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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have analyzed the type of cell death occurring in human normal ejaculated spermatozoa. Sperm cells were prepared either by centrifugation alone (group 1) or by density gradient centrifugation (group 2) and were cultured for 24 hrs. Cells were examined after 4 and 24 hrs. By comparison unprepared spermatozoa were used as a control group. Necrosis was investigated by intra-cellular vital stain penetration and electron microscopy. Apoptosis was researched by DAPI staining, annexin V-binding, electron microscopy, DNA fragmentation and PARP cleavage. In group 1, after 4 hrs., there was a mixture of spermatozoa dead either by necrosis or apoptosis while after 24 hrs., necrosis was prominent. Similar findings were observed in the control group. In contrast, in group 2 apoptosis was the major form of cell death of spermatozoa after 24 hrs. of culture. These findings suggest that apoptosis can be an important factor when spermatozoa are used for assisted reproductive technologies.
Cell Mol Biol (Noisy-le-grand) 2000 Feb
PMID:Morphological and biochemical analysis of cell death in human ejaculated spermatozoa. 1072 84

Phosphatidylserine (PS) exposure serves as a procoagulant stimulus and a signal for phagocytic clearance of apoptotic cells. In order to measure PS exposure in blood cells, we developed a flow-cytometric procedure to measure annexin V binding to leukocytes and platelets in whole-blood samples. Leukocytes were identified by CD45 and side-scatter gating, and platelets by CD6 1 and side-scatter gating. The absolute number of annexin V molecules bound per cell was determined from an independent calibration procedure. Normal populations had the following levels of annexin V binding (in molecules per cell): lymphocytes, 0.53 x 10(3) neutrophils, 1.75 x 10(3) monocytes, 2.45 x 10(3) platelets, 0.14 x 10(3). These levels represent </= 0.1% of the values obtained after maximal stimulation of PS exposure with calcium ionophore, confirming that virtually all PS is intracellular in normal circulating leukocytes and platelets. Pretreatment of whole-blood samples with ammonium chloride to lyse erythrocytes caused a 9- to 300-fold increase in annexin V binding to leukocytes, indicating that analysis of unlysed whole-blood samples is essential to avoid artifactual increases in annexin V binding to leukocytes. Comparison of annexin V with two other markers of platelet activation, CD62P and the activation-dependent epitope of glycoprotein IIb/IIIa detected by the PAC I antibody, indicated that platelets from normal donors showed the least amount of activation with the annexin V marker. Whole-blood flow cytometry with annexin V can reliably measure the state of PS exposure in platelets and leukocytes, and the results confirm that these cell
Blood Cells Mol Dis
PMID:Measurement of phosphatidylserine exposure in leukocytes and platelets by whole-blood flow cytometry with annexin V. 1074 22

Thrombosis is a major complication of human hemolytic anemias such as sickle cell disease, thalassemia, and severe hereditary spherocytosis (HS). Mice with severe HS and severe hereditary elliptocytosis (HE) also suffer from thrombosis, with incidences ranging from 15 and 22% in beta-spectrin- and ankyrin-deficient mice, respectively, to 85 to 100% in alpha-spectrin-deficient and band 3 knockout mice. A contributing factor to thrombosis could be loss of phospholipid asymmetry of the mutant red blood cells (RBCs), with concomitant exposure of the aminophospholipid phosphatidylserine (PS). Increased PS exposure occurs in RBCs from sickle cell and thalassemia patients and in RBCs from band 3-deficient mice. To determine if increased PS exposure correlates with thrombotic risk in HS and HE mice with ankyrin, beta-spectrin, and alpha-spectrin deficiencies, measurements of FITC-labeled annexin V binding to externalized PS on RBCs were performed. PS exposure is elevated in all mice with HS and HE, but the percentage of RBCs with exposed PS does not correlate with thrombotic risk in these mice.
Blood Cells Mol Dis 2000 Feb
PMID:Erythroid phosphatidyl serine exposure is not predictive of thrombotic risk in mice with hemolytic anemia. 1077 78

