23 results on '"Sukenaga Y"'
Search Results
2. Oral administration of a specific chymase inhibitor, NK3201, inhibits vascular proliferation in grafted vein
- Author
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Takai, S., Jin, D., Nishimoto, M., Yuda, A., Sakaguchi, M., Kamoshita, K., Ishida, K., Sukenaga, Y., Sasaki, S., and Miyazaki, M.
- Published
- 2001
- Full Text
- View/download PDF
3. Purification and characterization of a ubenimex (Bestatin)-sensitive aminopeptidase B-like enzyme from K562 human chronic myeloid leukemia cells
- Author
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Yamada, M., Sukenaga, Y., Fujii, H., and Abe, F.
- Published
- 1994
- Full Text
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4. Identification of SARS-CoV-2 M pro inhibitors containing P1' 4-fluorobenzothiazole moiety highly active against SARS-CoV-2.
- Author
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Higashi-Kuwata N, Tsuji K, Hayashi H, Bulut H, Kiso M, Imai M, Ogata-Aoki H, Ishii T, Kobayakawa T, Nakano K, Takamune N, Kishimoto N, Hattori SI, Das D, Uemura Y, Shimizu Y, Aoki M, Hasegawa K, Suzuki S, Nishiyama A, Saruwatari J, Shimizu Y, Sukenaga Y, Takamatsu Y, Tsuchiya K, Maeda K, Yoshimura K, Iida S, Ozono S, Suzuki T, Okamura T, Misumi S, Kawaoka Y, Tamamura H, and Mitsuya H
- Subjects
- Animals, Humans, Mice, Benzothiazoles, Molecular Docking Simulation, Viral Nonstructural Proteins chemistry, Antiviral Agents pharmacology, COVID-19, Protease Inhibitors pharmacology, SARS-CoV-2 drug effects, Coronavirus 3C Proteases antagonists & inhibitors
- Abstract
COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (M
pro ) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro , apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro 's active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)- Published
- 2023
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5. 4'-modified nucleoside analogs: potent inhibitors active against entecavir-resistant hepatitis B virus.
- Author
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Takamatsu Y, Tanaka Y, Kohgo S, Murakami S, Singh K, Das D, Venzon DJ, Amano M, Higashi-Kuwata N, Aoki M, Delino NS, Hayashi S, Takahashi S, Sukenaga Y, Haraguchi K, Sarafianos SG, Maeda K, and Mitsuya H
- Subjects
- Animals, Drug Resistance, Viral, Guanine analogs & derivatives, Guanine pharmacology, HIV-1 drug effects, Mice, Deoxyadenosines pharmacology, Hepatitis B virus drug effects
- Abstract
Unlabelled: Certain nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). However, both viruses often acquire NRTI resistance, making it crucial to develop more-potent agents that offer profound viral suppression. Here, we report that 4'-C-cyano-2-amino-2'-deoxyadenosine (CAdA) is a novel, highly potent inhibitor of both HBV (half maximal inhibitory concentration [IC50 ] = 0.4 nM) and HIV-1 (IC50 = 0.4 nM). In contrast, the approved anti-HBV NRTI, entecavir (ETV), potently inhibits HBV (IC50 = 0.7 nM), but is much less active against HIV-1 (IC50 = 1,000 nM). Similarly, the highly potent HIV-1 inhibitor, 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA; IC50 = 0.3 nM) is less active against HBV (IC50 = 160 nM). Southern analysis using Huh-7 cells transfected with HBV-containing plasmids demonstrated that CAdA was potent against both wild-type (IC50 = 7.2 nM) and ETV-resistant HBV (IC50 = 69.6 nM for HBVETV-RL180M/S202G/M204V), whereas ETV failed to reduce HBVETV-RL180M/S202G/M204V DNA even at 1 μM. Once-daily peroral administration of CAdA reduced HBVETV-RL180M/S202G/M204V viremia (P = 0.0005) in human-liver-chimeric/ HBVETV-RL180M/S202G/M204V-infected mice, whereas ETV completely failed to reduce HBVETV-RL180M/S202G/M204V viremia. None of the mice had significant drug-related body-weight or serum human-albumin concentration changes. Molecular modeling suggests that a shallower HBV-RT hydrophobic pocket at the polymerase active site can better accommodate the slightly shorter 4'-cyano of CAdA-triphosphate (TP), but not the longer 4'-ethynyl of EFdA-TP. In contrast, the deeper HIV-1-RT pocket can efficiently accommodate the 4'-substitutions of both NRTIs. The ETV-TP's cyclopentyl ring can bind more efficiently at the shallow HBV-RT binding pocket., Conclusion: These data provide insights on the structural and functional associations of HBV- and HIV-1-RTs and show that CAdA may offer new therapeutic options for HBV patients., (© 2015 by the American Association for the Study of Liver Diseases.)
