Kokai) No. 63-91085). Rice plants with a low glutelin content in the rice grain can now be produced more efficiently by down-regulating two or more of the the endogenous glutelin genes in rice seeds such as those provided in SEQ ID NOs: 223, 235, and 239 using methods known in the art including antisense and dsRNAi techniques. The invention thus also relates to a polynucleotide comprising a nucleotide sequence encoding a glutelin protein the expression of which is up-regulated during grain filling, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 224, 236, and 240. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a glutelin protein the expression of which is up-regulated during grain filling and which has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide of SEQ ID NOs: 224, 236, and 240, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a seed glutelin protein, the expression of which is up-regulated during grain filling and which is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 224, 236, and 240. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 223,235, and 239 or a part thereof which still encodes a partial length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: 223, 235, and 239, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). Another class of seed storage proteins are the prolamins, which are naturally rich in the essential amino acids lysine and methionine. Overexpressing said genes can thus increase the nutritional value of feeds and foods by producing said proteins at higher levels than those found in the unmodified wild-type plants. Another aspect of the present invention thus relates to providing genes that encode rice prolamin protein such as those given in SEQ ID NOs: 217, 219, 225 and 241. The invention thus also relates to a polynucleotide comprising a nucleotide sequence encoding a prolamin protein the expression of which is up-regulated during grain filling, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NOs: 218, 220, 226 and 242. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a prolamin protein, the expression of which is up-regulated during grain filling and which has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide of SEQ ID NOs: 218, 220, 226 and 242, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a prolamin protein, the expression of which is up-regulated during grain filling and which is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 218, 220, 226 and 242. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 217, 219, 225 and 241 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: 217, 219, 225 and 241, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). Gliadins are a further group of seed storage proteins that are of economic importance. Gliadin is a single-chained protein having an average molecular weight of about 30,000-40,000, with an isoelectric of pH 4.0-5.0. Gliadin proteins are extremely sticky when hydrated and have little or no resistance to extension. Gliadin is responsible for giving gluten dough its characteristic cohesiveness. Gliadin is a premium products, when available. Gliadin is known to improve the freeze-thaw stability of frozen dough and also improves microwave stability. This product is also used as an all-natural chewing gum base replacer, a pharmaceutical binder, and improves the texture and mouth feel of pasta products and has been found to improve cosmetic products. The invention provides a further subset of genes comprising a nucleotide sequence that encodes gliadin storage proteins. By overexpressing said genes in the plant, but preferably in the plant seed, the plant produces grain with an increased concentration of gliadin as compared to the unmodified wild-type plant. In a particular embodiment, the invention thus relates to a polynucleotide comprising a nucleotide sequence encoding a gliadin protein, the expression of which is up-regulated during grain filling, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NOs: 212, 219; 234, 248; and 250. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a gliadin protein, the expression of which is up-regulated during grain filling and which has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide of SEQ ID NOs: 212, 219; 234, 248; and 250, with any individual number within this range of between 700/o and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a seed gliadin protein, the expression of which is up-regulated during grain filling and which is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 212, 219; 234, 248; and 250. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence g) as given in any one of SEQ ID NOs: 211, 220; 233, 247; and 249 or a part thereof which still encodes a partial length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; h) having substantial similarity to (a); i) capable of hybridizing to (a) or the complement thereof; j) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: 211, 220; 233, 247; and 249, or the complement thereof; k) complementary to (a), (b) or (c); and l) which is the reverse complement of (a), (b) or (c). In a further embodiment the invention provides a subset of genes which encode polypeptides that are involved in or associated with the metabolism of fatty acids in the rice grain. Seed oil content has traditionally been modified by plant breeding. The use of recombinant DNA technology to alter seed oil composition can accelerate this process and in some cases alter seed oils in a way that cannot be accomplished by breeding alone. The oil composition of Brassica has been significantly altered by modifying the expression of a number of lipid metabolism genes. Such manipulations of seed oil composition have focused on altering the proportion of endogenous component fatty acids. For example, antisense repression of the .DELTA.12-desaturase gene in transgenic rapeseed has resulted in an increase in oleic acid of up to 83%. (Topfer et al. 1995 Science 268:681-686). There have been some successful attempts at modifying the composition of seed oil in transgenic plants by introducing new genes that allow the production of a fatty acid that the host plants were not previously capable of synthesizing. Van de Loo, et al. (1995 Proc. Natl. Acad. Sci USA 92:6743-6747) have been able to introduce a .DELTA.12-hydroxylase gene into transgenic tobacco, resulting in the introduction of a novel fatty acid, ricinoleic acid, into its seed oil. The reported accumulation was modest from plants carrying constructs in which transcription of the hydroxylase gene was under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Similarly, tobacco plants have been engineered to produce low levels of petroselinic acid by expression of an acyl-ACP desaturase from coriander (Cahoon et al. 1992 Proc. Natl. Acad. Sci USA 89:11184-11188). The long chain fatty acids (C18 and larger), have significant economic value both as nutritionally and medically important foods and as industrial commodities (Ohlrogge, J. B. 1994 Plant Physiol. 104:821-826). Linoleic (18:2.DELTA.9,12) and alpha.-linolenic acid (18:3 .DELTA.9,12,15) are essential fatty acids found in many seed oils. The levels of these fatty-acids have been manipulated in oil seed crops through breeding and biotechnology (Ohlrogge, et al. 1991 Biochim. Biophys. Acta 1082:1-26; Topfer et al. 1995 Science 268:681-686). Additionally, the production of novel fatty acids in seed oils can be of considerable use in both human health and industrial applications. Consumption of plant oils rich in .gamma.-linolenic acid (GLA) (18:3.DELTA.6,9,12) is thought to alleviate hypercholesterolemia and other related clinical disorders which correlate with susceptibility to coronary heart disease (Brenner R. R. 1976 Adv. Exp. Med. Biol. 83:85-101). The therapeutic benefits of dietary GLA may result from its role as a precursor to prostaglandin synthesis (Weete, J. D. 1980 in Lipid Biochemistry of Fungi and Other Organisms, eds. Plenum Press, New York, pp. 59-62). Linoleic acid(18:2) (LA) is transformed into gamma linolenic acid (18:3) (GLA) by the enzyme .DELTA.6-desaturase. Few seed oils contain GLA despite high contents of the precursor linoleic acid. This is due to the absence of .DELTA.6-desaturase activity in most plants. For example, only borage ( Borago officinalis ), evening primrose ( Oenothera biennis ), and currants ( Ribes nigrum ) produce appreciable amounts of linolenic acid. Of these three species, only Oenothera and Borage are cultivated as a commercial source for GLA. It would be beneficial if agronomic seed oils could be engineered to produce GLA in significant quantities by introducing a heterologous .DELTA.6-desaturase gene. It would also be beneficial if other expression products associated with fatty acid synthesis and lipid metabolism could be produced in plants at high enough levels so that commercial production of a particular expression product becomes feasible. As disclosed in U.S. Pat. No. 5,552,306, a cyanobacterial .DELTA.sup.6-desaturase gene has been recently isolated. Expression of this cyanobacterial gene in transgenic tobacco resulted in significant but low level GLA accumulation. (Reddy et al. 1996 Nature Biotech. 14:639-642). The present invention now provides a subset of genes encoding polypeptides that are involved in or associated with fatty acid metabolism, the expression of which is up-regulated during grain filling. In particular, the invention relates to a polynucleotide the expression of which is up-regulated during grain filling comprising a nucleotide sequence encoding a polypeptide that is involved in or associated with fatty acid synthesis or lipid metabolism, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in any one of the SEQ ID NOs of table 9 such as SEQ ID NOs: 252-280. More specifically, the invention relates to a polynucleotide the expression of which is up-regulated during grain filling comprising a nucleotide sequence encoding a polypeptide that is involved in or associated with fatty acid synthesis or lipid metabolism and has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide as given in any one of the SEQ ID NOs of table 9 such as SEQ ID NOs: 252-280, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide the expression of which is up-regulated during grain filling comprising a nucleotide sequence encoding a polypeptide that is involved in or associated with fatty acid synthesis or lipid metabolism and immunologically reactive with antibodies raised against a polypeptide as given in any one of the SEQ ID NOs of table 9 such as SEQ ID NOs: 252-280. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of the SEQ ID NOs of table 9 such as SEQ ID NOs: 251-279 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides as given in any one of the SEQ ID NOs of table 9 such as SEQ ID NOs: 251-279 or the complement thereof, e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). By providing this subset of genes it is now possible to modify the level and composition of grain lipids by modulating the expression of those genes in the plant seed. Expression can be modulated either by introducing at least one of the nucleic acid molecules from this subset into the plant, preferably under control of a seed specific promoter, and overexpressing said at least one to nucleic acid molecule in the plant seed, or, by down-regulating expression of the corresponding endogenous gene applying techniques know in the art including anti sense and dsRNAi techniques. In a specific embodiment, the invention relates to a subset of genes encoding oleosins as represented by SEQ ID NOs: 257 and 259. Oleosins are abundant seed proteins associated with the phospholipid monolayer membrane of oil bodies, which are a means for storing lipids in the plant cell. Analysis of the contents of lipid bodies has demonstrated that in addition to triglyceride and membrane lipids, there are also several polypeptides/proteins associated with the surface or lumen of the oil body (Bowman-Vance and Huang, 1987, J. Biol. Chem., 262:11275-11279, Murphy et al., 1989, Biochem. J., 258:285-293, Taylor et al., 1990, Planta, 181:18-26). Oil-body proteins have been identified in a wide range of taxonomically diverse species (Moreau et al., 1980, Plant Physiol., 65:1176-1180; Qu et al., 1986, Biochem. J., 235:57-65) and have been shown to be uniquely localized in oil-bodies and not found in organelles of vegetative tissues. In Brassica napus (rapeseed, canola) there are at least three polypeptides associated with the oil-bodies of developing seeds (Taylor et al., 1990, Planta, 181:18-26). One of the most abundant proteins associated with the phospholipid monolayer membrane of oil bodies are the oleosins. The first oleosin gene, L3, was cloned from maize by selecting clones whose in vitro translated products were recognized by an anti-L3 antibody (Vance et al. 1987 J. Biol. Chem. 262:11275-11279). Subsequently, different isoforms of oleosin genes from such different species as Brassica , soybean, carrot, pine, and Arabidopsis have been cloned (Huang, A. H. C., 1992, Ann. Reviews Plant Phys. and Plant Mol. Biol. 43:177-200; Kirik et al., 1996 Plant Mol. Biol. 31:413-417; Van Rooijen et al., 1992 Plant Mol. Biol. 18:1177-1179; Zou et al., Plant Mol. Biol. 31:429-433. Oleosin protein sequences predicted from these genes are highly conserved, especially for the central hydrophobic domain. All of these oleosins have the characteristic feature of three distinctive domains. An amphipathic domain of 40-60 amino acids is present at the N-terminus; a totally hydrophobic domain of 68-74 amino acids is located at the center; and an amphipathic .alpha.-helical domain of 33-40 amino acids is situated at the C-terminus (Huang, A. H. C. 1992). A maize oleosin has been expressed in seed oil bodies in Brassica napus transformed with a Zea mays oleosin gene. The gene was expressed under the control of regulatory elements from a Brassica gene encoding napin, a major seed storage protein. The temporal regulation and tissue specificity of expression was reported to be correct for a napin gene promoter/terminator (Lee et al., 1991, Proc. Natl. Acad. Sci. U.S.A., 88:6181-6185). By providing a subset of genes encoding oleosins, it is now possible to modify the oleosin content in the phospholipid monolayer membrane of oil bodies by either introducing the genes provided herein into a plant and overexpressing said gene in said plant or, in the alternative, by down-regulating expression of the endogenous oleosin encoding genes in the plant using method known in the art including anti-sense or dsRNAi techniques. In one specific embodiment, the present invention thus relates to a polynucleotide comprising a nucleotide sequence encoding an oleosin protein, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NOs: 258 and 260. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding an oleosin protein, which is up-regulated during grain filling and has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide of SEQ ID NOs: 258 and 260, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding an oleosin protein, which is up-regulated during grain filling and immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 258 and 260. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 257 and 259 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: 257 and 259, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). At least one of the genes provided herein, which is up-regulated during grain filling, encodes a phytoene dehydrogenase polypeptide that is involved in carotenoid biosynthesis and can thus be used to modify caroteinoid production in grain. Carotenoids are natural pigments that are essential to microbial, plant, and animal life. In photosynthetic organisms, they act as potent antioxidants that negate the lethal effects of singlet oxygen and superoxide formed during oxygen production. As human dietary constituents, these lipophilic antioxidants provide our cells with chemical protectants against the damaging effects of oxidation. Acting as chemical scavengers, carotenoids play roles in the prevention of cancer and chronic maladies, including heart disease. Phytoene (7,8,11,12,7′,8′,11′,12′-.omega. octahydro-.omega., omega.-carotene) is the first carotenoid in the carotenoid biosynthesis pathway and is produced by the dimerization of a 20-carbon atom precursor, geranylgeranyl pyrophosphate (GGPP). Phytoene has useful applications in treating skin disorders (U.S. Pat. No. 4,642,318) and is itself a precursor for colored carotenoids. Aside from certain mutant organisms, such as Phycomyces blakesleeanus carB, no current methods are available for producing phytoene via any biological process. In some organisms, the red carotenoid lycopene (.omega.,.omega.-carotene) is the next carotenoid produced in the phytoene in the pathway. Lycopene imparts the characteristic red color to ripe tomatoes. Lycopene has utility as a food colorant. It is also an intermediate in the biosynthesis of other carotenoids in some bacteria, fungi and green plants. Lycopene is prepared biosynthetically from phytoene through four sequential dehydrogenation reactions by the removal of eight atoms of hydrogen. The enzymes that remove hydrogen from phytoene are phytoene dehydrogenases. One or more phytoene dehydrogenases can be used to convert phytoene to lycopene and dehydrogenated derivatives of phytoene intermediate to lycopene are also known. For example, some strains of Rhodobacter sphaeroides contain a phytoene dehydrogenase that removes six atoms of hydrogen from phytoene to produce neurosporene. Lycopene is an intermediate in the biosynthesis of caaotenoids in some bacteria, fungi, and all green plants. Carotenoid-specific genes that can be used for synthesis of lycopene from the ubiquitous precursor farnesyl pyrophosphate include those for the enzymes GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H. In one specific embodiment the present invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in or associated with the dehydrogenation of phytoene and the expression of which is up-regulated during grain filling, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in SEQ ID NO: 278. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in or associated with the dehydrogenation of phytoene and the expression of which is up-regulated during grain filling and which has at least between 70%, and 99/o amino acid sequence identity to at least one polypeptide of SEQ ID NOs: 278, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in or associated with the dehydrogenation of phytoene and the expression of which is up-regulated during grain filling and which is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 278. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of SEQ ID NOs: 277 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in any one of SEQ ID NOs: 277, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). Another subset of genes that is provided as part of the invention comprises nucleic acid molecules that are involved in the transcriptional control of the highly coordinated grain filling process. Transcription factors are proteins that bind to the enhancer or promoter regions and interact such that transcription occurs from only a small group of promoters in any cell. Most transcription factors can bind to specific DNA sequences, and these trans-regulatory proteins can be grouped together in families based on similarities in structure. Within such a family, proteins share a common framework structure in their respective DNA-binding sites, and slight differences in the amino acids at the binding site can alter the sequence of the DNA to which it binds. In addition to having this sequence-specific DNA-binding domain, transcription factors contain a domain involved in activating the transcription of the gene whose promoter or enhancer it has bound. Usually, this trans-activating domain enables that transcription factor to interact with proteins involved in binding RNA polymerase. This interaction often enhances the efficiency with which the basal transcriptional complex can be built and bind RNA polymerase E. There are several families of transcription factors, and those discussed here are just some of the main types. The gene subset provided herein includes a gene which encodes a polypeptide that is similar to the CREB-binding protein from Mus sp (as represented by SEQ ID NO: 301), and is highly expressed in aleurone and endosperm tissues during grain filling. CREB-binding protein (CBP) is a necessary component of the CREB/PKA paradigm of gene regulation. The acetylation of histones and other proteins has been linked to gene regulation, and CBP has a potent intrinsic acetyltransferase (AT) enzymatic domain. CREB belongs to a class of proteins whose phosphorylation appears specifically to enhance their trans-activation potential (Arias J, et al Nature 1994 Jul. 21;370(6486):226-9). CBP possesses intrinsic histone acetyltransferase activity, and can acetylate not only histones but also certain transcriptional factors such as GATA1; p53 and also myb-type transcription factors such as c-Myb (Yuji Sano and Shunsuke Ishii J. Biol. Chem., Vol. 276, Issue 5, 3674-3682, Feb. 2, 2001). Acetylation of c-Myb by CBP increases the trans-activating capacity of c-Myb by enhancing its association with CBP. These results demonstrate a novel molecular mechanism of regulation of c-Myb activity. In rice, 70 known and putative MYB genes could be identified, some of which show interesting expression patterns such as those given in SEQ ID NOs: 311-321. The expression pattern of these transcription factors suggests that they play a key role during rice grain filling. Another transcription factor gene (as represented by SEQ ID NOs: 305) included in this subset encodes a protein that has structural similarity to the yeast HAP5 transcriptional activator protein. In yeast, the HAP5 protein is a component of the HAP (Hap2p-Hap3p-Hap4pHap5p) CCAAT-box-binding transcriptional activation complex and is essential for the binding activity of the complex. A further transcription factor gene within this subset is represented by SEQ ID NO: 307 which encodes a bZIP-type transcription factor similar to the plant G-box binding factor GBF4, that was found in Arabidopsis . GBF4, in a manner reminiscent of the Fos-related oncoproteins of mammalian systems, cannot bind to DNA as a homodimer, although it contains a basic region capable of specifically recognizing the G-box and G-box-like elements. However, GBF4 can interact with GBF2 and GBF3 to bind DNA as heterodimers. Mutagenesis of the leucine zipper of GBF4 indicates that the mutation of a single amino acid confers upon the protein the ability to recognize the G-box as a homodimer, apparently by altering the charge distribution within the leucine zipper (A E Menkens and A R Cashmore (1994) PNAS 91: 2522-2526). Another of the transcription factor genes within this subset encodes a protein that has a zinc finger domain and is similar to a zinc-finger type transcription factor found in Arabidopsis (gi|6899934). Zinc finger proteins include WT-1 (a important transcription factor critical in the formation of the kidney and gonads); the ubiquitous transcription factor Sp1; Xenopus 5S rRNA transcription factor TFIIIA; Krox 20 (a protein that regulates gene expression in the developing hindbrain); Egr-1 (which commits white blood cell development to the macrophage lineage); Krippel (a protein that specifes abdominal cells in Drosophila ); and numerous steroid-binding transcription factors. Each of these proteins has two or more “DNA-binding fingers,” a-helical domains whose central amino acids tend to be basic. These domains are linked together in tandem and are each stabilized by a centrally located zinc ion coordinated by two cysteines (at the base of the helix) and two internal histidines. The crystal structure shows that the zinc fingers bind in the major groove of the DNA. The expression pattern of these transcription factors during grain filling suggests that they play a key role during rice grain development. This is further supported by the fact that the AACA promoter element, which is known to be conserved in many seed storage protein genes, is over-represented in the promoters of the grain filling sub-set genes according to the invention. This subset comprises genes the protein products of which are involved in diverse cellular functions, including carbohydrate, protein and fatty acid metabolism, nutrient transportation, and transcription and translation. The ACCA promoter element was thus demonstrated to be likely one of the key elements in the coordination of different major pathways during grain development. In one embodiment the invention thus relates to a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that acts as a transcription factor and the expression of which is up-regulates during grain filling, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in any one of the SEQ ID NOs of table 11 such as SEQ ID NOs: 302-328. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encodes a polypeptide that acts as a transcription factor and the expression of which is up-regulated during grain filling and which has at least between 700%, and 99% amino acid sequence identity to at least one polypeptide as given in any one of the SEQ ID NOs of table 11 such as SEQ ID NOs: 302-328, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encodes a polypeptide that acts as a transcription factor and the expression of which is up-regulated during grain filling and which is immunologically reactive with antibodies raised against a polypeptide as given in any one of the SEQ ID NOs of table 11 such as SEQ ID NOs: 302-328. