UMLS:C0432222 - FACTA Search

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Query: UMLS:C0432222 (SEM)
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Annexin V is a human phospholipid binding protein (M(r) 36,000) that binds with high affinity to activated platelets in vitro. We studied the biodistribution and thrombus binding of annexin V in rabbit and swine models of fully occlusive arterial thrombi formed 1-2 h prior to injection of annexin V. Iodinated annexin V was cleared from blood in a rapid early phase (t1/2 = 6.4 min, 76% of radioactivity) and a slower late phase (t1/2 = 71 min, 24% of radioactivity). Organ uptake was highest in the kidney and spleen and lowest in heart and skeletal muscle. Thrombus/blood uptake ratios were (mean +/- SEM): 6.39 +/- 1.80 for rabbit iliac artery, 6.97 +/- 1.45 for swine carotid artery, and 7.68 +/- 1.70 for swine femoral artery (all p values < 0.01 versus control artery); a control protein, ovalbumin, showed an uptake ratio of 0.59 +/- 0.08 in swine femoral artery thrombi. These results indicate that annexin V is useful as an agent for selective targeting of platelet-containing thrombi.
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PMID:Evaluation of annexin V as a platelet-directed thrombus targeting agent. 799 50

Acute tubular injury in sepsis is associated with proximal tubular epithelial cell (PTEC) detachment into the lumen leading to back-leakage of glomerular ultrafiltrate and tubule obstruction. Inflammatory cytokines, such as IL-1alpha, IFNgamma and TNFalpha, are important mediators in sepsis-induced acute renal failure, although their precise role is unclear. We used primary cultures of human PTEC to investigate the hypothesis that inflammatory cytokines exert cytotoxic effects and cause detachment of cells from adherent monolayers, possibly through the intermediate nitric oxide (NO). At 5 days post-confluence, PTEC monolayers were stimulated for 24 hours with IL-1alpha (10 ng/ml), IFNgamma (200 u/ml) and TNFalpha (10 ng/ml). Monolayer viability was assessed by a live/dead dual fluorescence labeling technique. Apoptosis within monolayers was determined by morphological examination and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). PTEC in supernatants were counted and then analyzed by flow cytometry, using propidium iodide to assess cell viability and annexin V labeling to determine apoptosis. Results (mean +/- SEM; monolayers, n = 4; cell counts, n = 3; flow cytometry, n = 2) are shown below (at test, p < 0.05). Monolayers Supernatants Viable necrotic% of cells apoptotic countsx104/ml viable necrotic% of cells apoptotic Unstimulated 99.0+/-0.5 1.0+/-0.5 0 8.0+/-0.6a 64.6+/-2.5a 26.7+/-1.9a 6.2+/-0.6a Stimulated 92.4+/-3.2 7.6+/-3.2 0 14.7+/-0.6a 37.9+/-0.05a 48.0+/-0.3a 14.1+/-0.35a Following cytokine stimulation, there were significantly increased numbers of shed cells in supernatants. This cell population demonstrated significant loss of viability with increased numbers of both necrotic and apoptotic cells, as compared to unstimulated PTEC supernatants. Cytokine-stimulated monolayers maintained viability with no significant cell necrosis and no evidence of apoptosis. Preliminary experiments with the NO synthase inhibitor L-NMMA show that it reduces the number of cytokine-induced shed cells to the levels found in unstimulated cells (8.0 +/- 1.0 x 104/ml), although the percentages of necrotic and apoptotic cells are unchanged from cytokine-stimulated PTEC (44% and 15%, respectively). In conclusion, inflammatory cytokines induce necrotic and apoptotic cell shedding from PTEC monolayers with maintenance of monolayer viability. Preliminary data suggest that NO plays a cytotoxic role in this process.
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PMID:Inflammatory cytokines induce apoptotic and necrotic cell shedding from human proximal tubular epithelial cell monolayers 1035 8