- Published
- 2015
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6. Lysocellin, a metabolite of the novel drug 'alopestatin', induces G1 arrest and prevents cytotoxicity induced by etoposide.
- Author
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Takahara Y, Yogosawa S, Maruyama S, Watanabe N, Yokoyama H, Fukasawa K, Sukenaga Y, Kamiyama J, Izumi M, Wakada M, Zhang H, Yoshizawa K, Kawa S, Nikaido T, and Sakai T
- Subjects
- Alopecia chemically induced, Alopecia prevention & control, Animals, Animals, Newborn, Antineoplastic Agents, Phytogenic pharmacology, Antineoplastic Agents, Phytogenic toxicity, Area Under Curve, Blotting, Northern, Blotting, Western, Cell Cycle drug effects, Cell Line, Tumor, Cell Survival drug effects, Cyclin D1 genetics, Cyclin D1 metabolism, Cyclin-Dependent Kinase Inhibitor p21 genetics, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Dose-Response Relationship, Drug, Down-Regulation drug effects, Etoposide toxicity, Female, Furans administration & dosage, Furans blood, Furans metabolism, Furans pharmacokinetics, Furans pharmacology, Gene Expression drug effects, Humans, Male, Osteosarcoma genetics, Osteosarcoma metabolism, Osteosarcoma pathology, Proteasome Endopeptidase Complex metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Rats, Sprague-Dawley, Signal Transduction drug effects, Up-Regulation drug effects, Cell Proliferation drug effects, Etoposide pharmacology, G1 Phase drug effects
- Abstract
We report here that lysocellin, a polyether antibiotic from a streptomycete, induces G1 phase arrest in human osteosarcoma MG63 cells. Lysocellin up-regulates p21WAF1/Cip1 and down-regulates cyclin D1 at the mRNA level. In addition, cyclin D1 is down-regulated by the proteasome-dependent signal pathway in MG63 cells. In drug combination studies, we found that lysocellin treatment weakened the cytotoxic activity of etoposide in MG63 cells using a colony-formation assay. To study the in vivo efficacy of lysocellin, we isolated a novel compound related to lysocellin from the same streptomycete, and found that the novel drug is converted to lysocellin in vivo and decreases etoposide-induced alopecia in a neonatal rat model. We raise the possibility that this novel drug, named 'alopestatin', may be a promising agent against alopecia.
