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Sh3 domain binding inhibitors |
The present invention provides SH3 domain binding inhibitors comprising, as an active ingredient, a non-peptide compound exhibiting SH3 domain binding inhibitory activity, a low molecular weight compound with molecular weight less than 750 which exhibit SH3 domain binding inhibitory activity, in particular, a compound represented by the general formula (I) or (II) described above, a cytochalsin, etc., or pharmaceutically acceptable salts thereof. The present invention also provides compounds represented by the general formula (Va), (Vb) or (VI) described above or pharmaceutically acceptable salts thereof. |
1. A method for inhibiting SH3 domain binding, which comprises administering an effective amount of a non-peptide compound exhibiting SH3 domain binding inhibitory activity or a pharmaceutically acceptable salt thereof. 2. The method for inhibiting SH3 domain binding according to claim 1, wherein said non-peptide compound is a low molecular weight compound with molecular weight of less than 750. 3. The method for inhibiting SH3 domain binding inhibitor according to claim 1, wherein said non-peptide compound exhibiting SH3 domain binding inhibitory activity is a compound represented by the general formula (I): (wherein R1, R3a, R3b and R4 may be the same or different and represent a hydrogen atom, hydroxy, carboxy, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, or substituted or unsubstituted lower alkanoyloxy; or R3a and R3b are combined to represent an oxygen atom; R2a and R2b may be the same or different and represent a hydrogen atom, substituted or unsubstituted lower alkyl, or substituted or unsubstituted alkenyl; R5a and R5b may be the same or different and represent a hydrogen atom, hydroxy, carboxy, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkanoyloxy, substituted or unsubstituted lower alkenoyloxy, or substituted or unsubstituted lower alkanoylaminocarbonyloxy; or R5a and R5b are combined to represent an oxygen atom; and X represents an oxygen atom or —CH2—). 4. The method for inhibiting SH3 domain binding according to claim 1, wherein said non-peptide compound exhibiting SH3 domain binding inhibitory activity is a compound represented by the general formula (II): (wherein R6 represents a hydrogen atom, substituted or unsubstituted lower alkyl, or substituted or unsubstituted lower alkenyl; R7 and R9 may be the same or different and represent a hydrogen atom, formyl, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkanoyl, or substituted or unsubstituted lower aralkyl; and R8, R10 and R11 may be the same or different and represent a hydrogen atom, halogen, hydroxy, carboxy, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkoxy, substituted or unsubstituted lower alkanoyl, formyl, cyano, nitro, amino, mono- or di-lower alkylamino, substituted or unsubstituted lower alkanoylamino, or substituted or unsubstituted alkoxycarbonyl). 5. The method for inhibiting SH3 domain binding according to claim 1, wherein said non-peptide compound exhibiting SH3 domain binding inhibitory activity is a cytochalasin. 6. The method for inhibiting SH3 domain binding according to claim 1, wherein said non-peptide compound exhibiting SH3 domain binding inhibitory activity is a compound represented by the general formula (IIIa): (wherein R12a represents substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Q1 represents a single bond or an oxygen atom; represents a single bond or a double bond; and when the between Q2 and a carbon atom adjacent thereto represents a double bond, =Q2- represents ═C(CH3)—, and when the between Q2 and a carbon atom adjacent thereto represents a single bond, -Q2- represents —C(OH)(CH3)). 7. The method for inhibiting SH3 domain binding according to claim 1, wherein said non-peptide compound exhibiting SH3 domain binding inhibitory activity is a compound represented by the general formula (IIIb): (wherein R12b has the same meaning as the above-described R12a; Q3 and Q5 may be the same or different and represent a single bond or an oxygen atom; represents a single bond or a double bond; represents ═C(CH3)—, —C(═CH2)—, —CH(CH3)— or —C(CH3)═; R12c and R12h may be the same or different and represent a hydrogen atom or hydroxy; R12d and R12e may be the same or different and represent a hydrogen atom or methyl; and R12f and R12g represent formyl, or R12f and R12g are combined and and —CHR12eR12f form (wherein A and B may be the same or different and represent —CH(OH)—, —CH2— or —C(═O)—)). 8. The method for inhibiting SH3 domain binding according to claim 3, wherein X represents an oxygen atom; R2a, R3a, R4 and R5b are hydrogen atoms; R1 and R3b may be the same or different and represent hydroxy, carboxy, substituted or unsubstituted lower alkoxy, or substituted or unsubstituted lower alkanoyloxy; R2b represents substituted or unsubstituted lower alkyl; and R5a represents the general formula (IV): (wherein R5c represents substituted or unsubstituted lower alkyl; R5d and R5e may be the same or different and represent a hydrogen atom, hydroxy, or substituted or unsubstituted lower alkoxy, or R5d and R5e are combined to represent an oxygen atom; R5f and R5h represent a hydrogen atom, formyl, substituted or unsubstituted lower alkyl, or substituted or unsubstituted lower alkanoyl; R5g represents formyl, —CH═NQ (wherein Q represents substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted aralkyloxy, substituted or unsubstituted lower alkylamino, substituted or unsubstituted arylamino, or substituted or unsubstituted arylsulfonylamino), substituted or unsubstituted lower alkyl, or substituted or unsubstituted lower alkanoyl; and R5i and R5j may be the same or different and represent a hydrogen atom, hydroxy, or substituted or unsubstituted lower alkoxy, or R5i and R5j are combined to represent an oxygen atom). 9. A compound represented by the general formula (Va): (wherein represents a single bond or a double bond; and R12a, Q1 and Q2 have the same meanings as described above, respectively), or a pharmaceutically acceptable salt thereof. 10. A compound represented by the general formula (Vb): (wherein represents a single bond or a double bond; R12b, R12c, R12d, R12e, R12h, Q5 and have the same meanings as described above, respectively; and represents (wherein A and B have the same meanings as described above, respectively)), or a pharmaceutically acceptable salt thereof. 11. A compound represented by the general formula (VI) (wherein R1A, R3A and R3B may be the same or different and represent a hydrogen atom, hydroxy, carboxy, substituted or unsubstituted lower alkoxy, or substituted or unsubstituted lower alkanoyloxy, or R3A and R3B are combined to represent an oxygen atom; R2A represents substituted or unsubstituted lower alkyl; R5c, R5d, R5e, R5f, R5h, R5i and R5j have the same meanings as described above, respectively; and R5G represents formyl, hydroxymethyl, substituted or unsubstituted lower alkoxymethyl, substituted or unsubstituted lower alkanoyloxymethyl, substituted or unsubstituted lower alkanoylmethyl, or —CH═NQA (wherein QA represents substituted or unsubstituted lower alkoxy, substituted or unsubstituted aryloxy, or substituted or unsubstituted aralkyloxy), with the proviso that when R5G is formyl and one of R3A and R3B is a hydrogen atom, the other is not hydroxy], or a pharmaceutically acceptable salt thereof. 12. A pharmaceutical composition comprising the compound according to claim 9 or 10 or a pharmaceutically acceptable salt thereof as an active ingredient. 13. A pharmaceutical composition comprising the compound according to claim 11 or a pharmaceutically acceptable salt thereof as an active ingredient. 14. A method for inhibiting SH3 domain binding, which comprises administering an effective amount of the compound according to claim 9 or a pharmaceutically acceptable salt thereof. 15. A method for inhibiting SH3 domain binding, which comprises administering an effective amount of the compound according to claim 11 or a pharmaceutically acceptable salt thereof. 16. The method for inhibiting SH3 domain binding according to any one of claims 1-8 , wherein said SH3 domain binding is an interaction between a protein containing an SH3 domain and a protein containing a proline-rich sequence. 17. The method for inhibiting SH3 domain binding according to claim 16, wherein the protein containing an SH3 domain and/or the protein containing a proline-rich sequence are/is virus-derived protein(s). 18. The method for inhibiting SH3 domain binding according to claim 17, wherein said virus-derived protein is a retrovirus-derived protein, a hepatitis virus-derived protein or a herpes virus-derived protein. 19. The method for inhibiting SH3 domain binding according to claim 16, wherein said protein containing an SH3 domain is Src, Yes, Fgr, Hck, Lck, Abl, Fyn, Lyn, Blk, Yrk, Ras-GAP, PLCγ, P13K, Tec, Txk/Rlk, Tsk/Emt/Itk, Btk, Crk, Grb2, Nck, Vav, STAT, Cortactin, p40-phox, p67-phox, p47-phox, a TCR signaling molecule (TCRsm), or a β chain or a γ chain of IL-2R. 20. The method for inhibiting SH3 domain binding according to claim 16, wherein said protein containing a proline-rich sequence is HIV-1Nef, p22-phox, p47-phox, Sam68, Sos1, Dynamin, c-Cbl, ZO1, pX ORF I, LHDAg, NS5A, pORF3, ICP10, LMP2A, Tip or Tio. 21. The method for inhibiting SH3 domain binding according to claim 16, wherein said interaction between a protein containing an SH3 domain and a protein containing a proline-rich sequence is an interaction between Grb2 and Sos1, Fyn and Sam68, Src and Sam68, PLCγ and Sam68, Grb2 and Sam68, Lyn and HIV-1Nef, Hck and HIV-1Nef, TCRsm and HIV-1Nef, p47-phox and p22-phox, p67-phox and p47-phox, Lyn and Dynamin, Cortactin and ZO1, Lyn and c-Cb1, a β chain or a γ chain of IL-2R and pX ORF I, Grb2 and NS5A, Src and pORF3, Hck and pORF3, Fyn and pORF3, PI3K and pORF3, PLCγ and pORF3, Grb2 and pORF3, Grb2 and ICP10, Lyn and LMP2A, Lck and Tip, Lyn and Tio, Hck and Tio, Lck and Tio, Src and Tio, Fyn and Tio, or Yes and Tio. 22. The method for inhibiting SH3 domain binding according to claim 16, wherein said interaction between a protein containing an SH3 domain and a protein containing a proline-rich sequence is an interaction between Grb2 and Sos1, Fyn and Sam68, Src and Sam68, PLCγ and Sam68, Grb2 and Sam68, Lyn and HIV-1Nef, or Cortactin and ZO1. 23. A microorganism for producing the compound according to claim 9 or 10, selected from the group consisting of Xylariales filamentous fungus MPC1005 (Accession No.: FERM BP-7980), Aspergillus sp. MPC1006 (Accession No.: FERM BP-7899) and Aspergillus sp. MPC1009 (Accession No.: FERM BP-7900). 24-41. (canceled). 42. A method for treating and/or preventing a disease in which an SH3 domain binding is involved, which comprises administering an effective amount of the compound according to claim 9 or 10 or a pharmaceutically acceptable salt thereof. 43. A method for treating and/or preventing a disease in which an SH3 domain binding is involved, which comprises administering an effective amount of the compound according to claim 11 or a pharmaceutically acceptable salt thereof. 44. A method for treating a malignant tumor, which comprises administering an effective amount of the compound according to claim 9 or 10 or a pharmaceutically acceptable salt thereof. 45. A method for treating a malignant tumor, which comprises administering an effective amount of the compound according to claim 11 or a pharmaceutically acceptable salt thereof. 46. A method for treating and/or preventing an allergic disease, which comprises administering an effective amount of the compound according to claim 9 or 10 or a pharmaceutically acceptable salt thereof. 47. A method for treating and/or preventing an allergic disease, which comprises administering an effective amount of the compound according to claim 11 or a pharmaceutically acceptable salt thereof. 48. A method for treating a viral disease, which comprises administering an effective amount of the compound according to claim 9 or 10 or a pharmaceutically acceptable salt thereof. 49. A method for treating a viral disease, which comprises administering an effective amount of the compound according to claim 11 or a pharmaceutically acceptable salt thereof. 50. A method for treating AIDS, which comprises administering an effective amount of the compound according to claim 9 or 10 or a pharmaceutically acceptable salt thereof. 51. A method for treating AIDS, which comprises administering an effective amount of the compound according to claim 11 or a pharmaceutically acceptable salt thereof. 52. A method for treating AIDS, which comprises administering an effective amount of a compound exhibiting an SH3 domain binding inhibitory activity or a pharmaceutically acceptable salt thereof. 53. A method for treating a viral disease, which comprises administering an effective amount of a compound exhibiting an SH3 domain binding inhibitory activity or a pharmaceutically acceptable salt thereof. 54-57. (canceled). 58. A method for inhibiting SH3 domain binding, which comprises administering an effective amount of the compound according to claim 10 or a pharmaceutically acceptable salt thereof. 59. The method for inhibiting SH3 domain binding according to claim 19, wherein said protein containing a proline-rich sequence is HIV-1Nef, p22-phox, p47-phox, Sam68, Sos 1, Dynamin, c-Cbl, ZO1, pX ORF I, LHDAg, NS5A, pORF3, ICP10, LMP2A, Tip or Tio. 60. The method for inhibiting SH3 domain binding according to any one of claims 14, 15 and 58, wherein said SH3 domain binding is an interaction between a protein containing an SH3 domain and a protein containing a proline-rich sequence. 61. The method for inhibiting SH3 domain binding according to claim 60, wherein the protein containing an SH3 domain and/or the protein containing a proline-rich sequence are/is virus-derived protein(s). 62. The method for inhibiting SH3 domain binding according to claim 61, wherein said virus-derived protein is a retrovirus-derived protein, a hepatitis virus-derived protein or a herpes virus-derived protein. 63. The method for inhibiting_SH3 domain binding according to claim 60, wherein said protein containing an SH3 domain is Src, Yes, Fgr, Hck, Lck, Abl, Fyn, Lyn, Blk, Yrk, Ras-GAP, PLCγ, PI3K, Tec, Txk/Rlk, Tsk/Emt/Itk, Btk, Crk, Grb2, Nck, Vav, STAT, Cortactin, p40-phox, p67-phox, p47-phox, a TCR signaling molecule (TCRsm), or a β chain or a γ chain of IL-2R. 64. The method for inhibiting SH3 domain binding according to claim 60, wherein said protein containing a proline-rich sequence is HIV-1Nef, p22-phox, p47-phox, Sam68, Sos1, Dynamin, c-Cbl, ZO1, pX ORF I, LHDAg, NS5A, pORF3, ICP10, LMP2A, Tip or Tio. 65. The method for inhibiting SH3 domain binding according to claim 60, wherein said interaction between a protein containing an SH3 domain and a protein containing a proline-rich sequence is an interaction between Grb2 and Sos1, Fyn and Sam68, Src and Sam68, PLCγ and Sam68, Grb2 and Sam68, Lyn and HIV-1Nef, Hck and HIV-1Nef, TCRsm and HIV-1Nef, p47-phox and p22-phox, p67-phox and p47-phox, Lyn and Dynamin, Cortactin and ZO1, Lyn and c-Cbl, a 0 chain or a y chain of IL-2R and pX ORF I, Grb2 and NS5A, Src and pORF3, Hck and pORF3, Fyn and pORF3, P13K and pORF3, PLCγ and pORF3, Grb2 and pORF3, Grb2 and ICP10, Lyn and LMP2A, Lck and Tip, Lyn and Tio, Hck and Tio, Lck and Tio, Src and Tio, Fyn and Tio, or Yes and Tio. 66. The method for inhibiting SH3 domain binding according to claim 60, wherein said interaction between a protein containing an SH3 domain and a protein containing a proline-rich sequence is an interaction between Grb2 and Sos1, Fyn and Sam68, Src and Sam68, PLCγ and Sam68, Grb2 and Sam68, Lyn and HIV-1Nef, or Cortactin and ZO1. 67. The method for inhibiting SH3 domain binding according to claim 63, wherein said protein containing a proline-rich sequence is HIV-1Nef, p22-phox, p47-phox, Sam68, Sos1, Dynamin, c-Cbl, ZO1, pX ORF I, LHDAg, NS5A, pORF3, ICP10, LMP2A, Tip or Tio. |
<SOH> BACKGROUND TECHNOLOGY <EOH>There are many receptors on the cell membrane that are activated by the extracellular stimulus and transmit the signals into the cell. Various signaling molecules are assembled on the cytosol side of the receptors and form a complex. Depending on the kind of the stimulus, each signaling molecule induces different physiological action. The structure of the domain plays an important role in the formation of a complex of signaling molecules. Among them, the Src homology domain 3 (SH3 domain) was discovered as a region with a high homology among Src family and it has been known that the domain consists of about 60 amino acids, and is present in various proteins and bound to a sequence containing proline (proline-rich sequence) by recognizing the sequence [Science, Vol. 252, p. 668 (1991), Science, Vol. 257, p. 803 (1992), FASEB J., Vol. 14, p. 231 (2000)]. HIV-1 Nef, p22-phox, p47-phox, Som68, Sos1, Zo1, Dynamin, c-Cb1, etc., have been known as proteins containing the proline-rich sequence(s) [e.g. EMBO J., Vol. 14, p. 484(1995), Proc. Natl. Acad. Sci. USA, Vol. 91, p. 10650 (1994), J. Immunol., Vol. 157, p. 1226 (1996), J. Biol. Chem., Vol.270, p. 9115 (1995), etc.] Proteins containing SH3 domains were found in all eukaryotes, viruses such as HIV, etc., and are considered to have universal functions. For example, the following proteins are known to contain SH3 domain(s): Fyn, which is one of Src family kinase; Ras-GAP, PLCγ, PI3K, Abl, Btk, Lyn, Hck, Fgr, Yes, etc., which are known to have enzyme activity; Grb2, Nck, Vav, etc., which are adaptor proteins without enzyme activity; p40-phox, p47-phox, p67-phox, etc., which are components of a NADH oxidase complex [Proc. Natl. Acad. Sci. USA, Vol. 91, p. 10650 (1994)]; and some others, that is, Txk, Tec, Tsk, Crk, Cortactin, etc. It has been speculated that the protein-protein interaction mediated by an SH3 domain has a role in the protein complex formation through an interaction due to a suitable affinity. The protein-protein interactions mediated by an SH3 domain exist among the signaling molecules which have been considered as a cause of various diseases, such as cancer, AIDS (acquired immune deficiency syndrome), or allergy [Biopoly., Vol. 43, p. 383 (1997)]. These facts indicate that inhibitors of SH3 domain binding that inhibit the protein-protein interactions mediated by an SH3 domain (SH3 domain binding inhibitory activity) might be effective as a therapeutic agent for these diseases. The inhibitors may be used for various purposes, for example, for controlling a protein which cause a disease such as carcinogenesis in the body, in which SH3 domain binding is involved. As to peptides, the following are reported: peptides with mutation in an SH3 domain binding sequence [Cell, Vol. 76, p. 933 (1994), Chem. Biol., Vol. 7, p. R3-R8 (2000)]; a method for screening peptides, which bind to an SH3 domain of various proteins containing SH3 domains such as Src, PI3K, or Ab1, from a combinatorial phage display library (WO95/24419, Japanese Translation of PCT Application [Tokuhyo] 2000-506522); peptides, which specifically bind to an SH3 domain of c-Crk, have been also reported (WO96/21011). Among peptide-like compounds that are obtained by modification of a peptide, the following compounds are known: spirolactam compounds which inhibit SH3 domain binding (WO98/54208) and compounds which are isolated from a biased combinatorial library in which a synthetic peptide containing an amino acid sequence having a proline skeleton is fixed [J. Am. Chem. Soc., Vol. 118, p. 287 (1996)]. Among those reported compounds, the smallest one have molecular weight of 790. However, K d value thereof was 220 mmol/L and thus a binding ability thereof to the SH3 domain was week [J. Am. Chem. Soc., Vol. 118, p. 287 (1996)]. All the SH3 domain binding inhibitors reported so far are peptides or peptide-like compounds based on proline or other hydrophobic amino acids with 750 or more molecular weight. The peptides or the peptide-like compounds have been difficult to use as a therapeutic agent for a disease, in which SH3 domain binding is involved, because they are, for example, generally unstable in blood, poorly absorbed when orally administered, not easy to be taken into cells due to their large molecular weight, difficult to prepare and costly due to their structural complexity, etc. UCS15A (Luminacin C2/SI-4228A) has been reported as the compound exhibiting inhibitory activity against bone resorption, bactercidal activity, immunosuppressive activity, anti-tricophytosis activity and anti-tumor activity [The Journal of Antibiotics, Vol. 53, p. 579 (2000), Japanese Published Unexamined Patent Application Nos. 116686/83, 22583/88, 293920/86, 294619/87, 48213/88 and 268888/96]. Also, cytochalasins (cytochalasin, Rosellichalasin, epoxycytochalasin, chaetoglobosin, penochalasin, aspochalasin, etc.) are known to possess biological activities such as an inhibitory activity against angiogenesis (WO98/41205). |
<SOH> BRIEF DESCRIPTION OF THE FIGURES <EOH>FIG. 1 shows the results of in vitro assay for the Sam68ΔC and Fyn-SH3 binding inhibition when Compounds (I) and (VI) were added at different concentrations. The numbers on top of the lanes demonstrate the compound number and the concentration (μmol/L) of the compound added to the solution of Sam68ΔC. The name of each blot is shown in the left. FIG. 2 shows the results of in vitro assay for the Sam68ΔC and Fyn-SH3 binding inhibition when Compounds 1 and (IIIa) were added at different concentrations. The numbers on top of the lanes demonstrate the compound number and the concentration (μmol/L) of the compound added to the solution of Sam68ΔC. The name of each blot is shown in the left. FIG. 3 shows the results of in vitro assay for the Sam68ΔC and Fyn-SH3 binding inhibition when the Compounds (II) and (IIIa) were added at different concentrations. The numbers on top of the lanes demonstrate the compound number and the concentration (Pmol/L) of the compound added to the solution of Sam68ΔC. The name of each blot is shown in the left. FIG. 4 shows the results of the assay of the intracellular Src and Sam68 binding inhibition when the Compound 1 was added at different concentrations. The numbers on top of the lanes demonstrate the concentration (μmol/L) of the Compound 1 added to the HCT116 cells. The name of each blot is shown in the left. FIG. 5 shows the results of the assay of the intracellular PLCγ and Sam68 binding inhibition when Compound 1 was added at different concentrations. The numbers on top of the lanes demonstrate the concentration (μmol/L) of Compound 1 added to the HCT116 cells. The name of each blot is shown in the left. FIG. 6 shows the results of the assay of the intracellular Grb2 and Sos1 binding inhibition when Compound 1 was added at different concentrations. The numbers on top of the lanes demonstrate the concentration (μmol/L) of Compound 1 added to the HCT116 cells. The name of each blot is shown in the left. FIG. 7 shows the results of the assay of the intracellular Cortactin and Z1 binding inhibition when Compound 1 was added at different concentrations. The numbers on top of the lanes demonstrate the concentration (μmol/L) of Compound 1 added to the HCT116 cells. The name of each blot is shown in the left. FIG. 8 shows the results of in vitro assay for the Nef and Lyn binding inhibition when Compounds 1 and 3 were added at different concentrations. The numbers on top of the lanes demonstrate the compound number and the concentration (μmol/L) of each Compound added to the mixed solution of Nef and Lyn. The name of each blot is shown in the left. FIGS. 9-11 show the results of the HIV-1 growth inhibition assay (RT assay) when Compound 1 was added at different concentrations. Results of three experiments in the same condition are shown. The amount of HIV-1 is shown as the counts of RT assay (cpm/μL) at the left of the axis of ordinates. The time required since the infection of HIV-1 (days) are shown under the axis of abscissas. The plots in the graphs are as follows: □ white square]: positive control, × [cross]: negative control, ● [black circle]: Compound 1 (50 mmol/L), ▴ [black triangle]: Compound 1 (25 mmol/L), ▪ [black square]: Compound 1 (10 mmol/L), ∘ [white circle]: Compound 1 (5 mmol/L), Δ [white triangle]: Compound 1 (1 μmol/L) FIGS. 12-14 show the results of the HIV growth inhibition assay (RT assay) when PP2 was added at different concentrations. Results of three experiments in the same condition are shown. The amount of HIV-1 is shown as the counts of RT assay (cpm/μl) at the left of the axis of ordinates. The time required since theinfectionofHIV-1 (days) areshownundertheaxisofabscissas. The plots in the graphs are as follows: □ : positive control, × : negative control, ● [black circle]: PP2 (50 mmol/L), ▴ [black triangle]: PP2 (25 mmol/L), ▪ [black square] PP2 (10 μmol/L), ∘ [white circle]: PP2 (5 mmol/L), Δ [white triangle]: PP2 (1 μmol/L) detailed-description description="Detailed Description" end="lead"? |
Endoscopic image pickup method and magnetic resonance imaging device using the same |
An endoscope-like image taking method of the present invention includes a preparation step of providing at least one peculiar index, which can be discriminated from other portions on an MR image, at the tip of a catheter, a first step (S1) of previously inserting a metal guide wire for guiding the catheter into a body cavity of a patient into which the catheter is inserted, a second step (S2) of inserting the catheter into the body cavity along the guide wire, a third step (S3) of executing an MR imaging sequence of a plurality of sliced images intersecting the guide wire, a fourth step (S4) of reconstructing three-dimensional image data based upon the nuclear magnetic resonance signals, which are generated from the patient when the sequence is executed and are received by the guide wire, and determining the tip position and the inserting direction of the catheter by detecting the peculiar index provided at the tip of the catheter based upon the three-dimensional image data, and a fifth step (S5) of reconstructing the center projected image using the three-dimensional image data and setting the tip position and the inserting direction of the catheter as a view point and a line-of-sight direction and displaying the center projected image on a display means, thereby an endoscope-like image, which is observed from the catheter in the body cavity of the patient, is taken and displayed in real time or semi-real time so that the insertion of the catheter is supported. |
1. An endoscope-like image taking method, comprising: providing at least one peculiar index, which can be discriminated from other portions on an MR image, at the tip of a catheter; previously inserting a metal guide wire for guiding the catheter into a body cavity of a patient into which the catheter is inserted; executing an MR imaging sequence of a plurality of sliced images intersecting the guide wire while inserting the catheter into the body cavity along the guide wire; receiving the nuclear magnetic resonance signals, which are generated from the patient when the sequence is executed, by the guide wire; reconstructing three-dimensional image data using the nuclear magnetic resonance signals; determining the tip position and the inserting direction of the catheter by detecting the peculiar index provided at the tip of the catheter based on any one of the three-dimensional image data and image data imaged by an MR imaging method different from that of the three-dimensional image data; and rearranging a center projected image using the three-dimensional image data and setting the tip position and the inserting direction of the catheter as a view point and a line-of-sight direction and displaying the center projected image on a display means. 2. An endoscope-like image taking method comprising: a preparation step of providing at least one peculiar index, which can be discriminated from other portions on an MR image, at the tip of a catheter; a first step of previously inserting a metal guide wire for guiding the catheter into a body cavity of a patient into which the catheter is inserted; a second step of inserting the catheter into the body cavity along the guide wire; a third step of executing an MR imaging sequence of a plurality of sliced images intersecting the guide wire; a fourth step of reconstructing three-dimensional image data based upon the nuclear magnetic resonance signals, which are generated from the patient when the sequence is executed and are received by the guide wire, and determining the tip position and the inserting direction of the catheter by detecting the peculiar index provided at the tip of the catheter based on the three-dimensional image data; and a fifth step of rearranging a center projected image using the three-dimensional image data and setting the tip position and the inserting direction of the catheter as a view point and a line-of-sight direction and displaying the center projected image on a display means. 3. An endoscope-like image taking method comprising: a preparation step of providing at least one peculiar index, which can be discriminated from other portions on an MR image, at the tip of a catheter; a first step of previously inserting a metal guide wire for guiding the catheter into a body cavity of a patient into which the catheter is inserted; a second step of executing an MR imaging sequence of a plurality of sliced images intersecting the guide wire, reconstructing three-dimensional image data based upon the nuclear magnetic resonance signals, which are generated from the patient when the sequence is executed, received by the guide wire, and storing the three-dimensional image data; a third step of inserting the catheter into the body cavity along the guide wire; a fourth step of executing a measuring sequence to the tip of the catheter in the three-axis directions thereof to acquire nuclear magnetic resonance signals, receiving the nuclear magnetic resonance signals, which are generated from the patient when the sequence is executed, by a receiving coil disposed outside of the patient, and determining the tip position and the inserting direction of the catheter by detecting the peculiar index using the thus received nuclear magnetic resonance signals; and a fifth step of reconstructing a center projected image using the three-dimensional image data and setting the tip position and the inserting direction of the catheter as a view point and a line-of-sight direction and displaying the center projected image on a display means. 4. An endoscope-like image taking method comprising: a preparation step of providing at least one peculiar index, which can be discriminated from other portions on an MR image, at the tip of a catheter; a first step of previously inserting a metal guide wire for guiding the catheter into a body cavity of a patient into which the catheter is inserted; a second step of inserting the catheter into the body cavity along the guide wire; a third step of executing an MR measuring sequence to the tip of the catheter in each of the three-axis directions thereof, receiving the nuclear magnetic resonance signals, which are generated from the patient when the sequence is executed, by a receiving coil disposed outside of the patient, and determining the tip position and the inserting direction of the catheter by detecting the peculiar index provided at the tip of the catheter; a fourth step of executing an MR imaging sequence of a plurality of sliced images intersecting the guide wire; and a fifth step of reconstructing three-dimensional image data based upon the nuclear magnetic resonance signals, which are generated from the patient when the sequence is executed and are received by the guide wire, reconstructing a center projected image using the three-dimensional image data and setting the tip position and the inserting direction of the catheter determined above as a view point and a line-of-sight direction, and displaying the center projected image on a display means. 5. An endoscope-like image taking method according to claim 4, characterized in that the sliced positions imaged at the fourth step are set forward of the tip of the catheter. 6. An endoscope-like image taking method according to any one of claims 1 to 5, characterized in that the peculiar index is a ring-shaped marker provided at the tip of the catheter coaxially with the catheter. 7. An endoscope-like image taking method according to claim 6, characterized in that at least two markers are provided at the tip of the catheter in such a manner that the positions thereof are displaced in the axial direction of the catheter. 8. An endoscope-like image taking method according to claim 3, characterized in that the MR imaging sequence of the plurality of sliced images is executed once each time processing for rearranging the center projected image is executed a plurality of times. 9. A magnetic resonance imaging apparatus comprising: magnetic field generation means for generating the respective magnetic fields of a static magnetic field, a gradient magnetic field, and a high frequency magnetic field that are applied to a patient, receiving means for receiving the nuclear magnetic resonance signals generated from the patient, image reconstruction means for reconstructing the three-dimensional image data of the patient using the thus received nuclear magnetic resonance signals, display means for displaying a reconstructed image, and control means for controlling the magnetic field generation means, the receiving means, and the image reconstruction means, characterized in that: at least one peculiar index, which can be discriminated from other portions on an MR image, is provided at the tip of a catheter inserted into a body cavity of the patient as well as a metal guide wire for guiding the catheter is used as the receiving means; and the image reconstruction means reconstructs three-dimensional image data using the nuclear magnetic resonance signals received by the guide wire, detects the peculiar index using the reconstructed three-dimensional image data, determines the tip position and the inserting direction of the catheter based on the peculiar index, and reconstructs a center projected image using the three-dimensional image data and setting the tip position and the inserting direction of the catheter determined as described above as a view point and a line-of-sight direction and displays the center projected image on the display means. 10. A magnetic resonance imaging apparatus comprising: magnetic field generation means for generating the respective magnetic fields of a static magnetic field, a gradient magnetic field, and a high frequency magnetic field, receiving means for receiving the nuclear magnetic resonance signals generated from the patient, image reconstruction means for reconstructing an image based on the thus received nuclear magnetic resonance signals, display means for displaying the image, and control means for controlling the magnetic field generation means and the receiving means, applying the high frequency magnetic field and the gradient magnetic field to the patient placed in the static magnetic field, and causing an imaging sequence for receiving the nuclear magnetic resonance signals to be executed, characterized in that: the receiving means comprises a receiving coil disposed outside of the patient and a guide wire inserted into a body cavity of the patient; the catheter guided by the guide wire has at least one peculiar index, which can be discriminated from other portions on an MR image, at the tip thereof; and the control means has: a function for causing the image reconstruction means to reconstruct three-dimensional image data using the nuclear magnetic resonance signals received by the guide wire and causing the three-dimensional image data to be stored in a memory means; a function for causing a measuring sequence to be executed to measure NMR signals of the tip of the catheter in the three-axis directions thereof; a function for controlling the image reconstruction means and determining the tip position and the inserting direction of the catheter by detecting the peculiar index using the nuclear magnetic resonance signals received by the receiving coil when the measuring sequence is executed; a function for controlling the image reconstruction means and causing the image reconstruction means to reconstruct a center projected image using the three-dimensional image data and setting the tip position and the inserting direction of the catheter determined as described above as a view point and a line-of-sight direction; and a function for causing the thus reconstructed center projected image to be displayed on the display means. 11. A magnetic resonance imaging apparatus comprising: magnetic field generation means for generating the respective magnetic fields of a static magnetic field, a gradient magnetic field, and a high frequency magnetic field, receiving means for receiving the nuclear magnetic resonance signals generated from the patient, image reconstruction means for reconstructing an image based on the thus received nuclear magnetic resonance signals, display means for displaying the image, and control means for controlling the magnetic field generation means and the receiving means, applying the high frequency magnetic field and the gradient magnetic field to the patient placed in the static magnetic field, and causing an imaging sequence for receiving the nuclear magnetic resonance signals to be executed, characterized in that: the receiving means comprises a receiving coil disposed outside of the patient and a guide wire inserted into a body cavity of the patient; the catheter guided by the guide wire has at least one peculiar index, which can be discriminated from other portions on an MR image, at the tip thereof; and the control means has: a function for causing a measuring sequence to be executed to acquire the nuclear magnetic resonance signals of the tip of the catheter in the three-axis directions thereof; a function for controlling the image reconstruction means and determining the tip position and the inserting direction of the catheter by detecting the peculiar index using the nuclear magnetic resonance signals received by the receiving coil when the measuring sequence is executed; a function for causing an imaging sequence to be executed to image a plurality of a sliced plane intersecting the guide wire; a function for controlling the image reconstruction means and causing the image reconstruction means to reconstruct the three-dimensional image data using the nuclear magnetic resonance signals received by the guide wire; a function for controlling the image reconstruction means and causing the image reconstruction means to reconstruct a center projected image using the three-dimensional image data and setting the tip position and the inserting direction of the catheter determined as described above as a view point and a line-of-sight direction; and a function for causing the thus reconstructed center projected image to be displayed on the display means. 12. A magnetic resonance imaging apparatus according to any one of claims 9 to 11, characterized in that the peculiar index is a ring-shaped marker provided at the tip of the catheter coaxially With the catheter. 13. A magnetic resonance imaging apparatus according to claim 12, characterized in that at least two markers are provided at the tip of the catheter in such a manner that the positions thereof are displaced in the axial direction of the catheter. 14. A magnetic resonance imaging apparatus according to any one of claims 9 to 11, characterized in that the control means repeats a first step of executing an imaging sequence for acquiring the three-dimensional image data, a second step of determining the tip position and the inserting direction of the catheter, and a third step of rearranging and displaying the center projected image. 15. A magnetic resonance imaging apparatus according to claim 14, characterized in that the control means executes the first step once each time the first to third steps are repeated a plurality of times. 16. A magnetic resonance imaging apparatus according to any of claims 9 to 11, characterized in that the image of the wall surface in a body cavity forward of the inserting direction of the catheter is displayed on the display means by being varied according to an inserted position of the catheter. |
