| Project/Area Number |
08558084
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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 |
Neuroscience in general
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| Research Institution | TOKYO UNIVERSITY OF PHARMACY AND LIFE SCIENCE |
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Principal Investigator |
KUDO Yoshihisa TOKYO UNIVERSITY OF PHARMACY AND LIFE SCIENCE,PROFESSOR, 生命科学部, 教授 (20080179)
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| Co-Investigator(Kenkyū-buntansha) |
UCHIYAMA Shigeru HAMAMATSU PHOTONICS INC.RESEARCHER, システム事業部, 研究員
HIGASHI Hideyoshi MITSUBISHI-KASEI INSTITUTE OF LIFE SCIENCE CHIEF RESEARCHER, 主任研究員
MORITA Mitsuhiro TOKYO UNIV.OF PHARMACY AND LIFE SCI.RESEARCH ASSOCIATE, 生命科学部, 助手 (50297602)
NAKAMURA Takeshi TOKYO UNIV.OF PHARMACY AND LIFE SCI.RESEARCH ASSOCIATE, 生命科学部, 助手 (50227906)
MIYAKAWA Hiroyoshi TOKYO UNIV.OF PHARMACY AND LIFE SCI.ASSOCIATE PROFESSOR, 生命科学部, 助教授 (90166124)
片山 佳樹 九州大学, 工学部・応用物理化学, 助教授 (70284528)
高木 博 信州大学, 医学部・加齢研究所, 講師 (80247220)
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| Project Period (FY) |
1996 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥18,400,000 (Direct Cost: ¥18,400,000)
Fiscal Year 1998: ¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1997: ¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1996: ¥7,200,000 (Direct Cost: ¥7,200,000)
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| Keywords | FLUOROPROBE / IMAGING / CALCIUM CALMODULINE DEPENDENT PROTEIN KINASE / CYCLIC AMP DEPENDENT PROTEIN KINASE / CALCIUM / COOLED CCD / 冷却CCD / ペプチド試薬 / 神経細胞 / 可視化 / 偏光変調 / 蛍光色素 / イメージング / 情報伝達 / プロテインキナーゼ / cAMP / 細胞内情報伝達 / 海馬ニューロン |
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| Research Abstract |
The availability of imaging techniques for intracellular Ca^<2+> dynamics using fluorescent indicators prompted us to try to visualize the dynamic features of specific molecules in living cells, since it would provide us with new information permitting direct analysis of the role of given functional molecules in specific biological functions. Our aim in this project was to prepare reagents which could be used as indicators to direct visualize dynamic features of kinase activities and also to consist a system to detect and analyze faint fluorescence images. To monitor CaMKII activity, we synthesized a reagent, AS2, by conjugating a fluoroprobe, acrylodan to syntide 2, a specific substrate of CaMKII and showed it to be an effective indicator of the activation of calmodulin by Ca^<2+> and subsequent activation of CaMKII in cell-free conditions. In order to visualize dynamics of PKA activity, we constructed a fluorescence substrate for PKA consisting of a fluorescence probe acrylodan and a partial amino acid sequence of the one of the two isotypes of regulatory domain, regulatory domain II (RII) of PKA which contains a specific autophosphorylation site, a serine residue (ARII). ARII were found to be phosphorylated by PKA and its fluorescence intensity decreased in response to the phosphorylation. We developed a new imaging system with a cooled CCD device and an image analyzer. The newly developed fluoroprobes and imaging systems were applied to neuronal cells in primary culture and had desirable results to show the dynamic features of kinase activities in living cells. We further tried to improve these probes to make them much stable and sensitive, and got several promising dyes.
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