| Project/Area Number |
07557149
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Biological pharmacy
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| Research Institution | Tohoku University |
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Principal Investigator |
YAMAZOE Yasushi Tohoku Univ., Fac.of Pharm., Professor, 薬学部, 教授 (00112699)
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| Co-Investigator(Kenkyū-buntansha) |
KUMAGAI Hiromichi Asahi Glass Co., Researcher, 中央研究所, 主任研究員
TAINAKA Hitoshi Amersharm K.K.Japan, Researcher, 総合研究所, 研究員
SUZUKI Shinya Tohoku Univ., Fac.of Pharm., Research Ass., 薬学部, 教務職員 (40196829)
NAGATA Kiyoshi Tohoku Univ., Fac.of Pharm., Assistant Prof., 薬学部, 助手 (80189133)
DEGAWA Masakuni Tohoku Univ., Fac.of Pharm., Assistant Prof., 薬学部, 助手 (50134002)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥11,900,000 (Direct Cost: ¥11,900,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1995: ¥7,100,000 (Direct Cost: ¥7,100,000)
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| Keywords | Drug Metabolism / Cytochrome P450 / Heterologous expression / Stable Expression / ヒト薬物代謝 / 遺伝子発現系 / CYP2C9 / CYP2C18 / CYP2C19 / S.Pombe / P450還元酵素 / ミクロソーム / cDNA発現 |
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| Research Abstract |
Both Saccharomyces cerevisiae and Shizosaccharomyces pombe contain genome and organe systems similar to mammalian cells. The latter is rich in endoplasmic reticulum and shown to be applicable for gene modification using expression vector for mammalian cells. Therefore, we looked for the feasibly of S.pombe for expression of human P450. Human livers contain at least 4 different members of CYP2C subfamilies of P450 (CYP2C8, CYP2C9, CYP2C18 and CYP2C19). These forms are involved in the biotrans formation of varies chemicals including clinically important drugs. We, thus, isolated cDNA clones of CYP2C9, CYP2C18, and CYP2C19 from human liver cDNA libraties. The isolated clones were initiallry introduced into S.pombe using pTL2-vectors. The transformants produced respective human CYP2C forms in endoplasmic reticulum but has only trace amounts of their catalytic activities, because of low levels of an electron transport component, NADPH-cyt. P450 reductase. To overcome this situation, we introduced cDNA of NADPH-cyt. P450 reductase in S.pombe genome to obtain stable expression of the activity. The clone obtained showed fairly high level of P450 reducatase activity. Using the reductase expressing cells, we introduced again CYP2C forms and the resultant co-expressed cells showed high levels of production of cytochrome P450 and P450 reductase. Experiments using drugs including omeprazole and dizepam clearly indicate the usefulness of these cell lines for drug metabolism studies.
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