CALCIUM & CALCIUM BINDING PROTEINS IN THE EPIDERMIS

钙

基本信息

批准号：
3157509
负责人：
JANET A FAIRLEY
金额：
$ 13.41万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-09-30 至 1993-03-31
项目状态：
已结题

项目摘要

The skin is a constantly renewing organ that undergoes a series of regulated changes as the cells of the basal layer proliferate and differentiate into the anucleate stratum corneum. Failure of regulatory processes leads to abnormal growth in skin diseases such as psoriasis and neoplasia. We propose to define the role of free intracellular calcium, the calcium-binding protein calmodulin, and extracellular calcium in regulation of epidermal proliferation and differentiation. The free intracellular calcium level of keratinocytes is measured using the calcium-binding dye indo-1 and flow cytometry. Changes effected by agents such as TPA, retinoids, cyclosporin and vitamins D will be quantiated. Retinoid acid causes a decrease in the free intracellular calcium concentration of keratinocytes; cell lines free of retinoid binding proteins and inhibitors of calcium transports will be used to study the mechanism. Changes in intracellular calcium will be correlated with changes in cell envelope formation and transglutaminase activity. LTB4 and PGE2 increase the intracellular calcium concentration of keratinocytes and the mechanism will be determined. The calmodulin-binding proteins of the epidermis will be identified using biotinylated calmodulin staining of nitrocellulose blots from epidermal extracts. The effect of TPA, steroids, cyclosporin and vitamin D on the content of these calmodulin-binding proteins and their ability to bind calmodulin will be measured. Monoclonal antibodies we have generated to the calmodulin-binding proteins will be used for subcellular localization of the proteins and functional studies. The calmodulin-dependent protein kinase from epidermis will be isolated and its endogenous substrate (s) identified. The role of extracellular calcium in keratinocyte proliferation and differentiation will be studied. The effect of growth in low calcium medium on intracellular calcium concentration will be measured. Calmodulin binding proteins from low normal calcium keratinocytes will identified using the biotinylated probes and the amount, distribution and binding to calmodulin will be measured. Results of these studies and the technology employed will be applied to disease states such as psoriasis and skin neoplasms.

皮肤是一个不断更新的器官，经历一系列的随着基底层细胞增殖而发生调节变化分化为无核角质层。失败调节过程导致皮肤病的异常生长，例如如牛皮癣和肿瘤。我们建议定义免费的角色细胞内钙、钙结合蛋白钙调蛋白和细胞外钙对表皮增殖的调节差异化。细胞内游离钙水平使用钙结合染料 indo-1 和流式细胞术。受 TPA、类维生素A、环孢菌素和维生素 D 将进行定量。视黄酸导致细胞内游离钙浓度降低角质形成细胞；不含类视黄醇结合蛋白的细胞系和钙转运抑制剂将用于研究机制。细胞内钙的变化将相关随着细胞包膜形成和转谷氨酰胺酶的变化活动。 LTB4 和 PGE2 增加细胞内钙角质形成细胞的浓度及其机制决定。表皮的钙调蛋白结合蛋白会使用生物素化钙调蛋白染色进行鉴定来自表皮提取物的硝化纤维素印迹。 TPA的影响，类固醇、环孢素和维生素D对这些物质含量的影响钙调蛋白结合蛋白及其结合钙调蛋白的能力将被测量。我们生成的单克隆抗体钙调蛋白结合蛋白将用于亚细胞蛋白质定位和功能研究。这来自表皮的钙调蛋白依赖性蛋白激酶将被分离及其内源性底物的鉴定。的作用细胞外钙在角质形成细胞增殖中的作用将研究差异化。低增长的影响钙培养基对细胞内钙浓度的影响为测量。来自低正常钙的钙调蛋白结合蛋白将使用生物素化探针识别角质形成细胞钙调蛋白的数量、分布和结合将是测量。这些研究的结果和所采用的技术将应用于牛皮癣和皮肤病等疾病状态肿瘤。