WO1998032863A2 - Mammalian thioredoxin - Google Patents
Mammalian thioredoxin Download PDFInfo
- Publication number
- WO1998032863A2 WO1998032863A2 PCT/GB1998/000263 GB9800263W WO9832863A2 WO 1998032863 A2 WO1998032863 A2 WO 1998032863A2 GB 9800263 W GB9800263 W GB 9800263W WO 9832863 A2 WO9832863 A2 WO 9832863A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protein
- polypeptide
- trx2
- nucleic acid
- acid molecule
- Prior art date
Links
- 108060008226 thioredoxin Proteins 0.000 title abstract description 55
- 102000002933 Thioredoxin Human genes 0.000 title abstract description 43
- 229940094937 thioredoxin Drugs 0.000 title abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 23
- 101710168624 Thioredoxin 2 Proteins 0.000 claims description 100
- 108090000623 proteins and genes Proteins 0.000 claims description 82
- 102000004169 proteins and genes Human genes 0.000 claims description 81
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 44
- 229920001184 polypeptide Polymers 0.000 claims description 42
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 42
- 210000004027 cell Anatomy 0.000 claims description 30
- 150000007523 nucleic acids Chemical class 0.000 claims description 29
- 239000002299 complementary DNA Substances 0.000 claims description 28
- 102000039446 nucleic acids Human genes 0.000 claims description 25
- 108020004707 nucleic acids Proteins 0.000 claims description 25
- 239000000523 sample Substances 0.000 claims description 17
- 239000013598 vector Substances 0.000 claims description 17
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 16
- 230000000694 effects Effects 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 13
- 230000006378 damage Effects 0.000 claims description 11
- 150000001413 amino acids Chemical class 0.000 claims description 10
- 239000001301 oxygen Substances 0.000 claims description 10
- 229910052760 oxygen Inorganic materials 0.000 claims description 10
- 239000012634 fragment Substances 0.000 claims description 9
- 210000002569 neuron Anatomy 0.000 claims description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 8
- 238000003556 assay Methods 0.000 claims description 7
- 230000000302 ischemic effect Effects 0.000 claims description 6
- 230000036542 oxidative stress Effects 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 208000010125 myocardial infarction Diseases 0.000 claims description 4
- 230000005855 radiation Effects 0.000 claims description 4
- 241000238631 Hexapoda Species 0.000 claims description 3
- 206010063837 Reperfusion injury Diseases 0.000 claims description 3
- 231100000135 cytotoxicity Toxicity 0.000 claims description 3
- 230000003013 cytotoxicity Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 210000000056 organ Anatomy 0.000 claims description 3
- 210000001525 retina Anatomy 0.000 claims description 3
- 230000035882 stress Effects 0.000 claims description 3
- 230000000451 tissue damage Effects 0.000 claims description 3
- 231100000827 tissue damage Toxicity 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 230000004792 oxidative damage Effects 0.000 claims description 2
- 230000001681 protective effect Effects 0.000 claims description 2
- 230000004223 radioprotective effect Effects 0.000 claims description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 claims description 2
- 230000002207 retinal effect Effects 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 claims 5
- 239000008194 pharmaceutical composition Substances 0.000 claims 4
- 241000124008 Mammalia Species 0.000 claims 3
- 238000002405 diagnostic procedure Methods 0.000 claims 3
- 238000012360 testing method Methods 0.000 claims 2
- 206010057430 Retinal injury Diseases 0.000 claims 1
- 210000004102 animal cell Anatomy 0.000 claims 1
- 230000002906 microbiologic effect Effects 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 235000018102 proteins Nutrition 0.000 description 41
- 238000002372 labelling Methods 0.000 description 26
- 101150037769 TRX2 gene Proteins 0.000 description 21
- 241000700159 Rattus Species 0.000 description 20
- 239000000047 product Substances 0.000 description 19
- 210000001519 tissue Anatomy 0.000 description 17
- 238000004458 analytical method Methods 0.000 description 16
- 230000014509 gene expression Effects 0.000 description 16
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 16
- 238000009396 hybridization Methods 0.000 description 12
- 108020004999 messenger RNA Proteins 0.000 description 12
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 11
- 230000003647 oxidation Effects 0.000 description 11
- 238000007254 oxidation reaction Methods 0.000 description 11
- 101100153095 Dictyostelium discoideum trxB gene Proteins 0.000 description 10
- 102100036407 Thioredoxin Human genes 0.000 description 10
- 238000011065 in-situ storage Methods 0.000 description 10
- 210000004185 liver Anatomy 0.000 description 10
- 230000002438 mitochondrial effect Effects 0.000 description 10
- 238000010186 staining Methods 0.000 description 10
- 102000013090 Thioredoxin-Disulfide Reductase Human genes 0.000 description 9
- 108010079911 Thioredoxin-disulfide reductase Proteins 0.000 description 9
- 235000001014 amino acid Nutrition 0.000 description 9
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 9
- 238000013519 translation Methods 0.000 description 9
- 102000004877 Insulin Human genes 0.000 description 8
- 108090001061 Insulin Proteins 0.000 description 8
- 101000801893 Rattus norvegicus Thioredoxin, mitochondrial Proteins 0.000 description 8
- 210000000981 epithelium Anatomy 0.000 description 8
- 229940125396 insulin Drugs 0.000 description 8
- 239000002953 phosphate buffered saline Substances 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 7
- 101000852559 Homo sapiens Thioredoxin Proteins 0.000 description 7
- 230000003321 amplification Effects 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 210000000349 chromosome Anatomy 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 6
- 210000002216 heart Anatomy 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 210000003470 mitochondria Anatomy 0.000 description 6
- 150000003254 radicals Chemical class 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 102000007469 Actins Human genes 0.000 description 5
- 108010085238 Actins Proteins 0.000 description 5
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 108700026244 Open Reading Frames Proteins 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- 239000004365 Protease Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 5
- 210000002919 epithelial cell Anatomy 0.000 description 5
- -1 hydroxyl radicals Chemical class 0.000 description 5
- 238000007901 in situ hybridization Methods 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 4
- 101710168651 Thioredoxin 1 Proteins 0.000 description 4
- PTKRUDMLGIIORX-ITGWJZMWSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2r,3s,4r,5r)-5-(3-carbamoyl-4h-pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate;cyclohexanamine Chemical compound NC1CCCCC1.NC1CCCCC1.NC1CCCCC1.NC1CCCCC1.C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 PTKRUDMLGIIORX-ITGWJZMWSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 210000000988 bone and bone Anatomy 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- 102000056461 human TXN Human genes 0.000 description 4
- 238000003365 immunocytochemistry Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 210000002027 skeletal muscle Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 102100039556 Galectin-4 Human genes 0.000 description 3
- 101000608765 Homo sapiens Galectin-4 Proteins 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 238000000636 Northern blotting Methods 0.000 description 3
- 101710101078 Proton-activated chloride channel Proteins 0.000 description 3
- 108091023040 Transcription factor Proteins 0.000 description 3
- 102000040945 Transcription factor Human genes 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 238000000376 autoradiography Methods 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 210000004877 mucosa Anatomy 0.000 description 3
- 210000004165 myocardium Anatomy 0.000 description 3
- 230000037452 priming Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 210000005239 tubule Anatomy 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- UBOKASXZHPZFRZ-UHFFFAOYSA-N 2-phenyl-1h-indole-4,6-diamine Chemical compound N1C2=CC(N)=CC(N)=C2C=C1C1=CC=CC=C1 UBOKASXZHPZFRZ-UHFFFAOYSA-N 0.000 description 2
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000004568 DNA-binding Effects 0.000 description 2
- 230000010777 Disulfide Reduction Effects 0.000 description 2
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 101000614095 Homo sapiens Proton-activated chloride channel Proteins 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 108010057466 NF-kappa B Proteins 0.000 description 2
- 102000003945 NF-kappa B Human genes 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 108091036407 Polyadenylation Proteins 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 108010065868 RNA polymerase SP6 Proteins 0.000 description 2
- 238000010240 RT-PCR analysis Methods 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 206010070863 Toxicity to various agents Diseases 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 210000004404 adrenal cortex Anatomy 0.000 description 2
