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Molecular Biological Studies on the Function and Signal Transduction of Novel Brain and Cardiovascular Regulatory Factors

新型脑和心血管调节因子的功能和信号转导的分子生物学研究

基本信息

批准号：
09470033
负责人：
FUKUI Kiyoshi
金额：
$ 3.9万
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 (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470033/
关键词：
D-amino acid oxidase D-serine glia renin-binding protein renin-angiotensin system kidney D-アミノ酸血管内皮細胞

项目摘要

D-amino acid oxidase (DAO) is a flavoenzyme with FAD as its prosthetic group that catalyzes the oxidative deamination of wide range of D-amino acids. In mammals, DAO is found in highest concentrations in kidney, liver and brain. Recent reports that D-serine is present is brain and the distribution of DAO in the brain is inversely correlated to that of D-serine, prompted us to investigate the functional correlation of DAO with D-serine in brain.In search of nervous system specific expression of DAO as the first step, we have cloned the rat cerebellar cDNA and predicted the primary structure of brain DAO. Analysis of the nucleotide sequence (nt) revealed that full length cDNA has a 1547 nt sequence with a 5'-untranslated region of 199 nt, an open reading frame of 1041 nt, and 3'-untranslated region of 307 nt that contains the polyadenylation signal sequence. The deduced amino acid sequence consisting of 346 amino acids showed 93.1, 80.7, 77.8 and 79.0% identity with the mouse, human, por … More cine and rabbit kidney enzyme, respectively. Three catalytically important residuces, Tyr-224, Tyr-228 and Arg-283, of the porcine enzyme were all conserved in these 4 species. The targeting signal for the peroxisome localization of the brain enzyme was also present at the C-terminal sequence, Ser-His-Leu. Then we have established the culture systems of cerebral and cerebellar astrocytes and detected the gene expression of DAO in type 1 astrocyte. Moreover, the effect of D-serine on the cultured astrocytes was examined to investigate the induction of the gene expression of DAO and the cell death.Renin binding protein (RnBP) is a protein that binds to renin to form a protein complex called high molecular weight (HMW) renin. This protein inhibits renin activity. In search of its physiological function, gene expression of RnBP in rat aorta were examined by Northern blot hybridization. RnBP gene expression was observed in rat aorta, in addition to kidney, adrenal gland, brain and ovary.Using In Situ RT-PCR analysis to determine RnBP mRNA localization in rat kidney, it was suggested that RnBP mRNA was expressed in juxtamedullary glomerulus and vascular endothelial cells in glomerulus. The arterial endothelial cells derived from porcine aorta exhibited high level of RnBP gene expression, detected by Northern blot hybridization analysis. In order to study renin gene expression in relation to that of RnBP, porcine renin cDNA fragment was isolated by RT-PCR analysis of porcine kidney mRNA, followed by determination of the nucleotide sequence for porcine renin. Finally, RT-PCR analysis of porcine renin mRNA in endothelial cells has shown that renin gene is also expressed in the same endothelium. RnBP gene expression was shown to be upregulated by Angiotensin II and TGF-data, but noto by TNF-alfa in cultured endothelial cells. Therefore, RnBP regulation by angiotensin II sugggests that RnBP participate in blood pressure regulation at vascular endothelum as a member of vascular renin-angiotensin system. Less

D-氨基酸氧化酶（DAO）是一种以FAD为辅基的黄素酶，可催化多种D-氨基酸的氧化脱氨。在哺乳动物中，DAO 在肾脏、肝脏和大脑中的浓度最高。最近报道大脑中存在D-丝氨酸，并且DAO在大脑中的分布与D-丝氨酸呈负相关，促使我们研究DAO与D-丝氨酸在大脑中的功能相关性。以寻找DAO的神经系统特异性表达为第一步，我们克隆了大鼠小脑cDNA并预测了大脑DAO的一级结构。核苷酸序列（nt）分析表明，全长cDNA具有1547nt序列，其中5'-非翻译区为199nt，开放阅读框为1041nt，3'-非翻译区为307nt，包含多聚腺苷酸化信号序列。推导的氨基酸序列由 346 个氨基酸组成，与小鼠、人、电影和兔肾酶的一致性分别为 93.1%、80.7%、77.8% 和 79.0%。猪酶的三个重要催化残基 Tyr-224、Tyr-228 和 Arg-283 在这 4 个物种中均保守。脑酶过氧化物酶体定位的靶向信号也存在于 C 端序列 Ser-His-Leu 处。然后我们建立了大脑和小脑星形胶质细胞的培养体系，并检测了1型星形胶质细胞中DAO基因的表达。此外，还检查了 D-丝氨酸对培养的星形胶质细胞的影响，以研究 DAO 基因表达的诱导和细胞死亡。肾素结合蛋白 (RnBP) 是一种与肾素结合形成称为高分子量 (HMW) 肾素的蛋白质复合物的蛋白质。该蛋白抑制肾素活性。为了探寻其生理功能，通过 Northern blot 杂交检测了大鼠主动脉中 RnBP 的基因表达。在大鼠主动脉中观察到RnBP基因表达，此外在肾脏、肾上腺、脑和卵巢中也观察到RnBP基因表达。通过原位RT-PCR分析确定RnBP mRNA在大鼠肾脏中的定位，提示RnBP mRNA在近髓肾小球和肾小球血管内皮细胞中表达。通过Northern印迹杂交分析检测，源自猪主动脉的动脉内皮细胞表现出高水平的RnBP基因表达。为了研究与RnBP相关的肾素基因表达，通过对猪肾mRNA进行RT-PCR分析来分离猪肾素cDNA片段，然后测定猪肾素的核苷酸序列。最后，对内皮细胞中猪肾素mRNA的RT-PCR分析表明，肾素基因也在同一内皮细胞中表达。在培养的内皮细胞中，血管紧张素 II 和 TGF 数据显示 RnBP 基因表达上调，但 TNF-α 则不上调。因此，血管紧张素II对RnBP的调节表明RnBP作为血管肾素-血管紧张素系统的成员参与血管内皮的血压调节。较少的