Showing papers by "Andre E. Nel published in 1988"

Role of group-specific component (vitamin D binding protein) in clearance of actin from the circulation in the rabbit.

Abstract: The possible role of group specific component (Gc) (vitamin D-binding protein) in the clearance of cellular actin entering the circulation was examined with 125I-labeled Gc and actin injected into a rabbit model. Although filamentous F-actin is depolymerized primarily by plasma gelsolin, greater than or equal to 90% 125I-actin injected in either monomeric G- or F-form became complexed eventually with Gc (1:1 molar ratio). Clearance of Gc complexes was much faster (greater than 90% within 5 h) than that of native Gc (t1/2 = 17.2 h). Nephrectomy did not significantly alter the clearance of either Gc or actin. Since Gc complexes are dramatically increased in situations of tissue necrosis such as in fulminant hepatic failure, the current results suggest a crucial role for Gc in sequestration and clearance of released cellular actin.

Characterization of protein kinase C and its isoforms in human T lymphocytes.

Abstract: Protein kinase C (PKC) regulates numerous T cell functions and is present in abundance in normal human T cells and certain T cell lines. Although crude Triton X-100 soluble material obtained from T cell pellets contains minimal PKC activity, DEAE chromatography revealed that 12 to 37% of cellular PKC was membrane associated, probably due to removal of an inhibitor through column chromatography. As in other tissues, PKC from lymphoid tissue was phospholipid and Ca2+ dependent and diolein reduced the Ca2+ requirements for enzyme activity. Hydroxylapatite chromatography revealed that T cells possess two major peaks of PKC activity. Although, the enzyme in these peaks had similar m.w. and identical iso-electric mobility, the proteins differed with respect to their autophosphorylation sites and immunoreactivity toward an isoform specific antibody. Furthermore, differences in their activities in the presence of phospholipid, diolein, and limiting amounts of Ca2+ imply that these isoforms may be differentially activated. We discuss optimal conditions for activation of PKC and its isoforms for study of T lymphocyte cellular function.

Phosphorylation of human serum amyloid A protein by protein kinase C.

Abstract: Monokine-induced hepatic secretion of serum amyloid A protein (apo-SAA), an acute-phase reactant, is followed by rapid association with high-density lipoprotein (HDL) in plasma. Plasma clearance of apo-SAA is more rapid than any of the other HDL apolipoproteins. It has been shown that, of the acute-phase HDL3 apolipoproteins, apo-SAA preferentially associates with neutrophil membranes. HDL apolipoproteins have been shown to activate protein kinase C in endothelial cells. We therefore investigated potential phosphorylation of HDL3 apolipoproteins by protein kinase C. Apo-SAA was the only apolipoprotein phosphorylated (Km = 12 mM). Phosphorylation of the apo-SAA-containing HDL3 particle was selective for the more basic isoforms of apo-SAA (pI 7.0, 7.4, 7.5 and 8.0), with more acidic isoforms being phosphorylated when delipidated acute-phase apolipoproteins were used as substrate. However, phosphorylation was not in itself responsible for the establishment of the apo-SAA isoforms.

Cholera toxin partially inhibits the T-cell response to phytohaemagglutinin through the ADP-ribosylation of a 45 kDa membrane protein.

Abstract: This study examines the influence of cholera toxin (CT) on T lymphocyte activation by the mitogenic lectin phytohaemagglutinin (PHA). CT suppressed lectin-induced [3H]thymidine uptake in a dose-dependent fashion and acted synergistically with PHA in the generation of intracellular cyclic AMP. The toxin was assumed to act on Gs, because it also stimulated ADP-ribosylation of a 45 kDa membrane protein in vitro; no additional substrates were seen. The inhibitory effect of the adenylate cyclase/cyclic AMP pathway was shown to be directed at a concomitant stimulatory pathway, namely inositol phospholipid turnover. Lectin-stimulated 32P incorporation into both phosphatidylinositol as well as its 4,5-biphosphate derivative was depressed in the presence of CT or exogenous dibutyryl cyclic AMP. This, in turn, was associated with reduced activation of C-kinase as determined by decreased lectin-induced translocation from the cytosol to the surface membrane. These results indicate that Gs probably acts as a transducer between the PHA receptor and adenylate cyclase and may give rise to an exaggerated adenylate cyclase response in the presence of CT. It would seem as if reduction in inositol phospholipid turnover is related to the elevation of cyclic AMP rather than a CT effect on a putative transducer which acts directly on phospholipase C. Our study does not exclude the existence of non-CT-sensitive transducers in this capacity.

Promotion of DNA strand breaks in cocultured mononuclear leukocytes by protein kinase C-dependent prooxidative interactions of benoxaprofen, human polymorphonuclear leukocytes, and ultraviolet radiation.

Abstract: At concentrations of 5 µg/ml and greater the nonsteroidal antiinflammatory drug benoxaprofen caused dose-related activation of lucigeninenhanced chemiluminescence in human polymorphonuclear leukocytes (PMNL). Benoxaprofen-mediated activation of lucigenin-enhanced chemiluminescence by PMNL was increased by UV radiation and was particularly sensitive to inhibition by the selective protein kinase C inhibitor H-7. To identify the molecular mechanism of the prooxidative activity of benoxaprofen, the effects of the nonsteroidal antiinflammatory drug on the activity of purified protein kinase C in a cell-free system were investigated. Benoxaprofen caused a dose-related activation of protein kinase C by interaction with the binding site for the physiological activator phosphatidylserine, but could not replace diacylglycerol. When autologous mononuclear leukocytes (MNL) were cocultured with PMNL and benoxaprofen in combination, but not individually, the frequency of DNA strand breaks in MNL was markedly increased. UV radiation significantly potentiated damage to DNA mediated by benoxaprofen and PMNL. Inclusion of superoxide dismutase, H-7, and, to a much lesser extent, catalase during exposure of MNL to benoxaprofen-activated PMNL prevented oxidant damage to DNA. These results clearly demonstrate that potentially carcinogenic prooxidative interactions, which are unlikely to be detected by conventional assays of mutagenicity, may occur between phagocytes, UV radiation, and certain pharmacological agents.