1-12. (canceled)
13. A lithographic printing plate precursor comprising a support which includes a hydrophilic surface or which is provided with a hydrophilic layer and a coating thereon, said coating comprising an IR absorbing agent, a contrast enhancing compound and a polymeric binder that alters its solubility in an alkaline developing solution upon exposure, wherein said contrast enhancing compound has the structure of formula I:
wherein
R1 is hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl or heteroaryl group, halogen, \u2014NR4R5, \u2014CO\u2014NR4R5, \u2014SO2\u2014NR4R5, \u2014COR6, \u2014CN, \u2014NO2, \u2014COOR6, \u2014OR3, \u2014SR3, \u2014SOR3, \u2014SO2R6, \u2014SO3R6, \u2014PO4R4R5, \u2014PO3R4R5, \u2014NR6\u2014CO\u2014NR4R5, \u2014O\u2014COOR6, \u2014NR4\u2014COOR5, \u2014NR4\u2014CO\u2014R5 or a phosphoramidate group;
R2 is hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl or heteroaryl group, halogen, \u2014SO2\u2014NR4R5, \u2014CN, \u2014NO2, \u2014SOR3, \u2014SO2R6, \u2014SO3R6, \u2014PO4R4R5, \u2014PO3R4R5 or a phosphoramidate group;
R3 is an optionally substituted alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl or heteroaryl group;
R4, R5 and R6 independently are hydrogen or one of the groups as defined for R3, or wherein two groups selected from R4, R5 and R6 together constitute the atoms necessary to form a ring;
Q is one of the following groups which forms an optionally substituted 6 membered heteroaromatic ring, said groups being selected from **\u2014C(T2)-N\u2014N\u2014*, **\u2014N\u2014N\u2014C(T2)-*, **\u2014N\u2014C(T2)-C(T3)-*, **\u2014C(T2)-N\u2014C(T3)-*, **\u2014C(T2)-C(T3)-C(T4)-*, **\u2014C(T2)-C(T1)-N\u2014*, **\u2014N\u2014C(T1)-N\u2014* or **\u2014N\u2014N\u2014N\u2014*, or Q is one of the following groups which forms an optionally substituted 5 membered heteroaromatic ring, said groups being selected from **\u2014C(T1)-N(T2)-*, **\u2014C(T2)-S\u2014*, **\u2014C(T2)-O\u2014*, **\u2014N\u2014N(T2)-*, **\u2014N\u2014S\u2014*, **\u2014N\u2014O\u2014*, **\u2014N(T2)-C(T3)-*, **\u2014S\u2014N\u2014* or **\u2014O\u2014N\u2014*,
wherein
* indicates a binding site to the carbon atom between the two nitrogen atoms and ** indicates a binding site to the carbon atom substituted by R1; and
wherein
the symbol \u201cO\u201d in the middle of the ring comprising Q represents the pi-electrons necessary for the aromatic ring;
T1 is one of the groups as defined for R1;
T2, T3 and T4 independently are one of the groups as defined for R2; or
wherein one of the groups of T1, T2, T3 or T4 together with one of the groups of R1 comprise the atoms necessary to form a ring; or
wherein one of the groups of T1, T2, T3 or T4 together with one of the groups of R2 comprise the atoms necessary to form a ring; or
wherein two groups, selected from T1, T2, T3 or T4, comprise the atoms necessary to form a ring.
14. A lithographic printing plate precursor according to claim 13, wherein said contrast enhancing compound has the structure of formula III:
wherein
Y is a nitrogen atom or a carbon atom;
X is the atoms necessary to form an optionally-substituted five- or six-membered heteroaromatic ring;
Z is the atoms necessary to form an optionally-substituted five- to eight-membered ring; B1 is one of the groups as defined in formula I for R1; and
the symbol \u201cO\u201d in the middle of the ring comprising X and Y represents a number of pi-electrons necessary for the aromatic ring.
15. A lithographic printing plate precursor according to claim 13, wherein said contrast enhancing compound has the structure of formula IV
wherein
K1 is one of the groups as defined in formula I for R1; and
K2 to K5 independently are hydrogen, \u2014NR4R5, \u2014CO\u2014NR4R5, \u2014COR6, \u2014COOR6, \u2014OR3, \u2014NR6\u2014CO\u2014NR4R5, \u2014NR4\u2014COOR5, \u2014NR4\u2014CO\u2014R5, wherein R3, R4, R5 and R6 respectively are the groups R3, R4, R5 and R6 as defined in formula I, or wherein two groups selected from K2, K3, K4 and K5 together are the atoms necessary to form a ring.