- 210000004100 adrenal gland Anatomy 0.000 description 2
- 229940009456 adriamycin Drugs 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 208000030533 eye disease Diseases 0.000 description 2
- 108010081400 fluorescein isothiocyante avidin Proteins 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 229960004198 guanidine Drugs 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 210000003917 human chromosome Anatomy 0.000 description 2
- 210000004754 hybrid cell Anatomy 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 208000028867 ischemia Diseases 0.000 description 2
- 230000003859 lipid peroxidation Effects 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 2
- 210000001428 peripheral nervous system Anatomy 0.000 description 2
- 230000000858 peroxisomal effect Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000016314 protein import into mitochondrial matrix Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000001995 reticulocyte Anatomy 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 210000001044 sensory neuron Anatomy 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 230000004960 subcellular localization Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 210000001550 testis Anatomy 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- IMNIMPAHZVJRPE-UHFFFAOYSA-N triethylenediamine Chemical compound C1CN2CCN1CC2 IMNIMPAHZVJRPE-UHFFFAOYSA-N 0.000 description 2
- 241001515965 unidentified phage Species 0.000 description 2
- 210000004291 uterus Anatomy 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- 102100024341 10 kDa heat shock protein, mitochondrial Human genes 0.000 description 1
- 101710097567 12 kDa protein Proteins 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 102100039882 40S ribosomal protein S17 Human genes 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 101100000858 Caenorhabditis elegans act-3 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 206010048610 Cardiotoxicity Diseases 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 208000002177 Cataract Diseases 0.000 description 1
- 108091060290 Chromatid Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699662 Cricetomys gambianus Species 0.000 description 1
- 102000000634 Cytochrome c oxidase subunit IV Human genes 0.000 description 1
- 108090000365 Cytochrome-c oxidases Proteins 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 206010073306 Exposure to radiation Diseases 0.000 description 1
- 102400001064 Fibrinogen beta chain Human genes 0.000 description 1
- 101710170765 Fibrinogen beta chain Proteins 0.000 description 1
- 101150094690 GAL1 gene Proteins 0.000 description 1
- 102100028501 Galanin peptides Human genes 0.000 description 1
- 208000003098 Ganglion Cysts Diseases 0.000 description 1
- 102000003676 Glucocorticoid Receptors Human genes 0.000 description 1
- 108090000079 Glucocorticoid Receptors Proteins 0.000 description 1
- 101100121078 Homo sapiens GAL gene Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 1
- 125000003440 L-leucyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C(C([H])([H])[H])([H])C([H])([H])[H] 0.000 description 1
- 125000002707 L-tryptophyl group Chemical group [H]C1=C([H])C([H])=C2C(C([C@](N([H])[H])(C(=O)[*])[H])([H])[H])=C([H])N([H])C2=C1[H] 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 102000006404 Mitochondrial Proteins Human genes 0.000 description 1
- 108010058682 Mitochondrial Proteins Proteins 0.000 description 1
- 101710164418 Movement protein TGB2 Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 101100384865 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cot-1 gene Proteins 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 102000057361 Pseudogenes Human genes 0.000 description 1
- 108091008109 Pseudogenes Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- 101000800031 Rattus norvegicus Thioredoxin Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 102000000505 Ribonucleotide Reductases Human genes 0.000 description 1
- 108010041388 Ribonucleotide Reductases Proteins 0.000 description 1
- 230000006295 S-nitrosylation Effects 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 208000005400 Synovial Cyst Diseases 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 102100035140 Vitronectin Human genes 0.000 description 1
- 108010031318 Vitronectin Proteins 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- 101710185494 Zinc finger protein Proteins 0.000 description 1
- 102100023597 Zinc finger protein 816 Human genes 0.000 description 1
- ZHAFUINZIZIXFC-UHFFFAOYSA-N [9-(dimethylamino)-10-methylbenzo[a]phenoxazin-5-ylidene]azanium;chloride Chemical compound [Cl-].O1C2=CC(=[NH2+])C3=CC=CC=C3C2=NC2=C1C=C(N(C)C)C(C)=C2 ZHAFUINZIZIXFC-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 150000003838 adenosines Chemical class 0.000 description 1
- 210000003486 adipose tissue brown Anatomy 0.000 description 1
- 230000004103 aerobic respiration Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 210000003484 anatomy Anatomy 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 210000001052 bipolar neuron Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 231100000259 cardiotoxicity Toxicity 0.000 description 1
- 210000001011 carotid body Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- YDQXYRCYDMRJGD-UHFFFAOYSA-N chloroform;phenol;thiocyanic acid Chemical compound SC#N.ClC(Cl)Cl.OC1=CC=CC=C1 YDQXYRCYDMRJGD-UHFFFAOYSA-N 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 210000003737 chromaffin cell Anatomy 0.000 description 1
- 210000004756 chromatid Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000014107 chromosome localization Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000006552 constitutive activation Effects 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 210000004246 corpus luteum Anatomy 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000012973 diazabicyclooctane Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 108010046025 early pregnancy factor Proteins 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 210000000918 epididymis Anatomy 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000001752 female genitalia Anatomy 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 108700025906 fos Genes Proteins 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 210000000609 ganglia Anatomy 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 210000002837 heart atrium Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 239000000852 hydrogen donor Substances 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000002169 hydrotherapy Methods 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 210000002332 leydig cell Anatomy 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 210000000260 male genitalia Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 210000001501 megacaryocyte Anatomy 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 108010020410 methionine sulfoxide reductase Proteins 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000002161 motor neuron Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000000754 myometrium Anatomy 0.000 description 1
- 210000004897 n-terminal region Anatomy 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000004693 neuron damage Effects 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 210000000956 olfactory bulb Anatomy 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 229910000489 osmium tetroxide Inorganic materials 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000004783 oxidative metabolism Effects 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 210000001711 oxyntic cell Anatomy 0.000 description 1
- 210000005034 parasympathetic neuron Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 210000002824 peroxisome Anatomy 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 230000009993 protective function Effects 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 210000000449 purkinje cell Anatomy 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000006697 redox regulation Effects 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 108010093121 ribosomal protein S17 Proteins 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 210000002863 seminiferous tubule Anatomy 0.000 description 1
- 210000000717 sertoli cell Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 210000001057 smooth muscle myoblast Anatomy 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000000920 spermatogeneic effect Effects 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 210000003523 substantia nigra Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 210000004377 supraoptic nucleus Anatomy 0.000 description 1
- 210000000106 sweat gland Anatomy 0.000 description 1
- 210000000331 sympathetic ganglia Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 108091006106 transcriptional activators Proteins 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000010396 two-hybrid screening Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 238000001086 yeast two-hybrid system Methods 0.000 description 1
- 210000001235 zona fasciculata Anatomy 0.000 description 1
- 210000003368 zona glomerulosa Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to the isolation and characterisation of nucleic acid encoding for a new mammalian thioredoxin and to a protein or polypeptide produced from such nucleic acid.
