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

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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.