This project has established and characterized two endometrial epithelial cell lines which are temperature-sensitive for differentiation and express uteroglobin gene in response to steroid hormones. This confirmed preliminary observations that phospholipase A2 (PLA2) is activated by transglutaminase (TG)-mediated polyamination of this enzyme. The group has also identified a specific region of group I PLA2 (residues 21-40) interaction of which with a neutralizing antibody leads to complete inhibition of its activity, obtained overexpression of recombinant human uteroglobin (hUG) in E.coli, and demonstrated that this recombinant protein is a potent inhibitor of PLA2 activity and an excellent substrate of TG. They have performed site-directed mutagenesis of hUG and expression of recombinant mutated protein. Preliminary results suggest that Lys 43 of hUG is critical for its PLA2 inhibitory activity and established that hUG gene is expressed in various tissues and cells other than the pulmonary alveolar Clara cells as was originally thought; these results demonstrate the similarity between the rabbit and hUG expression. This project has also characterized a region of osteopontin (OP) which specifically binds Ca++ and confirmed preliminary results that in the milk of mothers who delivered premature infants, the OP level is significantly higher than that in the milk of mothers who delivered at term. The group discovered a novel splicing of OP mRNA with a possible regulatory role in transcriptional and/or posttranscriptional events, possibly explaining the multifunctional role of OP in diverse cell types and tissues. They also developed a rapid, inexpensive, non-radiometric and simple quantitative PCR-ELISA for quantitation of gene expression and detection of mutations. This assay is potentially applicable to prenatal genetic diagnosis and the diagnosis of viral (including HIV), pro- and eukaryotic infections. International and US patent applications have been filed.
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