- Published
- 2006
7. Impact of chymase inhibitor on cardiac function and survival after myocardial infarction.
- Author
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Jin D, Takai S, Yamada M, Sakaguchi M, Kamoshita K, Ishida K, Sukenaga Y, and Miyazaki M
- Subjects
- Animals, Cricetinae, Echocardiography, Doppler, Male, Myocardial Infarction blood, Myocardial Infarction diagnostic imaging, Peptidyl-Dipeptidase A blood, Renin blood, Survival Rate, Acetamides therapeutic use, Myocardial Infarction drug therapy, Myocardium enzymology, Pyrimidines therapeutic use, Serine Proteinase Inhibitors therapeutic use
- Abstract
Objectives: Recent studies have demonstrated that hamsters, like humans, possess both angiotensin converting enzyme (ACE)- and chymase-dependent angiotensin (Ang) II-forming pathways in cardiovascular tissues. We recently found that, after myocardial infarction (MI) in hamsters, cardiac chymase was significantly activated. In order to determine whether suppression of cardiac chymase activity could provide prognostic benefit after MI, we examined the effects of NK3201, a novel, orally active and specific chymase inhibitor, on cardiac function and survival during the acute phase of MI in hamsters., Methods: Two hundred and ten male Syrian hamsters were used in the present study. The left coronary artery of each hamster was ligated to induce MI. NK3201 at a dose of 30 mg/kg per day was administered orally by gastric gavage, starting either 3 days before or 1 day after MI., Results: ACE and chymase activities were significantly increased in the infarcted left ventricle 3 days after MI. NK3201 treatment starting 3 days before MI significantly inhibited the increase in cardiac chymase activity, while it did not affect ACE activity either in plasma or in heart 3 days after MI. A significant improvement in cardiac function was observed 3 and 14 days after MI in the group receiving NK3201. Compared with vehicle treatment, NK3201 treatment initiated either 3 days before or 1 day after MI significantly reduced the mortality rate during the 14 days of observation following MI., Conclusions: These findings indicate that cardiac chymase plays an important role after MI and this finding may provide a novel therapeutic target in post-MI treatment.
- Published
- 2003
- Full Text
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8. A novel chymase inhibitor, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[[,4-dioxo-1-phenyl-7-(2-pyridyloxy)]2-heptyl]acetamide (NK3201), suppressed intimal hyperplasia after balloon injury.
- Author
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Takai S, Sakonjo H, Fukuda K, Jin D, Sakaguchi M, Kamoshita K, Ishida K, Sukenaga Y, and Miyazaki M
- Subjects
- Acetamides therapeutic use, Animals, Carotid Artery, Common enzymology, Chymases, Dogs, Hyperplasia, Pyrimidines therapeutic use, Serine Proteinase Inhibitors therapeutic use, Tunica Intima enzymology, Tunica Intima injuries, Acetamides pharmacology, Carotid Artery, Common drug effects, Carotid Artery, Common pathology, Catheterization adverse effects, Pyrimidines pharmacology, Serine Endopeptidases metabolism, Serine Proteinase Inhibitors pharmacology, Tunica Intima drug effects, Tunica Intima pathology
- Abstract
In this study, we investigated whether an orally active chymase inhibitor, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[[3,4-dioxo-1-phenyl-7-(2-pyridyloxy)]-2-heptyl]acetamide (NK3201), prevents intimal hyperplasia in carotid arteries injured by a balloon catheter in dog. Each dog was administered NK3201 (1 mg/kg per day, p.o.) or placebo beginning 5 days before balloon injury and continuing through the experiments. Four weeks after balloon injury, NK3201 did not affect the plasma renin and angiotensin-converting enzyme activities. The chymase activity was significantly increased in the injured arteries, whereas the angiotensin-converting enzyme activity was not. NK3201 significantly reduced the chymase activity in the injured arteries. The intimal area in the placebo- and NK3201-treated group and was 0.46 +/- 0.06 and 0.24 +/- 0.04 mm2, respectively, and this difference was significant. In this study, we demonstrated for the first time that a chymase inhibitor prevented the development of intimal hyperplasia in the balloon-injured arteries.
- Published
- 2003
- Full Text
- View/download PDF
9. Development of the chymase inhibitor as an anti-tissue-remodeling drug: myocardial infarction and some other possibilities.