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of the SEQ ID NOs of table 11 such as SEQ ID NOs: 301-327 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence as given in any one of the SEQ ID NOs of table 11 such as SEQ ID NOs: 301-327, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). By changing the expression level and/or pattern of at least one transcription factor as provided herein, which is involved in the regulation and coordination of grain filling in plants, it is possible to modify the grain filling process to obtain grain with a modified nutritional composition and/or quality characteristics. A further subset of genes which is provided herein comprises genes encoding polypeptides the activity of which is involved in or associated with amino acid metabolism. In particular, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide the activity of which is involved or associated with the metabolism of amino acids and the expression of which is up-regulated during grain filling, which nucleotide sequence is substantially similar to a nucleic acid sequence encoding a polypeptide as given in any one of the SEQ ID NOs of table 10 such as SEQ ID NOs: 282-300. More specifically, the invention relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide the activity of which is involved or associated with the metabolism of amino acids and the expression of which is up-regulated during grain filling, which polypeptide has at least between 70%, and 99% amino acid sequence identity to at least one polypeptide as given in any one of the SEQ ID NOs of table 10 such as SEQ ID NOs: 282-300, with any individual number within this range of between 70% and 99% also being part of the invention. The invention further relates to a polynucleotide comprising a nucleotide sequence encoding a polypeptide the activity of which is involved or associated with the metabolism of amino acids and the expression of which is up-regulated during grain filling, which polypeptide is immunologically reactive with antibodies raised against a polypeptide as given in any one of the SEQ ID NOs of table 10 such as SEQ ID NOs: 282-300. More particularly, the invention relates to a polynucleotide comprising a nucleotide sequence a) as given in any one of the SEQ ID NOs of table 10 such as SEQ ID NOs: 281-299 or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence as given in any one of the SEQ ID NOs of table 10 such as SEQ ID NOs: 281-299, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c). In a final embodiment, the present invention provides a subset of genes encoding polypeptides for which no biological function is known so far. It is within the scope of this invention, that the expression products of these genes, respresentative examples of which are provided in column B of table 3, can for the first time be associated with a biological function. Based on their mRNA expression characteristics and their specific expression pattern during grain filling it is suggested that they are involved in or associated with nutrient partitioning during the grain filling process. By modifying the expression of at least one of the genes within this subgroup it is, therefore, possible to modify the compositional characteristics and thus the nutritional properties of the plant grain. The present invention provides a set of genes, which were shown to be preferentially up-regulated and to share a similar expression pattern during the process of grain filling as specified hereinbefore. The genes within this subgroup are useful tools for generating plants which produce grain with modified compositional characteristics leading to improved nutritional properties. According to one embodiment, the present invention is directed to a nucleic acid molecule comprising a nucleotide sequence isolated or obtained from any plant which encodes a polypeptide that has at least 70% amino acid sequence identity to a polypeptide encoded by a gene comprising any one of SEQ ID NOs provided in the Sequence Listing. Based on the Oryza nucleic acid sequences of the present invention as given in the SEQ ID NOs of the Sequence Listing, orthologs may be identified or isolated from the genome of any desired organism, preferably from another plant, according to well known techniques based on their sequence similarity to the Orya nucleic acid sequences, e.g., hybridization, PCR or computer generated sequence comparisons. For example, all or a portion of a particular Oryza nucleic acid sequence is used as a probe that selectively hybridizes to other gene sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen source organism. Further, suitable genomic and cDNA libraries may be prepared from any cell or tissue of an organism. Such techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g., Sambrook et al., 1989) and amplification by PCR using oligonucleotide primers preferably corresponding to sequence domains conserved among related polypeptide or subsequences of the nucleotide sequences provided herein (see, e.g., Innis et al., 1990). These methods are particularly well suited to the isolation of gene sequences from organisms closely related to the organism from which the probe sequence is derived. The application of these methods using the Oryza sequences as probes is well suited for the isolation of gene sequences from any source organism, preferably other plant species. In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker. Thus, for example, probes for hybridization can be made by is labeling synthetic oligonucleotides based on the sequence of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989). In general, sequences that hybridize to the sequences disclosed herein will have at least 40% to 50%, about 60% to 70% and even about 80% 85%, 90%, 95% to 98% or more identity with the disclosed sequences. That is, the sequence similarity of sequences may range, sharing at least about 40% to 50%, about 60% to 70%, and even about 80%, 85%, 900/0, 95% to 98% sequence similarity, with each individual number within the ranges given above also being part of the invention. The nucleic acid molecules of the invention can also be identified by, for example, a search of known databases for genes encoding polypeptides having a specified amino acid sequence identity or DNA having a specified nucleotide sequence identity. Methods of alignment of sequences for comparison are well known in the art and are described hereinabove. In a further embodiment, the invention provides isolated nucleic acid molecules comprising a plant nucleotide sequence that induces transcription of a linked nucleic acid segment in a plant or plant cell, e.g., a linked nucleic acid molecule comprising an open reading frame for or encoding a structural or regulatory gene, in a tissue specific or tissue preferential manner. In a specific embodiment, the invention provides isolated nucleic acid molecules comprising a plant nucleotide sequence that induces transcription of a linked nucleic acid segment in a plant or plant cell, e.g., a linked nucleic acid molecule comprising an open reading frame for or encoding a structural or regulatory gene, in a seed-specific or seed-preferential manner. In particular, the plant nucleotide sequence according to the invention is substantially less active in vegetative tissue as compared to seed and is most active in the endosperm. The transcription inducing activity icreases during seed development and reaches its peak at or around the time of grain filling. In particular, the nucleotide sequence of the invention directs seeds- (e.g. endosperm) specific or seeds- (e.g. endosperm) preferential transcription of a linked nucleic acid segment in a plant or plant cell and is preferably obtained or obtainable from plant genomic DNA having a gene comprising an open reading frame (ORF) encoding a polypeptide which is substantially similar, and preferably has at least 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%, amino acid sequence identity, to a polypeptide encoded by an Oryza , e.g., Oryza sativa , gene comprising any one of SEQ ID NOs: 2-462 (e.g., including a promoter obtained or obtainable from any one of SEQ ID NOs: 643-883) which directs seed-specific (or seed-preferential) transcription of a linked nucleic acid segment. The promoters of the invention include a consecutive stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500, contiguous nucleotides, e.g., 40 to about 750, 60 to about 750, 125 to about 750, 250 to about 750, 400 to about 750, 600 to about 750, of any one of SEQ ID NOs: 643-883, or the promoter orthologs thereof, which include the minimal promoter region. In a particular embodiment of the invention said consecutive stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500, contiguous nucleotides, e.g., 40 to about 750, 60 to about 750, 125 to about 750, 250 to about 750, 400 to about 750, 600 to about 750, has at least 75%, preferably 80%, more preferably 90% and most preferably 95%, nucleic acid sequence identity with a corresponding consecutive stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500, contiguous nucleotides, e.g., 40 to about 750, 60 to about 750, 125 to about 750, 250 to about 750, 400 to about 750, 600 to about 750, of any one of SEQ ID NOs: 643-883 or the promoter orthologs thereof, which include the minimal promoter region. The above defined stretch of contiguous nucleotides preferably comprises one or more promoter motifs, e.g., for seed-specific promoters, motifs selected from the group consisting of the P box and GCNA elements, including but not limited to TGTAAAG and TGA(G/C)TCA and a transcription start site. In case of promoters directing tissue-specific transcription of a linked nucleic acid segment in a plant or plant cell such as, for example, a promoter directing seed-specific or seed-preferential, but especially endosperm-specific or endosperm-preferential transcription, it is further preferred that previously defined stretch of contiguous nucleotides comprises further motifs that participate in the tissue specificity of said stretch(es) of nucleotides. Generally, the promoters of the invention may be employed to express a nucleic acid segment that is operably linked to said promoter such as, for example, an open reading frame, or a portion is thereof, an anti-sense sequence, or a transgene in plants. The open reading frame may be obtained from an insect resistance gene, a disease resistance gene such as, for example, a bacterial disease resistance gene, a fungal disease resistance gene, a viral disease resistance gene, a nematode disease resistance gene, a herbicide resistance gene, a gene affecting grain composition or quality, a nutrient utilization gene, a mycotoxin reduction gene, a male sterility gene, a selectable marker gene, a screenable marker gene, a negative selectable marker, a positive selectable marker, a gene affecting plant agronomic characteristics, i.e., yield, standability, and the like, or an environment or resistance gene, i.e., one or more genes that confer herbicide resistance or tolerance, insect resistance or tolerance, disease resistance or tolerance (viral, bacterial, fungal, oomycete, or nematode), stress tolerance or resistance (as exemplified by resistance or tolerance to drought, heat, chilling, fleezing, excessive moisture, salt stress, or oxidative stress), increased yields, food content and makeup, physical appearance, male sterility, drydown, standability, prolificacy, starch properties or quantity, oil quantity and quality, amino acid or protein composition, and the like. By “resistant” is meant a plant which exhibits substantially no phenotypic changes as a consequence of agent administration, infection with a pathogen, or exposure to stress. By “tolerant” is meant a plant which, although it may exhibit some phenotypic changes as a consequence of infection, does not have a substantially decreased reproductive capacity or substantially altered metabolism. For instance, seed-specific promoters may be useful for expressing genes as well as for producing large quantities of protein, for expressing oils or proteins of interest, e.g., antibodies, genes for increasing the nutritional value of the seed and the like. In particular, the seed-specific or seed-preferential promoters accroding to the invention such as those provided in SEQ ID NOs: 643-883 may be useful for expressing the Open Reading Frames which are represented by the nucleotide sequences of SEQ ID NOs: 1-461 and 501-511, respectively. Obtaining sufficient levels of transgene expression in the appropriate plant tissues is an important aspect in the production of genetically engineered crops. Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. It is specifically contemplated by the present invention that one could use any one of the promoters according to the present invention in unaltered or altered form. Mutagenization of a promoter of the present invention such as those provided in SEQ ID NOs: 643-883 may potentially improve the utility of the elements for the expression of transgenes in plants. The mutagenesis of these elements can be carried out at random and the mutagenized promoter sequences screened for activity in a trial-by-error procedure. Alternatively, particular sequences which provide the promoter with desirable expression characteristics, or the promoter with expression enhancement activity, could be identified and these or similar sequences introduced into the sequences via mutation. It is further contemplated that one could mutagenize these sequences in order to enhance their expression of transgenes in a particular species. The means for mutagenizing a DNA segment encoding a promoter sequence of the current invention are well-known to those of skill in the art. As indicated, modifications to promoter or other regulatory element may be made by random, or site-specific mutagenesis procedures. The promoter and other regulatory element may be modified by altering their structure through the addition or deletion of one or more nucleotides from the sequence which encodes the corresponding un-modified sequences. Mutagenesis may be performed in accordance with any of the techniques known in the art, such as, and not limited to, synthesizing an oligonucleotide having one or more mutations within the sequence of a particular regulatory region. In particular, site-specific mutagenesis is a technique useful in the preparation of promoter mutants, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered. In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids also are routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage. In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the promoter. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and cells are selected which include recombinant vectors bearing the mutated sequence arrangement. Vector DNA can then be isolated from these cells and used for plant transformation. A genetic selection scheme is devised by Kunkel et al. (1987) to enrich for clones incorporating mutagenic oligonucleotides. Alternatively, the use of PCR with commercially available thermostable enzymes such as Taq polymerase may be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an appropriate cloning or expression vector. The PCR-mediated mutagenesis procedures of Tomic et al. (1990) and Upender et al. (1995) provide two examples of such protocols. A PCR employing a thermostable ligase in addition to a thermostable polymerase also may be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment that may then be cloned into an appropriate cloning or expression vector. The mutagenesis procedure described by Michael (1994) provides an example of one such protocol. The preparation of sequence variants of the selected promoter-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of DNA sequences may be obtained. For example, recombinant vectors encoding the desired promoter sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. As used herein, the term “oligonucleotide directed mutagenesis procedure” refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term “oligonucleotide directed mutagenesis procedure” also is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template-dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson and Ramstad, 1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U.S. Pat. No. 4,237,224. A number of template dependent processes are available to amplify the target sequences of interest present in a sample, such methods being well known in the art and specifically disclosed herein below. Where a clone comprising a promoter has been isolated in accordance with the instant invention, one may wish to delimit the essential promoter regions within the clone. One efficient, targeted means for preparing mutagenizing promoters relies upon the identification of putative regulatory elements within the promoter sequence. This can be initiated by comparison with promoter sequences known to be expressed in similar tissue-specific or developmentally unique manner. Sequences which are shared among promoters with similar expression patterns are likely candidates for the binding of transcription factors and are thus likely elements which confer expression patterns. Confirmation of these putative regulatory elements can be achieved by deletion analysis of each putative regulatory region followed by functional analysis of each deletion construct by assay of a reporter gene which is functionally attached to each construct. As such, once a starting promoter sequence is provided, any of a number of different deletion mutants of the starting promoter could be readily prepared. As indicated above, deletion mutants, deletion mutants of the promoter of the invention also could be randomly prepared and then assayed. With this strategy, a series of constructs are prepared, each containing a different portion of the clone (a subclone), and these constructs are then screened for activity. A suitable means for screening for activity is to attach a deleted promoter or intron construct which contains a deleted segment to a selectable or screenable marker, and to isolate only those cells expressing the marker gene. In this way, a number of different, deleted promoter constructs are identified which still retain the desired, or even enhanced, activity. The smallest segment which is required for activity is thereby identified through comparison of the selected constructs. This segment may then be used for the construction of vectors for the expression of exogenous genes. Furthermore, it is contemplated that promoters combining elements from more than one promoter may be useful. For example, U.S. Pat. No. 5,491,288 discloses combining a Cauliflower Mosaic Virus promoter with a histone promoter. Thus, the elements from the promoters disclosed herein may be combined with elements from other promoters. The present invention further provides a composition, an expression cassette or a recombinant vector containing the nucleic acid molecule of the invention as discosed herinbefore, and host cells comprising the expression cassette or vector, e.g., comprising a plasmid. In particular, the present invention provides an expression cassette or a recombinant vector comprising a suitable promoter linked to a nucleic acid segment of the invention, representative examples of which are provided in the SEQ ID NOs of the Sequence Listing, which, when present in a plant, plant cell or plant tissue, results in transcription of the linked nucleic acid segment. Promoters which are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive (Odell et al., 1985), temporally regulated, spatially regulated, tissue-specific, and spatio-temporally regulated. Where expression in specific tissues or organs is desired, tissue-specific promoters may be used. In contrast, where gene expression in response to a stimulus is desired, inducible promoters are the regulatory elements of choice. Where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. Additional regulatory sequences upstream and/or downstream from the core promoter sequence may be included in expression constructs of transformation vectors to bring about varying levels of expression of heterologous nucleotide sequences in a transgenic plant. Suitable promoter and/or regulatory sequences further include those that are preferentially or specifically active in plant grain tissue such as, for example, the grain endosperm or the grain embryo. Further, the invention provides isolated polypeptides encoded by any one of the open reading frames of the invention, representative examples of which are provided in the SEQ ID NOs of the Sequence Listing, or a fragment thereof, which encodes a polypeptide which has substantially the same activity as the corresponding polypeptide encoded by an ORF given in the SEQ ID NOs of the Sequence Listing, or the orthologs thereof. Virtually any DNA composition may be used for delivery to recipient plant cells, e.g., monocotyledonous cells, to ultimately produce fertile transgenic plants in accordance with the present invention. For example, DNA segments or fragments in the form of vectors and plasmids, or linear DNA segments or fragments, in some instances containing only the DNA element to be expressed in the plant, and the like, may be employed. The construction of vectors which may be employed in conjunction with the present invention will be known to those of skill of the art in light of the present disclosure (see, e.g., Sambrook et al., 1989; Gelvin et al., 1990). It is one of the objects of the present invention to provide recombinant DNA molecules comprising a nucleotide sequence which directs transcription according to the invention operably linked to a nucleic acid segment or sequence of interest. The nucleic acid segment of interest can, for example, code for a ribosomal RNA, an antisense RNA or any other type of RNA that is not translated into protein. In another preferred embodiment of the invention, the nucleic acid segment of interest is translated into a protein product. The nucleotide sequence which directs transcription and/or the nucleic acid segment may be of homologous or heterologous origin with respect to the plant to be transformed. A recombinant DNA molecule useful for introduction into plant cells includes that which has been derived or isolated from any source, that may be subsequently characterized as to structure, size and/or function, chemically altered, and later introduced into plants. An example of a nucleotide sequence or segment of interest “derived” from a source, would be a nucleotide sequence or segment that is identified as a useful fragment within a given organism, and which is then chemically synthesized in essentially pure form. An example of such a nucleotide sequence or segment of interest “isolated” from a source, would be nucleotide sequence or segment that is excised or removed from said source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering. Such a nucleotide sequence or segment is commonly referred to as “recombinant.” Therefore a useful nucleotide sequence, segment or fragment of interest includes completely synthetic DNA, semi-synthetic DNA, DNA isolated from biological sources, and DNA derived from introduced RNA. Generally, the introduced DNA is not originally resident in the plant genotype which is the recipient of the DNA, but it is within the scope of the invention to isolate a gene from a given plant genotype, and to subsequently introduce multiple copies of the gene into the same genotype, e.g., to enhance production of a given gene product such as a storage protein or a protein that is involved in carbohydrate metabolism or any other gene of interest as provided in the SEQ ID NOs of the sequence listing. The introduced recombinant DNA molecule includes but is not limited to, DNA from plant genes, and non-plant genes such as those from bacteria, yeasts, animals or viruses. The introduced DNA can include modified genes, portions of genes, or chimeric genes, including genes from the same or different genotype. The term “chimeric gene” or “chimeric DNA” is defined as a gene or DNA sequence or segment comprising at least two DNA sequences or segments from species which do not combine DNA under natural conditions, or which DNA sequences or segments are positioned or linked in a manner which does not normally occur in the native genome of untransformed plant. The introduced recombinant DNA molecule used for transformation herein may be circular or linear, double-stranded or single-stranded. Generally, the DNA is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by regulatory sequences which promote the expression of the recombinant DNA present in the resultant plant. Generally, the introduced recombinant DNA molecule will be relatively small, i.e., less than about 30 kb to minimize any susceptibility to physical, chemical, or enzymatic degradation which is known to increase as the size of the nucleotide molecule increases. As noted above, the number of proteins, RNA transcripts or mixtures thereof which is introduced into the plant genome is preferably preselected and defined, e.g., from one to about 5-10 such products of the introduced DNA may be formed. This expression cassette or vector may be contained in a host cell. The expression cassette or vector may augment the genome of a transformed plant or may be maintained extrachromosomally. The expression cassette may be operatively linked to a structural gene, the open reading frame thereof, or a portion thereof. The expression cassette may further comprise a Ti plasmid and be contained in an Agrobacterium tumefaciens cell; it may be carried on a microparticle, wherein the microparticle is suitable for ballistic transformation of a plant cell; or it may be contained in a plant cell or protoplast. Further, the expression cassette or vector can be contained in a transformed plant or cells thereof, and the plant may be a dicot or a monocot. In particular, the plant may be a cereal plant. Obtaining sufficient levels of transgene expression in the appropriate plant tissues is an important aspect in the production of genetically engineered crops. Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. For example, for overexpression, a plant promoter fragment may be employed which will direct expression of the gene in all tissue; of a regenerated plant. Such promoters are referred to herein as “constitutive” promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35 S transcription initiation region, the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens , and other transcription initiation regions from various plant genes known to those of skill. Such genes include for example, the AP2 gene, ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104:1167-1176 (1994)), GPc1 from maize (GenBank No. X15596, Martinez et al. J. Mol. Biol. 208:551-565 (1989)), and Gpc2 from maize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol. 33:97-112 (1997)). Alternatively, the plant promoter may direct expression of the nucleic acid molecules of the invention in a specific tissue or may be otherwise under more precise environmental or developmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light. Such promoters are referred to here as “inducible” or “tissue-specific” promoters. One of skill will recognize that a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other tissues as well. Examples of promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as fruit, seeds, or flowers. Promoters that direct expression of nucleic acids in ovules, flowers or seeds are particularly useful in the present invention. As used herein a seed-specific or preferential promoter is one which directs expression specifically or preferentially in seed tissues, such promoters may be, for example, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed coat-specific, or some combination thereof. Examples include a promoter from the ovule-specific BEL1 gene described in Reiser et al. Cell 83:735-742 (1995) (GenBank No. U39944). Other suitable seed specific promoters are derived from the following genes: MAC1 from maize (Sheridan et al. Genetics 142:1009-1020 (1996), Cat3 from maize (GenBank No. L05934, Abler et al., Plant Mol. Biol. 22:10131-1038 (1993), the gene encoding oleosin 18 kD from maize (GenBank No, J05212, Lee et al., Plant Mol. Biol. 26:1981-1987 (1994)), vivparous-1 from Arabidopsis (Genbank No. U93215), the gene encoding oleosin from Arabidopsis (Genbank No. Z17657), Atmycl from Arabidopsis (Urao et al., Plant Mol. Biol. 32:571-576 (1996), the 2 s seed storage protein gene family from Arabidopsis (Conceicao et al. Plant 5:493-505 (1994)) the gene encoding oleosin 20 kD from Brassica napus (GenBank No. M63985), napA from Brassica napus (GenBank No. J02798, Josefsson et al. JBL 26:12196-1301 (1987), the napin gene family from Brassica napus (Sjodahl et al. Planta 197:264-271 (1995), the gene encoding the 2 S storage protein from Brassica napus (Dasgupta et al. Gene 133:301-302 (1993)), the genes encoding oleosin A (Genbank No. U09118) and oleosin B (Genbank No. U09119) from soybean and the gene encoding low molecular weight sulphur rich protein from soybean (Choi et al. Mol Gen, Genet. 