A critical step in early heart development is the elaboration of cardiac mesenchyme within the endocardial cushions. These cushions are located in the atrioventricular canal and proximal outflow tract and provide a framework for the elaboration of valves and for partitioning of the heart into its representative chambers. Mesenchyme formation is controlled by the secretion of a particulate form of extracellular matrix from the myocardium underlying these cushions. The particulate matrix consists of a multicomponent complex of proteins that includes fibronectin and at least 5-6 other components. The focus of my laboratory is in the isolation, purification and functional identification of these components.In pursuit of these proteins we have utilized lectin histochemistry and lectin affinity chromatography to isolate a group of proteins responsible for mesenchyme formation. While the molecular weight of these proteins is known, little information has been obtained as to the identity or functional role of these proteins in this process. To further characterize these proteins we have produced a series of monoclonal antibodies directed against the bound fraction from the lectin affinity column. We are currently using one of these antibodies to characterize a 283-kDa extracellular protein that is involved in this process.This antibody was also used to screen two cDNA expression libraries in an attempt to identify a clone for this protein. We have isolated a 500-bp fragment using this strategy. Sequence analysis of this probe revealed a unique sequence not found in the database. This probe was also used to isolate two 1-Kb fragments that are now being sequenced.In addition we are separating the media into components using size exclusion chromatography utilizing both low and high pressure systems so that we can test individual proteins or groups of proteins for biological activity in a bioassay of mesenchyme formation. Finally we are examining the affect of retinoic acid, a known teratogen of heart development, on the expression of this protein to determine if the defects associated with this teratogen are manifested by changes in the extracellular matrix.
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