Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P08758 (annexin V)
 9,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lymphocyte granule-mediated apoptosis is postulated to entail the formation of membrane pores by perforin. Then soluble granzyme reaches the cytosol either through these pores or by reparative pinocytosis. We demonstrate here that Jurkat cells bind and internalize granzyme B via high affinity binding sites without toxic consequence. Apoptosis occurs, however, if sublytic perforin is added to targets washed free of soluble granzyme B. We suggest that granule-mediated apoptosis mimics viral strategies for cellular entry. Accordingly, co-internalization of granzyme B with adenovirus, a virus that escapes endosomes to reach the cytosol, also induced apoptosis. Poly(ADP-ribose) polymerase cleavage and processing of CPP32, ICE-LAP3, and Mch2 were detected at 30 min, while cytosolic acidification and DNA fragmentation occurred at 60 min. Annexin V binding and membrane permeabilization arose at 4 h. The concurrent activation of the Ced-3 proteases differed from the rate at which each cysteine protease is cleaved in vitro by granzyme B. Thus, granzyme B may not directly process these proteases in whole cells but rather may function by activating a more proximal enzyme. These results indicate that adenovirus-mediated delivery of granzyme B is suitable for elucidating biochemical events that accompany granule-mediated apoptosis.
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PMID:New paradigm for lymphocyte granule-mediated cytotoxicity. Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis. 891 May 61

Apoptosis of glomerular cells (GMC) has been observed in the early phase as well as the resolution phase of Thy-1 nephritis. Recently, we and others reported that IgG2a (ER4G) and IgG1 (OX7) monoclonal mouse anti-Thy-1 antibodies (anti-Thy-1 MoAb) are able to induce apoptosis of rat GMC in vivo. The purpose of this study was to investigate whether cross-linking of Thy-1 would influence the degree of apoptosis in cultured rat GMC using monomeric and dimeric IgA anti-Thy-1 MoAb. IgA anti-Thy-1 MoAb (ER4A) was generated by class switching of the IgG producing ER4 (ER4G) hybridoma. The ER4A clone spontaneously produces monomeric (m-ER4A) and dimeric IgA anti-Thy-1 MoAb *di-ER4A). Unaltered epitope specificity of ER4A was confirmed by blocking experiments of the binding of fluorescence labeled ER4G to cultured rat GMC with unlabeled ER4A on FACS. For the experiments of apoptosis, quiescent rat GMC were incubated for eight hours with medium alone or with medium in the presence of 10 micrograms/ml of m-ER4A, di-ER4A or control IgA MoAb of corresponding sizes. Apoptosis was assessed by morphological studies, agarose gel electrophoresis and quantitative FACS analyses using terminal deoxynucleotidyl transferase (TDT) method and the annexin V method. The TDT method detects specific-DNA nicking in apoptosis. The annexin V method detects early membrane changes during apoptosis. In morphological studies, cells incubated with m-ER4A and di-ER4A showed typical apoptotic features such as nuclear condensation and fragmentation. DNA isolated from the cells incubated with di-ER4A was cleaved into a distinctive ladder pattern compatible with apoptosis. In contrast, both medium alone and control IgA MoAb did not reveal detectable changes in morphological studies and agarose gel electrophoresis. In quantitative analyses by FACS using the TDT method and the annexin method, both m-ER4A and di-ER4A induced significantly higher percentages of apoptosis in rat GMC as compared to the controls. Furthermore, di-ER4A was considerably more efficient than m-ER4A in inducing apoptosis possibly through additional cross-linking of Thy-1 on the cell surface. This notion was confirmed by experiments, in which the addition of goat anti-mouse kappa antibodies enhanced apoptosis of rat GMC pre-sensitized with m-ER4A. Taken together, our results indicate that apoptosis of rat GMC by anti-Thy-1 antibodies is enhanced by cross-linking of Thy-1 on the cell surface. These studies are of importance for our understanding of mechanisms that may play a role in glomerular diseases.
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PMID:Efficient induction of apoptosis in cultured rat glomerular mesangial cells by dimeric monoclonal IgA anti-Thy-1 antibodies. 899 31

