REGULATION OF ERYTHROPOIESIS BY CELL SURFACE COMPONENTS

细胞表面成分对红细胞生成的调节

• 批准号: 3229831
• 负责人: Nicholas Dainiak
• 金额: $ 9.86万
• 依托单位: ST. ELIZABETH'S MEDICAL CENTER OF BOSTON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1988-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3229831
• 关键词: affinity chromatography; bone marrow; cell differentiation; cell growth regulation; cell membrane; chromatography; density gradient ultracentrifugation; erythropoiesis; glycoproteins; growth factor; hematopoietic stem cells; human tissue; lymphocyte; membrane activity; membrane proteins; monocyte; protein biosynthesis; protein structure; tissue /cell culture

We have found that erythroid burst promoting activity (BPA) is expressed by lymphocyte plasma membranes. We now propose to utilize a biochemically defined culture system to: 1. Purify membrane-associated BPA. We will utilize a variety of biochemical methods, including immunoaffinity, lectin affinity and gel filtration high-performance liquid chromatography (HPLC) and reverse-phase HPLC, to purify BPA from extracts of lymphocyte plasma membranes and shed vesicles. Purified fractions will be characterized by isoelectric focusing and sucrose gradient fractionation. Ultimately, purified BPA will be subjected to amino acid analysis. 2. Determine relatedness of soluble and vesicular BPA. We will employ antisera and monoclonal antibodies that react with BPA to detect whether purified BPA from membranes and in solution are antigenically related. Studies will be performed to determine if both physical forms of BPA are released by similar mechanisms from similar cell types, and if they act on similar marrow subpopulations. Target cells for each growth factor will be characterized histochemically and morphologically. 3. Define kinetics of vesicle-associated BPA release. Cumulative amounts and rates of vesiculation and BPA release by BPA-producing cell populations will be determined. Our findings will be correlated with studies aimed at measuring the biosynthesis of selected membrane components in culture. 4. Examine modes of BPA action. We will determine whether BPA shedding is a constitutive process or part of an immune reaction. Studies will be performed to examine whether vesiculation involves cytoskeletal components and whether membrane lipid and phospholipid composition is important to the shedding process. We will determine whether vesicles associate preferentially with marrow cells and, if so, what the nature of this association might be using electronmicroscopic methods. Ultimately, we will locate and purify BPA receptors with the projected long-range goal to examine intracellular mechanisms for receptor activation.

我们发现红细胞爆裂促进活性（BPA）的表达方式为 淋巴细胞质膜。 我们现在建议利用生化方法 将文化系统定义为： 1. 纯化膜相关的 BPA。 我们将利用各种 生化方法，包括免疫亲和法、凝集素亲和法和凝胶法 过滤 高效液相色谱 (HPLC) 和反相 HPLC，从淋巴细胞质膜和脱落物提取物中纯化 BPA 囊泡。 纯化的馏分将通过等电聚焦进行表征 和蔗糖梯度分馏。 最终，纯化的 BPA 将被 进行氨基酸分析。 2. 确定可溶性 BPA 和囊泡 BPA 的相关性。 我们将聘用 抗血清和单克隆抗体与 BPA 发生反应以检测是否 从膜和溶液中纯化的 BPA 具有抗原相关性。 将进行研究以确定 BPA 的两种物理形式是否 由相似的细胞类型通过相似的机制释放，如果它们作用于 相似的骨髓亚群。 每种生长因子的靶细胞将是 具有组织化学和形态学特征。 3. 定义囊泡相关 BPA 释放的动力学。 累计金额 以及产生 BPA 的细胞群的囊泡形成和 BPA 释放率 将被确定。 我们的研究结果将与旨在 测量培养物中选定膜成分的生物合成。 4. 检查 BPA 作用模式。 我们将确定 BPA 是否脱落 免疫反应的组成过程或一部分。 研究将是 进行检查囊泡形成是否涉及细胞骨架成分 以及膜脂和磷脂的组成是否对 脱落过程。 我们将确定囊泡是否缔合 优先使用骨髓细胞，如果是这样，其性质是什么 协会可能正在使用电子显微镜方法。 最终，我们 将定位并纯化 BPA 受体，预计的长期目标是 检查受体激活的细胞内机制。