- Thioredoxin is a 12-kDa protein, known to be present in many prokaryotes and eukaryotes and appears to be truly ubiquitous in all living cells (Holmgren, A. (1984). Methods Enzymol. 107, 295-300, Holmgren, A. (1985). Ann. Rev. Biochem. 54, 237-271). It is characterized by an active site sequence -Trp-Cys-Gly-Pro-Cys-Lys-, which has been conserved throughout evolution. The active site of thioredoxin is localised in a protrusion of its three dimensional structure (Jeng. M. F.
- Trx-S 2 Oxidized thioredoxin, Trx-S 2 , is reduced to Trx-(SH) 2 by the flavoenzyme thioredoxin reductase (TR) and NADPH (the thioredoxin system) (Holmgren, A. (1985) supra).
- Mammalian thioredoxins isolated from several sources have certain structural differences with respect to those from prokaryotes.
- two or three (depending on the Trx source) additional structural cysteine residues exist in the C-terminal half of the molecule. Oxidation of these residues leads to a loss of its enzymatic activity (Ren. X et al. (1993) Biochemistry 32, 9701-9708).
- thioredoxin exists in many eukaryotes, e.g. yeast (Muller E. G. (1992) Yeast 8, 117-120). However, only one thioredoxin (Trx 1) has thus far been cloned from mammalian cells.
- Mammalian thioredoxin has been implicated in a wide variety of biochemical functions acting as hydrogen donor for ribonucleotide reductase (Thelander, L., Reichard, P. (1979) Ann. Rev. Biochem. 48, 133-158) and methionine sulfoxide reductase (Holgren, A. (1985) Supra), facilitating refolding of disulphide-containing proteins (Holmgren, A. (1988).
- ribonucleotide reductase Thelander, L., Reichard, P. (1979) Ann. Rev. Biochem. 48, 133-158
- methionine sulfoxide reductase Holgren, A. (1985) Supra
- Thioredoxin can be secreted by cells using a leaderless pathway (Ericson, M.L., et al (1992) Lumphokine & Cytokine Research 11, 201-207; Rubartelli, A., et al (1992) J. Biol. Chem. 267, 24161-24164; Rubartelli, A., et al (1995) Cancer Res. 55, 675-680) and stimulate the proliferation of lymphoid cells, fibroblasts and a variety of human solid tumour cell lines (Wakasugi, N., et al (1990). Proc. Natl. Acad. SCI. USA 87, 8282-8286; Oblong, J. E., et al (1994) J. Biol.
- Trx is an essential component of the early pregnancy factor (Clarke F. M. et al. (1991) Reproduction & Fertility 93, 525-539), it inhibits HIV expression in macrophages (Newman, G.W. et al (1994) J. Experim. Medicine 180, 359-363), can reduce H 2 O 2 (Spector A et al (1988) J. Biol. Chem.
- Trx2 The inventors have identified a novel mammalian thioredoxin which is functionally and structurally distinct from the previously known mammalian thioredoxin. This novel thioredoxin is known as Trx2.
- a first aspect of the invention provides an isolated nucleic acid molecule encoding the Trx2 gene product or a polypeptide which is functionally similar to Trx2.
- the nucleic acid molecule comprises the nucleic acid sequence shown in Figure 1A or Figure 9.
- the nucleic acid molecule may be cDNA.
- the nucleic acid has at least 60%, 70% or 80% homology, more preferably 90% homology, to the nucleic acid sequences of Figure 1 A or Figure 9 encoding the Trx2 gene product or a polypeptide which is functionally similar to Trx2.
- This allows, for example, for variations in the sequence which still allows the production of Trx2, by virtue of degeneracy of the genetic code.
- Trx2 functionally similar to Trx2
- the polypeptide has thioredoxin activity, as indicated, for example by the catalysis of disulphide reduction or insulin with NADPH in the presence of mammalian thioredoxin reductase which is resistant against oxidation compared to Trx 1.
- Trx 1 exhibits oxidation of cysteine residues which results in a loss of its enzymatic activity (Ren X et al. (1993) Biochemistry 32, 9701-9708). Trx2, however, lacks these cysteine residues.
- a second aspect of the invention provides polypeptides, preferably mature proteins, encoded by nucleic acid according to the first aspect of the invention.
- the polypeptide comprises the amino acid sequence of Figure 1A or Figure 10.
- Trx2 Analysis of the amino acid sequence of Trx2 indicates that it may be processed into a mature protein by enzymatic action. Accordingly, the invention also provides a mature protein, fragment, homologue or analogue which is at least functionally similar to Trx2. The invention also includes recombinant or synthetic polypeptides having the same or preferably better, functionality compared to native Trx2.
- the mature protein comprises the amino acid sequence of Figure 1 A from Thr 59 to Asp 166, or the sequence of Figure 10 from Leu 59 to He 166.
- a further aspect of the invention provides plasmids or other vectors comprising nucleic acids according to the first aspect of the invention. Such plasmids or other vectors are preferably expression vectors of the sort known in the art, into which a nucleic acid according to the invention can be inserted.
- Such plasmids or vectors may be inserted into a suitable microbial host, such as E.coli, or a suitable cell of the type known in the art for expression of the nucleic acid sequences of the invention such as a mammalian or insect cell. Accordingly, the invention provides bacterial, mammalian or insect cells comprising vectors or plasmids according to the invention.
- Thioredoxin 2 (Trx2) is advantageous in that it is resistant to oxidation, and is translocated to mitochondria.
- Mitochondria are the sites of vital cellular functions such as lipid metabolism and aerobic respiration (oxidative phosphorylation).
- ROIs reactive oxygen intermediates
- Oxidative stress can result in lipid peroxidation, inactivation of proteins and strand breakage in DNA.
- Thioredoxin can: act as an antioxidative molecule and scavenge hydroxyl radicals, reduce hydrogen peroxide and reactivate proteins inactivated by oxidation.
- Trx2 provides therapeutic applications of Trx2 including protection against oxidative stress induced cytotoxicity and tissue damage. More specifically Trx2 may be used to protect against ischaemic damage, eye disease, radiation, and drug toxicity.
- Trx2 may be used as protective compound to attenuate ischaemia reperfusion injury.
- many diseases of the eye including cataract involve photo-oxidative stress. Trx2 may be used for protection of retinal cells from damage caused by active oxygen species generated during oxygen stress to the retina.
- Trx2 may be used as a radio-protective compound to attenuate the effects of radiation.
- Trx2 can be used to eliminate side effects.
- Figure 1 shows cDNA, deduced amino acid sequence and predicted secondary structure of rat Trx2.
- Figure 2 shows in vitro translation of Trx2 cDNA using the Sp6 RNA polymerase, the TNT coupled reticulocyte transcription translation system and [ 35 S] methionine. The product was analyzed by SDS-PAGE;
- Figure 3 shows alignment of the predicted amino acid sequence of rat Trx2 with that of human and rat Trxl.