In many cell types, DNA fragmentation is a late event of apoptosis which may be lacking. This contrasts with the early translocation of phosphatidylserine (PS) from the internal to the external leaflet of the cell membrane. We examined whether an early PS translocation also occurs during apoptosis induced in adult rat ventricular myocytes grown in the presence of 10% fetal calf serum (FCS), by the protein kinase inhibitor staurosporine. Apoptosis was assessed by the observation of: (i) typical alterations in cell morphology; (ii) nuclear alterations visualized using the permeant intercalating agent Hoechst 33258; (iii) DNA fragmentation detected by the TUNEL method. PS translocation was detected using annexin V binding. Data are expressed as means +/- SEM. Prolonged exposure of myocytes to 10 microM staurosporine from day 3 to day 7 of culture resulted in cell shrinkage, typical nuclear alterations, membrane protrusions and fragmentation of the sarcomeric apparatus in the vast majority of myocytes. At this time, 52.4 +/- 5.7% of staurosporine-treated myocytes were TUNEL positive (vs 6.1 +/- 2.0% in control cultures (CC), p < 0.001) and 69.7 +/- 1.7% were annexin V positive (vs 21.1 +/- 1.0% in CC, p < 0.001). Importantly, PS translocation was detected as early as 35 minutes following staurosporine addition, the percentage of annexin V positive myocytes reaching 10 times the control value (19.2 +/- 2.7 vs 1.8 +/- 0.8%, p < 0.001) after 3 hours. A 18-hour staurosporine exposure of freshly isolated myocytes resulted, at the end of exposure, in 24.3 +/- 1.7% annexin V positive myocytes (vs 9.6 +/- 0.5% in CC, p < 0.05), whereas a marked increase in the percentage of TUNEL positive myocytes was observed only from day 5. Finally, myocyte exposure to the membrane-permeant ceramide analog, C2-ceramide (50 microM), resulted in 63.2 +/- 3.5% annexin V positive myocytes 4 hours later (vs 17.8 +/- 4.4% in CC, p < 0.001), whereas a significant increase in the percentage of TUNEL positive myocytes was detected only the next day (43.7 +/- 3.4 vs 9.9 +/- 1.3%, p < 0.001). Taken together, these results strongly suggest that the loss of PS asymmetry is an early event of cardiac myocyte apoptosis which precedes DNA fragmentation.
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PMID:Early redistribution of plasma membrane phosphatidylserine during apoptosis of adult rat ventricular myocytes in vitro. 1042 35

A recombinant immunotoxin was constructed from the hybridoma antibody TH-69 directed against human CD7, a surface antigen of leukemic T cells. The antibody was subcloned as a single chain Fv (scFv) fragment and genetically linked to a truncated Pseudomonas exotoxin A fragment containing the catalytic domains II and III but lacking the receptor binding domain I. Domain I was replaced by the scFv, thus conferring restricted specificity for CD7-positive cells. The bacterially expressed and purified toxin retained binding specificity for CD7-positive cells. It promoted apoptosis in two CD7-positive cell lines derived from T-lineage acute lymphoblastic leukemias, CEM and Jurkat, but not in the CD7-negative B-lymphoid lines REH, Nalm-6, and SEM. Maximum killing in excess of 95% was reached after 96 h in CEM and Jurkat cells with a single dose of 100 ng/ml. Cells treated with a similarly constructed scFv-exotoxin A immunotoxin against melanoma-associated chondroitin sulfate proteoglycan, an antigen absent from leukemic T cells, remained unaffected. Lysis of target cells occurred via apoptosis as evidenced by staining with Annexin V and specific cleavage of poly(ADP-ribose) polymerase. Approximately 20% of leukemic cells from a patient with CD7-positive acute T-cell leukemia kept in long-term primary culture for 30 cell generations were killed within 96 h after treatment with the toxin. These findings justify further evaluation of the agent in view of potential therapeutic applications.
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PMID:A recombinant CD7-specific single-chain immunotoxin is a potent inducer of apoptosis in acute leukemic T cells. 1201 63