246:266-268 (1995)). It is specifically contemplated that one could use one of the promoters that are disclosed in co-pending provisional U.S. application Ser. No. 60/325,448, filed Sep. 26, 2001 in unaltered or altered form. Especially preferred are promoters that direct transcription of an associated nucleic acid molecule specifically or preferentially in tissues of the plant grain such as those provided in SEQ ID NOs: 2275-2672. Mutagenization of a promoter such as those mentioned hereinbefore or those provided in provisional U.S. application Ser. No. 60/325,448 may potentially improve the utility of the elements for the expression of transgenes in plants. The mutagenesis of these elements can be carried out at random and the mutagenized promoter sequences screened for activity in a trial-by-error procedure. Alternatively, particular sequences which provide the promoter with desirable expression characteristics, or the promoter with expression enhancement activity, could be identified and these or similar sequences introduced into the sequences via mutation. It is further contemplated that one could mutagenize these sequences in order to enhance their expression of transgenes in a particular species. Furthermore, it is contemplated that promoters combining elements from more than one promoter may be useful. For example, U.S. Pat. No. 5,491,288 discloses combining a Cauliflower Mosaic Virus promoter with a histone promoter. Thus, the elements from the promoters disclosed herein may be combined with elements from other promoters. A variety of 5N and 3N transcriptional regulatory sequences are available for use in the present invention. Transcriptional terminators are responsible for the termination of transcription and correct mRNA polyadenylation. The 3N nontranslated regulatory DNA sequence preferably includes from about 50 to about 1,000, more preferably about 100 to about 1,000, nucleotide base pairs and contains plant transcriptional and translational termination sequences. Appropriate transcriptional terminators and those which are known to function in plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator, the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens , and the 3N end of the protease inhibitor 1 or 11 genes from potato or tomato, although other 3N elements known to those of skill in the art can also be employed. Alternatively, one also could use a gamma coixin, oleosin 3 or other terminator from the genus Coix. Preferred 3N elements include those from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan et al., 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens , and the 3′ end of the protease inhibitor 1 or 11 genes from potato or tomato. As the DNA sequence between the transcription initiation site and the start of the coding sequence, i.e., the untranslated leader sequence, can influence gene expression, one may also wish to employ a particular leader sequence. Preferred leader sequences are contemplated to include those which include sequences predicted to direct optimum expression of the attached gene, i.e., to include a preferred consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation. The choice of such sequences will be known to those of skill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in plants will be most preferred. Other sequences that have been found to enhance gene expression in transgenic plants include intron sequences (e.g., from Adh1, bronze1, actin1, actin 2 (WO 00/760067), or the sucrose synthase intron) and viral leader sequences (e.g., from TMV, MCMV and AMV). For example, a number of non-translated leader sequences derived from viruses are known to enhance expression. Specifically, leader sequences from Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g., Gallie et al., 1987; Skuzeski et al., 1990). Other leaders known in the art include but are not limited to: Picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5 noncoding region) (Elroy-Stein et al., 1989); Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus); MDMV leader (Maize Dwarf Mosaic Virus); Human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak et al., 1991); Untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling et al., 1987; Tobacco mosaic virus leader (TMV), (Gallie et al., 1989; and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel et al., 1991. See also, Della-Cioppa et al., 1987. Regulatory elements such as Adh intron 1 (Callis et al., 1987), sucrose synthase intron (Vasil et al., 1989) or TMV omega element (Gallie, et al., 1989), may further be included where desired. Examples of enhancers include elements from the CaMV 35S promoter, octopine synthase genes (Ellis et al., 1987), the rice actin I gene, the maize alcohol dehydrogenase gene (Callis et al., 1987), the maize shrunken I gene (Vasil et al., 1989), TMV omega element (Gallie et al., 1989) and promoters from non-plant eukaryotes (e.g. yeast; Ma et al., 1988). Two principal methods for the control of expression are known, viz.: overexpression and underexpression. Overexpression can be achieved by insertion of one or more than one extra copy of the selected gene. It is, however, not unknown for plants or their progeny, originally transformed with one or more than one extra copy of a nucleotide sequence, to exhibit the effects of underexpression as well as overexpression. For underexpression there are two principle methods which are commonly referred to in the art as “antisense downregulation” and “sense downregulation” (sense downregulation is also referred to as “cosuppression”). Generically these processes are referred to as “gene silencing”. Both of these methods lead to an inhibition of expression of the target gene. Within the scope of the present invention, the alteration in expression of the nucleic acid molecule of the present invention may be achieved in one of the following ways: (1) “Sense” Suppression Alteration of the expression of a nucleotide sequence of the present invention, preferably reduction of its expression, is obtained by “sense” suppression (referenced in e.g. Jorgensen et al. (1996) Plant Mol. Biol. 31, 957-973). In this case, the entirety or a portion of a nucleotide sequence of the present invention is comprised in a DNA molecule. The DNA molecule is preferably operatively linked to a promoter functional in a cell comprising the target gene, preferably a plant cell, and introduced into the cell, in which the nucleotide sequence is expressible. The nucleotide sequence is inserted in the DNA molecule in the “sense orientation”, meaning that the coding strand of the nucleotide sequence can be transcribed. In a preferred embodiment, the nucleotide sequence is fully translatable and all the genetic information comprised in the nucleotide sequence, or portion thereof, is translated into a polypeptide. In another preferred embodiment, the nucleotide sequence is partially translatable and a short peptide is translated. In a preferred embodiment, this is achieved by inserting at least one premature stop codon in the nucleotide sequence, which bring translation to a halt. In another more preferred embodiment, the nucleotide sequence is transcribed but no translation product is being made. This is usually achieved by removing the start codon, e.g. the “ATG”, of the polypeptide encoded by the nucleotide sequence. In a further preferred embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated in the genome of the plant cell. In another preferred embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is comprised in an extrachromosomally replicating molecule. In transgenic plants containing one of the DNA molecules described immediately above, the expression of the nucleotide sequence corresponding to the nucleotide sequence comprised in the DNA molecule is preferably reduced. Preferably, the nucleotide sequence in the DNA molecule is at least 70% identical to the nucleotide sequence the expression of which is reduced, more preferably it is at least 80% identical, yet more preferably at least 90% identical, yet more preferably at least 95% identical, yet more preferably at least 99% identical. (2) “Antisense” Suppression In another preferred embodiment, the alteration of the expression of a nucleotide sequence of the present invention, preferably the reduction of its expression is obtained by “anti-sense” suppression. The entirety or a portion of a nucleotide sequence of the present invention is comprised in a DNA molecule. The DNA molecule is preferably operatively linked to a promoter functional in a plant cell, and introduced in a plant cell, in which the nucleotide sequence is expressible. The nucleotide sequence is inserted in the DNA molecule in the “anti-sense orientation”, meaning that the reverse complement (also called sometimes noncoding strand) of the nucleotide sequence can be transcribed. In a preferred embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated in the genome of the plant cell. In another preferred embodiment the DNA molecule comprising the nucleotide sequence, or a portion thereof, is comprised in an extrachromosomally replicating molecule. Several publications describing this approach are cited for further illustration (Green, P. J. et al., Ann. Rev. Biochem. 55:569-597 (1986); van der Krol, A. R. et al, Antisense Nuc. Acids & Proteins, pp. 125-141 (1991); Abel, P. P. et al., Proc. Natl. Acad. Sci. USA 86:6949-6952 (1989); Ecker, J. R. et al., Proc. Natl. Acad. Sci. USA 83:5372-5376 (August 1986)). In transgenic plants containing one of the DNA molecules described immediately above, the expression of the nucleotide sequence corresponding to the nucleotide sequence comprised in the DNA molecule is preferably reduced. Preferably, the nucleotide sequence in the DNA molecule is at least 70% identical to the nucleotide sequence the expression of which is reduced, more preferably it is at least 80% identical, yet more preferably at least 90% identical, yet more preferably at least 95% identical, yet more preferably at least 99% identical. (3) Homologous Recombination In another preferred embodiment, at least one genomic copy corresponding to a nucleotide sequence of the present invention is modified in the genome of the plant by homologous recombination as further illustrated in Paszkowski et al., EMBO Journal 7:4021-26 (1988). This technique uses the property of homologous sequences to recognize each other and to exchange nucleotide sequences between each by a process known in the art as homologous recombination. Homologous recombination can occur between the chromosomal copy of a nucleotide sequence in a cell and an incoming copy of the nucleotide sequence introduced in the cell by transformation. Specific modifications are thus accurately introduced in the chromosomal copy of the nucleotide sequence. In one embodiment, the regulatory elements of the nucleotide sequence of the present invention are modified. Such regulatory elements are easily obtainable by screening a genomic library using the nucleotide sequence of the present invention, or a portion thereof, as a probe. The existing regulatory elements are replaced by different regulatory elements, thus altering expression of the nucleotide sequence, or they are mutated or deleted, thus abolishing the expression of the nucleotide sequence. In another embodiment, the nucleotide sequence is modified by deletion of a part of the nucleotide sequence or the entire nucleotide sequence, or by mutation. Expression of a mutated polypeptide in a plant cell is also contemplated in the present invention. More recent refinements of this technique to disrupt endogenous plant genes have been described (Kempin et al., Nature 389:802-803 (1997) and Miao and Lam, Plant J., 7:359-365 (1995). In another preferred embodiment, a mutation in the chromosomal copy of a nucleotide sequence is introduced by transforming a cell with a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. An additional feature of the oligonucleotide is for example the presence of 2′-O-methylation at the RNA residues. The RNA/DNA sequence is designed to align with the sequence of a chromosomal copy of a nucleotide sequence of the present invention and to contain the desired nucleotide change. For example, this technique is further illustrated in U.S. Pat. No. 5,501,967 and Zhu et al. (1999) Proc. Natl. Acad. Sci. USA 96: 8768-8773. (4) Ribozymes In a further embodiment, the RNA coding for a polypeptide of the present invention is cleaved by a catalytic RNA, or ribozyme, specific for such RNA. The ribozyme is expressed in transgenic plants and results in reduced amounts of RNA coding for the polypeptide of the present invention in plant cells, thus leading to reduced amounts of polypeptide accumulated in the cells. This method is further illustrated in U.S. Pat. No. 4,987,071. (5) Dominant-Negative Mutants In another preferred embodiment, the activity of the polypeptide encoded by the nucleotide sequences of this invention is changed. This is achieved by expression of dominant negative mutants of the proteins in transgenic plants, leading to the loss of activity of the endogenous protein. (6) Aptamers In a further embodiment, the activity of polypeptide of the present invention is inhibited by expressing in transgenic plants nucleic acid ligands, so-called aptamers, which specifically bind to the protein. Aptamers are preferentially obtained by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) method. In the SELEX method, a candidate mixture of single stranded nucleic acids having regions of randomized sequence is contacted with the protein and those nucleic acids having an increased affinity to the target are partitioned from the remainder of the candidate mixture. The partitioned nucleic acids are amplified to yield a ligand enriched mixture. After several iterations a nucleic acid with optimal affinity to the polypeptide is obtained and is used for expression in transgenic plants. This method is further illustrated in U.S. Pat. No. 5,270,163. (7) Zinc Finger Proteins A zinc finger protein that binds a nucleotide sequence of the present invention or to its regulatory region is also used to alter expression of the nucleotide sequence. Preferably, transcription of the nucleotide sequence is reduced or increased. Zinc finger proteins are for example described in Beerli et al. (1998) PNAS 95:14628-14633, or in WO 95/19431, WO 98/54311, or WO 96/06166, all incorporated herein by reference in their entirety. (8) dsRNA Alteration of the expression of a nucleotide sequence of the present invention is also obtained by dsRNA interference as described for example in WO 99/32619, WO 99/53050 or WO 99/61631, all incorporated herein by reference in their entirety. (9) Insertion of a DNA Molecule (Insertional Mutagenesis) In another preferred embodiment, a DNA molecule is inserted into a chromosomal copy of a nucleotide sequence of the present invention, or into a regulatory region thereof. Preferably, such DNA molecule comprises a transposable element capable of transposition in a plant cell, such as e.g. Ac/Ds, Em/Spm, mutator. Alternatively, the DNA molecule comprises a T-DNA border of an Agrobacterium T-DNA. The DNA molecule may also comprise a recombinase or integrase recognition site which can be used to remove part of the DNA molecule from the chromosome of the plant cell. An example of this method is set forth in Example 2. Methods of insertional mutagenesis using T-DNA, transposons, oligonucleotides or other methods known to those skilled in the art are also encompassed. Methods of using T-DNA and transposon for insertional mutagenesis are described in Winkler et al. (1989) Methods Mol. Biol. 82:129-136 and Martienssen (1998) PNAS 95:2021-2026, incorporated herein by reference in their entireties. (10) Deletion Mutagenesis In yet another embodiment, a mutation of a nucleic acid molecule of the present invention is created in the genomic copy of the sequence in the cell or plant by deletion of a portion of the nucleotide sequence or regulator sequence. Methods of deletion mutagenesis are known to those skilled in the art. See, for example, Miao et al, (1995) Plant J. 7:359. In yet another embodiment, this deletion is created at random in a large population of plants by chemical mutagenesis or irradiation and a plant with a deletion in a gene of the present invention is isolated by forward or reverse genetics. Irradiation with fast neutrons or gamma rays is known to cause deletion mutations in plants (Silverstone et al, (1998) Plant Cell, 10:155-169; Bruggemann et al., (1996) Plant J., 10:755-760; Redei and Koncz in Methods in Arabidopsis Research , World Scientific Press (1992), pp. 16-82). Deletion mutations in a gene of the present invention can be recovered in a reverse genetics strategy using PCR with pooled sets of genomic DNAs as has been shown in C. elegans (Liu et al., (1999), Genome Research, 9:859-867.). A forward genetics strategy would involve mutagenesis of a line displaying PTGS followed by screening the M2 progeny for the absence of PTGS. Among these mutants would be expected to be some that disrupt a gene of the present invention. This could be assessed by Southern blot or PCR for a gene of the present invention with genomic DNA from these mutants. (11) Overexpression in a Plant Cell In yet another preferred embodiment, a nucleotide sequence of the present invention encoding a polypeptide comprising a 3′-5′ exonuclease domain and/or activity in a plant cell is overexpressed. Examples of nucleic acid molecules and expression cassettes for overexpression of a nucleic acid molecule of the present invention are described above. Methods known to those skilled in the art of over-expression of nucleic acid molecules are also encompassed by the present invention. In still another embodiment, the expression of the nucleotide sequence of the present invention is altered in every cell of a plant. This is for example obtained though homologous recombination or by insertion in the chromosome. This is also for example obtained by expressing a sense or antisense RNA, zinc finger protein or ribozyme under the control of a promoter capable of expressing the sense or antisense RNA, zinc finger protein or ribozyme in every cell of a plant. Constitutive expression, inducible, tissue-specific or developmentally-regulated expression are also within the scope of the present invention and result in a constitutive, inducible, tissue-specific or developmentally-regulated alteration of the expression of a nucleotide sequence of the present invention in the plant cell. Constructs for expression of the sense or antisense RNA, zinc finger protein or ribozyme, or for overexpression of a nucleotide sequence of the present invention, are prepared and transformed into a plant cell according to the teachings of the present invention, e.g. as described infra. The invention hence also provides sense and anti-sense nucleic acid molecules corresponding to the open reading flames identified in the SEQ ID NOs of the Sequence Listing as well as their orthologs. The genes and open reading frames according to the present invention which are substantially similar to a nucleotide sequence encoding a polypeptide as given in any one of the SEQ ID NOs of the Sequence Lisiting including any corresponding antisense constructs can be operably linked to any promoter that is functional within the plant host including the promoter sequences according to the invention or mutants thereof. The present invention further provides a method of augmenting a plant genome by contacting plant cells with a nucleic acid molecule of the invention, e.g., one having a nucleotide sequence that directs tissue-specific, tissue-preferential transcription of a linked nucleic acid segment isolatable or obtained from a plant gene encoding a polypeptide that is substantially similar to a polypeptide encoded by the an Oryza gene having a sequence according to any one of SEQ ID NOs provided in the Sequence Listing so as to yield transformed plant cells; and regenerating the transformed plant cells to provide a differentiated transformed plant, wherein the differentiated transformed plant expresses the nucleic acid molecule in the cells of the plant, preferably in the appropriate tissues of the plant grain. The nucleic acid molecule may be present in the nucleus, chloroplast, mitochondria and/or plastid of the cells of the plant. Plant species may be transformed with the DNA construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art. Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector of the present invention. The term “organogenesis,” as used herein, means a process by which shoots and roots are developed sequentially from meristematic centers; the term “embryogenesis,” as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and ultilane meristem). Plants of the present invention may take a variety of forms. The plants may be chimeras of transformed cells and nor-transformed cells; the plants may be clonal transformants (e.g., all cells transformed to contain the expression cassette); the plants may comprise grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species). The transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, first generation (or T1) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques. A dominant selectable marker (such as npt II) can be associated with the expression cassette to assist in breeding. Thus, the present invention provides a transformed (transgenic) plant cell in planta or ex planta, including a transformed plastid or other organelle, e.g., nucleus, mitochondria or chloroplast. The present invention may be used for transformation of any plant species, including, but not limited to, cells from corn ( Zea mays ), Brassica sp. (e.g., B. napus, B. rapa, B. juncea ), particularly those Brassica species useful as sources of seed oil, alfalfa ( Medicago sativa ), rice ( Oryza sativa ), rye ( Secale cereale ), sorghum ( Sorghum bicolor, Sorghum vulgare ), millet (e.g., pearl millet ( Pennisetum glaucum ), proso millet ( Panicum miliaceum ), foxtail millet ( Setaria italica ), finger millet ( Eleusine coracana )), sunflower ( Helianthus annuus ), safflower ( Carthamus tinctorius ), wheat ( Triticum aestivum ), soybean ( Glycine max ), tobacco ( Nicotiana tabacum ), potato ( Solanum tuberosum ), peanuts ( Arachis hypogaea ), cotton ( Gossypium barbadense, Gossypium hirsutum ), sweet potato ( Ipomoea batatus ), cassaya ( Manihot esculenta ), coffee ( Cofea spp.), coconut ( Cocos nucifera ), pineapple ( Ananas comosus ), citrus trees ( Citrus spp.), cocoa ( Theobroma cacao ), tea ( Camellia sinensis ), banana ( Musa spp.), avocado ( Persea ultilane ), fig ( Ficus casica ), guava ( Psidium guajava ), mango ( Mangifera indica ), olive ( Olea europaea ), papaya ( Carica papaya ), cashew ( Anacardium occidentale ), macadamia ( Macadamia integrifolia ), almond ( Prunus amygdalus ), sugar beets ( Beta vulgaris ), sugarcane ( Saccharum spp.), oats, duckweed ( Lemna ), barley, vegetables, ornamentals, and conifers. Duckweed ( Lemna , see WO 00/07210) includes members of the family Lemnaceae. There are known four genera and 34 species of duckweed as follows: genus Lemna ( L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L.turionifera, L. valdiviana ); genus Spirodela ( S. intermedia, S. polyrrhiza, S. punctata ); genus Woffia ( Wa. Angusta, Wa. Arrhiza, Wa. Australina, Wa. Borealis, Wa. Brasiliensis, Wa. Columbiana, Wa. Elongata, Wa. Globosa, Wa. Microscopica, Wa. Neglecta ) and genus Wofiella ( Wl. ultila, Wl. ultilanen, Wl. gladiala, Wl. ultila, Wl. lingulata, Wl. repunda, Wl. rotunda , and Wl. neotropica ). Any other genera or species of Lemnaceae , if they exist, are also aspects of the present invention. Lemna gibba, Lemnaminor , and Lemna miniscula are preferred, with Lemnaminor and Lemna miniscula being most preferred. Lemna species can be classified using the taxonomic scheme described by Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae—A Monograph Study. Geobatanischen Institut ETH, Stiflung Rubel, Zurich (1986)). Vegetables within the scope of the invention include tomatoes ( Lycopersicon esculentum ), lettuce (e.g., Lactuca sativa ), green beans ( Phaseolus vulgaris ), lima beans ( Phaseolus limensis ), peas ( Lathyrus spp.), and members of the genus Cucumis such as cucumber ( C. sativus ), cantaloupe ( C. cantalupensis ), and musk melon ( C. melo ). Ornamentals include azalea ( Rhododendron spp.), hydrangea ( Macrophylla hydrangea ), hibiscus ( Hibiscus rosasanensis ), roses ( Rosa spp.), tulips ( Tulipa spp.), daffodils ( Narcissus spp.), petunias ( Petunia hybrida ), carnation ( Dianthus caryophyllus ), poinsettia ( Euphorbia pulcherrima ), and chrysanthemum. Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine ( Pinus taeda ), slash pine ( Pinus elliotii ), ponderosa pine ( Pinus ponderosa ), lodgepole pine ( Pinus contorta ), and Monterey pine ( Pinus radiata ), Douglas-fir ( Pseudotsuga menziesii ); Western hemlock ( Tsuga ultilane); Sitka spruce ( Picea glauca ); redwood ( Sequoia sempervirens ); true firs such as silver fir ( Abies amabilis ) and balsam fir ( Abies balsamea ); and cedars such as Western red cedar ( Thuja plicata ) and Alaska yellow-cedar ( Chamaecyparis nootkatensis ). Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc. Legumes include, but are not limited to, Arachis , e.g., peanuts, Vicia , e.g., crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea, Lupinus, e.g., lupine, trifolium, Phaseolus , e.g., common bean and lima bean, Pisum , e.g., field bean, Melilotus, e.g., clover, Medicago , e.g., alfalfa, Lotus , e.g., trefoil, lens, e.g., lentil, and false indigo. Preferred forage and turf grass for use in the methods of the invention include alfalfa, orchard grass, tall fescue, perennial ryegrass, creeping bent grass, and redtop. Other plants within the scope of the invention include Acacia , aneth, artichoke, arugula, blackberry, canola, cilantro, clementines, escarole, eucalyptus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon, lime, mushroom, nut, okra, orange, parsley, persimmon, plantain, pomegranate, poplar, radiata pine, radicchio, Southern pine, sweetgum, tangerine, triticale, vine, yams, apple, pear, quince, cherry, apricot, melon, hemp, buckwheat, grape, raspberry, chenopodium, blueberry, nectarine, peach, plum, strawberry, watermelon, eggplant, pepper, cauliflower, Brassica , e.g., broccoli, cabbage, ultilan sprouts, onion, carrot, leek, beet, broad bean, celery, radish, pumpkin, endive, gourd, garlic, snapbean, spinach, squash, turnip, ultilane, and zucchini. Ornamental plants within the scope of the invention include impatiens, Begonia, Pelargonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia. Other plants within the scope of the invention are shown in Table 1 (above). Preferably, transgenic plants of the present invention are crop plants and in particular cereals (for example, corn, alfalfa, sunflower, rice, Brassica , canola, soybean, barley, soybean, sugarbeet, to cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), and even more preferably corn, rice and soybean. The present invention also provides a transgenic plant prepared by this method, a seed from such a plant and progeny plants from such a plant including hybrids and inbreds. Preferred transgenic plants are transgenic maize, soybean, barley, alfalfa, sunflower, canola, soybean, cotton, peanut, sorghum, tobacco, sugarbeet, rice, wheat, rye, turfgrass, millet, sugarcane, tomato, or potato. A transformed (transgenic) plant of the invention includes plants, the genome of which is augmented by a nucleic acid molecule of the invention, or in which the corresponding gene has been disrupted, e.g., to result in a loss, a decrease or an alteration, in the function of the product encoded by the gene, which plant may also have increased yields and/or produce a better-quality product than the corresponding wild-type plant. The nucleic acid molecules of the invention are thus useful for targeted gene disruption, as well as markers and probes. The invention also provides a method of plant breeding, e.g., to prepare a crossed fertile transgenic plant. The method comprises crossing a fertile transgenic plant comprising a particular nucleic acid molecule of the invention with itself or with a second plant, e.g., one lacking the particular nucleic acid molecule, to prepare the seed of a crossed fertile transgenic plant comprising the particular nucleic acid molecule. The seed is then planted to obtain a crossed fertile transgenic plant. The plant may be a monocot or a dicot. In a particular embodiment, the plant is a cereal plant. The crossed fertile transgenic plant may have the particular nucleic acid molecule inherited through a female parent or through a male parent. The second plant may be an inbred plant. The crossed fertile transgenic may be a hybrid. Also included within the present invention are seeds of any of these crossed fertile transgenic plants. Transformation of plants can be undertaken with a single DNA molecule or multiple DNA molecules (i.e., co-transformation), and both these techniques are suitable for use with the expression cassettes of the present invention. Numerous transformation vectors are available for plant transformation, and the expression cassettes of 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. A variety of techniques are available and known to those skilled in the art for introduction of constructs into a plant cell host. These techniques generally include transformation with DNA employing A. tumefaciens or A. rhizogenes as the transforming agent, liposomes, PEG precipitation, electroporation, DNA injection, direct DNA uptake, microprojectile bombardment, particle acceleration, and the like (See, for example, EP 295959 and EP 138341) (see below). However, cells other than plant cells may be transformed with the expression cassettes of the invention. The general descriptions of plant expression vectors and reporter genes, and Agrobacterium and Agrobacterium -mediated gene transfer, can be found in Gruber et al. (1993). Expression vectors containing genomic or synthetic fragments can be introduced into protoplasts or into intact tissues or isolated cells. Preferably expression vectors are introduced into intact tissue. General methods of culturing plant tissues are provided for example by Maki et al., (1993); and by Phillips et al. (1988). Preferably, expression vectors are introduced into maize or other plant tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation and the like. More preferably expression vectors are introduced into plant tissues using the microprojectile media delivery with the biolistic device. See, for example, Tomes et al. (1995). The vectors of the invention can not only be used for expression of structural genes but may also be used in exon-trap cloning, or promoter trap procedures to detect differential gene expression in varieties of tissues, (Lindsey et al., 1993; Auch & Reth et al.). It is particularly preferred to use the binary type vectors of Ti and Ri plasmids of Agrobacterium spp. Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti et al., 1985: Byrne et al., 1987; Sukhapinda et al., 1987; Lorz et al., 1985; Potrykus, 1985; Park et al., 1985: Hiei et al., 1994). The use of T-DNA to transform plant cells has received extensive study and is amply described (EP 120516; Hoekema, 1985; Knauf, et al., 1983; and An et al., 1985). For introduction into plants, the chimeric genes of the invention can be inserted into binary vectors as described in the examples. Other transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs (see EP 295959), techniques of electroporation (Fromm et al., 1986) or high velocity ballistic bombardment with metal particles coated with the nucleic acid constructs (Kine et al., 1987, and U.S. Pat. No. 4,945,050). Once transformed, the cells can be regenerated by those skilled in the art. Of particular relevance are the recently described methods to transform foreign genes into commercially important crops, such as rapeseed (De Block et al., 1989), sunflower (Everett et al., 1987), soybean (McCabe et al., 1988; Hinchee et al., 1988; Chee et al., 1989; Christou et al., 1989; EP 301749), rice (Hiei et al., 1994), and corn (Gordon Kamm et al., 1990; Fromm et al., 1990). Those skilled in the art will appreciate that the choice of method might depend on the type of plant, i.e., monocotyledonous or dicotyledonous, targeted for transformation. Suitable methods of transforming plant cells include, but are not limited to, microinjection (Crossway et al., 1986), electroporation (Riggs et al., 1986), Agrobacterium -mediated transformation (Hinchee et al., 1988), direct gene transfer (Paszkowski et al., 1984), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wis. And BioRad, Hercules, Calif. (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; and McCabe et al., 1988). Also see, Weissinger et al., 1988; Sanford et al., 1987 (onion); Christou et al., 1988 (soybean); McCabe et al., 1988 (soybean); Datta et al., 1990 (rice); Klein et al., 1988 (maize); Klein et al., 1988 (maize); Klein et al., 1988 (maize); Fromm et al., 1990 (maize); and Gordon-Kamm et al., 1990 (maize); Svab et al., 1990 (tobacco chloroplast); Koziel et al., 1993 (maize); Shimamoto et al., 1989 (rice); Christou et al., 1991 (rice); European Patent Application EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al., 1993 (wheat); Weeks et al., 1993 (wheat). In one embodiment, the protoplast transformation method for maize is employed (European Patent Application EP 0 292 435, U.S. Pat. No. 5,350,689). In another embodiment, a nucleotide sequence of the present invention is directly transformed into the plastid genome. Plastid transformation technology is extensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride et al., 1994. The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest 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 orthologous 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 et al., 1990; Staub et al., 1992). This resulted in stable homoplasmic as transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub et al., 1993). 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-3N-adenyltransferase (Svab et al., 1993). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the invention. Typically, approximately 15-20 cell division cycles following transformation are required to reach a homoplastidic state. Plastid expression, in which genes are inserted by orthologous recombination into all of 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, a nucleotide sequence of the present invention is inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleotide sequence of the present invention are obtained, and are preferentially capable of high expression of the nucleotide sequence. Agrobacterium tumefaciens cells containing a vector comprising an expression cassette of the present invention, wherein the vector comprises a Ti plasmid, are useful in methods of making transformed plants. Plant cells are infected with an Agrobacterium tumefaciens as described above to produce a transformed plant cell, and then a plant is regenerated from the transformed plant cell. Numerous Agrobacterium vector systems useful in carrying out the present invention are known. For example, vectors are available for transformation using Agrobacterium tumefaciens . These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, 1984). In one preferred embodiment, the expression cassettes of the present invention may be inserted into either of the binary vectors pCIB200 and pCIB2001 for use with Agrobacterium . These vector cassettes for Agrobacterium -mediated transformation wear constructed in the following manner. PTJS75kan was created by Narl digestion of pTJS75 (Schmidhauser & Helinski, 1985) allowing excision of the tetracycline-resistance gene, followed by insertion of an Accl fragment from pUC4K carrying an NPTh (Messing & Vierra, 1982; Bevan et al., 1983; McBride et al., 1990). XhoI linkers were ligated to the EcoRV fragment of pCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptII chimeric gene and the pUC polylinker (Rothstein et al., 1987), and the XhoI-digested fragment was cloned into SalI-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). PCIB200 contains the following unique polylinker restriction sites: EcoRI, SstI, KpnI, BgfII, XbaI, and SalI. The plasmid pCIB2001 is a derivative of pCIB200 which was created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI, BglII, XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI, and StuI. PCIB2001, in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium -mediated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the OnT and OriV functions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals. An additional vector useful for Agrobacterium -mediated transformation is the binary vector pCIB 10, which contains a gene encoding kanamycin resistance for selection in plants, T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium . Its construction is described by Rothstein et al., 1987. Various derivatives of pCIB10 have been constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al., 1983. These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kananycin (pCIB715, pCIB717). Methods using either a form of direct gene transfer or Agrobacterium -mediated transfer usually, but not necessarily, are undertaken with a selectable marker which may provide resistance to an antibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide (e.g., phosphinothricin). The choice of selectable marker for plant transformation is not, however, critical to the invention. For certain plant species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptlI gene which confers resistance to kanamycin and related antibiotics (Messing & Vierra, 1982; Bevan et al., 1983), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., 1990, Spencer et al., 1990), the hph gene which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., 1983). Selection markers resulting in positive selection, such as a phosphomannose isomerase gene, as described in patent application WO 93/05163, are also used. Other genes to be used for positive selection are described in WO 94/20627 and encode xyloisomerases and phosphomanno-isomerases such as mannose-6-phosphate isomerase and mannose-1-phosphate isomerase; phosphomanno mutase; mannose epimerases such as those which convert carbohydrates to mannose or mannose to carbohydrates such as glucose or galactose; phosphatases such as mannose or xylose phosphatase, mannose-6-phosphatase and mannose-1-phosphatase, and permeases which are involved in the transport of mannose, or a derivative, or a precursor thereof into the cell. The agent which reduces the toxicity of the compound to the cells is typically a glucose derivative such as methyl-3-O-glucose or phloridzin. Transformed cells are identified without damaging or killing the non-transformed cells in the population and without co-introduction of antibiotic or herbicide resistance genes. As described in WO 93/05163, in addition to the fact that the need for antibiotic or herbicide resistance genes is eliminated, it has been shown that the positive selection method is often far more efficient than traditional negative selection. One vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is pCIB3064. This vector is based on the plasmid pCIB246, which comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278, herein incorporated by reference. One gene useful for conferring resistance to phosphinothricin is the bar gene from Streptomyces viridochromogenes (Thompson et al., 1987). This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals. An additional transformation vector is pSOG35 which utilizes the E. coli gene dihydrofolate reductase (DHFR) as a selectable marker conferring resistance to methotrexate. PCR was used to amplify the 35S promoter (about 800 bp), intron 6 from the maize Adh1 gene (about 550 bp) and 18 bp of the GUS untranslated leader sequence from pSOG10. A 250 bp fragment encoding the E. coli dihydrofolate reductase type II gene was also amplified by PCR and these two PCR fragments were assembled with a SacI-PstI fragment from pB1221 (Clontech) which comprised the pUC19 vector backbone and the nopaline synthase terminator. Assembly of these fragments generated pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus check (MCMV) generated the vector pSOG35. pSOG19 and pSOG35 carry the pUC-derived gene for ampicillin resistance and have HindIII, SphI, PstI and EcoRI sites available for the cloning of foreign sequences. Binary backbone vector pNOV2117 contains the T-DNA portion flanked by the right and left border sequences, and including the Positcch™ (Syngenta) plant selectable marker and the “grain filling candidate gene” gene expression cassette. The Positech™ plant selectable marker confers resistance to mannose and in this instance consists of the maize ubiquitin promoter driving expression of the PMI (phosphomannose isomerase) gene, followed by the cauliflower mosaic virus transcriptional terminator. Transgenic plant cells are then placed in an appropriate selective medium for selection of transgenic cells which are then grown to callus. Shoots are grown from callus and plantlets generated from the shoot by growing in rooting medium. The various constructs normally will be joined to a marker for selection in plant cells. Conveniently, the marker may be resistance to a biocide (particularly an antibiotic, such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide, or the like). The particular marker used will allow for selection of transformed cells as compared to cells lacking the DNA which has been introduced. Components of DNA constructs including transcription cassettes of this invention may be prepared from sequences which are native (endogenous) or foreign (exogenous) to the host. By “foreign” it is meant that the sequence is not found in the wild-type host into which the construct is introduced. Heterologous constructs will contain at least one region which is not native to the gene from which the transcription-initiation-region is derived. To confirm the presence of the transgenes in transgenic cells and plants, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, in situ hybridization and nucleic acid-based amplification methods such as PCR or RT-PCR; “biochemical” assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as seed assays; and also, by analyzing the phenotype of the whole regenerated plant, e.g., for disease or pest resistance. DNA may be isolated from cell lines or any plant parts to determine the presence of the preselected nucleic acid segment through the use of techniques well known to those skilled in the art. Note that intact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell. The presence of nucleic acid elements introduced through the methods of this invention may be determined by polymerase chain reaction (PCR). Using this technique discreet fragments of nucleic acid are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a preselected nucleic acid segment is present in a stable transformant, but does not prove integration of the introduced preselected nucleic acid segment into the host cell genome. In addition, it is not possible using PCR techniques to determine whether transformants have exogenous genes introduced into different sites in the genome, i.e., whether transformants are of independent origin. It is contemplated that using PCR techniques it would be possible to clone fragments of the host genomic DNA adjacent to an introduced preselected DNA segment. Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition it is possible through Southern hybridization to demonstrate the presence of introduced preselected DNA segments in high molecular weight DNA, i.e., confirm that the introduced preselected DNA segment has been integrated into the host cell genome. The technique of Southern hybridization provides information that is obtained using PCR, e.g., the presence of a preselected DNA segment, but also demonstrates integration into the genome and characterizes each individual transformant. It is contemplated that using the techniques of dot or slot blot hybridization which are modifications of Southern hybridization techniques one could obtain the same information that is derived from PCR, e.g., the presence of a preselected DNA segment. Both PCR and Southern hybridization techniques can be used to demonstrate transmission of a preselected DNA segment to progeny. In most instances the characteristic Southern hybridization pattern for a given transformant will segregate in progeny as one or more Mendelian genes (Spencer et al., 1992); Laursen et al., 1994) indicating stable inheritance of the gene. The nonchimeric nature of the callus and the parental transformants (R 0 ) was suggested by germline transmission and the identical Southern blot hybridization patterns and intensities of the transforming DNA in callus, R 0 plants and R 1 progeny that segregated for the transformed gene. Whereas DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA may only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues. PCR techniques may also be used for detection and quantitation of RNA produced from introduced preselected DNA segments. In this application of PCR it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. In most instances PCR techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species. While Southern blotting and PCR may be used to detect the preselected DNA segment in question, they do not provide information as to whether the preselected DNA segment is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced preselected DNA segments or evaluating the phenotypic changes brought about by their expression. Assays for the production and identification of specific proteins may make use of physical chemical, structural, functional, or other properties of the proteins. Unique physicachemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used. Assay procedures may also be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed. Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Morphological changes may include greater stature or thicker stalks. Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays. The compositions of the invention include plant nucleic acid molecules, and the amino acid sequences for the polypeptides or partial-length polypeptides encoded by the nucleic acid molecule which comprises an open reading frame. These sequences can be employed to alter expression of a particular gene corresponding to the open reading frame by decreasing or eliminating expression of that plant gene or by overexpressing a particular gene product. Methods of this embodiment of the invention include stably transforming a plant with the nucleic acid molecule of the invention which includes an open reading frame operably linked to a promoter capable of driving expression of that open reading frame (sense or antisense) in a plant cell. By “portion” or “fragment”, as it relates to a nucleic acid molecule which comprises an open reading frame or a fragment thereof encoding a partial-length polypeptide having the activity of the full length polypeptide, is meant a sequence having at least 80 nucleotides, more preferably at least 150 nucleotides, and still more preferably at least 400 nucleotides. If not employed for expressing, a “portion” or “fragment” means at least 9, preferably 12, more preferably 15, even more preferably at least 20, consecutive nucleotides, e.g., probes and primers (oligonucleotides), corresponding to the nucleotide sequence of the nucleic acid molecules of the invention. Thus, to express a particular gene product, the method comprises introducing to a plant, plant cell, or plant tissue an expression cassette comprising a promoter linked to an open reading frame so as to yield a transformed differentiated plant, transformed cell or transformed tissue. Transformed cells or tissue can be regenerated to provide a transformed differentiated plant. The transformed differentiated plant or cells thereof preferably expresses the open reading frame in an amount that alters the amount of the gene product in the plant or cells thereof, which product is encoded by the open reading frame. The present invention also provides a transformed plant prepared by the method, progeny and seed thereof. The invention further includes a nucleotide sequence which is complementary to one (hereinafter “test” sequence) which hybridizes under stringent conditions with a nucleic acid molecule of the invention as well as RNA which is transcribed from the nucleic acid molecule. When the hybridization is performed under stringent conditions, either the test or nucleic acid molecule of invention is preferably supported, e.g., on a membrane or DNA chip. Thus, either a denatured test or nucleic acid molecule of the invention is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of, e.g., between 55 and 70° C., in double strength citrate buffered saline (SC) containing 0.1% SDS followed by rinsing of the support at the same temperature but with a buffer having a reduced SC concentration. Depending upon the degree to of stringency required such reduced concentration buffers are typically single strength SC containing 0.1% SDS, half strength SC containing 0.1% SDS and one-tenth strength SC containing 0.1% SDS. In a further embodiment, the present invention provides a transformed plant host cell, or one obtained through breeding, capable of over-expressing, under-expressing, or having a knock out of amino acid genes and/or their gene products. The plant cell is transformed with at least one such expression vector wherein the plant host cell can be used to regenerate plant tissue or an entire plant, or seed there from, in which the effects of expression, including overexpression or underexpression, of the introduced sequence or sequences can be measured in vitro or in planta. Polynucleotides derived from the nucleic acid molecules of the present invention having any of the nucleotide sequences of SEQ ID NO: 1 to 461 and 501 to 511, respectively, encoding a polypeptide the expression of which is up-regulated during grain filling, are useful to detect the presence in a test sample of at least one copy of a nucleotide sequence containing the same or substantially the same sequence, or a fragment, complement, or variant thereof. The sequence of the probes and/or primers of the instant invention need not be identical to those provided in the Sequence Listing or the complements thereof. Some variation in probe or primer sequence and/or length can allow additional family members to be detected, as well as orthologous genes and more taxonomically distant related sequences. Similarly probes and/or primers of the invention can include additional nucleotides that serve as a label for detecting duplexes, for isolation of duplexed polynucleotides, or for cloning purposes. Preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides containing a contiguous span of between at least 12 to at least 1000 nucleotides of any nucleotid sequence which is substantially similar, and preferably has at least between 70% and 99% sequence identity to any one of SEQ ID NO: 1 to 461, 501-511, and 513-641, respectively, encoding a polypeptide the expression of which is up-regulated during grain filing, or the complements thereof, with each individual number of nucleotides within this range also being part of the invention. Preferred are isolated, purified, or recombinant polynucleotides containing a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 750, or 1000 nucleotides of any nucleotide sequence which is substantially similar, and preferably has at least between 70% and 99%, sequence identity to any one of SEQ ID NO: 1 to 461, 501-511, and 513-641, respectively, encoding a polypeptide the expression of which is up-regulated during grain filling, or the complements thereof. The appropriate length for primers and probes will vary depending on the application. For use as PCR primers, probes are 12-40 nucleotides, preferably 18-30 nucleotides long. For use in mapping, probes are 50 to 500 nucleotides, preferably 100-250 nucleotides long. For use in Southern hybridizations, probes as long as several kilobases can be used. The appropriate length for primers and probes under a particular set of assay conditions may be empirically determined by one of skill in the art. The primers and probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al. ( Meth Enzymol 68: 90 (1979)), the diethylphosphoramidite method, the triester method of Matteucci et al. ( J Am Chem Soc 103: 3185 (1981)), or according to Urdea et al. ( Proc Natl Acad 80: 7461 (1981)), the solid support method described in EP 0 707 592, or using commercially available automated oligonucleotide synthesizers. Detection probes are generally nucleotide sequences or uncharged nucleotide analogs such as, for example peptide nucleotides which are disclosed in International Patent Application WO 92/20702, morpholino analogs which are described in U.S. Pat. Nos. 5,185,444, 5,034,506 and 5,142,047. The probe may have to be rendered “non-extendable” such that additional dNTPs cannot be added to the probe. Analogs are usually nonextendable, and nucleotide probes can be rendered non-extendable by modifying the 3′ end of the probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3′ end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl group simply can be cleaved, replaced or modified so as to render the probe non-extendable. Any of the polynucleotides of the present invention can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive substances ( 32 P, 35 S, 3 H, 25 I), fluorescent dyes (5-bromodesoxyuridine, fluorescein, acetylaminofluorene, digoxigenin) or biotin. Preferably, polynucleotides are labeled at their 3′ and 5′ ends. Examples of non-radioactive labeling of nucleotide fragments are described in the French patent No. FR-7810975 and by Urdea et al. ( Nuc Acids Res 16:4937 (1988)). In addition, the probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as described in EP 0 225 807. A label can also be used to capture the primer so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support. A capture label is attached to the primers or probes and can be a specific binding member that forms a binding pair with the solid's phase reagent's specific binding member, for example biotin and streptavidin. Therefore depending upon the type of label carried by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, primers or probes provided herein, may, themselves, serve as the capture label. For example, in the case where a solid phase reagent's binding member is a nucleotide sequence, it may be selected such that it binds a complementary portion of a primer or probe to thereby immobilize the primer or probe to the solid phase. In cases where a polynucleotide probe itself serves as the binding member, those skilled in the art will recognize that the probe will contain a sequence or “ail” that is not complementary to the target. In the case where a polynucleotide primer itself serves as the capture label, at least a portion of the primer will be free to hybridize with a nucleotide on a solid phase. DNA labeling techniques are well known in the art. Any of the polynucleotides, primers and probes of the present invention can be conveniently immobilized on a solid support. Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others. The solid support is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing nucleotides on solid phases include ionic, hydrophobic, covalent interactions and the like. A solid support, as used herein, refers to any material that is insoluble, or can be made insoluble by a subsequent reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor that has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes and other configurations known to those of ordinary skill in the art. The polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support. In addition, polynucleotides other than those of the invention may be attached to the same solid support as one or more polynucleotides of the invention. The polynucleotides of the invention that are expressed or repressed in response to environmental stimuli such as, for example, biotic or abiotic stress or treatment with chemicals or pathogens or at different developmental stages can be identified by employing an array of nucleic acid samples, e.g., each sample having a plurality of oligonucleotides, and each plurality corresponding to a different plant gene, on a solid substrate, e.g., a DNA chip, and probes corresponding to nucleic acid expressed in, for example, one or more plant tissues and/or at one or more developmental stages, e.g., probes corresponding to nucleic acid expressed in seed of a plant relative to control nucleic acid from sources other than seed. Thus, genes that are upregulated or downregulated in the majority of tissues at a majority of developmental stages, or upregulated or downregulated in one tissue such as in seed, can be systematically identified. The probes may also correspond to nucleic acid expressed in respone to a defined treatment such as, for example, a treatment with a variety of plant hormones or the exposure to specific environmental conditions involving, for example, an abiotic stress or exposure to light. Specifically, labeled rice cRNA probes were hybridized to the rice DNA array, expression levels were determined by laser scanning and then rice genes were identified that had a particular expression pattern. The rice oligonucleotide probe array consists of probes from over 18,000 unique rice genes, which covers approximately 40-50% of the genome. This genome array permits a broader, more complete and less biased analysis of gene expression. As described herein, GeneChip® technology was utilized to discover rice genes that are preferentially (or exclusively) expressed during the grain filling process in specific tissues of the plant grain such as, for example, the aleurone, embryo, endosperm, seed coat, etc. Using this approach, 461 genes were identified, the expression of which was specifically elevated during the grain filling process. Consequently, the invention also deals with a method for detecting the presence of a polynucleotide including a nucleotide sequence which is substantially similar, and preferably has at least between 70% and 99% sequence identity to any one of SEQ ID NO: 1 to 461, 501-511, and 513-641, respectively, encoding a polypeptide the expression of which is up-regulated during grain filing, or a fragment or a variant thereof, or a complementary sequence thereto in a sample, the method including the following steps of: (a) bringing into contact a nucleotide probe or a plurality of nucleotide probes which can hybridize with polynucleotide having a nucleotide sequence which is substantially similar, and preferably has at least between 70% and 99% sequence identity to any one of SEQ ID NO: 1 to 461, 501-511, and 513-641, respectively, encoding a polypeptide the expression of which is up-regulated during grain filling, or a fragment or a variant thereof, or a complementary sequence thereto and the sample to be assayed. (b) detecting the hybrid complex formed between the probe and a nucleotide in the sample. The invention further concerns a kit for detecting the presence of a polynucleotide including a nucleotide sequence which is substantially similar, and preferably has at least between 70% and 99% sequence identity to any one of SEQ ID NO: 1 to 461, 501-511, and 513-641, respectively, encoding a polypeptide the expression of which is up-regulated during grain filling, or a fragment or a variant thereof, or a complementary sequence thereto in a sample, the kit including a nucleotide probe or a plurality of nucleotide probes which can hybridize with a nucleotide sequence included in a polynucleotide including a nucleotide sequence which is substantially similar, and preferably has at least between 70% and 99% sequence identity to any one of SEQ ID NO: 1 to 461, 501-511, and 513-641, respectively, encoding a polypeptide the expression of which is up-regulated during grain filing, or a fragment or a variant thereof, or a complementary sequence thereto and, optionally, the reagents necessary for performing the hybridization reaction. In a first preferred embodiment of this detection method and kit, the nucleotide probe or the plurality of nucleotide probes are labeled with a detectable molecule. In a second preferred embodiment of the method and kit, the nucleotide probe or the plurality of nucleotide probes has been immobilized on a substrate. The isolated polynucleotides of the invention can be used to create various types of genetic and physical maps of the genome of rice or other plants. Such maps are used to devise positional cloning strategies for isolating novel genes from the mapped crop species. The sequences of the present invention are also useful for chromosome mapping, chromosome identification, tagging of genes that are involved in the grain filling process. The isolated polynucleotides of the invention can further be used as probes for identifying polymorphisms associated with phenotypes of interest such as, for example, enhanced phosphate utilization, and higher yield. Briefly, total DNA is isolated from an individual or isogenic line, cleaved with one or more restriction enzymes, separated according to mass, transferred to a solid support, and hybridized with a probe molecule according to the invention. The pattern of fragments hybridizing to a probe molecule is compared for DNA from different individuals or lines, where differences in fragment size signals a polymorphism associated with a particular nucleotide sequence according to the present invention. After identification of polymorphic sequences, linkage studies can be conducted. After identification of many polymorphisms using a nucleotide sequence according to the invention, linkage studies can be conducted by using the individuals showing polymorphisms as parents in crossing programs. Recombinants, F 2 progeny recombinants or recombinant inbreds, can then be analyzed using the same restriction enzyme/hybridization procedure. The order of DNA polymorphisms along the chromosomes can be inferred based on the frequency with which they are inherited together versus inherited independently. The closer together two polymorphisms occur in a chromosome, the higher the probability that they are inherited together. Integration of the relative positions of polymorphisms and associated marker sequences produces a genetic map of the species, where the distances between markers reflect the recombination frequencies in that chromosome segment. Preferably, the polymorphisms and marker sequences are sufficiently numerous to produce a genetic map of sufficiently high resolution to locate one or more loci of interest. The use of recombinant inbred lines for such genetic mapping is described for rice (Oh et al, Mol Cells 8:175 (1998); Nandi et al, Mol Gen Genet 255:1 (1997); Wang et al, Genetics 136:1421 (1994)), sorghum (Subudhi et al, Genome 43:240 (2000)), maize (Burr et al., Genetics 118:519 (1998); Gardineret al, Genetics 134:917 (1993)), and Arabidopsis ( Methods in Molecular Biology , Martinez-Zapater and Salinas, eds., 82:137-146, (1998)). However, this procedure is not limited to plants and can be used for other organisms such as yeast or other fungi, or for oomycetes or other protistans. The nucleotide sequences of the present invention can also be used for simple sequence tppeat identification, also known as single sequence repeat, (SSR) mapping. SSR mapping in rice has been described by Miyao et al. ( DNA Res 3:233 (1996)) and Yang et al. ( Mol Gen Genet 245:187 (1994)), and in maize by Ahn et al. ( Mol Gen Genet 241:483 (1993)). SSR mapping can be achieved using various methods. In one instance, polymorphisms are identified when sequence specific probes flanking an SSR contained within an sequence of the invention are made and used in polymerase chain reaction (PCR) assays with template DNA from two or more individuals or, in plants, near isogenic lines. A change in the number of tandem repeats between the SSR-flanking sequence produces differently sized fragments (U.S. Pat. No. 5,766,847). Alternatively, polymorphisms can be identified by using the PCR fragment produced from the SSR-flanking sequence specific primer reaction as a probe against Southern blots representing different individuals (Refseth et al., Electrophoresis 18:1519 (1997)). Rice SSRs were used to map a molecular marker closely linked to a nuclear restorer gene for fertility in rice as described by Akagi et al. ( Genome 39:205 (1996)). The nucleotide sequences of the present invention can be used to identify and develop a variety of microsatellite markers, including the SSRs described above, as genetic markers for comparative analysis and mapping of genomes. The nucleotide sequences of the present invention can be used in a variation of the SSR technique known as inter-SSR (ISSR), which uses microsatellite oligonucleotides as primers to amplify genomic segments different from the repeat region itself (Zietkiewicz et al., Genomics 20:176 (1994)). ISSR employs oligonucleotides based on a simple sequence repeat anchored or not at their 5′- or 3′-end by two to four arbitrarily chosen nucleotides, which triggers site-specific annealing and initiates PCR amplification of genomic segments which are flanked by inversely orientated and closely spaced repeat sequences. In one embodiment of the present invention, microsatellite markers derived from the nucleotide sequences disclosed in the Sequence Listing, or substantially similar sequences or allelic variants thereof, may be used to detect the appearance or disappearance of markers indicating genomic instability as described by Leroy et al. ( Electron. J. Biotechnol, 3(2), at http://www.ejb.org (2000)), where alteration of a fingerprinting pattern indicated loss of a marker corresponding to a part of a gene involved in the regulation of cell proliferation. Microsatellite markers derived from nucleotide sequences as provided in the Sequence Listing will be useful for detecting genomic alterations such as the change observed by Leroy et al. ( Electron. J Biotechnol, 3(2), supra (2000)) which appeared to be the consequence of microsatellite instability at the primer binding site or modification of the region between the microsatellites, and illustrated somaclonal variation leading to genomic instability. Consequently, the nucleotide sequences of the present invention are useful for detecting genomic alterations involved in somaclonal variation, which is an important source of new phenotypes. In addition, because the genomes of closely related species are largely syntenic (that is, they display the same ordering of genes within the genome), these maps can be used to isolate novel alleles from wild relatives of crop species by positional cloning strategies. This shared synteny is very powerful for using genetic maps from one species to map genes in another. For example, a gene mapped in rice provides information for the gene location in maize and wheat. The various types of maps discussed above can be used with the nucleotide sequences of the invention to identify Quantitative Trait Loci (QTLs) for a variety of uses, including marker-assisted breeding. Many important crop traits are quantitative traits and result from the combined interactions of several genes. These genes reside at different loci in the genome, often on different chromosomes, and generally exhibit multiple alleles at each locus. Developing markers, tools, and methods to identify and isolate the QTLs involved regulating the content and composition of the plant grain, enables marker-assisted breeding to enhance the nutritional value of the grain or suppress undesirable traits that interfere with an efficient grain filling process. The nucleotide sequences as provided in the Sequence Listing can be used to generate markers, including single-sequence repeats (SSRs) and microsatellite markers for QTLs and utilization to assist marker-assisted breeding. The nucleotide sequences of the invention can be used to identify QTLs regulating the grain filling process and isolate alleles as described by Li et al. in a study of QTLs involved in resistance to a pathogen of rice. (Li et al., Mol Gen Genet 261:58 (1999)). In addition to isolating QTL alleles in rice, other cereals, and other monocot and dicot crop species, the nucleotide sequences of the invention can also be used to isolate alleles from the corresponding QTL(s) of wild relatives. Transgenic plants having various combinations of QTL alleles can then be created and the effects of the combinations measured. Once an ideal allele combination has been identified, crop improvement can be accomplished either through biotechnological means or by directed conventional breeding programs. (Flowers et al., J Exp Bot 51:99 (2000); Tanksley and McCouch, Science 277:1063 (1997)). In another embodiment the nucleotide sequences of the invention can be used to help create physical maps of the genome of maize, Arabidopsis and related species. Where the nucleotide sequences of the invention have been ordered on a genetic map, as described above, then the nucleotide sequences of the invention can be used as probes to discover which clones in large libraries of plant DNA fragments in YACs, PACs, etc. contain the same nucleotide sequences of the invention or similar sequences, thereby facilitating the assignment of the large DNA fragments to chromosomal positions. Subsequently, the large BACs, YACs, etc. can be ordered unambiguously by more detailed studies of their sequence composition and by using their end or other sequence to find the identical sequences in other cloned DNA fragments (Mozo et al., Nat Genet 22:271 (1999)). Overlapping DNA sequences in this way allows assembly of large sequence contigs that, when sufficiently extended, provide a complete physical map of a chromosome. The nucleotide sequences of the invention themselves may provide the means of joining cloned sequences into a contig, and are useful for constructing physical maps. In another embodiment, the nucleotide sequences of the present invention may be useful in mapping and characterizing the genomes of other cereals. Rice has been proposed as a model for cereal genome analysis Havukkala, Curr Opin Genet Devel 6:711 (1996)), based largely on its smaller genome size and higher gene density, combined with the considerable conserved gene order among cereal genomes (Ahn et al., Mol Gen Genet 241:483 (1993)). The cereals demonstrate both general conservation of gene order (synteny) and considerable sequence homology among various cereal gene families. This suggests that studies on the functions of genes or proteins from rice according to the present invention could lead to elucidation of the functions of orthologous genes or proteins in other cereals, including maize, wheat, secale, sorghum, barley, millet, teff, milo, triticale, flax, gramma grass, Tripsacum sp., and teosinte. The nucleotide sequences according to the invention can also be used to physically characterize homologous chromosomes in other cereals, as described by Sarma et al. ( Genome 43:191 (2000)), and their use can be extended to non-cereal monocots such as sugarcane, grasses, and lilies. Given the synteny between rice and other cereal genomes, the nucleotide sequences of the present invention can be used to obtain molecular markers for mapping and, potentially, for positional cloning. Kilian et al. described the use of probes from the rice genomic region of interest to isolate a saturating number of polymorphic markers in barley, which were shown to map to syntenic regions in rice and barley, suggesting that the nucleotide sequences of the invention derived from the rice genome would be useful in positional cloning of syntenic grain-filling genes of interest from other cereal species. (Kilian, et al., Nucl Acids Res 23:2729 (1995); Kilian, et al, Plant Mol Biol 35:187 (1997)). Synteny between rice and barley has recently been reported in the area of the carrying malting quality QTLs (Han, et al., Genome 41:373 (1998)), and use of synteny between cereals for positional cloning efforts is likely to add considerable value to rice genome analysis. Likewise, mapping of the ligules region of sorghum was facilitated using molecular markers from a syntenic region of the rice genome. (Zwick, et al., Genetics 148:1983 (1998)). Rice marker technology utilizing the nucleotide sequences of the present invention can also be used to identify QTL alleles from a wild relative of cultivated rice, for example as described by Xiao, et al. ( Genetics 150:899 (1998)). Wild relatives of domesticated plants represent untapped pools of genetic resources for abiotic and biotic stress resistance, apomixis and other breeding strategies, plant architecture, determinants of yield, secondary metabolites, and other valuable traits. In rice, Xiao et al. (supra) used molecular markers to introduce an average of approximately 5% of the genome of a wild relative, and the resulting plants were scored for phenotypes such as plant height, panicle length and 1000-grain weight. Trait-improving alleles were found for all phenotypes except plant height, where any change is considered negative. Of the 35 trait-improving alleles, Xiao et al. found that 19 had no effect on other phenotypes whereas 16 had deleterious effects on other traits. The nucleotide sequences of the invention such as those provided in the Sequence Listing can be employed as molecular markers to identify QTL alleles involved in the regulation of the grain filling process from a wild relative, by which these valuable traits can be introgressed from wild relatives using methods including, but not limited to, that described by Xiao et al. ((1998) supra). Accordingly, the nucleotide sequences of the invention can be employed in a variety of molecular marker technologies for yield improvement. Following the procedures described above to identify polymorphisms, and using a plurality of the nucleotide sequences of the invention, any individual (or line) can be genotyped. Genotyping a large number of DNA polymorphisms such as single nucleotide polymorphisms (SNPs), in breeding lines makes it possible to find associations between certain polymorphisms or groups of polymorphisms, and certain phenotypes. In addition to sequence polymorphisms, length polymorphisms such as triplet repeats are studied to find associations between polymorphism and phenotype. Genotypes can be used for the identification of particular cultivars, varieties, lines, ecotypes, and genetically modified plants or can serve as tools for subsequent genetic studies of complex traits involving multiple phenotypes. The patent publication WO95/35505 and U.S. Pat. Nos. 5,445,943 and 5,410,270 describe scanning multiple alleles of a plurality of loci using hybridization to arrays of oligonucleotides. The nucleotide sequences of the invention are suitable for use in genotyping techniques useful for each of the types of mapping discussed above. In a preferred embodiment, the nucleotide sequences of the invention are useful for identifying and isolating a least one unique stretch of protein-encoding nucleotide sequence. The nucleotide sequences of the invention are compared with other coding sequences having sequence similarity with the sequences provided in the Sequence Listing, using a program such as BLAST. Comparison of the nucleotide sequences of the invention with other similar coding sequences permits the identification of one or more unique stretches of coding sequences encoding polypeptides that are up-regulated during grain filling that are not identical to the corresponding coding sequence being screened. Preferably, a unique stretch of coding sequence of about 25 base pairs (bp) long is identified, more preferably 25 bp, or even more preferably 22 bp, or 20 bp, or yet even more preferably 18 bp or 16 bp or 14 bp. In one embodiment, a plurality of nucleotide sequences is is screened to identify unique coding sequences accroding to the invention. In one embodiment, one or more unique coding sequences accroding to the invention can be applied to a chip as part of an array, or used in a non-chip array system. In a further embodiment, a plurality of unique coding sequences accroding to the invention is used in a screening array. In another embodiment, one or more unique coding sequences accroding to the invention can be used as immobilized or as probes in solution. In yet another embodiment, one or more unique coding sequences accroding to the invention can be used as primers for PCR. In a further embodiment, one or more unique coding sequences accroding to the invention can be used as organism specific primers for PCR in a solution containing DNA from a plurality of sources. In another embodiment unique stretches of nucleotide sequences according to the invention are identified that are preferably about 30 bp, more preferably 50 bp or 75 bp, yet more preferably 100 bp, 150 bp, 200 bp, 250, 500 bp, 750 bp, or 1000 bp. The length of an unique coding sequence may be chosen by one of skill in the art depending on its intended use and on the characteristics of the nucleotide sequence being used. In one embodiment, unique coding sequences accroding to the invention may be used as probes to screen libraries to find homologs, orthologs, or paralogs. In another embodiment, unique coding sequences accroding to the invention may be used as probes to screen genomic DNA or cDNA to find homologs, orthologs, or paralogs. In yet another embodiment, unique coding sequences accroding to the invention may be used to study gene evolution and genome evolution. detailed-description description="Detailed Description" end="lead"? |
Proteins associated with cell growth, differentiation, and death |
Various embodiments of the invention provide human proteins associated with cell growth, differentiation, and death (CGDD) and polynucleotides which identify and encode CGDD. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of CGDD. |
1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2-6 and SEQ ID NO:8-19, c) a polypeptide comprising a naturally occurring amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:7, d) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and e) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. 3. An isolated polynucleotide encoding a polypeptide of claim 1. 4. An isolated polynucleotide encoding a polypeptide of claim 2. 5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3. 7. A cell transformed with a recombinant polynucleotide of claim 6. 8. (canceled) 9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed. 10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. 11. An isolated antibody which specifically binds to a polypeptide of claim 1. 12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, c) a polynucleotide complementary to a polynucleotide of a), d) a polynucleotide complementary to a polynucleotide of b), and e) an RNA equivalent of a)-d). 13. (canceled) 14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof. 15. (canceled) 16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof. 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. 19. (canceled) 20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample. 21-22. (canceled) 23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample. 24-25. (canceled) 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. 27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1. 28. (canceled) 29. A method of assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. 30-93. (canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>Human growth and development requires the spatial and temporal regulation of cell differentiation, cell proliferation, and apoptosis. These processes coordinately control reproduction, aging, embryogenesis, morphogenesis, organogenesis, and tissue repair and maintenance. At the cellular level, growth and development is governed by the cell's decision to enter into or exit from the cell division cycle and by the cell's commitment to a terminally differentiated state. These decisions are made by the cell in response to extracellular signals and other environmental cues it receives. The following discussion focuses on the molecular mechanisms of cell division, embryogenesis, cell differentiation and proliferation, and apoptosis, as well as disease states such as cancer which can result from disruption of these mechanisms. Cell Cycle Cell division is the fundamental process by which all living things grow and reproduce. In unicellular organisms such as yeast and bacteria, each cell division doubles the number of organisms. In multicellular species many rounds of cell division are required to replace cells lost by wear or by programmed cell death, and for cell differentiation to produce a new tissue or organ. Progression through the cell cycle is governed by the intricate interactions of protein complexes. This regulation depends upon the appropriate expression of proteins which control cell cycle progression in response to extracellular signals, such as growth factors and other mitogens, and intracellular cues, such as DNA damage or nutrient starvation. Molecules which directly or indirectly modulate cell cycle progression fall into several categories, including cyclins, cyclin-dependent protein kinases, growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, and tumor-suppressor proteins. Details of the cell division cycle may vary, but the basic process consists of three principle events. The first event, interphase, involves preparations for cell division, replication of the DNA, and production of essential proteins. In the second event, mitosis, the nuclear material is divided and separates to opposite sides of the cell. The final event, cytokinesis, is division and fission of the cell cytoplasm. The sequence and timing of cell cycle transitions is under the control of the cell cycle regulation system which controls the process by positive or negative regulatory circuits at various check points. Mitosis marks the end of interphase and concludes with the onset of cytokinesis. There are four stages in mitosis, occurring in the following order: prophase, metaphase, anaphase and telophase. Prophase includes the formation of bi-polar mitotic spindles, composed of microtubules and associated proteins such as dynein, which originate from polar mitotic centers. During metaphase, the nuclear material condenses and develops kinetochore fibers which aid in its physical attachment to the mitotic spindles. The ensuing movement of the nuclear material to opposite poles along the mitotic spindles occurs during anaphase. Telophase includes the disappearance of the mitotic spindles and kinetochore fibers from the nuclear material. Mitosis depends on the interaction of numerous proteins. For example, centromere-associated proteins such as CENP-A, -B, and -C, play structural roles in kinetochore formation and assembly (Saffery, R. et al. (2000) Human Mol. Gen. 9:175-185). During the M phase of eukaryotic cell cycling, structural rearrangements occur ensuring appropriate distribution of cellular components between daughter cells. Breakdown of interphase structures into smaller subunits is common. The nuclear envelope breaks into vesicles, and nuclear lamins are disassembled. Subsequent phosphorylation of these lamins occurs and is maintained until telophase, at which time the nuclear lamina structure is reformed. cDNAs responsible for encoding M phase phosphorylation (MPPs) are components of U3 small nucleolar ribonucleoprotein (snoRNP), and relocalize to the nucleolus once mitosis is complete (Westendorf, J. M. et al. (1998) J. Biol. Chem. 9:437-449). U3 snoRNPs are essential mediators of RNA processing events. Proteins involved in the regulation of cellular processes such as mitosis include the Ser/Thr-protein phosphatases type 1 (PP-1). PP-1s act by dephosphorylation of key proteins involved in the metaphase-anaphase transition. The gene PP1R7 encodes the regulatory polypeptide sds22, having at least six splice variants (Ceulemans, H. et al. (1999) Eur. J. Biochem. 