- Figure 4 shows a phylogenetic tree.
- the phylogenetic tree was constructed by the Clustal method with the PAM250 residue weight table;
- FIG. 5 shows Northern blot analysis of Trxl and Trx2 in rat tissues. Each lane contains 2 ⁇ g poly(A)+ RNA. The blot was hybridized with the Trxl and Trx2 probes as described under "Experimental Procedures". Tissues used for analysis are shown at the top. The estimated size of the mRNAs was 0.6 kb and 1.3 kb for Trxl and Trx2, respectively;
- Figure 6 shows RT-PCR analysis of transcripts encoding Trxl, and Trx2.
- One ⁇ g of total RNA was reverse transcribed and one ⁇ l was used as template for PCR with specific primers for Trxl, Trx2 and ⁇ -actin.
- the products were separated by agarose gel electrophoresis, transferred to membranes and probed with specific oligonucleotide probes;
- Figure 7 shows immunoblotting analysis of cell fractions.
- Cell fractions were analyzed by SDS/PAGE and developed with anti-Trx2 antibodies.
- Lane 1 ⁇ -Trx2 (5ng).
- Lane 2 total rat liver cell extract (15 ⁇ g)
- Lane 5 peroxisomal fraction (15 ⁇ g).
- FIG. 8 shows analysis of Trx activity.
- Oxidized Trx2 and human thioredoxin were assayed for their ability to reduce insulin in the presence or absence of DTT.
- the reaction was stopped after 20 min by the addition of 6 M guanidine-HCl, ImM DTNB;
- Figure 9 shows human Trx2 cDNA sequence
- Figure 10 shows the predicted human Trx2 amino acid sequence based on the cDNA sequence of Fig. 9;
- Figure 11 shows two human liver cDNA sequences that code for proteins which interact with Trx2;
- Figure 12 shows five rat brain cDNA sequences that code for proteins which interact with Trx2;
- Figure 13 shows the human trx2 gene
- Figure 14 shows a gel picture of a PCR product in chromosome 22.
- Figure 15 shows the mRNA levels of the human Trx2 analysed by using an RNA master blot (Clontech).
- VVVDFSATWCGPCK The primary structure of the active site of thioredoxin (VVVDFSATWCGPCK), which is conserved throughout evolution was used to design degenerate primers which were labelled with 32 P and used as probes for screening a rat heart cDNA library (Clontech) for novel thioredoxin genes. Approximately lxlO 6 plaques were screened according the instructions of the manufacturer (Amersham) and a positive bacteriophage was isolated. The insert from the bacteriophage was excised, cloned into the TA-vector (Invitrogen) and sequenced.
- a 392 bp portion of the above clone was amplified by PCR (30 cycles at; 94°C for 1 min, 52°C for 1 min and 72°C for 1 min) with specific primers (trx2fl: 5'-AACCTTTATCGTCCAGGATGGAC-3' and trx2rl: 5'-GCTGGGAGTTCTACTAGGTTCC-3').
- the PCR product was 32 P-labelled by random priming and used to rescreen the same library under high-stringency conditions. Hybridization was performed at 60°C in o
- the overall composite sequence consists of 1276 bp, including a stretch of 20 adenosines corresponding to the poly( A) tail and an AATAAAA motif, 18 bp upstream from the poly(A) tail.
- the open reading frame encodes a protein of 166 amino acids with a predicted mass of 18.2 kDa and a pi of 7.9 ( Figures 1A, IB).
- the cDNA was transcribed from the SP6 promoter of the TA-Trx2 clone and 0.5 ⁇ g was translated using the TNT coupled reticulocyte lysate system (Promega) and Sp6 RNA polymerase with incorporation of [ 35 S]methionine for 60 min at 30°C.
- the translation products were analyzed by 15% SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The results showed a 20-kDa translation product, indicating the presence of translatable, functional coding sequence ( Figure 2).
- the N-terminal region of the protein has a high content of positively charged residues and secondary structure prediction indicated a potential ⁇ -helix followed by ⁇ -sheets ( Figure IB).
- Figure IB These features are common to most mitochondrial targeting signal peptides (Neupert, W. (1994). Clinical Investigator 72, 251-261) and an algorithm analysis of the partial amino acid composition, indicated mitochondrial intracellular localization (Newman, G. W. et al (1994). J. Experim. Medicine 180, 359-363).
- a motif for mitochondrial prepeptide proteases Hendrick J. P., Hodges, P.E., Rosenberg, L.E. (1989) Proc. Natl. Acad. SCI. USA.
- the C-terminal half of the protein contained the active site found in all thioredoxins with the characteristic amino acid sequence, Trp-Cys-Gly-Pro-Cys-Lys.
- the molecule showed a 35% homology with other mammalian thioredoxins and many of the structural amino acids that are conserved in mammalian thioredoxins, i.e Phe-12, Pro-40, Asp-59, Lys-82, were also conserved in Trx2 ( Figure 3B). However, amino acids participating in protein-protein interactions such as Ala-93 and Glu-57 are changed to He and Lys, respectively.
- Trx2 and mammalian thioredoxins is the absence of structural cysteines, residues which are present in all mammalian thioredoxins.
- Trx2 has higher homology with the E.coli thioredoxin than with the known mammalian proteins and a phylogenetic analysis places Trx2 in a different branch of the tree, distant from the mammalian proteins and closer to the prokaryotic and lower eukaryotic ones.
- RNA from different rat tissues was hybridized with two probes, one of 360 bp specific for rat thioredoxin (Trxl) and one of 392 bp specific for Trx2 as described in Spyrou et al (1996) J. Biol Chem 272, 2936-2941.
- Rat Trxl and Trx2 open reading frame probes were labelled with [ 32 P]dCTP by a random priming procedure and hybridized in ExpressHyb solution (Clontech). The Trxl probe hybridized to an RNA of 0.6 kb with highest levels in lung, liver and kidney ( Figure 5).
- Trx2 mRNA was detected as a 1.3 kb band, in agreement with the size of cDNA (1,276 bp) and it was highly expressed in heart, liver, skeletal muscle and kidney.
- RT-PCR was used to compare the relative expression of Trxl and Trx2 in tissues where the expression of these proteins may be very low.
- Figure 6 For RT-PCR analysis, male and female rats (6-8 weeks old) were killed by cervical dislocation, tissues were collected and samples were immediately processed for total RNA isolation according to the acid guanidium thiocyanate-phenol-chloroform single step extraction protocol (Chomczynski, P., Sacchi, N. (1987) J. Biol. Chem. 162, 156-159). The integrity and quality of the purified RNA was controlled by formaldehyde denaturing agarose gel electrophoresis and by measuring the A260/A280 ratio.
- RNA total RNA (l ⁇ g) was dissolved in 10 ⁇ l water, heated to 70°C for 5 min and then chilled on ice. The volume was increased to 20 ⁇ l, giving a final concentration of 1 mM each dATP dGTP, dCTP, dTTP, 10 mM DTT, 5 pmol random hexamers/1 (Promega), 1 U RNAsin/ ⁇ l, 200 U Superscript RT (GIBCO-BRL) and the incubation buffer recommended by the supplier.
