PROTEOGLYCANS IN CHONDRODYSPLASIA

骨骼发育不良由一组不同的遗传性 以软骨内显著改变为特征的缺陷 颅骨长骨骨化，及各种椎骨病变 包括人类在内的物种。除粘液多发性硬脑膜病外， 这些患者软骨内骨化改变的分子基础 个人在很大程度上是未知的。一种已被证明有用的动物模型 研究软骨发育不良的是阿拉斯加马拉穆特人，因为 这些动物的软骨发育不良具有临床和放射学特征 与人类形而上学的软骨发育不良相似。以前的生化 我们实验室对这些动物生长板的研究失败了。 以揭示胶原代谢的任何原发缺陷，但超微结构 领土和领土间汇总表研究，包括 受影响的生长板显示出异常的组织和分布 蛋白多糖基质颗粒。这项提议的目标将是 确定矮小病毒的蛋白多糖成分是否存在缺陷 生长板，利用生物化学和形态学技术。 具体地说，我们将确定是否存在定性和 在物理化学性质上的定量差异 蛋白多糖存在于矮小生长板通过检查大小类别 完整的蛋白多糖单体、糖胺多糖成分，以及 构成骨骼的蛋白质核心的整体大小。 单体。这将通过使用传统的 4M奎尼丁盐酸法提取蛋白多糖 酶抑制剂，密度梯度法分离纯化蛋白多糖 通过分子筛色谱对分子进行离心和分级， 高压液相色谱、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法 电子显微镜。此外，还将进行研究，以确定 矮人体内蛋白多糖聚集状态是否发生改变 生长板。原生PG聚合体的存在、存在和 连接蛋白的性质及其蛋白多糖单体的能力 矮小和正常的生长板，以形成聚合体(自由连接或 稳定的链接)将使用密度梯度超速离心法进行评估， 聚集体的分子筛组分(即连接蛋白)以及 免疫电子显微镜观察矮小生长的单体 PG聚集性改变的证据。最后，我们将建立 对正常和矮小生长板进行器官培养，以确定是否有 在蛋白多糖的合成和/或降解过程中的变化 利用35S-硫酸盐、~3H氨基葡萄糖、~3H丝氨酸掺入的碎屑 变成蛋白质多聚糖。还将进行放射自显影。

The skeletal dysplasias consist of a heterogeneous group of inherited defects which are characterized by significantly altered endochondral ossification in the long bones of skull, and vertabrate of various affected species including man. With the exception of the mucopolysacharidoses, the molecular basis for the altered endochondral ossification in these affected individuals is largely unknown. One animal model which has proven useful in the study of chondrodysplasia is the Alaskan Malamute, since chondrodysplasia in these animals bears clinical and radiographic resemblance to human metaphysical chondrodysplasias. Previous biochemical studies in our laboratory of the growth plates in these animals have failed to reveal any primary defect in collagen metabolism, but ultrastructural studies of the territorial and interterritorial matrix comprising the affected growth plates reveal an abnormal organization and distribution of proteoglycan matrix granules. The objective of this proposal will be to determine whether there is a defect in proteoglycan components of the dwarf growth plates by using the techniques of biochemistry and morphology. Specifically, we will determine whether there are qualitative and quantitative differences in the physical-chemical nature of the proteoglycans present in dwarf growth plate by examining the size class of the entire proteoglycan monomers, the glycosaminoglycan constituents, and the overall size of the protein core which constitutes the back bone of the monomer. This will be accomplished by using conventional methods of extracting the proteoglycans with 4M quanidine HCl in the presence of protease inhibitors, purifying the proteoglycans by density gradient centrifugation and sizing the molecules by molecular sieve chromatography, high pressure liquid chromatography, SDS polyacrylamide electrophoresis and electron microscopy. In addition, studies will be conducted to determine whether the state of proteoglycan aggregation is altered in the dwarf growth plate. The presence of native PG aggregate, the presence and character of link proteins, and the ability of proteoglycan monomers from dwarf and normal growth plates to form aggregate (either "link" free or "link" stable) will be assessed using density gradient ultracentrifugation, molecular sieve components of the aggregate (i.e. link protein) as well as the monomer to immuno-electron microscopically examine the dwarf growth plate for evidence of altered PG aggregation. Finally, we will establish organ cultures for normal and dwarf growth plates to determine if there is in alteration in the synthesis and/or degradation of proteoglycans in the swarf utilizing incorporation of 35S-sulfate, 3H glucosamine, 3H serine into proteoglycans. Autoradiography will also be done.