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D K Perry

Publications and source records attributed to D K Perry.

28 records · Page 2Linked to original sources
PubMed1997

Cytokine response modifier A (CrmA) inhibits ceramide formation in response to tumor necrosis factor (TNF)-alpha: CrmA and Bcl-2 target distinct components in the apoptotic pathway.

Proteases are now firmly established as major regulators of the "execution" phase of apoptosis. Here, we examine the role of proteases and their relationship to ceramide, a proposed mediator of apoptosis, in the tumor necrosis factor-alpha (TNF-alpha)-induced pathway of cell death. Ceramide induced activation of prICE, the protease that cleaves the death substrate poly(ADP-ribose) polymerase. Bcl-2 inhibited ceramide-induced death, but not ceramide generation. In contrast, Cytokine response modifier A (CrmA), a potent inhibitor of Interleukin-1 beta converting enzyme and related proteases, inhibited ceramide generation and prevented TNF-alpha-induced death. Exogenous ceramide could overcome the CrmA block to cell death, but not the Bcl-2 block. CrmA, however, did not inhibit the activation of nuclear factor (NF)-kappa B by TNF-alpha, demonstrating that other signaling functions of TNF-alpha remain intact and that ceramide does not play a role in the activation of NF-kappa B. These studies support a distinct role for proteases in the signaling/activation phase of apoptosis acting upstream of ceramide formation.

Apoptosis↗
PubMed1997

Bcl-2 acts upstream of the PARP protease and prevents its activation.

Apoptosis has recently been extensively studied and multiple factors have been implicated in its regulation. It remains unclear how these factors are ordered in the cell death pathway. Here we investigate the relationship between the inhibitor of apoptosis, bcl-2, and the PARP protease, prlCE/CPP32, recently implicated in apoptosis. Using PARP proteolysis as an indicator of the activation of the PARP protease, we find that the chemotherapeutic agent, etoposide, induces apoptosis and PARP proteolysis in Molt4 cells as early as 4 h with cell death lagging behind this event. In contrast, Molt4 cells that over-express bcl-2 show no PARP proteolysis or cell death. In order to determine if bcl-2 inhibits the PARP protease or its activation, we developed a cell-free system. Using this system with extracts from etoposide-treated cells and purified bovine PARP, we demonstrate that extracts from bcl-2 over-expressing cells cause little or no PARP proteolysis. Whereas, extracts from control vector cells contain an active PARP protease. This protease is inhibited by the tetrapeptide ICE-like protease inhibitor, YVAD-chloromethylketone. Interestingly, this protease is not inhibited by the addition of purified bcl-2 protein. These results rule out that bcl-2 directly inhibits the active protease or that it has an effect downstream of prlCE/CPP32 such as preventing access to the PARP substrate. These results also demonstrate a role of bcl-2 in interfering with an upstream signal required to activate the PARP protease and allow us to begin to order the components in the apoptotic pathway.

Journal Article↗
PubMed1996

prICE: a downstream target for ceramide-induced apoptosis and for the inhibitory action of Bcl-2.

The novel lipid second messenger, ceramide, specifically induced poly(ADP-ribose) polymerase cleavage through activation of the protease prICE. Over-expression of Bcl-2 inhibited ceramide-induced poly(ADP-ribose) polymerase proteolysis and protected cells from ceramide-induced death. These data provide the first insight into the mechanism by which ceramide mediates apoptosis and suggest a mechanism by which Bel-2 protects from cell death.

Apoptosis↗
PubMed1993

A novel ecto-phosphatidic acid phosphohydrolase activity mediates activation of neutrophil superoxide generation by exogenous phosphatidic acid.

Phosphatidic acid (PA) added to intact cells activates a variety of processes including mitogenesis in fibroblasts and superoxide generation in neutrophils. We have investigated the mechanism of activation of superoxide generation in intact human neutrophils by a short-chain (dioctanoyl) PA (diC8PA). After a lag, diC8PA caused a high rate of superoxide production (19.6 nmol of cytochrome c reduced/min/10(6) cells). Activation did not require extracellular Ca2+ and coincided with near quantitative conversion of diC8PA to dioctanoylglycerol (diC8-glycerol). diC8PA also activated cellular phospholipase D with release of long-chain PA and secondary production of long-chain diradylglycerol (sn-1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol). The metabolism of diC8PA to diC8-glycerol was catalyzed by a novel PA phosphohydrolase on the outer leaflet of the plasma membrane as demonstrated by the exclusive release of Pi into the extracellular medium. This enzyme also showed activity toward PA containing long-chain unsaturated fatty acids. The ecto-PA phosphohydrolase differed from the intracellular PA phosphohydrolase based on its relative insensitivity to desipramine and N-ethylmaleimide. The enzyme was also present in Chinese hamster ovary (CHO) cells and its activity did not change in transfected CHO cells expressing the two membrane-associated isoforms of alkaline phosphatase, indicating that the PA phosphohydrolase was not alkaline phosphatase. Non-hydrolyzable phosphonate analogs of diC8PA poorly stimulated superoxide production. Activation of superoxide generation by diC8PA was inhibited by staurosporine, suggesting a protein kinase C-dependent mechanism. We suggest that the action of a novel ecto-PA phosphohydrolase permits exogenously added short-chain PA to serve as "timed-release diacylglycerol" and that its biological effects in neutrophils are secondary to diacylglycerol-mediated protein kinase C activation.