<SOH> BACKGROUND ART <EOH>An MRI apparatus is an apparatus for observing the inside of a patient by acquiring a tomogram and a frequency spectrum of a patient making use of a nuclear magnetic resonance phenomenon and includes a static magnetic field generator, a gradient magnetic field coil, a transmitting coil, and a receiving coil. The static magnetic field generator aligns the directions of the spins of nuclei (ordinarily, protons) that constitute the patient, the gradient magnetic field coil identifies the imaging slice of the patient as well as encodes position information to the nuclear magnetic resonance signals acquired from the patient, the transmitting coil generates a high frequency magnetic field having the same frequency as the resonance frequency of the protons, and the receiving coil receives the signals from the protons. The MRI apparatus arranged as described above can selectively image any of arbitrary regions and tissues, and various imaging methods have been proposed according to patients to be imaged. For example, imaging can be executed using a two- or three-dimensional measurement. Further, in recent years, as an important field to which the MRI apparatus is applied, there has been developed a method (IV-MRI) of utilizing the MRI apparatus as the monitor of a catheter while needling or introducing the catheter into a blood vessel. In this IV-MRI, it is required to execute imaging and to display images in real time so that, for example, the catheter can be inserted to a target position without fail, and various types of a high speed imaging method such as EPI and the like have been in practical use. In contrast, various shapes of the receiving coil have been developed and practically used according to portions to be imaged, and an RF receiving antenna, which also acts as a guide wire of the catheter, has been proposed as a receiving coil preferably used when the catheter is inserted as described above (for example, Japanese Unexamined Patent Publication No. 10-179550, PCT Japanese Translation Patent Publication No. 2000-509276, a document “Intravascular Magnetic Resonance Imaging Using a Loopless Catheter Antenna”, MRM 37: 112-118 (1997), and the like). Note that since the measurable sensitivity range of the guide-wire-shaped RF receiving antenna is limited to the vicinity of the guide wire, a tomogram that can be imaged is limited to a small region (for example, several millimeters). However, an image obtained by conventional MRI apparatuses is mainly a tomogram. Accordingly, the conventional MRI apparatuses are disadvantageous in an application for confirming the position of a catheter inserted into a body cavity such as a blood vessel having a curving portion because they cannot uniquely determine a sliced plane including the catheter. In contrast, as to a straight needle, the conventional MRI apparatuses can automatically take a tomogram on a plane including the needle or on a plane orthogonal to the needle by attaching an active or passive marker to the needle, and many conventional technologies exist. Further, as to the catheter, there is known a method of executing imaging by providing a marker, which can be identified by the MRI apparatuses, in the catheter. Since, however, a sliced plane including the catheter having a curved portion cannot be uniquely determined, it is not always easy to confirm the inserted position of the catheter. Incidentally, the applicant has proposed a method of creating an endoscope-like image making use of three-dimensional image data acquired by an X-ray CT apparatus and an MRI apparatus as a method of displaying an image of the inside wall of a blood vessel, and the like in place of a conventional tomogram (Japanese Unexamined Patent Publications Nos. 7-210704 and 8-16813). According to the method, it is possible to convert three-dimensional tomogram data of a region including a blood vessel and the like into an image (endoscope-like image) of the inside wall of a body cavity, and the like observed from the inside by a center projecting method and to display the image, and this image is effective to diagnosis. In this case, the image may be processed according to particular shading algorithm. However, the conventional endoscope-like image is created by a method of creating it based on three-dimensional image data that has been acquired, and further a view point and a line-of-sight direction must be input by a mouse or a track ball. Accordingly, the method cannot be applied to the IV-MRI which executes imaging and display of an image in real time. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 shows the overall arrangement of an MRI apparatus of an embodiment to which the present invention is applied; FIG. 2 is a view explaining a guide wire also acting as an RF receiving antenna according to the present invention and a state of use of a catheter guided by the guide wire; FIG. 3 is a flowchart showing a procedure of an embodiment according to an endoscope-like image taking method of the present invention; FIG. 4 is a view showing an example of three-dimensional image data acquired by the endoscope-like image taking method of the present invention; FIG. 5 is a view showing an example of three-dimensional image data acquired by the endoscope-like image taking method of the present invention; FIG. 6 is a view explaining center projecting processing in the endoscope-like image taking method of the present invention; FIG. 7 is a view explaining a method of creating a center projected image in the endoscope-like image taking method of the present invention; FIG. 8 is a view schematically showing an endoscope-like image acquired by the center projecting processing; FIG. 9 is a flowchart showing a procedure of another embodiment of the endoscope-like image taking method according to the present invention; and FIG. 10 is a flowchart showing a procedure of still another embodiment of the endoscope-like image taking method according to the present invention. detailed-description description="Detailed Description" end="lead"? |
Plant transcriptional repressor, proteic nuclear factors binding thereto, and uses thereof |
The present application describes a plant gene regulatory element and the uses thereof. More particularly a silencing element for modulating plant responses to pathogens, auxin and ethylene is described. The invention also describes transcriptional repressors which specifically binds onto the silencing element of the invention. Nucleotide and amino acid sequences of a novel transcriptional repressor referred as “SEBF” are given. |
1-79. (Canceled). 80. A transformed or transfected cell comprising: a nucleic acid sequence selected from the group consisting of a nucleic acid sequence having at least 96% nucleotide sequence identity with SEQ ID NO: 21; and a nucleic acid sequence having at least 75% nucleotide sequence identity with a nucleic acid encoding an amino acid sequence of SEQ ID NO: 22. 81. A transformed or transfected cell comprising an isolated or purified protein comprising: an amino acid sequence selected from the group consisting of a sequence having at least 85% sequence similarity to amino acid sequences encoded by nucleotides 68 to 937 of SEQ ID NO: 21; and a sequence having at least 85% similarity to SEQ ID NO: 22. 82. A cloning or expression vector comprising a nucleic acid sequence having at least 96% nucleotide sequence identity with SEQ ID NO: 21, or a nucleic acid sequence having at least 75% nucleotide sequence identity with a nucleic acid encoding an amino acid sequence of SEQ ID NO: 22. 83. The cloning or expression vector of claim 82, wherein it further comprises an inducible or constitutive promoter. 84. A method for obtaining a result in a plant, wherein the result is modulating resistance or tolerance to a pathogen, comprising modulating expression or biological activity of SEBF (Silencing Element Binding Factor) protein or of a SEBF functional homologue in said plant. 85. The method of claim 84, wherein the result is increasing resistance or tolerance to the pathogen, the method comprises reducing expression or biological activity of SEBF (Silencing Element Binding Factor) protein or of a SEBF functional homologue in said plant. 86. The method of claim 84, wherein expression or biological activity of said SEBF protein or said SEBF functional homologue in said plant is reduced by expressing a SEBF antisense molecule, expressing proteins inducing co-suppression of SEBF level, expressing proteins inducing co-suppression of SEBF activity, knocking out a gene encoding the SEBF protein, chemically mutating a gene encoding the SEBF protein, expressing a ribozyme cleaving a SEBF mRNA, or introducing RNAi molecules in said plant. 87. The method of claim 85, wherein chemically mutating comprises TILLING (Target Induced Local Lesion in Genomes). 88. The methods of claims 84, wherein the result is decreasing resistance or tolerance to the pathogen, the method comprises increasing expression or biological activity of said SEBF protein or SEBF functional homologue by introducing a SEBF coding sequence in said plant. 89. The method of claim 88, comprising overexpressing the SEBF protein or functional homologue thereof. 90. The method of claim 88, comprising increasing binding affinity of the SEBF protein or functional homologue thereof to the promoter region by mutating the SEBF coding sequence. 91. The method as in any one of claims 84 to 90 wherein the SEBF protein is an endogenous SEBF protein. 92. An isolated or purified gene regulatory element, comprising a nucleic acid selected from the group consisting of nucleic acid sequence GACTGTCAC (SEQ ID NO: 26); and nucleic acid sequence BTGTCNC (SEQ ID NO: 23). 93. The regulatory element of claim 92, wherein said regulatory element consists of a silencing element. 94. The regulatory element of claim 92, wherein in the sequence BTGTCNC (SEQ ID NO: 23), B is a pyrimidine. 95. The regulatory element of claim 92, wherein in the sequence BTGTCNC (SEQ ID NO: 23), B is C and N is A. 96. A method for obtaining a result in a plant, wherein the result is modulating resistance or tolerance to a pathogen, said plant comprising nucleic acid sequence BTGTCNC (SEQ ID NO: 23) in a promoter region of a gene, the method comprising altering binding of an endogenous nuclear DNA-binding protein to the sequence BTGTCNC (SEQ ID NO: 23) in said plant. 97. The method of claim 96, wherein the result is increasing resistance or tolerance to a pathogen, said plant having a sequence BTGTCNC (SEQ ID NO: 23) in a promoter region of a gene, the method comprising reducing or preventing binding of an endogenous nuclear DNA-binding protein to the sequence BTGTCNC (SEQ ID NO: 23) in said plant. 98. The method of claim 97, comprising mutating or deleting the sequence BTGTCNC (SEQ ID NO: 23). 99. The method of claims 97, wherein the sequence BTGTCNC (SEQ ID NO: 23) is located in the promoter region of a defense gene. 100. The method of claim 99, wherein said defense gene is selected from the group of genes listed in Table 5 consisting of Pr-10, Pr-10 (ypr10a), Pr10 (ypr10b), Pr-10a, Pr-10b, chitinase, glucanase, PR-1, osmotin (PR-5), peroxidase, alternative oxidase, and anti-fungal protein. 101. The method of claim 96, wherein the result is reducing resistance or tolerance to a pathogen, comprising increasing binding of an endogenous nuclear DNA-binding protein to the promoter region of a gene including sequence BTGTCNC (SEQ ID NO: 23) in said plant. 102. The method of claim 96, wherein said plant is selected from the group consisting of vegetables, leguminous plants, trees, and cereals. 103. The method of claim 96, wherein said plant is selected from the group consisting of potato, tomato, tobacco, cotton, rice, wheat, corn, barley, oat, canola, soybean, pea, sugar cane, sugar beet, strawberry, and banana. 104. A vegetal host genetically modified for exhibiting an altered expression or biological activity of a proteinic nuclear factor having a specific binding activity to BTGTCNC (SEQ ID NO: 23), wherein said altered level is compared to an endogenous level that has not been altered in a corresponding genetically unmodified vegetal host. 105. The vegetal host of claim 104, wherein said factor is a polypeptide encoded by a nucleic acid sequence selected from the group consisting of a nucleic acid sequence having at least 96% nucleotide sequence identity with SEQ ID NO: 21; and a nucleic acid sequence having at least 75% nucleotide sequence identity with a nucleic acid encoding an amino acid sequence of SEQ ID NO: 22. 106. The vegetal host of claim 104, wherein said factor is a SEBF protein comprising an amino acid sequence selected from the group consisting of a sequence having at least 85% sequence similarity to amino acid sequences encoded by nucleotides 68 to 937 of SEQ ID NO: 21; and a sequence having at least 85% similarity to SEQ ID NO: 22. |
<SOH> BACKGROUND OF THE INVENTION <EOH>A) Field of the Invention The present invention relates to a plant gene regulatory element and its uses, and more particularly to a silencing element for modulating plant responses to pathogens, auxin and ethylene. The invention also relates to transcriptional repressors which specifically binds onto the silencing element of the invention, including a protein referred herein after as “SEBF”. B) Brief Description of the Prior Art A variety of defense specific events are induced in plants in response to pathogen infection. Although key components of the signaling cascades are being discovered, few transcription factors that integrate these signals at the transcriptional level have been identified up to date. PR genes are plant genes that are induced by pathogen invasion. These genes are subdivided into 11 classes. Since PR genes are well characterized, they provide excellent models to study transcriptional regulation of defense genes. The PR-10 gene family is one of the classes of PR genes. Expression studies have identified cis-acting elements involved in PR-10a gene regulation, a member of the PR-10 gene family. An elicitor response element (ERE) located between nucleotides −135 and −105 is essential and sufficient for elicitor induced expression of PR-10a. PBF-2, a single-stranded DNA binding factor, appears to play a role in activation of PR-10a from the ERE. It has been shown that the presence of the ERE is sufficient for PR-10a activation, removal of the silencing element (SE), located between −52 and −27, leads to further activation, suggesting that SE participates, with the ERE, in the regulation of PR-10a (Matton et al, 1993; Després et al., 1995). However, the exact nucleic acid sequence required for full SE activity has never been given. Furthermore, the identity of the transcriptional repressor specifically binding to the silencing element (SE) of PR-10a is also unknown. Accordingly, there is a need for an isolated or purified nucleic acid comprising a sequence coding for full of SE activity and to the use thereof for modulating activity of genes, and more particularly genes involved in plant responses to pathogen such as PR-10a gene. There is also a need for methods and genetically modified plants wherein the nucleic acid of the invention has been introduced or wherein the sequence coding for full SE activity has been mutated, deleted, or silenced, thereby modulating the plant defense mechanisms and resistance to pathogens. There is also a long felt need for a transcriptional repressor that is capable of modulating plant defense mechanisms and resistance to pathogens and more particularly for a transcriptional repressor capable to specifically bind the isolated or purified nucleic acid of the invention. There is also a need for methods and genetically modified plants wherein levels of the transcriptional repressor of the invention have been modulated. There is a further need for effective methods and compositions to modulate plant resistance or tolerance to pathogens, and/or to modulate plant response to auxin and/or to ethylene. The present invention fulfils these needs and also other needs which will be apparent to those skilled in the art upon reading the following specification. |
<SOH> SUMMARY OF THE INVENTION <EOH>According to a first aspect, the present invention relates to an isolated or purified nucleic acid molecule comprising a sequence selected from the group consisting of: a) sequence set forth in SEQ ID NO: 21; b) a nucleotide sequence having at least 96% nucleotide sequence identity with SEQ ID NO: 21; and c) a nucleotide sequence having at least 75% nucleotide sequence identity with a nucleic acid encoding an amino acid sequence of SEQ ID NO:22. The invention also concerns transformed or transfected cells as well as cloning or expression vector that contains such a nucleic acid. Preferably, the cell and the vector express or are capable of directing expression of the peptide encoded by the nucleic acid. In a related aspect, the invention concerns an isolated or purified protein comprising an amino acid sequence selected from the group consisting of: a) sequences encoded by a nucleic acid as defined previously; b) sequences having at least 85% identity to SEQ ID NO: 22; c) sequences having at least 87% similarity to SEQ ID NO: 22; d) sequence set forth in SEQ ID NO: 22; e) sequences having at least 85% identity to amino acid sequences encoded by nucleotides 68 to 937 of SEQ ID NO: 21; and f) sequences having at least 87% sequence similarity to amino acid sequences encoded by nucleotides 68 to 937 of SEQ ID NO: 21. The inventions also concerns compositions comprising any of the nucleic acids or protein defined previously. In another aspect, the invention relates to plant proteinic nuclear factors that are capable, among other things, of mediating repression of a silencing element involves in plant defense mechanisms. Preferably, the proteinic nuclear factor is a plant transcriptional repressor which specifically binds onto the sequence BTGTCNC or YTGTCNC. Most preferred transcriptional repressor consists of a protein referred herein after as “SEBF” for Silencing Element Binding Factor. In one embodiment there is described a composition comprising an isolated or purified SEBF protein or a functional homologue thereof. Preferably, the SEBF protein or homologue comprises an amino acid sequence selected from the group consisting of: a) sequences encoded by a nucleic acid having a sequence at least 85% identical to nucleotides 68 to 937 of SEQ ID NO: 21; b) sequences having at least 85% identity to SEQ ID NO:22; c) sequences having at least 87% similarity to SEQ ID NO:22; and d) sequence provided in SEQ ID NO:2. More preferably, the SEBF protein is purified from potato, and wherein it has a purification factor of about 90 to about 20 700 fold. According to another aspect, the present invention relates to an isolated or purified nucleic acid comprising a binding sequence onto which proteinic nuclear factors, such as the transcriptional repressor of the silencing element (SE) of PR-10a, specifically binds. Preferably, the binding sequence comprises sequence BTGTCNC (SEQ ID NO:23), more preferably sequence YTGTCNC (SEQ ID NO:24). The invention also concerns an isolated or purified gene regulatory element comprising a nucleic acid sequence that is essential for the full activity in plant of the silencing element (SE) of PR-10a. Preferably, the gene regulatory element consists of a silencing element and it comprises sequence GACTGTCAC (SEQ ID NO:26) or sequence BTGTCNC (SEQ ID NO:23), and more preferably sequence YTGTCNC (SEQ ID NO:24). The invention also concerns a DNA construct comprising the gene regulatory element and genetically modified plant entities comprising the gene regulatory element or the DNA construct. According to a related aspect, the present invention concerns a method for altering gene expression in a plant. The method comprises the step of altering in the plant binding of a nuclear DNA-binding protein to sequence BTGTCNC. A non-limitative list of preferred endogenous DNA-binding proteins includes those having at least 48% identity or similarity to SEBF. More preferably, the DNA-binding protein consists of SEBF or of a functional homologue thereof having at least 90% identity or similarity to SEBF. In a preferred embodiment, there is described a method for increasing the expression of a gene of interest, this gene having a promoter region comprising sequence BTGTCNC. The method comprises the step of mutating the promoter region of the gene for mutating or deleting the sequence BTGTCNC. The gene maybe PR gene. In another preferred embodiment, there is described a method for reducing the expression of a gene of interest, this gene having a promoter region devoid of sequence BTGTCNC. The method comprises the step of introducing in an operable linked manner the sequence BTGTCNC into the promoter region. In a more specific embodiment, there is described a vegetal host (e.g. algae, plant) genetically modified for exhibiting an altered expression or biological activity of a proteinic nuclear factor having a specific binding activity to sequence BTGTCNC (SEQ ID NO:23), preferably YTGTCNC (SEQ ID NO:24), the altered level being compared to a corresponding genetically unmodified vegetal host in which the endogenous level has not been altered. Preferred proteinic nuclear factors are those having at least 48% identity or similarity to SEBF. More preferably, the proteinic nuclear factors consists of SEBF or of a functional homologue thereof having at least 90% identity or similarity to SEBF. In an even more specific embodiment, the expression or biological activity of SEBF or homologue has been increased in the vegetal host such that it exhibits a phenotype selected from the group consisting of: reduced resistance or tolerance to a pathogen; reduced growth, rooting and/or fruit production; increased resistance to an auxinic herbicide; reduced ethylene production; delay in ripening of its fruit(s) and/or protection of its fruit(s) against over-ripening. In an other specific embodiment, the expression or biological activity of SEBF or homologue has been reduced in the vegetal host such that it exhibits a phenotype selected from the group consisting of increased resistance or tolerance to a pathogen; increased growth, rooting and/or fruit production; increased sensitivity to an auxinic herbicide; increased ethylene production; and early fruit maturation. The invention also encompasses methods for obtaining the vegetal host having the phenotype(s) described previously. Typically these methods comprises the step of modulating (typically reducing or increasing) in the plant expression or biological activity of SEBF or of a SEBF functional homologue. Another related aspect of the invention concerns a genetically modified vegetal host comprising a genome, wherein transcriptional activity of a gene associated with presence or absence of sequence BTGTCNC (SEQ ID NO:23), preferably YTGTCNC (SEQ ID NO:24), in a promoter region this gene has been altered. Of course, the altered biological activity is compared to a corresponding genetically unmodified vegetal host in which the endogenous biological activity has not been altered. In one embodiment, the promoter region of the gene comprises the sequence BTGTCNC, and the promoter region has been genetically modified (e.g. mutation, deletion) for inactivating a repressive transcriptional activity associated with the presence of the sequence BTGTCNC. In another embodiment, the promoter region is devoid of sequence BTGTCNC (SEQ ID NO:23), and this region has been genetically modified for inserting therein in an operable linked manner the sequence BTGTCNC. In another aspect, the present invention relates to methods for obtaining a particular phenotype in plants, and more particularly plants which are used in agriculture and plants with a horticultural value. In one embodiment, there is described methods for: the modulation of a plant resistance or tolerance to pathogens; the modulation of induction of genes of a plant controlled by auxins; the modulation of a plant auxins-controlled genes induction; the augmentation of growth, rooting and/or fruit production in a plant; the modulation of a plant auxin-inducted ACC synthase gene; the modulation of a plant ethylene production; and the modulation of a plant plastid mRNAs stability, expression or activity. All these methods comprises the step of modulating in the plant expression or biological activity of an endogenous SEBF protein or of a SEBF functional homologue. In one preferred embodiment, there is described a method for obtaining a genetically modified plant exhibiting a phenotype selected from the group consisting of: increased resistance or tolerance to a pathogen; increased growth, rooting and/or fruit production; increased sensitivity to an auxinic herbicide; increased ethylene production; early fruit maturation; altered plastid mRNAs stability, expression or activity; a promoter region of a gene involved in the phenotype comprising sequence BTGTCNC (SEQ ID NO:23), the phenotype of the plant being compared to a corresponding genetically unmodified plant; the method comprising the step of genetically modifying the genome of this plant for inactivating an endogenous biological activity associated with the presence of the sequence BTGTCNC. In one preferred embodiment, there is described a method for obtaining a genetically modified plant exhibiting a phenotype selected from the group consisting of: reduced resistance or tolerance to a pathogen; reduced growth, rooting and/or fruit production; increased resistance to an auxinic herbicide; reduced ethylene production; delay in ripening of its fruit(s) and/or protection of its fruit(s) against over-ripening; and altered plastid mRNAs stability, expression or activity; a promoter region of a gene involved in the phenotype being devoid of sequence BTGTCNC (SEQ ID NO:23), the phenotype of the plant being compared to a corresponding genetically unmodified plant; the method comprising the step of genetically modifying the genome of said plant for inserting therein in an operably linked manner the sequence BTGTCNC. In further embodiments of the invention, there is described plants, and methods for obtaining the same, the plants exhibiting an increased (or decreased) resistance or tolerance to pathogens; a faster (or lower) induction of its defense response; an increased (or decreased) sensitivity for endogenous auxins; and/or a delayed ripening or an advanced fruit maturation. In one specific embodiment, there is provided a method for modulating a plant resistance or tolerance to a pathogen, comprising modulating in the plant expression or biological activity of an endogenous SEBF protein. More particularly, the is described a method for increasing a plant resistance or tolerance to a pathogen, comprising reducing in the plant expression or biological activity of an endogenous SEBF protein. Expression or biological activity of the endogenous SEBF protein may be reduced increased for instance by expressing in the plant SEBF antisense molecules; by expressing proteins inducing a co-suppression of SEBF level or activity; by a knock out or a chemical mutagenesis of a gene encoding the SEBF protein; of by expressing a ribozyme cleaving a SEBF mRNA. The is also described a method for reducing a plant resistance or tolerance to a pathogen, comprising increasing in the plant expression or biological activity of a SEBF protein. The expression or biological activity of the endogenous SEBF protein may increased for instance by introducing in the plant a expressible SEBF coding sequence. The SEBF coding sequence may be under control of an inducible or constitutive promoter. The invention also encompasses plants genetically modified for having an increased (or reduced) resistance or tolerance to a pathogen when compared to a corresponding plant not genetically modified, wherein expression or biological activity of an endogenous SEBF protein is reduced (or increased) in the genetically modified plant as compared to a corresponding genetically unmodified plant. In a another specific embodiment, there is provided a method for modulating a plant resistance or tolerance to a pathogen, the plant having sequence BTGTCNC (SEQ ID NO:23) in a promoter region of a gene, the method comprising altering in this plant the binding of an endogenous nuclear DNA-binding protein to the sequence BTGTCNC. More particularly, the is described a method for increasing a plant resistance or tolerance to a pathogen, the plant having sequence BTGTCNC (SEQ ID NO:23) in a promoter region of a gene, the method comprising reducing or preventing in the plant binding of an endogenous nuclear DNA-binding protein to the sequence BTGTCNC. The is also described a method for reducing a plant resistance or tolerance to a pathogen, comprising permitting or increasing in said plant binding of an endogenous nuclear DNA-binding protein to a promoter region of a gene including sequence BTGTCNC (SEQ ID NO:23). The invention also encompasses plants genetically modified by these methods for having an increased (or reduced) resistance or tolerance to a pathogen. In a further specific embodiment, there is provided a method for modulating induction of genes controlled by auxins in plants, the method comprising modulating in the plant expression or biological activity of an endogenous SEBF protein or of a SEBF functional homologue. More particularly, the is described a method for increasing growth, rooting and/or fruit production in a plant, the method comprising reducing in the plant expression or biological activity of an endogenous SEBF protein or of a SEBF functional homologue. There is also described a method for increasing a plant resistance to an auxinic herbicide, comprising reducing in the plant expression or biological activity of an endogenous SEBF protein or of a SEBF functional homologue. The invention also encompasses plants genetically modified by these methods. In still a further specific embodiment, there is provided a method for modulating a plant ethylene production, comprising modulating in the plant expression or biological activity of an endogenous SEBF protein or of a SEBF functional homologue. More particularly, the is described a method for increasing production of ethylene by a plant, comprising reducing in the plant expression or biological activity of an endogenous SEBF protein or of a SEBF functional homologue. There is also described a method for reducing production of ethylene by a plant, comprising increasing in the plant expression or biological activity of an endogenous SEBF protein or of a SEBF functional homologue. The invention also encompasses plants genetically modified by these methods for having an increased (or reduced) production of ethylene. As used hereinbefore, the vegetal host preferably consists of a plant (monocotyledon or dicotyledon), more preferably a vegetable, a leguminous plant, a tree, a fruit tree, grass, a cereal, and even more preferably it consists of a potato, a tomato, tobacco, cotton, rice, wheat, corn, barley, oat, canola, soybean, pea, sugar cane, sugar beet, strawberry, and banana. Other objects and advantages of the present invention will be apparent upon reading the following non-restrictive description of several preferred embodiments, made with reference to the accompanying drawings. |
Efficient and scalable parametric stereo coding for low bitrate applications |
The present invention provides improvements to prior art audio codecs that generate a stereo-illusion through post-processing of a received mono signal. These improvements are accomplished by extraction of stereo-image describing parameters at the encoder side, which are transmitted and subsequently used for control of a stereo generator at the decoder side. Furthermore, the invention bridges the gap between simple pseudo-stereo methods, and current methods of true stereo-coding, by using a new form of parametric stereo coding. A stereo-balance parameter is introduced, which enables more advanced stereo modes, and in addition forms the basis of a new method of stereo-coding of spectral envelopes, of particular use in systems where guided HFR (High Frequency Reconstruction) is employed. As a special case, the application of this stereo-coding scheme in scalable HFR-based codecs is described. |
