Cloning of novel anti-oxidative-stress genes form hydrogen peroxide-and nitric oxide-resistant cells
| Project/Area Number |
12670146
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Wakayama Medical University |
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Principal Investigator |
NISHIKIMI Morirnitsu Wakayama Medical University Medicine, Professor, 医学部, 教授 (20022816)
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| Co-Investigator(Kenkyū-buntansha) |
YAMADA Michio Yokohama City University, Science, Professor, 理学部, 教授 (10076995)
INAI Yoko Wakayama Medical University Medicine, Research associate, 医学部, 助手 (20316087)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | hydrogen peroxide / nitric oxide / HL 60 / PCR-select subtraction / Rpn 9 / proteasome / alkaline phosphatase / oxidative stress / 過酸化水素耐性 / HL60細胞 / 差次的クローニング / NO耐性 / 抗酸化ストレス遺伝子 / サブトラクション法 |
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| Research Abstract |
This study was aimed at cloning of genes functioning to resist against oxidative stress in hydrogen peroxide- and nitric oxide-resistant cells. We attempted to screen such genes by PCR-select subtraction between hydrogen peroxide- or nitrogen oxide-resistant HL60 cells and their parental cells. In the former screening, the mRNA level of catalase was found to markedly elevated in the resistant cells, whereas expression of the myeloperoxidase gene was greatly suppressed. In this study, we focused on Rpn 9, one of the subunits of 19 S regulatory unit of the proteasome, whose clone was picked up in the first screening but eventually determined false positive, because the proteasome plays an important role in the degradation of oxidatively damaged proteins. It was found that a yeast mutant lacking the Rpn9-gene was more resistant to hydrogen peroxide than the wild type. Investigation of the basis of this phenomenon revealed that in the mutant yeast, the 20 S proteasome and the species migrating slightly slowly on native gel were increased in both protein amount and activity. Screening from nitrogen oxide-resistant HL60 cells led to an isolation of a clone coding for one of the subunits of cytochrome oxidase. Additionally, in view of the importance of sequestration of heavy metal for protection from oxidative stress, we investigated the copper-binding capacity and affinity of mouse brain cytosolic proteins and disclosed their remarkably large capacity and high affinity. To work out a system to quantitatively measure oxidative stress within the cell, we further constructed CHO cells that express bacterial alkaline phosphatase inside the cell, and found that they can be used for detection of oxidative stress.
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Report
(3 results)
Research Products
(3 results)