- PCR amplification 1 ⁇ l of cDNA (total 20 ⁇ l) was subjected to PCR and amplified for 24 cycles by incubation at 94°C for 10 sec, 54°C for 30 sec and 72°C for 60 sec in a PCR9600 thermocycler (Perkin-Elmer, Norwalk, CT).
- the oligonucleotides trx2fl: 5'-AACCTTTATCGTCCAGGATGGAC-3' and trx2rl: 5'-GCTGGGAGTTCTACTAGGTTCC-3' were used for the amplification of a 392 bp fragment of the Trx2 mRNA.
- TGATTAGGCAAACTCCGTAATAGTG-3' were used for the amplification of a 360 bp fragment of the Trxl mRNA.
- the oligonucleotides Act 5', CTGGCACCACACCTTCTA and Act 3', GGGCACAGTGTGGGTGAC were used for the amplification of a 238 bp fragment from ⁇ -actin mRNA.
- After agarose gel electrophoresis and blotting to nitrocellulose filters the PCR products were hybridized to 32 P-labelled internal oligonucleotides: trx2r2: 5'-
- Trxl and actin primer 5'- GATGACCCAGATCATGTTTGA-3' for Trxl and actin primer 5'- GATGACCCAGATCATGTTTGA-3'.
- Hybridization was performed at 50°C in Expresshyb hybridization solution followed by five 10 min washes in 2xSSC-0.1%SDS at room temperature and finally two 40 min washes in 0.1%SSC-0.1%SDS at 50°C. While Trxl is found to follow the ⁇ -actin expression with higher expression in colon and liver, Trx2 gave a completely different pattern. It was highly expressed in cerebellum, heart, skeletal muscle, kidney, adrenal gland and testis. No cross hybridization between Trxl and Trx2 was observed.
- the C-terminal part of the cDNA encoding a part of rat Trx2 was amplified by PCR from the TA-Trx2 plasmid by using two mutagenic primers that introduce a Ndel (trx2pl: 5'-ACCACCAGAGTCCATATGACAACCTTTAACGTC-3') and a BamH I (trx2p2: 5'-CTGGCCGGATCCCTGCTTATCAGCCAATTAGC-3') site at the N-terminus and C-terminus of the polypeptide respectively.
- the amplified DNA was cloned into the Ndel-Bam l sites of the pET-15b expression vector (Novagen) under the control of a T7 promoter, and the resulting plasmid, pET-trx2 was transformed into the E. coli strain BL21 (DE3).
- the cells were harvested by centrifugation at 10,000 x g for 10 min, the pellet was resuspended in 50 ml of 20 mM Tris-HCl pH 8.0, 100 mM NaCl and 1 mM PMSF. Lysozyme was added to a final concentration of 0.5 mg/ml with stirring for 30 min on ice. Subsequently, MgCl 2 (10 mM), MnCl 2 (lmM), DNAse I (10 ⁇ g/ml), and RNAse (10 ⁇ g/ml) were added and the incubation was continued for another 45 min on ice.
- the cells were disrupted by sonication for 8 min, the supernatant was cleared by centrifugation at 15,000 x g for 30 min and loaded onto a Talon resin column (Clontech) and the protein was eluted with 20 mM imidazole. The size and purity of the eluted protein was determined by SDS-PAGE on a 15% gel. A single band of 15 kD was detected after the Talon chromatography (data not shown).
- the inventors next analyzed the subcellular localization of Trx2 using affinity purified polyclonal antibodies obtained from immunized rabbits (Zeneca Research Biochemicals, England). After 6 immunizations, serum from the rabbits was purified by ammonium sulfate precipitation. Affinity-purified antibodies were prepared using a cyanogen bromide-activated Sepharose 4B column, onto which 0.5 mg of Trx2 had been coupled using the procedure recommended by the manufacturer (Pharmacia). Specificity of the antibodies was tested by western blotting using recombinant Trx2 and total cell extracts.
- Mitochondrial, peroxisomal and cytosolic fractions were prepared from rat liver as described (Svensson, L.T., Alexson, S.E.H., Hiltunen, J.K. (1995) J, Biol. Chem. 270, 12177-12183).
- For immunoblotting analysis samples were subjected to 15% SDS-PAGE and the separated proteins were electrophoretically transferred to nitrocellulose membranes (Hybond-C Super, Amersham). The membranes were blocked with PBS containing 5% dry fat free milk powder and 0.05% Tween 80 and further incubated with affinity purified anti-Trx2 antibodies.
- Trx2 is only present in mitochondrial fractions as neither cytosolic nor peroxisome enriched fractions displayed any signal. Rat Trxl did not cross-react with the affinity purified antibodies against Trx2.
- Trx2 content in total cell-free extracts from rat liver was estimated to be around 0.1 ⁇ g/mg protein (data not shown). The transient preprotein with the mitochondrial translocation peptide was not detected indicating that the translocation process is very fast.
- the recombinant Trx2 in lane 1 has a higher molecular weight because the His-tag was not removed by thrombin.
- Trx2 In order to confirm the specificity of the recombinant Trx2, the inventors examined the reduction of insulin, a classical assay in which thioredoxin catalyzes disulfide reduction of insulin with NADPH in the presence of mammalian thioredoxin reductase (TR). They compared the activities of human thioredoxin with the recombinant Trx2.
- the insulin disulfide reduction assay was essentially performed as described (Holgrem, A., BjUrnstedt, M. (1995) Methods Enzymol 252, 199-208)with a slight modification to activate Trxl and Trx2 by reduction.
- Trxl and Trx2 were preincubated at 37°C for 20 min with 2 ⁇ l of: 50 mM Hepes pH 7.6, 100 ⁇ g/ml BSA and 2 mM DTT, in a total volume of 70 ⁇ l. Then, 40 ⁇ l of a reaction mixture composed of 200 ⁇ l Hepes (1M) pH 7.6, 40 ⁇ l EDTA (0.2 M), 40 ⁇ l NADPH (40 mg/ml) and 500 ⁇ l insulin (10 mg/ml) was added. The reaction started with the addition of 10 ⁇ l of TR from calf-thymus (3.0 A 4 ⁇ 2 unit) and incubation continued for 20 min at 37°C.
- Trx2 cDNA encoding Trx2 has been isolated. This is shown in Figure 9. This is 87.4 % homologous to rat Trx2 cDNA.
- Trx2 The predicted amino acid sequence of human Trx2 is shown in Figure 10. This also contains a putative mitochondrial prepeptide protease cleavage site between Ser58 and Leu59.
- Trx2 In order to determine which proteins Trx 2 interacts with, a human liver cDNA library was used to isolate Trx2 interacting proteins with the yeast two hybrid system. Trx2 was cloned into the pGBT9y vector (Clontech), expressing the DNA binding domain of GAL4 transcriptional activator fused with Trx2. The liver cDNA library was cloned into the pGADIO vector (Clontech) that expresses the cloned cDNA fused with the DNA activation domain of GAL4.
- the proteins isolated were fibrinogen ⁇ -chain precursor, plasminogen, vitronectin, PDI, ⁇ -actin, serine hydroxymetyltransferase, NF-kB p65 and two cDNA sequences that code for proteins with no apparent homology to known proteins in a protein sequence data base (Figure 11).
- a rat brain cDNA library screened in the same way as above, the following clones were isolated: cytochrome c oxidase, rat ribosomal protein S17, c-myc intron binding protein (zinc finger protein), and five cDNA sequences that code for proteins with no apparent homology in the database.