1. A method for coding of stereo properties of an input signal, characterised by: at an encoder, calculate a width-parameter that signals a stereo-width of said input signal, and at a decoder, generate a stereo output signal, using said width-parameter to control a stereo-width of said output signal. 2. A method according to claim 1, characterised by: at said encoder, form a mono signal from said input signal, and at said decoder, said generation implies a pseudo-stereo method operating on said mono signal. 3. A method according to claim 2, characterised in that said pseudo-stereo method implies splitting of said mono signal into two signals as well as addition of delayed version(s) of said mono signal to said two signals, at level(s) controlled by said width-parameter. 4. A method according to claim 3, characterised in that said delayed version(s) are high-pass filtered and progressively attenuated at higher frequencies prior to being added to said two signals. 5. A method according to claim 1, characterised in that said width-parameter is a vector, and the elements of said vector correspond to separate frequency bands. 6. A method according to claims 1-5, characterised in that if said input signal is of type dual mono, said output signal is also of type dual mono. 7. A method for coding of stereo properties of an input signal, characterised by: at an encoder, calculate a balance-parameter that signals a stereo-balance of said input signal, and at a decoder, generate a stereo output signal, using said balance-parameter to control a stereo-balance of said output signal. 8. A method according to claim 7, characterised by: at said encoder, form a mono signal from said input signal, and at said decoder, said generation implies splitting of said mono signal into two signals, and said control implies adjustment of levels of said two signals. 9. A method according to claim 7, characterised in that a power for each channel of said input signal is calculated, and said balance-parameter is calculated from a quotient between said powers. 10. A method according to claim 9, characterised in that said powers and said balance-parameter are vectors where every element corresponds to a specific frequency band. 11. A method according to claim 7, characterised in at said decoder interpolating between two in time consequtive values of said balance-parameters in a way that the momentary value of the corresponding power of said mono signal controls how steep the momentary interpolation should be. 12. A method according to claim 11, characterised in that said interpolation method is performed on balance values represented as logarithmic values. 13. A method according to claim 7, characterised in that said values of balance-parameters are limited to a range between a previous balance value, and a balance value extracted from other balance values by a median filter or other filter process, where said range can be further extended by moving the borders of said range by a certain factor. 14. A method according to claim 13, characterised in that said method of extracting limiting borders for balance values, is, for a multiband system, frequency dependent. 15. A method according to claim 10, characterised in that an additional level-parameter is calculated as a vector sum of said powers and sent to said decoder, thereby providing said decoder a representation of a spectral envelope of said input signal. 16. A method according to claim 15, characterised in that said level-parameter and said balance-parameter adaptively are replaced by said powers. 17. A method according to claim 16, characterised in that said spectral envelope is used to control a HFR-process in a decoder. 18. A method according to claim 15, characterised in that said level-parameter is fed into a primary bitstream of a scalable HFR-based stereo codec, and said balance-parameter is fed into a secondary bitstream of said codec. 19. A method according to claims 2 and 18, characterised in that said mono signal and said width-parameter are fed into said primary bitstream. 20. A method according to claims 5 and 16, characterised in that said width-parameters are processed by a function that gives smaller values for a balance value that corresponds to a balance position further from the center position. 21. A method according to any of claims 7-18, characterised in that a quantization of said balance-parameter employs smaller quantization steps around a center position and larger steps towards outer positions. 22. A method according to claims 5 and 21, characterised in that said width-parameters and said balance-parameters are quantized using a quantization method in terms of resolution and range which, for a multiband system, is frequency dependent. 23. A method according to any of claims 10-18, characterised in that said balance-parameter adaptively is delta-coded either in time or in frequency. 24. A method according to any of claims 2 and 8, characterised in that said input signal is passed though a Hilbert transformer prior to forming said mono signal. 25. An apparatus for parametric stereo coding, characterised by: at an encoder, means for calculation of a width-parameter that signals a stereo-width of an input signal, and means for forming a mono signal from said input signal, at a decoder, means for generating a stereo output signal from said mono signal, using said width-parameter to control a stereo-width of said output signal. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Audio source coding techniques can be divided into two classes: natural audio coding and speech coding. At medium to high bitrates, natural audio coding is commonly used for speech and music signals, and stereo transmission and reproduction is possible. In applications where only low bitrates are available, e.g. Internet streaming audio targeted at users with slow telephone modem connections, or in the emerging digital AM broadcasting systems, mono coding of the audio program material is unavoidable. However, a stereo impression is still desirable, in particular when listening with headphones, in which case a pure mono signal is perceived as originating from “within the head”, which can be an unpleasant experience. One approach to address this problem is to synthesize a stereo signal at the decoder side from a received pure mono signal. Throughout the years, several different “pseudo-stereo” generators have been proposed. For example in [U.S. Pat. No. 5,883,962], enhancement of mono signals by means of adding delayed/phase shifted versions of a signal to the unprocessed signal, thereby creating a stereo illusion, is described. Hereby the processed signal is added to the original signal for each of the two outputs at equal levels but with opposite signs, ensuring that the enhancement signals cancel if the two channels are added later on in the signal path. In [PCT WO 98/57436] a similar system is shown, albeit without the above mono-compatibility of the enhanced signal. Prior art methods have in common that they are applied as pure post-processes. In other words, no information on the degree of stereo-width, let alone position in the stereo sound stage, is available to the decoder. Thus, the pseudo-stereo signal may or may not have a resemblance of the stereo character of the original signal. A particular situation where prior art systems fall short, is when the original signal is a pure mono signal, which often is the case for speech recordings. This mono signal is blindly converted to a synthetic stereo signal at the decoder, which in the speech case often causes annoying artifacts, and may reduce the clarity and speech intelligibility. Other prior art systems, aiming at true stereo transmission at low bitrates, typically employ a sum and difference coding scheme. Thus, the original left (L) and right (R) signals are converted to a sum signal, S=(L+R)/2, and a difference signal, D=(L−R)/2, and subsequently encoded and transmitted. The receiver decodes the S and D signals, whereupon the original L/R-signal is recreated through the operations L=S+D, and R=S−D. The advantage of this, is that very often a redundancy between L and R is at hand, whereby the information in D to be encoded is less, requiring fewer bits, than in S. Clearly, the extreme case is a pure mono signal, i.e. L and R are identical. A traditional UR-codec encodes this mono signal twice, whereas a S/D codec detects this redundancy, and the D signal does (ideally) not require any bits at all. Another extreme is represented by the situation where R=−L, corresponding to “out of phase” signals. Now, the S signal is zero, whereas the D signal computes to L. Again, the S/D-scheme has a clear advantage to standard L/R-coding. However, consider the situation where e.g. R=0 during a passage, which was not uncommon in the early days of stereo recordings. Both S and D equal L/2, and the S/D-scheme does not offer any advantage. On the contrary, L/R-coding handles this very well: The R signal does not require any bits. For this reason, prior art codecs employ adaptive switching between those two coding schemes, depending on what method that is most beneficial to use at a given moment. The above examples are merely theoretical (except for the dual mono case, which is common in speech only programs). Thus, real world stereo program material contains significant amounts of stereo information, and even if the above switching is implemented, the resulting bitrate is often still too high for many applications. Furthermore, as can be seen from the resynthesis relations above, very coarse quantization of the D signal in an attempt to further reduce the bitrate is not feasible, since the quantization errors translate to non-neglectable level errors in the L and R signals. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention employs detection of signal stereo properties prior to coding and transmission. In the simplest form, a detector measures the amount of stereo perspective that is present in the input stereo signal. This amount is then transmitted as a stereo width parameter, together with an encoded mono sum of the original signal. The receiver decodes the mono signal, and applies the proper amount of stereo-width, using a pseudo-stereo generator, which is controlled by said parameter. As a special case, a mono input signal is signaled as zero stereo width, and correspondingly no stereo synthesis is applied in the decoder. According to the invention, useful measures of the stereo-width can be derived e.g. from the difference signal or from the cross-correlation of the original left and right channel. The value of such computations can be mapped to a small number of states, which are transmitted at an appropriate fixed rate in time, or on an as-needed basis. The invention also teaches how to filter the synthesized stereo components, in order to reduce the risk of unmasking coding artifacts which typically are associated with low bitrate coded signals. Alternatively, the overall stereo-balance or localization in the stereo field is detected in the encoder. This information, optionally together with the above width-parameter, is efficiently transmitted as a balance-parameter, along with the encoded mono signal. Thus, displacements to either side of the sound stage can be recreated at the decoder, by correspondingly altering the gains of the two output channels. According to the invention, this stereo-balance parameter can be derived from the quotient of the left and right signal powers. The transmission of both types of parameters requires very few bits compared to full stereo coding, whereby the total bitrate demand is kept low. In a more elaborate version of the invention, which offers a more accurate parametric stereo depiction, several balance and stereo-width parameters are used, each one representing separate frequency bands. The balance-parameter generalized to a per frequency-band operation, together with a corresponding per band operation of a level-parameter, calculated as the sum of the left and right signal powers, enables a new, arbitrary detailed, representation of the power spectral density of a stereo signal. A particular benefit of this representation, in addition to the benefits from stereo redundancy that also S/D-systems take advantage of, is that the balance-signal can be quantized with less precision than the level ditto, since the quantization error, when converting back to a stereo spectral envelope, causes an “error in space”, i.e. perceived localization in the stereo panorama, rather than an error in level. Analogous to a traditional switched L/R- and S/D-system, the level/balance-scheme can be adaptively switched off, in favor of a levelL/levelR-signal, which is more efficient when the overall signal is heavily offset towards either channel. The above spectral envelope coding scheme can be used whenever an efficient coding of power spectral envelopes is required, and can be incorporated as a tool in new stereo source codecs. A particularly interesting application is in HFR systems that are guided by information about the original signal highband envelope. In such a system, the lowband is coded and decoded by means of an arbitrary codec, and the highband is regenerated at the decoder using the decoded lowband signal and the transmitted highband envelope information [PCT WO 98/57436]. Furthermore, the possibility to build a scalable HFR-based stereo codec is offered, by locking the envelope coding to level/balance operation. Hereby the level values are fed into the primary bitstream, which, depending on the implementation, typically decodes to a mono signal. The balance values are fed into the secondary bitstream, which in addition to the primary bitstream is available to receivers close to the transmitter, taking an IBOC (In-Band On-Channel) digital AM-broadcasting system as an example. When the two bitstreams are combined, the decoder produces a stereo output signal. In addition to the level values, the primary bitstream can contain stereo parameters, e.g. a width parameter. Thus, decoding of this bitstream alone already yields a stereo output, which is improved when both bitstreams are available. |
Device and methods for detecting the response of a plurality of cells to at least one analyte of interest |
An apparatus and methods for detecting at least one analyte of interest either produced or consumed by a plurality of cell. In one embodiment of the present invention, the method includes the steps of providing a housing defining a chamber, placing a plurality of cells in the chamber, and simultaneously detecting at least two analytes of interest either produced or consumed by the plurality of cells in the chamber. |
1. A method for detecting at least one analyte of interest either produced or consumed by a plurality of cell, comprising the steps of: a. providing a housing defining a chamber, b. placing a plurality of cells in the chamber; and c. simultaneously detecting at least two analytes of interest either produced or consumed by the plurality of cells in the chamber. 2. The method of claim 1, wherein the detecting step comprises the step of using a first electrode to detect one of the at least two analytes of interest and a second electrode to detect another of the at least two analytes of interest. 3. The method of claim 2, wherein the first electrode and the second electrode have different electrochemical characteristics. 4. The method of claim 2, wherein the first electrode comprises a gold electrode and the second electrode comprises a platinum electrode. 5. The method of claim 2, further comprising the step of providing a potentiostat electrically coupled to the first electrode and the second electrode for detecting a voltage as a function of the two analytes of interest either produced or consumed by the plurality of cells in the chamber. 6. The method of claim 2, further comprising the step of providing a reference electrode, and an amperemeter electrically coupled to the first electrode and the second electrode for detecting a current as a function of the two analytes of interest either produced or consumed by the plurality of cells in the chamber. 7. The method of claim 1, wherein the detecting step comprises the step of using an optical detector to detect one of the at least two analytes of interest. 8. The method of claim 6, wherein the optical detector comprises an optical fiber. 9. A device for detecting at least one analyte of interest either produced or consumed by a plurality of cell, wherein the plurality of cells is placed in a chamber, comprising means for simultaneously detecting at least two analytes of interest either produced or consumed by the plurality of cells in the chamber. 10. The device of claim 9, wherein the simultaneously detecting means comprises a first electrode to detect one of the at least two analytes of interest and a second electrode to detect another of the at least two analytes of interest, the first electrode and the second electrode positioned apart from each other. 11. The device of claim 10, wherein the first electrode and the second electrode have different electrochemical characteristics. 12. The device of claim 11, wherein the first electrode comprises a gold electrode and the second electrode comprises a platinum electrode. 13. The method of claim 10, further comprising a potentiostat electrically coupled to the first electrode and the second electrode for detecting a voltage as a function of the two analytes of interest either produced or consumed by the plurality of cells in the chamber. 14. The device of claim 10, further comprising a reference electrode, and an amperemeter electrically coupled to the first electrode and the second electrode for detecting a current as a function of the two analytes of interest either produced or consumed by the plurality of cells in the chamber. 15. The device of claim 9, further comprising an inlet in fluid communication with the chamber for introducing a medium into the chamber. 16. The device of claim 9, further comprising an outlet in fluid communication with the chamber for introducing a medium away from the chamber. 17. The device of claim 9, further comprising an optical detector to detect one of the at least two analytes of interest 18. The device of claim 17, wherein the optical detector comprises an optical fiber. 19. A method for detecting a plurality of analytes of interest either produced or consumed by a plurality of cell, comprising the steps of: a. providing a housing defining a chamber; b. placing a plurality of cells in the chamber; and c. simultaneously detecting a plurality of analytes of interest either produced or consumed by the plurality of cells in the chamber. 20. The method of claim 19, wherein the detecting step comprises the step of using a plurality of electrodes to detect the plurality of analytes of interest, respectively. 21. The method of claim 20, wherein the plurality of electrodes each has different electrochemical characteristics. 22. The method of claim 21, wherein the plurality of electrodes comprise a gold electrode. 23. The method of claim 21, wherein the plurality of electrodes comprise a platinum electrode. 24. The method of claim 19, wherein the detecting step comprises the step of using an optical detector to detect the plurality of analytes of interest. 25. The method of claim 24, wherein the optical detector comprises an optical fiber. 26. A device for detecting a plurality of analytes of interest either produced or consumed by a plurality of cell, wherein the plurality of cells is placed in a chamber, comprising means for simultaneously detecting a plurality of analytes of interest either produced or consumed by the plurality of cells in the chamber. 27. The device of claim 26, wherein the simultaneously detecting means comprises a plurality of electrodes to detect the plurality of analytes of interest, respectively. 28. The device of claim 27, wherein the plurality of electrodes each has different electrochemical characteristics. 29. The device of claim 27, wherein the plurality of electrodes comprise a gold electrode. 30. The device of claim 27, wherein the plurality of electrodes comprise a platinum electrode. 31. The device of claim 27, further comprising a reference electrode, and an amperemeter electrically coupled to the plurality of electrodes, respectively, for detecting a current as a function of the plurality of analytes of interest either produced or consumed by the plurality of cells in the chamber. 32. The device of claim 27, further comprising a potentiostat electrically coupled to the plurality of electrodes, respectively, for detecting a voltage as a function of the plurality of analytes of interest either produced or consumed by the plurality of cells in the chamber. 33. The device of claim 27, further comprising an inlet in fluid communication with the chamber for introducing a medium into the chamber. 34. The device of claim 27, further comprising an outlet in fluid communication with the chamber for introducing a medium away from the chamber. 35. The device of claim 27, further comprising an optical detector to detect the at least one of the plurality of analytes of interest. 36. The device of claim 35, wherein the optical detector comprises an optical fiber. 37. A device for detecting at least one analyte of interest either produced or consumed by at least one cell wherein the at least one cell is placed in a chamber, comprising: a. an inlet in fluid communication with the chamber, b. a first electrode having a first electrochemical characteristic; and c. a second electrode positioned away from the first electrode and having a second electrochemical characteristic, wherein the first electrode detects a first analyte of interest either produced or consumed by at least one cell, and the second electrode detects a second analyte of interest by at least one cell, respectively and simultaneously. 38. The device of claim 37, further comprising a reference electrode, and an amperemeter electrically coupled to the first electrode and the second electrode for detecting a current as a function of the two analytes of interest either produced or consumed by at least one cell in the chamber. 39. The device of claim 37, further comprising a potentiostat electrically coupled to the first electrode and the second electrode for detecting a voltage as a function of the two analytes of interest either produced or consumed by at least one cell in the chamber. 40. The device of claim 37, further comprising additional electrodes, each having a different electrochemical characteristic and being positioned away from the first and second electrodes. 41. The device of claim 37, further comprising an outlet in fluid communication with the chamber for introducing a medium away from the chamber. 42. The device of claim 37, further comprising an optical detector to detect optical signatures of intracellular physiological processes of the cell. 43. The device of claim 37, further comprising an optical detector to detect at least one of the analytes of interest. 44. The device of claim 43, wherein the optical detector comprises an optical fiber having a first end, a second end and a body portion defined therebetween. 45. The device of claim 43, wherein the first end of the optical fiber reaches in the chamber capable of detecting an optical signal related to the two analytes of interest either produced or consumed by at least one cell. 46. The device of claim 43, wherein the optical detector further comprises: a. a cover slip member having a first surface and a second surface, wherein the first surface of the cover slip is underneath the chamber and the second surface of the cover slip is optically coupled to the first end of the optical fiber; and b. a light source optically coupled to the second end of the optical fiber. 47. The device of claim 46, further comprising: a. a beam splitter optically coupled to the optical fiber and positioned between the light source and the cover slip for directing optical signals transmitted through the optical fiber corresponding to the optical response from a first direction to a second direction; and b. an analyzer for receiving the optical signals directed by the beam splitter. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The biological cell may act as a parallel processing, non-linear, multistate, analog computer. This analog computer can occupy a volume of less than 10 −46 m 3 and is primarily powered only by sugars, fats, and oxygen. The complexity of these computers is evidenced by the attempts to model ongoing biochemical processes based on Mycoplasma genitalium , a microbe with the smallest known gene set of any self-replicating organism (http:www.e-cell.org). However, even this simplest model requires hundreds of variables and reaction rules, and a complete model even for a mammalian cell would be much more complex, requiring in excess of 10 5 variables and equations. Because the cell behaves as an analog computer, it can be programmed. Historically, a limited set of interventions has allowed physiologists and engineers to study living cells and characterize the feedback control systems that govern cell function. With the advent of genetic engineering, it is now possible to reprogram the genetic machinery of a cell, for example to turn a particular gene on or off, or to produce large quantities of a particular biochemical. However, there has been little efforts and progress for inserting man-made devices into the control system of a single living cell so as to convert the cell into a programmable computational engine. Therefore, among other things, there is a need to merge cellular biophysics, microcircuits and microfluidics, and information technology to create, among other things, programmable microsystems that can be used for sensing, feedback, control and analysis of a single cell and/or an array of interconnected and instrumented living cells. Additionally, current bio-sensors use biological molecules for specific agent detection via specific binding reactions. However, wide-spectrum detection is expensive, requiring a priori threat knowledge and a large quantity of specific cells. Assays are susceptible to overload from multiple threats and false detection and from non-pathogenic “spoof” organisms. Furthermore, addressing new threats involves a lengthy, costly design process. In addition, conventional assays lack cellular machinery to increase sensitivity. Therefore, among other things, there is also a need to develop new systems and methods that are capable of providing a complete bio-functional signature of a CBW agent, environmental contaminant, unknown drug, or other threats for better, fast, sensitive accurate and efficient detection. |
<SOH> SUMMARY OF THE INVENTION <EOH>In one aspect, the present invention relates to a method for detecting at least one analyte of interest either produced or consumed by a plurality of cell. In one embodiment, the method includes the steps of providing a housing defining a chamber, placing a plurality of cells in the chamber, and simultaneously detecting at least two analytes of interest either produced or consumed by the plurality of cells in the chamber. The detecting step comprises the step of using a first electrode to detect one of the at least two analytes of interest and a second electrode to detect another of the at least two analytes of interest, wherein the first electrode and the second electrode have different electrochemical characteristics. For examples, the first electrode may comprises a gold electrode and the second electrode may comprise a platinum electrode, or vice versa Additionally, the first electrode and the second electrode each can have different surface film, coating, shape, material modifications to accommodate the needs for detecting one or more desired analytes of interest. The method further includes the step of providing a potentiostat electrically coupled to the first electrode and the second electrode for detecting a voltage as a function of the two analytes of interest either produced or consumed by the plurality of cells in the chamber. Alternatively, the method further includes the step of providing a reference electrode, and an amperemeter electrically coupled to the first electrode and the second electrode for detecting a current as a function of the two analytes of interest either produced or consumed by the plurality of cells in the chamber. The detecting step additionally may include the step of using an optical detector to detect the optical response of the plurality of cells to one of the at least two analytes of interest, wherein the optical detector comprises an optical fiber. In another aspect, the present invention relates to a device for detecting at least one analyte of interest either produced or consumed by a plurality of cell, wherein the plurality of cells is placed in a chamber. In one embodiment, the device includes means for simultaneously detecting the response of the plurality of cells to at least two analytes of interest. The simultaneously detecting means includes a first electrode to detect one of the at least two analytes of interest, and a second electrode to detect another of the at least two analytes of interest, the first electrode and the second electrode positioned apart from each other. The first electrode and the second electrode have different electrochemical characteristics, which can be achieved by several ways. One way is to use different materials to make the first electrode and the second electrode. For example, the first electrode can be a gold electrode and the second electrode can be a platinum electrode. Other materials known to people skilled in the art can also be used. The device further may include a reference electrode, and an amperemeter that is electrically coupled to the first electrode and the second electrode for detecting a current as a function of the two analytes of interest either produced or consumed by the plurality of cells in the chamber. Moreover, the device may include a potentiostat that is electrically coupled to the first electrode and the second electrode for detecting a voltage as a function of the two analytes of interest either produced or consumed by the plurality of cells in the chamber. The contacting means includes an inlet in fluid communication with the chamber for introducing a medium into the chamber. Additionally, the device of has an outlet in fluid communication with the chamber for introducing a medium away from the chamber. Moreover, the device may have an optical detector to detect one of the at least two analytes of interest, wherein the optical detector comprises an optical fiber. In a further aspect, the present invention relates to a method for detecting a plurality of analytes of interest either produced or consumed by a plurality of cell. In one embodiment, the method includes the steps of providing a housing defining a chamber, placing a plurality of cells in the chamber, and simultaneously detecting a plurality of analytes of interest either produced or consumed by the plurality of cells in the chamber. The detecting step includes the step of using a plurality of electrodes to detect the plurality of analytes of interest, respectively, wherein the plurality of electrodes each has different electrochemical characteristics. In yet another aspect, the present invention relates to a device for detecting a plurality of analytes of interest either produced or consumed by a plurality of cell, wherein the plurality of cells is placed in a chamber. In one embodiment, the device includes means for simultaneously detecting a plurality of analytes of interest either produced or consumed by the plurality of cells in the chamber, wherein the simultaneously detecting means has a plurality of electrodes to detect the plurality of analytes of interest, respectively. The plurality of electrodes each has different electrochemical characteristics. In another aspect, the present invention relates to a device for detecting at least one analyte of interest either produced or consumed by at least one cell, wherein the at least one cell is placed in a chamber. In one embodiment, the device includes an inlet in fluid communication with the chamber, a first electrode having a first electrochemical characteristic, and a second electrode positioned away from the first electrode and having a second electrochemical characteristic. The first electrode detects a first analyte of interest either produced or consumed by at least one cell, and the second electrode detects a second analyte of interest by at least one cell, respectively and simultaneously. An outlet is in fluid communication with the chamber for introducing medium away from the chamber. The device may further have a reference electrode, and an amperemeter electrically coupled to the first electrode and the second electrode for detecting a current as a function of the two analytes of interest either produced or consumed by at least one cell in the chamber. Alternatively, the device has a potentiostat electrically coupled to the first electrode and the second electrode for detecting a voltage as a function of the two analytes of interest either produced or consumed by at least one cell in the chamber. Moreover, the device may further has additional electrodes, each having a different electrochemical characteristic and being positioned away from the first and second electrodes. Furthermore, the device may have an optical detector to detect at least one of the analytes of interest, wherein the optical detector comprises an optical fiber having a first end, a second end and a body portion defined therebetween. The first end of the optical fiber reaches in the chamber capable of detecting an optical signal related to the two analytes of interest either produced or consumed by at least one cell. Moreover, in one embodiment, the optical detector additionally has a cover slip member having a first surface and a second surface, wherein the first surface of the cover slip is underneath the chamber and the second surface of the cover slip is optically coupled to the first end of the optical fiber, and a light source optically coupled to the second end of the optical fiber. A beam splitter is optically coupled to the optical fiber and positioned between the light source and the cover slip for directing optical signals transmitted through the optical fiber corresponding to the optical response from a first direction to a second direction. And the optical detector further has an analyzer for receiving the optical signals directed by the beam splitter. Among other things, the optical detector can also be used to detect and measure optical signatures of intracellular physiological processes such as the transmembrane resting potential, or the transmembrane action potential of a cell. These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure. |
Electrical field stimulation of eukaryotic cells |
Methods of identifying activators and inhibitors of voltage-gated ion channels are provided in which the methods employ electrical field stimulation of the cells in order to manipulate the open/close state transition of the voltage-gated ion channels. This allows for more convenient, more precise experimental manipulation of these transitions, and, coupled with efficient methods of detecting the result of ion flux through the channels, provides methods that are especially suitable for high throughput screening. |
