| Project/Area Number |
13670143
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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 |
Pathological medical chemistry
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| Research Institution | THE UNIVERSITY OF TOKUSHIMA |
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Principal Investigator |
FUKUI Kiyoshi THE INSTITUTE FOR ENZYME RESEARCH, THE UNIVERSITY OF TOKUSHIMA, Professor, 分子酵素学研究センター, 教授 (00175564)
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| Co-Investigator(Kenkyū-buntansha) |
TOMITA Yumiko THE UNIVERSITY OF TOKUSHIMA, THE INSTITUTE FOR ENZYME RESERCH, Research Associate, 分子酵素学研究センター, 助手 (00089913)
SAKAI Takashi THE INSTITUTE FOR ENZYME RESEARCH, THE UNIVERSITY OF TOKUSHIMA, Associate Professor, 分子酵素学研究センター, 助教授 (80284321)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | D-serine / D-amino acid ozidase / glutamate receptor / astrocyte / microglia / RT-PCR / cerebral ischemia / gene expression / CG-4 / C-6 |
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| Research Abstract |
D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidative deamination of D-amino acids. Histochemical studies revealed that DAO activity was located to some types of astrocytes. Although the existence of in vivo substrate of DAO was not known for many years, recently it had been demonstrated that substantial amounts of free D-serine are present in mammalian brain. D-serine as well as giycine acts as co-agonists at glycine site of N-methyi-D-aspartate(NMDA) type of glutamate receptor which plays an important role in nurotoxicity in cerebral ischemia. Extracelluiar free D-serine can modulate neurotransmission. D-serine was found to be localized to type-2 astrocytes in culture, and released by glutamate stimulation.
We examined the DAO gene expression in cultured rat astrocytes to investigate the physiological role of DAO in the context of free D-serine metabolism. We examined the DAO gene expression in cultured rat astrocytes by reverse transcriptase-polymerase chain reaction. We established a method to prepare highly purified cuiture of type-1 and type-2 astrocytes from any brain region. This method utilizes combinaion of cell type spesific separaton by shaking and subsequent purification by immunopanning or trearment with cytosine arabinoside. We detected higher DAO gene expression in type-1 astrocyte cultures from cerebellum than that in type-2, We also revealed that DAO expression in C6, corresponding to type-1astrocyte, was higher than that in CG-4 derived type-2 astrocytes. Gene expression of DAO in microglia was also demonstrated with the use of MG5 cell line and primary culture.
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