16. A lithographic printing plate precursor according to claim 13, wherein said contrast enhancing compound has the structure of formula V
wherein
M1 is one of the groups as defined in formula I for R1; and
M2 to M6 independently are hydrogen, \u2014NR4R5, \u2014CO\u2014NR4R5, \u2014COR6, \u2014COOR6, \u2014OR3, \u2014NR6\u2014CO\u2014NR4R5, \u2014NR4\u2014COOR5, \u2014NR4\u2014CO\u2014R5, wherein R3, R4, R5 and R6 respectively are the groups R3, R4, R5 and R6 as defined in formula I, or are one of the groups as defined in formula I for R3, R4, R5 and R6; or wherein M1 and M2 together are the atoms necessary to form a ring; or wherein two groups, selected from M2 to M6, together are the atoms necessary to form a ring.
17. A lithographic printing plate precursor according to claim 13, wherein said contrast enhancing compound has the structure of formula VI
wherein
V1 is one of the groups as defined in formula I for R1; and
V2 and V3 independently are hydrogen or one of the groups as defined in formula I for R3; and
V4 is hydrogen or one of the groups as defined in formula I for R3; or wherein two groups, selected from V1 to V3, together represent the atoms necessary to form a ring.
18. The lithographic printing plate precursor according to claim 1, wherein said binder is a polymer comprising a sulphonamide group.
19. The lithographic printing plate precursor according to claim 18, wherein said sulphonamide group is present in the side chain of said polymer.
20. The lithographic printing plate precursor according to claim 13, wherein said binder is a polymer comprising a contrast enhancing compound present in the side chain of said polymer.
21. The lithographic printing plate precursor according to claim 13, wherein said binder is a phenolic resin.
22. A lithographic printing plate precursor comprising a support which includes a hydrophilic surface or which is provided with a hydrophilic layer and a coating thereon, said coating comprising an IR absorbing agent, a contrast-enhancing compound and a polymeric binder that alters its solubility in an alkaline developing solution upon exposure, wherein said contrast-enhancing compound has the structure of formula I
wherein in formula I
R1 is hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl or heteroaryl group, halogen, \u2014NR4R5, \u2014CO\u2014NR4R5, \u2014SO2\u2014NR4R5, \u2014COR6, \u2014CN, \u2014NO2, \u2014COOR6, \u2014OR3, \u2014SR3, \u2014SOR3, \u2014SO2R6, \u2014SO3R6, \u2014PO4R4R5, \u2014PO3R4R5, \u2014NR6\u2014CO\u2014NR4R5, \u2014O\u2014COOR6, \u2014NR4\u2014COOR5, \u2014NR4\u2014CO\u2014R5 or a phosphoramidate group;
R2 is hydrogen, an optionally substituted alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl or heteroaryl group, halogen, \u2014SO2\u2014NR4R5, \u2014CN, \u2014NO2, \u2014SOR3, \u2014SO2R6, \u2014SO3R6, \u2014PO4R4R5, \u2014PO3R4R5 or a phosphoramidate group;
R3 is an optionally substituted alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl or heteroaryl group;
R4, R5 and R6 independently are hydrogen or one of the groups as defined for R3, or wherein two groups selected from R4, R5 and R6 together constitute the atoms necessary to form a ring;
Q is one of the following groups which forms an optionally substituted 6 membered heteroaromatic ring, said groups being selected from **\u2014C(T2)-N\u2014N\u2014*, **\u2014N\u2014N\u2014C(T2)-*, **\u2014N\u2014C(T2)-C(T3)-*, **\u2014C(T2)-N\u2014C(T3)-*, **\u2014C(T2)-C(T3)-C(T4)-*, **\u2014C(T2)-C(T1)-N\u2014*, **\u2014N\u2014C(T1)-N\u2014* or **\u2014N\u2014N\u2014N\u2014*, or Q is one of the following groups which forms an optionally substituted 5 membered heteroaromatic ring, said groups being selected from **\u2014C(T1)-N(T2)-*, **\u2014C(T2)-S\u2014*, **\u2014C(T2)-O\u2014*, **\u2014N\u2014N(T2)-*, **\u2014N\u2014S\u2014*, **\u2014N\u2014O\u2014*, **\u2014N(T2)-C(T3)-*, **\u2014S\u2014N\u2014* or **\u2014O\u2014N\u2014*,
wherein
* indicates a binding site to the carbon atom between the two nitrogen atoms and ** indicates a binding site to the carbon atom substituted by R1; and
wherein
the symbol \u201cO\u201d in the middle of the ring comprising Q represents the pi-electrons necessary for the aromatic ring;
T1 is one of the groups as defined for R1;
T2, T3 and T4 independently are one of the groups as defined for R2; or
wherein one of the groups of T1, T2, T3 or T4 together with one of the groups of R1 comprise the atoms necessary to form a ring; or
wherein one of the groups of T1,
T2, T3 or T4 together with one of the groups of R2 comprise the atoms necessary to form a ring; or
wherein two groups, selected from T1, T2, T3 or T4, comprise the atoms necessary to form a ring,
and wherein the contrast-enhancing compound is linked to a polymer by a chemical bound formed between at least one atom of a group selected from R1, R2 and T1 to T4, and at least one atom of said polymer.