1-74. (canceled) 75. A method for identifying modulators of the activity of a voltage-gated ion channel comprising: (a) altering the transmembrane potential of at least a portion of the membrane of a cell expressing the voltage-gated ion channel by applying a voltage to the cells through extracellular electrodes while monitoring ion flow through the voltage-gated ion channel; (b) exposing the cell in step (a) to a substance and monitoring ion flow through the voltage-gated ion channel; (c) comparing the ion flow through the voltage-gated ion channel in step (a) and step (b); where a difference in the ion flow through the voltage-gated ion channel in step (a) and step (b) indicates that the substance is a modulator of the voltage-gated ion channel. 76. The method of claim 75 wherein a plurality of cells expressing the voltage-gated ion channel in step (a) are divided into a control portion and a test portion and where a difference in the ion flow through the voltage-gated ion channel of the control portion as compared to the test portion indicates that the substance is modulator of the voltage-gated ion channel. 77. The method of claim 76 wherein a pre-selected voltage is applied through positive and negative electrodes to said plurality of cells in step (a) and where a difference in the ion flow after the pre-selected voltage is applied to the control portion as compared to the test portion indicates that the substance is a modulator of the voltage-gated ion channel. 78. The method of claim 77 wherein if the value of the control portion is greater than the value of the test portion, then the substance is an inhibitor of the voltage-gated ion channel. 79. The method of claim 77 wherein if the value of the control portion is less than the value of the test portion, then the substance is an activator of the voltage-gated ion channel. 80. The method of claim 77 wherein the plurality of cells are on a substrate that is a glass or a multiwell tissue culture plate and is not silicon or a field effect transistor. 81. The method of claim 80 wherein the multiwell tissue culture plate has a plurality of wells that contain one positive and one negative electrode. 82. The method of claim 81 wherein the positive or negative electrodes form the bottom of the wells and the other of the positive or negative electrode enters the wells from above. 83. The method of claim 81 wherein the electrodes are interdigitating. 84. The method of claim 77 where the cells are selected from the group consisting of: L cells L-M(TK−) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), HEK293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Ki (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), CPAE (ATCC CCL 209), Saos-2 (ATCC HTB-85), ARPE-19 human retinal pigment epithelium (ATCC CRL-2302), GH3 cells, and primary cardiac myocytes. 85. The method of claim 77 where the cells do not naturally express a voltage-gated ion channel but have been transfected with an expression vector that encodes a voltage-gated ion channel. 86. The method of claim 77 where the cells contain a fluorescent indicator compound selected from the group consisting of: fluo-3, fura-2, fluo-4, fluo-5, calcium green-1, Oregon green, 488 BAPTA, SNARF-1, and indo-1. 87. An apparatus for use in identifying activators or inhibitors of voltage-gated ion channels comprising: (a) a substrate having an upper surface upon which are present at least 103 living eukaryotic cells which have a voltage-gated ion channel of interest in their plasma membranes; (b) a plurality of positive electrodes and a plurality of negative electrodes positioned either on or near the substrate such that when a voltage is applied through the positive and negative electrodes the transmembrane potential of the cells is controlled; (c) at least one substance that is suspected of being an activator or an inhibitor of the voltage-gated ion channel; and (d) where said cells contain a fluorescent indicator compound. 88. The apparatus of claim 87 wherein: the substrate is a multiwell tissue culture plate having a plurality of wells in which are present at least 103 living eukaryotic cells per well of the plurality which cells have a voltage-gated ion channel of interest in their plasma membranes; the plurality of positive electrodes and a plurality of negative electrodes are positioned such that when a pre-selected voltage is applied through the positive and negative electrodes, the transmembrane potential of the cells is altered; at least one substance that is suspected of being an activator or an inhibitor of the voltage-gated ion channel is in at least one of the wells; and the cells contain a fluorescent indicator compound or a voltage sensitive membrane dye. 89. The apparatus of claim 88 wherein a pre-selected voltage applied across the electrodes alters the transmembrane potential of cells within the wells. 90. The apparatus of claim 88 wherein the multiwell tissue culture plate contains one of the pair of electrodes on the bottom of the wells and the other of the pair of electrodes on the side of the wells. 91. The apparatus of claim 88 wherein the multiwell tissue culture plate contains both of the pair of electrodes on the bottom of the wells. 92. The apparatus of claim 88 wherein the wells of the plate contain interdigitating electrodes. 93. The apparatus of claim 92 wherein the interdigitating electrodes comprise an electrode array that has been chemically etched onto an indium tin oxide coated glass plate. 94. A system for applying electrical field stimulation to cells, said system comprising: (a) a multiwell tissue culture plate, wherein the bottom of the wells is comprised of an optically transparent filter membrane upon which cells can be grown; (b) a trough suitable for containing fluid and configured such that said multiwell tissue culture plate may sit therein; (c) at least one first electrode disposed in said trough; and (d) an electrode head comprising a plurality of second electrodes in an amount corresponding to the number of wells in said multiwell tissue culture plate, wherein said electrode head and said plurality of electrodes are configured such that said electrodes are disposed in the wells of the multiwell tissue culture plate upon positioning said electrode head onto said multiwell tissue culture plate; and wherein said first electrode and said plurality of second electrodes are so disposed that when a pre-selected voltage is applied across the electrodes the transmembrane potential of cells within the wells is altered. 95. The system of claim 94 further comprising a waveform generator that is in electrical communication with said first electrode or said plurality of second electrodes, or both, whereby electric pulse signals are generated by said waveform generator. 96. The system of claim 95 further comprising a computer electrically connected to said waveform generator, said computer comprising software for coordinating said pulse signals produced by said waveform generator. 97. The system of claim 95 wherein said waveform generator generates a binary value that represents the address of the well to be excited by said pulse signals. 98. The system of claim 95 further comprising electrical relays upstream of said plurality of second electrodes. 99. The system of claim 98 further comprising a microcontroller in electrical communication with said waveform generator and said electrical relays so disposed such that upon receiving a trigger pulse and a particular binary value from said waveform generator, said microcontroller switches on the appropriate relay thereby directing a pulse to the particular electrode corresponding to the said particular binary value. 100. The system of claim 94 wherein: said trough comprises a plurality of individual troughs suitable for containing fluid, where the number of plurality of troughs corresponds to the number of wells in said multiwell tissue culture plate, where the plurality of troughs are so disposed to individually contain each well of said multiwell tissue culture plate; and where said plurality of troughs my be accessed by a port defined in said multiwell tissue culture plate and disposed laterally to each well; and said conductive electrode head comprises a conductive electrode plate configured to be mounted above said multiwell tissue culture plate, wherein said electrode plate comprises a plurality of apertures configured to allow the wells of the multiwell tissue culture plate to pass through said electrode plate, where said electrode plate comprises a plurality of conductive pins integral or attached to said electrode plate, and where the individual pins of said plurality of conductive pins pass through said port to be disposed in individual troughs upon mounting said electrode plate on top of said multiwell tissue culture plate. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to methods of identifying activators and inhibitors of voltage-gated ion channels in which the methods employ electrical field stimulation of the cells via extracellular electrodes in order to manipulate the open/close state transitions of the voltage-gated ion channels. This allows for more convenient, more precise manipulation of these transitions, and, coupled with efficient methods of detecting ion flux or membrane potential, results in methods that are especially suitable for high throughput screening in order to identify substances that are activators or inhibitors of voltage-gated ion channels. The present invention also provides apparatuses for use in the above- described methods. In particular, modifications of standard multiwell tissue culture plates are provided where the modified multiwell tissue culture plates have electrodes that can alter the transmembrane electric potential of cells in the wells of the plates, thus altering the ratio of open/close states of voltage-gated ion channels in the cells. |
Novel process for the preparation of tert-butyl 6-cyano-5-hydroxy-3-oxohexanoate |
In one aspect, the present invention provides a novel process for the preparation of tert-butyl 6-cyano-5-hydroxy-3-oxohexanoate, a key intermediate for the preparation of HMG-CoA reductase inhibitor. |
1. A process for the preparation of a compound of Formula comprising removing the tert-butyl diphenyl silyl group of a compound of Formula II wherein the compound of Formula II is obtained by treating a compound of Formula III wherein Alk is a straight or branched C1-C6 alkyl, with a carbanion, generated by the reaction of tert-butyl acetate with lithium diisopropylamide, wherein the compound of Formula III is obtained from reacting a compound of Formula IV wherein Alk is a straight or branched C1-C6 alkyl, with tert-butyl diphenyl silyl chloride. 2. A compound of Formula II 3. A novel compound of Formula III wherein Alk is a straight or branched C1-C6 alkyl. 4. The process of claim 1, wherein the compound of Formula I is used for the preparation of atorvastatin. |
<SOH> BACKGROUND OF THE INVENTION <EOH>HMG CoA reductase inhibitors are pharmaceutically active compounds used for inhibition of cholesterol biosynthesis. A group of compounds called ‘statins’ comprising lovastatin, simvastatin, mevastatin, pravastatin, atorvastatin, rosuvastatin, cerivastatin and fluvastatin show antilipidemic activity and are widely known HMG CoA reductase inhibitors. Ester Derivative of the Formula I is a valuable chiral synthon for synthesizing atorvastatin. |
<SOH> SUMMARY OF THE INVENTION <EOH>In one aspect, the present invention provides a novel process for the preparation of the compound of Formula I. In another aspect, the process employs novel intermediates. The present invention relates to a novel process for preparing a compound of Formula I comprising hydrolysis of a compound of Formula II wherein, the compound of Formula II is obtained from a compound of Formula III wherein Alk is a straight or branched C 1 -C 6 alkyl; and wherein the compound of Formula III is obtained from a compound of Formula IV wherein Alk is a straight or branched C 1 -C 6 alkyl. The process of the present invention has several advantages over the prior art including: higher yield during conversion of the compound of Formula III to the compound of Formula II as the hydroxy group is protected, reactions after protection with TBDPS can be followed by TLC, low consumption of reagents as the hydroxy group is protected, and reduced levels of undesired side products. detailed-description description="Detailed Description" end="lead"? |
Method for treating medical devices using glycerol and an antimicrobial agent |
A non-metallic medical device treated with a antimicrobial agents is provided. Different combinations of antimicrobial agents can be used for different types of non-metallic medical devices depending on the types of infections related to each device. The combination of different antimicrobial substances has a synergistic effect against certain bacteria and fungi. An antimicrobial agent can be used to treat a non-metallic medical device by mixing the antimicrobial agent with an acid solution and glycerol and exposing the non-metallic medical device to the resulting mixture such that an enough of the antimicrobial agent binds to a portion of the non-metallic medical device to inhibit the growth of bacterial and fungal organisms. |
1. A method of treating a non-metallic medical device with an antimicrobial agent comprising the steps of: mixing at least an antimicrobial agent, an acid solution, wherein said acid solution is comprised of short chain monocarboxylic acid and ortho-phosphoric acid, and glycerol to form an antimicrobial composition; and applying the antimicrobial composition to at least a portion of the non-metallic medical device under conditions wherein an effective concentration of the antimicrobial composition binds to the non-metallic medical device. 2-23. (canceled) 24. An implantable medical device comprising: a body with one or more non-metallic surfaces, wherein at least one of the one or more non-metallic surfaces has been treated with an effective concentration of antimicrobial agent, acid solution, wherein said acid solution is comprised of short chain monocarboxylic acid and ortho-phosphoric acid, and glycerol. 25-29. (canceled) 30. The device of claim 24, wherein at least one of the one or more non-metallic surfaces comprises a biomaterial. 31. The device of claim 30, wherein the biomaterial may be human tissue, non-human tissue, or any combination thereof. 32. The device of claim 31, wherein the human tissue is a small intestinal submucosa or skin. 33. The device of claim 31, wherein the non-human tissue is a small intestinal submucosa or skin. 34. The device of claim 31, wherein the combination of human tissue and non-human tissue is a small intestinal submucosa or skin. 35. A method of treating a non-metallic medical device with an antimicrobial agent comprising the steps of: mixing at least an antimicrobial agent, an acid solution, and glycerol to form an antimicrobial composition; applying the antimicrobial composition to at least a portion of the non-metallic medical device under conditions wherein an effective concentration of the antimicrobial composition binds to the non-metallic medical device; and wherein the non-metallic medical device comprises a biomaterial. 36. The method according to claim 35, wherein the biomaterial may be human tissue, non-human tissue, or any combination thereof. 37. The method according to claim 36, wherein the human tissue is a small intestinal submucosa or skin. 38. The method according to claim 36, wherein the non-human tissue is a small intestinal submucosa or skin. 39. The method according to claim 36, wherein the combination of human tissue and non-human tissue is a small intestinal submucosa or skin. 40. The method according to claim 35, wherein the acid solution comprises formic acid, potassium chloride and phosphoric acid. 41. The method according to claim 40, wherein the ratio of formic acid to potassium chloride to phosphoric acid is about 20 ml:20 mg: 1.4 ml. 42. The method according to claim 40, wherein the ratio of formic acid to potassium chloride to phosphoric acid to glycerol is about 20 ml:20 mg: 1.4 ml: 1.9 ml. 43. The method according to claim 35, wherein the antimicrobial agent comprises minocycline and rifampin. 44. The method according to claim 35, wherein the biomaterial is immersed in the antimicrobial composition for about 30 minutes. 45. The method according to claim 44, wherein the biomaterial is immersed in the antimicrobial composition in the dark. 46. The method according to claim 35, wherein the antimicrobial composition has a temperature of about 5° C. 47. The method according to claim 1, wherein the acid solution comprises formic acid, potassium chloride and phosphoric acid. 48. The method according to claim 47, wherein the ratio of formic acid to potassium chloride to phosphoric acid is about 30 ml:400 mg:3 ml. 49. The method according to claim 47, wherein the ratio of formic acid to potassium chloride to phosphoric acid to Glycerol is about 30 ml:400 mg:3 ml:5 ml. 50. The method according to claim 1, wherein the antimicrobial agent comprises chlorhexidine and chlorohexidin. 51. (canceled) 52. An implantable medical device comprising: a body with one or more non-metallic surfaces, glycerol, and an antimicrobial agent, wherein the glycerol and an effective concentration of the antimicrobial agent coat the one or more non-metallic surfaces, and wherein at least one of the one or more non-metallic surfaces comprises a biomaterial. 53. The device of claim 52, wherein the antimicrobial agent is selected from the group consisting of: chlorhexidine and methylisothiazolone; chlorhexidine and α-terpineol; thymol and chloroxylenol; thymol and methylisothiazolone; chlorhexidine and cetylpyridinium chloride; chlorhexidine and chloroxylenol; chlorhexidine, methylisothiazolone and thymol; methylisothiazolone and α-terpineol; minocycline and rifampin; and chlorhexidine, methylisothiazolone and α-terpineol. 54. The device of claim 52, wherein the antimicrobial agent is comprised of a minocycline and rifampin. 55. The device of claim 52, wherein the biomaterial may be human tissue, non-human tissue, or any combination thereof. 56. The device of claim 55, wherein the human tissue is a small intestinal submucosa or skin. 57. The device of claim 55, wherein the non-human tissue is a small intestinal submucosa or skin. 58. The device of claim 55, wherein the combination of human tissue and non-human tissue is a small intestinal submucosa or skin. |
<SOH> BACKGROUND <EOH>Indwelling medical devices such as catheters are becoming essential to patient care. The benefit derived from these catheters, orthopedic devices, and other types of medical implants, however, is often offset by infectious complications. Some of the common organisms causing infectious complications associated with indwelling medical devices are Staphylococcus epidermidis and Staphylococcus aureus . In the case of vascular catheters, these two organisms account for almost 70-80% of all infectious organisms, with Staphylococcus epidermidis being the most common organism. Gram-negative bacilli cause about 15-20% of the infections, and Candida species, a fungal agent, accounts for about 10-15% of the vascular catheter infections. Other gram-negative bacteria and fungal organisms ( Candida ) account for the remaining one-third of cases. Another common hospital-acquired infection is a urinary tract infection (UTI). The majority of UTI cases are associated with the use of urinary catheters, including transurethral foley, suprapubic and nephrostomy catheters. These urinary catheters are inserted in a variety of populations, including the elderly, stroke victims, spinal cord-injured patients, postoperative patients and those with obstructive uropathy. Despite adherence to sterile guidelines for the insertion and maintenance of urinary catheters, catheter-associated UTI continues to pose a major problem. In the U.S. alone, about 1 million cases of hospital-acquired cases of UTI occur annually. For instance, it is estimated that almost one-quarter of hospitalized spinal cord-injured patients develop symptomatic UTI during their hospital course. Gram-negative bacilli account for almost 60-70%, enterococci for about 25% and Canada species for about 10% of cases of UTI. Colonization of bacteria on the surfaces of the implant or other parts of the device can produce serious patient problems, including the need to remove and/or replace the implanted device and to vigorously treat secondary infective conditions. A considerable amount of attention and study has been directed toward preventing such colonization by the use of antimicrobial agents, such as antibiotics, bound to the surface of the materials employed in such devices. In such attempts, the objective has been to produce a sufficient bacteriostatic or bactericidal action to prevent colonization. Various methods have previously been employed to prevent infection of medical devices. A simple method is to flush the surfaces of a device with an antimicrobial solution. Generally, this flushing technique requires convenient access to the implantable device. For example, catheters are generally amenable to flushing with a solution of rifampin and minocycline or rifampin and novobiocin. For use in flushing solutions, the effective concentration of the antibiotic range from about 1 to 10 mg/ml for minocycline, preferably about 2 mg/ml; 1 to 10 mg/ml for rifampin, preferably about 2 mg/ml; and 1 to 10 mg/ml for novobiocin, preferably about 2 mg/ml. The flushing solution is normally composed of sterile water or sterile saline solutions. Other methods of coating surfaces of medical devices with antimicrobial agents are taught in U.S. Pat. No. 4,895,566 (a medical device substrate carrying a negatively charged group having a pKa of less than 6 and a cationic antibiotic bound to the negatively charged group); U.S. Pat. No. 4,917686 (antibiotics are dissolved in a swelling agent which is absorbed into the matrix of the surface material of the medical device); U.S. Pat. No. 4,107,121 (constructing the medical device with ionogenic hydrogels, which thereafter absorb or ironically bind antibiotics); U.S. Pat. No. 5,013,306 (laminating an antibiotic to a polymeric surface layer of a medical device); U.S. Pat. No. 4,952,419 (applying a film of silicone oil to the surface of an implant and then contacting the silicone film bearing surface with antibiotic powders); and U.S. Pat. No. 4,442,133. These and other methods of coating medical devices with antimicrobial agents appear in numerous patents and medical journal articles. However, these methods also have significant drawbacks in that they can alter the integrity of non-metallic medical devices or result in residual antimicrobial material precipitating within the device. Accordingly, there is a need for a non-metallic medical device treated with an antimicrobial agent to provide a broad range of antimicrobial activity while minimizing the harmful side effects noted above. Further, there is a need for a method that results in low residual coating material left on the surface of the medical device, which reduces complications arising from precipitation of coating material within the device. There is also a need to enhance the versatility of the treatment to accommodate higher concentrations of antimicrobial agents if needed. |
<SOH> SUMMARY OF THE INVENTION <EOH>One aspect of the present invention is a method for treating non-metallic medical devices with an antimicrobial agent comprising the steps of mixing at least an antimicrobial agent, an acid solution, and glycerol to form an antimicrobial composition and applying the antimicrobial composition to at least a portion of the non-metallic medical device under conditions wherein an effective concentration of the antimicrobial composition binds to the non-metallic medical device. In a specific embodiment, the antimicrobial composition may be formed by mixing antimicrobial agents and an acid solution and then adding glycerol. In another specific embodiment, the antimicrobial agent may be selected from the chlorhexidine and methylisothiazolone; chlorhexidine and α-terpineol; thymol and chloroxylenol; thymol and methylisothiazolone; chlorhexidine and cetylpyridinium chloride; chlorhexidine and chloroxylenol; chlorhexidine, methylisothiazolone and thymol; methylisothiazolone and α-terpineol; minocycline and rifampin; and chlorhexidine, methylisothiazolone and α-terpineol. In another specific embodiment, the portion of the non-metallic medical device treated may be made from rubber, plastic, nylon, silicone, polyurethane, polyethylene, polyvinyl chloride, polytetrafluoroethylene tetraphthalate, polyethylene tetraphthalate, polytetrafluoroethylene, latex, elastomers, polymers, and materials sealed with gelatin, collagen or alumin. In another specific embodiment, the non-metallic medical device may be a peripherally insertable central venous catheter, dialysis catheter, long term tunneled central venous catheter, peripheral venous catheter, short-term central venous catheter, arterial catheter, pulmonary artery Swan-Ganz catheter, urinary catheter, long term non-tunneled central venous catheters, peritoneal catheters, ventricular catheters, long term urinary devices, tissue bonding urinary devices, penile prostheses, vascular grafts, extravascular grafts, urinary stints, vascular catheter ports, wound drain tubes, hydrocephalus shunts, pacemaker systems, artificial urinary sphincters, vascular dialators, extravascular dialators, vascular stints, extravascular stints, small joint replacements, temporary joint replacements, urinary dilators, heart valves, orthopedic implants, heart assist devices, stents, penial implants, mammary implants, and dental devices. In another specific embodiment, the acid solution may be a short chain monocarboxylic acid and ortho-phosphoric acid. The short chain monocarboxylic acid may be formic acid, acetic acid, or propionic acid. A further specific embodiment includes the ratio of monocarboxylic acid to ortho-phosphoric acid to glycerol may be about 79:8:13. In another specific embodiment, the antimicrobial composition has a temperature that is between 2° C. to 75° C. at some point during the treatment, preferably about 45° C. In another specific embodiment, the acid solution may also contain potassium chloride. In another specific embodiment, the antimicrobial composition may be applied by exposing the non-metallic medical device to the antimicrobial composition for about 10 minutes to about 18 hours, preferably about 60 minutes. In another specific embodiment, the method of treating the non-metallic medical device may further comprise the step of removing excess antimicrobial composition from the non-metallic medical device after the application step and then drying the non-metallic medical device. The non-metallic medical device may be dried for about 16 hours. In another specific embodiment, the non-metallic medical device may be flushed with water after the drying step and may then be dried again for about 10 hours to about 24 hours. A further embodiment of the invention provides for an implantable medical device comprising a body; one or more non-metallic surfaces on said body, glycerol, and an antimicrobial agent, wherein the glycerol and an effective concentration of the antimicrobial agent coat the one or more non-metallic surfaces. In another specific embodiment, the antimicrobial agent may be selected from the group consisting of chlorhexidine and methylisothiazolone; chlorhexidine and α-terpineol; thymol and chloroxylenol; thymol and methylisothiazolone; chlorhexidine and cetylpyridinium chloride; chlorhexidine and chloroxylenol; chlorhexidine, methylisothiazolone and thymol; methylisothiazolone and α-terpineol; minocycline and rifampin; and chlorhexidine, methylisothiazolone and α-terpineol. In another specific embodiment, the device may consist, at least in part, of rubber, plastic, silicone, polyurethane, polyethylene, polytetrafluoroethylene and polyethylene tetraphthalate and polyethylene tetraphthalate sealed with gelatin, collagen or albumin. In another specific embodiment, the non-metallic medical device may be a peripherally insertable central venous catheter, dialysis catheter, long term tunneled central venous catheter, peripheral venous catheter, short-term central venous catheter, arterial catheter, pulmonary artery Swan-Ganz catheter, urinary catheter, long term non-tunneled central venous catheters, peritoneal catheters, ventricular catheters, long term urinary devices, tissue bonding urinary devices, penile prostheses, vascular grafts, extravascular grafts, urinary stints, vascular catheter ports, wound drain tubes, hydrocephalus shunts, pacemaker systems, artificial urinary sphincters, vascular dialators, extravascular dialators, vascular stints, extravascular stints, small joint replacements, temporary joint replacements, urinary dilators, heart valves, orthopedic implants, heart assist devices, stents, penial implants, mammary implants, and dental devices. detailed-description description="Detailed Description" end="lead"? |
Method and system for providing formatted data to image processing means in accordance with a standard format |
A method and system for providing, in accordance with a standard format, formatted information to an image-processor, especially software and/or components. The formatted information is related to defects of a chain of appliances including an image-capture appliance and/or an image-restitution appliance. The image-processor uses the formatted data to modify the quality of at least one image derived from or addressed to the chain of appliances. The formatted information includes data characterizing defects of the image-capture appliance, especially distortion characteristics, and/or data characterizing defects of the image-restitution appliance, especially distortion characteristics. The method fills in at least one field of the standard format with the formatted information. This field is designated by a field name and contains at least one field value. |
1-32. (Canceled). 33. A method for providing formatted information in a standard format to an image-processor, the formatted information being related to defects of an appliance chain, the appliance chain including at least one image-capture appliance and/or one image-restitution appliance, the image-processor using the formatted information to modify quality of at least one image derived from or addressed to the appliance chain; the formatted information including: data characterizing the defects of the image-capture appliance; and/or data characterizing the defects of the image-restitution appliance; the method comprising: filling in at least one field of the standard format with the formatted information, the field being designated by a field name, the field containing at least one field value. 34. A method according to claim 33, wherein the field is related to sharpness defects of the image-capture appliance and/or of the image-restitution appliance, wherein the field contains at least one value related to the sharpness defects of the image-capture appliance and/or of the image-restitution appliance. 35. A method according to claim 33, wherein the field is related to colorimetry defects of the image-capture appliance and/or of the image-restitution appliance, wherein the field contains at least one value related to the colorimetry defects of the image-capture appliance and/or of the image-restitution appliance. 36. A method according to claim 33, wherein the field is related to geometric distortion defects and/or to geometric chromatic aberration defects of the image-capture appliance and/or of the image-restitution appliance, wherein the field contains at least one value related to the geometric distortion defects and/or to the geometric chromatic aberration defects of the image-capture appliance and/or of the image-restitution appliance. 37. A method according to claim 33, wherein the field is related to geometric vignetting defects and/or to contrast defects of the image-capture appliance and/or of the image-restitution appliance, wherein the field contains at least one value related to the geometric vignetting defects and/or to the contrast defects of the image-capture appliance and/or of the image-restitution appliance. 38. A method according to claim 33, wherein the field contains at least one value related to deviations. 39. A method according to claim 34, wherein the formatted information is composed at least partly of parameters of a parameterizable transformation model representative of the sharpness defects of the image-capture appliance and/or of the image-restitution appliance, wherein the value or values contained in the field related to the sharpness defects are composed at least partly of parameters of the parameterizable transformation model; wherein the image-processor can use the parameters of the parameterizable transformation model to calculate a corrected shape or corrected restitution shape of a point of the image. 40. A method according to claim 35, wherein the formatted information is composed at least partly of parameters of a parameterizable transformation model representative of the colorimetry defects of the image-capture appliance and/or of the image-restitution appliance, wherein the value or values contained in the field related to the colorimetry defects are composed at least partly of parameters of the parameterizable transformation model; wherein the image-processor can use the parameters of the parameterizable transformation model to calculate a corrected color or corrected restitution color of a point of the image. 41. A method according to claim 36, wherein the formatted information is composed at least partly of parameters of a parameterizable transformation model representative of the geometric distortion defects and/or of the geometric chromatic aberration defects of the image-capture appliance and/or of the image-restitution appliance, wherein the value or values contained in the field related to the geometric distortion defects and/or to the geometric chromatic aberration defects are composed at least partly of parameters of the parameterizable transformation model; wherein the image-processor can use the parameters of the parameterizable transformation model to calculate a corrected position or the corrected restitution position of a point of the image. 42. A method according to claim 37, wherein the formatted information is composed at least partly of parameters of a parameterizable transformation model representative of the geometric vignetting defects and/or of the contrast defects of the image-capture appliance and/or of the image-restitution appliance, wherein the value or values contained in the field related to the geometric vignetting defects and/or to the contrast defects are composed at least partly of parameters of the parameterizable transformation model; wherein the image-processor can use the parameters of the parameterizable transformation model to calculate a corrected intensity or the corrected restitution intensity of a point of the image. 43. A method according to claim 33, wherein to provide the formatted information in a standard format to the image-processor, the method further comprises associating the formatted information with the image. 44. A method according to claim 43, wherein the image is transmitted in a form of a file, the file further containing the formatted information. 45. A method according to claim 33, wherein the image-capture appliance and/or the image-restitution appliance includes at least one variable characteristic depending on the image, at least one of the defects of the image-capture appliance and/or of the image-restitution appliance depending on the at least one variable characteristic, wherein at least one of the fields contains at least one value that is a function of the at least one variable characteristic depending on the image; wherein the image-processor can process the image as a function of the variable characteristics. 46. A method according to claim 33, wherein the formatted information is measured formatted information, at least in part. 47. A method according to claim 33, wherein the formatted information is extended formatted information, at least in part. 48. A method according to claim 33, wherein the image is composed of color planes, the formatted information being at least partly related to the color planes. 49. A system for providing formatted information in a standard format to an image-processor, the formatted information being related to defects of an appliance chain, the appliance chain including at least one image-capture appliance and/or one image-restitution appliance, the image-processor using the formatted information to modify quality of at least one image derived from or addressed to the appliance chain; the formatted information including: data characterizing the defects of the image-capture appliance; and/or data characterizing the defects of the image-restitution appliance; the system comprising: a data-processor configured to fill in at least one field of the standard format with the formatted information, the field being designated by a field name, the field containing at least one field value. 50. A system according to claim 49, wherein the field is related to sharpness defects of the image-capture appliance and/or of the image-restitution appliance, wherein the field contains at least one value related to the sharpness defects of the image-capture appliance and/or of the image-restitution appliance. 51. A system according to claim 49, wherein the field is related to colorimetry defects of the image-capture appliance and/or of the image-restitution appliance, wherein the field contains at least one value related to the colorimetry defects of the image-capture appliance and/or of the image-restitution appliance. 52. A system according to claim 49, wherein the field is related to geometric distortion defects and/or to geometric chromatic aberration defects of the image-capture appliance and/or of the image-restitution appliance, wherein the field contains at least one value related to the geometric distortion defects and/or to the geometric chromatic aberration defects of the image-capture appliance and/or of the image-restitution appliance. 53. A system according to claim 49, wherein the field is related to geometric vignetting defects and/or to contrast defects of the image-capture appliance and/or of the image-restitution appliance, wherein the field contains at least one value related to the geometric vignetting defects and/or to the contrast defects of the image-capture appliance and/or of the image-restitution appliance. 54. A system according to claim 49, wherein the field contains at least one value related to deviations. 55. A system according to claim 50, wherein the formatted information is composed at least partly of parameters of a parameterizable transformation model representative of the sharpness defects of the image-capture appliance and/or of the image-restitution appliance, wherein the value or values contained in the field related to the sharpness defects are composed at least partly of parameters of the parameterizable transformation model. 56. A system according to claim 51, wherein the formatted information is composed at least partly of parameters of a parameterizable transformation model representative of the colorimetry defects of the image-capture appliance and/or of the image-restitution appliance, wherein the value or values contained in the field related to the colorimetry defects are composed at least partly of parameters of the parameterizable transformation model. 57. A system according to claim 52, wherein the formatted information is composed at least partly of parameters of a parameterizable transformation model representative of the geometric distortion defects and/or of the geometric chromatic aberration defects of the image-capture appliance and/or of the image-restitution appliance, wherein the value or values contained in the field related to the geometric distortion defects and/or to the geometric chromatic aberration defects are composed at least partly of parameters of the parameterizable transformation model. 58. A system according to claim 53, wherein the formatted information is composed at least partly of parameters of a parameterizable transformation model representative of the geometric vignetting defects and/or of the contrast defects of the image-capture appliance and/or of the image-restitution appliance, wherein the value or values contained in the field related to the geometric vignetting defects and/or to the contrast defects are composed at least partly of parameters of the parameterizable transformation model. 59. A system according to claim 49, wherein to provide the formatted information in a standard format to the image-processor, the system further comprises a data-processor configured to associate the formatted information with the image. 60. A system according to claim 59, further comprising a transmission device to transmit the image in a form of a file, the file further containing the formatted information. 61. A system according to claim 49, wherein the image-capture appliance and/or the image-restitution appliance include at least one variable characteristic depending on the image, at least one of the defects of the image-capture appliance and/or of the image-restitution appliance depending on the at least one variable characteristic, wherein at least one of the fields contains at least one value that is a function of the at least one variable characteristic depending on the image. 62. A system according to claim 49, wherein the formatted information is measured formatted information, at least in part. 63. A system according to claim 49, wherein the formatted information is extended formatted information, at least in part. 64. A system according to claim 49, wherein the image is composed of color planes, the formatted information being at least partly related to the color planes. |