- the cDNA sequences can be seen in Figure 12.
- the human PAC library (No. 704 purchased from the Resource Center/Primary Database of the German Human Genome Project, Berlin), constructed from a human male fibroblast cell line and ligated into pCYPAC-2 (Lehrach et al, (1990); Loannou et al, (1994)), was screened with the genomic DNA probe H6-2. Trx2 from the human Trx2 gene. The 4.2 kb fragment was labelled with 2 P by random priming and hybridized to the filter bound PAC clones under stringent conditions (45% formamide, 42°C) over night.
- Somatic cell hybrid panel Fluorescence in situ Hybridization (FISH)
- the PAC clone of trx2 was used as a probe after labelling with biotin- 16-dUTP by nick-translation.
- slides with human metaphase chromosomes were prepared using standard procedures. The slides were postfixed, Rnase treated and denatured as previously described (Pinkel et al (1986) Proc. Natl. Acad. Sci USA 83, 2934-2938).
- the PAC clone for Trx2 was used as a probe after labelling with biotin- 16-dUTP by nick-translation.
- the probe (50ng, lOOng) was pre-annealed with Cot-1 DNA (2.5-3.5 (g) for 30-60 min at 37°C after denaturing in 68°C for 10 min. Hybridization was performed in 50% formamide at 37°C overnight in a moist chamber. The slides were then washed three times for 5 min in 50% formamide, 2XSSC at 42»C and three times in 0.1XSSC at 60 « C. After washing, the hybridized probe was coupled to fluorescein-isothiocyanate-avidin D and the fluorescent signal was amplified by three successive treatments with biotinylated anti-avidin antibodies alternated with fluorescein-isothiocyanate-avidin D (Vector Lab).
- the slides were mounted in glycerol containing 2.3% DABCO (1,4-diazabicyclo- (2,2,2) octane) as antifade, and DAPI (4,6- diamino-2-phenyl-indole) at 0.5(g/ml as counterstain.
- the signal was visualized using a Zeiss Axioskop fluorescence microscope equipped with a cooled CCD-camera (Photometries Sensys) controlled by a Power Macintosh
- Quadra 950 computer Gray scale images were captured, pseudocolored and merged using the SmartCapture software (Vysis). The hybridisation signal was detected as symmetrical spots on both chromatids of the homologues chromosomes 22 at ql 31.1.
- trx2 cDNA probe labelled with 32 P dUTP was hybridized to a human mRNA master blot (Clontech). After exposing the blot to autoradiography film, densitometric measurements were performed on the dots appearing on the film. The values measured were then corrected with G3PDH (glyceraldehyde-3-phosphate dehydrogenase). The highest value was set as 100% and the rest were then compared to the set value. The relative values for the various tissue mRNAs blotted can be seen in Fig 15.
- Trx2 is located in the inner mitochondrial membrane.
- the rats were anesthesized with pentobarbital and perfused transcardially first with 100ml of saline followed by a mixture of 4% paraformaldehyde and 0,1% picric acid in 0.1 M phosphate buffered saline (PBS; pH. 7.3) for 4-5 minutes. After perfusion the tissues were excised and further fixed by immersion in the same solution for 60 min. The samples were cryoprotected with 15% sucrose in PBS before sectioning in the cryostat. Immunocytochemistry was performed using ABC-method. The sections were first incubated with rabbit antiserum against Trx2 (dil. 1:250 in PBS containing 1% BSA and 0.3% Triton X-100) for 12-24h.
- the anesthesized animals were perfused with a mixture of 4% paraformaldehyde, 0.1% picric acid and 0.2% glutaraldehyde in PBS for 5 min.
- the tissues were postfixed by immersion for 5-6 h.
- the tissues were cryoprotected with 50% sucrose and 10% glycerol in PBS for several days.
- the tissues were rapidly frozen in liquid nitrogen and 50um frozen sections were cut with the cryostat.
- the sections were processed free floating.
- the Trx2 antibody was diluted (1:50) with PBS containing 1% BSA and 0.2% Saponin and the sections were incubated for 4-6 days with the antibody. After several washes the sections were incubated with secondary antibody and ABC-complex for 12 h each.
- Diaminobenzidine was used as chromogen. Subsequently the section were fixed with 2% glutaraldehyde, 1% osmium tetraoxide and 1% uranyl acetate for 30 min each. The tissues were dehydrated with ethanol and embedded in Epon. The samples were processed for electron microscopy and the thin sections were examined in Jeol 1200 electron microscope.
- Nitric acid is a short lived free radical gas with a variety of physiological roles which include S-nitrosylation of proteins in vivo. Since Thioredoxin 1 is well known to scavenge free radicals (Schallreuter, K.V., Wood J. M. (1986) Biochem. Biophys. Res. Commun 136, 630-637) and can reverse the action of NO in API in vitro. (Nikitorite et al (1998) Biochem. Biophys Res. Commun. 242, 109-112), it is therefore possible that trx2 located in mitochandria may have a similar function to trxl, thus reversing the effect of NO on mitochondrial proteins. 13. Immunocytochemistry and in situ hybridization in different tissues of adult male and female rat
- the sections were incubated in humidified boxes at 42°C for 18h with 5ng/ml of the labeled probe in the hybridization cocktail, washed, dried and covered with Amersham B-max autoradiograph film for 30-60 days.
- the sections were dipped in Kodak NTB2 nuclear track emulsion (Rochester, NY) and exposed for 90 days at 4°C.
- the sections were examined in a Nikon Microphot-FXA microscope equipped for dark-field and epipolarization microscopy. Finally, the sections were stained with cresyl violet and analyzed under brightfield conditions.
- Immuno The epithelial cells are clearly labelled.
- Immuno Strong labelling in collapsing follicles. Some labelling in corpus luteum. Labelling in Oocytes in primordial follicles. Clear signal in epithelium in uterus and also staining in smooth muscle cells in myometrium.
- Immuno Labelling in keratinocytes in the basal epidermis. Labelling in some cells in hair follicles. Labelling in epithelial cells in sweat glands.
- Adrenal gland Strong signal with in situ in the adrenal cortex, low signal in medulla.
- Salivary gland Some labelled cells in secretory alveoli.
- Stomach Strong labelling in the mucosa, in parietal cells (secrete acid) of gastric glands and in the epithelial cells of the mucosa.
- Duodenum Strong labelling in epithelial cells in the mucosa.
- Liver Uniform clear signal with in situ. Clear labelling in most of the hepatocytes, some are very strongly stained.
- Lens Very strong staining in subcapsular epithelium of the lens.
- Retina Staining in ganglion neurons and in bipolar neurons (rods and cones negative).
- lymph nodes Clear signal with in lymph nodes, spleen, thymus and bone marrow. Strong staining in large number of lymphocytes (proliferating cells in germinal centres) in lymph nodes with immuno. Many cells stained both in spleen and in thymus. In bone marrow several celltypes, megacaryocytes are easily identified.
- Labelling in chondrocytes in cartilage Labelling in cells lining bone. Strong labelling in some cells around developing bone. With in situ very strong signal in some cells in developing bone.
- Motoneurons in spinal cord are labelled. Large number of the sensory neurons labelled, strongest labelling in small neurons (related to pain) and middle-sized neurons.
