Analysis of proteins specific to sinlge neurons and small brain regions : Application to diseased brains

一维和二维凝胶电泳的超微方法一直发展到研究的最后一年，这些方法被用于分析中枢神经系统的几种蛋白质。1)测定了s抗原（一种诱导实验性自身免疫性视网膜炎（EAR）的蛋白）的分布，并研究了SAg在EAR大鼠视网膜中的行为。我们的微western blot结果显示，SAg在EAR高潮期（注射SAg后14天）下降到对照水平的2.6%，然后在沉降期（注射SAg后28天）恢复到29%的水平。2)双向电泳显示，与人红细胞质膜相比，脑毛细血管内皮中GLUT1糖基化程度较低。微western blot分析表明，分化发育的小脑内颗粒层含有nNOS（神经元NOS）和eNOS（内皮NOS）。在外部颗粒层。发现eNOS为主要成分，同时检测到eNOS mRNA的表达。3)在原代培养的颗粒细胞中发现了nNOS- mrna，但其活性极低，提示培养细胞中可能表达了失活的nNOS。4)将抗syntaxin及其反义DND抗体加入到视网膜外植体的生长锥中，可显著促进生长锥出芽。这表明了质膜合成素的稳定作用。5)对小脑发育特定阶段发现的V1和富含亮氨酸的酸性核蛋白（LANP）进行了表征，并确定了脑钙网蛋白的共价结构。

Ultramicromethods for one-dimensional and two-dimensional gel electrophoresis has been developed until the final reaerch year and these methods were utilized to analyze several kinds of proteins in the central nervous system. 1) The distribution of S-antigen (SAg ; a protein to induce experimental autoimmune retinouveitis, EAR) had been determined and the behavior of SAg in EAR rat retinas was investigated. Our micro-Western blot revealed that SAg was decreased to 2.6% of the control level in the culminant EAR phase (14 days after SAg injection), and then recovered to 29% level at the subsiding phase (28 days after SAg inj.). 2) Two-dimensional electrophoresis showed that GLUT1 in the cerebral capillary-endothelium is less glicosylated compared with plasma membrane of human erythrocytes. The micro-Wester blot analyzes indicated that the inner granular layrs of the differentiating and developing cerebellum contianed nNOS (neuronal NOS) and eNOS (endotherial NOS). In the external granular layrs. eNOS was found as a main component and mRNA of eNOS was also detected. 3) In the primary cultured granule cells, nNOS-mRNA was found, but its activity was exrtremely low, indicating that inactive nNOS might be possibly expressed in the cultured cells. 4) When the antibody against syntaxin and its antisense DND were incorporated into the growth cones of the retinal explatants, the budding from the growth cones was markedly promoted. This indicates the stabilizing effect by syntaxin of the plasma membrane. 5) The characterization of V1 and LANP (leucine-rich acidic nulcear protein), which have been found in specified phases of cerebellar development, had been performed and the covalant structure of brain calreticulin was determined.