<SOH> Fields <EOH>Referring to FIG. 8 , a definition will now be given of the concept of fields 91 . The formatted information 15 associated with image 103 can be recorded in several forms and structured into one or more tables, but it corresponds logically to all or part of fields 91 , comprising: (a) the focal distance, (b) the depth of field (c) the geometric defects. The said geometric defects include geometric defects of image 103 characterized by the parameters associated with the filming characteristics 74 and a parameterizable transformation representing the characteristics of image-capture appliance 1 at the moment of filming. By means of the said parameters and of the said parameterizable transformation, it is possible to calculate the corrected position of a point of image 103 . The said geometric defects also include the vignetting characterized by the parameters associated with filming characteristics 74 and a parameterizable transformation representing the characteristics of image-capture appliance 1 at the moment of filming. By means of the said parameters and the said parameterizable transformation, it is possible to calculate the corrected intensity of a point of image 103 . The said geometric defects also include the color cast characterized by the parameters associated with filming characteristics 74 and a parameterizable transformation representing the characteristics of image-capture appliance 1 at the moment of filming. By means of the said parameters and the said parameterizable transformation, it is possible to calculate the corrected color of a point of image 103 . The said fields 91 also include (d) the sharpness of image 103 . The said sharpness includes the blurring in resolution of image 103 characterized by the parameters associated with filming characteristics 74 and a parameterizable transformation representing the characteristics of image-capture appliance 1 at the moment of filming. By means of the said parameters and the said parameterizable transformation, it is possible to calculate the corrected shape of a point of image 103 . Blurring covers in particular coma, spherical aberration, astigmatism, grouping into pixels 104 , chromatic aberration, depth of field, diffraction, parasitic reflections and field curvature. The said sharpness also includes the blurring in depth of field, in particular spherical aberrations, coma and astigmatism. The said blurring depends on the distance of the points of scene 3 relative to image-capture appliance 1 , and it is characterized by the parameters associated with filming characteristics 74 and a parameterizable transformation representing the characteristics of image-capture appliance 1 at the moment of filming. By means of the said parameters and of the said parameterizable transformation, it is possible to calculate the corrected shape of a point of image 103 . The said fields 91 also include (e) parameters of the quantization method. The said parameters depend on the geometry and physics of sensor 101 , on the architecture of electronic unit 102 and on any processing software that may be used. The said parameters include a function that represents the variations of intensity of a pixel 104 as a function of wavelength and light flux derived from the said scene 3 . The said function includes in particular gamma information. The said parameters also include: the geometry of the said sensor 101 , especially the shape, the relative position and the number of sensitive elements of the said sensor 101 , a function representative of the spatial and temporal distribution of noise of image-capture appliance 1 , a value representative of the exposure time for image capture. The said fields 91 also include (f) parameters of the digital-processing operations performed by image-capture appliance 1 , especially digital zoom and compression. These parameters depend on the processing software of image-capture appliance 1 and on the user's adjustments. The said fields 91 also include: (g) parameters representative of the user's preferences, especially as regards the degree of blurring and the resolution of image 103 . (h) the deviations 14 . |
Composition for use in prophylaxis and or treatment |
The present invention relates generally to a composition for use in the prophylaxis and/or treatment of pain. More particularly, the present invention relates to a composition for use in the prophylaxis and/or treatment of chronic neuromuscular pain. The composition of the present invention is particularly useful in the prophylaxis and/or treatment of chronic neuromuscular pain such as, for example, fibromylagia, myofascial pain, repetitive strain injury or overuse syndromes, and cytokine-mediated cancer and chemotherapy associated pain. |
1. A composition comprising an amino acid for use in the prophylaxis and/or treatment of one or more symptoms of chronic neuromuscular pain. 2. A composition comprising an amino acid together with an oligosaccharide and/or monosaccharide for use in the prophylaxis and/or treatment of one or more symptoms of chronic neuromuscular pain. 3. A composition according to claim 1 comprising: i) asparagine; ii) a branched chain amino acid; iii) an amino acid selected from the group consisting of alanine, aspartate and glutamine; and iv) an amino acid selected from the group consisting of arginine, lysine and ornithine; optionally together with at least one of: v) a glucose-containing oligosaccharide; and vi) a monosaccharide for use in the prophylaxis and/or treatment of one of more symptoms of chronic neuromuscular pain. 4. A composition according to claim 3 wherein said branched chain amino acid is leucine, valine and/or isoleucine. 5. A composition according to claim 3 wherein said glucose-containing oligosaccharide is dextrose and/or maltodextrin. 6. A composition according to claim 3 wherein said monosaccharide is fructose and/or glucose. 7. A composition according to claim 1 when used in the prophylaxis and/or treatment of one of more symptoms of chronic neuromuscular pain. 8. A method for the prophylaxis and/or treatment of one or more symptoms of chronic neuromuscular pain in a subject, said method comprising administering to said subject an effective amount of a composition comprising an amino acid. 9. A method for the prophylaxis and/or treatment of one or more symptoms of chronic neuromuscular pain in a subject, said method comprising administering to said subject an effective amount of a composition comprising an amino acid together with an oligosaccharide and/or monosaccharide. 10. A method according to claim 8 wherein said composition comprises: i) asparagine; ii) a branched chain amino acid; iii) an amino acid selected from the group consisting of alanine, aspartate and glutamine; and iv) an amino acid selected from the group consisting of arginine, lysine and ornithine; optionally together with at least one of: v) a glucose-containing oligosaccharide; and vi) a monosaccharide for a time and under conditions sufficient to treat or prevent one of more symptoms of chronic neuromuscular pain. 11. A method according to 10 wherein said branched chain amino acid is leucine, valine and/or isoleucine. 12. A method according to claim 10 wherein said glucose-containing oligosaccharide is dextrose and/or maltodextrin. 13. A method according to claim 10 wherein said monosaccharide is fructose and/or glucose. 14. A method according to claim 13 wherein said monosaccharide is fructose. 15. A method according to claim 10 wherein said subject is a human subject. 16. Use of: i) asparagine; ii) a branched chain amino acid, iii) an amino acid selected from the group consisting of alanine, aspartate and glutamine; and iv) an amino acid selected from the group consisting of arginine, lysine and ornithine; optionally together with at least one of: v) a glucose-containing oligosaccharide; and vi) a monosaccharide in the manufacture of a medicament for the prophylaxis and/or treatment of one or more symptoms of chronic neuromuscular pain. 17. A use according to claim 16 wherein said branched chain amino acid is leucine, valine and/or isoleucine. 18. A use according to claim 16 wherein said glucose-containing oligosaccharide is dextrose and/or maltodextrin. 19. A use according to claim 16 wherein said monosaccharide is fructose and/or glucose. 20. A use according to claim 19 wherein said monosaccharide is fructose. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Reference to any prior art, in this specification is not, and should not be taken as an acknowledgment or any form of suggestion that this prior art is common general knowledge or forms a part of the common general knowledge in Australia or any other country. Chronic neuromuscular pain is a very common clinical and therapeutic problem (de Girolamo, 1991) which affects up to 70% of the population, with severe forms, such as fibromyalgia, occurring between 5-10%. Myofascial pain syndrome occurs in approximately 45-50% of the community with pain of sufficient intensity being noted in 15-20% of the population. The degree of pain or discomfort appears to dictate patient reporting and treatment presentation. These syndromes occur with low prevalence until post puberty, after which they increase in prevalence and severity in the 30 to 50 year age group with a small decline following 50 years of age. There is a female:male ratio between 3.5:1 and 4:1 with the percentage of females increasing with increasing severity of the pain. In patients with fibromyalia, a broad range of symptoms are also reported including: sleep disturbance, fatigue, depression, gastrointestinal disturbance, sinus and thyroid problems, concentration problems, swollen glands, tachycardia and weakness as well as joint hypermobility indicating a whole body metabolic problem (Waylonis & Heck, 1992). Between 60-65% of these chronic neuromuscular pain patients have a slow or gradual onset of symptoms with a progression of the condition from a localised condition to a widespread one involving the whole body in patients with fibromyalgia. Overuse syndromes represent the low-grade spectrum of these conditions with expression determined by an increase in muscle activity. The increase in activity of the muscles results in increased energy use which is reduced due to the underlying problems. The muscles expressing most of the problems are those with the highest energy demand, such as shoulders, neck, arms and wrists in typists, elbows in people playing tennis, facial and neck muscles in violinists and musicians people playing brass and woodwind instruments, etc. Despite the prevalence of chronic neuromuscular pain, there is currently no effective therapy and accordingly, there is a need for new methods and agents for preventing and/or treating conditions characterised by chronic neuromuscular pain. In the work leading up to the present invention the instant inventor has developed a composition which is effective in alleviating one or more of the symptoms associated with chronic neuromuscular pain. |
<SOH> SUMMARY OF THE INVENTION <EOH>Throughout this specification, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or integer or step or group of elements or integers or steps but not the exclusion of any other elements or integer or step or group of elements or integers or steps. In one aspect of the invention there is provided a composition comprising an amino acid for use in the prophylaxis and/or treatment of one or more symptoms of chronic neuromuscular pain. Another aspect of the present invention provides a composition comprising an amino acid together with an oligosaccharide and/or monosaccharide for use in the prophylaxis and/or treatment of one or more symptoms of chronic neuromuscular pain. In a related aspect, the present invention provides a composition comprising: i) asparagine; ii) a branched chain amino acid; iii) an amino acid selected from the group consisting of alanine, aspartate and glutamine; and iv) an amino acid selected from the group consisting of arginine, lysine and ornithine; optionally together with at least one of: v) a glucose-containing oligosaccharide; and vi) a monosaccharide for use in the prophylaxis and/or treatment of one of more symptoms of chronic neuromuscular pain. In another related aspect, the present invention provides a method for the prophylaxis and/or treatment of one or more symptoms of chronic neuromuscular pain in a subject, said method comprising administering to said subject an effective amount of a composition comprising an amino acid. In a further related aspect, the present invention provides a method for the prophylaxis and/or treatment of one or more symptoms of chronic neuromuscular pain in a subject, said method comprising administering to said subject an effective amount of a composition comprising an amino acid together with an oligosaccharide and/or monosaccharide. In yet a further related aspect, the present invention provides a method for the prophylaxis and/or treatment of one or more symptoms of chronic neuromuscular pain in a subject, said method comprising administering to said subject an effective amount of a composition comprising: i) asparagine; ii) a branched chain amino acid; iii) an amino acid selected from the group consisting of alanine, aspartate and glutamine; and iv) an amino acid selected from the group consisting of arginine, lysine and ornithine; optionally together with at least one of: v) a glucose-containing oligosaccharide; and vi) a monosaccharide for a time and under conditions sufficient to prevent or treat one or more symptoms of chronic neuromuscular pain. Still even yet a further aspect of the present invention provides the use of a composition comprising; i) asparagine; ii) a branched chain amino acid; iii) an amino acid selected from the group consisting of alanine, aspartate and glutamine; and iv) an amino acid selected from the group consisting of arginine, lysine and ornithine; optionally together with at least one of: v) a glucose-containing oligosaccharide; and vi) a monosaccharide for the prophylaxis and/or treatment of one or more symptoms of chronic neuromuscular pain. Yet even a further aspect of the present invention contemplates the use of: i) asparagine; ii) a branched chain amino acid; iii) an amino acid selected from the group consisting of alanine, aspartate and glutamine; and iv) an amino acid selected from the group consisting of arginine, lysine and ornithine; optionally together with at least one of: v) a glucose-containing oligosaccharide; and vi) a monosaccharide in the manufacture of a medicament for prophylaxis and/or treatment of one of more symptoms of chronic neuromuscular pain. detailed-description description="Detailed Description" end="lead"? |
Wear resistant tubular connection |
A wear resistant casing connection including a wear resistant portion on its exterior surface is taught. A casing connection having a controlled bend angle is also taught. |
1. A wear resistant casing connection comprising an outer surface and a wear resistant material on at least a portion of the outer surface. 2. The wear resistant casing connection of claim 1 wherein the casing connection is selected to be useful for drilling with casing. 3. The wear resistant casing connection of claim 1 wherein the wear resistant material is positioned on the outside of any bend that is accidentally or intentionally imposed across the connection. 4. The wear resistant casing connection of claim 1 wherein the wear resistant material is integral to the connection 5. The wear resistant casing connection of claim 1 wherein the wear resistant material is formed by surface hardness treatment. 6. The wear resistant casing connection of claim 1 wherein the wear resistant material is applied to the connection as a coating 7. The wear resistant casing connection of claim 6 wherein the coating includes hardfacing. 8. The wear resistant casing connection of claim 7 wherein the hardfacing is formed as at least one hard band. 9. The wear resistant casing connection of claim 1 further comprising an upset interval having a leading edge and the wear resistant material being positioned adjacent the leading edge. 10. The wear resistant casing connection of claim 1 further comprising a casing coupling having a leading edge and the wear resistant material being positioned adjacent the leading edge. 11. The wear resistant casing connection of claim 10 wherein the wear resistant material is disposed axi-symmetrically on said external surface over one or more axial intervals to form one or more hardbands of diameter somewhat greater than the diameter of the generally cylindrical exterior surface. 12. The wear resistant casing connection of claim 10 wherein the coupling includes adjacent its leading edge, a box end including a threaded interval therein and an extension between the leading edge and the threaded interval and wherein the wear resistant material is disposed on the extension. 13. The wear resistant casing connection of claim 10 wherein the coupling includes a mill end box having a center axis and a field end box having a center axis and the mill end box center axis being angularly offset from the field end box center axis to form a bend angle in the coupling. 14. The wear resistant casing connection of claim 10 wherein the bend angle defines an outer bend area on the coupling outer surface and the wear resistant material is disposed on the outer bend area. 15. The wear resistant casing connection of claim 10 further comprising a shoulder ring in the coupling. 16. A casing string comprising: an interval; a bend angle in the interval, the bend angle being selected to control the lateral reaction force of the casing string against the borehole wall in which the casing string is intended to extend. 17. The casing string of claim 16 further comprising a second bend angle in the interval and the spacing between the bend angle and the second bend angle. 18. The casing string of claim 16 wherein the directional orientation of the bend angle is selected. 19. The casing string of claim 16 wherein the bend angle is generated at a connection between an upper casing joint and a lower casing joint. 20. The casing string of claim 19 wherein connection includes a threaded coupling having a mill end box having a center axis and a field end box having a center axis and the mill end box center axis being angularly offset from the field end box center axis to form a coupling bend angle and the bend angle is controlled by selecting the coupling bend angle. 21. The casing string of claim 16 wherein the bend angle is selected by incorporating into the interval a casing joint having a characteristic capable of generating a bend angle in a casing string. 22. The casing string of claim 16 wherein the bend angle is selected by incorporating into the interval a casing coupling having a characteristic capable of generating a bend angle in a casing string. 23. A casing coupling comprising: an outer surface, a box end including an end edge and a threaded interval therein, and an extension between the end edge and the threaded interval. 24. The casing coupling of claim 23 wherein the extension extends beyond any load bearing threads of the threaded interval. 25. The casing coupling of claim 23 wherein the extension is formed to be generally cylindrical. 26. The casing coupling of claim 23 wherein the end edge defines a leading edge of the casing coupling during insertion of the casing coupling into a well. 27. The casing coupling of claim 23 wherein the box end is on the mill end. 28. A casing coupling comprising: a main body formed as a cylinder and including an upper end, a lower end, an outer surface and an axial inner bore defined by an inner surface, load bearing threads formed on the inner surface adjacent a selected end selected from one of the upper end or the lower end, an extension of the main body between the load bearing threads and the selected end. 29. The casing coupling of claim 28 wherein the load bearing threads are part of a threaded interval and the extension extends beyond any threads of the threaded interval. 30. The casing coupling of claim 28 wherein the selected end is the lower end. 31. The casing coupling of claim 28 wherein the selected end is the mill end. 32. The casing coupling of claim 28 wherein the extension includes an outer surface. 33. The casing coupling of claim 32 wherein the outer surface is generally cylindrical. 34. The casing coupling of claim 32 wherein the outer surface supports a hardband. 35. The casing coupling of claim 28 wherein the extension extends axi-symmetrically about the selected end relative to an axis defined by the axial inner bore. 36. A casing string comprising: at least one casing joint including an externally threaded pin end and a casing coupling including a box end including an end edge and a threaded interval therein, and an extension between the end edge and the threaded interval, the casing coupling threadedly engaged by load bearing threads of the threaded interval to the pin end of the at least one casing joint with the extension extending out beyond any load bearing threads of the coupling. 37. The casing string of claim 36 wherein the end edge defines a leading edge of the casing coupling during insertion of the casing string into a well. 38. The casing string of claim 36 wherein the box end is on the mill end. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Lengths of tubulars used to drill and complete bore holes in earth materials, referred to as joints, are typically joined by threaded connections to form a long assembly referred to as a drill string. Numerous threaded connection geometries are employed to provide sealing and load carrying capacities to meet drilling, installation and operating requirements. Of these geometries, connections having an external diameter greater than the pipe body are the most widely used. Thus the majority length of a typical drill string is comprised of alternating long lengths of generally cylindrical pipe separated by relatively short externally upset intervals at the connections. Within the context of petroleum drilling and well completion, wells are typically constructed by drilling the well bore using one tubular string, largely comprised of drill pipe, then removing the drill pipe string and completing the well by installing a second tubular string, referred to as casing, which is subsequently permanently cemented in place. The tubular strings are formed by connecting joints of pipe with threaded connections. With this historic method of well construction, both the drill pipe and casing joint designs are separately optimized for the different performance requirements of the drilling and completion operations respectively. More specifically, the drill pipe connections must typically accommodate more torque to drill, than is required during completion, and must resist wear that occurs where the connection is in contact with the abrasive borehole wall during extended periods of drilling rotation. The tendency toward wear is strongly dependent on the lateral forces that arise at the points of contact between the drill string and borehole. These contact forces result from the interaction of several variables, but may be generally attributed to: inertia loads required to react the tendency of the rotating drill string to whirl, reaction of lateral load induced by the axial load transferred along the string through intervals of curvature and gravity loads in deviated intervals. Concentration of all or a majority of the wall contact load over the short upset interval containing the connection tends to exacerbate wear at these locations. This wear has the effect of generally reducing the diameter of the connections. For that reason, it is common industry practice to apply bands or zones of abrasion resistant coatings around the circumference of the drill pipe connections, referred to as hardbanding or hardfacing, to build up the diameter of the connection and thus provide a sacrificial layer of slow wearing material. U.S. Pat. Nos. 4,665,996 and 6,375,895 are two examples describing the materials and application methods used to apply such surface preparations to drill pipe tool joints. Recent advances in drilling technology have enabled wells to be drilled and completed with a single casing string, eliminating the need to ‘trip’ the drill pipe in and out of the hole to service the bit and make room for the casing upon completion of drilling. This technology employs a wireline retrievable bottom hole drilling assembly capable of deployment on the distal end of casing strings. Development of the technology was initially motivated by potential cost savings arising from reduced drilling time and the expense of providing and maintaining the drill string, plus various technical advantages, such as reduced risk of well caving before installation of the casing. In addition to drilling, this technology finds utility in casing running applications where reaming is required to resize the borehole. The established performance requirements for casing are only those required to meet the needs of historic well construction methods. The new use of casing to drill, naturally changes the performance requirements of the casing string. Such changes include increased torque capacity required to drill with the casing connections, but did not initially anticipate the need for increased wear resistance particularly in relatively straight wells where lateral forces arising from curvature and gravity are minimal. This expectation was based on the shorter exposure time to conditions of rotating wear likely for casing strings compared to drill pipe. (Drill pipe is used to drill many wells, resulting in extended exposure of drill pipe connections to conditions of rotating wear. In contrast, the application using a casing string to drill, deliberately only intends to expose the connections to rotating wear conditions for the time required to drill the single well interval to be cased by that string.) However, it has been discovered that drilling with casing strings using industry standard threaded and coupled buttress (BTC) connections, having tapered pipe thread geometries specified by the American Petroleum Institute (API) and equipped with shoulder rings such as, for example, those described in Canadian Patent Application 2,311,156, frequently causes eccentric wear in the region of the connection. This wear may locally reduce the coupling side wall thickness until the coupling radius, in the region of wear, is little more than the pipe body. This amount of wear may occur during even a fraction of the relatively short period required to drill a single well interval in a nearly vertical well. As will be appreciated by one skilled in the art, this wear substantially compromises the load and sealing capacity of the connection. This eccentric wear mechanism arises because the straightness of these connections are not as tightly controlled as in drill pipe, since the historic use of casing only contemplates the requirements of running, cementing and well access and not drilling. Thus a small bend in the string axis often occurs across the connections. Such bends tend to preferentially force the connections against the borehole wall at the ‘outside’ of the bends. The lateral wall contact force arising at these points of contact is strongly dependent on whether or not the lateral deflection imposed by the bend angles in the axially loaded casing is sufficient to interfere with the confining bore hole. This lateral interference acts to displace the casing string from its neutral position at the points of contact with the borehole, the casing string behaving as a long beam bent at the connections and restrained by the borehole. Particularly, where such lateral interference occurs between connections spaced one joint apart, the lateral load and hence wear rate is much greater than occurs over comparably ‘straight’ intervals. For example, the connection bend angles were inferred a sample of typical 7 inch (178 mm) API buttress threaded and coupled (BTC) casing joints. These magnitudes were used to calculate the possible maximum lateral load arising from this load mechanism, were such casing joints assembled into a casing string and placed in a borehole drilled with a bit size of 8.5 inches (216 mm). It was found that, with negligible axial load, a lateral force of at least 1000 lbf (4450N) could be present if the casing string were so confined in an interval. As described above, this lateral load mechanism is not normally present in drill pipe strings placed in a bore hole because the connections in those strings are typically straighter and the tube bodies flexurally less rigid than the same respective components of a casing string assembly. Furthermore, unlike the other lateral loading mechanisms which result in relatively axi-symmetric wear of the connection, wear resulting from the connection bend angle is non-axi-symmetric or eccentric. This eccentric wear could be mitigated by providing connections with increased straightness. In certain applications this alternative may be preferable. However in general this will increase manufacturing cost and prevent the use of readily available tubulars. Furthermore, the presence of this new lateral wall contact load, while discovered to produce an unfavorable tendency toward excess wear, was simultaneously discovered to have a beneficial effect by improving borehole wall stability and reducing the risk of lost circulation when compared to drilling with straight drill pipe strings. Excess wear can be avoided by use of a separate device, termed a wear band, as disclosed in Cdn. Patent App 2,353,249. The disclosed wear band includes a band of wear resistant material and is structurally attached to the casing adjacent the connection by crimping. This solution is effective and provides a readily implemented means enhance the usefulness of casing joints having standard non-wear resistant connections for casing drilling or reaming. However, the method requires additional handling and operations to crimp the wear bands to the casing joints with associated labour, capital and logistical overburden costs, plus introducing a longer upset interval length in the region of the connection, which longer interval must be accommodated by the pipe handling, running and drilling equipment. |
<SOH> SUMMARY OF THE INVENTION <EOH>A wear resistant connection has been invented for joining lengths of casing tubulars into assemblies referred to as strings. The wear resistant connection of the present invention provides a means to substantially prevent loss of material from the exterior surface of the tube wall, in the region of the connection, caused by rotating wear mechanisms present where such strings are placed in boreholes and rotated. In one embodiment, this wear resistant connection provides resistance to eccentric rotating wear mechanisms arising from the bend angle either accidentally or deliberately present in casing connections. For the purpose of this invention, a connection is understood broadly to mean any arrangement or device that joins the ends of casing tubulars to create a section over which a structural union is arranged so that the axes of the joined tubulars is substantially continuous across the connection interval, and while generally straight, may have a small bend either accidentally or deliberately introduced. Understood thus, the connection of the present invention includes but is not limited to welded connections, integral connections and threaded and coupled connections. Where an upset interval is associated with such a connection, references to the connection are understood to include this upset interval. Where the connection is made without an upset interval, i.e., an externally flush connection, reference to the connection is understood to include a section of the joined casing tubulars having a length of at least 10 casing diameters on each side of the actual joint (i.e. the weld) between the casing tubulars. Thus in accordance with a broad aspect of the present invention, a casing connection is provided having an exterior surface, at least some portion of which includes a wear resistant material. The casing connection is preferably selected to be useful for drilling with casing. The wear resistant material can be arranged to at least overlap the circumferentially oriented location forming the outside of any bend that may be accidentally or intentionally imposed across the connection. The wear resistant material may be integral to the connection, obtained by surface hardness treatment such as boronizing, nitriding or case hardening or applied thereto such as by use of a coating such as hardfacing. The relatively high cost of the applying, working with and forming wear resistant materials encourages a reduction in the size of the area covered and thickness of material. The vast majority of well bores are lined with metal casing strings having threaded connections. Therefore to be most readily implemented, wear resistance of metal casing connections is best provided in a manner which accommodates existing thread-forms, sealing geometries and bend magnitude tolerances. Such existing threaded connections include the thread-forms and sealing geometries comprising so called premium connections, in addition to both integral and threaded and coupled American Petroleum Institute (API) specified geometries. (Reference herein to a ‘thread-form’ is generally understood to include the seal geometry if present, unless these two components of the connection geometry are specifically separated in the context.) This accommodation of existing geometry extends to the connection diameter where it is preferable to provide wear resistance without a significant increase in outside diameter to avoid correlatively increasing the annular flow resistance, where such a wear resistant connection is deployed in a casing string within a well bore. It is advantageous to adapt existing threaded connection geometries to provide locations where wear resistant materials can be most economically and least invasively applied to the connection, i.e., without significantly altering the existing connections with respect to seal and structural performance, while providing adequate protection against wear from rotation while drilling. In particular, preferably the wear resistant material is provided at the lower or leading end of the coupling (leading is defined with respect to the axial direction of travel while drilling), as the upset diameter change from the pipe body to the coupling occurring at this location tends to promote preferential wear while drilling with casing. Threaded and coupled connections according to the present invention can include an internally threaded coupling having an upper end, a lower end and generally cylindrical exterior surface, as typically provided for such couplings, where wear resistant surface treatment or coating material is disposed axi-symmetrically on said external surface over one or more axial intervals to form one or more hardbands of diameter somewhat greater than the diameter of the generally cylindrical exterior surface. Said axial interval length and coating thickness are chosen, based on application requirements, to provide sufficient volume of material to resist wearing through to the base metal. Wear resistant surface treatment or coating material is axi-symmetrically distributed to accommodate the random distribution of bend angle and hence circumferential location of connection contact with the well bore. For most of these geometries, wear resistance can be provided by applying coatings resistant to abrasive wear to the exterior surface of the connection. Such coatings are commonly referred to as hardfacing. These coatings are applied using a variety of techniques and materials, but typically the bond chemistry and mechanics require heat input to obtain the elevated temperature required to create a strong bond between the coating and metal substrate. It is therefore necessary to consider the effects of this heat input and bond chemistry on the metal substrate, and in particular to allow for any changes in structural or mechanical performance the heat input and bond chemistry might have. In addition, the choice of axial interval location where wear resistant surface treatment or coating is provided is preferably selected to occur at locations where stresses induced by structural and pressure loads are lowest. Such choice of location reduces the risk of connection failure due to crack initiation within the typically brittle coating material. However such a suitable region of low stress is often not available for many of the threaded and coupled connection geometries employed by industry. It is therefore a further purpose of the present invention to provide such a suitable region