REGULATION OF MYELINATION

控制髓鞘形成过程的调节机制可以是 以至少两个阶段为特征。第一阶段包括 雪旺细胞控制神经生长因子生物合成的调控机制 在没有髓鞘组装的情况下，髓鞘特异的成分，以及 第二阶段涉及雪旺细胞的调节机制 控制髓鞘膜的组装。将对这两个阶段进行评估 在这项拨款提案中，通过检查挤压损伤的成年大鼠坐骨神经 有髓鞘组装的神经并将其与永久性的 切断成年大鼠坐骨神经，其中没有髓鞘组装。这个 实验方法涉及以下具体目标。1)至 确定与生物合成相关的事件的时间序列 周围神经主要髓鞘糖蛋白(Po)在挤压中的表达 损伤的神经和永久切断的神经来评估是否 甘露糖的相对分子质量漂移和递增 合并到PO中参与了在 这种糖蛋白的生物合成以及这些事件是否也发挥了作用 在随后的髓鞘组装的调节中。2)确定是否 在完成后翻译之间存在联系 Po糖蛋白的处理事件和随后的启动 评价永久性切断神经后髓鞘组装的研究 神经切断后35d处理成年雪旺细胞的实验研究 用甲状腺激素、轴膜制剂或 皮质类固醇。3)评估其他翻译后处理事件 Po糖蛋白生物合成和组装中的糖基化作用 在挤压损伤的神经和永久切断的神经中。 具体地说，将评估酰化、硫酸盐化和磷酸化。 确定相对于以下各项的翻译后修改的顺序 糖基化事件。4)评估中存在的差异 关于髓鞘雪旺细胞缺乏表达的文献 特定成分在胚胎或新生儿雪旺细胞的表达 当胚胎或新生儿雪旺细胞 在培养中生长，我们的发现是关于在体内表达 成年永久切断神经中Po糖蛋白的基础水平。 培养物中Po糖蛋白的表达将通过 评价以下技术：a)放射性碘标记凝集素结合 经十二烷基硫酸钠-孔梯度电泳法，b)放射性前驱体 结合到该糖蛋白中，c)内切糖苷酶-H消化和d) 链霉蛋白酶消化放射性系列凝集素亲和层析 经电泳法分离得到的标记糖肽。

The regulatory mechanisms which control the process of myelination can be characterized by at least two phases. The first phase includes the regulatory mechanisms by which Schwann cells control the biosynthesis of myelin specific components in the absence of myelin assembly, and the second phase involves the regulatory mechanisms by which Schwann cells control the assembly of the myelin membrane. Both phases will be evaluated in this grant proposal by examining the crush injured adult rat sciatic nerve where there is myelin assembly and comparing it to the permanently transected adult rat sciatic nerve where there is no myelin assembly. The experimental approach involves the following specific aims. 1) To determine the temporal sequence of events associated with the biosynthesis of the major myelin glycoprotein (Po) from peripheral nerve in the crush injured nerve and the permanently transected nerve to assess whether the molecular weight shift and the progressive increase in mannose incorporation into Po are involved in the down regulation mechanism in the biosynthesis of this glycoprotein and whether these events also play a role in the subsequent regulation of myelin assembly. 2) To determine if there's an association between completion of the post-translational processing events for Po glycoprotein and the subsequent initiation of myelin assembly by evaluating the permanently transected nerve after treating the adult Schwann cells at 35 days after nerve transection in culture with thyroid hormone, axolemma membrane preparations, or corticosteroids. 3) To evaluate other post-translational processing events besides glycosylation in the biosynthesis and assembly of Po glycoprotein in both the crush injured nerve and the permanently transected nerve. Specifically, acylation, sulfation, and phosphorylation will be evaluated to determine the sequence of post-translational modifications relative to the glycosylation events. 4) To evaluate to discrepancies that exist in the literature regarding a lack of Schwann cell expression of myelin specific components when embryonic or neonatal Schwann cell expression of myelin specific components when embryonic or neonatal Schwann cells are grown in culture with our findings concerning the in vivo expression of basal levels of Po glycoprotein in the adult permanently transected nerve. The expression of Po glycoprotein in culture will be assessed through an evaluation of the following techniques: a) Radioiodinated lectin binding after SDS-pore gradient electrophoresis, b) radioactive precursor incorporation into this glycoprotein, c) endoglycosidase-H digestion and d) serial lectin affinity chromatography of pronase digested radioactive labeled glycopeptides obtained after electrophoretic separation.