262:3642). Sds22 modulates the activity of the catalytic subunit of PP-1s, and enhances the PP-1-dependent dephosphorylation of mitotic substrates. Cell cycle regulatory proteins play an important role in cell proliferation and cancer. For example, failures in the proper execution and timing of cell cycle events can lead to chromosome segregation defects resulting in aneuploidy or polyploidy. This genomic instability is characteristic of transformed cells (Luca, F. C. and M. Winey (1998) Mol. Biol. Cell. 9:2946). A recently identified protein, mMOB1, is the mammalian homolog of yeast MOB 1, an essential yeast gene required for completion of mitosis and maintenance of ploidy. The mammalian mMOB1 is a member of protein complexes including protein phosphatase 2A (PP2A), and its phosphorylation appears to be regulated by PP2A (Moreno, C. S. et al. (2001) J. Biol. Chem. 276:24253-24260). PP2A has been implicated in the development of human cancers, including lung and colon cancers and leukemias. Cell cycle regulation involves numerous proteins interacting in a sequential manner. The eukaryotic cell cycle consists of several highly controlled events whose precise order ensures successful DNA replication and cell division. Cells maintain the order of these events by making later events dependent on the successful completion of earlier events. This dependency is enforced by cellular mechanisms called checkpoints. Examples of additional cell cycle regulatory proteins include the histone deacetylases (HDACs). HDACs are involved in cell cycle regulation, and modulate chromatin structure. Human HDAC1 has been found to interact in vitro with the human Hus1 gene product, whose Schizosaccharomyces pombe homolog has been implicated in G 2 /M checkpoint control (Cai, R. L. et al. (2000) J. Biol. Chem. 275:27909-27916). DNA damage (G 2 ) and DNA replication (S-phase) checkpoints arrest eukaryotic cells at the G 2 /M transition. This arrest provides time for DNA repair or DNA replication to occur before entry into mitosis. Thus, the G 2 /M checkpoint ensures that mitosis only occurs upon completion of DNA replication and in the absence of chromosomal damage. The Hus1 gene of Schizosaccharomyces pombe is a cell cycle checkpoint gene, as are the rad family of genes (e.g., rad1 and rad9) (Volkmer, E. and L. M. Karnitz (1999) J. Biol. Chem. 274:567-570; Kostrub C. P. et al. (1998) EMBO J. 17:2055-2066). These genes are involved in the mitotic checkpoint, and are induced by either DNA damage or blockage of replication. Induction of DNA damage or replication block leads to loss of function of the Hus1 gene and subsequent cell death. Human homologs have been identified for most of the rad genes, including ATM and ATR, the human homologs of rad3p. Mutations in the ATM gene are correlated with the severe congenital disease ataxia-telagiectasia (Savitsky, K. et al. (1995) Science 268:1749-1753). The human Hus1 protein has been shown to act in a complex with rad1 protein which interacts with rad9, making them central components of a DNA damage-responsive protein complex of human cells (Volkmer and Karnitz, supra). The entry and exit of a cell from mitosis is regulated by the synthesis and destruction of a family of activating proteins called cyclins. Cyclins act by binding to and activating a group of cyclin-dependent protein kinases (Cdks) which then phosphorylate and activate selected proteins involved in the mitotic process. Cyclins are characterized by a large region of shared homology that is approximately 180 amino acids in length and referred to as the “cyclin box” (Chapman, D. L. and D. J. Wolgemuth (1993) Development 118:229-240). In addition, cyclins contain a conserved 9 amino acid sequence in the N-terminal region of the molecule called the “destruction box.” This sequence is believed to be a recognition code that triggers ubiquitin-mediated degradation of cyclin B (Hunt, T. (1991) Nature 349:100-101). Several types of cyclins exist (Ciechanover, A. (1994) Cell 79:13-21). Progression through G1 and S phase is driven by the G1 cyclins and their catalytic subunits, including Cdk2-cyclin A, Cdk2-cyclin E, Cdk4-cyclin D and Cdk6-cyclin D. Progression through the G2-M transition is driven by the activation of mitotic CDK-cyclin complexes such as Cdc2-cyclin A, Cdc2-cyclin B1 and Cdc2-cyclin B2 complexes (reviewed in Yang, J. and S. Kornbluth (1999) Trends Cell Biol. 9:207-210). Cyclins are degraded through the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaroytic cells and in some bacteria. The UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control cellular processes such as gene transcription and cell cycle progression. The UCS is implicated in the degradation of mitotic cyclin kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins, cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, supra). The process of ubiquitin conjugation and protein degradation occurs in five principle steps (Jentsch, S. (1992) Annu. Rev. Genet. 26:179-207). First ubiquitin (Ub), a small, heat stable protein is activated by a ubiquitin-activating enzyme (E1) in an ATP dependent reaction which binds the C-terminus of Ub to the thiol group of an internal cysteine residue in E1. Second, activated Ub is transferred to one of several Ub-conjugating enzymes (E2). Different ubiquitin-dependent proteolytic pathways employ structurally similar, but distinct ubiquitin-conjugating enzymes that are associated with recognition subunits which direct them to proteins carrying a particular degradation signal. Third, E2 transfers the Ub molecule through its C-terminal glycine to a member of the ubiquitin-protein ligase family, E3. Fourth, E3 transfers the Ub molecule to the target protein. Additional Ub molecules may be added to the target protein forming a multi-Ub chain structure. Fifth, the ubiquinated protein is then recognized and degraded by the proteasome, a large, multisubunit proteolytic enzyme complex, and Ub is released for re-utilization. Prior to activation, Ub is usually expressed as a fusion protein composed of an N-terminal ubiquitin and a C-terminal extension protein (CEP) or as a polyubiquitin protein with Ub monomers attached head to tail. CEPs have characteristics of a variety of regulatory proteins; most are highly basic, contain up to 30% lysine and arginine residues, and have nucleic acid-binding domains (Monia, B. P. et al. (1989) J. Biol. Chem. 264:4093-4103). The fusion protein is an important intermediate which appears to mediate co-regulation of the cell's translational and protein degradation activities, as well as localization of the inactive enzyme to specific cellular sites. Once delivered, C-terminal hydrolases cleave the fusion protein to release a functional Ub (Monia et al., supra). Ub-conjugating enzymes (E2s) are important for substrate specificity in different UCS pathways. All E2s have a conserved domain of approximately 16 kDa called the UBC domain that is at least 35% identical in all E2s and contains a centrally located cysteine residue required for ubiquitin-enzyme thiolester formation (Jentsch, supra). A well conserved proline-rich element is located N-terminal to the active cysteine residue. Structural variations beyond this conserved domain are used to classify the E2 enzymes. Class I E2s consist almost exclusively of the conserved UBC domain. Class II E2s have various unrelated C-terminal extensions that contribute to substrate specificity and cellular localization. Class m E2s have unique N-terminal extensions which are believed to be involved in enzyme regulation or substrate specificity. A mitotic cyclin-specific E2 (E2-C) is characterized by the conserved UBC domain, an N-terminal extension of 30 amino acids not found in other E2s, and a 7 amino acid unique sequence adjacent to this extension. These characteristics together with the high affinity of E2-C for cyclin identify it as a new class of E2 (Aristarkhov, A. et al. (1996) Proc. Natl. Acad. Sci.93:4294-99). Ubiquitin-protein ligases (E3s) catalyze the last step in the ubiquitin conjugation process, covalent attachment of ubiquitin to the substrate. E3 plays a key role in determining the specificity of the process. Only a few E3s have been identified so far. One type of E3 ligases is the HECT (homologous to E6-AP C-terminus) domain protein family. One member of the family, E6-AP (E6-associated protein) is required, along with the human papillomavirus (HPV) E6 oncoprotein, for the ubiquitination and degradation of p53 (Scheffner, M. et al. (1993) Cell 75:495-505). The C-terminal domain of HECT proteins contains the highly conserved ubiquitin-binding cysteine residue. The N-terminal region of the various HECT proteins is variable and is believed to be involved in specific substrate recognition (Huibregtse, J. M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:3656-3661). The SCF (Skp1-Cdc53/Cullin-F box receptor) family of proteins comprise another group of ubiquitin ligases (Deshaies, R. (1999) Annu. Rev. Dev. Biol. 15:435-467). Multiple proteins are recruited into the SCF complex, including Skp1, cullin, and an F box domain containing protein. The F box protein binds the substrate for the ubiquitination reaction and may play roles in determining substrate specificity and orienting the substrate for reaction. Skp1 interacts with both the F box protein and cullin and may be involved in positioning the F box protein and cullin in the complex for transfer of ubiquitin from the E2 enzyme to the protein substrate. Substrates of SCF ligases include proteins involved in regulation of CDK activity, activation of transcription, signal transduction, assembly of kinetochores, and DNA replication. Sgt1 was identified in a screen for genes in yeast that suppress defects in kinetochore function caused by mutations in Skp1 (Kitagawa, K. et al. (1999) Mol. Cell 4:21-33). Sgt1 interacts with Skp1 and associates with SCF ubiquitin ligase. Defects in Sgt1 cause arrest of cells at either G1 or G2 stages of the cell cycle. A yeast Sgt1 null mutant can be rescued by human Sgt1, an indication of the conservation of Sgt1 function across species. Sgt1 is required for assembly of kinetochore complexes in yeast. Abnormal activities of the UCS are implicated in a number of diseases and disorders. These include, e.g., cachexia (Llovera, M. et al. (1995) Int. J. Cancer 61:138-141), degradation of the tumor-suppressor protein, p53 (Ciechanover, supra), and neurodegeneration such as observed in Alzheimer's disease (Gregori, L. et al. (1994) Biochem. Biophys. Res. Commun. 203:1731-1738). Since ubiquitin conjugation is a rate-limiting step in antigen presentation, the ubiquitin degradation pathway may also have a critical role in the immune response (Grant, E. P. et al. (1995) J. Immunol. 155:3750-3758). Certain cell proliferation disorders can be identified by changes in the protein complexes that normally control progression through the cell cycle. A primary treatment strategy involves reestablishing control over cell cycle progression by manipulation of the proteins involved in cell cycle regulation (Nigg, E. A. (1995) BioEssays 17:471-480). Embryogenesis Mammalian embryogenesis is a process which encompasses the first few weeks of development following conception. During this period, embryogenesis proceeds from a single fertilized egg to the formation of the three embryonic tissues, then to an embryo which has most of its internal organs and all of its external features. The normal course of mammalian embryogenesis depends on the correct temporal and spatial regulation of a large number of genes and tissues. These regulation processes have been intensely studied in mouse. An essential process that is still poorly understood is the activation of the embryonic genome after fertilization. As mouse oocytes grow, they accumulate transcripts that are either translated directly into proteins or stored for later activation by regulated polyadenylation. During subsequent meiotic maturation and ovulation, the maternal genome is transcriptionally inert, and most maternal transcripts are deadenylated and/or degraded prior to, or together with, the activation of the zygotic genes at the two-cell stage (Stutz, A. et al. (1998) Genes Dev. 12:2535-2548). The maternal to embryonic transition involves the degradation of oocyte, but not zygotic transcripts, the activation of the embryonic genome, and the induction of cell cycle progression to accommodate early development. MATER (Maternal Antigen That Embryos Require) was initially identified as a target of antibodies from mice with ovarian immunity (Tong, Z-B. and L. M. Nelson (1999) Endocrinology 140:3720-3726). Expression of the gene encoding MATER is restricted to the oocyte, making it one of a limited number of known maternal-effect genes in mammals (Tong, Z-B. et al. (2000) Mamm. Genome 11:281-287). The MATER protein is required for embryonic development beyond two cells, based upon preliminary results from mice in which this gene has been inactivated. The 1111-amino acid MATER protein contains a hydrophilic repeat region in the amino terminus, and a region containing 14 leucine-rich repeats in the carboxyl terminus. These repeats resemble the sequence found in porcine ribonuclease inhibitor that is critical for protein-protein interactions. The degradation of maternal transcripts during meiotic maturation and ovulation may involve the activation of a ribonuclease just prior to ovulation. Thus the function of MATER may be to bind to the maternal ribonuclease and prevent degradation of zygotic transcripts (Tong et al., supra). In addition to its role in oocyte development and embryogenesis, MATER may also be relevant to the pathogenesis of ovarian immunity, as it is a target of autoantibodies in mice with autoimmune oophoritis (Tong and Nelson, supra). The maternal mRNA D7 is a moderately abundant transcript in Xenopus laevis whose expression is highest in, and perhaps restricted to, oogenesis and early embryogenesis. The D7 protein is absent from oocytes and first begins to accumulate during oocyte maturation. Its levels are highest during the first day of embryonic development and then they decrease, The loss of D7 protein affects the maturation process itself, significantly delaying the time course of germinal vesicle breakdown. Thus, D7 is a newly described protein involved in oocyte maturation (Smith, R. C. et al. (1988) Genes Dev. 2(10):1296-306.) Many other genes are involved in subsequent stages of embryogenesis. After fertilization, the oocyte is guided by fimbria at the distal end of each fallopian tube into and through the fallopian tube and thence into the uterus. Changes in the uterine endometrium prepare the tissue to support the implantation and embryonic development of a fertilized ovum. Several stages of division have occurred before the dividing ovum, now a blastocyst with about 100 cells, enters the uterus. Upon reaching the uterus, the developing blastocyst usually remains in the uterine cavity an additional two to four days before implanting in the endometrium, the inner lining of the uterus. Implantation results from the action of trophoblast cells that develop over the surface of the blastocyst. These cells secrete proteolytic enzymes that digest and liquefy the cells of the endometrium. The invasive process is reviewed in Fisher, S. J. and C. H. Damsky (1993; Semin Cell Biol 4:183-188) and Graham, C. H. and P. K. Lala (1992; Biochem Cell Biol 70:867-874). Once implantation has taken place, the trophoblast and other sublying cells proliferate rapidly, forming the placenta and the various membranes of pregnancy. (See Guyton, A. C. (1991) Textbook of Medical Physiology, 8 th ed. W. B. Saunders Company, Philadelphia Pa., pp. 915-919.) The placenta has an essential role in protecting and nourishing the developing fetus. In most species the syncytiotrophoblast layer is present on the outside of the placenta at the fetal-maternal interface. This is a continuous structure, one cell deep, formed by the fusion of the constituent trophoblast cells. The syncytiotrophoblast cells play important roles in maternal-fetal exchange, in tissue remodeling during fetal development, and in protecting the developing fetus from the maternal immune response (Stoye, J. P. and J. M. Coffin (2000) Nature 403:715-717). A gene called syncytin is the envelope gene of a human endogenous defective provirus. Syncytin is expressed in high levels in placenta, and more weakly in testis, but is not detected in any other tissues (Mi, S. et al. (2000) Nature 403:785-789). Syncytin expression in the placenta is restricted to the syncytiotrophoblasts. Since retroviral env proteins are often involved in promoting cell fusion events, it was thought that syncytin might be involved in regulating the fusion of trophoblast cells into the syncytiotrophoblast layer. Experiments demonstrated that syncytin can mediate cell fusion in vitro, and that anti-syncytin antibodies can inhibit the fusion of placental cytotrophoblasts (Mi et al., supra). In addition, a conserved immunosuppressive domain present in retroviral envelope proteins, and found in syncytin at amino acid residues 373-397, might be involved in preventing maternal immune responses against the developing embryo. Syncytin may also be involved in regulating trophoblast invasiveness by inducing trophoblast fusion and terminal differentiation (Mi et al., supra). Insufficient trophoblast infiltration of the uterine wall is associated with placental disorders such as preeclampsia, or pregnancy induced hypertension, while uncontrolled trophoblast invasion is observed in choriocarcinoma and other gestational trophoblastic diseases. Thus syncytin function may be involved in these diseases. Cell Differentiation Multicellular organisms are comprised of diverse cell types that differ dramatically both in structure and function, despite the fact that each cell is like the others in its hereditary endowment. Cell differentiation is the process by which cells come to differ in their structure and physiological function. The cells of a multicellular organism all arise from mitotic divisions of a single-celled zygote. The zygote is totipotent, meaning that it has the ability to give rise to every type of cell in the adult body. During development the cellular descendants of the zygote lose their totipotency and become determined. Once its prospective fate is achieved, a cell is said to have differentiated. All descendants of this cell will be of the same type. Human growth and development requires the spatial and temporal regulation of cell differentiation, along with cell proliferation and regulated cell death. These processes coordinate to control reproduction, aging, embryogenesis, morphogenesis, organogenesis, and tissue repair and maintenance. The processes involved in cell differentiation are also relevant to disease states such as cancer, in which case the factors regulating normal cell differentiation have been altered, allowing the cancerous cells to proliferate in an anaplastic, or undifferentiated, state. The mechanisms of differentiation involve cell-specific regulation of transcription and translation, so that different genes are selectively expressed at different times in different cells. Genetic experiments using the fruit fly Drosophila melanogaster have identified regulated cascades of transcription factors which control pattern formation during development and differentiation. These include the homeotic genes, which encode transcription factors containing homeobox motifs. The products of homeotic genes determine how the insect's imaginal discs develop from masses of undifferentiated cells to specific segments containing complex organs. Many genes found to be involved in cell differentiation and development in Drosophila have homologs in mammals. Some human genes have equivalent developmental roles to their Drosophila homologs. The human homolog of the Drosophila eyes absent gene (eya) underlies branchio-oto-renal syndrome, a developmental disorder affecting the ears and kidneys (Abdelhak, S. et al. (1997) Nat. Genet. 15:157-164). The Drosophila slit gene encodes a secreted leucine-rich repeat containing protein expressed by the midline glial cells and required for normal neural development. At the cellular level, growth and development are governed by the cell's decision to enter into or exit from the cell cycle and by the cell's commitment to a terminally differentiated state. Differential gene expression within cells is triggered in response to extracellular signals and other environmental cues. Such signals include growth factors and other mitogens such as retinoic acid; cell-cell and cell-matrix contacts; and environmental factors such as nutritional signals, toxic substances, and heat shock. Candidate genes that may play a role in differentiation can be identified by altered expression patterns upon induction of cell differentiation in vitro. The final step in cell differentiation results in a specialization that is characterized by the production of particular proteins, such as contractile proteins in muscle cells, serum proteins in liver cells and globins in red blood cell precursors. The expression of these specialized proteins depends at least in part on cell-specific transcription factors. For example, the homeobox-containing transcription factor PAX-6 is essential for early eye determination, specification of ocular tissues, and normal eye development in vertebrates. In the case of epidermal differentiation, the induction of differentiation-specific genes occurs either together with or following growth arrest and is believed to be linked to the molecular events that control irreversible growth arrest. Irreversible growth arrest is an early event which occurs when cells transit from the basal to the innermost suprabasal layer of the skin and begin expressing squamous-specific genes. These genes include those involved in the formation of the cross-linked envelope, such as transglutaminase I and III, involucrin, loricin, and small proline-rich repeat (SPRR) proteins. The SPRR proteins are 8-10 kDa in molecular mass, rich in proline, glutamine, and cysteine, and contain similar repeating sequence elements. The SPRR proteins may be structural proteins with a strong secondary structure or metal-binding proteins such as metallothioneins. (Jetten, A. M. and B. L. Harvat (1997) J. Dermatol. 24:711-725; PRINTS Entry PR00021 PRORICH Small proline-rich protein signature.) The Wnt gene family of secreted signaling molecules is highly conserved throughout eukaryotic cells. Members of the Wnt family are involved in regulating chondrocyte differentiation within the cartilage template. Wnt-5a, Wnt-5b and Wnt-4 genes are expressed in chondrogenic regions of the chicken limb, Wnt-5a being expressed in the perichondrium (mesenchymal cells immediately surrounding the early cartilage template). Wnt-5a misexpression delays the maturation of chondrocytes and the onset of bone collar formation in chicken limb (Hartmann, C. and C. J. Tabin (2000) Development 127:3141-3159). Glypicans are a family of cell surface heparan sulfate proteoglycans that play an important role in cellular growth control and differentiation. Cerebroglycan, a heparan sulfate proteoglycan expressed in the nervous system, is involved with the motile behavior of developing neurons (Stipp, C. S. et al. (1994) J. Cell Biol. 124:149-160). Notch plays an active role in the differentiation of glial cells, and influences the length and organization of neuronal processes (for a review, see Frisen, J. and U. Lendahl (2001) Bioessays 23:3-7). The Notch receptor signaling pathway is important for morphogenesis and development of many organs and tissues in multicellular species. Drosophila fringe proteins modulate the activation of the Notch signal transduction pathway at the dorsal-ventral boundary of the wing imaginal disc. Mammalian fringe-related family members participate in boundary determination during segmentation (Johnston, S. H. et al. (1997) Development 124:2245-2254). Recently a number of proteins have been found to contain a conserved cysteine-rich domain of about 60 amino-acid residues called the LIM domain (for Lin-11 Isl-1 Mec-3) (Freyd, G. et al. (1990) Nature 344:876-879; Baltz, R. et al. (1992) Plant Cell 4:1465-1466). In the LIM domain, there are seven conserved cysteine residues and a histidine. The LIM domain binds two zinc ions (Michelsen, J. W. et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:4404-4408). LIM does not bind DNA; rather, it seems to act as an interface for protein-protein interaction. WD repeats represent a common motif in regulatory proteins first identified in the β-subunit of G proteins (Neer, E. J. et al. (1994) Nature 371: 297-300). These repeats comprise about 40 amino acid residues and end with a Trp-Asp (WD) motif. WD repeats appear to be associated with protein-protein interactions rather than enzymatic activity and typically appear in multiples, with 5-7 repeats per protein being average, although proteins containing 11 (e.g., GenBank Accession Nos. P74442 and 018215) and 16 (e.g., GenBank Accession No. Q55563) repeats have been identified. More recently, a polypeptide harboring 30 WD repeats was identified. This 380 kDa polypeptide is encoded by the DMX gene of Drosophila melanogaster and is expressed in embryos, larvae, adults of both sexes, and in adult ovaries (Kraemer, C. et al. (1998) Gene 216:267-276). DMX1 has been identified as a human homologue of DMX that is expressed in bone, breast, eye, foreskin, heart, parathyroid, small intestine, testis, tonsils, uterus, placenta, and in whole embryo preparations. Similar to DMX, DMXL1 comprises at least 28 WD repeats. Structural predictions suggest that DMX/DMXL1 forms N-terminal and C-terminal propeller structures. Human diseases associated with WD repeat-containing polypeptides include, but are not limited to, essential hypertension, rhizomelic chondrodysplasia punctata, Cockayne syndrome, holoprosencephaly, and potentially DiGeorge syndrome. WD repeat-containing proteins are also candidate nuclear retinoblastoma-binding proteins, apoptotic factors, chromatin assembly factors, and TNF signaling factors (Kraemer, C. et al. (2000) Genomics 64:97-101; and references within). The chick embryo has been particularly valuable for the study of developmental biology. Birds have evolved acute vision which requires a significant allocation of resources in terms of the avian central nervous system. In the developing embryo, the forebrain, midbrain, and hindbrain are formed between 26-33 hours after incubation. The structures in the brain required for visual perception are formed from the posterior part of the forebrain at 33-38 hours of incubation and result from the effects of carefully regulated morphogenetic gradients. Many of the factors that regulate the programmed differentiation of the developing chick central nervous system are homeodomain-containing transcription factors (e.g., the Pax-6, Lhx2, Prox-1, Chx10, Msx-1, and Msx-2 genes). Homeobox proteins comprise helix-turn-helix motifs consisting of two a helices connected at a fixed angle by a short amino acid chain. One of the helices binds to the major groove of target DNA. These proteins are critical for specifying the anterior-posterior body axis during development and are conserved throughout the animal kingdom (Pabo, C. O. and R. T. Sauer (1992) Ann. Rev. Biochem. 