- Trx2 is mainly localised in special sets of neurons (mitral cells in olfactory bulb, magnocellular neurons in supraoptic nucleus, substantia nigra, Purkinje cells).
- Heart In situ: Strong signal both in atrium and in ventrical (in cardiac muscle). Immuno: Strong staining in most of the muscle cells.
- Blood vessels Clear staining of the smooth muscle cells and in small blood vessels.
- Trx2 may have a role in the limitation and/or reversal of neuron damage since a common factor in a variety of neurodegenerative diseases is the production of free radicals.
- Trx2 may also play a significant role in the control of pain, since there are a large number of pain-related sensory neurons in the peripheral nervous system which are strongly labelled with the Trx2 antibody.
- Trx2 shows an interesting two-domain structure consisting of a N-terminal part of a 60 amino acid region rich in basic amino acids with a theoretical pi of 12.1 and a C-terminal part homologous to thioredoxin with a pi of 4.8.
- the N-terminal of Trx2 has characteristic properties of a mitochondrial translocation peptide and a proposed protease cleavage site which may give a mature protein of 12.2 kD.
- a slightly larger mitochondrial form of Trx compared to the cytosolic Trx, has been reported to be present in pig heart based on electrophoretic mobility (Bodenstein, J., Follman, H. (1990) Z.
- Trx2 is phylogenetically closer to prokaryotic than mammalian thioredoxin some amino acids conserved in all prokaryotes like Trp-28 are not conserved in Trx2. Also the differences in amino acids involved in protein interaction such as Ala-93 and Glu-57 will probably confer a different specificity for Trx2 compared to Trx 1. All previously described mammalian thioredoxins have 2-3 additional cysteine residues to the two located in the active site. These structural or non catalytic cysteine residues can undergo oxidation, a process which leads to inactivation. From the structure of reduced human thioredoxin Cys-72 is located in a loop in proximity to the active site. Xilin et al.
- Trx2 shows that Cys-72 is responsible for dimer formation and subsequent loss of activity.
- the absence of corresponding structural cysteines in Trx2 confers a resistance to oxidation. This property might have important physiological implications for the role of Trx2.
- Trx Mammalian Trx can be found in many different cellular compartments including nucleus, endoplasmic reticulum, mitochondria and plasma membrane (Martin, H., Dean, M. (1991) Biochem. Biophys. Res. Commun 175, 123-128), (Holgrem, A., Luthman, M. (1978) Biochemistry 17, 4071-4077)). Also Trx is differentially regulated and has separate functions including promotion of cell growth to transcription factor activation and radical scavenging activities. Trx2 is highly expressed in tissues such as heart and skeletal muscle where Trxl protein is not detectable (Fujii, S. et al (1991). Virchows Archiv A, Pathological Anatomy & Histopathology 419, 317-326).
- ROI Reactive oxygen intermediates
- An important source of ROI are mitochondria (Turrens, J. F., Boveris, R. (1980). Biochem. J. 191, 421-427).
- ROI are regarded as toxic and harmful metabolites and when their formation occurs in an uncontrolled fashion, they may be implicated in several diseases by inducing lipid peroxidation and disruption of structural proteins, enzymes and nucleic acids.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Toxicology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Ophthalmology & Optometry (AREA)
- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU57745/98A AU5774598A (en) | 1997-01-28 | 1998-01-28 | Mammalian thioredoxin |
CA002279241A CA2279241A1 (en) | 1997-01-28 | 1998-01-28 | Mammalian thioredoxin |
JP53176098A JP2001510997A (en) | 1997-01-28 | 1998-01-28 | Mammalian thioredoxin |
EP98901415A EP1012296A2 (en) | 1997-01-28 | 1998-01-28 | Mammalian thioredoxin |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9701710.7A GB9701710D0 (en) | 1997-01-28 | 1997-01-28 | Mammalian protein |
GB9701710.7 | 1997-01-28 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1998032863A2 true WO1998032863A2 (en) | 1998-07-30 |
WO1998032863A3 WO1998032863A3 (en) | 1998-11-05 |
Family
ID=10806693
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1998/000263 WO1998032863A2 (en) | 1997-01-28 | 1998-01-28 | Mammalian thioredoxin |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP1012296A2 (en) |
JP (1) | JP2001510997A (en) |
KR (1) | KR20000070582A (en) |
AU (1) | AU5774598A (en) |
CA (1) | CA2279241A1 (en) |
GB (1) | GB9701710D0 (en) |
WO (1) | WO1998032863A2 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001288103A (en) * | 2000-02-02 | 2001-10-16 | Oriental Yeast Co Ltd | Thioredoxin composition stable in solution and method for producing the same |
WO2002000260A1 (en) * | 2000-06-29 | 2002-01-03 | Mitsubishi Pharma Corporation | Remedial agent for optic nerve disease and the like |
WO2002081513A3 (en) * | 2001-04-06 | 2003-05-01 | Novartis Ag | Disease-associated protein |
US7825161B2 (en) * | 2003-12-15 | 2010-11-02 | Nano-C, Inc. | Higher fullerenes useful as radical scavengers |
CN104062294A (en) * | 2014-07-11 | 2014-09-24 | 青岛千士医疗科技有限公司 | Method for detecting mitochondria damage of epithelial cells of bronchia/lung |
WO2019168364A1 (en) * | 2018-03-02 | 2019-09-06 | 사회복지법인 삼성생명공익재단 | Method for upregulation of thioredoxin expression in stem cells |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4925155B2 (en) * | 2002-06-10 | 2012-04-25 | レドックス・バイオサイエンス株式会社 | Retinal nerve cell function recovery agent |
WO2005087249A1 (en) * | 2004-03-11 | 2005-09-22 | Kurume University | Protease inhibitor and preventives or remedies for diseases |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0425821A1 (en) * | 1989-09-29 | 1991-05-08 | Ajinomoto Co., Inc. | The use of human ADF (=Adult T-cell leukemia-derived factor) for producing a medicament |
-
1997
- 1997-01-28 GB GBGB9701710.7A patent/GB9701710D0/en active Pending
-
1998
- 1998-01-28 EP EP98901415A patent/EP1012296A2/en not_active Withdrawn
- 1998-01-28 KR KR1019997006829A patent/KR20000070582A/en not_active Application Discontinuation
- 1998-01-28 JP JP53176098A patent/JP2001510997A/en active Pending
- 1998-01-28 CA CA002279241A patent/CA2279241A1/en not_active Abandoned
- 1998-01-28 WO PCT/GB1998/000263 patent/WO1998032863A2/en not_active Application Discontinuation
- 1998-01-28 AU AU57745/98A patent/AU5774598A/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0425821A1 (en) * | 1989-09-29 | 1991-05-08 | Ajinomoto Co., Inc. | The use of human ADF (=Adult T-cell leukemia-derived factor) for producing a medicament |
Non-Patent Citations (7)
Title |
---|
EMBL Database Entry HS898283 Accession number N41898; 27 January 1996 HILLIER L. ET AL.:"The WashU-Merck EST Project" XP002067407 * |
EMBL Database Entry MMW027 Accession number W91027; 9 July 1996 MARRA M. ET AL.:"The WashU-HHMI Mouse EST Project" XP002067408 * |
