MODULATION OF GLUCOSE TRANSPORTERS IN BRAIN

描述(改编自申请人的摘要)：完整的 对解释跨细胞疾病的分子事件的理解 葡萄糖从血浆到脑间质液的运输将 包括运输者的身份，他们的本地化地点， 以及调节它们活动的机制。在这场竞争中 继续提案，申请人概述了这样一项调查 大脑的葡萄糖转运蛋白在mRNA和蛋白质水平上。他会的 合成与已知氨基酸相对应的12-25个氨基酸多肽 C-末端和GLUT3的序列(转运蛋白亚型发现于 脑)以及之前任何未知转运蛋白的推导序列 (通过聚合酶链式反应扩增35个碱基的共同转运蛋白基因进行鉴定 从犬脑mRNA制备中获得的序列)。这些合成的 多肽和鉴定的cDNA将被用于制备多克隆 用于光镜和电子显微镜的抗体和标记寡核苷酸 免疫细胞化学/ELISA法和原位杂交/Northern印迹法， 这些试剂将分别允许(A)测定 转运蛋白的定位、丰度和亚细胞分布 海马区内皮细胞、神经细胞和神经胶质细胞；和(B) 缺血对双侧颈动脉转运体的调节作用 动脉阻塞模型(沙土鼠)与胰岛素低血糖的关系 渗透泵模型(大鼠)。最后，GLUT1(一种转运蛋白)的诱导 在脑内皮细胞中发现的异构体)将通过以下方法在体外进行评估 将培养的犬脑内皮细胞联合应用于 细胞外基质蛋白和/或星形胶质细胞调节的Matrigel。 活性提取物将按大小和层析性质进行分级。 并以此为特征。 人们希望，描述血脑分子事件的研究 葡萄糖的转运将在理解大范围的 神经紊乱。

DESCRIPTION (Adapted from the applicant's abstract): A complete understanding of the molecular events that account for the transcellular transport of glucose from plasma to the interstitial fluid of brain will include identification of the transporters, their sites of localization, and the mechanisms that regulate their activities. In this competing continuation proposal, the applicant outlines such an investigation of the brain's glucose transporters at the mRNA and protein levels. He will synthesize 12-25 amino acid polypeptides corresponding to the known sequence of the C-terminal and of GLUT3 (a transporter isoform found in brain) and to the deduced sequences of any previous unknown transporters (identified by PCR amplification of a 35-base consensus transporter cDNA sequence derived from a canine brain mRNA preparation). These synthesized polypeptides and identified cDNAs will be used to prepare polyclonal antibodies and labeled oligonucleotides for light and electron microscopic immunocytochemistry/ELISA and in situ hybridization/Northern blots, respectively These reagents will allow (a) determination of the localization, abundance, and subcellular distribution of transporters in hippocampal endothelial, neuronal, and glial cells; and (b) study of the regulation of the transporters by ischemia in the bilateral carotid arterial occlusion model (gerbil) and by hypoglycemia in the insulin osmotic pump model (rat). Finally, the induction of GLUT1 (a transporter isoform found in brain endothelial cells) will be evaluated in vitro by subjecting cultured canine brain endothelial cells to a combination of extracellular matrix proteins and/or astrocyte-conditioned Matrigel. Active extracts will be fractionated by size and chromatographic properties and characterized. It is hoped that research describing the molecular events of blood-brain glucose transport will be valuable in the understanding of a wide range of neurological disorders.