Spermatozoa with deteriorated plasma membranes can be separated by magnetic-activated cell sorting (MACS) after binding superparamagnetic annexin V-conjugated microbeads (ANMBs) to membrane phosphatidylserine (PS). Semen samples from 15 donors and 25 infertile patients were divided into 2 spermatozoal fractions by annexin V-MACS. Activated caspases (aCPs), which mediate degradations of cell quality, were determined by CaspaTag in the 2 subpopulations. Spermatozoa from donors showed lower levels of bound annexin V (3.6% +/- 0.5% vs 11.9% +/- 1.1%; P <.01) and aCPs (21.8% +/- 2.6% vs 43.2% +/- 2.1%; P <.01) than did spermatozoa from infertile patients. MACS resulted in a decrease of spermatozoa with aCPs from 21.8% +/- 2.6% (before separation) to 9.2% +/- 1.4% (in the ANMB-negative fraction) in donors and from 43.2% +/- 2.1% to 18.8% +/- 2.6% in infertile patients (mean +/- SEM; P <.01). Separation effects of the MACS technique were confirmed with flow cytometry using anti-annexin V antibodies and with electron microscopy. ANMB-MACS removes spermatozoa with PS-bound annexin V and produces a higher quality spermatozoal fraction. Spermatozoa with a deteriorated membrane are characterized by an increase in aCPs. A higher percentage of spermatozoa with ANMBs bound to PS and with aCPs were found in infertile patients.
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PMID:Deterioration of plasma membrane is associated with activated caspases in human spermatozoa. 1263 12

Women with systemic lupus erythematosus (SLE) are at risk for premature atherothrombosis independent of Framingham risk factors. We investigated whether endothelial cell (EC) apoptosis predicts abnormal vasomotor tone and contributes to circulating tissue factor (TF) levels in this disease. Brachial artery flow-mediated dilation (FMD) and nitroglycerin-mediated dilation were determined in women with SLE, healthy control subjects, and subjects with coronary artery disease (CAD) (n = 43/group). Quantification of circulating apoptotic ECs was performed by flow cytometry (CD146(+) cells that stained for Annexin V [CD146(AnnV+)]) and immunofluorescent microscopy. Plasma TF was measured by enzyme-linked immunosorbent assay (ELISA). Compared with healthy control and CAD subjects, patients with SLE had higher numbers of circulating CD146(AnnV+) cells (10 +/- 3, 18 +/- 5, and 89 +/- 32 cells/mL, respectively, mean +/- SEM; P <.01). Increased CD146(AnnV+) cells correlated strongly with abnormal vascular function (P =.037). After adjusting for known predictors of endothelial function, CD146(AnnV+) was the only variable that predicted FMD (beta = -4.5, P <.001). Increased CD146(AnnV+) was strongly associated with elevated levels of circulating TF (r =.46, P =.002). Circulating apoptotic ECs are elevated in young women with SLE and strongly correlate with markedly abnormal vascular function and elevated TF levels. Heightened endothelial apoptosis may represent an important mechanism for development of atherothrombosis in SLE.
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PMID:Endothelial cell apoptosis in systemic lupus erythematosus: a common pathway for abnormal vascular function and thrombosis propensity. 1472 73

