Deciphering the plasma membrane hallmarks of apoptotic cells: Phosphatidylserine transverse redistribution and calcium entry

BACKGROUND: During apoptosis, Ca(2+)-dependent events participate in the regulation of intracellular and morphological changes including phosphatidylserine exposure in the exoplasmic leaflet of the cell plasma membrane. The occurrence of phosphatidylserine at the surface of specialized cells, such as platelets, is also essential for the assembly of the enzyme complexes of the blood coagulation cascade, as demonstrated by hemorrhages in Scott syndrome, an extremely rare genetic deficiency of phosphatidylserine externalization, without other apparent pathophysiologic consequences. We have recently reported a reduced capacitative Ca(2+) entry in Scott cells which may be part of the Scott phenotype. RESULTS: Taking advantage of these mutant lymphoblastoid B cells, we have studied the relationship between this mode of Ca(2+) entry and phosphatidylserine redistribution during apoptosis. Ca(2+) ionophore induced apoptosis in Scott but not in control cells. However, inhibition of store-operated Ca(2+) channels led to caspase-independent DNA fragmentation and decrease of mitochondrial membrane potential in both control and Scott cells. Inhibition of cytochrome P450 also reduced capacitative Ca(2+) entry and induced apoptosis at comparable extents in control and Scott cells. During the apoptotic process, both control and more markedly Scott cells externalized phosphatidylserine, but in the latter, this membrane feature was however dissociated from several other intracellular changes. CONCLUSIONS: The present results suggest that different mechanisms account for phosphatidylserine transmembrane migration in cells undergoing stimulation and programmed death. These observations testify to the plasticity of the plasma membrane remodeling process, allowing normal apoptosis even when less fundamental functions are defective

L'elevage sur lies des vins blancs de Bourgogne II. Evolution des macromolécules: polysaccharides et protéines

La conservation sur lies des vins blancs de Bourgogne se traduit dans un premier temps par un enrichissement de ceux-ci en composés macromoléculaires précipitables à l'éthanol; puis dans un second temps la teneur en ces composés diminue à la suite d'une hydrolyse des polysaccharides d'origine pariétale.Un étude en milieu modèle permet de préciser que les principaux produits de l'autolyse provenant des parois de levures sont des mannoprotéines qui sont libérées après une hydrolyse enzymatique des glucanes. Les ß(1→3 )-glucanases responsables de cette hydrolyse sont libérées dans le milieu d'autolyse et il en est de même d'une protéase de type A. Ainsi l'hydrolyse des macromolécules se poursuit dans le milieu extracellulaire

Increased Oxidative Stress Induces Apoptosis in Human Cystic Fibrosis Cells

Oxidative stress results in deleterious cell function in pathologies associated with inflammation. Here, we investigated the generation of superoxide anion as well as the anti-oxidant defense systems related to the isoforms of superoxide dismutases (SOD) in cystic fibrosis (CF) cells. Pro-apoptotic agents induced apoptosis in CF but not in control cells that was reduced by treatment with SOD mimetic. These effects were associated with increased superoxide anion production, sensitive to the inhibition of IκB-α phosphorylation, in pancreatic but not tracheal CF cells, and reduced upon inhibition of either mitochondrial complex I or NADPH oxidase. CF cells exhibited reduced expression, but not activity, of both Mn-SOD and Cu/Zn-SOD when compared to control cells. Although, expression of EC-SOD was similar in normal and CF cells, its activity was reduced in CF cells. We provide evidence that high levels of oxidative stress are associated with increased apoptosis in CFTR-mutated cells, the sources being different depending on the cell type. These observations underscore a reduced anti-oxidant defense mechanism, at least in part, via diminished EC-SOD activity and regulation of Cu/Zn-SOD and Mn-SOD expressions. These data point to new therapeutic possibilities in targeting anti-oxidant pathways to reduce oxidative stress and apoptosis in CF cells

Microparticles from patients with metabolic syndrome induce vascular hypo-reactivity via Fas/Fas-ligand pathway in mice

Peer reviewedPublisher PD

Stability of the thrombin-thrombomodulin complex on the surface of endothelial cells from human saphenous vein or from the cell line EA.hy 926

Protein C activation by alpha-thrombin on the surface of endothelial cells depends on an essential membrane-glycoprotein cofactor, thrombomodulin. In the present study we have monitored the activity of thrombin-thrombomodulin complexes on human saphenous-vein endothelial cells (HSVEC) or on the endothelial cell line EA.hy 926. Cell monolayers were exposed for 5 min to 8.5 nM human alpha-thrombin and then washed to remove unbound thrombin. The cells were then incubated at 37 degrees C for 5-180 min. At the end of the respective incubation periods, purified human protein C (120 nM) was added in order to assay the activity of the thrombin-thrombomodulin complexes present on the cell surface. HSVEC pre-exposed to thrombin retained their full capacity to promote protein C activation up to 90 min after free thrombin was removed. This capacity then decreased slowly to reach 56% of control value after 180 min of incubation. Original activity was 3.8 +/- 0.9 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 5). The capacity of protein C activation of EA.hy 926 cells remained constant for 120 min after free thrombin was removed, then decreased to 76% of control after 180 min. Original activity was 2.0 +/- 0.4 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 3). Similar results were obtained with cells fixed with 3% paraformaldehyde. However, during the 5-180 min incubation period, non-fixed cells of both types were capable of significantly internalizing fluorescent acetylated low-density lipoprotein. In the experimental protocol used here, an eventual inhibition of thrombin internalization by protein C can be excluded, as protein C is only added at the end of the incubation period. We conclude that there is no evidence of rapid internalization of thrombin-thrombomodulin complexes on HSVEC or the EA.hy 926 cell line, as assessed by the ability of membrane-bound thrombin to activate protein C