GIANNIS SPYROU ET AL.: "Cloning and expression of a novel mammalian thioredoxin" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 5, 31 January 1997, MD US, pages 2936-2941, XP002067405 * |
HARIHARAN J ET AL: "Alternative forms of the human thioredoxin mRNA: identification and characterization" GENE, vol. 173, no. 2, 16 September 1996, page 265-270 XP004043228 * |
JOHANNA BODENSTEIN ET AL.: "characterization of two thioredoxins in pig heart including a new mitochondrial protein" ZEITSCHRIFT F]R NATURFORSCHUNG, vol. 46, no. 3-4, 1991, pages 270-279, XP002067404 * |
SHOJI WATABE ET AL.: "SP-22 is a thioredoxin-dependent peroxide reductase in mitochondria" EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 249, no. 1, October 1997, pages 52-60, XP002067406 * |
XILIN REN ET AL.: "Mutagenesis of structural half-cystine residues in human thioredoxin and effects on the regulation of activity by selenodiglutathione" BIOCHEMISTRY, vol. 32, no. 37, 21 September 1993, EASTON, PA US, pages 9701-9708, XP002067403 cited in the application * |
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001288103A (en) * | 2000-02-02 | 2001-10-16 | Oriental Yeast Co Ltd | Thioredoxin composition stable in solution and method for producing the same |
US7638516B2 (en) | 2000-06-29 | 2009-12-29 | Mei Co., Ltd. | Agent for therapeutic treatment of optic nerve diseases and the like |
WO2002000260A1 (en) * | 2000-06-29 | 2002-01-03 | Mitsubishi Pharma Corporation | Remedial agent for optic nerve disease and the like |
US8394756B2 (en) | 2001-04-06 | 2013-03-12 | Novartis Ag | Methods of increasing RDCVF 1 or RDCVF 2 polypeptides in retinal cells |
CN1529753A (en) * | 2001-04-06 | 2004-09-15 | ��˹��ŵ�� | Disease-associated protein |
US7795387B2 (en) | 2001-04-06 | 2010-09-14 | Novartis Ag | Rod-derived cone viability factor (RDCVF) and a method of enhancing cone cell survival by RDCVF |
US8071745B2 (en) | 2001-04-06 | 2011-12-06 | Novartis Ag | Polynucleotides encoding rod-derived cone viability factor (rdcvf) and methods of using the same |
US8114849B2 (en) | 2001-04-06 | 2012-02-14 | Novartis Ag | Retinal dystrophy-associated protein and uses thereof |
WO2002081513A3 (en) * | 2001-04-06 | 2003-05-01 | Novartis Ag | Disease-associated protein |
CN1529753B (en) * | 2001-04-06 | 2013-06-12 | 诺瓦提斯公司 | Disease-associated protein |
US8518695B2 (en) | 2001-04-06 | 2013-08-27 | Novartis Ag | Compositions comprising polynucleotides encoding RDCVF1 or RDCVF2 |
US8957043B2 (en) | 2001-04-06 | 2015-02-17 | Novartis Ag | Methods of treating retinitis pigmentosa using nucleic acids encoding RDCVF1 or RDCVF2 |
US7825161B2 (en) * | 2003-12-15 | 2010-11-02 | Nano-C, Inc. | Higher fullerenes useful as radical scavengers |
CN104062294A (en) * | 2014-07-11 | 2014-09-24 | 青岛千士医疗科技有限公司 | Method for detecting mitochondria damage of epithelial cells of bronchia/lung |
WO2019168364A1 (en) * | 2018-03-02 | 2019-09-06 | 사회복지법인 삼성생명공익재단 | Method for upregulation of thioredoxin expression in stem cells |
Also Published As
Publication number | Publication date |
---|---|
CA2279241A1 (en) | 1998-07-30 |
AU5774598A (en) | 1998-08-18 |
WO1998032863A3 (en) | 1998-11-05 |
KR20000070582A (en) | 2000-11-25 |
EP1012296A2 (en) | 2000-06-28 |
JP2001510997A (en) | 2001-08-07 |
GB9701710D0 (en) | 1997-03-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Perry et al. | Genetic evidence for an androgen-regulated epididymal secretory glutathione peroxidase whose transcript does not contain a selenocysteine codon | |
Eum et al. | HIV-1 Tat-mediated protein transduction of Cu, Zn-superoxide dismutase into pancreatic β cells in vitro and in vivo | |
EP2902035B1 (en) | Cell-permeable peptide inhibitors of the JNK signal transduction pathway for use in the treatment of inflammatory eye diseases. | |
US9150618B2 (en) | Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of chronic or non-chronic inflammatory eye diseases | |
CZ90999A3 (en) | Pharmaceutical preparation containing leptin antagonist, suitable for treating insulin resistance in diabetes mellitus of the type ii | |
CZ20011608A3 (en) | Nucleotide and protein sequences of Nogo genes and methods based thereon | |
IL199316A (en) | Purified antibody and kit comprising it for diagnosis of retinal dystrophy | |
DE69731463T2 (en) | RECOMBINANT RIBONUCLEASE PROTEINS | |
WO1998032863A2 (en) | Mammalian thioredoxin | |
JPH08283296A (en) | Human apolipoproteine analog and human superoxide dismutase analog | |
EP1692187B1 (en) | EC SOD and cell transducing EC SOD and use thereof | |
JP2016536306A (en) | Human relaxin analog, pharmaceutical composition thereof and pharmaceutical use thereof | |
AU765741B2 (en) | Sag: sensitive to apoptosis gene | |
JPH08504580A (en) | Recombinant dog gastric lipase and pharmaceutical composition | |
US20040152115A1 (en) | Manganese superoxide dismutase exon 3-deleted isoforms and nucleic acid molecules encoding the isoforms | |
EP0727490B1 (en) | Human-origin prostacyclin synthase | |
Granger et al. | Identification of a neutrophil chemotactic factor from Tritrichomonas foetus as superoxide dismutase | |
US6531305B1 (en) | Sperm associated protein kinase polypeptides, corresponding nucleic acids, and methods of use | |
US8748568B2 (en) | Isolated A-type FHF N-terminal domain peptides and methods of use | |
JPH02249487A (en) | Sheep growth hormone | |
JPH06245763A (en) | Human variant manganese superoxide dismutase | |
JP3328260B2 (en) | Recombinant DNA obtained by incorporating DNA encoding myosin heavy chain SM1 isoform protein into vector DNA, and microorganism and therapeutic agent for atherosclerosis containing the recombinant DNA | |
FR2857598A1 (en) | Use of fibronectin fragments for prevention or treatment of angiogenesis-related diseases, e.g. cancer, act by binding fibroblast growth factor-2, also new peptides and related nucleic acids and antibodies | |
AU754446B2 (en) | Novel NPY family member | |
JP3904584B2 (en) | Human prostacyclin synthase |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AU CA JP KR US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2279241 Country of ref document: CA Ref country code: CA Ref document number: 2279241 Kind code of ref document: A Format of ref document f/p: F Ref country code: JP Ref document number: 1998 531760 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1019997006829 Country of ref document: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1998901415 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 57745/98 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09355397 Country of ref document: US |
|
WWP | Wipo information: published in national office |
Ref document number: 1998901415 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1019997006829 Country of ref document: KR |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1019997006829 Country of ref document: KR |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1998901415 Country of ref document: EP |