| Project/Area Number |
07458206
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | YOKOHAMA CITY UNIVERSITY |
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Principal Investigator |
KATO Takahiko YOKOHAMA CITY UNIVERSITY SCHOOL OF MEDICINE,PROFESSOR, 医学部, 教授 (80010023)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAYAMA Takashi YOKOHAMA CITY UNIVERSITY,SCHOOL OF MEDICINE,LECTURER, 医学部, 講師 (90150060)
INOUE Nobuo YOKOHAMA CITY UNIVERSITY,SCHOOL OF MEDICINE,ASSOCIATE PROFESSOR, 医学部, 助教授 (50159985)
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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 |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1996: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | NITRIC OXIDE / NITRIC OXIDE SYNTHASE (NOS) / NOS-ISOFORMS / NOS-ISOFORM mRNAs / MICRO-RT-PCR / CEREBELLAR GRANULAR LAYER / PRIMARY CULTURE / ORGANOTYPIC CULTURE / 一酸化窒素生成酵素(N0S) / 脳領野 / 小脳 organotypic culture / 一酸化窒素生成酵素 / 小脳顆粒細胞初代培養 / Micro-Western blot / nNOS mRNA / nNOS / 一酸化窒素生成酵素同位体 / 定量的RT-PCR / 栄養因子 / 小脳顆粒細胞 |
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| Research Abstract |
Nitric oxide synthase (NOS), which catalyzes the formation of the information molecule nitric oxide (NO), is widely distributed in general tissue cells and its high concentrations are found in the cerebellar granular layr. Cerebellar granule cells differentiate postnatally and the cerebellar layrs are known to develop accordingly. Thus, in order to investigate the differentiation and maturation process of granule cells in the postnatal development of rat cerebellum, NOS activities and isoforms were analyzed in the developing layrs. We developed a microassay method for NOS,using an enzymatic amplification reaction NADP cycling. NOS activities were increased in the molecular and internal granular layrs during development and the activity was detected in the external granular layrs. Our newly developed micro-immunoblot showed neuronal NOS (nNOS) and endothelial NOS (eNOS) in the molecular layr and internal granular layr, and the nNOS immunostaining intensity increased during the postnatal development, representing differentiation and maturation in both layrs. eNOS and nNOS were stained in immunoblot of the neuroblastic external granular layr. Further-more, nNOS mRNA was also detected in this germinal layr, using our newly devised micro-immunoblot. Intraperitoneal administration of N^G-nitro-L-arginine inhibited NOS activity in vivo and disturbed the layr formation in developing cerebellum ; thus NO seems to support the differentiation and maturation of granule cells. In the primary culture of granules cells, isolated from the postnatal day 9 cerebella, NOS activity and nNOS mRNA increased during maturation in vitro. The organotypic culture of the developing slices, prepared from the postnatal day 9 cerebella, showed migration and maturation of granule cells to from the cerebellar layrs, but the layr structure was irregular and the preparation of good microsamples form the slices was hardly possible.
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