| Project/Area Number |
09670123
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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 |
General medical chemistry
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| Research Institution | Nagoya University |
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Principal Investigator |
WATANABE Yasuo School of Medicine, Nagoya University Assistant Professor, 医学部, 講師 (10273228)
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| Co-Investigator(Kenkyū-buntansha) |
NAITO Yasuhito School of Medicine, Resaerch Associate, 医学部, 助手 (80303618)
横倉 久幸 名古屋大学, 医学部, 助手 (90273242)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1998: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | calcium signal / CaM dependent enzyme / NO synthase / inactive mutant / activation mechanism / molecular biology / cell biology / molecular pharmacology / 一酸化窒素合成酵素(NOS) / 非活性型ミュータントNOS / リン酸化による機能制御 |
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| Research Abstract |
Purpose Calcium ion acts as a ubiquitous second messenger, especially in neural tissues where many of its physiological responses are mediated by the Ca^2-binding protein calmodulin (CaM). The actions of CaM are mediated by its association with specific target proteins, some of which are known as CaM-binding proteins, including the nitric oxide synthases (NOSs) which catalyze the formation of nitric oxide (NO) and L-citrulline from L-arginine. In order to elucidate the activation mechanisms of neuronal NOS (nNOS) at the molecular level, the following projects were undertaken : 1. Structure-activity relationship of nNOS.2. Discovery of a small molecule inhibitor for nNOS.3. Molecular cloning and characterization of novel Ca^<2+>/CaM-dependent proteins.
Results and discussions 1. A specific hydrophobic/basic amino acid cluster in the rat nNOS sequence, Lys^<732>LysLeu, critical for its CaM and membrane binding is identified. We also show intracellular nNOS-iNOS dimerization within engineered cells and would suggest that nNOS-iNOS dimerization might result in generating Ca^<2+>/CaM stimulated activity of the heteromeric enzyme. 2. A newly synthesized isoquinolinesulfonamide, HMN-1180 (1-(5-isoquinolinylsulfonyl)-7-methylhomopiperazine), was shown to have selective inhibitory action against nNOS with a Ki value of 5.4 muM.HMN-1180 was also found to inhibit glutamate stimulated NO production generated by nNOS in the human neuroblastoma cell line SK-N-MC.3. We report the molecular cloning and expression of a cDNA encoding bovine brain NCalpha, a neuron specific Ca^<2+>-binding protein with three EF-hand motifs. We also demonstrate the existence of an isoform-specific activation mechanism of CaM-kinase I.Future prospect Based on our present studies, the analyses for an important component of the "cross-talk" between NO and kinases or Ca^<2+>-binding proteins in calcium signal will need to be determined.
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