Basic study for dunction and gene regulation of human fetal liver P-450

人胎肝P-450功能及基因调控的基础研究

• 批准号: 02454482
• 负责人: KAMATAKI Tetsuya
• 金额: $ 3.9万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454482/
• 关键词: P-450IIIA; Fetus; Liver; Aflatoxin B_1; SRalpha promoter; Dexamethasone; Cytochrome P-450; Nuclear polyhedorosis virus; チトクロムPー450; Pー450IIIA; SF9細胞; nuclear polyhedorosis virus; umu試験; 薬物代謝; チトクロ-ムPー450; ヒト胎児肝; 細胞毒性試験; 転写調節機構; MCFー7; P

1.A pM56 expression vector, which was ligated with P-450IIIA7 cDNA downstream of SRalpha promoter, was constructed. The pM56 was introduced into mammary tumor cell line, MCF-7, two transformants, M21 and M27 were obtained. We examined whether or not these transformants were capable of activating aflatoxin B_1, and lead to the cell death. The 50% lethal dose of aflatoxin B_1 for MCF-7 was 17.0 mug/ml, while that for M21 and M27 was 1.4 mug/ml. This result indicated that M21 and M27 became thirtween times more sensitive to aflatoxin B_1 than MCF-7. It was suggested that P-450IIIA7 protein expressed in M21 and M27 activated the the micotoxin to result in the cell death.2.We tried to express P-450lllA7 protein in an insect cell to obtain a larger amounts of P-450IIIA7 protein. The construction procedure was as follows ; P-450IIIA7 cDNA was inserted to the polyhedorin gene of nuclear polyhedorosis virus (NPV), and recombinant Virus (NPVHP1) was prepared. The insect cell (Sf9 cell) was infected with the NPVHP1. Then, the whole cell extracts from the infected Sf9 cells were prepared. One miligram of the whole extracts contained 1.10 nmol P-450IIIA7 protein. The umu test using the cell lysates showed that P-450IIA7 expressed in Sf9 cells converted aflatoxin B_1 to a mutagen.3.P-450IIIA4 (adult form) and P-450IIIA7 (fetal form) genes were isolated from human genomic library, and the sequences analyzed. Both genes had 13 exons over 20kb. Moreover, 5'flanking sequences between both genes had high similarity (91%).4.We isolated cDNAs of mouse P-450IIIA, which were termed as P-450IIIA_<M1> (P-450IIIA11) and P-450IIIA_<M2>, from liver cDNA library of ddY strain mouse. P-450IIIA_<M1> (P-450IIIA11) and P-450IIIA_<M2>, from liver cDNA library of ddY strain mouse. P-450IIIA_<M1> was constitutively expressed in mouse livers and induced by dexamethasone, while P-450IIIA_<M2> was expressed at a very low level.

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项目成果

Masayuki Komori: "Fetus-specific expression of a form of cytochrome P-450 in human livers." Biochemistry. 29. 4430-4433 (1990)

Masayuki Komori：“人类肝脏中某种形式的细胞色素 P-450 的胎儿特异性表达。”

Masayuki Komori: "Fetusーspecific Expression of a Form of Cytochrome Pー450 in Human Livers." Biochemistry. 29. 4430-4433 (1990)

Masayuki Komori：“人类肝脏中细胞色素 P-450 的胎儿特异性表达。”29. 4430-4433 (1990)。

Tetsuya Kamataki: "Comparative biochemistry of cytochrome P-450 species for the activation of mutagenic food-derived heterocyclic amines.in “N-Oxidation of drugs"" Chapman & Hall,London, 487 (1991)

Tetsuya Kamataki：“细胞色素 P-450 物质用于激活诱变食品衍生杂环胺的比较生物化学。在“药物的 N 氧化”中” Chapman & Hall，伦敦，487 (1991)

Susumu Itoh: "Genomic organization of human fetal specific P-450IIIA7 (cytochrome P-450HFLa)-related gene(s) and interaction of transcriptional regulatory factor with its DNA element in the 5'flanking region." Biochim.Biophys.Acta. in press. (1992)

Susumu Itoh：“人类胎儿特异性 P-450IIIA7（细胞色素 P-450HFLa）相关基因的基因组组织以及转录调节因子与其 5 侧翼区域 DNA 元件的相互作用。”

Taisuke Uchida: "Isolation of cDNAs Coding for Three Different Forms of Liver Microsomal Cytochrome Pー450 from Polychlorinated BiphenylーTreated Beagle Dogs." Mol.Pharmacol.38. 644-651 (1990)

Taisuke Uchida：“从多氯联苯处理的小猎犬中分离编码三种不同形式的肝微粒体细胞色素 P-450 的 cDNA。”Mol.Pharmacol.38（1990）。