MEMBRANE GLYCOPROTEINS AND GLIAL DIFFERENTIATION IN CULT

本项目将强调定性与定量的关系 胶质细胞膜糖蛋白低聚糖的变化 大脑发育过程中的分化。 本研究的目的是：（1）分析 与胶质细胞分化相关的膜寡糖模式 原代培养的大鼠脑细胞和C-6胶质细胞，2）研究 膜糖肽变化与胶质细胞分化的关系 以及，3）提供了一个洞察机制，在这种变化的运作 并确定生物合成途径中可能的调控步骤 低聚糖。 随着神经胶质细胞分化的变化将在培养的 C-6神经胶质细胞和纯化星形胶质细胞的原代培养物中， 从新生大鼠大脑中制备的少突胶质细胞。 神经胶质细胞会 在各种实验条件下诱导分化 并将用糖蛋白的糖前体3 H-葡糖胺标记 和3 H-甘露糖以及32 p-磷酸（用于磷酸糖蛋白）， 与35 S-硫酸盐（用于磺基糖蛋白）。 通过以下方法制备的糖肽： 胰蛋白酶和神经胶质细胞膜的蛋白水解消化 将通过1）凝胶过滤，2）凝集素亲和 色谱法，3）离子交换色谱法和4）HPLC。 功能 胶质细胞膜寡糖表达意义 将通过测试a）以下抑制剂的作用来评估分化： 糖基化：衣霉素、康帕汀和苦马豆素，B）分离的膜 糖肽对神经胶质细胞特性（2 '，3'，环 核苷酸磷酸水解酶、谷氨酰胺合成酶和硫脂 合成）。 N-连接寡糖途径的调控 将研究参与神经胶质分化的生物合成，1）通过 分析途径的中间体，和2）通过测定特定的 参与加工的糖苷酶和糖基转移酶， 寡糖的修饰。

This project will emphasize the relation of qualitative and quantitative changes in the oligosaccharides of membrane glycoproteins to glial cell differentiation during the development of the brain. The objectives of this research are to: 1) analyze the changes in the membrane oligosaccharide patterns associated with glial differentiation in primary cultured rat brain cells and in C-6 glial cells, 2) investigate the relation of changes in the membrane glycopeptides to glial differentiation and, 3) provide an insight into the mechanisms operative in such changes and to identify the possible regulatory steps in the biosynthetic pathway of oligosaccharides. The changes accompanying glial differentiation will be studied in cultured C-6 glial cells and in primary cultures of purified astrocytes and oligodendrocytes prepared from newborn rat brain. The glial cells will be induced to undergo differentiation under various experimental conditions and will be labeled with suger precursors of glycoproteins, 3H-glucosamine and 3H-mannose as well as with 32p-phosphate (for phosphoglycoproteins) and with 35S-sulfate (for sulfoglycoproteins). The glycopeptides prepared by proteolytic digestion of the trypsinates and membranes of the glial cells will be fractionated by 1) gel filtration, 2) lectin affinity chromatography, 3) ion exchange chromatography and 4) HPLC. The functional significance of membrane oligosaccharide expression during glial differentiation will be assessed by testing the effect of a) inhibitors of glycosylation: tunicamycin, compactin and swainsonine, b) isolated membrane glycopeptides on biochemical expression of glial properties (2',3', cyclic nucleotide phosphohydrolase, glutamine synthetase and sulfolipid synthesis). The regulation of the pathway of N-linked oligosaccharide biosynthesis attending glial differentiation will be studied, 1) by analyzing the intermediates of the pathway and 2) by assaying the specific glycosidases and glycosyltransferases involved in the processing and modification of the oligosaccharides.