The metabolic cocktail of glucose-insulin-potassium (GIK) has been shown to reduce mortality in humans and reduce infarct size in the rat when administered from the onset of reperfusion following an ischemic insult. The mechanisms underlying GIK mediated cardioprotection are, however, still unclear. Recent data implicates insulin "alone" as the major protagonist of cardioprotection when administered at the time of reperfusion. We have therefore begun to investigate an insulin activated signalling pathway and the putative role of apoptosis in this insulin-induced cardioprotection. Simulated ischemia and reoxygenation were induced in rat neonatal cardiocyte experiments. The administration of insulin [0.3 mU/ml] at the moment of reoxygenation (Ins(R)) enhanced myocardial cell viablility as assessed by trypan blue exclusion compared to vehicle alone treated control myocytes (Ins(R)50+/-2%v controls 70+/-1%, P<0.001). This insulin-mediated cardioprotection was due, in part to a reduction in myocyte apoptosis as measured by TUNEL (Ins(R)29+/-2%v controls 49+/-3%, P<0.001) and Annexin V staining (Ins(R)34+/-2%v controls 65+/-3%, P<0.001). These cardioprotective and anti-apoptotic effects of insulin were completely abolished by the tyrosine kinase inhibitor lavendustin A and by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin. Thus, we conclude that the early administration of insulin appears to be an effective modality to reduce reoxgygenation injury in cardiocytes, in part, via the attenuation of ischemia/reoxygenation-induced apoptosis. Moreover, the cardioprotective and anti-apoptotic effects of insulin are mediated via tyrosine kinase and PI3-kinase signalling pathways.
J Mol Cell Cardiol 2000 May
PMID:Insulin administered at reoxygenation exerts a cardioprotective effect in myocytes by a possible anti-apoptotic mechanism. 1077 81

Overexpression of proapoptotic Bax favors death in cells resistant to ionizing radiation. We hypothesized that expression of Bax via adenoviral-mediated gene delivery could sensitize radiation-refractory cells to radiotherapy. An inducible Bax recombinant adenovirus (Ad/Bax) had been generated using the Cre/loxp system. Human ovarian cancer cell lines and primary, patient-derived cancer cells from ascites were irradiated and infected with the Ad/Bax and an expression-inducing vector, Ad/Cre. Cell death was evaluated by crystal violet staining, fluorescence-activated cell sorter analysis of Annexin V, and colony formation assay (cell lines only). To further characterize the mechanism of death, cell morphology was examined by nuclear staining with Hoechst 33258. Lastly, to evaluate the capacity of the combined treatment to inhibit tumor growth, mice were injected subcutaneously with ovarian cancer cells exposed to Bax, radiation therapy (RT), or both, and tumor size was measured periodically. Infection of the cancer cell lines and primary cells with both Ad/Bax and Ad/Cre significantly enhanced sensitivity to ionizing radiation, achieving high levels of cell killing in short-term assays. In addition, the combination of Bax and radiotherapy reduced the survival fraction of cell lines 2 logs in standard colony-forming assays. Investigation into the involved mechanism suggests that Bax-mediated radiosensitization occurs through both apoptosis and necrosis pathways. Further, mice subcutaneously injected with ovarian tumor cells previously treated with radiation, or with radiation and irrelevant viruses, consistently developed tumor nodules. In addition, approximately 80% of injections were followed by tumor formation after treatment with Ad/Bax and Ad/Cre alone. In contrast, tumor formation was completely inhibited after combined treatment with Ad/Bax and Ad/Cre and radiation. Augmentation of the effect of radiotherapy on human ovarian cancer cells and primary cancer cells from patients via a recombinant adenovirus encoding Bax is feasible.
Mol Ther 2000 Jun
PMID:An adenovirus encoding proapoptotic Bax induces apoptosis and enhances the radiation effect in human ovarian cancer. 1093 79