- Author
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Sukenaga Y, Kamoshita K, Takai S, and Miyazaki M
- Subjects
- Acetamides pharmacokinetics, Acetamides therapeutic use, Animals, Chymases, Cricetinae, Myocardial Infarction metabolism, Myocardial Infarction pathology, Pyrimidines pharmacokinetics, Pyrimidines therapeutic use, Serine Proteinase Inhibitors pharmacokinetics, Serine Proteinase Inhibitors therapeutic use, Acetamides pharmacology, Myocardial Infarction drug therapy, Pyrimidines pharmacology, Serine Endopeptidases metabolism, Serine Proteinase Inhibitors pharmacology, Ventricular Remodeling drug effects
- Abstract
Chymase leading to tissue remodeling is expected to be a potent pharmaceutical target. Its functions in vivo are still unclear, because of lack of orally available inhibitors. Recently, however, the chymase inhibitor NK3201 (2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidin-1-yl)-N-[[3,4-dioxo-1-phenyl-7-(2-pyridyloxy)]-2-heptyl] acetamide) was demonstrated to have oral activity against neointimal hyperplasia in dog models (Takai S. et al., Life Sci 69, 1725 - 1732 (2001)). In this review, by showing the efficacy of NK3201 in some hamster models, chymase functions in vivo are summarized, and the potency of this chymase inhibitor is introduced. In vitro study, NK3201 showed potent chymase specific inhibitory activity, and Dixon plot analysis indicated competitive inhibition. Oral administration of NK3201 into normal rats resulted in rapid spread over every tissue except the brain, and sufficient activity to inhibit tissue chymase was detected even after 24 h. In passive cutaneous anaphylaxis, myocardial infarction and bleomycin-induced pulmonary fibrosis models, orally administered NK3201 showed potent inhibition of inflammatory response, tissue angiotensin II formation, and fibrosis, respectively. These data suggest that chymase has a vital role in tissue remodeling through promotion of the inflammatory response, tissue angiotensin II and tissue fibrosis. Our recent data indicated chymase participation in bladder fibrosis, like interstitial cystitis. Therefore, the orally active chymase inhibitor NK3201 may have protective effects on tissue remodeling in several diseases.
- Published
- 2002
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10. Oxidative modification of NK30424A and B enhance inhibitory effect on lipopolysaccharide-induced tumour necrosis factor-alpha promoter activity.
- Author
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Takayasu Y, Tsuchiya K, and Sukenaga Y
- Subjects
- Animals, Cell Line, Cysteine chemistry, Cysteine pharmacology, Lipopolysaccharides pharmacology, Macrophages, Magnetic Resonance Spectroscopy, Mice, Oxidation-Reduction, Promoter Regions, Genetic genetics, Transfection, Tumor Necrosis Factor-alpha metabolism, Cysteine analogs & derivatives, Lactones chemistry, Lactones pharmacology, Lipopolysaccharides antagonists & inhibitors, Promoter Regions, Genetic drug effects, Tumor Necrosis Factor-alpha genetics
- Published
- 2002
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11. NK30424A and B, novel inhibitors of lipopolysaccharide-induced tumour necrosis factor alpha production, produced by Streptomyces sp. NA30424.
- Author
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Takayasu Y, Tsuchiya K, Aoyama T, and Sukenaga Y
- Subjects
- Humans, Lipopolysaccharides pharmacology, Stereoisomerism, Lactones blood, Lipopolysaccharides antagonists & inhibitors, Tumor Necrosis Factor-alpha biosynthesis
- Published
- 2001
- Full Text
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12. Bronchodilator and cardiovascular effects of NKH477, a novel water-soluble forskolin derivative, in guinea pigs.
