1992 Fiscal Year Final Research Report Summary
Development by Gene Engineering of Kits for Screening the Enzymes of the Arachidonate Cascade
| Project/Area Number |
03557014
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | The University of Tokushima |
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Principal Investigator |
YAMAMOTO Shozo Tokushima University School of Medicine Department of Biochemistry, Professor, 医学部, 教授 (50025607)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIMOTO Tanihiro Tokushima University School of Medicine Department of Biochemistry, Associate Pr, 医学部, 助教授 (60127876)
MATSUMOTO Takashi Japan Tabacco Inc., Life Science Research Laboratory, Chief Researcher, 生命科学研究所, 主席研究員
TANABE Tadashi National Cardiovascular Center Research Institute Department of Pharmacology, He, 部長 (60025624)
SHIMIZU Takao University of Tokyo School of Medicine Department of Biochemistry, Professor, 医学部, 教授 (80127092)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Arachidonic acid / Cyclooxygenase / Thromboxane A2 synthase / 12-Lipoxygenase / 5-Lipoxygenase / Leukotriene A4 hydrolase / Leukotriene C4 synthase / Arachidonate cascade |
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| Research Abstract |
A variety of bioactive compounds are produced in a metabolic pathway referred to as the arachidonate cascade. Several key enzymes of the cascade must be studied to elucidate the physiological and pathological roles of these compounds. These enzymes are also the targets in the development of various drugs. The purpose of this research project is the development of the assay kits for the arachidonate cascade enzymes by gene engineering techniques.
1) Cloning of cDNAs for the arachidonate cascade enzymes: In addition to previous cDNA clonings for 5- and 12-lipoxygenases, cyclooxygenase and leukotriene A hydrolase, we cloned cDNAs for human platelet and vascular endothelial cyclooxygenases and for human platelet thromboxane A synthase.
2) Expression of the enzyme cDNAs: 5-Lipoxygenase cDNA was expressed in E. coli and yeast, 12-lipoxygenase in E. coli, thromboxane A synthase in insect cells and leukotriene A hydrolase in E. coli.
3) Quantitative assay of the enzyme mRNA: By the use of 12-lipoxygenase cDNA as a probe, the 12-lipoxygenase mRNA in human platelets was PCR-amplified and quantitated with a cRNA as an internal standard. The assay was applied to leukemia patients with reduced 12-lipoxygenase activity.
4) Enzyme immunoassays for the arachidonate cascade enzymes: A peroxidase-linked immunoassay was developed for human platelet 12-lipoxygenase and applied to the leukemia patients with 12-lipoxygenase deficiency. An enzyme immunoassay was also set up for leukotriene A hydrolase.
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[Publications] Oshima,T., Yoshimoto,T., Yamamoto,S., Kumegawa,M., Yokoyama,C., and Tanabe,T.: "cAMP-dependent induction of fatty acid cyclooxygenase mRNA in mouse osteoblastic cells (MC3T3-E1)." J.Biol.Chem.266. 13621-13626 (1991)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Takahashi,Y., Ueda,N., Yoshimoto,T., Yamamoto,S., Yokoyama,C., Miyata,A., Tanabe,T., Fuse,I., Hattori,A., and Shibata,A.: "Immunoaffinity purification and cDNA cloning of human platelet prostaglandin endoperoxide synthase (cyclooxygenase)." Biochem.Biophys.Res.Commun.182. 433-438 (1992)
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「研究成果報告書概要(欧文)」より
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[Publications] Honda,Z., Nakamura,M., Miki,I., Minami,M., Watanabe,T., Seyama,Y., Okado,H., Toh,H., Ito,K., Miyamoto,T., and Shimizu,T.: "Cloning by functional expression of guinea-pig lung platelet activating factor (PAF) receptor." Nature. 349. 342-346 (1991)
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[Publications] Ihara,H., Yokoyama,C., Miyata,A., Kosaka,T., Nusing,R., Ullrich,V., and Tanabe,T.: "Induction of thromboxane synthase and prostaglandin endoperoxide synthase mRNAs in human erythroleukemia cells by phorbol ester." FEBS Lett.306. 1479-1484 (1992)
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[Publications] Honda,I., Noguchi,M., Furuno,M., Matsumoto,T., Shibagaki,M., Noma,M., Yoneyama,K., Hayashi,T., Iwamura,H., Takahashi,N., and Yoshida,S.: "Inhibition of human 5-lipoxygenase by 3-nitor-2,4,6-trihydroxybenzamide derivatives." Agric.Biol.Chem.55. 833-836 (1991)
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