BLOOD BRAIN BARRIER LARGE NEUTRAL AMINO ACID TRANSPORTER

血脑屏障大型中性氨基酸转运蛋白

• 批准号: 6529545
• 负责人: RUBEN J. BOADO
• 金额: $ 22.88万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6529545
• 关键词: Xenopus oocyte; aminoacid transport; blood brain barrier; brain neoplasms; confocal scanning microscopy; developmental genetics; developmental neurobiology; enzyme linked immunosorbent assay; gene induction /repression; hypothyroidism; hypoxia; immunocytochemistry; immunoelectron microscopy; in situ hybridization; laboratory rabbit; laboratory rat; membrane transport proteins; molecular cloning; neurogenetics; neurotransmitter transport; site directed mutagenesis; tissue /cell culture; vascular endothelium; western blottings

DESCRIPTION (adapted from applicant's abstract): The availability of large neutral amino acids in brain cells is crucial to the regulation of both cerebral protein metabolism and neurotransmitter synthesis. The transport of circulating amino acids from blood to brain involves transport through two membranes in series: (i) the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo, and (ii) the brain cell (neuron, glial) plasma membrane. Since the surface area of the brain cell membrane is log orders greater than the surface area of the blood-brain barrier, the rate limiting step in amino acid movement from plasma to brain intracellular space is the BBB transport step. This work will study the pathophysiological expression of the large neutral amino acid transporter (LAT) at the blood-brain barrier. The preliminary data show that the full length LAT-cDNA encodes a protein that expresses LAT activity in frog oocytes. Antisera will be produced with synthetic peptides encoding either the carboxyl terminus or predicted extracellular domains of the bovine BBB LAT. The abundance and cellular localization of this transporter will be studied by Western blotting, ELISA, in situ hybridization, immunocytochemistry, and confocal and immunogold electron microscopy, respectively. The modulation of gene expression of BBB LAT will be studied under different pathophysiologic conditions known to modify the transporter activity, i.e. brain tumors, development, hypothyroidism and hypoxia. The mechanisms of gene regulation of BBB LAT will be investigated in brain endothelial cultured cells measuring the abundance of its protein and transcript, and the transcriptional rate and decay of the BBB LAT mRNA. The cloning of cDNAs encoding the rabbit BBB LAT will also be performed. Because the in vivo Km for the BBB LAT markedly differs among species (i.e. human BBB < rat BBB < rabbit BBB), comparison of the predicted amino acid sequence of BBB LAT from these 3 species will provide insight into the amino acids comprising the active site of the LAT protein, which will be confirmed by site-directed mutagenesis studies. These studies will provide new molecular biological information on a crucial transporter at the blood-brain barrier that regulates the supply in the brain of essential amino acids.

描述（改编自申请人的摘要）：大型的可用性 脑细胞中的中性氨基酸对两者的调节至关重要 脑蛋白质代谢和神经递质合成。运输 从血到大脑循环氨基酸涉及通过两个 串联的膜：（i）构成脑毛细管内皮壁 体内的血脑屏障（BBB）和（ii）脑细胞（神经元，神经胶质） 质膜。由于脑细胞膜的表面积是原木 订单大于血脑屏障的表面积，速率 限制从血浆到大脑细胞内空间的氨基酸运动的限制步骤 是BBB运输步骤。这项工作将研究病理生理学 在血脑内表达大型中性氨基酸转运蛋白（LAT） 障碍。初步数据显示全长lat-cDNA编码A 在青蛙卵母细胞中表达LAT活性的蛋白质。将产生安提拉 与编码羧基末端或预测的合成肽 牛BBB LAT的细胞外域。丰度和细胞 该运输蛋白的定位将通过Western blotting（Elisa）研究 原位杂交，免疫细胞化学以及共聚焦和免疫元电子 显微镜分别。 BBB LAT基因表达的调节将是 在不同的病理生理状况下研究以改变 转运蛋白活性，即脑肿瘤，发育，甲状腺功能减退症和 缺氧。 BBB LAT的基因调节机制将在 测量其蛋白质丰度的脑内皮培养细胞和 转录本，以及BBB LAT mRNA的转录率和衰减。这 还将执行编码兔BBB LAT的CDNA的克隆。因为 BBB LAT的体内km在物种之间明显不同（即人bbb < 大鼠BBB <兔BBB），比较BBB的预测氨基酸序列 来自这三个物种的纬度将提供有关包含的氨基酸的洞察力 LAT蛋白的活性位点，将通过位置确认 诱变研究。这些研究将提供新的分子生物学 有关调节血脑屏障的关键转运蛋白的信息 必需氨基酸的大脑供应。