BIOLOGY OF HEMATOPOIETIC MEMBRANE REGULATORY MOLECULES

描述：(改编自调查人员摘要)校长 研究人员发现，B细胞上表达了造血调节因子 淋巴细胞膜。明确描述红血球的特征 调节分子和研究正常生产的生物学， 释放和行动，以下研究将在 生物化学定义的培养系统：1.生化和结构 膜相关调节剂的特性。调查人员将 从B细胞质膜中提取红系调节剂 B淋巴母细胞系。调整器将被提纯并 以标准的生化方法为特征的。为了提高效率，一个 将开发使用单抗的ELISA法，该单抗 已经并将针对红系调节器产生。之后 收集纯化的双酚A，部分氨基末端序列用于 将获得双酚A。如果氨基末端被阻断， 蛋白质分解消化将在测序前进行。理性与理性 将设计反义寡核苷酸，并将设计BPA的cDNA 用标准方法制备，使用Lambda GT11淋巴细胞cDNA 表达式库。抗双酚A抗体也将用于筛查 图书馆。在获得BPA的氨基酸序列后，搜索 同源蛋白质将会被制造出来。2.调节剂的生产和释放。 从单个核细胞亚群释放调节剂的速度将是 作为种子浓度和细胞混合物的函数进行评估 不同种类的老鼠。这些研究将评估类旁分泌细胞 细胞间的通信和/或免疫反应的产生 对双酚A生产很重要。3.响应性因素的识别 细胞。表达调节因子的脱落膜小泡将是 标记和混合骨髓单个核亚群，以便 识别目标单元格。纯化的调节剂与靶细胞的结合将 将确定其特征，并评估有约束力后的影响，包括 细胞周期状态和蛋白质合成的改变。4.决心 体内效应。柱提纯，膜红系调节剂将 给小鼠体内注射，以及对外周血细胞计数的影响， 将测定骨髓和脾的造血活性。 这些实验的目的是扩大调查人员的早期发现 体内的红细胞生成是由调节剂刺激的。使用这个 方法，他们希望阐明膜相关的生理作用 调节分子。

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.