Phagocytosis of asbestos fibers may be a necessary step for asbestos-induced injury to mesothelial cells, but this has not been established because quantification of fiber uptake is difficult and ways to increase fiber phagocytosis without also increasing total dose were not available. We quantified phagocytosis by counting intracellular fibers after removing adherent fibers with trypsin; we selectively increased fiber phagocytosis by coating crocidolite asbestos fibers with the adhesive serum protein vitronectin (VN), which we have shown increases fiber uptake via integrins. We measured various aspects of asbestos-induced cytotoxicity: intracellular oxidation by the shift of fluorescence of cells loaded with an oxidative probe, DNA strand breakage by the alkaline unwinding ethidium bromide fluorometric assay, apoptosis by annexin V binding and by nuclear morphology, and cell-cycle progression. We found that, compared with control fibers or particles, asbestos increased intracellular oxidation, DNA strand breakage, and apoptosis. Selective increases in fiber uptake by VN-coating of the fibers further increased the oxidation, DNA strand breakage, and apoptosis, and induced a cell-cycle arrest in G2/M. Selective decreases in fiber uptake by cytochalasin or by integrin blockade with RGD peptides inhibited several of these measures of injury. We conclude that phagocytosis is important and perhaps necessary for asbestos-induced injury to mesothelial cells.
Am J Respir Cell Mol Biol 2000 Sep
PMID:Phagocytosis of crocidolite asbestos induces oxidative stress, DNA damage, and apoptosis in mesothelial cells. 1097 Aug 29

Recent work from this laboratory demonstrated that apoptosis of pulmonary alveolar epithelial cells (AEC) in response to Fas requires angiotensin II (ANGII) generation de novo and binding to its receptor (Wang et al., 1999b, Am J Physiol Lung Cell Mol Physiol 277:L1245-L1250). These findings led us to hypothesize that a similar mechanism might be involved in the induction of AEC apoptosis by TNF-alpha. Apoptosis was detected by assessment of nuclear and chromatin morphology, increased activity of caspase 3, binding of annexin V, and by net cell loss inhibitable by the caspase inhibitor ZVAD-fmk. Purified human TNF-alpha induced dose-dependent apoptosis in primary type II pneumocytes isolated from rats or in the AEC-derived human lung carcinoma cell line A549. Apoptosis in response to TNF-alpha was inhibited in a dose-dependent manner by the nonselective ANGII receptor antagonist saralasin or by the nonthiol ACE inhibitor lisinopril; the inhibition of TNF-induced apoptosis was maximal at 50 microgram/ml saralasin (101% inhibition) and at 0.5 microgram/ml lisinopril (86% inhibition). In both cell culture models, purified TNF-alpha caused a significant increase in the mRNA for angiotensinogen (ANGEN), which was not expressed in unactivated cells. Transfection of primary cultures of rat AEC with antisense oligonucleotides against ANGEN mRNA inhibited the subsequent induction of TNF-stimulated apoptosis by 72% (P < 0.01). Exposure to TNF-alpha increased the concentration of ANGII in the serum-free extracellular medium by fivefold in A549 cell cultures and by 40-fold in primary AEC preparations; further, exposure to TNF-alpha for 40 h caused a net cell loss of 70%, which was completely abrogated by either the caspase inhibitor ZVAD-fmk, lisinopril, or saralasin. Apoptosis in response to TNF-alpha was also completely inhibited by neutralizing antibodies specific for ANGII (P < 0.01), but isotype-matched nonimmune immunoglobulins had no significant effect. These data indicate that the induction of AEC apoptosis by TNF-alpha requires a functional renin/angiotensin system (RAS) in the target cell. They also suggest that therapeutic control of AEC apoptosis in response to TNF-alpha is feasible through pharmacologic manipulation of the local RAS.
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PMID:Apoptosis of lung epithelial cells in response to TNF-alpha requires angiotensin II generation de novo. 1102 47

