THE RED CELL CYTOSKELETON AND SICKLE CELL DISEASE

镰状细胞性贫血的特征是大量的临床和 由于病毒的存在而引起的细胞异常 突变基因产生血红蛋白S(HBs)。尽管很可能有很多人 随之而来的细胞缺陷，其机制是 它们的出现仍是未知的。这项提议的长期目标是 就是确定膜的形成机制 SS细胞异常。为了实现这一目标，我们将测试 氧化损伤在糖尿病的发展中起作用的假说 SS细胞膜和细胞骨架蛋白功能异常。 我们将重点研究氧化对大豆主要蛋白质成分的影响。 红细胞膜和细胞骨架，包括血影蛋白、锚蛋白和 带3.蛋白质氧化损伤将通过结合到 巯基特异性化学探针和蛋白质的直接分析 氨基酸。其他研究将检查蛋白质的功能，以 确定功能是否因氧化修饰而改变。 将研究血影蛋白和钙调蛋白之间的相互作用以确定 如果这些蛋白质之间的异常相互作用在 产生不可逆转的镰状细胞。功能研究将 确定维持蛋白质巯基的酶是否参与 在SS细胞中，动态平衡，如硫醇转移酶，受到破坏。角色 镰状事件本身在蛋白质氧化发展中的作用 损伤将在氧合-去氧循环研究中进行检查。这个 确定的SS细胞群体的抗氧化能力将被测量为 确定氧化损伤是否是由于某些 SS细胞修复氧化损伤。蛋白质氧化在蛋白质合成中的作用 形态异常的发展将通过 使用巯基特定化学物质的荧光和电子显微镜 探测器。蛋白质氧化在糖尿病发生发展中的作用 细胞体积调节异常的研究将使用定义的 高K：C_1共转运活性的细胞。哈佛商学院的角色 细胞膜结合与铁的分解作用 细胞形态和蛋白质功能异常的发展将 在含有一定数量的HBS的转基因HBS小鼠身上进行测试。一起， 这些研究可能有助于阐明导致高血压的机制。 SS细胞膜异常的发生发展及其临床意义 对这种疾病和其他疾病的病理生理学的理解 以不稳定的血红蛋白为特征的血红蛋白病。

Sickle cell anemia is characterized by a multitude of clinical and cellular abnormalities that arise as a consequence of the presence of the mutant gene produce hemoglobin S (HbS). Although it is likely that many of the attendant cellular defects are acquired, the mechanisms by which they arise are still unknown. The long-term objective of this proposal is to define the mechanisms responsible for the development of membrane abnormalities in SS cells. To achieve this objective we will test the hypothesis that oxidative damage plays a role in the development of membrane and cytoskeletal protein functional abnormalities in SS cells. We will focus on the effects of oxidation on key protein components of the red cell membrane and cytoskeleton, including spectrin, ankyrin and band 3. Protein oxidative damage will be assessed by binding to sulfhydryl-specific chemical probes and by direct analysis of protein amino acids. Additional studies will examine protein function to determine if function has been altered by oxidative modifications. Interactions between spectrin and calmodulin will be studied to determine if abnormal interactions between these proteins plays a role in the generation of irreversibly-sickled cells. Functional studies will determine if enzymes involved in maintaining protein sulfhydryl homeostasis, such as thioltransferase, are damaged in SS cells. The role of the sickling event per se in the development of protein oxidative lesions will be examined in oxygenated-deoxygenated cycling studies. The antioxidant capacity of defined SS cell populations will be measured to determine if oxidative damage results from a decreased capacity of some SS cells to repair oxidative lesions. The role of protein oxidation in the development of morphologic abnormalities will be studied by fluorescent and electron microscopy using sulfhydryl-specific chemical probes. The role of protein oxidation in the development of abnormalities in cell volume regulation will be studied using defined cells with high of low K:C1 cotransport activity. The role of HbS binding to the membrane and iron decompartmentalization in the development of cell morphologic and protein functional abnormalities will be tested in transgenic HbS mice with defined amounts of HbS. Together, these studies may help to elucidate the mechanisms responsible for the development of membrane abnormalities in SS cells and better our understanding of the pathophysiology of this disease and of other hemoglobinopathies characterized by unstable hemoglobins.