23. The lithographic printing plate precursor according to claim 22, wherein said contrast-enhancing compound is chemically bound to a side chain of said polymer and wherein said chemical bound is formed between at least one atom of a group selected from R1, R2 and T1 to T4 and at least one atom of the side chain of said polymer, optionally by a linking group L between said side chain and said contrast-enhancing compound.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID NO:1,
b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1,
c) a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:1, wherein said fragment binds to microtubules and
d) an immunogenic fragment of a polypeptide having the amino acid sequence of SEQ ID NO:1.
2. An isolated polypeptide of claim 1 comprising the amino acid sequence of SEQ ID NO:1.
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 the polynucleotide sequence of SEQ ID NO:2.
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 for producing a polypeptide encoded by the polynucleotide of claim 3, 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 the polynucleotide of claim 3, and
b) recovering the polypeptide so expressed.
10. An isolated antibody which specifically binds to a polypeptide of claim 1.
11. An isolated polynucleotide selected from the group consisting of:
a) a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2,
b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to the polynucleotide sequence of SEQ ID NO:2,
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).
12. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 11.
13. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, 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.
14. A method of claim 13, wherein the probe comprises at least 60 contiguous nucleotides.
15. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, 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.
16. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.
17. A composition of claim 16, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:1.
18. A method for treating a disease or condition associated with decreased expression of functional hLC3, comprising administering to a patient in need of such treatment the composition of claim 16.
19. A method for 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.
20. A composition comprising an agonist compound identified by a method of claim 19 and a pharmaceutically acceptable excipient.
21. A method for treating a disease or condition associated with decreased expression of functional hLC3, comprising administering to a patient in need of such treatment a composition of claim 20.
22. A method for 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.
23. A composition comprising an antagonist compound identified by a method of claim 22 and a pharmaceutically acceptable excipient.
24. A method for treating a disease or condition associated with overexpression of functional hLC3, comprising administering to a patient in need of such treatment a composition of claim 23.
25. A method of screening for a compound that specifically binds to the polypeptide of claim 1, said method comprising the steps of:
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.
26. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, said 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.
27. A method for 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.
28. 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 of claim 11 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 11 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.
29. A diagnostic test for a condition or disease associated with the expression of hLC3 in a biological sample comprising the steps of:
a) combining the biological sample with an antibody of claim 10, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex; and
b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.
30. The antibody of claim 10, wherein the antibody is:
a) a chimeric antibody,
b) a single chain antibody,
c) a Fab fragment,
d) a F(ab)2 fragment, or
e) a humanized antibody.
31. A composition comprising an antibody of claim 10 and an acceptable excipient.
32. A method of diagnosing a condition or disease associated with the expression of hLC3 in a subject, comprising administering to said subject an effective amount of the composition of claim 31.
33. A composition of claim 31, wherein the antibody is labeled.
34. A method of diagnosing a condition or disease associated with the expression of hLC3 in a subject, comprising administering to said subject an effective amount of the composition of claim 33.
35. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 10 comprising:
a) immunizing an animal with a polypeptide having the amino acid sequence of SEQ ID NO:1, or an immunogenic fragment thereof, under conditions to elicit an antibody response;
b) isolating antibodies from said animal; and
c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide having the amino acid sequence of SEQ ID NO:1.
36. An antibody produced by a method of claim 35.
37. A composition comprising the antibody of claim 36 and a suitable carrier.
38. A method of making a monoclonal antibody with the specificity of the antibody of claim 10 comprising:
a) immunizing an animal with a polypeptide having the amino acid sequence of SEQ ID NO:1, or an immunogenic fragment thereof, under conditions to elicit an antibody response;
b) isolating antibody producing cells from the animal;
c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells;
d) culturing the hybridoma cells; and
e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide having the amino acid sequence of SEQ ID NO:1.
39. A monoclonal antibody produced by a method of claim 38.
40. A composition comprising the antibody of claim 39 and a suitable carrier.
41. The antibody of claim 10, wherein the antibody is produced by screening a Fab expression library.
42. The antibody of claim 10, wherein the antibody is produced by screening a recombinant immunoglobulin library.
43. A method for detecting a polypeptide having the amino acid sequence of SEQ ID NO:1 in a sample, comprising the steps of:
a) incubating the antibody of claim 10 with a sample under conditions to allow specific binding of the antibody and the polypeptide; and
b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide having the amino acid sequence of SEQ ID NO:1 in the sample.
44. A method of purifying a polypeptide having the amino acid sequence of SEQ ID NO:1 from a sample, the method comprising:
a) incubating the antibody of claim 10 with a sample under conditions to allow specific binding of the antibody and the polypeptide; and
b) separating the antibody from the sample and obtaining the purified polypeptide having the amino acid sequence of SEQ ID NO:1.