Our objectives are to determine: a) whether our newly discovered 5 kDa peptide (Fx) is identical to thymosin beta4; we will compare their sequences, amino acid compositions and interaction with actin in several functional assays; b) whether the Fx-actin complex is the source of G-actin for actin polymerization when platelets and neutrophils are activated; we will use immunoprecipitation, immunoblotting, DNase assay, gel filtration, gel electrophoresis and high performance liquid chromatography to study the ratio of free Fx to the Fx-actin complex and the G/F actin ratio in the cells before and after stimulation with agonists. c) the structure of the Fx-actin complex and the major interacting amino acids, using labelled Fx, chemical crosslinking, sequencing of the cross linked peptide and other structural approaches; how the Fx-actin complex is regulated; d) whether Fx binds to cell components, and whether it is secreted; e) whether known analogues of thymosin beta4 also interact with actin; f) whether analogues peptides occur in non-mammalian cells which also sequester actin monomers; g) whether newly synthesized actin is complexed with Fx or with other actin binding proteins. The results of our studies should shed light on the mechanisms by which cells maintain pools of G-actin in their cytoplasms. This mechanism is of fundamental importance in hemostasis, chemotaxis, nerve cell growth, phagocytosis, and formation of scar tissue. It may also be involved in the process of cell division which is a requisite for survival.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR040840-03
Application #
3161297
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1991-04-01
Project End
1994-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
3
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Safer, D; Chowrashi, P K (1997) Beta-thymosins from marine invertebrates: primary structure and interaction with actin. Cell Motil Cytoskeleton 38:163-71
Sanger, J M; Golla, R; Safer, D et al. (1995) Increasing intracellular concentrations of thymosin beta 4 in PtK2 cells: effects on stress fibers, cytokinesis, and cell spreading. Cell Motil Cytoskeleton 31:307-22

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