Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., & Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1 beta (IL-1 beta) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 X 10(-8) M TPA. Higher concentrations of TPA, which partially or totally depleted protein kinase C levels in the cells (10(-9)-2 X 10(-5) M), had an inhibitory effect on IL-1 beta mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1 beta mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 X 10(-8) M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the alpha, beta, and gamma isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC alpha was particulate, and PKC beta was cytosolic, while there was no detectable PKC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)
Escherichia coli expression vectors encoding an acid-labile aspartyl-proline (Asp-Pro) dipeptide bridging two protein sequences were constructed and used to synthesize two different bovine growth hormone (bGH) fusion proteins. The codons GAT-CCX coding for Asp-Pro are provided by the recognition site for Bam HI (GGATCC). Treatment of the bGH fusion proteins at low pH in the presence of guanidine hydrochloride releases the bGH moiety from the fusion protein. The release of the bGH from the fusion protein specifically requires the Asp-Pro dipeptide linking the bGH sequence to the fusion protein. The bGH released from the fusion protein retains anti-bGH immunoreactivity as well as the ability to bind to growth hormone receptor in vitro.
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