This study involved DNA analysis of bone marrow cells of 15 patients with megaloblastic anaemia. The diagnosis was based on the morphological changes seen in the bone marrow, associated with either a low red cell folate or serum vitamin B12 level and an adequate response to appropriate therapy as confirmation of the diagnosis. Flow cytometric DNA analysis showed an increase in the S and G2 phases of the cell cycle, but conventional agarose gel electrophoretic DNA analysis did not confirm the characteristic 'ladder pattern' which might have been expected in classic apoptosis. In addition, cells showing morphological changes suggestive of apoptosis, such as nuclear condensation and fragmentation, did not show evidence of DNA fragmentation using the ApopTag in situ digoxigenin nucleotide labelled, peroxidase detection system. Further studies using annexin V flow cytometric analysis and pulsed field gel electrophoresis were also unable to detect evidence of apoptosis as a significant cause of cell death in megaloblastic anaemia.
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PMID:Evaluation of DNA analysis for evidence of apoptosis in megaloblastic anaemia. 905 66

Whereas unperturbed endothelial cells provide potent anticoagulant properties, exposure to inflammatory and atherogenic stimuli can rapidly lead to a procoagulant behavior. Because recent studies provide evidence that apoptosis of vascular cells may occur under conditions such as atherosclerosis and inflammation, we investigated whether apoptotic endothelial cells may contribute to the development of a prothrombotic state. In this report, it is shown that both adherent and detached apoptotic human umbilical vein endothelial cells (HUVECs) become procoagulant. Apoptosis was induced by staurosporine, a nonspecific protein kinase inhibitor, or by culture in suspension with serum deprivation. Both methods resulted in similar findings. As assessed by flow cytometric determination of annexin V binding, HUVECs undergoing cell death exhibited typically a more rapid exposure of membrane phosphatidylserine (PS) than DNA fragmentation. Depending on the stage of apoptosis, this redistribution of phospholipids was found to induce an increase of the activity of the intrinsic tenase complex by 25% to 60%. Although apoptotic cells did not show antigenic or functional tissue factor (TF) activity, when preactivated with lipopolysaccharide, TF procoagulant activity increased by 50% to 70%. At 8 hours after apoptosis induction, antigenic thrombomodulin, heparan sulfates, and TF pathway inhibitor decreased by about 83%, 80%, and 59%, respectively. The functional activity of these components was reduced by about 36%, 52%, and 39%, respectively. Moreover, the presence of apoptotic HUVECs led to a significant increase of thrombin formation in recalcified citrated plasma. In conclusion, apoptotic HUVECs, either adherent or in suspension, become procoagulant by increased expression of PS and the loss of anticoagulant membrane components.
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PMID:Apoptotic vascular endothelial cells become procoagulant. 911 87

Oxidative stress is a potential component of the final common pathway leading to apoptosis following many diverse stimuli. Here, we document that the oxidant paraquat caused apoptosis in mouse 32D cells. We examined early paraquat-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid (cis-PA) into phospholipids and high-performance liquid chromatography separation of specific phospholipid classes. Paraquat induced peroxidation of cis-PA primarily in phosphatidylserine (PS) and to a lesser extent in phosphatidylinositol (PI) within 2 h. The selective oxidation of PS occurred before signs of cytotoxicity and preceded the externalization of PS as assessed by annexin V binding. Overexpression of Bcl-2 afforded significant protection against paraquat-induced apoptosis, early PS and PI oxidation, and PS externalization but not the ultimate formation of high-molecular-weight DNA fragments. Therefore, both selective phospholipid peroxidation and DNA damage occurred after paraquat exposure, but only the former was specifically associated with apoptosis. We suggest Bcl-2 may inhibit oxidant-induced apoptosis by preventing the peroxidation of specific membrane phospholipids.
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PMID:Bcl-2 inhibits selective oxidation and externalization of phosphatidylserine during paraquat-induced apoptosis. 912 12