- Author
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Iida M, Fujita N, Hosono M, and Sukenaga Y
- Subjects
- Animals, Blood Pressure drug effects, Colforsin pharmacology, Dose-Response Relationship, Drug, Guinea Pigs, Heart Rate drug effects, Injections, Intravenous, Male, Time Factors, Bronchodilator Agents pharmacology, Colforsin analogs & derivatives, Muscle, Smooth, Vascular drug effects, Vasodilator Agents pharmacology
- Abstract
The bronchodilator and cardiovascular effects of NKH477 (6-(3-dimethylaminopropionyl)forskolin hydrochloride) were evaluated. In anesthetized guinea pigs, i.v. bolus injections of NKH477 (1-100 micrograms/kg) inhibited the bronchoconstriction induced by inhaled leukotriene D4, increased the heart rate (HR) and decreased the diastolic arterial blood pressure (DBP) in a dose-dependent manner. The bronchodilator effect of NKH477 was 1500 times more potent than that of aminophylline and 17 times less potent than that of isoproterenol. The selectivity of NKH477 for bronchodilation vs an increase in HR was 15 times higher than that of isoproterenol and similar to that of aminophylline; and vs a decrease in DBP, the selectivity was 4 times higher than that of aminophylline and similar to that of isoproterenol. I.v. infusion of NKH477 (0.1-3 micrograms/kg/min) for 2 hr dose-dependently inhibited the bronchoconstriction induced by i.v. histamine. Isoproterenol (0.1 microgram/kg/min, i.v.) enhanced the bronchoconstriction after termination of the infusion, whereas NKH477 did not. In conscious guinea pigs, inhalation of NKH477 (0.1-5 mg/ml) concentration-dependently inhibited the bronchoconstriction induced by inhaled histamine, and a high concentration of NKH477 (35.4 mg/ml) increased the HR. The bronchodilator effect of inhaled NKH477 was 15 times less potent than that of isoproterenol. The selectivity of inhaled NKH477 was similar to that of isoproterenol. These results indicate that NKH477 may be useful as a bronchodilator.
- Published
- 1995
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13. Purification and molecular cloning of chymase from human tonsils.
- Author
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Sukenaga Y, Kido H, Neki A, Enomoto M, Ishida K, Takagi K, and Katunuma N
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Child, Chymases, Cloning, Molecular, DNA, Humans, Molecular Sequence Data, Polymerase Chain Reaction, Sequence Homology, Amino Acid, Serine Endopeptidases isolation & purification, Palatine Tonsil enzymology, Serine Endopeptidases genetics
- Abstract
A chymotrypsin-like protease was purified to homogeneity from human tonsils by a series of chromatographic procedures. The purified enzyme gave a single protein band with an apparent molecular mass of 30 kDa on SDS-PAGE. The sequence of the first 21 amino acids at the N-terminus of the enzyme was determined. A cDNA for the enzyme was cloned by PCR amplification from extracted tonsillar mRNA using a supposed N-terminal oligonucleotide primer and a conserved C-terminal primer of the chymase family. The deduced amino acid sequence of the isolated clone was identical to that of human chymase in connective tissue-type mast cells from heart except for a Ser instead of a Cys at the N-terminal 7th position.
- Published
- 1993
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14. The 5' untranslated region of the human cellular glutathione peroxidase gene is indispensable for its expression in COS-7 cells.
- Author
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Kurata H, Kamoshita K, Kawai E, Sukenaga Y, and Mizutani T
- Subjects
- Amino Acid Sequence, Animals, Base Composition, Base Sequence, Blotting, Western, Cell Line, Exons, Gene Expression, Genetic Vectors, Humans, Introns, Molecular Sequence Data, Mutagenesis, Site-Directed, Nucleic Acid Conformation, Protein Biosynthesis, RNA, Messenger genetics, RNA, Messenger metabolism, Genes, Glutathione Peroxidase genetics, Transfection
- Abstract
We studied the expression of the human cellular glutathione peroxidase (GPx) gene, from which a key enzyme containing selenocysteine (Scy) at the active site is produced. Expression of some human GPx gene mutants in COS-7 cells revealed that the 5' untranslated region (utr) was necessary for expression of the GPx gene, since mutant genes having 10 base pairs (bps) at the 5'utr (the complete had 311 bps) expressed GPx at very low levels. The genes with 311 or 408 bps at the 5'utr were better expressed than those having 257 bps. The GPx gene having 133 bps at the 3'utr (80 bps shorter than the entire length) was highly expressed. This deletion did not influence expression. We constructed some mutants in which 3 bases were altered at the upstream region of the Scy UGA codon in the frame of the GPx gene, by site-directed mutagenesis. GPx expression decreased but the expression was restored. Therefore, the upstream region of the in-frame Scy codon was not essential in the Scy decoding mechanisms. Finally, the 5'utr was essential for the expression of GPx gene. However, the deletion of a part of the 3'utr and the site-directed mutation upstream of the Scy codon did not show drastic effects on the expression.
