The long-term objective of this proposal is to study extensively the molecular biology and the biological activity of lipokinins (LPK), natural activators of phospholipase A2 (PLA2) discovered in our laboratory. PLA2 releases arachidonic acid from the cell membrane and therefore is stimulatory to the arachidonic acid cascade. There has been increasing evidence to suggest that the arachidonic acid cascade and its product, the prostaglandins, play major roles in normal development, and that a disturbance of this pathway at any point may lead to teratogenesis. Study of the roles of LPK during normal and abnormal development is therefore of considerable importance. Recently we have succeeded in purifying a 55 kDa LPK. In the proposed study, other LPK will also be purified, and the amino acid sequences and polyclonal antibodies of each will be obtained. Bovine and human LPK cDNAs will be isolated, and the human cDNA will be used to determine the chromosome localization of the LPK gene or genes and for the isolation and characterization of human LPK genomic clones. Production of LPK in prokaryote expression system will be attempted. LPK gene expression will be determined in the developing mouse embryo by Northern blot analysis of RNA isolated from embryos at different developmental stages. Difference in LPK gene expression between normal and drug-treated embryos will also be studied. Biological effects of purified LPK on arachidonic acid release from thymocytes will be tested. Preliminary studies show that partially purified lipokinin prevent hyperglycemia-induced teratogenesis in cultured mouse embryos, and induces growth/differentiation in pre-somite stage mouse embryos in culture. It will be determined whether purified LPK also has these effects on the mouse embryos. The proposed studies should provide important information about the regulations of PLA2 and LPK in normal and abnormal development.