Although several lines of investigation demonstrate that many heavy metals are cytotoxic to host defense cells, the mechanism of killing is poorly understood. The major focus of this investigation was to determine if organic mercuric compounds kill human lymphocytes by inducing the cells to undergo apoptosis and to evaluate possible flow cytometric systems for assessing cell death. T-cells were exposed to 0.6-5 microM MeHgCl, EtHgCl, or PhHgCl for up to 24 hr and then analyzed by flow cytometry. Mercury-treated cells exhibited increased Hoechst 33258 and 33342 fluorescence while maintaining their ability to exclude the vital stain 7-AAD. Furthermore, T-cells exposed to mercury exhibited changes in light scatter patterns that included decreased forward light scatter and increased side scatter. The light scatter and fluorescent changes were consistent with changes that cells display during apoptosis. To further evaluate cell death and to distinguish between apoptosis and necrosis, merocyanine 540 staining and annexin V binding to the plasma membrane as well as DNA fragmentation were assessed. Mercury-treated cells exhibited increased merocyanine 540 fluorescence and annexin V binding along with changes in nuclear morphology consistent with the notion of apoptosis. Conventional agarose gel electrophoresis failed to demonstrate low-molecular-weight DNA bands; however, when probed by flow cytometry using both nick translation and a modified TUNEL assay, patterns consistent with nuclear fragmentation were evident. We noted that the percentage of T-cells undergoing apoptosis was dependent upon the amount of serum present in the medium; as serum concentrations were increased from 0 to 10%, cell death declined. Apoptosis (33%) was observed within 1 hr of exposure to MeHgCl; maximum cell death (67%) occurred after 24 hr exposure. Induction of apoptosis was dependent on the mercury concentration and independent of the hydrophobicity of the mercury ligand. Finally, we assessed mercury-dependent apoptosis in activated T-cells. When treated with mitogen, mercury failed to induce apoptosis in these cells. Indeed, there was no evidence of either apoptosis nor necrosis in these populations. It was concluded that the activation process prevented development of a metabolic state that was required for induction of apoptogenic genes.
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PMID:Induction of apoptosis in human T-cells by organomercuric compounds: a flow cytometric analysis. 914 56

Quantification of apoptotic cell death in vivo has become an important area of investigation in patients with acute lymphoblastic leukemia (ALL). We have devised a noninvasive analytical method to estimate the percentage of apoptotic lymphoblasts in doxorubicin-treated Jurkat T-cell ALL cultures, using proton nuclear magnetic resonance spectroscopy (1H NMR). We have found that the ratio of the methylene (CH2) resonance (at 1.3 ppm) to the methyl (CH3) resonance (at 0.9 ppm) signal intensity, as observed by 1H NMR, is directly proportional to the percentage of apoptotic lymphoblasts in vitro. The correlation between the CH2/CH3 signal intensity ratio and the percentage of apoptotic lymphoblasts was optimal 24 to 28 hours after doxorubicin treatment (r2 = .947, N = 27 samples). There was also a direct temporal relationship between an increase in the CH2/CH3 signal intensity ratio and the onset of apoptosis as detected by nuclear morphologic analysis, fluorescein-annexin V flow cytometry, and DNA gel electrophoresis. Thin-layer chromatography confirmed that a dynamic and/or compositional change of the plasma membrane, rather than increases in lipase activity or fatty acid production, appears to account for the increase in the CH2/CH3 signal intensity ratio during apoptosis. 1H NMR may have clinical utility for the early noninvasive assessment of chemotherapeutic efficacy in patients with ALL.
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PMID:Quantitative analysis of apoptotic cell death using proton nuclear magnetic resonance spectroscopy. 916 Jun 84