- Published
- 1992
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15. Changes in Cu,Zn-superoxide dismutase gene during induced erythroid and myeloid differentiation.
- Author
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Tomoda T, Nomura I, Kurashige T, Kubonishi I, Miyoshi I, Sukenaga Y, and Taniguchi T
- Subjects
- Blotting, Southern methods, Bone Marrow Cells, Cell Differentiation genetics, Cell Line, DNA isolation & purification, Erythroid Precursor Cells cytology, Humans, Leukemia, Myelogenous, Chronic, BCR-ABL Positive enzymology, Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Leukemia, Promyelocytic, Acute enzymology, Leukemia, Promyelocytic, Acute genetics, Leukemia, Promyelocytic, Acute pathology, RNA, Messenger analysis, Superoxide Dismutase analysis, Superoxide Dismutase isolation & purification, Tumor Cells, Cultured cytology, Tumor Cells, Cultured enzymology, Bone Marrow enzymology, Erythroid Precursor Cells enzymology, Genes, Superoxide Dismutase genetics
- Abstract
We investigated the alteration of Cu,Zn-superoxide dismutase during erythroid and myeloid differentiation in order to elucidate its physiological significance in different types of cells. We measured enzyme activity and mRNA levels of superoxide dismutase in the process of differentiation to erythroid cells or myeloid cells. When human leukemia K562 cells are incubated in the presence of 80 microM hemin, benzidine-positive cells appear on day 1 and 80% of the cells become positive on day 5. During hemin-induced erythroid differentiation, Cu,Zn-superoxide dismutase activity increases 3.5-fold of the initial value and mRNA for Cu,Zn-superoxide dismutase increases prior to the activity to the same extent. On the other hand, when human promyelocytic leukemia HL-60 cells are incubated in the presence of 1.3% dimethyl sulfoxide, nitroblue tetrazolium-positive cells reach approximately 90% on day 5. During dimethyl sulfoxide-induced myeloid differentiation, the activity of Cu,Zn-superoxide dismutase decreases below 15% of the initial value on day 5 and mRNA for Cu,Zn-superoxide dismutase decreases as well. The results indicate that the synthesis of superoxide dismutase is linked with either the erythroid or myeloid differentiation program.
- Published
- 1991
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16. Ligand bindings of bovine carboxypeptidase B. III. Hydrophobic activators in dipeptide hydrolysis.
- Author
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Kuroda K, Akanuma H, Sukenaga Y, Sugihara H, and Yamasaki M
- Subjects
- Animals, Carboxypeptidase B, Cattle, Enzyme Activation, Kinetics, Ligands, Pancreas enzymology, Protein Binding, Structure-Activity Relationship, Alcohols pharmacology, Carboxypeptidases metabolism, Dipeptides pharmacology
- Abstract
Several hydrophobic compounds acted as activators in dipeptide (Bz-Gly-L-Arg-OH, Z-Gly-L-Phe-OH) hydrolysis by bovine carboxypeptidase B. These hydrophobic compounds include Bz-Gly-OH, Z-Gly-OH, Z-L-Phe-OH, and Z-L-Phe-GLy-OH. These compounds were indicated to bind to the secondary substrate binding sites which is proposed to be responsible for substrate activation kinetics in dipeptide hydrolysis. Of the compounds Z-L-Phe-OH alone acted also as a inhibitor at higher concentrations, indicating that it binds to both primary and secondary sites as the dipeptide substrates do. Comparison of the activation effects of the compounds employed indicated that hydrophobic interaction played an important role in binding to the secondary site. Substrate and modifier binding constants were also determined and the results indicated that modifier binding increased both affinity and catalytic rate constant of the primary site. On the other hand, Z-Gly-OH and Z-L-Phe-Gly-OH inhibited the hydrolyses of tri and tetrapeptide substrates. This observation suggests that the secondary site is contained in the extended active center which the enzyme possibly has.
