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.
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