REGULATION OF MYELINATION

控制髓鞘形成过程的调节机制可以是 其特征在于至少两相。 第一阶段包括 施旺细胞控制生物合成的调节机制 髓鞘特异性组分在缺乏髓鞘组装的情况下， 第二阶段涉及许旺细胞的调节机制， 控制髓鞘膜的组装。 两个阶段都将进行评估 在这项拨款申请中，通过检查挤压受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 横切的成年大鼠坐骨神经，其中没有髓鞘组装。 的 实验方法涉及以下具体目标。 1)到 确定与生物合成相关的事件的时间顺序 周围神经中主要髓鞘糖蛋白（Po）的表达 损伤的神经和永久横断的神经，以评估是否 分子量变化和甘露糖的逐渐增加 在下调机制中， 这种糖蛋白的生物合成以及这些事件是否也起作用 在随后的髓鞘组装调节中。 2)以确定是否 翻译后阶段的完成与 Po糖蛋白的加工事件和随后的 髓鞘组装通过评估永久横断神经后， 在神经切断后35天处理成年雪旺细胞， 用甲状腺激素、轴膜制备物培养，或 皮质类固醇 3)评估其他翻译后加工事件 除了在Po糖蛋白的生物合成和组装中的糖基化之外， 在挤压损伤的神经和永久性切断的神经中。 具体而言，将评估酰化、硫酸化和磷酸化 以确定相对于以下的翻译后修饰序列： 糖基化事件。 4)评估存在的差异， 关于缺乏髓鞘的雪旺细胞表达的文献 当胚胎或新生儿雪旺细胞表达特定成分时， 当胚胎或新生的雪旺细胞被 我们的研究发现，在体内表达的 成人永久性横断神经中Po糖蛋白的基础水平。 Po糖蛋白在培养物中的表达将通过免疫组织化学方法进行评估。 评价以下技术：a）放射性碘标记的凝集素结合 SDS-孔梯度电泳后，B）放射性前体 c）内切糖基化酶-H消化和d） 链霉蛋白酶消化的放射性系列凝集素亲和层析 电泳分离后获得的标记糖肽。

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.