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

d -氨基酸氧化酶（DAO）是一种以FAD为配基的黄酶，可催化多种d -氨基酸的氧化脱胺反应。在哺乳动物中，DAO在肾脏、肝脏和大脑中含量最高。最近有报道称d -丝氨酸存在于大脑中，并且DAO在大脑中的分布与d -丝氨酸的分布呈负相关，这促使我们研究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并预测了脑DAO的初级结构。核苷酸序列（nt）分析显示全长cDNA为1547 nt，其中5'-未翻译区199 nt，开放阅读框1041 nt， 3'-未翻译区307 nt包含多聚腺苷化信号序列。该序列包含346个氨基酸，与小鼠肾酶、人肾酶、猪肾酶和兔肾酶的同源性分别为93.1、80.7、77.8和79.0%。猪酶的三个催化重要残基，tyrr -224、tyrr -228和Arg-283，在这4个物种中都是保守的。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上。建立了大脑和小脑星形胶质细胞培养体系，检测了DAO在1型星形胶质细胞中的基因表达。此外，我们还观察d -丝氨酸对培养的星形胶质细胞的影响，探讨其诱导DAO基因表达和细胞死亡的作用。肾素结合蛋白（RnBP）是一种与肾素结合形成一种称为高分子量肾素（HMW）的蛋白质复合物的蛋白质。这种蛋白质抑制肾素的活性。采用Northern blot杂交技术检测RnBP在大鼠主动脉组织中的基因表达，探讨其生理功能。RnBP基因在大鼠主动脉、肾脏、肾上腺、脑和卵巢中均有表达。利用原位RT-PCR分析RnBP mRNA在大鼠肾脏中的定位，提示RnBP mRNA在肾小球近髓和肾小球血管内皮细胞中表达。Northern blot杂交分析显示，猪主动脉内皮细胞RnBP基因表达水平较高。为了研究肾素基因与RnBP基因表达的关系，采用RT-PCR方法对猪肾mRNA进行分析，分离猪肾素cDNA片段，并测定猪肾素的核苷酸序列。最后，对猪内皮细胞肾素mRNA的RT-PCR分析表明，肾素基因在同一内皮细胞中也有表达。血管紧张素II和tgf数据显示RnBP基因表达上调，但tnf - α在培养的内皮细胞中没有上调。因此，血管紧张素II对RnBP的调控提示RnBP作为血管肾素-血管紧张素系统的一员参与血管内皮的血压调节。少

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