of low stress at one or both ends of the coupling by more preferably providing a coupling having its length and interval of internal threading arranged so that the end hardband interval does not overlap with the internal threaded interval of the coupling. Otherwise stated, relative to the ‘standard’ non-wear resistant coupling geometry a coupling is provided where at least one end and preferably the lower end is modified to provide a generally cylindrical extension which extension or extensions having external and internal surfaces without load bearing threads on which said external surface or surfaces wear resistant surface treatment or coating material such as hardfacing is applied to create a hardband or hardbands of upset diameter. Where only one hardband is required, the lower end is preferred as this end forms the leading edge of the coupling while drilling with casing and protects this region from preferential wear. Application of these teachings for placement of wear resistant surface treatment or coating material on the couplings of threaded and coupled connections may be extended to integral connections and externally upset integral connections. As commonly understood in the industry, an integral connection is comprised of an externally threaded pin formed on the end of one tubular screwed into a mating internally threaded box formed on the end of a second tubular. Said internally threaded box having an external largely cylindrical surface and proximal end. Particularly where the connection design is arranged to shoulder on said proximal end when made up to the pin, the stress state in this region is less prone to crack initiation and propagation. To best serve the purposes of the present invention a wear resistant integral connection is therefore provided having a hardband of wear resistant surface treatment or coating material disposed on its proximal end. Relative to the ‘standard’ non-wear resistant geometry of an integral connection box it is more preferable if the proximal end of the box is modified to provide a generally cylindrical extension which extension having external and internal surfaces without load bearing threads on which said external surface wear resistant surface treatment or coating material such as hardfacing is applied axi-symmetrically to create a hardband or hardbands of upset diameter. Where the integral connection is formed on externally upset tubulars, such externally upset interval typically extends beyond the depth required to carry the box or pin threaded connection geometry, and in certain applications it may be preferable to provide a hardband on the connection exterior surface at or near the leading end of the upset interval either separately or in combination with a hardband placed at the proximal end of the box. The leading end of the upset interval, thus carrying the hardband, occurs at a location of significantly greater thickness than the pipe body and therefore of significantly reduced stress, but having the further advantage of being positioned at the location of preferential wear. It is therefore an additional purpose of the present invention to provide a wear resistance externally upset tubular connection having an externally upset interval with leading and trailing ends comprising the connection, and having at least one hardband positioned on said leading end. The bend magnitude occurring across the connection interval is a function of the pipe end straightness and combined thread axis angle alignment with the pipe axes for integral connections and additionally the coupling thread axes with respect to the coupling for threaded and coupled connections. For industry typical casing connections, the bend magnitude or axis misalignment is not tightly controlled, as for example described in the API Specification 5CT and Standard 5B. Furthermore the bend direction is randomly oriented. The wear resistant casing connections of the present invention enjoy further utility when also deliberately provided with a small bend in tubular axis across the connection interval. Where such connections are employed to assemble a plurality of tubular joints to form at least one interval of a casing string placed in a borehole, the bend angle and direction controls the local lateral stess of the casing string within the confines of the borehole. The bend angle and direction may thus be arranged to deflect some or all of the connections into generally radially opposed contact with the borehole wall over an interval of several joints. As will be apparent to one skilled in the art, the lateral forces arising from this contact will tend to increase with increasing bend angle. It will also be apparent that control of the bend direction provides a further means to control this force compared to random orientation of connection bend direction. When such a string is rotated within the confining borehole, the region of connection contact rotates with respect to the borehole causing an axi-symmetric ‘wiping’ action on the interior of the borehole wall, but does not rotate with respect to the connection causing the associated wear mechanism to be non-axi-symmetric, i.e., eccentric. In certain applications, the wiping action thus provided results in axi-symmetric consolidation of the near well bore earth material, reducing risk of sloughing and lost circulation. The degree of consolidation and associated benefits depends on the lateral force generated by the casing as it bears against the borehole wall. Control of the connection bend magnitude, and preferably also the bend direction, enables control of said lateral force exerted and is thus a means to balance the benefits gained by wiping action on the borehole wall against the eccentric wear rate of the connection. This then is the basis for the further utility obtained for the present invention of a wear resistant connection having a controlled small bend. In accordance with this further purpose, in one embodiment of the present invention, a wear resistant casing connection is provided having at least some portion of its exterior surface provided with a wear resistant coating or surface treatment and arranged to provide a controlled bend in the axes of the tubulars joined by the connection. Said bend magnitude is selected such that when said bent wear resistant connections are employed to assemble at least some portion of a plurality of tubular joints to form at least one interval of a casing string placed in a borehole, the resulting local directional variations introduced by the bend magnitudes will induce some or all of the bent wear resistant connections to at least contact the borehole wall and induce generally radially opposed contact forces. As a means to more predictably control said radially opposed contact forces, in a further embodiment, said wear resistant casing connection of controlled bend is provided having the circumferential direction of the bend controlled with respect to a casing string assembled from such connections. Such control of circumferential direction is preferably selected to provide a repeating pattern between bent connections comprising an interval of an assembled casing string. As will be apparent to one skilled in the art, the teachings of the present invention with respect to placement of wear resistant surface treatment or coatings on typical threaded connection geometries to form wear resistant connection where the bend angle and direction is allowed to vary randomly according to existing industry practice apply equally well to connections where the bend angle is controlled. However, where the bend angle is introduced deliberately in the manufacturing process the circumferential location corresponding to the outside of the bend may be readily identified. Since contact with the borehole must occur at this location wear resistant surface treatment or coating material need only be disposed over this region and need not be disposed axi-symmetrically, thus requiring less volume of wear resistant material with consequent opportunity for cost saving. In accordance with another aspect of the present invention, there is provided a casing string including an interval over which the bend angle is selected to control the lateral reaction force of the casing string against the borehole wall in which the casing string is intended to extend. |
Nucleic acid-associated proteins |
Various embodiments of the invention provide human nucleic acid-associated proteins (NAAP) and polynucleotides which identify and encode NAAP. 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 NAAP. |
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: 1-35, 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:1-2, SEQ ID NO:4-7, SEQ ID NO:9-16, SEQ ID NO:18-19, SEQ ID NO:21-22, SEQ ID NO:24, SEQ ID NO:27-35, c) 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:3, SEQ ID NO: 17, and SEQ ID NO:25, d) a polypeptide comprising a naturally occurring amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:8, e) a polypeptide comprising a naturally occurring amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:20, f) a polypeptide comprising a naturally occurring amino acid sequence at least 94% identical to the amino acid sequence of SEQ ID NO:23, g) a polypeptide comprising a naturally occurring amino acid sequence at least 91% identical to the amino acid sequence of SEQ ID NO:26, h) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and i) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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:36-70. 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. (canceled) 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:36-70, 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:36-70, 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. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 12. 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. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides. 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. 18-19. (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. 24-26. (canceled) 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. 30-45. (canceled) 46. A microarray wherein at least one element of the microarray is a polynucleotide of claim 13. 47-125. (canceled) |
<SOH> BACKGROUND OF THE INVENTION <EOH>Multicellular organisms are comprised of diverse cell types that differ dramatically both in structure and function. The identity of a cell is determined by its characteristic pattern of gene expression, and different cell types express overlapping but distinctive sets of genes throughout development. Spatial and temporal regulation of gene expression is critical for the control of cell proliferation, cell differentiation, apoptosis, and other processes that contribute to organismal development. Furthermore, gene expression is regulated in response to extracellular signals that mediate cell-cell communication and coordinate the activities of different cell types. Appropriate gene regulation also ensures that cells function efficiently by expressing only those genes whose functions are required at a given time. Transcription Factors Transcriptional regulatory proteins are essential for the control of gene expression. Some of these proteins function as transcription factors that initiate, activate, repress, or terminate gene transcription. Transcription factors generally bind to the promoter, enhancer, and upstream regulatory regions of a gene in a sequence-specific manner, although some factors bind regulatory elements within or downstream of a gene coding region. Transcription factors may bind to a specific region of DNA singly or as a complex with other accessory factors. (Reviewed in Lewin, B. (1990) Genes IV , Oxford University Press, New York, N.Y., and Cell Press, Cambridge, Mass., pp. 554-570.) The double helix structure and repeated sequences of DNA create topological and chemical features which can be recognized by transcription factors. These features are hydrogen bond donor and acceptor groups, hydrophobic patches, major and minor grooves, and regular, repeated stretches of sequence which induce distinct bends in the helix. Typically, transcription factors recognize specific DNA sequence motifs of about 20 nucleotides in length. Multiple, adjacent transcription factor-binding motifs may be required for gene regulation. Many transcription factors incorporate DNA-binding structural motifs which comprise either a helices or β sheets that bind to the major groove of DNA. Four well-characterized structural motifs are helix-turn-helix, zinc finger, leucine zipper, and helix-loop-helix. Proteins containing these motifs may act alone as monomers, or they may form homo- or heterodimers that interact with DNA. The helix-turn-helix motif consists of two a helices connected at a fixed angle by a short chain of amino acids. One of the helices binds to the major groove. Helix-turn-helix motifs are exemplified by the homeobox motif which is present in homeodomain proteins. These proteins are critical for specifying the anterior-posterior body axis during development and are conserved throughout the animal kingdom. The Antennapedia and Ultrabithorax proteins of Drosophila melanogaster are prototypical homeodomain proteins. (Pabo, C. O. and R. T. Sauer (1992) Annu. Rev. Biochem. 61:1053-1095.) 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, designated C2H2 and C3HC4 (“RING” finger), have been described. (Lewin, supra.) Zinc finger proteins each contain an a helix and an antiparallel β sheet whose proximity and conformation are maintained by the zinc ion. Contact with DNA is made by the arginine preceding the a helix and by the second, third, and sixth residues of the a helix. Variants of the zinc finger motif include poorly defined cysteine-rich motifs which bind zinc or other metal ions. These motifs may not contain histidine residues and are generally nonrepetitive. The zinc finger motif may be repeated in a tandem array within a protein, such that the a helix of each zinc finger in the protein makes contact with the major groove of the DNA double helix. This repeated contact between the protein and the DNA produces a strong and specific DNA-protein interaction. The strength and specificity of the interaction can be regulated by the number of zinc finger motifs within the protein. Though originally identified in DNA-binding proteins as regions that interact directly with DNA, zinc fingers occur in a variety of proteins that do not bind DNA (Lodish, H. et al. (1995) Molecular Cell Biology , Scientific American Books, New York, N.Y., pp. 447-451). For example, Galcheva-Gargova, Z. et al. ((1996) Science 272:1797-1802) have identified zinc finger proteins that interact with various cytokine receptors. The C2H2-type zinc finger signature motif contains a 28 amino acid sequence, including 2 conserved Cys and 2 conserved His residues in a C-2-C-12-H-3-H type motif. The motif generally occurs in multiple tandem repeats. A cysteine-rich domain including the motif Asp-His-His-Cys (DHHC-CRD) has been identified as a distinct subgroup of zinc finger proteins. The DHHC-CRD region has been implicated in growth and development. One DHHC-CRD mutant shows defective function of Ras, a small membrane-associated GTP-binding protein that regulates cell growth and differentiation, while other DHHC-CRD proteins probably function in pathways not involving Ras (Bartels, D. J. et al. (1999) Mol. Cell Biol. 19:6775-6787). Zinc-finger transcription factors are often accompanied by modular sequence motifs such as the Kruppel-associated box (KRAB) and the SCAN domain. For example, the hypoalphalipoproteinemia susceptibility gene ZNF202 encodes a SCAN box and a KRAB domain followed by eight C2H2 zinc-finger motifs (Homer, C. et al. (2001) Biochim. Biophys. Acta 1517:441-448). The SCAN domain is a highly conserved, leucine-rich motif of approximately 60 amino acids found at the amino-terminal end of zinc finger transcription factors. SCAN domains are most often linked to C2H2 zinc finger motifs through their carboxyl-terminal end. Biochemical binding studies have established the SCAN domain as a selective hetero- and homotypic oligomerization domain. SCAN domain-mediated protein complexes may function to modulate the biological function of transcription factors (Schumacher, C. et al. (2000) J. Biol. Chem. 275:17173-17179). The KRAB (Kruppel-associated box) domain is a conserved amino acid sequence spanning approximately 75 amino acids and is found in almost one-third of the 300 to 700 genes encoding C2H2 zinc fingers. The KRAB domain is found N-terminally with respect to the finger repeats. The KRAB domain is generally encoded by two exons; the KRAB-A region or box is encoded by one exon and the KRAB-B region or box is encoded by a second exon. The function of the KRAB domain is the repression of transcription. Transcription repression is accomplished by recruitment of either the KRAB-associated protein-1, a transcriptional corepressor, or the KRAB-A interacting protein. Proteins containing the KRAB domain are likely to play a regulatory role during development (Williams, A. J. et al. (1999) Mol. Cell Biol. 19:8526-8535). A subgroup of highly related human KRAB zinc finger proteins detectable in all human tissues is highly expressed in human T lymphoid cells (Bellefroid, E. J. et al. (1993) EMBO J. 12:1363-1374). The ZNF85 KRAB zinc finger gene, a member of the human ZNF91 family, is highly expressed in normal adult testis, in seminomas, and in the NT2/D1 teratocarcinoma cell line (Poncelet, D. A. et al. (1998) DNA Cell Biol. 17:931-943). Additional zinc finger-associated proteins include the sprouty (SPRY) protein, which was first identified in a genetic screen in Drosophila . SPRY proteins are classified by virtue of their characteristic cysteine-rich residues located in their carboxyl termini (Wong, E. S. M., et al. (2001) J. Biol. Chem. 276:5866-5875). Zinc-binding B-box motifs are located within the B30.2-like domain, constituting a diverse family of proteins (Seto, M. H., et al. (1999) Proteins 35:235-249). The functions of these domains include regulation of cell growth and differentiation. The SPRY domain has been identified as a subdomain within the B30.2-like domain (Torok, M. and Etkin, L. D. (2001) Differentiation 67:63-71). The B-box domain itself is involved in growth control and transcriptional regulation. These genes possess several conserved motifs that always include a B-box zinc binding motif associated with various other motifs such as the RING zinc finger. The RING finger domain is a zinc-binding Cys-His protein motif found in various proteins involved in signal transduction, gene transcription, differentiation, and morphogenesis. A RING-B-box-coiled-coil (RBCC) subclass of RING-finger proteins contains an NH 2 -terminal RING-finger followed by either single or multiple additional B-box zinc finger domains (Spencer, J. A., et al. (2000) J. Cell Biol. 150:771-784). Several RBCC proteins have been implicated in oncogenesis. The RET finger protein (RFP) also belongs to the B-box zinc finger protein family. RFPs possess a tripartite motif consisting of a RING finger, a B-box finger, and a coiled-coil domain. RFP may become oncogenic when its tripartite motif becomes fused with the tyrosine kinase domain of the RET protein (Tezel, G., et al. (1999) Pathol. Int. 49:881-886). The C4 motif is found in hormone-regulated proteins. The C4 motif generally includes only 2 repeats. A number of eukaryotic and viral proteins contain a conserved cysteine-rich domain of 40 to 60 residues (called C3HC4 zinc-finger or RING finger) that binds two atoms of zinc, and is probably involved in mediating protein-protein interactions. The 3D “cross-brace” structure of the zinc ligation system is unique to the RING domain. The spacing of the cysteines in such a domain is C-x(2)-C-x(9 to 39)-C-x(1 to 3)-H-x(2 to 3)-C-x(2)-C-x(4 to 48)-C-x(2)-C. The PHD finger is a C4HC3 zinc-finger-like motif found in nuclear proteins thought to be involved in chromatin-mediated transcriptional regulation. GATA-type transcription factors contain one or two zinc finger domains which bind specifically to a region of DNA that contains the consecutive nucleotide sequence GATA. NMR studies indicate that the zinc finger comprises two irregular anti-parallel b sheets and an a helix, followed by a long loop to the C-terminal end of the finger (Ominchinsid, J. G. (1993) Science 261:438-446). The helix and the loop connecting the two b-sheets contact the major groove of the DNA, while the C-terminal part, which determines the specificity of binding, wraps around into the minor groove. The LIM motif consists of about 60 amino acid residues and contains seven conserved cysteine residues and a histidine within a consensus sequence (Schmeichel, K. L. and M. C. Beckerle (1994) Cell 79:211-219). The LIM family includes transcription factors and cytoskeletal proteins which may be involved in development, differentiation, and cell growth. One example is actin-binding LIM protein, which may play roles in regulation of the cytoskeleton and cellular morphogenesis (Roof, D. J. et al. (1997) J. Cell Biol. 138:575-588). The N-terminal domain of actin-binding LIM protein has four double zinc finger motifs with the LIM consensus sequence. The C-terminal domain of actin-binding LIM protein shows sequence similarity to known actin-binding proteins such as dematin and villin. Actin-binding LIM protein binds to F-actin through its dematin-like C-terminal domain. The LIM domain may mediate protein-protein interactions with other LIM-binding proteins. Myeloid cell development is controlled by tissue-specific transcription factors. Myeloid zinc finger proteins (MZF) include MZF-1 and MZF-2. MZF-1 functions in regulation of the development of neutrophilic granulocytes. A murine homolog MZF-2 is expressed in myeloid cells, particularly in the cells committed to the neutrophilic lineage. MZF-2 is down-regulated by G-CSF and appears to have a unique function in neutrophil development (Murai, K. et al. (1997) Genes Cells 2:581-591). The leucine zipper motif comprises a stretch of amino acids rich in leucine which can form an amphipathic a helix. This structure provides the basis for dimerization of two leucine zipper proteins. The region adjacent to the leucine zipper is usually basic, and upon protein dimerization, is optimally positioned for binding to the major groove. Proteins containing such motifs are generally referred to as bZIP transcription factors. The leucine zipper motif is found in the proto-oncogenes Fos and Jun, which comprise the heterodimeric transcription factor AP1 involved in cell growth and the determination of cell lineage (Papavassiliou, A. G. (1995) N. Engl. J. Med. 332:45-47). Many neoplastic disorders in humans can be attributed to inappropriate gene expression. 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. Such an arrangement likely results in inappropriate gene transcription, potentially contributing to malignancy. 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. supra). The helix-loop-helix motif (HLH) consists of a short a helix connected by a loop to a longer a helix. The loop is flexible and allows the two helices to fold back against each other and to bind to DNA. The transcription factor Myc contains a prototypical BLH motif. The NF-kappa-B/Rel signature defines a family of eukaryotic transcription factors involved in oncogenesis, embryonic development, differentiation and immune response. Most transcription factors containing the Rel homology domain (RHD) bind as dimers to a consensus DNA sequence motif termed kappa-B. Members of the Rel family share a highly conserved 300 amino acid domain termed the Rel homology domain. The characteristic Rel C-terminal domain is involved in gene activation and cytoplasmic anchoring functions. Proteins known to contain the RHD domain include vertebrate nuclear factor NF-kappa-B, which is a heterodimer of a DNA-binding subunit and the transcription factor p65, mammalian transcription factor RelB, and vertebrate proto-oncogene c-rel, a protein associated with differentiation and lymphopoiesis (Kabrun, N. and P. J. Enrietto (1994) Semin. Cancer Biol. 5:103-112). A DNA binding motif termed ARID (AT-rich interactive domain) distinguishes an evolutionarily conserved family of proteins. The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression. ARID proteins include Bright (a regulator of B-cell-specific gene expression), dead ringer (involved in development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) (Dallas, P. B. et al. (2000) Mol. Cell Biol. 20:3137-3146). The ELM2 (Egl-27 and MTA1 homology 2) domain is found in metastasis-associated protein MTA1 and protein ER1. The Caenorhabditis elegans gene egl-27 is required for embryonic patterning MTA1, a human gene with elevated expression in metastatic carcinomas, is a component of a protein complex with histone deacetylase and nucleosome remodelling activities (Solari, F. et al. (1999) Development 126:2483-2494). The ELM2 domain is usually found to the N terminus of a myb-like DNA binding domain. ELM2 is also found associated with an ARID DNA. The Iroquois (Irx) family of genes are found in nematodes, insects and vertebrates. Irx genes usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The Irx genes function early in development to specify the identity of diverse territories of the body. Later in development in both Drosophila and vertebrates, the Irx genes function again to subdivide those territories into smaller domains. (For a review of Iroquois genes, see Cavodeassi, F. et al. (2001) Development 128:2847-2855.) For example, mouse and human Irx4 proteins are 83% conserved and their 63-aa homeodomain is more than 93% identical to that of the Drosophila Iroquois patterning genes. Irx4 transcripts are predominantly expressed in the cardiac ventricles. The homeobox gene Irx4 mediates ventricular differentiation during cardiac development (Bruneau, B. G. et al. (2000) Dev. Biol. 217:266-77). Histidine triad (HIT) proteins share residues in distinctive dimeric, 10-stranded half-barrel structures that form two identical purine nucleotide-binding sites. Hint (histidine triad nucleotide-binding protein)-related proteins, found in all forms of life, and fragile histidine triad (Fhit)-related proteins, found in animals and fungi, represent the two main branches of the HIT superfamily. Fhit homologs bind and cleave diadenosine polyphosphates. Fhit-Ap(n)A complexes appear to function in a proapoptotic tumor suppression pathway in epithelial tissues (Brenner C. et al. (1999) J. Cell Physiol. 181:179-187). Most transcription factors contain characteristic DNA binding motifs, and variations on the above motifs and new motifs have been and are currently being characterized. (Faisst, S. and S. Meyer (1992) Nucleic Acids Res. 20:3-26.) Most transcription factors contain characteristic DNA binding motifs, and variations on the above motifs and new motifs have been and are currently being characterized (Paisst, S. and S. Meyer (1992) Nucl. Acids Res. 20:3-26). These include the forkhead motif, found in transcription factors involved in development and oncogenesis (Hacker, U. et al. (1995) EMBO J. 14:5306-5317). Foxj2 is a human forkhead transcriptional activator that binds DNA with a dual sequence specificity. Foxj2 expression is activated early in zygotic development (Granadino, B. et al. (2000) Mech. Dev. 97:157-160). Cold-shock proteins (Csp) are involved in a specific pattern of gene expression in response to abrupt shifts to lower temperatures. This pattern includes the induction of cold-shock proteins, synthesis of proteins involved in transcription and translation, and repression of heat-shock proteins. The major cold-shock protein, cold-shock protein A (CspA), has high sequence similarity with three other proteins—CspB, CspC, and CspD. The Csp proteins share sequence similarity with other prokaryotic proteins and with the ‘cold-shock domain’ of eukaryotic Y-box proteins (Jones, P. G. and Inouye, M. (1994) Mol. Microbiol. 11:811-818). Chromatin Associated Proteins In the nucleus, DNA is packaged into chromatin, the compact organization of which limits the accessibility of DNA to transcription factors and plays a key role in gene regulation. (Lewin, supra, pp. 409-410.) The compact structure of chromatin is determined and influenced by chromatin-associated proteins such as the histones, the high mobility group (HMG) proteins, and the chromodomain proteins. There are five classes of histones, H1, H2A, H2B, H3, and H4, all of which are highly basic, low molecular weight proteins. The fundamental unit of chromatin, the nucleosome, consists of 200 base pairs of DNA associated with two copies each of H2A, H2B, H3, and H4. H1 links adjacent nucleosomes. HMG proteins are low molecular weight, non-histone proteins that may play a role in unwinding DNA and stabilizing single-stranded DNA. Chromodomain proteins play a key role in the formation of highly compacted heterochromatin, which is transcriptionally silent. Diseases and Disorders Related to Gene Regulation Many neoplastic disorders in humans can be attributed to inappropriate gene expression. 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). The zinc finger-type transcriptional regulator WT1 is 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. 332:45-47). Chromosomal translocations may also produce chimeric loci that fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in inappropriate gene transcription, potentially contributing to malignancy. In Burkitt's lymphoma, for example, the transcription factor Myc is translocated to the immunoglobulin heavy chain locus, greatly enhancing Myc expression and resulting in rapid cell growth leading to leukemia (Latchman, D. S. (1996) N. Engl. J. Med. 334:28-33). In addition, the immune system responds to infection or trauma by activating a cascade of events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms. A complex and balanced program of gene activation and repression is involved in this process. However, hyperactivity of the immune system as a result of improper or insufficient regulation of gene expression may result in considerable tissue or organ damage. This damage is well-documented in immunological responses associated with arthritis, allergens, heart attack, stroke, and infections (Isselbacher, K. J. et al. Harrison's Principles of Internal Medicine, 13/e, McGraw Hill, Inc. and Teton Data Systems Software, 1996). The causative gene for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) was recently isolated and found to encode a protein with two PHD-type zinc finger motifs (Bjorses, P. et al. (1998) Hum. Mol. Genet. 7:1547-1553). Furthermore, the generation of multicellular organisms is based upon the induction and coordination of cell differentiation at the appropriate stages of development. Central to this process is differential gene expression, which confers the distinct identities of cells and tissues throughout the body. Failure to regulate gene expression during development could result in developmental disorders. Human developmental disorders caused by mutations in zinc finger-type transcriptional regulators include: urogenital developmental abnormalities associated with WT1; Greig cephalopolysyndactyly, Pallister-Hall syndrome, and postaxial polydactyly type A (GL13), and Townes-Brocks syndrome, characterized by anal, renal, limb, and ear abnormalities (SALL1) (Engelkemp, D. and V. van Heyningen (1996) Curr. Opin. Genet. Dev. 6:334-342; Kohlhase, J. et al. (1999) Am. J. Hum. Genet. 64:435-445). Human acute leukemias involve reciprocal chromosome translocations that fuse the ALL-1 gene located at chromosome region 11q23 to a series of partner genes positioned on a variety of human chromosomes. The fused genes encode chimeric proteins. The AF17 gene encodes a protein of 1093 amino acids, containing a leucine-zipper dimerization motif located 3′ of the fusion point and a cysteine-rich domain at the N terminus that shows homology to a domain within the protein Br140 (peregrin) (PrasadR. et al. (1994) Proc. Natl. Acad. Sci. USA 91:8107-8111). Synthesis of Nucleic Acids Polymerases DNA and RNA replication are critical processes for cell replication and function. DNA and RNA replication are mediated by the enzymes DNA and RNA polymerase, respectively, by a “templating” process in which the nucleotide sequence of a DNA or RNA strand is copied by complementary base-pairing into a complementary nucleic acid sequence of either DNA or RNA. However, there are fundamental differences between the two processes. DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the 3′-OH end of a polynucleotide strand (the primer strand) that is paired to a second (template) strand. The new DNA strand therefore grows in the 5′ to 3′ direction (Alberts, B. et al. (1994) The Molecular Biology of the Cell , Garland Publishing Inc., New York, N.Y., pp 251-254). The substrates for the polymerization reaction are the corresponding deoxynucleotide triphosphates which must base-pair with the correct nucleotide on the template strand in order to be recognized by the polymerase. Because DNA exists as a double-stranded helix, each of the two strands may serve as a template for the formation of a new complementary strand. Each of the two daughter cells of a dividing cell therefore inherits a new DNA double helix containing one old and one new strand. Thus, DNA is said to be replicated “semiconservatively” by DNA polymerase. In addition to the synthesis of new DNA, DNA polymerase is also involved in the repair of damaged DNA as discussed below under “Ligases.” In contrast to DNA polymerase, RNA polymerase uses a DNA template strand to “transcribe” DNA into RNA using ribonucleotide triphosphates as substrates. Like DNA polymerization, RNA polymerization proceeds in a 5′ to 3′ direction by addition of a ribonucleoside monophosphate to the 3′-OH end of a growing RNA chain. DNA transcription generates messenger RNAs (mRNA) that carry information for protein synthesis, as well as the transfer, ribosomal, and other RNAs that have structural or catalytic functions. In eukaryotes, three discrete RNA polymerases synthesize the three different types of RNA (Alberts, supra, pp. 367-368). RNA polymerase I makes the large ribosomal RNAs, RNA polymerase II makes the mRNAs that will be translated into proteins, and RNA polymerase m makes a variety of small, stable RNAs, including 5S ribosomal RNA and the transfer RNAs (tRNA). In all cases, RNA synthesis is initiated by binding of the RNA polymerase to a promoter region on the DNA and synthesis begins at a start site within the promoter. Synthesis is completed at a stop (termination) signal in the DNA whereupon both the polymerase and the completed RNA chain are released. Ligases DNA repair is the process by which accidental base changes, such as those produced by oxidative damage, hydrolytic attack, or uncontrolled methylation of DNA, are corrected before replication or transcription of the DNA can occur. Because of the efficiency of the DNA repair process, fewer than one in a thousand accidental base changes causes a mutation (Alberts, supra, pp. 245-249). The three steps common to most types of DNA repair are (1) excision of the damaged or altered base or nucleotide by DNA nucleases, (2) insertion of the correct nucleotide in the gap left by the excised nucleotide by DNA polymerase using the complementary strand as the template and, (3) sealing the break left between the inserted nucleotide(s) and the existing DNA strand by DNA ligase. In the last reaction, DNA ligase uses the energy from ATP hydrolysis to activate the 5′ end of the broken phosphodiester bond before forming the new bond with the 3′-OH of the DNA strand. In Bloom's syndrome, an inherited human disease, individuals are partially deficient in DNA ligation and consequently have an increased incidence of cancer (Alberts, supra p. 247). Nucleases Nucleases comprise enzymes that hydrolyze both DNA (DNase) and RNA (Rnase). They serve different purposes in nucleic acid metabolism. Nucleases hydrolyze the phosphodiester bonds between adjacent nucleotides either at internal positions (endonucleases) or at the terminal 3′ or 5′ nucleotide positions (exonucleases). A DNA exonuclease activity in DNA polymerase, for example, serves to remove improperly paired nucleotides attached to the 3′-OH end of the growing DNA strand by the polymerase and thereby serves a “proofreading” function. As mentioned above, DNA endonuclease activity is involved in the excision step of the DNA repair process. RNases also serve a variety of functions. For example, RNase P is a ribonucleoprotein enzyme which cleaves the 5′ end of pre-tRNAs as part of their maturation process. RNase H digests the RNA strand of an RNA/DNA hybrid. Such hybrids occur in cells invaded by retroviruses, and RNase H is an important enzyme in the retroviral replication cycle. Pancreatic RNase secreted by the pancreas into the intestine hydrolyzes RNA present in ingested foods. RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases (Schein, C. H. (1997) Nat. Biotechnol. 