- Published
- 1980
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17. Ligand binding of bovine carboxypeptidase B. IV. Oligopeptide substrates and extended active center.
- Author
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Sukenaga Y, Akanuma H, and Yamasaki M
- Subjects
- Animals, Binding Sites, Binding, Competitive, Carboxypeptidase B, Cattle, Chromatography, Affinity, Kinetics, Ligands, Pancreas enzymology, Protein Binding, Substrate Specificity, Carboxypeptidases metabolism, Oligopeptides
- Abstract
Catalytic and binding properties of bovine carboxypeptidase B were studied by kinetic and affinity chromatographic methods both using several oligopeptides as substrates or immobilized ligands. These oligopeptides contained either arginines or phenylalanines at carboxy termini as well as phenylalanyl residues in one of the other positions. The chromatographic studies showed that the phenylalanyl residues in endo-positions play a significant role in binding of the immobilized peptides to the enzyme, while the kinetics studies indicated further that the presence of an internal hydrophobic residue in a substrate was advantageous for the catalytic release of the carboxyl terminal residue from the substrate. These observations support the supposition that the enzyme has an extended active center which contains an extended hydrophobic binding site. Several hydrophobic compounds, which have been shown to act as activators in dipeptide substrate hydrolyses, showed inhibitory effect on hydrolyses of oligopeptide substrates. This observation suggests that these hydrophobic compounds bind to a portion of the hydrophobic site in the active center.
- Published
- 1980
- Full Text
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18. cDNA sequence coding for human glutathione peroxidase.
- Author
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Sukenaga Y, Ishida K, Takeda T, and Takagi K
- Subjects
- Amino Acid Sequence, Base Sequence, Humans, Liver enzymology, Molecular Sequence Data, DNA analysis, Genes, Glutathione Peroxidase genetics
- Published
- 1987
- Full Text
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19. [Recombinant human superoxide dismutase (r-hSOD)].
- Author
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Sukenaga Y and Katoh K
- Subjects
- Animals, Base Sequence, Chemical Phenomena, Chemistry, Physical, Cloning, Molecular, DNA genetics, Escherichia coli genetics, Genetic Code, Genetic Vectors, Humans, Molecular Sequence Data, Recombination, Genetic, Superoxide Dismutase genetics
- Published
- 1988
20. Nucleotide sequence of a human gene for glutathione peroxidase.
- Author
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Ishida K, Morino T, Takagi K, and Sukenaga Y
- Subjects
- Amino Acid Sequence, Base Sequence, Humans, Molecular Sequence Data, Genes, Glutathione Peroxidase genetics