Animals↗
PubMed1992

Role of phospholipase D-derived diradylglycerol in the activation of the human neutrophil respiratory burst oxidase. Inhibition by phosphatidic acid phosphohydrolase inhibitors.

An agonist-activated phospholipase D/phosphatidic acid phosphohydrolase (PAH) pathway was recently demonstrated in human neutrophils, and evidence suggests that phosphatidic acid (PA) and/or diradylglycerol (DG) generated from this pathway participates in activation of the O2(-)-generating respiratory burst. We have used a series of cationic amphiphilic compounds (sphingosine, propranolol, chlorpromazine, and desipramine) and antibiotics (clindamycin, trimethoprim, and roxithromycin) all of which inhibit the respiratory burst, to investigate the role of the phospholipase D/PAH pathway in neutrophil activation. The phosphatidylcholine (PC) pool in intact cells was first labeled using [3H]-1-O-alkyl-lysoPC; released [3H]-PA and [3H]-DG were then quantified after the addition of either chemo-attractant or PMA. Using either agonist, all compounds showed a dose-dependent inhibition of [3H]-DG generation which correlated with inhibition of O2- generation, but compounds failed to inhibit directly the NADPH oxidase in a cell-free system. For either activator, a plot of the ID50 values for O2- generation vs those for DG generation was linear over four orders of magnitude. In many cases, inhibition of [3H]-DG generation corresponded to an increase in [3H]-PA, implicating PAH as the locus of inhibition. Superoxide generation was inhibited under conditions where PA was either elevated or minimally affected. Neither O2- release nor DG generation showed any selectivity for stereoisomers of propranolol, suggesting that this inhibition does not act via a specific binding site on PAH. No evidence was obtained for an effect of the inhibitors on PA mobility as monitored by electron spin resonance studies of spin-labeled PA in a model membrane system. Data are consistent with an effect of the inhibitors at the level of the interaction of PAH with the membrane and/or its substrate. These data imply that DG produced via the phospholipase D/PAH pathway functions in the activation or maintenance of the respiratory burst.

Diglycerides↗
PubMed1992

A carboxy-terminal peptide from p47-phox is a substrate for phosphorylation by protein kinase C and by a neutrophil protein kinase.

Agonist-activated phosphorylation of neutrophil proteins including p47-phox, a cytosolic component of the respiratory burst oxidase, has been implicated in the signal transduction cascade which leads to activation of the superoxide generating respiratory burst. We have previously reported (J. Biol. Chem. 265, 17550-59) that in a cell-free activation system consisting of cytosol plus plasma membrane from human neutrophils, diacylglycerol acts synergistically with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to augment superoxide generation and assembly of the oxidase, and that p47 phosphorylation can occur under these conditions. Herein, we show that a peptide corresponding to a carboxy terminal sequence of p47-phox is a substrate for phosphorylation both by purified protein kinase C (a mixture of alpha, beta, and gamma forms) and by a distinct kinase or kinases present in neutrophil cytosol. Based on its activator requirements, the neutrophil kinase differs from classical protein kinase C, but may be a protein kinase C variant, based on inhibition by a protein kinase C peptide. Although in the cell-free system phosphorylation occurs under conditions which are similar to those for activation of superoxide generation, phosphorylation is not required for activation (1). Rather, protein assembly or aggregation which occurs under activation conditions may also promote phosphorylation.

Adenosine Triphosphate↗
PubMed1986

Cigarette smoking behavior among Rhode Island physicians, 1963-83.

A survey of practicing physicians in Rhode Island revealed a cigarette smoking rate of only 8.3 per cent in 1983. This represents a continuation of the consistent decrease seen in the percentage of physician smokers in each such survey since 1963. Under age 30, the rate was only 4.5 per cent. An exponential model suggests that the overall rate for Rhode Island physicians will be below 3 per cent by the year 2000.

Adult↗