15:529-536). Regulation of RNase activity is being investigated as a means to control tumor angiogenesis, allergic reactions, viral infection and replication, and fungal infections. Modification of Nucleic Acids Methylases Methylation of specific nucleotides occurs in both DNA and RNA, and serves different functions in the two macromolecules. Methylation of cytosine residues to form 5-methyl cytosine in DNA occurs specifically in CG sequences which are base-paired with one another in the DNA double-helix. The pattern of methylation is passed from generation to generation during DNA replication by an enzyme called “maintenance methylase” that acts preferentially on those CG sequences that are base-paired with a CG sequence that is already methylated. Such methylation appears to distinguish active from inactive genes by preventing the binding of regulatory proteins that “turn on” the gene, but permitting the binding of proteins that inactivate the gene (Alberts, supra pp. 448-451). In RNA metabolism, “tRNA methylase” produces one of several nucleotide modifications in tRNA that affect the conformation and base-pairing of the molecule and facilitate the recognition of the appropriate mRNA codons by specific tRNAs. The primary methylation pattern is the dimethylation of guanine residues to form N,N-dimethyl guanine. Helicases and Single-Stranded Binding Proteins Helicases are enzymes that destabilize and unwind double helix structures in both DNA and RNA. Since DNA replication occurs more or less simultaneously on both strands, the two strands must first separate to generate a replication “fork” for DNA polymerase to act on. Two types of replication proteins contribute to this process, DNA helicases and single-stranded binding proteins. DNA helicases hydrolyze ATP and use the energy of hydrolysis to separate the DNA strands. Single-stranded binding proteins (SSBs) then bind to the exposed DNA strands, without covering the bases, thereby temporarily stabilizing them for templating by the DNA polymerase (Alberts, supra, pp. 255-256). RNA helicases also alter and regulate RNA conformation and secondary structure. Like the DNA helicases, RNA helicases utilize energy derived from ATP hydrolysis to destabilize and unwind RNA duplexes. The most well-characterized and ubiquitous family of RNA helicases is the DEAD-box family, so named for the conserved B-type ATP-binding motif which is diagnostic of proteins in this family. Over 40 DEAD-box helicases have been identified in organisms as diverse as bacteria, insects, yeast, amphibians, mammals, and plants. DEAD-box helicases function in diverse processes such as translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability. Examples of these RNA helicases include yeast Drs1 protein, which is involved in ribosomal RNA processing; yeast TIP1 and TIP2 and mammalian eIF4A; which are essential to the initiation of RNA translation; and human p68 antigen, which regulates cell growth and division (Ripmaster, T. L. et al. (1992) Proc. Natl. Acad. Sci. USA 89:11131-11135; Chang, T.-H. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1571-1575). These RNA helicases demonstrate strong sequence homology over a stretch of some 420 amino acids. Included among these conserved sequences are the consensus sequence for the A motif of an ATP binding protein; the “DEAD box” sequence, associated with ATPase activity; the sequence SAT, associated with the actual helicase unwinding region; and an octapeptide consensus sequence, required for RNA binding and ATP hydrolysis (Pause, A. et al. (1993) Mol. Cell Biol. 13:6789-6798). Differences outside of these conserved regions are believed to reflect differences in the functional roles of individual proteins (Chang et al., supra). Some DEAD-box helicases play tissue- and stage-specific roles in spermatogenesis and embryogenesis. Overexpression of the DEAD-box 1 protein (DDX1) may play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors (Godbout, R. et al. (1998) J. Biol. Chem. 273:21161-21168). These observations suggest that DDX1 may promote or enhance tumor progression by altering the normal secondary structure and expression levels of RNA in cancer cells. Other DEAD-box helicases have been implicated either directly or indirectly in tumorigenesis. (Discussed in Godbout et al., supra.) For example, murine p68 is mutated in ultraviolet light-induced tumors, and human DDX6 is located at a chromosomal breakpoint associated with B-cell lymphoma. Similarly, a chimeric protein comprised of DDX10 and NUP98, a nucleoporin protein, may be involved in the pathogenesis of certain myeloid malignancies. Topoisomerases Besides the need to separate DNA strands prior to replication, the two strands must be “unwound” from one another prior to their separation by DNA helicases. This function is performed by proteins known as DNA topoisomerases. DNA topoisomerase effectively acts as a reversible nuclease that hydrolyzes a phosphodiesterase bond in a DNA strand, permits the two strands to rotate freely about one another to remove the strain of the helix, and then rejoins the original phosphodiester bond between the two strands. Topoisomerases are essential enzymes responsible for the topological rearrangement of DNA brought about by transcription, replication, chromatin formation, recombination, and chromosome segregation. Superhelical coils are introduced into DNA by the passage of processive enzymes such as RNA polymerase, or by the separation of DNA strands by a helicase prior to replication. Knotting and concatenation can occur in the process of DNA synthesis, storage, and repair. All topoisomerases work by breaking a phosphodiester bond in the ribose-phosphate backbone of DNA. A catalytic tyrosine residue on the enzyme makes a nucleophilic attack on the scissile phosphodiester bond, resulting in a reaction intermediate in which a covalent bond is formed between the enzyme and one end of the broken strand. A tyrosine-DNA phosphodiesterase functions in DNA repair by hydrolyzing this bond in occasional dead-end topoisomerase I-DNA intermediates (Pouliot, J. J. et al. (1999) Science 286:552-555). Two types of DNA topoisomerase exist, types I and II. Type I topoisomerases work as monomers, making a break in a single strand of DNA while type II topoisomerases, working as homodimers, cleave both strands. DNA Topoisomerase I causes a single-strand break in a DNA helix to allow the rotation of the two strands of the helix about the remaining phosphodiester bond in the opposite strand. DNA topoisomerase II causes a transient break in both strands of a DNA helix where two double helices cross over one another. This type of topoisomerase can efficiently separate two interlocked DNA circles (Alberts, supra, pp. 260-262). Type II topoisomerases are largely confined to proliferating cells in eukaryotes, such as cancer cells. For this reason they are targets for anticancer drugs. Topoisomerase II has been implicated in multi-drug resistance (MDR) as it appears to aid in the repair of DNA damage inflicted by DNA binding agents such as doxorubicin and vincristine (DNA topoisomerases are reviewed in Wang, J. C. (1996) Annu. Rev. Biochem. 65:635-692.). The topoisomerase I family includes topoisomerases I and III (topo I and topo III). The crystal structure of human topoisomerase I suggests that rotation about the intact DNA strand is partially controlled by the enzyme. In this “controlled rotation” model, protein-DNA interactions limit the rotation, which is driven by torsional strain in the DNA (Stewart, L. et al. (1998) Science 379:1534-1541). Structurally, topo I can be recognized by its catalytic tyrosine residue and a number of other conserved residues in the active site region. Topo I is thought to function during transcription. Two topo III are known in humans, and they are homologous to prokaryotic topoisomerase I, with a conserved tyrosine and active site signature specific to this family. Topo III has been suggested to play a role in meiotic recombination. A mouse topo III is highly expressed in testis tissue and its expression increases with the increase in the number of cells in pachytene (Seki, T. et al. (1998) J. Biol. Chem. 273:28553-28556). The topoisomerase II family includes two isozymes (IIa and IIb) encoded by different genes. Topo II cleaves double stranded DNA in a reproducible, nonrandom fashion, preferentially in an AT rich region, but the basis of cleavage site selectivity is not known. Structurally, topo II is made up of four domains, the first two of which are structurally similar and probably distantly homologous to similar domains in eukaryotic topo I. The second domain bears the catalytic tyrosine, as well as a highly conserved pentapeptide. The IIa isoform appears to be responsible for unlinking DNA during chromosome segregation. Cell lines expressing IIa but not IIb suggest that IIb is dispensable in cellular processes; however, IIb knockout mice died perinatally due to a failure in neural development. That the major abnormalities occurred in predominantly late developmental events (neurogenesis) suggests that IIb is needed not at mitosis, but rather during DNA repair (Yang, X. et al. (2000) Science 287:131-134). Topoisomerases have been implicated in a number of disease states, and topoisomerase poisons have proven to be effective anti-tumor drugs for some human malignancies. Topo I is mislocalized in Fanconi's anemia, and may be involved in the chromosomal breakage seen in this disorder (Wunder, E. (1984) Hum. Genet. 68:276-281). Overexpression of a truncated topo III in ataxia-telangiectasia (A-T) cells partially suppresses the A-T phenotype, probably through a dominant negative mechanism. This suggests that topo m is deregulated in A-T (Fritz, E. et al. (1997) Proc. Natl. Acad. Sci. USA 94:4538-4542). Topo III also interacts with the Bloom's Syndrome gene product, and has been suggested to have a role as a tumor suppressor (Wu, L. et al. (2000) J. Biol. Chem. 275:9636-9644). Aberrant topo II activity is often associated with cancer or increased cancer risk. Greatly lowered topo II activity has been found in some, but not all A-T cell lines (Mohamed, R. et al. (1987) Biochem. Biophys. Res. Commun. 149:233-238). On the other hand, topo H can break DNA in the region of the A-T gene (ATM), which controls all DNA damage-responsive cell cycle checkpoints (Kaufmann, W. K. (1998) Proc. Soc. Exp. Biol. Med. 217:327-334). The ability of topoisomerases to break DNA has been used as the basis of antitumor drugs. Topoisomerase poisons act by increasing the number of dead-end covalent DNA-enzyme complexes in the cell, ultimately triggering cell death pathways (Fortune, J. M. and N. Osheroff (2000) Prog. Nucleic Acid Res. Mol. Biol. 64:221-253; Guichard, S. M. and M. K. Danks (1999) Curr. Opin. Oncol. 11:482-489). Antibodies against topo I are found in the serum of systemic sclerosis patients, and the levels of the antibody may be used as a marker of pulmonary involvement in the disease (Diot, E. et al. (1999) Chest 116:715-720). Finally, the DNA binding region of human topo I has been used as a DNA delivery vehicle for gene therapy (Chen, T. Y. et al. (2000) Appl. Microbiol. Biotechnol. 53:558-567). Recombinases Genetic recombination is the process of rearranging DNA sequences within an organism's genome to provide genetic variation for the organism in response to changes in the environment. DNA recombination allows variation in the particular combination of genes present in an individual's genome, as well as the tiring and level of expression of these genes. (See Alberts, supra pp. 263-273.) Two broad classes of genetic recombination are commonly recognized, general recombination and site-specific recombination. General recombination involves genetic exchange between any homologous pair of DNA sequences usually located on two copies of the same chromosome. The process is aided by enzymes, recombinases, that “nick” one strand of a DNA duplex more or less randomly and permit exchange with a complementary strand on another duplex. The process does not normally change the arrangement of genes in a chromosome. In site-specific recombination, the recombinase recognizes specific nucleotide sequences present in one or both of the recombining molecules. Base-pairing is not involved in this form of recombination and therefore it does not require DNA homology between the recombining molecules. Unlike general recombination, this form of recombination can alter the relative positions of nucleotide sequences in chromosomes. RNA Metabolism Ribonucleic acid (RNA) is a linear single-stranded polymer of four nucleotides, ATP, CTP, UTP, and GTP. In most organisms, RNA is transcribed as a copy of deoxyribonucleic acid (DNA), the genetic material of the organism. In retroviruses RNA rather than DNA serves as the genetic material. RNA copies of the genetic material encode proteins or serve various structural, catalytic, or regulatory roles in organisms. RNA is classified according to its cellular localization and function. Messenger RNAs (mRNAs) encode polypeptides. Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins, into ribosomes, which are cytoplasmic particles that translate mRNA into polypeptides. Transfer RNAs (tRNAs) are cytosolic adaptor molecules that function in mRNA translation by recognizing both an mRNA codon and the amino acid that matches that codon. Heterogeneous nuclear RNAs (hnRNAs) include mRNA precursors and other nuclear RNAs of various sizes. Small nuclear RNAs (snRNAs) are a part of the nuclear spliceosome complex that removes intervening, non-coding sequences (introns) and rejoins exons in pre-mRNAs. Proteins are associated with RNA during its transcription from DNA, RNA processing, and translation of mRNA into protein. Proteins are also associated with RNA as it is used for structural, catalytic, and regulatory purposes. RNA Processing Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins, into ribosomes, which are cytoplasmic particles that translate messenger RNA (mRNA) into polypeptides. The eukaryotic ribosome is composed of a 60S (large) subunit and a 40S (small) subunit, which together form the 80S ribosome. In addition to the 18S, 28S, 5S, and 5.8S rRNAs, ribosomes contain from 50 to over 80 different ribosomal proteins, depending on the organism. Ribosomal proteins are classified according to which subunit they belong (i.e., L, if associated with the large 60S large subunit or S if associated with the small 40S subunit). E. coli ribosomes have been the most thoroughly studied and contain 50 proteins, many of which are conserved in all life forms. The structures of nine ribosomal proteins have been solved to less than 3.0 D resolution (i.e., S5, S6, S17, L1, L6, L9, L12, L14, L30), revealing common motifs, such as b-a-b protein folds in addition to acidic and basic RNA-binding motifs positioned between b-strands. Most ribosomal proteins are believed to contact rRNA directly (reviewed in Liljas, A. and M. Garber (1995) Curr. Opin. Struct. Biol. 5:721-727; see also Woodson, S. A. and N. B. Leontis (1998) Curr. Opin. Struct. Biol. 8:294-300; Ramakrishnan, V. and S. W. White (1998) Trends Biochem. Sci. 23:208-212). Ribosomal proteins may undergo post-translational modifications or interact with other ribosome-associated proteins to regulate translation. For example, the highly homologous 40S ribosomal protein S6 kinases (S6K1 and S6K2) play a key role in the regulation of cell growth by controlling the biosynthesis of translational components which make up the protein synthetic apparatus (including the ribosomal proteins). In the case of S6K1, at least eight phosphorylation sites are believed to mediate kinase activation in a hierarchical fashion (Dufner and Thomas (1999) Exp. Cell. Res. 253:100-109). Some of the ribosomal proteins, including L1, also function as translational repressors by binding to polycistronic mRNAs encoding ribosomal proteins (reviewed in Liljas and Garber, supra). Recent evidence suggests that a number of ribosomal proteins have secondary functions independent of their involvement in protein biosynthesis. These proteins function as regulators of cell proliferation and, in some instances, as inducers of cell death. For example, the expression of human ribosomal protein L13a has been shown to induce apoptosis by arresting cell growth in the G2/M phase of the cell cycle. Inhibition of expression of L13a induces apoptosis in target cells, which suggests that this protein is necessary, in the appropriate amount, for cell survival. Similar results have been obtained in yeast where inactivation of yeast homologues of L13a, rp22 and rp23, results in severe growth retardation and death. A closely related ribosomal protein, L7, arrests cells in G1 and also induces apoptosis. Thus, a subset of ribosomal proteins may function as cell cycle checkpoints and compose a new family of cell proliferation regulators. Mapping of individual ribosomal proteins on the surface of intact ribosomes is accomplished using 3D immunocryoelectron microscopy, whereby antibodies raised against specific ribosomal proteins are visualized. Progress has been made toward the mapping of L1, L7, and L12 while the structure of the intact ribosome has been solved to only 20-25D resolution and inconsistencies exist among different crude structures (Frank, J. (1997) Curr. Opin. Struct. Biol. 7:266-272). Three distinct sites have been identified on the ribosome. The aminoacyl-tRNA acceptor site (A site) receives charged tRNAs (with the exception of the initiator-tRNA). The peptidyl-tRNA site (P site) binds the nascent polypeptide as the amino acid from the A site is added to the elongating chain. Deacylated tRNAs bind in the exit site (E site) prior to their release from the ribosome. The structure of the ribosome is reviewed in Stryer, L. (1995) Biochemistry , W. H. Freeman and Company, New York N.Y., pp. 888-9081; Lodish, supra, pp. 119-138; and Lewin, B (1997) Genes VI , Oxford University Press, Inc. New York, N.Y.). Various proteins are necessary for processing of transcribed RNAs in the nucleus. Pre-mRNA processing steps include capping at the 5′ end with methylguanosine, polyadenylating the 3′ end, and splicing to remove introns. The primary RNA transcript from DNA is a faithful copy of the gene containing both exon and intron sequences, and the latter sequences must be cut out of the RNA transcript to produce a mRNA that codes for a protein. This “splicing” of the mRNA sequence takes place in the nucleus with the aid of a large, multicomponent ribonucleoprotein complex known as a spliceosome. The spliceosomal complex is comprised of five small nuclear ribonucleoprotein particles (snRNPs) designated U1, U2, U4, U5, and U6. Each snRNP contains a single species of snRNA and about ten proteins. The RNA components of some snRNPs recognize and base-pair with intron consensus sequences. The protein components mediate spliceosome assembly and the splicing reaction. Autoantibodies to snRNP proteins are found in the blood of patients with systemic lupus erythematosus (Stryer, supra, p. 863). Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified that have roles in splicing, exporting of the mature RNAs to the cytoplasm, and mRNA translation (Biamonti, G. et al. (1998) Clin. Exp. Rheumatol. 16:317-326). Some examples of hnRNPs include the yeast proteins Hrp1p, involved in cleavage and polyadenylation at the 3′ end of the RNA; Cbp80p, involved in capping the 5′ end of the RNA; and Np13p, a homolog of mammalian hnRNP A1, involved in export of mRNA from the nucleus (Shen, B. C. et al. (1998) Genes Dev. 12:679-691). HnRNPs have been shown to be important targets of the autoimmune response in rheumatic diseases (Biamonti, supra). Many snRNP and hnRNP proteins are characterized by an RNA recognition motif (RRM). (Reviewed in Birney, E. et al. (1993) Nucleic Acids Res. 21:5803-5816.) The RRM is about 80 amino acids in length and forms four b-strands and two a-helices arranged in an a/b sandwich. The RRM contains a core RNP-1 octapeptide motif along with surrounding conserved sequences. In addition to snRNP proteins, examples of RNA-binding proteins which contain the above motifs include heteronuclear ribonucleoproteins which stabilize nascent RNA and factors which regulate alternative splicing. Alternative splicing factors include developmentally regulated proteins, specific examples of which have been identified in lower eukaryotes such as Drosophila melanogaster and Caenorhabditis elegans . These proteins play key roles in developmental processes such as pattern formation and sex determination, respectively. (See, for example, Hodgkin, J. et al. (1994) Development 120:3681-3689.) The 3′ ends of most eukaryote mRNAs are also posttranscriptionally modified by polyadenylation. Polyadenylation proceeds through two enzymatically distinct steps: (i) the endonucleolytic cleavage of nascent mRNAs at cis-acting polyadenylation signals in the 3′-untranslated (non-coding) region and (ii) the addition of a poly(A) tract to the 5′ mRNA fragment. The presence of cis-acting RNA sequences is necessary for both steps. These sequences include 5′-AAUAAA-3′ located 10-30 nucleotides upstream of the cleavage site and a less well-conserved GU- or U-rich sequence element located 10-30 nucleotides downstream of the cleavage site. Cleavage stimulation factor (CstF), cleavage factor I (CF I), and cleavage factor II (CF II) are involved in the cleavage reaction while cleavage and polyadenylation specificity factor (CPSF) and poly(A) polymerase (PAP) are necessary for both cleavage and polyadenylation. An additional enzyme, poly(A)-binding protein I (PAB II), promotes poly(A) tract elongation (Rüegsegger, U. et al. (1996) J. Biol. Chem. 271:6107-6113; and references within). YT521-B is a nuclear protein that was identified by using a yeast two-hybrid screen for proteins that interact with known mRNA splicing factors (Hartmann, A. M. et al. (1999) Mol. Biol. Cell 10:3909-3926). The protein contains four nuclear localization signals, an N-terminal glutamic acid-rich region, a glutamic acid/arginine-rich region, and a C-terminal proline-rich region. YT521 associates with the nuclear transcriptosomal component scaffold attachment factor B and with the Src kinase substrate, Sam68. Phosphorylation of Sam68 by Src family kinase p59 fyn reduces the association of Sam68 with YT521-B. Both YT521 and Sam68 may participate in a signal transduction pathway that controls alternative splice site selection. Translation Correct translation of the genetic code depends upon each amino acid forming a linkage with the appropriate transfer RNA (tRNA). The aminoacyl-tRNA synthetases (aaRSs) are essential proteins found in all living organisms. The aaRSs are responsible for the activation and correct attachment of an amino acid with its cognate tRNA, as the first step in protein biosynthesis. Prokaryotic organisms have at least twenty different types of aaRSs, one for each different amino acid, while eukaryotes usually have two aaRSs, a cytosolic form and a mitochondrial form, for each different amino acid. The 20 aaRS enzymes can be divided into two structural classes. Class I enzymes add amino acids to the 2′ hydroxyl at the 3′ end of tRNAs while Class II enzymes add amino acids to the 3′ hydroxyl at the 3′ end of tRNAs. Each class is characterized by a distinctive topology of the catalytic domain. Class I enzymes contain a catalytic domain based on the nucleotide-binding Rossman ‘fold’. In particular, a consensus tetrapeptide motif is highly conserved (Prosite Document PDOC00161, Aminoacyl-transfer RNA synthetases class-I signature). Class I enzymes are specific for arginine, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, tyrosine, tryptophan, and valine. Class II enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel β-sheet domain, as well as N- and C-terminal regulatory domains. Class II enzymes are separated into two groups based on the heterodimeric or homodimeric structure of the enzyme; the latter group is further subdivided by the structure of the N- and C-terminal regulatory domains (Hartlein, M. and S. Cusack (1995) J. Mol. Evol. 40:519-530). Class II enzymes are specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine, and threonine. Certain aaRSs also have editing functions. IleRS, for example, can misactivate valine to form Val-tRNA Ile , but this product is cleared by a hydrolytic activity that destroys the mischarged product. This editing activity is located within a second catalytic site found in the connective polypeptide 1 region (CP1), a long insertion sequence within the Rossman fold domain of Class I enzymes (Schimmel, P. et al. (1998) FASEB J. 12:1599-1609). AaRSs also play a role in tRNA processing. It has been shown that mature tRNAs are charged with their respective amino acids in the nucleus before export to the cytoplasm, and charging may serve as a quality control mechanism to insure the tRNAs are functional (Martinis, S. A. et al. (1999) EMBO J. 18:4591-4596). Under optimal conditions, polypeptide synthesis proceeds at a rate of approximately 40 amino acid residues per second. The rate of misincorporation during translation in on the order of 10 −4 and is primarily the result of aminoacyl-t-RNAs being charged with the incorrect amino acid. Incorrectly charged tRNA are toxic to cells as they result in the incorporation of incorrect amino acid residues into an elongating polypeptide. The rate of translation is presumed to be a compromise between the optimal rate of elongation and the need for translational fidelity. Mathematical calculations predict that 10 −4 is indeed the maximum acceptable error rate for protein synthesis in a biological system (reviewed in Stryer, supra; and Watson, J. et al. (1987) The Benjamin/Cummings Publishing Co., Inc. Menlo Park, Calif.). A particularly error prone aminoacyl-tRNA charging event is the charging of tRNA Gln with Gln. A mechanism exits for the correction of this mischarging event which likely has its origins in evolution. Gln was among the last of the 20 naturally occurring amino acids used in polypeptide synthesis to appear in nature. Gram positive eubacteria, cyanobacteria, Archeae, and eukaryotic organelles possess a noncanonical pathway for the synthesis of Gln-tRNA Gln based on the transformation of Glu-tRNA Gln (synthesized by Glu-tRNA synthetase, GluRS) using the enzyme Glu-tRNA Gln amidotransferase (Glu-AdT). The reactions involved in the transamidation pathway are as follows (Curnow, A. W. et al. (1997) Nucleic Acids Symposium 36:24): |
<SOH> SUMMARY OF THE INVENTION <EOH>Various embodiments of the invention provide purified polypeptides, nucleic acid-associated proteins, referred to collectively as “NAAP” and individually as “NAAP-1,” “NAAP-2,” “NAAP-3,” “NAAP-4,” “NAAP-5,” “NAAP-6,” “NAAP-7,” “NAAP-8,” “NAAP-9, ” “NAAP-10” “NAAP-11,” “NAAP-12,” “NAAP-13,” “NAAP-14, ” “NAAP-15,” “NAAP-16,” “NAAP-17,” “NAAP-18,” “NAAP-19,” “NAAP-20,” “NAAP-21,” “NAAP-22,” “NAAP-23,” “NAAP-24,” “NAAP-25,” “NAAP-26,” “NAAP-27,” “NAAP-28,” “NAAP-29,” “NAAP-30,” “NAAP-31,” “NAAP-32,” “NAAP-33,” “NAAP-34,” and “NAAP-35,” 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 nucleic acid-associated proteins 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 nucleic acid-associated proteins 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-35. 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-35. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:36-70. 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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:36-70, 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:36-70, 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:36-70, 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:36-70, 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:36-70, 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:36-70, 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, 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:1-35. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional NAAP, 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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 NAAP, 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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 NAAP, 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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:36-70, 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:36-70, 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:36-70, 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:36-70, 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:36-70, 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. detailed-description description="Detailed Description" end="lead"? |
Sensor and ink-jet print-head |
The invention relates to a sensor and ink-jet print-head assembly comprised in a housing for a hand-held and hand-operated printing device controlled by a processor, and a method therefore. It provides a control for navigation with coordinate systems and angles on a print medium that preferably is bigger than the assembly. |
1. A sensor and ink-jet print-head (2) assembly comprised in a housing (1) for a hand-held and hand-operated printing device controlled by a processor (4), comprising: two position sensor means (S0, S1) at least one sensor means being related to a first coordinate system, having one axis in a relation to said print-head array, and one axis (62) in a direction through both sensor means (S0, S1); a print-head array (60) attached in a fixed position to said sensor means (S0, S1); input means (6) on said housing (1) connected to said processor (4) for input of control commands; determining means for reference coordinates in a second coordinate system provided in relation to a print medium, said reference coordinates being established by a control command through said input means (6) with the thus read sensor means signals; integrating means for keeping track of the assemblies position related to said reference coordinates in said second coordinate system by integrating displacement of sensor means (S0, S1) position in the first coordinate system; computing means for transforming the sensor means coordinates to coordinates in the second coordinate system, whereby the assemblies position on the print medium is determined in relation to the reference coordinates. 2. An assembly according to claim 1, wherein a look-up table comprises normalized sensor steps with a predetermined resolution between sensor steps, one of said sensor steps determining a minimum movement of the assembly. 3. An assembly according to claim 1, wherein a position is expressed through the coordinates of the sensor means and the angle between the prior position and the current position of the sensor means. 4. An assembly according to claim 1, wherein said transforming of the sensor means coordinates is derived through the position of the sensor means related to the first coordinate system and the angle of the print-head array in relation to the second coordinate system. 5. An assembly according to claim 1, wherein an angular change is computed as the difference of the sensor means movement in the y-direction of the first coordinate system multiplied with a constant which is determined in relation to the distance between the two sensor means. 6. An assembly according to claim 1, wherein the position of the sensor means in the second coordinate system at an angle ‘alpha’ is calculated as: deltaX=S0DiffX*cos(alpha)−S0DiffY*sin(alpha); deltaY=S0DiffX*sin(alpha)+S0DiffY*cos(alpha); and where S0DiffX and S0DiffY are the movements of the sensor means in x- and y-directions respectively, in the first coordinate system. 7. An assembly according to claim 1, wherein the print-head nozzle position is computed from the knowledge of the position of one sensor means and the tilt angle of the assembly, by calculating the position of the first and last nozzle in said array. 8. An assembly according to claim 1, wherein the positions of the print head nozzles are calculated as follows: PNfirstX=S0x+Ho*cosine(alpha)−Vo*sine(alpha); PNfirstY=S0y+Ho*sine(alpha)+Vo*cosine(alpha); PNlastX=S0x+Ho*cosine(alpha)−Ve*sine(alpha); and PNlastY=S0y+Ho*sine(alpha)+Ve*cosine(alpha). 9. An assembly according to claim 7, wherein remaining nozzle positions are computed by starting from the first nozzle positions and adding up the difference in x- and y-directions between the nozzles, whereby the x- and y-distance between the first and last nozzle is divided by the number of nozzles. 10. An assembly according to claim 7, wherein remaining nozzle positions are calculated as follows: PN(n)X=PNfirstX+n*deltaX PN(n)Y=PNlast+n*deltaY where deltaX=PNlastX−PNfirstY deltaY=PNlastY−PnfirstY 11. An assembly according to claim 1, wherein its width is smaller then the width of the print medium. 12. An assembly according to claim 1, wherein a positioning means is provided to position the assembly in a correct starting position in relation to the print medium. 13. An assembly according to claim 1, wherein a, not visible for a human eye, pattern provided by injected ink-jet drops in even intervals is used as reference points to adjust for possible sensor means position dislocations. 14. A method for a sensor and ink-jet print-head (2) assembly comprised in a housing (1) for a hand-held and hand-operated printing device controlled by a processor (4), comprising the steps of: providing two position sensor means (S0, S1), whereby at least one sensor means being related to a first coordinate system, having one axis in relation to said print-head array, and one axis (62) in a direction through both sensor means (S0, S1); providing a print-head array (60) attached in a fixed position to said sensor means (S0, S1); providing input means (6) on said housing (1) connected to said processor (4) for input of control commands; providing determining means for reference coordinates in a second coordinate system provided in relation to a print medium, said reference coordinates being established by a control command through said input means (6) with the thus read sensor means (S0, S1) signals; providing integrating means for keeping track of the assemblies position related to said reference coordinates in said second coordinate system by integrating displacement of sensor means (S0, S1) position in the first coordinate system; providing computing means for transforming the sensor means (S0, S1) coordinates to coordinates in the second coordinate system, whereby the assemblies position on the print medium is determined in relation to the reference coordinates. 15. A method according to claim 14, wherein a look-up table comprises normalized sensor steps with a predetermined resolution between sensor steps, one of said sensor steps determining a minimum movement of the assembly. 16. A method according to claim 14, wherein a position is expressed through the coordinates of the sensor means and the angle between the prior position and the current position of the sensor means. 17. A method according to claim 14, wherein said transforming of the sensor means coordinates is derived through the position of the sensor means related to the first coordinate system and the angle of the print-head array in relation to the second coordinate system. 18. A method according to claim 14, wherein an angular change is computed as the difference of the sensor means movement in the y-direction of the first coordinate system multiplied with a constant which is determined in relation to the distance between the two sensor means. 19. A method according to claim 14, wherein the position of the sensor means in the second coordinate system at an angle ‘alpha’ is calculated as: deltaX=S0DiffX*cos(alpha)−S0DiffY*sin(alpha); deltaY=S0DiffX*sin(alpha)+S0DiffY*cos(alpha); and where S0DiffX and S0DiffY are the movements of the sensor means in x- and y-directions respectively, in the first coordinate system. 20. A method according to claim 14, wherein the print-head nozzle position is computed from the knowledge of the position of one sensor means and the tilt angle of the assembly, by calculating the position of the first and last nozzle in said array. 21. A method according to claim 14, wherein the positions of the print head nozzles are calculated as follows: PNfirstX=S0x+Ho*cosine(alpha)−Vo*sine(alpha); PNfirstY=S0y+Ho*sine(alpha)+Vo*cosine(alpha); PNlastX=S0x+Ho*cosine(alpha)−Ve*sine(alpha); and PNlastY=S0y+Ho*sine(alpha)+Ve*cosine(alpha). 22. A method according to claim 20, wherein remaining nozzle positions are computed by starting from the first nozzle positions and adding up the difference in x- and y-directions between the nozzles, whereby the x- and y-distance between the first and last nozzle is divided by the number of nozzles. 23. A method according to claim 22, wherein remaining nozzle positions are calculated as follows: PN(n)X=PNfirstX+n*deltaX PN(n)Y=PNlast+n*deltaY where deltaX=PNlastX−PNfirstY deltaY=PNlastY−PnfirstY 24. A method according to claim 14, wherein its width is smaller then the width of the print medium. 25. A method according to claim 14, wherein a positioning means is provided to position the assembly in a correct starting position in relation to the print medium. 26. A method according to claim 14, wherein a, not visible for a human eye, pattern provided by injected ink-jet drops in even intervals is used as reference points to adjust for possible sensor means position dislocations. |
