BIOLOGY OF HEMATOPOIETIC MEMBRANE REGULATORY MOLECULES

造血膜调节分子的生物学

• 批准号: 3229833
• 负责人: Nicholas Dainiak
• 金额: $ 13.4万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 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 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3229833
• 关键词: B lymphocyte; T lymphocyte; affinity chromatography; antigen receptors; antisense nucleic acid; binding proteins; bone marrow; cell cycle; cell membrane; colony stimulating factor; complementary DNA; enzyme linked immunosorbent assay; erythrocyte membrane; erythroid stem cell; erythropoiesis; gel electrophoresis; glycoprotein biosynthesis; glycosylation; hematopoiesis; hematopoietic growth factor; hematopoietic stem cells; human subject; immunofluorescence technique; interleukin 3; lymphoblast; membrane lipids; membrane proteins; molecular cloning; monoclonal antibody; monocyte; neoplastic cell; oligonucleotides; protein biosynthesis; protein purification; radioimmunoassay; surface antigens; vesicle /vacuole

DESCRIPTION: (Adapted from investigator's abstract) The Principal Investigator has found that regulators of hematopoiesis are expressed on B lymphocyte plasma membranes. To definitely characterize an erythroid regulatory molecule and to examine the biology of regular production, release and action, the following studies will be performed in a biochemically defined culture system: 1. Biochemical and structural characterization of membrane-associated regulators. The investigators will extract an erythroid regulator from plasma membranes of B-cells and B-lymphoblastoid cell lines. The regulator will be purified and characterized by standard biochemical methods. To improve efficiency, an ELISA assay will be developed that will employ monoclonal antibodies which have been and will be produced against the erythroid regulator. After collection of purified BPA, a portion of the amino terminal sequence for BPA will be obtained. If the amino terminal is blocked, limited proteolytic digestion will be performed prior to sequencing. Sense and antisense oligonucleotides will be designed and cDNA for BPA will be prepared by standard methods, using a Lambda GT11 lymphocyte cDNA expression library. Antibody to BPA will be used as well to screen the library. After the amino acid sequence for BPA is obtained, a search for homologous proteins will be made. 2. Regulator production and release. Rates of regulator release from mononuclear cell subpopulations will be assessed as a function of seeding concentration and mixture of cells from various species of mice. These studies will assess whether paracrine-like cell-cell communication and/or generation of an immune response are important for BPA production. 3. Identification of factor responsive cells. Exfoliated membrane vesicles expressing regulatory factors will be labeled and mixed with marrow mononuclear subpopulations in order to identify target cells. Binding of purified regulators to target cells will be characterized and post-binding effects will be assessed, including alterations in cell cycle status and protein synthesis. 4. Determination of in vivo effects. Column-purified, membrane erythroid regulator will be administered to mice in vivo, and effects on peripheral blood counts, hematopoietic activity in the bone marrow and spleen will be determined. These experiments are aimed to extend the investigators' early finding that erythropoiesis in vivo is stimulated by the regulator. Using this approach, they hope to clarify the physiologic role of membrane-associated regulatory molecules.

描述：（改编自研究者摘要）负责人 研究者发现造血调节因子在B上表达 淋巴细胞质膜 明确描述红细胞的特征 调节分子和检查生物学的定期生产， 发布和行动，以下研究将在 生物化学确定的培养系统：1. 生物化学和结构 膜相关调节剂的表征。 调查人员将 从B细胞的质膜中提取红细胞调节剂， B淋巴母细胞样细胞系。 调节器将被净化， 以标准生物化学方法为特征。 为了提高效率， 将开发采用单克隆抗体的ELISA测定法， 已经产生并将产生对抗红细胞调节因子的抗体 后 收集纯化的BPA，一部分氨基末端序列， BPA将被使用。 如果氨基末端被封闭， 在测序之前进行蛋白水解消化。 理智与 将设计反义寡核苷酸， 使用λ GT 11淋巴细胞cDNA通过标准方法制备 表达式库。 BPA抗体也将用于筛选 图书馆 在获得BPA的氨基酸序列后，搜索 将产生同源蛋白质。 2. 调节剂的生产和释放。 调节剂从单核细胞亚群释放的速率将是 作为接种浓度和细胞混合物的函数进行评估 不同种类的老鼠 这些研究将评估旁分泌样 细胞-细胞通讯和/或免疫应答的产生被 对BPA生产很重要。 3. 响应因子的识别 细胞 表达调节因子的脱落膜囊泡将被 标记并与骨髓单核细胞亚群混合， 识别靶细胞。 纯化的调节剂与靶细胞的结合将 将进行表征并评估绑定后的影响，包括 细胞周期状态和蛋白质合成的改变。 4. 测定 体内效应。 将柱纯化的膜红细胞调节剂 在体内给予小鼠，以及对外周血细胞计数的影响， 将测定骨髓和脾中的造血活性。 这些实验旨在扩展研究人员的早期发现， 体内红细胞生成受调节剂刺激。 使用此 通过这种方法，他们希望澄清膜相关的生理作用， 调节分子