61:1053-1095). The expression of homeodomain transcription factors is affected by retinoic acid, synthesized from retinaldehyde. This oxidation event is mediated by localized enzyme activities that produces the all-trans isomer of retinoic acid. Similarly, retinoic acid-degrading p450 oxidase activity is localized in regions where control of gene expression by retinoic acid is undesirable. Lessons learned form the study of chick embryos have recently been reviewed by Mey, J. and Thanos, S. ((2000) Brain Research Reviews 32:343-379). Identification of human homologues of these factors is essential for the understanding of human development and genetic diseases. Apoptosis Apoptosis is the genetically controlled process by which unneeded or defective cells undergo programmed cell death. Selective elimination of cells is as important for morphogenesis and tissue remodeling as is cell proliferation and differentiation. Lack of apoptosis may result in hyperplasia and other disorders associated with increased cell proliferation. Apoptosis is also a critical component of the immune response. Immune cells such as cytotoxic T-cells and natural killer cells prevent the spread of disease by inducing apoptosis in tumor cells and virus-infected cells. In addition, immune cells that fail to distinguish self molecules from foreign molecules must be eliminated by apoptosis to avoid an autoimmune response. Apoptotic cells undergo distinct morphological changes. Hallmarks of apoptosis include cell shrinkage, nuclear and cytoplasmic condensation, and alterations in plasma membrane topology. Biochemically, apoptotic cells are characterized by increased intracellular calcium concentration, fragmentation of chromosomal DNA, and expression of novel cell surface components. The molecular mechanisms of apoptosis are highly conserved, and many of the key protein regulators and effectors of apoptosis have been identified. Apoptosis generally proceeds in response to a signal which is transduced intracellularly and results in altered patterns of gene expression and protein activity. Signaling molecules such as hormones and cytokines are known both to stimulate and to inhibit apoptosis through interactions with cell surface receptors. Transcription factors also play an important role in the onset of apoptosis. A number of downstream effector molecules, especially proteases, have been implicated in the degradation of cellular components and the proteolytic activation of other apoptotic effectors. The Bcl-2 family of proteins, as well as other cytoplasmic proteins, are key regulators of apoptosis. There are at least 15 Bcl-2 family members within 3 subfamilies. These proteins have been identified in mammalian cells and in viruses, and each possesses at least one of four Bcl-2 homology domains (BH1 to BH4), which are highly conserved. Bcl-2 family proteins contain the BH1 and BH2 domains, which are found in members of the pro-survival subfamily, while those proteins which are most similar to Bcl-2 have all four conserved domains, enabling inhibition of apoptosis following encounters with a variety of cytotoxic challenges. Members of the pro-survival subfamily include Bcl-2, BCl-x L , Bcl-w, Mcl-1, and A1 in mammals; NF-13 (chicken); CED-9 ( Caenorhabditis elegans ); and viral proteins BHRF1, LMW5-HL, ORF16, KS-Bcl-2, and E1B-19K. The BH3 domain is essential for the function of pro-apoptosis subfamily proteins. The two pro-apoptosis subfamilies, Bax and BH3, include Bax, Bak, and Bok (also called Mtd); and Bik, Blk, Hrk, BNIP3, Bim L , Bad, Bid, and Egl-1 ( C. elegans ); respectively. Members of the Bax subfamily contain the BH1, BH2, and BH3 domains, and resemble Bcl-2 rather closely. In contrast, members of the BH3 subfamily have only the 9-16 residue BH3 domain, being otherwise unrelated to any known protein, and only Bik and Blk share sequence similarity. The proteins of the two pro-apoptosis subfamilies may be the antagonists of pro-survival subfamily proteins. This is illustrated in C. elegans where Egl-1, which is required for apoptosis, binds to and acts via CED-9 (for review, see Adams, J. M. and S. Cory (1998) Science 281:1322-1326). Heterodimerization between pro-apoptosis and anti-apoptosis subfamily proteins seems to have a titrating effect on the functions of these protein subfamilies, which suggests that relative concentrations of the members of each subfamily may act to regulate apoptosis. Heterodimerization is not required for a pro-survival protein; however, it is essential in the BH3 subfamily, and less so in the Bax subfamily. The Bcl-2 protein has 2 isoforms, alpha and beta, which are formed by alternative splicing. It forms homodimers and heterodimers with Bax and Bak proteins and the Bcl-X isoform Bcl-x S . Heterodimerization with Bax requires intact BH1 and BH2 domains, and is necessary for pro-survival activity. The BH4 domain seems to be involved in pro-survival activity as well. Bcl-2 is located within the inner and outer mitochondrial membranes, as well as within the nuclear envelope and endoplasmic reticulum, and is expressed in a variety of tissues. Its involvement in follicular lymphoma (type II chronic lymphatic leukemia) is seen in a chromosomal translocation T(14;18) (q32;q21) and involves immunoglobulin gene regions. The Bcl-x protein is a dominant regulator of apoptotic cell death. Alternative splicing results in three isoforms, Bcl-xB, a long isoform, and a short isoform. The long isoform exhibits cell death repressor activity, while the short isoform promotes apoptosis. Bcl-xL forms heterodimers with Bax and Bak, although heterodimerization with Bax does not seem to be necessary for pro-survival (anti-apoptosis) activity. Bcl-xS forms heterodimers with Bcl-2. Bcl-x is found in mitochondrial membranes and the perinuclear envelope. Bcl-xS is expressed at high levels in developing lymphocytes and other cells undergoing a high rate of turnover. Bcl-xL is found in adult brain and in other tissues' long-lived post-mitotic cells. As with Bcl-2, the BH1, BH2, and BH4 domains are involved in pro-survival activity. The Bcl-w protein is found within the cytoplasm of almost all myeloid cell lines and in numerous tissues, with the highest levels of expression in brain, colon, and salivary gland. This protein is expressed in low levels in testis, liver, heart, stomach, skeletal muscle, and placenta, and a few lymphoid cell lines. Bcl-w contains the BH1, BH2, and BH4 domains, all of which are needed for its cell survival promotion activity. Although mice in which Bcl-w gene function was disrupted by homologous recombination were viable, healthy, and normal in appearance, and adult females had normal reproductive function, the adult males were infertile. In these males, the initial, prepuberty stage of spermatogenesis was largely unaffected and the testes developed normally. However, the seminiferous tubules were disorganized, contained numerous apoptotic cells, and were incapable of producing mature sperm. This mouse model may be applicable to some cases of human male sterility and suggests that alteration of programmed cell death in the testes may be useful in modulating fertility (Print, C. G. et al. (1998) Proc. Natl. Acad. Sci. USA 95:12424-12431). Studies in rat ischemic brain found Bcl-w to be overexpressed relative to its normal low constitutive level of expression in nonischemic brain. Furthermore, in vitro studies to examine the mechanism of action of Bcl-w revealed that isolated rat brain mitochondria were unable to respond to an addition of recombinant Bax or high concentrations of calcium when Bcl-w was also present. The normal response would be the release of cytochrome c from the mitochondria. Additionally, recombinant Bcl-w protein was found to inhibit calcium-induced loss of mitochondrial transmembrane potential, which is indicative of permeability transition. Together these findings suggest that Bcl-w may be a neuro-protectant against ischemic neuronal death and may achieve this protection via the mitochondrial death-regulatory pathway (Yan, C. et al. (2000) J. Cereb. Blood Flow Metab. 20:620-630). The bfl-1 gene is an additional member of the Bcl-2 family, and is also a suppressor of apoptosis. The Bfl-1 protein has 175 amino acids, and contains the BH1, BH2, and BH3 conserved domains found in Bcl-2 family members. It also contains a Gln-rich NH2-terminal region and lacks an NH domain 1, unlike other Bcl-2 family members. The mouse A1 protein shares high sequence homology with Bfl-1 and has the 3 conserved domains found in Bfl-1. Apoptosis induced by the p53 tumor suppressor protein is suppressed by Bfl-1, similar to the action of Bcl-2, Bcl-xL, and EBV-BHRF1 (D'Sa-Eipper, C. et al. (1996) Cancer Res. 56:3879-3882). Bfl-1 is found intracellularly, with the highest expression in the hematopoietic compartment, i.e. blood, spleen, and bone marrow; moderate expression in lung, small intestine, and testis; and minimal expression in other tissues. It is also found in vascular smooth muscle cells and hematopoietic malignancies. A correlation has been noted between the expression level of bfl-1 and the development of stomach cancer, suggesting that the Bfl-1 protein is involved in the development of stomach cancer, either in the promotion of cancerous cell survival or in cancer (Choi, S. S. et al. (1995) Oncogene 11:1693-1698). Cancers are characterized by continuous or uncontrolled cell proliferation. Some cancers are associated with suppression of normal apoptotic cell death. Strategies for treatment may involve either reestablishing control over cell cycle progression, or selectively stimulating apoptosis in cancerous cells (Nigg, E. A. (1995) BioEssays 17:471-480). Immunological defenses against cancer include induction of apoptosis in mutant cells by tumor suppressors, and the recognition of tumor antigens by T lymphocytes. Response to mitogenic stresses is frequently controlled at the level of transcription and is coordinated by various transcription factors. For example, the Rel/NF-kappa B family of vertebrate transcription factors plays a pivotal role in inflammatory and immune responses to radiation. The NF-kappa B family includes p50, p52, RelA, RelB, cRel, and other DNA-binding proteins. The p52 protein induces apoptosis, upregulates the transcription factor c-Jun, and activates c-Jun N-terminal kinase 1 (JNK1) (Sun, L. et al. (1998) Gene 208:157-166). Most NF-kappa B proteins form DNA-binding homodimers or heterodimers. Dimerization of many transcription factors is mediated by a conserved sequence known as the bZIP domain, characterized by a basic region followed by a leucine zipper. The Fas/Apo-1 receptor (FAS) is a member of the tumor necrosis factor (TNF) receptor family. Upon binding its ligand (Fas ligand), the membrane-spanning FAS induces apoptosis by recruiting several cytoplasmic proteins that transmit the death signal. One such protein, termed FAS-associated protein factor 1 (FAF1), was isolated from mice, and it was demonstrated that expression of FAF1 in L cells potentiated FAS-induced apoptosis (Chu, K. et al. (1995) Proc. Natl. Acad. Sci. USA 92:11894-11898). Subsequently, FAS-associated factors have been isolated from numerous other species, including fruit fly and quail (Frohlich, T. et al. (1998) J. Cell Sci. 111:2353-2363). Another cytoplasmic protein that functions in the transmittal of the death signal from Fas is the Fas-associated death domain protein, also known as FADD. FADD transmits the death signal in both FAS-mediated and TNF receptor-mediated apoptotic pathways by activating caspase-8 (Bang, S. et al. (2000) J. Biol. Chem. 275:36217-36222). Fragmentation of chromosomal DNA is one of the hallmarks of apoptosis. DNA fragmentation factor (DFF) is a protein composed of two subunits, a 40-kDa caspase-activated nuclease termed DFF40/CAD, and its 45-kDa inhibitor DFF45/ICAD. Two mouse homologs of DFF45/ICAD, termed CIDE-A and CIDE-B, have recently been described (Inohara, N. et al. (1998) EMBO J. 17:2526-2533). CIDE-A and CIDE-B expression in mammalian cells activated apoptosis, while expression of CIDE-A alone induced DNA fragmentation. In addition, FAS-mediated apoptosis was enhanced by CIDE-A and CIDE-B, further implicating these proteins as effectors that mediate apoptosis. Transcription factors play an important role in the onset of apoptosis. A number of downstream effector molecules, particularly proteases such as the cysteine proteases called caspases, are involved in the initiation and execution phases of apoptosis. The activation of the caspases results from the competitive action of the pro-survival and pro-apoptosis Bcl-2-related proteins (Print, C. G. et al. (1998) Proc. Natl. Acad. Sci. USA 95:12424-12431). A pro-apoptotic signal can activate initiator caspases that trigger a proteolytic caspase cascade, leading to the hydrolysis of target proteins and the classic apoptotic death of the cell. Two active site residues, a cysteine and a histidine, have been implicated in the catalytic mechanism. Caspases are among the most specific endopeptidases, cleaving after aspartate residues. Caspases are synthesized as inactive zymogens consisting of one large (p20) and one small (p10) subunit separated by a small spacer region, and a variable N-terminal prodomain. This prodomain interacts with cofactors that can positively or negatively affect apoptosis. An activating signal causes autoproteolytic cleavage of a specific aspartate residue (D297 in the caspase-1 numbering convention) and removal of the spacer and prodomain, leaving a p10/p20 heterodimer. Two of these heterodimers interact via their small subunits to form the catalytically active tetramer. The long prodomains of some caspase family members have been shown to promote dimerization and auto-processing of procaspases. Some caspases contain a “death effector domain” in their prodomain by which they can be recruited into self-activating complexes with other caspases and FADD protein-caspase family members can associate, changing the substrate specificity of the resultant tetramer. Tumor necrosis factor (TNF) and related cytokines induce apoptosis in lymphoid cells. (Reviewed in Nagata, S. (1997) Cell 88:355-365.) Binding of TNF to its receptor triggers a signal transduction pathway that results in the activation of a proteolytic caspase cascade. One such caspase, ICE (Interleukin-1β converting enzyme), is a cysteine protease comprised of two large and two small subunits generated by ICE auto-cleavage (Dinarello, C. A. (1994) FASEB J. 8:1314-1325). ICE is expressed primarily in monocytes. ICE processes the cytokine precursor, interleulin-1β, into its active form, which plays a central role in acute and chronic inflammation, bone resorption, myelogenous leukemia, and other pathological processes. ICE and related caspases cause apoptosis when overexpressed in transfected cell lines. A caspase recruitment domain (CARD) is found within the prodomain of several apical caspases and is conserved in several apoptosis regulatory molecules such as Apaf-2, RAIDD, and cellular inhibitors of apoptosis proteins (IAPs) (Hofmann, K. et al. (1997) Trends Biochem. Sci. 22:155-157). The regulatory role of CARD in apoptosis may be to allow proteins such as Apaf-1 to associate with caspase-9 (Li, P. et al. (1997) Cell 91:479-489). A human cDNA encoding an apoptosis repressor with a CARD (ARC) which is expressed in both skeletal and cardiac muscle has been identified and characterized. ARC functions as an inhibitor of apoptosis and interacts selectively with caspases (Koseki, T. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5156-5160). All of these interactions have clear effects on the control of apoptosis (reviewed in Chan S. L. and M. P. Mattson (1999) J. Neurosci. Res. 58:167-190; Salveson, G. S. and V. M. Dixit (1999) Proc. Natl. Acad. Sci. USA 96:10964-10967). ES18 was identified as a potential regulator of apoptosis in mouse T-ells (Park, E. J. et al. (1999) Nuc. Acid. Res. 27:1524-1530). ES18 is 428 amino acids in length, contains an N-terminal proline-rich region, an acidic glutamic acid-rich domain, and a putative LXXLL nuclear receptor binding motif. The protein is preferentially expressed in lymph nodes and thymus. The level of ES18 expression increases in T-ell thymoma S49.1 in response to treatment with dexamethasone, staurosporine, or C2-ceramide, which induce apoptosis. ES 18 may play a role in stimulating apoptotic cell death in T-cells. The rat ventral prostate (RVP) is a model system for the study of hormone-regulated apoptosis. RVP epithelial cells undergo apoptosis in response to androgen deprivation. Messenger RNA (mRNA) transcripts that are up-regulated in the apoptotic RVP have been identified (Briehl, M. M. and R. L. Miesfeld (1991) Mol. Endocrinol. 5:1381-1388). One such transcript encodes RVP.1, the precise role of which in apoptosis has not been determined. The human homolog of RVP.1, hRVP1, is 89% identical to the rat protein (Katahira, J. et al. (1997) J. Biol. Chem. 272:26652-26658). hRVP1 is 220 amino acids in length and contains four transmembrane domains. hRVP1 is highly expressed in the lung, intestine, and liver. Interestingly, hRVP1 functions as a low affinity receptor for the Clostridium perfringens enterotoxin, a causative agent of diarrhea in humans and other animals. Cytoline-mediated apoptosis plays an important role in hematopoiesis and the immune response. Myeloid cells, which are the stem cell progenitors of macrophages, neutrophils, erythrocytes, and other blood cells, proliferate in response to specific cytokines such as granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3). When deprived of GM-CSF or IL-3, myeloid cells undergo apoptosis. The murine requiem (req) gene encodes a putative transcription factor required for this apoptotic response in the myeloid cell line FDCP-1 (Gabig, T. G. et al. (1994) J. Biol. Chem. 269:29515-29519). The Req protein is 371 amino acids in length and contains a nuclear localization signal, a single Kruppel-type zinc finger, an acidic domain, and a cluster of four unique zinc-finger motifs enriched in cysteine and histidine residues involved in metal binding. Expression of req is not myeloid- or apoptosis-specific, suggesting that additional factors regulate Req activity in myeloid cell apoptosis. Dysregulation of apoptosis has recently been recognized as a significant factor in the pathogenesis of many human diseases. For example, excessive cell survival caused by decreased apoptosis can contribute to disorders related to cell proliferation and the immune response. Such disorders include cancer, autoimmune diseases, viral infections, and inflammation. In contrast, excessive cell death caused by increased apoptosis can lead to degenerative and immunodeficiency disorders such as AIDS, neurodegenerative diseases, and myelodysplastic syndromes. (Thompson, C. B. (1995) Science 267:1456-1462.) Impaired regulation of apoptosis is also associated with loss of neurons in Alzheimer's disease. Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by the formation of senile plaques and neurofibrillary tangles containing amyloid beta peptide. These plaques are found in limbic and association cortices of the brain, including hippocampus, temporal cortices, cingulate cortex, amygdala, nucleus basalis and locus caeruleus. B-amyloid peptide participates in signaling pathways that induce apoptosis and lead to the death of neurons (Kajkowski, C. et al. (2001) J. Biol. Chem. 276:18748-18756). Early in Alzheimer's pathology, physiological changes are visible in the cingulate cortex (Minoshima, S. et al. (1997) Annals of Neurology 42:85-94). In subjects with advanced Alzheimer's disease, accumulating plaques damage the neuronal architecture in limbic areas and eventually cripple the memory process. Cancer Cancers are characterized by continuous or uncontrolled cell proliferation. Some cancers are associated with suppression of normal apoptotic cell death. Understanding of the neoplastic process can be aided by the identification of molecular markers of prognostic and diagnostic importance. Cancers are associated with oncoproteins which are capable of transforming normal cells into malignant cells. Some oncoproteins are mutant isoforms of the normal protein while others are abnormally expressed with respect to location or level of expression. Normal cell proliferation begins with binding of a growth factor to its receptor on the cell membrane, resulting in activation of a signal system that induces and activates nuclear regulatory factors to initiate DNA transcription, subsequently leading to cell division. Classes of oncoproteins known to affect the cell cycle controls include growth factors, growth factor receptors, intracellular signal transducers, nuclear transcription factors, and cell-cycle control proteins. Several types of cancer-specific genetic markers, such as tumor antigens and tumor suppressors, have also been identified. Oncogenes Oncoproteins are encoded by genes, called oncogenes, that are derived from genes that normally control cell growth and development. Many oncogenes have been identified and characterized. These include growth factors such as sis, receptors such as erbA, erbB, neu, and ros, intracellular receptors such as src, yes, fps, abl, and met, protein-serine/threonine kinases such as mos and raf, nuclear transcription factors such as jun, fos, nyc, N-inyc, myb, ski, and rel, cell cycle control proteins such as RB and p53, mutated tumor-suppressor genes such as mdm2, Cip1, p16, and cyclin D, ras, set, can, sec, and gag R10. Viral oncogenes are integrated into the human genome after infection of human cells by certain viruses. Examples of viral oncogenes include v-src, v-abl, and v-fps. Transformation of normal genes to oncogenes may also occur by chromosomal translocation. The Philadelphia chromosome, characteristic of chronic myeloid leukemia and a subset of acute lymphoblastic leukemias, results from a reciprocal translocation between chromosomes 9 and 22 that moves a truncated portion of the proto-oncogene c-abl to the breakpoint cluster region (bcr) on chromosome 22. The hybrid c-abl-bcr gene encodes a chimeric protein that has tyrosine kinase activity. In chronic myeloid leukemia, the chimeric protein has a molecular weight of 210 kd, whereas in acute leukemias a more active 180 kd tyrosine kinase is formed (Robbins, S. L. et al. (1994) Pathologic Basis of Disease , W. B. Saunders Co., Philadelphia Pa.). The Ras superfamily of small GTPases is involved in the regulation of a wide range of cellular signaling pathways. Ras family proteins are membrane-associated proteins acting as molecular switches that bind GTP and GDP, hydrolyzing GTP to GDP. In the active GTP-bound state Ras family proteins interact with a variety of cellular targets to activate downstream signaling pathways. For example, members of the Ras subfamily are essential in transducing signals from receptor tyrosine kinases (RTKs) to a series of serine/threonine kinases which control cell growth and differentiation. Activated Ras genes were initially found in human cancers and subsequent studies confirmed that Ras function is critical in the determination of whether cells continue to grow or become terminally differentiated (Barbacid, M. (1987) Annu. Rev. Biochem. 56:779-827; Treisman, R. (1994) Curr. Opin. Genet. Dev. 4:96-98). Mutant Ras proteins, which bind but can not hydrolyze GTP, are permanently activated, and cause continuous cell proliferation or cancer. Activation of Ras family proteins is catalyzed by guanine nucleotide exchange factors (GEFs) which catalyze the dissociation of bound GDP and subsequent binding of GTP. A recently discovered RalGEF-like protein, RGL3, interacts with both Ras and the related protein Rit. Constitutively active Rit, like Ras, can induce oncogenic transformation, although since Rit fails to interact with most known Ras effector proteins, novel cellular targets may be involved in Rit transforming activity. RGL3 interacts with both Ras and Rit, and thus may act as a downstream effector for these proteins (Shao, H. and D. A. Andres (2000) J. Biol. Chem. 275:26914-26924). Tumor Antigens Tumor antigens are cell surface molecules that are differentially expressed in tumor cells relative to non-tumor tissues. Tumor antigens make tumor cells immunologically distinct from normal cells and are potential diagnostics for human cancers. Several monoclonal antibodies have been identified which react specifically with cancerous cells such as T-cell acute lymphoblastic leukemia and neuroblastoma (Minegishi, M. et al. (1989) Leukemia Res. 13:43-51; Takagi, S. et al. (1995) Int. J. Cancer 61:706-715). In addition, the discovery of high level expression of the ER2 gene in breast tumors has led to the development of therapeutic treatments (Liu, E. et al. (1992) Oncogene 7:1027-1032; Kern, J. A. (1993) Am. J. Respir. Cell Mol. Biol. 9:448-454). Tumor antigens are found on the cell surface and have been characterized either as membrane proteins or glycoproteins. For example, MAGE genes encode a family of tumor antigens recognized on melanoma cell surfaces by autologous cytolytic T lymphocytes. Among the 12 human MAGE genes isolated, half are differentially expressed in tumors of various histological types (De Plaen, E. et al. (1994) Immunogenetics 40:360-369). None of the 12 MAGE genes, however, is expressed in healthy tissues except testis and placenta. Tumor Suppressors Tumor suppressor genes are generally defined as genetic elements whose loss or inactivation contributes to the deregulation of cell proliferation and the pathogenesis and progression of cancer. Tumor suppressor genes normally function to control or inhibit cell growth in response to stress and to limit the proliferative life span of the cell. Several tumor suppressor genes have been identified including the genes encoding the retinoblastoma (Rb) protein, p53, and the breast cancer 1 and 2 proteins (BRCA1 and BRCA2). Mutations in these genes are associated with acquired and inherited genetic predisposition to the development of certain cancers. The role of p53 in the pathogenesis of cancer has been extensively studied. (Reviewed in Aggarwal, M. L. et al. (1998) J. Biol. Chem. 273:14; Levine, A. (1997) Cell 88:323-331.) About 50% of all human cancers contain mutations in the p53 gene. These mutations result in either the absence of functional p53 or, more commonly, a defective form of p53 which is overexpressed. p53 is a transcription factor that contains a central core domain required for DNA binding. Most cancer-associated mutations in p53 localize to this domain. In normal proliferating cells, p53 is expressed at low levels and is rapidly degraded. p53 expression and activity is induced in response to DNA damage, abortive mitosis, and other stressful stimuli. In these instances, p53 induces apoptosis or arrests cell growth until the stress is removed. Downstream effectors of p53 activity include apoptosis-specific proteins and cell cycle regulatory proteins, including Rb, oncogene products, cyclins, and cell cycle-dependent kinases. The metastasis-suppressor gene KAI1 (CD82) has been reported to be related to the tumor suppressor gene p53. KAI1 is involved in the progression of human prostatic cancer and possibly lung and breast cancers when expression is decreased. KAI1 encodes a member of a structurally distinct family of leukocyte surface glycoproteins. The family is known as either the tetraspan transmembrane protein family or transmembrane 4 superfamily (TM4SF) as the members of this family span the plasma membrane four times. The family is composed of integral membrane proteins having a N-terminal membrane-anchoring domain which functions as both a membrane anchor and a translocation signal during protein biosynthesis. The N-terminal membrane-anchoring domain is not cleaved during biosynthesis. TM4SF proteins have three additional transmembrane regions, seven or more conserved cysteine residues, are similar in size (218 to 284 residues), and all have a large extracellular hydrophilic domain with three potential N-glycosylation sites. The promoter region contains many putative binding motifs for various transcription factors, including five AP2 sites and nine SpI sites. Gene structure comparisons of KAI1 and seven other members of the TM4SF indicate that the splicing sites relative to the different structural domains of the predicted proteins are conserved. This suggests that these genes are related evolutionarily and arose through gene duplication and divergent evolution (Levy, S. et al. (1991) J. Biol. Chem. 266:14597-14602; Dong, J. T. et al. (1995) Science 268:884-886; Dong, J. T. et al., (1997) Genomics 41:25-32). The Leucine-rich gene-Glioma Inactivated (LGI1) protein shares homology with a number of transmembrane and extracellular proteins which function as receptors and adhesion proteins. LGI1 is encoded by an LLR (leucine-rich, repeat-containing) gene and maps to 10q24. LGI1 has four LLRs which are flanked by cysteine-rich regions and one transmembrane domain (Somerville, R. P. et al. (2000) Mamm. Genome 11:622-627). LGI1 expression is seen predominantly in neural tissues, especially brain. The loss of tumor suppressor activity is seen in the inactivation of the LGI1 protein which occurs during the transition from low to high-grade tumors in malignant gliomas. The reduction of LGI1 expression in low grade brain tumors and its significant reduction or absence of expression in malignant gliomas suggests that it could be used for diagnosis of glial tumor progression (Chernova, O. B. et al. (1998) Oncogene 17:2873-2881). The ST13 tumor suppressor was identified in a screen for factors related to colorectal carcinomas by subtractive hybridization between cDNA of normal mucosal tissues and mRNA of colorectal carcinoma tissues (Cao, J. et al. (1997) J. Cancer Res. Clin. Oncol. 