Molecular Enzymological Studies on the Pathophysiological Significance of Brain Endogeneous D-amino Acids and Their Metabolizing Enzyme

脑内源性D-氨基酸及其代谢酶病理生理意义的分子酶学研究

• 批准号: 13670143
• 负责人: FUKUI Kiyoshi
• 金额: $ 2.3万
• 依托单位: THE UNIVERSITY OF TOKUSHIMA
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670143/
• 关键词: D-serine; D-amino acid ozidase; glutamate receptor; astrocyte; microglia; RT-PCR; cerebral ischemia; gene expression; CG-4; C-6

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.

D-氨基酸氧化酶（DAO）是一种催化D-氨基酸氧化脱氨的黄素酶。组织化学研究显示DAO活性定位于某些类型的星形胶质细胞。虽然DAO的体内底物的存在多年来并不为人所知，但最近已经证明在哺乳动物脑中存在大量的游离D-丝氨酸。D-丝氨酸和甘氨酸是N-甲基-D-天冬氨酸（NMDA）型谷氨酸受体甘氨酸位点的共激动剂，在脑缺血的神经毒性中起重要作用。细胞外游离D-丝氨酸可调节神经传递。D-丝氨酸被发现定位于2型星形胶质细胞在培养中，并释放谷氨酸stimulation.We检测DAO基因在培养的大鼠星形胶质细胞中的表达，探讨DAO在游离D-丝氨酸代谢的背景下的生理作用。我们用逆转录-聚合酶链反应检测了培养的大鼠星形胶质细胞中DAO基因的表达。我们建立了一种从任何脑区制备高纯度1型和2型星形胶质细胞培养物的方法。该方法利用通过振荡进行细胞类型特异性分离和随后通过免疫淘洗或用阿糖胞苷处理进行纯化的组合。我们检测到DAO基因在小脑1型星形胶质细胞中的表达高于2型星形胶质细胞。我们还发现DAO基因在对应于1型星形胶质细胞的C6中的表达高于CG-4来源的2型星形胶质细胞。还使用MG 5细胞系和原代培养证明了DAO在小胶质细胞中的基因表达。

项目成果

Ming Li: "Minoxidil-Induced Hair Growth is mediated by adenosine in Cultured Dermal Papilla Cells : Possible Involvement of Sulfonylurea Reporter 2B as a Target of Minoxidil"The Journal of Investigative Dermatology. 117. 1594-1600 (2001)

李明：“米诺地尔诱导的毛发生长是由培养的真皮乳头细胞中的腺苷介导的：磺酰脲报告基因 2B 作为米诺地尔的靶标可能参与其中”《皮肤病学研究杂志》。

Atsuhiko Suzue et al.: "Astroglial Expression of D-amino Acid Oxidase Gene"Flavins and Flavoprotains 2002. 853-856 (2002)

Atsuhiko Suzue 等：“D-氨基酸氧化酶基因的星形胶质细胞表达”Flavins and Flavoprotains 2002. 853-856 (2002)

Atsuhiko Suzue: "Astroglial Expression of D-amino Acid Oxidase Gene"FLAVINS AND FLAVOPROTEINS 2002. 853-856 (2002)

Atsuhiko Suzue：“D-氨基酸氧化酶基因的星形胶质细胞表达”FLAVINS AND FLAVOPROTEINS 2002. 853-856 (2002)

Yoshiteru Urai et al.: "Gene expression of D-amino Acid oxidase in cultured rat astrocytes;regional and cell type specific expression"Neuroscience Letters. 324. 101-104 (2002)

Yoshiteru Urai 等人：“培养的大鼠星形胶质细胞中 D-氨基酸氧化酶的基因表达；区域和细胞类型特异性表达”神经科学快报。

Takashi Sakai: "Identification of a Novel, Embryonal Carcinoma Cell-associated Molecule, Nucling, That Is Up-regulated during Cardiac Muscle Differentiation"The Journal of Biochemistry. (印刷中).

Takashi Sakai：“鉴定一种新型胚胎癌细胞相关分子，成核，在心肌分化过程中上调”《生物化学杂志》（正在出版）。