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为辅基的黄素酶，催化多种D-氨基酸的氧化脱氨反应。在哺乳动物中，DAO在肾脏、肝脏和脑中的浓度最高。最近有报道指出D-丝氨酸存在于脑内，并且DAO在脑内的分布与D-丝氨酸的分布呈负相关，这促使我们研究DAO与D-丝氨酸在脑内的功能相关性。核苷酸序列分析表明，该基因全长为1547个核苷酸，5‘端非翻译区为199个核苷酸，开放阅读框为1041个核苷酸，3’端非翻译区为307个核苷酸，包含多聚腺苷化信号序列。推导的氨基酸序列与小鼠、人、POR…的同源性分别为93.1%、80.7%、77.8%和79.0%较多的电影和兔肾的酶分别。猪酶的三个重要催化残基Tyr-224、Tyr-228和Arg-283在这4个物种中都是保守的。脑酶的过氧化物酶体定位的靶向信号也存在于C-末端序列Ser-His-Leu。建立了脑和小脑星形胶质细胞的培养体系，检测了DAO基因在1型星形胶质细胞中的表达。此外，还观察了D-丝氨酸对培养的星形胶质细胞DAO基因表达和细胞死亡的诱导作用。肾素结合蛋白(RnBP)是一种与肾素结合的蛋白质，形成高分子量肾素(HMW)肾素复合体。这种蛋白质会抑制肾素活性。为探讨其生理功能，采用Northern印迹杂交法检测了RnBP在大鼠主动脉中的基因表达。RnBP基因在大鼠主动脉、肾脏、肾上腺、脑和卵巢中均有表达。通过原位RT-PCR检测RnBP基因在大鼠肾脏中的定位，结果表明RnBP基因在延髓旁肾小球和肾小球血管内皮细胞中均有表达。Northern印迹杂交分析显示，猪主动脉内皮细胞表达高水平的RnBP基因。为了研究猪肾素基因表达与RnBP基因表达的关系，通过对猪肾mRNA进行RT-PCR分析，获得了猪肾素基因片段，并测定了猪肾素基因的核苷酸序列。最后，对猪血管内皮细胞肾素基因的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和转化生长因子-α上调培养内皮细胞的RNBP基因表达，而肿瘤坏死因子-α上调RNBP基因的表达。因此，血管紧张素II对RnBP的调节提示RnBP作为血管肾素-血管紧张素系统的一员，参与了血管内皮细胞的血压调节。较少

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