Stomatocytosis is absent in "stomatin"-deficient murine red blood cells. (9/2206)

To examine the relationship between erythrocyte membrane protein 7. 2b deficiency and the hemolytic anemia of human hereditary stomatocytosis, we created 7.2b knock-out mice by standard gene targeting approaches. Immunoblots showed that homozygous knock-out mice completely lacked erythrocyte protein 7.2b. Despite the absence of protein 7.2b, there was no hemolytic anemia and mouse red blood cells (RBCs) were normal in morphology, cell indices, hydration status, monovalent cation content, and ability to translocate lipids. The absence of the phenotype of hereditary stomatocytosis implies that protein 7.2b deficiency plays no direct role in the etiology of this disorder and casts doubt on the previously proposed role of this protein as a mediator of cation transport in RBC.  (+info)

Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs. (10/2206)

The phospholipid composition and phospholipid fatty acid composition of purified Rickettsia prowazeki were determined. The lipid phosphorous content was 6.8 +/- 1.3 microgram/mg of total rickettsial protein. The major phospholipid was phosphatidylethanolamine (60 to 70%); phosphatidylglycerol constituted 20%, and phosphatidylcholine constituted 15%. Small amounts of phosphatidylserine and cardiolipin were detected. The principal fatty acids were 18:1, 16:1, and 16:0. The fatty acid composition of the phosphatidylcholine in the rickettsial extracts was very different than that of the other rickettsial phosphatides and very similar to that of normal yolk sac phosphatidylcholine. The specific of the phosphatidylcholine of rickettsiae grown in the presence of 32P was markedly lower than that of phosphatidylethanolamine and phosphatidylglycerol. It is suggested that the phosphatidylcholine in the rickettsial extract is yolk sac derived and either tightly absorbed or exchanged into the rickettsial membrane.  (+info)

Inhibition of poly(ADP-ribose) synthetase (PARS) and protection against peroxynitrite-induced cytotoxicity by zinc chelation. (11/2206)

Peroxynitrite, a potent oxidant formed by the reaction of nitric oxide and superoxide causes thymocyte necrosis, in part, via activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS). The cytotoxic PARS pathway initiated by DNA strand breaks and excessive PARS activation has been shown to deplete cellular energy pools, leading to cell necrosis. Here we have investigated the effect of tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) a heavy metal chelator on peroxynitrite-induced cytotoxicity. TPEN (10 microM) abolished cell death induced by authentic peroxynitrite (25 microM) and the peroxynitrite generating agent 3-morpholinosidnonimine (SIN-1, 250 microM). Preincubation of TPEN with equimolar Zn2+ but not Ca2+ or Mg2+ blocked the cytoprotective effect of the chelator. TPEN (10 microM) markedly reduced the peroxynitrite-induced decrease of mitochondrial transmembrane potential, secondary superoxide production and mitochondrial membrane damage, indicating that it acts proximal to mitochondrial alterations. Although TPEN (1 - 300 microM) did not scavenge peroxynitrite, it inhibited PARS activation in a dose-dependent manner. The cytoprotective effect of TPEN is only partly mediated via PARS inhibition, as the chelator also protected PARS-deficient thymocytes from peroxynitrite-induced death. While being cytoprotective against peroxynitrite-induced necrotic death, TPEN (10 microM), similar to other agents that inhibit PARS, enhanced apoptosis (at 5-6 h after exposure), as characterized by phosphatydilserine exposure, caspase activation and DNA fragmentation. In conclusion, the current data demonstrate that TPEN, most likely by zinc chelation, exerts protective effects against peroxynitrite-induced necrosis. Its effects are, in part, mediated by inhibition of PARS.  (+info)

Anti-phospholipid antibodies and CD5+ B cells in HIV infection. (12/2206)

This cross-sectional study evaluates the correlation between anti-phospholipid antibodies and CD5+ B cells in 110 patients infected with HIV-1. There were 89.1% of the patients who had IgG antibodies against cardiolipin and phosphatidylserine. The prevalence of IgM and IgA antibodies was < 22%. AIDS was associated with lower frequencies of IgM antibodies against cardiolipin (P = 0.05) and IgG-antibodies against cardiolipin and phosphatidylserine (P = 0.011). Drug users had higher IgM antibodies against phospholipids than patients from other risk groups (P = 0.02). A history of thromboembolic events was not accompanied by higher levels of anti-phospholipid antibodies (P > 0.2). No correlation between anti-phospholipid antibodies and CD5+ B cells was detected. Percentage part of CD5+ B lymphocytes was elevated in all patients and absolute CD4+ T lymphocyte counts and HIV p24 antigen were inversely correlated. In advanced disease a significant reduction of anti-phospholipid antibodies was contrasted with persistent elevation of CD5+ B lymphocytes. These observations may reflect immunological dysfunction involving apoptosis and endothelial damage rather than polyclonal B cell hyperstimulation. A possible explanation would be that in HIV infection an increased rate of spontaneous apoptosis in peripheral blood lymphocytes is accompanied by functional and structural changes of mitochondria. Therefore, structurally altered mitochondrial phospholipids could serve as antigen to induce specific humoral immune responses.  (+info)

An alternative splicing form of phosphatidylserine-specific phospholipase A1 that exhibits lysophosphatidylserine-specific lysophospholipase activity in humans. (13/2206)