- Published
- 1987
- Full Text
- View/download PDF
21. Ligand bindings of bovine carboxypeptidase B. II. Affinity chromatography and cooperative ligations.
- Author
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Sukenaga Y, Akanuma H, Suekane C, and Yamasaki M
- Subjects
- Animals, Arginine, Binding Sites, Carboxypeptidase B, Cattle, Chromatography, Affinity, Kinetics, Ligands, Models, Biological, Protein Binding, Solubility, Carboxypeptidases metabolism
- Abstract
Affinity chromatography was used to characterize the binding properties of carboxypeptidase B with its ligands. The affinity adsorbents employed included arginine directly attached to agarose beads, arginine attached to the same support through hydrophilic and hydrophobic spacers, and immobilized caproylphenylalanine. The enzyme showed marked retardation on all of the arginine columns but only slight retardation on the phenylalanine column. Several hydrophobic ligands in solution enhanced to some extent the enzyme retardation on columns having arginine directly attached to the solid support, while several amino- and guanidino-alkylcarboxylic acids (lysine and arginine analogs) greatly enhanced the enzyme retardation on the phenylalanine column and somewhat enhanced it on the other columns having hydrophobic spacer arms. These observations confirmed that the enzyme has twobinding sites for the soluble and immobilized ligands and that these two sites exhibit cooperative ligations. Binding constants of the enzyme for various soluble ligands were also calculated from their chromatographic effects and the resulting values were interpreted in terms of the cooperative action of the two bindings sites, i.e., one for the primary binding of basic amino acid analogs (SITE I) and the other for hydrophobic compounds (Site II). In this chromatographic study, however, such cooperation of the two sites was obscure when arginine, acylarginine, or alkylarginine was the ligand directing to Site I.
- Published
- 1980
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22. The detection of natural opal suppressor seryl-tRNA in Escherichia coli by the dot blot hybridization and phosphorylation by a tRNA kinase [corrected] .
- Author
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Mizutani T, Maruyama N, Hitaka T, and Sukenaga Y
- Subjects
- Chromatography, DNA Probes, Formate Dehydrogenases genetics, Nucleic Acid Hybridization, Phosphorylation, RNA, Bacterial metabolism, RNA, Transfer, Amino Acyl metabolism, Escherichia coli genetics, Phosphotransferases metabolism, Phosphotransferases (Alcohol Group Acceptor), RNA, Bacterial analysis, RNA, Transfer, Amino Acyl analysis
- Abstract
It was believed that there was no natural suppressor tRNA in Escherichia coli, however, it has been suggested that selC, relating to the synthesis of formate dehydrogenase of a selenoprotein [(1988) Nature 331, 723-725], codes for tRNA, even though the presence of tRNA has not yet been demonstrated. We detected the product of selC in the tRNA preparation of the E. coli MC 4100 strain by the dot blot hybridization method with a DNA probe (ACCGCTGGCGGC) corresponding to the extra arm of selC tRNA. Two hybridization peaks were found in the chromatographic pattern from Sephadex A50. The amount of tRNA was estimated to be about 0.03% of the total tRNA. Some tRNA [corrected] was phosphorylated by a tRNA kinase in E. coli B. These results suggest that the opal suppressor seryl-tRNA in E. coli should be converted to selenocysteyl-tRNA [corrected] and occurs in vertebrates as a general phenomenon.
- Published
- 1989
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23. Physicochemical properties of charge isomers of recombinant human superoxide dismutase.
- Author
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Kajihara J, Enomoto M, Seya K, Sukenaga Y, and Katoh K
- Subjects
- Amino Acids analysis, Chemical Phenomena, Chemistry, Humans, Metals analysis, Spectrum Analysis, Isoenzymes analysis, Recombinant Proteins analysis, Superoxide Dismutase analysis
- Abstract
Recombinant human Cu2Zn2SOD expressed in Escherichia coli consisted of mainly three isomers with isoelectric points of 5.14 (A), 5.06 (B), and 4.99 (C). Each isomer was isolated by DEAE-Toyopearl chromatography and the physiochemical properties were investigated. No significant differences in chemical and spectrophotometric properties, such as specific activity, metal contents, amino acid composition, and UV and ESR spectra, were found. The result of labeling of free cysteine residues with ABD-F showed the disulfide bond to be formed between 57Cys and 146Cys in every isomer. A few differences were found in the CD spectrum around 260 nm and in the elution patterns on reverse-phase HPLC. The isoelectric points of the three isomers became the same after treatment by reduction and carboxymethylation and even after reduction only, pI of isomers tended to be at the value of component (A). These results suggest that the three isomers are identical in primary structure but slightly different in secondary or tertiary structure. These differences are probably derived from structural alterations around 111Cys.
- Published
- 1988
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