MEGAKARYOCYTE AND PLATELET PROTEOGLYCANS

这是竞争性续约提案的第三次修订。 本提案的长期目标是了解 丝甘素的结构、功能及表达调控 蛋白聚糖。 已知它由巨核细胞产生， 骨髓细胞 它包含在血小板α颗粒中， 肥大细胞分泌颗粒，并在刺激时释放。 由淋巴细胞组成性分泌， 在激活期间。 该实验室最近发现， 这种分子也在人内皮细胞、鼠内皮细胞和人内皮细胞中表达。 卵黄囊和胚胎干细胞。 丝甘肽与 几种基质分子和细胞因子，并可能参与 调节细胞活化和粘附。 丝甘肽可能是 对胎儿早期发育很重要。 糖胺聚糖 (GAG)链的长度、数量和组成在细胞中不同 类型，是约束力的重要决定因素 不同细胞所制造的分子的特性。 的 建议将试图建立在体内的功能作用 的serglycin。 在具体目标1中，研究人员将 表征来自不同来源的血清甘氨酸的GAG链含量 细胞来源，以确定其结合倾向 不同的分子。 在具体目标2中，他们将执行 通过分析丝氨酸蛋白酶的能力， 来自不同来源的天然和修饰的血清甘氨酸， 基质分子和细胞因子。 因此，这些实验可能 阐明的作用机制的结构基础， 丝氨酸蛋白聚糖家族，并可能导致 治疗药物的开发。 他们将准备 胚胎干细胞中基因的纯合敲除， 监测对胚状体形成的影响， 造血细胞 在《特定目标3》中， 在胎儿和成人组织中原位检测serglycin蛋白和mRNA， 确定体内合成和沉积的位点。 这将 使他们能够更好地理解这个分子的功能。 在具体目标4中，他们将制备以下启动子构建体： 鼠血清甘氨肽并比较几种细胞类型中的调节 与他们的人类基因的细胞特异性表达的数据。 已发现在体外有活性的启动子构建体 将用于产生转基因小鼠，以分析 体内启动子调控的细胞特异性。 进一步研究 可以产生独特的方法，用于细胞特异性敲除 小鼠中的基因，以确定该分子在 不同的细胞

This is the third revision of a competitive renewal proposal. The long-range goal of this proposal is to understand the structure, function and regulation of expression of the serglycin proteoglycan. It is known to be made by megakaryocytes and myeloid cells. It is contained in platelet alpha granules and mast cell secretory granules, and is released upon stimulation. It is constitutively secreted from lymphocytes and upregulated during activation. This laboratory has recently found that the molecule is also expressed in human endothelial cells, in murine yolk sac, and in embryonic stem cells. Serglycin binds to several matrix molecules and cytokines, and could be involved in modulating cell activation and adhesion. Serglycin may be important for early fetal development. The glycosaminoglycan (GAG) chain length, number and composition differ amongst cell types and are important determinants of the binding characteristics of molecules made by different cells. The proposal will attempt to establish the in vivo functional roles of serglycin. In Specific Aim 1, the investigators will characterize the GAG chain content of serglycins from different cell sources in order to determine their propensity for binding to different molecules. In specific Aim 2, they will perform functional analyses of serglycin by analyzing the ability of native and modified serglycins from different sources to bind to matrix molecules and cytokines. These experiments may thus elucidate the structural basis for the mechanism of action of the serglycin family of proteoglycans, and possibly lead to the development of the therapeutic agents. They will prepare homozygous knockouts of the gene in embryonic stem cells and monitor the effects on formation of embryoid bodies and hematopoietic cells. In Specific Aim 3, they will localize serglycin protein and mRNA in fetal and adult tissues in situ to determine sites of synthesis and deposition in vivo. This will enable them to better understand the function f this molecule. In Specific Aim 4, they will prepare promoter constructs of murine serglycin and compare regulation in several cell types with their data on cell-specific expression of the human gene. Promoter construct which have been found to be active in vitro will be used to generate transgenic mice in order to analyze the cell specificity of promoter regulation in vivo. Further studies could generate unique methods for cell-specific knockouts of the gene in mice to establish the function of this molecule in different cells.