Previous studies revealed that expression and activation of cyclooxygenase-2 (Cox-2) conveyed a protective principle in murine macrophages, thus attenuating pro-apoptotic actions of chemotherapeutic agents or programmed cell death as a result of massive nitric oxide (NO) generation. Expression of Cox-2 was achieved by treatment of cells with lipopolysaccharide/interferon-gamma or nontoxic doses of NO releasing agents. We reasoned E-type prostanoid formation, and in turn an intracellular cAMP increase as the underlying protective mechanism. To prove our hypothesis, we analyzed the effects of lipophilic cAMP-analogs on NO, cisplatin, or etoposide induced apoptosis in RAW 264.7 macrophages. Selected apoptotic parameters comprised DNA fragmentation (diphenylamine assay), annexin V staining of phosphatidylserine, caspase activity (quantitated by the cleavage of a fluorogenic caspase-3-like substrate Ac-DEVD-AMC), and mitochondrial membrane depolarisation (delta psi). Western blots detected accumulation of the tumor suppressor protein p53, relocation of cytochrome c to the cytosol, and expression of the anti-apoptotic protein Bcl-xL. Prestimulation with lipophilic cAMP-analogs attenuated apoptosis with the notion that cell death parameters were basically absent. To verify gene induction by cAMP in association with protection we established activation of cAMP response element binding protein (CREB) by gel-shift analysis and moreover, treated macrophages with oligonucleotides containing a cAMP-responsive element (CRE) in order to scavenge CREB. Decoy oligonucleotides, but not control oligonucleotides, attenuated cAMP-evoked protection and reestablished pro-apoptotic parameters. We conclude that gene induction by cAMP protects macrophages towards apoptosis that occurs as a result of excessive NO formation or addition of chemotherapeutica. Attenuating programmed cell death by the cAMP-signaling system may be found in association with Cox-2 expression and tumor formation.
Mol Cell Biochem 2000 Sep
PMID:Attenuation of macrophage apoptosis by the cAMP-signaling system. 1110 34

Etoposide, a clinically useful anticancer drug, is a potent inhibitor of topoisomerase II. The DNA strand breaks caused by this epipodophyllotoxin lead to apoptotic death of tumor cells. Flow cytometry was used to investigate the relationship between the effects of the drug on the cell cycle of human leukemia HL-60 cells and the variations of the mitochondrial transmembrane potential (DeltaPsi(mt)). Three cationic fluorescent probes, DiOC(6), JC-1, and TMRM, were used to measure drug-induced changes of DeltaPsi(mt). In all three cases, we found that the arrest in the G2/M phase of the cells treated with 0.5 microM etoposide is associated with an increase in the potential of mitochondrial membranes whereas treatment with a tenfold higher drug concentration trigger massive apoptosis and a collapse of DeltaPsi(mt). DNA fragmentation (TUNEL assay) and externalization of phosphatidylserine residues in the outer leaflet of the plasma membrane (annexin V binding) were measured to characterize the apoptotic cell population.
Mol Cell Biol Res Commun 2000 Jul
PMID:Relationship between cell cycle changes and variations of the mitochondrial membrane potential induced by etoposide. 1115 26

The objective of this study was to elucidate the role of annexin V, an endogenous inhibitor of protein kinase C (PKC), with regard to the antiproliferative effect of gonadotrophin-releasing hormone (GnRH) agonist (buserelin) on cultured human uterine leiomyoma cells. Uterine leiomyoma tissue was collected from the surgical specimens of patients and cells from 37 specimens (15 cases) were cultured. For up to 96 h after the addition of buserelin to the cultured cells, a time-dependent antiproliferative effect was noted in the group to which 10(-5) mol/l buserelin was added. Both the intracellular concentration of annexin V and the expression of annexin V mRNA increased time-dependently with the addition of buserelin. The intracellular concentration of annexin V increased with the addition of PKC activator (12-O:-tetradecanoylphorbor-13-acetate; TPA) much as it did with the addition of buserelin, and the rise in the concentration caused by the addition of buserelin was completely attenuated by pretreatment with PKC inhibitor (calphostin C). Our findings suggest that buserelin inhibits cell proliferation in cultured human uterine leiomyoma cells accompanied with an increase in the intracellular concentration of annexin V, mediated, at least in part, by the activation of PKC.
Mol Hum Reprod 2001 Feb
PMID:Involvement of annexin V in the antiproliferative effect of GnRH agonist on cultured human uterine leiomyoma cells. 1116 Aug 43


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