Paul P Shields

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The export of heterologous products into the conditioned medium of an Escherichia coli culture offers the advantages of a higher product yield, an increased probability of recovering an intact recombinant protein, proper folding for biological activity, and greater stability of a secreted product. In this report, we describe the development of an optimized(More)
Ascorbate is an important cofactor in the biosynthesis of alpha-amidated endocrine and neural peptides. Peptidylglycine alpha-amidating monooxygenase (PAM) is the enzyme responsible for the generation of mature COOH-terminal alpha-amidated peptides from COOH-terminal glycine-extended peptides, and this enzyme requires ascorbate for full activity in vitro.(More)
Aspartic acid proteases have been implicated in the processing of ET-1(1-39) to ET-1(1-21). To further understand the role of this class of enzymes in ET-1 synthesis, cultured vascular endothelial cells were incubated with pepstatin, and the accumulation of the inhibitor and its effect on the processing of ET-1(1-39) was examined. Pepstatin accumulated in(More)
Atrial natriuretic factor (ANF) is stored in mammalian atria primarily as ANF-(1-126), the precursor to the known circulating form of the hormone ANF-(99-126). When primary cultures of atrial myocytes were maintained in a complete serum-free medium, they contained and secreted an ANF-(1-126)-like peptide. The addition of dexamethasone to the culture medium,(More)
Atrial natriuretic factor (ANF) is stored in atrial myocytes as a prohormone (ANF-(1-126] and is cosecretionally processed to the circulating ANF-related peptides, ANF-(1-98) and ANF-(99-126). Recently, we have shown that the cosecretional processing of ANF can be replicated in primary cultures of neonatal rat atrial myocytes maintained under serum-free(More)
Atrial myocytes cultured for 7 days in serum-free medium secrete a 15K form of atrial natriuretic peptide (15K ANP), but isolated perfused rat hearts secrete the major circulating form of the hormone, a 3K peptide, 3K ANP. This difference was examined in the present study. 15K ANP was purified from rat atria, and sequencing analysis demonstrated that this(More)
Salmon calcitonin (sCT) is a 32 amino acid peptide hormone that requires C-terminal amidation for full biological activity. We have produced salmon calcitonin by in vitro amidation of an E. coli produced precursor peptide. Glycine-extended sCT, the substrate for amidation, was produced in recombinant E. coli as part of a fusion with(More)
Primary cultures of neonatal rat atrial myocytes were maintained in two different serum-free media for up to 25 days. Reversed-phase high performance liquid chromatography coupled with atrial natriuretic factor (ANF)-specific radioimmunoassay demonstrated that the cultures maintained in our previously described serum-free medium (Glembotski, C.C., and(More)
Although cultures of neonatal rat atria and ventricles have been widely used to study ANP biosynthesis and secretion, little is known regarding the circulating form of ANP in neonatal animals. To begin to address this issue, we have developed a method for perfusing isolated neonatal rat hearts. Reversed phase-HPLC analysis of the heart effluents coupled(More)
The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of(More)