BLOOD BRAIN BARRIER LARGE NEUTRAL AMINO ACID TRANSPORTER

描述（改编自申请人的摘要）：大型 脑细胞中的中性氨基酸对调节这两者都至关重要。 脑蛋白质代谢和神经递质合成。运输 从血液到大脑的氨基酸循环涉及通过两个 膜系列：（一）脑毛细血管内皮细胞壁，这使得 体内血脑屏障（BBB），和（ii）脑细胞（神经元、神经胶质） 质膜因为脑细胞膜的表面积是 数量级大于血脑屏障的表面积， 氨基酸从血浆向脑细胞内空间移动的限制步骤 是BBB运输步骤。这项工作将研究的病理生理 大分子中性氨基酸转运蛋白（LAT）在血脑中的表达 屏障初步数据表明，LAT-cDNA全长编码一个 在青蛙卵母细胞中表达LAT活性的蛋白质。将产生抗血清 用合成肽编码羧基末端或预测的 牛BBB LAT的细胞外结构域。这些细胞的丰度和 该转运蛋白的定位将通过蛋白质印迹，ELISA， 原位杂交、免疫细胞化学、共聚焦和免疫金电子显微镜 显微镜，分别。BBB LAT的基因表达的调节将是 在不同的病理生理条件下研究已知修改 转运蛋白活性，即脑肿瘤、发育、甲状腺功能减退和 缺氧BBB LAT的基因调控机制将在 脑内皮培养细胞，测量其蛋白质的丰度， 转录，以及BBB LAT mRNA的转录速率和衰减。的 还将进行编码兔BBB LAT的cDNA的克隆。因为 BBB LAT的体内Km在物种之间显著不同（即人BBB < 大鼠BBB <兔BBB），BBB的预测氨基酸序列的比较 来自这3个物种的LAT将提供对氨基酸的深入了解， LAT蛋白的活性位点，将通过定点确认 诱变研究。这些研究将提供新的分子生物学 关于血脑屏障上一种重要转运蛋白的信息， 大脑中必需氨基酸的供应。

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.