<SOH> BACKGROUND ART <EOH>Hand-held and hand-operated printing devices with an ink-jet print-head are known through various documents. U.S. Pat. No. 5,927,872 by Yamada discloses a system and a method of printing an image represented by a frame of image data utilizing a hand-held printer having optical sensor means for tracking positions of the hand-held printer relative to the surface of a print medium during a printing process. It is monitored in real time using navigation information generated by the optical sensor. Each optical sensor comprises an array of opto-electronic elements to capture images of the surface of a print medium at fixed time intervals. Preferably, the optical sensor means can detect slight pattern variations on the print medium, such as paper fibers or illumination pattern formed by highly reflective surface features and shadowed areas between raised surface features.These features can then be used as references for determining the position and the relative movement of the hand-held printer. During the printing process, the printed portions of the image can also be used as reference positions by the hand-held printer. In the preferred embodiment, the hand-held printer contains a navigation processor and a printer driver. Using the printer driver, the navigation processor drives the hand-held printer to print segments of the image onto a print medium as the hand-held printer travels across the print medium during a printing process. Each segment of the image is printed onto a particular location on the print medium to form a composite of the image. In the U.S. Pat. No. 6,233,368 B1 by Badyal et al it is taught a CMOS digital integrated circuit (IC) chip on which an image is captured, digitized, and then processed on-chip in substantially the digital domain. A preferred embodiment comprises imaging circuitry including a photo cell array for capturing an image and generating a representative analog signal, conversion circuitry including an n-bit successive approximation register (SAR) analog-to-digital converter for converting the analog signal to a corresponding digital signal, filter circuitry including a spatial filter for edge and contrast enhancement of the corresponding image, compression circuitry for reducing the digital signal storage needs, correlation circuitry for processing the digital signal to generate a result surface on which a minima resides representing a best fit image displacement between the captured image and previous images, interpolation circuitry for mapping the result surface into x- and y-coordinates, and an interface with a device using the chip, such as a hand-held scanner. The filter circuitry, the compression circuitry, the correlation circuitry and the interpolation circuitry are all embodied in an on-chip digital signal processor (DSP). The DSP embodiment allows precise algorithmic processing of the digitized signal with almost infinite hold time, depending on storage capability. The corresponding mathematical computations are thus no longer subject to the vagaries of CMOS chip structure processing analog signals. Parameters may also be programmed into the DSP's software making the chip tunable, as well as flexible and adaptable for different applications. U.S. Pat. No. 5,644,139 by Allen et al discloses a scanning device and a method for forming a scanned electronic image including the use of navigation information that is acquired along with image data, and then rectifying the image data based upon the navigation and image information. The navigation information is obtained in frames. The differences between consecutive frames are detected and accumulated, and this accumulated displacement value is representative of a position of the scanning device relative to a reference. The image data is then positioned-tagged using the position data obtained from the accumulated displacement value. To avoid the accumulation of errors, the accumulated displacement value obtained from consecutive frames is updated by comparing a current frame with a much earlier frame stored in memory and using the resulting difference as the displacement from the earlier frame. These larger displacement steps are then accumulated to determine the relative position of the scanning device. The above documents do only teach how to determine the position in a conceptual generation of navigation information. In this context the U.S. Pat. No. 5,927,872 by Yamada uses the navigation information for a hand-held scanner disclosed in U.S. Pat. No. 5,644,139 by Allen et al. The invention according to Allen et al teaches navigation through comparison of pixels on a frame basis. By analyzing the state of the art through the above documents a need of providing a navigation control through a coordinate system emerges, which does not need to compare prior position information with current position information for a hand-held printer. |
<SOH> SUMMARY OF THE DISCLOSED INVENTION <EOH>The present invention relates to a new sensor and an ink-jet print-head assembly for a hand-held and hand-operated printing on a print medium controlled by a processor and a method therefore. One aim of the present invention is to provide a new navigation control for print-outs accomplished by the assembly. Hence, the present invention sets forth a sensor and an ink-jet print-head assembly comprised in a housing for a hand-held and hand-operated printing device controlled by a processor. Thereby it comprises: two position sensor means at least one sensor means being related to a first coordinate system, having one axis in a relation to the print-head assembly, and one axis in a direction through both sensor means; a print-head array attached in a fixed position to the sensor means; input means on the housing connected to the processor for input of control commands; determining means for reference coordinates in a second coordinate system provided in relation to a print medium, the reference coordinates being established by a control command through the input means with the thus read sensor means signals; integrating means for keeping track of the assemblies position related to the reference coordinates in the second coordinate system by integrating displacement of sensor means position in the first coordinate system; computing means for transforming the sensor means coordinates to coordinates in the second coordinate system, whereby the assemblies position on the print medium is determined in relation to the reference coordinates. In one embodiment of the present invention a look-up table comprises normalized sensor steps with a predetermined resolution between sensor steps, one of the sensor steps determining a minimum movement of the assembly. One embodiment comprises that a position is expressed through the coordinates of the sensor means and the angle between the prior position and the current position of the sensor means. Another embodiment comprises that the transforming of the sensor means coordinates is derived through the position of the sensor means related to the first coordinate system and the angle of the print-head array in relation to the second coordinate system. A further embodiment comprises that an angular change is computed as the difference of the sensor means movement in the y-direction of the first coordinate system multiplied with a constant which is determined in relation to the distance between the two sensor means. A still further embodiment comprises that the print-head nozzle position is computed from the knowledge of the position of one sensor means and the tilt angle of the assembly, by calculating the position of the first and last nozzle in the array. Yet one other embodiment comprises that remaining nozzle positions are computed by starting from the first nozzle positions and adding up the difference in x- and y-directions between the nozzles, whereby the x- and y-distance between the first and last nozzle is divided by the number of nozzles. A yet further embodiment comprises that its width is smaller then the width of the print medium. A still further embodiment comprises that a positioning means is provided to position the assembly in a correct starting position in relation to the print medium. Yet another embodiment comprises that a, not visible for a human eye, pattern provided by injected ink-jet drops in even intervals is used as reference points to adjust for possible sensor means position dislocations. Furthermore the present invention sets forth method for a sensor and ink-jet print-head assembly comprised in a housing for a hand-held and hand-operated printing device controlled by a computer processor. It comprises the steps of: providing two position sensor means, whereby at least one sensor means being related to a first coordinate system, having one axis in a relation to the print-head assembly, and one axis in a direction through both sensor means; providing a print-head array attached in a fixed position to the sensor means; providing input means on the housing connected to the processor for input of control commands; providing determining means for reference coordinates in a second coordinate system provided in relation to a print medium, the reference coordinates being established by a control command through the input means; providing integrating means for keeping track of the assemblies position related to the reference coordinates in the second coordinate system by integrating displacement of sensor means position in the first coordinate system; providing computing means for transforming the sensor means coordinates to coordinates in the second coordinate system, whereby the assemblies position on the print medium is determined in relation to the reference coordinates. The method of the present invention is able to perform method steps of the above assembly embodiments in accordance with attached method sub-claims. |
Hand-held and hand-operated device and printing method for such a device |
A hand-held and/or hand-operated random movement printing device is controlled by at least one processor. The printing device provides a control to determine the position of an ink-jet print head assembly on a print medium. Specifically the assembly prints a pattern that it uses to determine its position. |
1. A hand-held and hand-operated random movement printing device controlled by at least one processor (4), controlling comprised means to perform their intended tasks, having an ink-jet print-head (2) assembly comprised in a housing (1), further comprising: at least one ink container; a transmitter for transmission of electromagnetic radiation on a print medium; a receiver for receiving reflected electromagnetic radiation from said print medium; digital signal processing means providing a digital signal of the inherent information received by reflected radiation; a memory storing a digitised raster of a code pattern for printing a plurality of marks, each mark being related to a coordinate in an imaginary coordinate system made up of the raster to be printed on said print medium, said memory also storing an image to be printed on the print medium, said image being associated to coordinates in the imaginary coordinate system; a print-head array disposed to have nozzles for printing a raster of a code pattern, and print nozzles for the image to be printed, printing a part of the pattern through spray dozes from the nozzles and related parts belonging to the image; and whereby said raster of a code pattern enables the printer to be randomly moved over a print medium still keeping track of the print-head arrays position, and integrates the image to the coordinate system to be coherently printed. 2. A printing device according to claim 1, wherein an image is printed on the print medium by preventing the print-head to spray ink on the positions coinciding with marks of the code pattern, said non-printed positions thus making up a negative raster of a code pattern. 3. A printing device according to claim 1, wherein the position of the printer using the coded pattern is updated only when the position of the image on the print medium does not coincide with marks of the code pattern. 4. A printing device according to claim 1, wherein said at least one ink container is separated to accommodate at least a first and a second type of ink and wherein said print-head array is disposed to have a first set of print nozzles for said first type of ink to print said raster of the code pattern and a second set of print nozzles for said second type of ink to print said image. 5. A printing device according to claim 4, wherein an image is printed on the print medium by preventing the print-head to spray ink on positions coinciding with marks of the code pattern, thereby enabling said marks to be identified by the receiver due to its different reflection/absorption characteristics. 6. A printing device according to claim 1, wherein the raster of a code pattern to be printed on a print medium is printed at least in part on the medium before the image is printed, whereby the image to be printed is printed when the printer re-captures an already printed raster by random movement. 7. A printing device according to claim 1, wherein the printing device uses the microstructure of the print medium to determine its relative movement. 8. A printing device according to claim 7, wherein the printing device uses the raster for error correction of the movement determined by using the microstructure of the print medium. 9. A method for a hand-held and hand-operated random movement printing device controlled by at least one processor (4) controlling comprised means to perform their intended tasks, having an ink-jet print-head (2) assembly comprised in a housing (1), at least one ink container, and a memory storing a digitised raster of a code pattern for printing a plurality of marks, each mark being related to a coordinate in an imaginary coordinate system made up of the raster to be printed on a print medium, and also storing an image to be printed on the print medium, said image being associated to coordinates in the imaginary coordinate system, comprising the steps of: transmitting electromagnetic radiation on a print medium through a comprised transmitter; receiving reflected electromagnetic radiation from said print medium through a comprised receiver; providing a digital signal of the inherent information received by reflected radiation by comprised digital signal processing means; printing, through print nozzles in the print-head array, a part of a raster of a code pattern, and printing related parts of the image; whereby said raster of a code pattern enables the printer to be randomly moved over a print medium still keeping track of the print-head arrays position, and integrates the image to the coordinate system to be printed in a coherent manner. 10. A method according to claim 9 wherein an image is printed on the print medium by preventing the print-head to spray ink on the positions coinciding with marks of the code pattern, said non-printed positions thus making up a negative raster of a code pattern. 11. A method according to claim 9 wherein the position of the printer using the coded pattern is updated only when the position of the image on the print medium does not coincide with marks of the code pattern. 12. A method according to claim 9 wherein said at least one ink container used accommodates at least a first and a second type of ink separated, said raster of the code pattern is printed through a first set of print nozzles disposed in said print-head using a first type of ink, and said image is printed through a second set of print nozzles disposed in said print-head using a second type of ink 13. A method according to claim 12 wherein an image is printed on the print medium by preventing the print-head to spray ink on positions coinciding with marks of the code pattern, thereby enabling said marks to be identified by the receiver due to its different reflection/absorption characteristics. 14. A method according to claim 9 wherein the raster of a code pattern to be printed on the print medium is printed at least in part on the medium, whereby the image to be printed is printed when the printer re-captures an already printed raster by random movement. 15. A method according to claim 9, wherein the microstructure of the print medium is used to determine the relative movement of the printing device. 16. A method according to claim 15, wherein the raster is used for error correction of the movement determined by using the microstructure of the print medium. |
<SOH> SUMMARY OF THE DISCLOSED INVENTION <EOH>A hand-held printer according to the present invention uses a positioning method for printing on a print medium without the drawbacks of the prior art. Specifically the hand-held printer prints a simple navigation pattern and uses the pattern for determining positions on the print medium. Hence, the present invention sets forth a hand-held and hand-operated random movement printing device controlled by at least one processor controlling comprised means to perform their intended tasks, having an ink-jet print-head assembly comprised in a housing. It further comprises: an ink container; a transmitter for transmission of electromagnetic radiation on a print medium; a receiver for receiving reflected electromagnetic radiation from said print medium; digital signal processing means providing a digital signal of the inherent information received by reflected radiation; a memory storing a digitised raster of a code pattern for printing a plurality of marks, each mark being related to a coordinate in an imaginary coordinate system made up of the raster to be printed on said print medium, said memory also storing an image to be printed on the print medium, said image being associated to coordinates in the imaginary coordinate system; a print-head array disposed to have print nozzles for printing a raster of a code pattern, and for the image to be printed, printing a part of the pattern through spray dozes from the nozzles and related parts belonging to the image; and whereby said raster of a code pattern enables the printer to be randomly moved over a print medium still keeping track of the print-head arrays position, and integrates the image to the coordinate system to be coherently printed. One embodiment of the present invention provides that the position of the printer, using the coded pattern, is updated only when the position of the image on the print medium does not coincide with marks of the code pattern. One embodiment of the present invention presents that an image is printed on the print medium by not spraying ink on the positions coinciding with marks of the code pattern, said non-printed positions thus making up a negative raster of a code pattern. Another embodiment of the present invention provides the at least one ink container separated to accommodate at least a first and a second type of ink and wherein said print-head array is disposed to have a first set of print nozzles for said first type of ink to print said raster of the code pattern and a second set of print nozzles for said second type of ink to print said image. A further embodiment provides that the raster of a code pattern to be printed on a print medium is printed at least in part on the medium, whereby the image to be printed is printed when the printer re-captures an already printed raster by random movement. In a further embodiment the printing device uses the microstructure of the print medium to determine its relative movement, and preferably the printing device uses the raster for error correction of the movement determined by using the microstructure of the print medium. Furthermore, the present invention sets forth a method for a hand-held and hand-operated random movement printing device controlled by at least one processor controlling comprised means to perform their intended tasks. It provides an ink-jet print-head assembly comprised in a housing, at least one ink container, and a memory storing a digitised raster of a code pattern for printing a plurality of marks, each mark being related to a coordinate in an imaginary coordinate system made up of the raster to be printed on a print medium, and also storing an image to be printed on the print medium, said image being associated to coordinates in the imaginary coordinate system. Further comprising the steps of: transmitting electromagnetic radiation on a print medium through a comprised transmitter; receiving reflected electromagnetic radiation from said print medium through a comprised receiver; providing a digital signal of the inherent information received by reflected radiation by comprised digital signal processing means; printing, through print nozzles in the print-head array, a part of a raster of a code pattern, and printing related parts of the image; whereby said raster of a code pattern enables the printer to be randomly moved over a print medium still keeping track of the print-head arrays position, and integrates the image to the coordinate system to be printed in a coherent manner. The method and device according to the invention combines to different ways of optical navigation and could be described as operating on relative coordinates which are provided with a main navigation subsystem using microstructures of the navigation surface and on absolute coordinates provided by said raster of a code pattern. The code pattern thereby correcting possible accumulated errors made by the main navigation subsystem. The method of the present invention is able to perform method steps of the above assembly embodiments in accordance with attached method sub-claims. |
Novel halo-substituted active methylene compounds |
A novel process for the preparation of compounds of formula I by employing novel halo-substituted active methylene compounds of formula III and process of preparation thereof. |
1. A process for preparing compound of formula I comprising the steps of reaction of the compound of formula III with compounds of formula IV to obtain compound of formula I 2. A process as in claim 1, wherein the reaction between compounds of formula III and formula IV is carried out in the presence of reagents selected from lithium diisopropylamide, sodium hydride n-butyllithium, sodium ethoxide or any such suitable base. 3. A process as in claim 1, wherein the compounds of formula III is prepared by halogenation of compound of formula II to afford a compound of formula II. 4. A process as in claim 3, wherein the halogenation is carried out in the presence of reagents selected from Bromine, N-bromosuccinimide, thionyl chloride, Br2(CN)2, 4-(dimethylamino)pyridinium bromide or any such suitable halogenating agent. 5. An intermediate of formula III |
<SOH> BACKGROUND <EOH>U.S. Pat. No. 5,124,482 and U.S. Pat. No. 5,216,174 discloses the manufacture and use of 4-Fluoro-α-[2-methyl-1-oxopropyl]γ-oxo-N-β-diphenylbenzenebutane amide for preparation of [R-(R*,R*)]-2-(4-Fluorophenyl)-B,D-Dihydroxy-5-(1-Methylethyl)-3-Phenyl-4-[(Phenylamino) Carbonyl]-1h-Pyrrole-1-Heptanoic Acid. [R-(R*,R*)]-2-(4-Fluorophenyl)-B,D-Dihydroxy-5-(1-Methylethyl)-3-Phenyl-4-[(Phenylamino)Carbonyl]-1h-Pyrrole-1-Heptanoic Acid is inhibitor of HMG CoA reductase and thus is used as antihypercholesterolemic agent. Hitherto unknown compounds of the formula III are extremely useful novel intermediates for an improved process for the preparation of 4-Fluoro-α-[2-methyl-1-oxopropyl]γ-oxo-N-β-diphenylbenzenebutaneamide (Scheme 1). |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention also relates to a process for preparation of novel intermediates of formula III. The present invention also relates to novel process for preparation compounds of formula I. As mentioned earlier the compounds of formula I can be prepared by a novel process comprising, a) a) halogenation of compound of formula II to afford a compounds of formula III, b) reaction of compounds of formula III with compounds of formula IV. detailed-description description="Detailed Description" end="lead"? |
Dynamic traffic bandwidth management system and method for a communication network |
An apparatus and a method for managing service traffic in a communications network capable of providing voice, data, and A/V services to a plurality of customers. In particular, the present invention is a digital subscriber line system (DSL), preferably based on ADSL, having a plurality of customer premise equipment (CPEs) coupled to voice, data and A/V services via network system equipment comprising a DSLAM, an ATM switch and a DSL terminator for connecting the system to the internet. The invention provides a network control system that includes a plurality of databases including a provisioning database and a real time database indicative of the actual bandwidth being utilized. A service control processor coupled to the plurality of databases and the network system equipment periodically polls the databases to determine the amount of bandwidth being used, and if the usage exceeds a predetermined level, throttles the internet data entering the system through the DSL terminator and/or the user data entering the system through the CPEs. Preferrably, throttling is performed using a leaky-bucket algorithm. By dynamically managing the traffic flow in the network, the present invention reduces the potential for dropped calls and similar service restrictions. |
1. A communications system, comprising: network system equipment coupled to, and providing connections between, a circuit switched telephone network, a plurality of customer premise equipment (“CPE”), and the internet via a network terminator, for providing voice and data services to a plurality of customer premises; and network control system, coupled to the network system equipment, for coordinating connections between the plurality of CPEs and the telephone network and the internet, and for managing traffic therebetween, the network control system including a first database that includes system configuration information and configuration information for each CPE coupled to the network system equipment, a second database that includes information indicative of service connections currently being utilized in the communications system and the usage by each CPE, and a control processor for periodically polling the first database and the second database to determine the bandwidth capacity currently being utilized in the communications system and throttling the amount of data services entering the communications system in response to the determined bandwidth utilization. 2. The system according to claim 1, wherein the communications system is a DSL system and the network system equipment comprises a DSLAM coupled to the plurality of CPEs, and an ATM switch coupled to the DSLAM, the telephone network, and to the ISP via a DSL terminator, and the control processor is coupled to the ATM switch for throttling the amount of data services entering the DSL system, wherein the control processor reduces the amount of data services entering the DSL system when the determined bandwidth utilization exceeds a first predetermined level. 3. The system according to claim 2, wherein the ATM switch is further coupled to an A/V streaming source for providing streaming media data to the CPEs. 4. The system according to claim 2, wherein the control processor throttles the data services entering the DSL system by controlling the amount of data entering via the DSL terminator. 5. The system according to claim 4, wherein the control processor throttles the data services entering the DSL system via DSL terminator using a leaky-bucket algorithm. 6. The system according to claim 5, wherein the control processor throttles the data services entering the DSL system using SNMP. 7. The system according to claim 4, wherein the control processor throttles the data services entering the DSL system by controlling the data entering via the CPEs. 8. The system according to claim 7, wherein the control processor throttles the data services entering DSL system via the CPEs using a leaky-bucket algorithm. 9. The system according to claim 8, wherein the control processor throttles the data services entering the DSL system using SNMP. 10. The system according to claim 1, wherein if the amount of data services entering the DSL system has been reduced, the control processor moves any restrictions on the amount of data services entering the DSL system when the determined bandwidth utilization is below a second predetermined utilization level, which is lower than the first predetermined utilization level. 11. In a communication system comprising: network system equipment coupled to, and providing connections between, a circuit switch telephone network, a plurality of customer premise equipment (“CPE”), and the internet via a network terminator, for providing voice and data services to a plurality of customer premises; and a network control system, coupled to the network system equipment, for coordinating connections between the plurality of CPEs and the telephone network and the internet, and for managing traffic therebetween, a method for controlling the communications system comprising the steps of: providing a first database that includes system configuration information and configuration information for each CPE coupled to the network system equipment; providing second database that includes information indicative of service connections currently being utilized in the communications system and the usage by each CPE; periodically polling the first database and the second database to determine the bandwidth capacity currently being utilized in the communications system; and throttling the amount of data services entering the communications system in response to the determined bandwidth utilization level. 12. The method according to claim 11, wherein the communications system is a DSL system and the network system equipment comprises a DSLAM coupled to the plurality of CPEs, and an ATM switch coupled to the DSLAM, the telephone network, and to the internet via a DSL terminator, and the throttling step comprises reducing the amount of data services entering the DSL system when the determined bandwidth utilization exceeds a first predetermined level. 13. The method according to claim 12, wherein the throttling step comprises controlling the amount of data services entering the DSL system by controlling the amount of data entering via the DSL terminator. 14. The method according to claim 13, wherein the throttling step comprises throttling the data services entering via the DSL terminator using a leaky-bucket algorithm. 15. The method according to claim 14, wherein the throttling step comprises throttling the data services entering the DSL system using SNMP. 16. The method according to claim 13, wherein the throttling step comprises throttling the data services entering the DSL system by controlling the data entering via the CPEs. 17. The system according to claim 16, wherein the throttling step comprises throttling the data services entering via the CPEs using a leaky-bucket algorithm. 18. The system according to claim 17, wherein the throttling step comprises throttling the data services entering the DSL system using SNMP. 19. The method according to claim 18, wherein the throttling step comprises, if the amount of data services entering the DSL system has been reduced, removing any restrictions on the amount of data services entering the DSL system when the determined bandwidth utilization is below a second predetermined level, which is lower than the first predetermined level. |
Methods for diagnosing and treating diseases and conditions of the digestive system and cancer |
The invention provides methods of diagnosing diseases and conditions of the digestive system and cancer, methods for identifying compounds that can be used to treat or to prevent such diseases and conditions, and methods of using these compounds to treat or to prevent such diseases and conditions. Also provided in the invention are animal model systems that can be used in screening methods. |
1. A method of determining whether a test subject has, or is at risk of developing, a disease or condition related to a nil per os protein, said method comprising analyzing a nucleic acid molecule of a sample from the test subject to determine whether the test subject has a mutation in a gene encoding said protein, wherein the presence of a mutation indicates that said test subject has, or is at risk of developing, a disease or condition related to a nil per os protein. 2. The method of claim 1, wherein said test subject is a human. 3. The method of claim 1, wherein said disease or condition is a disease or condition of the digestive system or cancer. 4. The method of claim 3, wherein said disease or condition is of the intestine, the liver, the bile duct, the pancreas, the stomach, the gall bladder, or the esophagus. 5. A method for identifying a compound that can be used to treat or to prevent a disease or condition of the digestive system or cancer, said method comprising contacting an organism comprising a mutation in a gene encoding a nil per os protein and having a phenotype characteristic of a disease or condition of the digestive system or cancer with said compound, and determining the effect of said compound on said phenotype, wherein detection of an improvement in said phenotype indicates the identification of a compound that can be used to treat or to prevent a disease or condition of the digestive system or cancer. 6. The method of claim 5, wherein said disease or condition of the digestive system is of the intestine, the liver, the bile duct, the pancreas, the stomach, the gall bladder, or the esophagus. 7. The method of claim 5, wherein said organism is a zebrafish. 8. The method of claim 5, wherein said mutation in the gene encoding the nil per os protein is the nil per os mutation. 9. A method of treating or preventing a disease or condition of the digestive system or cancer in a patient, said method comprising administering to said patient a compound identified using the method of claim 5. 10. The method of claim 9, wherein said disease or condition of the digestive system is digestive organ failure. 11. The method of claim 9, wherein said patient has a mutation in a gene encoding a nil per os protein. 12. A method of treating or preventing a disease or condition of the digestive system in a patient, said method comprising administering to said patient a functional nil per os protein or an expression vector comprising a nucleic acid molecule encoding said protein. 13. A method of treating or preventing cancer in a patient, said method comprising administering to said patient a compound or molecule that inhibits the activity or expression of nil per os in said patient. 14. A substantially pure nil per os polypeptide. 15. The polypeptide of claim 14, wherein said polypeptide is a zebrafish polypeptide or a human polypeptide. 16. The polypeptide of claim 14, wherein said polypeptide comprises an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5 or comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5. 17. A substantially pure nucleic acid molecule comprising a sequence encoding a nil per os polypeptide. 18. The nucleic acid molecule of claim 17, wherein said nucleic acid molecule encodes a zebrafish polypeptide or a human polypeptide. 19. The nucleic acid molecule of claim 17, wherein said nucleic acid molecule encodes a polypeptide that comprises an amino sequence that is substantially identical to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5, or comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5. 20. The nucleic acid molecule of claim 17, wherein said nucleic acid molecule is DNA. 21. A vector comprising the nucleic acid molecule of claim 17. 22. A cell comprising the vector of claim 21. 23. A non-human transgenic animal comprising the nucleic acid molecule of claim 17. 24. The non-human transgenic animal of claim 23, wherein said animal is a zebrafish. 25. A non-human animal having a knockout mutation in one or both alleles encoding a nil per os polypeptide. 26. A cell from the non-human knockout animal of claim 25. 27. A non-human transgenic animal comprising a nucleic acid molecule encoding a mutant nil per os polypeptide. 28. The non-human transgenic animal of claim 27, wherein the non-human transgenic animal is a zebrafish. 29. The non-human transgenic animal of claim 28, wherein the non-human transgenic animal comprises the nil per os mutation. 30. An antibody that specifically binds to a nil per os polypeptide. 31. A method of identifying a stem cell of the gastrointestinal tract, said method comprising analyzing a pool of candidate cells for expression of nil per os. 32. The method of claim 31, further comprising separating cells that express nil per os from said pool of candidate cells. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The cells that line the digestive organs, such as the intestine, pancreas, and liver, arise from a part of the early embryo called the endoderm. The endodermal cells undergo defined movements and changes in cell shape that ultimately lead to the formation of highly organized structures that collectively constitute mature, functioning organs. The individual steps that lead to organ formation have been described by microscopic analysis of developing embryos, but the molecules that are responsible for guiding these steps are largely unknown. The zebrafish, Danio rerio, is a convenient organism to use in genetic analysis of development. In addition to having a short generation time and being fecund, it has an accessible and transparent embryo, allowing direct observation of organ function from the earliest stages of development. |
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