Cryopreservation increases the rate of apoptotic spermatozoa with decreased capability to fertilise oocytes. In order to optimise the fertilisation rates, especially in assisted reproduction the use of apoptotic sperms should be avoided. Early events of apoptosis in cryopreserved spermatozoa are not detectable by conventional methods. However, the surface of apoptotic spermatozoa is characterised by externalisation of phosphatidylserine (PS), which has a high affinity to Annexin V. Therefore, colloid paramagnetic Annexin-V-conjugated microbeads (AN-MB) were tested for their ability to eliminate apoptotic spermatozoa from a total of 40 fresh and in TEST yolk buffer cryopreserved semen samples which were provided by 15 healthy volunteers. By passing through a magnetic field (MiniMACS, Miltenyi Biotec) the sperm suspensions were divided into 2 sperm fractions depending on bound magnetic Annexin V-microbeads (AN-MB) to spermatozoa. As additional markers of apoptosis CD95 (Fas, APO-1) on the sperm surface and activated caspases in the cytosol were detected in both fractions. Supplementary investigations comprised eosin-supravital staining and computer assisted sperm motion analysis. The separation was supervised by flow cytometric analysis of spermatozoa labelled with FITC-conjugated anti Annexin V-antibodies. Analyses of the magnetic inactive sperm fraction (AN-MB-negative) showed CD95 on 0.6 +/- 0.3% (X +/- SEM) of spermatozoa and only 3.2 +/- 0.5% were stainable with eosin, whereas, 40.6 +/- 6.7% of the remaining cells in the column appeared to be CD95 positive and 99.8 +/- 0.1% stainable with eosin after cryopreservation. Indeed the overall amount of CD95 positive spermatozoa did not significantly increase after cryopreservation (2.5 +/- 0.5% vs. 4.3 +/- 1.2%; p > 0.05). Activated caspases were found in 21.8 +/- 2.6% of the spermatozoa in fresh and in 47.7 +/- 5.8% of cryopreserved semen samples (p < 0.01). The separation procedure of the cryopreserved spermatozoa reduced significantly the quantity of those containing activated caspases to 9.3 +/- 2.2% within the AN-MB-negative fraction. In contrast 89.1 +/- 2.3% of AN-MB-positive sperms showed activation of these proteolytic enzymes. Flow cytometric analyses using FITC-conjugated anti Annexin V-antibodies for monitoring of AN-MB-binding to spermatozoa showed 5.2 +/- 1.0% labelled spermatozoa in the AN-MB negative fraction and 72.6 +/- 2.7% labelled spermatozoa in the AN-MB positive one. There was no significant influence of the separation column and the magnetic field on the sperm functions. The passage through the column led to a sperm loss of 0.8 +/- 1.2%. Conclusion: The binding of paramagnetic Annexin V-conjugated microbeads is an excellent method to eliminate spermatozoa at early apoptotic stages from cryopreserved semen samples. A deleterious influence of the separation column and the magnetic field on the spermatozoa was not observed.
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PMID:Enrichment of non-apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting. 1525 10

The effects of cryopreservation on two characteristics of human spermatozoa were investigated: the early phases of disturbed plasma membrane function and the activity of enzymes in intact spermatozoa. The membrane function was detected by means of the calcium-dependent binding of fluorescein isothiocyanate (FITC)-conjugated Annexin V to sperm plasma membranes. Annexin V monitors the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane, which is one of the earliest features of membrane disintegration. For the second aim synthetic fluorogenic substrates for peptidases, proteinases, esterases, elastases and collagenases were applied. These substrates, CellProbe trade mark reagents consist of different peptide sequences, specific for the enzymes, and a fluorescein- or rhodamine 110-dye moiety. They enter the cells without previous membrane permeabilisation and exhibit fluorescence after cleavage depending on enzyme activity. The number of positive cells and the intensity of the fluorescence were determined by flow cytometric analysis comparing fresh spermatozoa with cryopreserved ones. Thirty-five semen samples collected from 35 donors were cryopreserved using the freezing medium TEST yolk buffer. All specimens showed normal spermiogram parameters. Twenty-five of the samples were used for detection of Annexin V-FITC binding and 10 semen samples for investigations of the intracellular enzymes. The Annexin V-assay applied two fluorescent dyes (Annexin V, AN and propidium iodide, PI) which led to three groups of spermatozoa (a) viable spermatozoa (AN V-negative and PI-negative), (b) dead spermatozoa (AN V-positive and PI-positive) and (c) cells with impaired but integer plasma membrane (AN V-positive and PI-negative). The percentage of vital Annexin V-negative spermatozoa (x +/- SEM) decreased significantly (p < 0.001) from fresh spermatozoa (51.6 +/- 3.1) to cryopreserved spermatozoa (26.6 +/- 2.2%) and was associated with their motility (57.9 +/- 1.9% motile fresh spermatozoa vs. 22.6 +/- 3.9% motile sperm after cryopreservation). Of the spermatozoa 28.2% were Annexin V-positive before and 44.4% after cryostorage even though they did not bind to PI. Thus, vital spermatozoa showed a disturbed membrane function indicating viability before as well as after cryostorage. Moreover, after cryopreservation the spermatozoal fluorescence increased applying substrates for butyryl esterase (p < 0.05), prolyl-aminopeptidase (p < 0.001) and val-lys-(VK)-cathepsin (p < 0.001). In contrast, the activities of fluorescein diacetate (FDA)- and FDA/sodium fluoride (NAF)-esterase (p < 0.05), ala-ala-pro-val-(AAPV)-elastase (p < 0.001), gly-pro-leu-gly-pro-(GPLGP)-collagenase (p < 0.05) and gly-gly-leu-(GGL)-subtilisin (p < 0.001) decreased after cryopreservation. The substrates for arg-gly-glut-ser-(RGES)-elastase, gly-phenyl-gly-ala-(GFGA)-collagenase and threo-pro-(TP)-cathepsin were not cleaved before as well as after cryostorage. In addition to the known effects of sperm cryopreservation our results showed two further alterations of human ejaculated spermatozoa: (a) disturbed plasma membrane function, which is not detectable by supravital staining and (b) a changed pattern of intracellular enzyme activities.
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PMID:Hidden effects of cryopreservation on quality of human spermatozoa. 1525 59