Phosphatidylserine-specific phospholipase A1 (PS-PLA1), which acts specifically on phosphatidylserine (PS) and 1-acyl-2-lysophosphatidylserine (lyso-PS) to hydrolyze fatty acids at the sn-1 position of these phospholipids, was first identified in rat platelets (Sato, T., Aoki, J., Nagai, Y., Dohmae, N., Takio, K., Doi, T., Arai, H., and Inoue, K. (1997) J. Biol. Chem. 272, 2192-2198). In this study we isolated and sequenced cDNA clones encoding human PS-PLA1, which showed 80% homology with rat PS-PLA1 at the amino acid level. In addition to an mRNA encoding a 456-amino acid product (PS-PLA1), an mRNA with four extra bases inserted at the boundary of the exon-intron junction was detected in human tissues and various human cell lines. This mRNA is most probably produced via an alternative use of the 5'-splicing site (two consensus sequences for RNA splicing occur at the boundary of the exon-intron junction) and encodes a 376-amino acid product (PS-PLA1DeltaC) that lacks two-thirds of the C-terminal domain of PS-PLA1. Unlike PS-PLA1, PS-PLA1DeltaC hydrolyzed exclusively lyso-PS but not PS appreciably. Any other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and their lyso derivatives were not hydrolyzed at all. These data demonstrated that PS-PLA1DeltaC exhibits lyso-PS-specific lysophospholipase activity and that the C-terminal domain of PS-PLA1 is responsible for recognizing diacylphospholipids. In addition, human PS-PLA1 gene was mapped to chromosome 3q13.13-13.2 and was unexpectedly identical to the nmd gene, which is highly expressed in nonmetastatic melanoma cell lines but poorly expressed in metastatic cell lines (van Groningen, J. J., Bloemers, H. P., and Swart, G. W. (1995) Cancer Res. 55, 6237-6243).  (+info)

Co-expression of Fas and Fas-ligand on the surface of influenza virus-infected cells. (14/2206)

Influenza virus-infected cultured cells undergo apoptosis after an increment of Fas (APO-1/CD95) on the cell surface. By flow cytometry, cell surface Fas-ligand was detected in virus-infected cells with a time course similar to that of Fas. Moreover, Fas and Fas-ligand were co-expressed in those cells. The mode of induction, however, appeared to be distinct for the two proteins. Influenza virus infection induced the externalization of phosphatidylserine on the cell surface at the early stage of apoptosis, an event that has been observed in cells undergoing Fas-mediated apoptosis. In fact, apoptosis of the virus-infected cells was inhibited in the presence of an antagonistic anti-Fas-ligand monoclonal antibody. These results suggest that influenza virus infection causes augmented expression of both Fas and Fas-ligand and apoptosis is induced when the infected cells come into contact with each other.  (+info)

The role of phosphatidylserine in recognition of apoptotic cells by phagocytes. (15/2206)

Exposure of phosphatidylserine on the outer leaflet of the plasma membrane is a surface change common to many apoptotic cells. Normally restricted to the inner leaflet, phosphatidylserine appears as a result of decreased aminophospholipid translocase activity and activation of a calcium-dependent scramblase. Phosphatidylserine exposure has several potential biological consequences, one of which is recognition and removal of the apoptotic cell by phagocytes. It is still not clear which receptors mediate PS recognition on apoptotic cells; however, several interesting candidates have been proposed. These include the Class B scavenger and thrombospondin receptor CD36, an oxLDL receptor (CD68), CD14, annexins, beta2 glycoprotein I, gas-6 and a novel activity expressed on macrophages stimulated with digestible particles such as beta-glucan. Whether PS is the sole ligand recognized by phagocytes or whether it associated with other molecules to form a complex ligand remains to be determined.  (+info)

Apoptosis in hematopoietic cells (FL5.12) caused by interleukin-3 withdrawal: relationship to caspase activity and the loss of glutathione. (16/2206)

The mechanism of cell death caused by cytokine deprivation remains largely unknown. FL5.12 cells (a murine prolymphocytic cell line), following interleukin-3 (IL-3) withdrawal, undergo a decrease in intracellular glutathione (GSH) that precedes the onset of apoptosis. In the present study, the induction of apoptosis following IL-3 withdrawal or GSH depletion with DL-buthionine-[S,R,]-sulfoximine (BSO) was examined. Both conditions caused time-dependent increases in phosphatidylserine externalization, acridine orange and ethidium bromide staining, decreases in mitochondrial membrane potential, processing and activation of caspase-3 and proteolysis of the endogenous caspase substrate poly(adenosine diphosphate ribose)polymerase (PARP). Apoptosis induced by IL-3 deprivation but not BSO also caused lamin B1 cleavage, suggesting activation of caspase-6. Despite a more profound depletion of GSH after BSO than withdrawal of IL-3, the extent of apoptosis was somewhat lower. Benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone (z-VAD.fmk) blocked this caspase activity and prevented cell death after BSO exposure but not after IL-3 deprivation. Following IL-3 withdrawal, the caspase inhibitors z-VAD.fmk and boc-asp(OMe)fluoromethylketone (boc-asp.fmk) prevented the cleavage and activation of caspase-3, the breakdown of lamin B1 and partially mitigated PARP degradation. However, the externalization of phosphatidylserine, the fall in mitochondrial membrane potential and subsequent apoptotic cell death still occurred. These results suggest that IL-3 withdrawal may mediate cell death by a mechanism independent of both caspase activation and the accompanying loss of GSH.  (+info)