We have previously demonstrated that CD40 stimulation induced a cellular growth arrest of the highly CD40-positive myeloma cell line XG2. To further characterize this inhibition of proliferation, we looked for a possible induction of apoptosis. Since no DNA fragmentation could be detected, we used newly described techniques that enable detection of apoptosis independently of DNA degradation, i.e. supravital exposure to propidium iodide (PI) and Annexin V labelling. We demonstrated that CD40 effectively induced programmed cell death. Furthermore, we have shown that CD95 (Fas) stimulation significantly enhanced the CD40-induced apoptosis.
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PMID:CD40 and CD95 induce programmed cell death in the human myeloma cell line XG2. 920 15

Apoptosis is a form of programmed cell death serving physiologic and homeostatic functions. However, recent evidence implicating apoptosis in the etiology and pathophysiology of known human diseases, such as heart diseases, cancer, AIDS, and neurodegenerative and autoimmune diseases are continually surfacing. This has spawned the need for identifying which methods are the most effective and well accepted to decipher its presence in a variety of research settings. We have therefore detailed the morphology and biochemical features of apoptotic cell death, with an emphasis on discriminating it from necrosis. In addition, we describe specific and selective techniques which are optimal to target hallmark apoptotic features, such as microscopy, Annexin V labeling, in situ nick-end labeling (TUNEL), and DNA fragmentation analysis by gel electrophoresis and ELISA for oligonucleosome-sized DNA. The advantages and disadvantages of each technique are discussed, as well as their experimental importance relative to one another. The methods have been described in a stepwise fashion, and can readily be applied in the majority of cell systems. Whether working on the tissue or single cell level, these methods are highly effective in qualifying and quantifying apoptosis. The application of these methods in conjunction with molecular techniques can further delineate the underlying mechanisms of apoptosis.
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PMID:Morphological and biochemical characterization and analysis of apoptosis. 927 77

Spontaneous and experimental changes in arterial blood flow rates affect tissue accumulation in developing arteries. To examine whether cell proliferation and/or cell death are affected by alterations in blood flow, we ligated the left external carotid artery of 3-week-old rabbits, which reduces left common carotid blood flow by 71%. In control arteries and after 2 days of flow reduction, agarose gel electrophoresis of DNA extracted from all carotid arteries resolved multiple low molecular weight bands characteristic of apoptosis; however, DNA fragmentation in arteries carrying reduced blood flow was 2.5-fold higher than that of control arteries. The effect of reduced blood flow on cell death subsequently waned but remained significant at 7 days. Cell death in carotid arteries was also detected by in vivo uptake of propidium iodide, a DNA-binding fluorescent dye that labels the nuclei of nonviable cells. Both smooth muscle and endothelial cells exhibited large and statistically significant increases in labeling index in the flow-reduced artery. Propidium iodide-labeled cells were cleared from the vessel wall within 1 to 4 hours of labeling, and nuclear staining displayed condensation (clumping) of chromatin in all labeled cells at later time points. This time course and nuclear morphology and the rapid clearance of labeled cells are consistent with death via apoptosis. Many propidium iodide-positive cells did not display chromatin condensation immediately after labeling; however, this was also true of cultured endothelial cells that were driven into apoptosis with sphingomyelinase treatment and then double-labeled with propidium iodide and the apoptosis marker annexin V. We infer that propidium iodide can label apoptotic vascular cells before these cells display chromatin condensation that is detectable with fluorescence labeling of DNA. Replication rates of smooth muscle and endothelial cells, determined by 5-bromo-2'-deoxyuridine uptake, were inhibited by >75% with decreased blood flow. The inhibition of proliferation was unabated after 7 days of reduced flow. These findings indicate that the coordinated regulation of cell death and cell proliferation, in response to changes in arterial blood flow rates, contributes to arterial remodeling during development.
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PMID:Effects of changes in blood flow rate on cell death and cell proliferation in carotid arteries of immature rabbits. 928 34


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