Lakhdar Gasmi

The Saccharomyces cerevisiae PCD1 Gene Encodes a Peroxisomal Nudix Hydrolase Active toward Coenzyme A and Its Derivatives

The PCD1 nudix hydrolase gene ofSaccharomyces cerevisiae has been cloned and the Pcd1p protein characterized as a diphosphatase (pyrophosphatase) with specificity for coenzyme A and CoA derivatives. Oxidized CoA disulfide is preferred over CoA as a substrate with K m andk cat values of 24 μm and 5.0 s− 1, respectively, compared with values for CoA of 280 μm and 4.6 s− 1respectively. The products of CoA hydrolysis were 3′-phosphoadenosine 5′-monophosphate and 4′-phosphopantetheine. F− ions inhibited the activity with an IC50 of 22 μm. The sequence of Pcd1p contains a potential PTS2 peroxisomal targeting signal. When fused to the N terminus of yeast-enhanced green fluorescent protein, Pcd1p was shown to locate to peroxisomes by confocal microscopy. It was also shown to co-localize with peroxisomal thiolase by immunofluorescence microscopy. N-terminal sequence analysis of the expressed protein revealed the loss of 7 or 8 amino acids, suggesting processing of the proposed PTS2 signal after import. The function of Pcd1p may be to remove potentially toxic oxidized CoA disulfide from peroxisomes in order to maintain the capacity for β-oxidation of fatty acids.

Stimulation of Intracellular Ca2+ Levels in Human Neutrophils by Soluble Immune Complexes FUNCTIONAL ACTIVATION OF FcγRIIIb DURING PRIMING

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor α or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcγRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcγRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcγRII, but not FcγRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcγRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcγRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcγRII or FcγRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcγRIIIb.

Neutrophil apoptosis is delayed by the diadenosine polyphosphates, Ap5A and Ap6A: synergism with granulocyte-macrophage colony-stimulating factor.

In addition to ATP, platelets and other cell types can secrete high quantities of diadenosine polyphosphates Ap3A, Ap4A, Ap5A and Ap6A. There is increasing evidence to show that these molecules can function as novel modulators of cell function. For this report we have measured the effects of the diadenosine polyphosphates Ap5A and Ap6A on neutrophil apoptosis. These molecules can themselves delay neutrophil apoptosis (as assessed by morphology, function, CD16 expression and chromatin integrity), and are as effective on a molar basis as ATP, Ap3A and Ap4A. Moreover, these dinucleotides act synergistically with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) to delay neutrophil apoptosis. Thus, diadenosine polyphosphates may act, in concert with cytokines, as novel modulators of neutrophil function and survival in certain types of inflammatory conditions.

Diadenosine polyphosphates induce intracellular Ca2+ mobilization in human neutrophils via a pertussis toxin sensitive G-protein‘pa

The diadenosine polyphosphates diadenosine 5′,5′′′‐P1,P3‐triphosphate(Ap3A), diadenosine 5′,5′′′‐P1,P4‐tetraphosphate (Ap4A), diadenosine 5′,5′′′‐P1,P5‐pentaphosphate (Ap5A) and diadenosine 5′,5′′′‐P1,P6‐hexaphosphate (Ap6A) all stimulated increases in intracellular Ca2+ in human neutrophils. Maximal increases in intracellular Ca2+ of 650 nm were obtained at dinucleotide concentrations of 500–700 μm. These increases in intracellular Ca2+ were completely abolished by pre‐treatment of the neutrophils with pertussis toxin and were hardly affected when the extracellular buffer was devoid of Ca.2+ On the other hand, adenosine triphosphate (ATP) could stimuate much greater increases in intracellular Ca2+ (up to 1·1 μm) at much lower concentrations (half maximal responses obtained at around 5 μm ATP). Receptor de‐sensitization experiments indicate that human neutrophils may possess two types of P2‐purinoceptors. The first of these may bind ATP (but not the dinucleotides) with high affinity whilst the second may bind the dinucleotides with lower affinity and also bind ATP.