123:447-451). ST13 is down-regulated in human colorectal carcinomas. Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are associated with retinal and central nervous system hemangioblastomas, clear cell renal carcinomas, and pheochromocytomas (Hoffman, M. et al. (2001) Hum. Mol. Genet. 10: 1019-1027; Kamada, M. (2001) Cancer Res. 61:4184-4189). Tumor progression is linked to defects or inactivation of the VHL gene. VHL regulates the expression of transforming growth factor-a, the GLUT-1 glucose transporter and vascular endothelial growth factor. The VHL protein associates with elongin B, elongin C, CuI2 and Rbx1 to form a complex that regulates the transcriptional activator hypoxia-inducible factor (HIF). HIF induces genes involved in angiogenesis such as vascular endothelial growth factor and platelet-derived growth factor B. Loss of control of HIF caused by defects in VHL results in the excessive production of angiogenic peptides. VHL may play roles in inhibition of angiogenesis, cell cycle control, fibronectin matrix assembly, cell adhesion, and proteolysis. Mutations in tumor suppressor genes are a common feature of many cancers and often appear to affect a critical step in the pathogenesis and progression of tumors. Accordingly, Chang, F. et al. (1995; J. Clin. Oncol. 13:1009-1022) suggest that it may be possible to use either the gene or an antibody to the expressed protein 1) to screen patients at increased risk for cancer, 2) to aid in diagnosis made by traditional methods, and 3) to assess the prognosis of individual cancer patients. In addition, Hamada, K. et al. (1996; Cancer Res. 56:3047-3054) are investigating the introduction of p53 into cervical cancer cells via an adenoviral vector as an experimental therapy for cervical cancer. The PR-domain genes were recently recognized as playing a role in human tumorigenesis. PR-domain genes normally produce two protein products: the PR-plus product, which contains the PR domain, and the PR-minus product which lacks this domain. In cancer cells, PR-plus is disrupted or overexpressed, while PR-minus is present or overexpressed. The imbalance in the amount of these two proteins appears to be an important cause of malignancy (Jiang, G. L. and S. Huang (2000) Histol. Histopathol. 15:109-117). Many neoplastic disorders in humans can be attributed to inappropriate gene transcription. Malignant cell growth may result from either excessive expression of tumor promoting genes or insufficient expression of tumor suppressor genes (Cleary, M. L. (1992) Cancer Surv. 15:89-104). Chromosomal translocations may also produce chimeric loci which fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. An important class of transcriptional regulators are the zinc finger proteins. The zinc finger motif, which binds zinc ions, generally contains tandem repeats of about 30 amino acids consisting of periodically spaced cysteine and histidine residues. Examples of this sequence pattern include the C 2 H 2 -type, C4-type, and C3HC4-type zinc fingers, and the PHD domain (Lewin, B. (1990) Genes IV , Oxford University Press, New York, N.Y., and Cell Press, Cambridge, Mass., pp. 554-570; Aasland, R., et al. (1995) Trends Biochem. Sci. 20:56-59). One clinically relevant zinc-finger protein is WT1, a tumor-suppressor protein that is inactivated in children with Wilm's tumor. The oncogene bcl-6, which plays an important role in large-cell lymphoma, is also a zinc-finger protein (Papavassiliou, A. G. (1995) N. Engl. J. Med. 332:4547). Tumor Responsive Proteins Cancers, also called neoplasias, are characterized by continuous and uncontrolled cell proliferation. They can be divided into three categories: carcinomas, sarcomas, and leukemias. Carcinomas are malignant growths of soft epithelial cells that may infiltrate surrounding tissues and give rise to metastatic tumors. Sarcomas may be of epithelial origin or arise from connective tissue. Leukemias are progressive malignancies of blood-forming tissue characterized by proliferation of leukocytes and their precursors, and may be classified as myelogenous (granulocyte- or monocyte-derived) or lymphocytic (lymphocyte-derived). Tumorigenesis refers to the progression of a tumor's growth from its inception. Malignant cells may be quite similar to normal cells within the tissue of origin or may be undifferentiated (anaplastic). Tumor cells may possess few nuclei or one large polymorphic nucleus. Anaplastic cells may grow in a disorganized mass that is poorly vascularized and as a result contains large areas of ischemic necrosis. Differentiated neoplastic cells may secrete the same proteins as the tissue of origin. Cancers grow, infiltrate, invade, and destroy the surrounding tissue through direct seeding of body cavities or surfaces, through lymphatic spread, or through hematogenous spread. Cancer remains a major public health concern and current preventative measures and treatments do not match the needs of most patients. Understanding of the neoplastic process of tumorigenesis can be aided by the identification of molecular markers of prognostic and diagnostic importance. Current forms of cancer treatment include the use of immunosuppressive drugs (Morisaki, T. et al. (2000) Anticancer Res. 20:3363-3373; Geoerger, B. et al. (2001) Cancer Res. 61:1527-1532). The identification of proteins involved in cell signaling, and specifically proteins that act as receptors for immunosuppressant drugs, may facilitate the development of anti-tumor agents. For example, immunophilins are a family of conserved proteins found in both prokaryotes and eukaryotes that bind to immunosuppressive drugs with varying degrees of specificity. One such group of immunophilic proteins is the peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) family (PPIase, rotamase). These enzymes, first isolated from porcine kidney cortex, accelerate protein folding by catalyzing the cis-trans isomerization of proline imidic peptide bonds in oligopeptides (Fischer, G. and F. X. Schmid (1990) Biochemistry 29:2205-2212). Included within the immunophilin family are the cyclophilins (e.g., peptidyl-prolyl isomerase A or PPIA) and FK-binding protein (e.g., FKBP) subfamilies. Cyclophilins are multifunctional receptor proteins which participate in signal transduction activities, including those mediated by cyclosporin (or cyclosporine). The PPIase domain of each family is highly conserved between species. Although structurally distinct, these multifunctional receptor proteins are involved in numerous signal transduction pathways, and have been implicated in folding and trafficking events. The immunophilin protein cyclophilin binds to the immunosuppressant drug cyclosporin A. FKBP, another immunophilin, binds to FK506 (or rapamycin). Rapamycin is an immunosuppressant agent that arrests cells in the G 1 phase of growth, inducing apoptosis. Like cyclophilin, this macrolide antibiotic (produced by Streptomyces tsukubaensis ) acts by binding to ubiquitous, predominantly cytosolic immunophilin receptors. These immunophilin/immunosuppressant complexes (e.g., cyclophilin A/cyclosporin A (CypA/CsA) and FKBP12/FK506) achieve their therapeutic results through inhibition of the phosphatase calcineurin, a calcium/calnodulin-dependent protein kinase that participates in T-cell activation (Hamilton, G. S. and J. P. Steiner (1998) J. Med. Chem. 41: 5119-5143). The murine fkbp51 gene is abundantly expressed in immunological tissues, including the thymus and T lymphocytes (Baughman, G. et al. (1995) Molec. Cell. Biol. 15: 4395-4402). FKBP12/rapamycin-directed immunosuppression occurs through binding to TOR (yeast) or FRAP (FKBP12-rapamycin-associated protein, in mammalian cells), the kinase target of rapamycin essential for maintaining normal cellular growth patterns. Dysfunctional TOR signaling has been linked to various human disorders including cancer (Metcalfe, S. M. et al. (1997) Oncogene 15:1635-1642; Emami, S. et al. (2001) FASEB J. 15:351-361), and autoimmunity (Damoiseaux, J. G. et al. (1996) Transplantation 62:994-1001). Several cyclophilin isozymes have been identified, including cyclophilin B, cyclophilin C, mitochondrial matrix cyclophilin, bacterial cytosolic and periplasmic PPIases, and natural-killer cell cyclophilin-related protein possessing a cyclophilin-type PPIase domain, a putative tumor-recognition complex involved in the function of natural killer (NK) cells. These cells participate in the innate cellular immune response by lysing virally-infected cells or transformed cells. NK cells specifically target cells that have lost their expression of major histocompatibility complex (MHC) class I genes (common during tumorigenesis), endowing them with the potential for attenuating tumor growth. A 150-kDa molecule has been identified on the surface of human NK cells that possesses a domain which is highly homologous to cyclophilin/peptidyl-prolyl cis-trans isomerase. This cyclophilin-type protein may be a component of a putative tumor-recognition complex, a NK tumor recognition sequence (NK-TR) (Anderson, S. K. et al. (1993) Proc. Natl. Acad. Sci. USA 90:542-546). The NKTR tumor recognition sequence mediates recognition between tumor cells and large granular lymphocytes (LGLs), a subpopulation of white blood cells (comprised of activated cytotoxic T cells and natural killer cells) capable of destroying tumor targets. The protein product of the NKTR gene presents on the surface of LGLs and facilitates binding to tumor targets. More recently, a mouse Nktr gene and promoter region have been located on chromosome 9. The gene encodes a NK-cell-specific 150-kDa protein (NK-TR) that is homologous to cyclophilin and other tumor-responsive proteins (Simons-Evelyn, M. et al. (1997) Genomics 40:94-100). Other proteins that interact with tumorigenic tissue include cytokines such as tumor necrosis factor (TNF). The TNF family of cytokines are produced by lymphocytes and macrophages, and can cause the lysis of transformed (tumor) endothelial cells. Endothelial protein 1 (Edp1) has been identified as a human gene activated transcriptionally by TNF-alpha in endothelial cells, and a TNF-alpha inducible Edp1 gene has been identified in the mouse (Swift, S. et al. (1998) Biochim Biophys. Acta 1442:394-398). Expression Profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. Colorectal cancer is the fourth most common cancer and the second most common cause of cancer death in the United States with approximately 130,000 new cases and 55,000 deaths per year. Colon and rectal cancers share many environmental risk factors and both are found in individuals with specific genetic syndromes. (See Potter, J D (1999) J. Natl Cancer Institute 91:916-932 for a review of colorectal cancer.) Colon cancer is the only cancer that occurs with approximately equal frequency in men and women, and the five-year survival rate following diagnosis of colon cancer is around 55% in the United States (Ries et al. (1990) National Institutes of Health, DHHS Publ No. (NIH)90-2789). Colon cancer is causally related to both genes and the environment. Several molecular pathways have been linked to the development of colon cancer, and the expression of key genes in any of these pathways may be lost by inherited or acquired mutation or by hypermethylation. Two of these molecular pathways are associated with inherited genetic syndromes that carry a markedly elevated risk of developing colon cancer. For example, it is well known that abnormal patterns of DNA methylation occur consistently in human tumors and include, simultaneously, widespread genomic hypomethylation and localized areas of increased methylation. In colon cancer in particular, it has been found that these changes occur early in tumor progression such as in premalignant polyps that precede colon cancer. Indeed, DNA methyltransferase, the enzyme that performs DNA methylation, is significantly increased in histologically normal mucosa from patients with colon cancer or the benign polyps that precede cancer, and this increase continues during the progression of colonic neoplasms (Wafik, S et al. (1991) Proc Natl Acad Sci USA 88:3470-3474). Increased DNA methylation occurs in G+C rich areas of genomic DNA termed “CpG islands” that are important for maintenance of an “open” transcriptional conformation around genes, and that hypermethylation of these regions results in a “closed” conformation that silences gene transcription. It has been suggested that the silencing or downregulation of differentiation genes by such abnormal methylation of CpG islands may prevent differentiation in immortalized cells (Anteguera, F. et al. (1990) Cell 62:503-514). Familial Adenomatous Polyposis (FAP) is a rare autosomal dominant syndrome that precedes colon cancer and is caused by an inherited mutation in the adenomatous polyposis coli (APC) gene. PAP is characterized by the early development of multiple colorectal adenomas that progress to cancer at a mean age of 44 years. The APC gene is a part of the APC-β-catenin-Tcf (T-cell factor) pathway. Impairment of this pathway results in the loss of orderly replication, adhesion, and migration of colonic epithelial cells that results in the growth of polyps. A series of other genetic changes follow activation of the APC-β-catenin-Tcf pathway and accompanies the transition from normal colonic mucosa to metastatic carcinoma. These changes include mutation of the K-Ras proto-oncogene, changes in methylation patterns, and mutation or loss of the tumor suppressor genes p53 and Smad4/DPC4. While the inheritance of a mutated APC gene is a rare event, the loss or mutation of APC and the consequent effects on the APC-β-catenin-Tcf pathway is believed to be central to the majority of colon cancers in the general population. Hereditary nonpolyposis Colorectal Cancer (HNPCC) is another inherited autosomal dominant syndrome with a less well defined phenotype than FAP. HNPCC, which accounts for about 2% of colorectal cancer cases, is distinguished by the tendency to early onset of cancer and the development of other cancers, particularly those involving the endometrium, urinary tract, stomach and biliary system. HNPCC results from the mutation of one or more genes in the DNA mis-match repair (MMR) pathway. Mutations in two human MMR genes, MSH2 and MLH1, are found in a large majority of HNPCC families identified to date. The DNA MMR pathway identifies and repairs errors that result from the activity of DNA polymerase during replication. Furthermore, loss of MMR activity contributes to cancer progression through accumulation of other gene mutations and deletions, such as loss of the BAX gene which controls apoptosis, and the TGFβ receptor II gene which controls cell growth. Because of the potential for irreparable damage to DNA in an individual with a DNA MMR defect, progression to carcinoma is more rapid than usual. Although ulcerative colitis is a minor contributor to colon cancer, affected individuals have about a 20-fold increase in risk for developing cancer. Progression is characterized by loss of the p53 gene which may occur early, appearing even in histologically normal tissue. The progression of the disease from ulcerative colitis to dysplasia/carcinoma without an intermediate polyp state suggests a high degree of mutagenic activity resulting from the exposure of proliferating cells in the colonic mucosa to the colonic contents. Almost all colon cancers arise from cells in which the estrogen receptor (ER) gene has been silenced. The silencing of ER gene transcription is age related and linked to hypermethylation of the ER gene, a modification of DNA known to correlate closely with silencing of gene transcription (Issa, J-P J et al. (1994) Nature Genetics 7:536-540). Introduction of an exogenous ER gene into cultured colon carcinoma cells results in marked growth suppression. The conection between the loss of the ER protein in colonic epithelial cells and the consequent development of cancer has not been established. Clearly there are a number of genetic alterations associated with colon cancer, particularly the downregulation or deletion of genes, that potentially provide early indicators of cancer development, that may be used to monitor disease progression or that are possible therapeutic targets. The specific genes affected in a given case of colon cancer depends on the molecular progression of the disease. Identification of additional genes associated with colon cancer would provide more reliable diagnostic patterns associated with development and progression of the disease. PRAME encodes an HLA-A24-restricted CTL (autologous cytolytic T lymphocytes) clone that lysed melanoma line B (MEL.B) cells. MEL.B cells have lost expression of all class I molecules except for HLA-A24. This novel CTL, which is active against tumor cells showing partial HLA loss, is thought to be an intermediate line of anti-tumor defense between the CTL, which recognize highly specific tumor antigens, and the natural killer cells, which recognize HLA loss variants. The antigen is expressed in a large proportion of tumors and at lower concentrations in normal tissues (Ikeda, H. et al. (1997) Immunity 6:199-208). The expression of the PB9/POV1 gene is up-regulated in human prostate cancer. The human cDNA is 2317 nucleotides in length and contains an open reading frame of 559 amino acids. The protein is not homologous with any reported human genes. The N-terminus contains charged amino acids and a helical loop pattern suggestive of an srp leader sequence for a secreted protein. The gene has been mapped to chromosome 11p11.1-p11.2. PB39 has a unique splice variant mRNA that appears to be primarily associated with fetal tissues and tumors. This splice variant appears in prostatic intraepithelial neoplasia, a microscopic precursor lesion of prostate cancer (Cole, K. A. (1998) Genomics 51:282-287). Translocated in liposarcoma (TLS) protein, or FUS, is an interacting molecule of the p65 (ReIA) subunit of the transcription factor nuclear factor kappaB (NF-kappaB). TLS acts as part of a fusion protein with CHOP arising from chromosomal translocation in human myxoid liposarcomas. TLS is involved in TFIID complex formation and is associated with RNA polymerase II. TLS acts as a coactivator of NF-kappaB and plays a pivotal role in NF-kappaB-mediated transactivation (Uranishi H. et al. (2001) J. Biol. Chem.276:13395-13401). The novel cDNA, LDOC1, is down-regulated in some cancer cell lines. It is expressed in normal human tissue but has no expression in pancreatic and gastric cancer cell lines. The gene was mapped to chromosome Xq27 and is probably a nuclear protein. Down-regulation of LDOC1 may have an important role in the development and/or progression of some cancer (Nagasaki K. et al. (1999) Cancer Lett. 140:227-234). Lung Cancer Lung cancer is the leading cause of cancer death for men and the second leading cause of cancer death for women in the U.S. The vast majority of lung cancer cases are attributed to smoking tobacco, and increased use of tobacco products in third world countries is projected to lead to an epidemic of lung cancer in these countries. Exposure of the bronchial epithelium to tobacco smoke appears to result in changes in tissue morphology, which are thought to be precursors of cancer. Lung cancers are divided into four histopathologically distinct groups. Three groups (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) are classified as non-small cell lung cancers (NSCLCs). With squamous cell carcinoma, a series of changes occur over time from an early loss of the ciliated columnar epithelium, basal cell hyperplasia, and the formation of a low columnar epithelium without cilia, to a squamous metaplasia, then mild, moderate and severe dysplasia, and finally to carcinoma. The fourth group of cancers is referred to as small cell lung cancer (SCLC). Collectively, NSCLCs account for approximately 70% of cases while SCLCs account for approximately 18% of cases. The molecular and cellular biology underlying the development and progression of lung cancer are incompletely under-stood. Deletions on chromosome 3 are common in this disease and are thought to indicate the presence of a tumor suppressor gene in this region. Activating mutations in K-ras are commonly found in lung cancer and are the basis of one of the mouse models for the disease. There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of cell proliferative disorders including cancer, developmental disorders, neurological disorders, autoimmune/inflammatory disorders, reproductive disorders, disorders of the placenta, and metabolic disorders. |
<SOH> SUMMARY OF THE INVENTION <EOH>Various embodiments of the invention provide purified polypeptides, proteins associated with cell growth, differentiation, and death, referred to collectively as “CGDD” and individually as “CGDD-I,” “CGDD-2,” “CGDD-3,” “CGDD-4,” “CGDD-5,” “CGDD-6,” “CGDD-7,” “CGDD-8,” “CGDD-9,” “CGDD-10,” “CGDD-1,” “CGDD-12,” “CGDD-13,” “CGDD-14,” “CGDD-15,” “CGDD-16,” “CGDD-17,” “CGDD-18,” and “CGDD-19,” and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified proteins associated with cell growth, differentiation, and death and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified proteins associated with cell growth, differentiation, and death and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions. An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-19. Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-19. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:20-38. Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed. Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof. Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional CGDD, comprising administering to a patient in need of such treatment the composition. Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional CGDD, comprising administering to a patient in need of such treatment the composition. Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional CGDD, comprising administering to a patient in need of such treatment the composition. Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide. Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-19. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide. Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound. Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:20-38, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound. |
Anti-collision light for aircraft |
Anticollision-lamp operable in day-light and at night. It is known to provide separate day-light lamps with a high light output, and night-lamps with little infrared radiation in order not to dazzle night vision goggles. The anticollision light according to the invention comprizes at least one optical interference filter (11) with an average transmission factor in the infrared which is less than 4% of its value in the visible white or red. Preferred embodiments comprize among others: an average transmission factor in a spectral domain larger than 80%, preferably 90% and most desirably 95%; the combination of an interference filter (11) with an absorption filter (10) transparent for red light; an interference filter (11) consisting of several plane elements arranged octagonally; and a band-pass interference filter with a transmission domain situated in the visible red and the transmission factor of which is at least 104 times larger than in an infrared domain. |
1. Anticollision light for aircrafts characterized in that it comprises at least one optical filter (11) the average optical transmission factor of which in a first spectral domain is below 4% of its value in a second spectral domain, where the first domain lies on the longwave side and the second domain lies on the shortwave side of a transition interval situated between the visible red and a boundary located in the infrared region. 2. Anticollision light for aircrafts according to claim 1, characterized in that the average transmission factor in the first spectral domain is below 1%, preferably below 0.4%, and most desirably below 0.1% of its value in the second spectral domain. 3. Anticollision light for aircrafts according to claim 1, characterized in that the average transmission factor in the second spectral domain exceeds 80%, preferably 90%, and most desirably 95%. 4. Anticollision light for aircrafts according to claim 1, characterized in that the average transmission factor in the first spectral domain is less than 2−7 and that it sinks at least by a factor of 104 between 600 nm and 700 nm. 5. Anticollision light for aircrafts according to claim 1, characterized in that the boundary lies between 640 nm and 700 nm, and preferably between 660 nm and 680 nm. 6. Anticollision light for aircrafts according to claim 1, characterized in that the first domain extends at least up to 850 nm, and preferably as far as 1000 nm into the infrared. 7. Anticollision light for aircrafts according to claim 1, characterized in that the second spectral domain extends at least over a visible white spectral domain, or at least over a visible red spectral domain. 8. Anticollision light for aircrafts according to claim 1, characterized in that it comprises a further filter (10) which attenuates wave lengths below the visible red light. 9. Anticollision light for aircrafts according to claim 8, characterized in that the further filter (10) is placed between the light source (6) of the anticollison light and the at least one optical filter (11). 10. Anticollision light for aircrafts according to claim 1, characterized in that the one filter (11) is an interference filter (11), and in particular that the further filter (10) is an absorption filter (10). 11. Anticollision light for aircrafts according to claim 10, characterized in that the interference filter (11) consists of several flat elements (11). 12. Anticollision light for aircrafts according to claim 1, characterized in that the one filter (11) is an interference band-pass filter with transmission band situated in the region of visible red light. 13. Anticollision light for aircrafts according to claim 10, characterized in that the band-pass filter consists of several flat elements. 14. Anticollision light for aircrafts according to claim 1, characterized in that it comprizes a flashing light source (6) in the shape of a fluorescent tube which winds torus-like over more than 200° around an axis (2), and is surrounded by a reflector (7, 71) which also extends in an essentially torus-like shape over more than 200° and exhibits a slit along its outer periphery, where the aperture of the slit, as seen from the circular longitudinal axis of the fluorescent tube (6) in a plane containing the axis, has a maximal aperture angle (α) of 180°, preferably of 130°, and most desirably of 90°. |
<SOH> TECHNICAL FIELD <EOH>The invention relates to an anticollision light for aircrafts. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>Further implementations, advantages and applications of the invention can be derived from the dependent claims and from the following description made with the help of the drawings. Therein, FIG. 1 shows a simplified perspective view of an embodiment of the invention; FIG. 2 shows a schematic axial section through the embodiment of FIG. 1 ; FIG. 3 shows a very schematic section along III-III of FIG. 2 ; FIG. 4 shows an instance of a transmission diagram of the infrared filter of the embodiment according to FIG. 1 ; and FIG. 5 shows an example of further a preferred transmission diagram of the infrared filter. detailed-description description="Detailed Description" end="lead"? |
Medication compositions |
A medicinal composition comprising: (a) a core comprising a medicinally effective unit dose of one or more active medicaments; and (b) said medicament(s) being enclosed within a film material which comprises at least 40% by weight hydroxypropylmethyl cellulose. |
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