We have previously shown that resveratrol can induce apoptotic cell death in cell lines established from patients with acute lymphoblastic leukemia (ALL). Cyclosporin A (CsA) and PK11195 are modulators of the mitochondrial permeability transition pore (MPTP) which has been proposed to play a critical role in regulating survival and death. Using SEM and RS4;11 lines with the t(4;11) translocation, the B-ALL line REH, and the T-ALL line Jurkat, we show that pre-treatment with CsA or PK11195 significantly enhances resveratrol-mediated apoptosis and mitochondrial membrane depolarization in these cells, as measured by annexin V and JC-1 staining, respectively. No significant multi-drug resistance efflux of the fluorescent substrate calcein was observed in these ALL lines, indicating that CsA and PK11195 were acting at the level of the mitochondria to enhance loss of mitochondrial membrane potential and induction of apoptosis. These data suggest targeting the MPTP sensitizes B- and T-cell ALL to the anti-cancer activity of resveratrol, and may be particularly useful for the treatment of high-risk t(4;11) ALL.
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PMID:Resveratrol-induced apoptosis is enhanced in acute lymphoblastic leukemia cells by modulation of the mitochondrial permeability transition pore. 1622 72

The neuropeptide vasoactive intestinal peptide (VIP) is anti-inflammatory and protective in the immune and nervous systems, respectively. This study demonstrated in corneal endothelial (CE) cells injured by severe oxidative stress (1.4 mM H(2)O(2)) in bovine corneal organ cultures that VIP pre-treatment (0, 10(-10), 10(-8), and 10(-6) M; 15 min), in a VIP concentration-dependent manner, switched the inflammation-causing necrosis to inflammation-neutral apoptosis (showing annexin V-binding, chromatin condensation, and DNA fragmentation) and upheld ATP levels in a VIP antagonist (SN)VIPhyb-sensitive manner, while up-regulated mRNA levels of the anti-apoptotic Bcl-2 and the differentiation marker N-cadherin in a kinase A inhibitor-sensitive manner. As a result, VIP, in a concentration-dependent and VIP antagonist-sensitive manners, promoted long-term CE cell survival. ATP levels, a determining factor in the choice of apoptosis versus necrosis, measured after VIP pre-treatment and 0.5 min post-H(2)O(2) were 39.6 +/- 3.3, 50.8 +/- 6.2, 60.1 +/- 4.8, and 53.6 +/- 5.3 pmoles/microg protein (mean +/- SEM), respectively (p < 0.05, anova). VIP treatment alone concentration-dependently increased levels of N-cadherin (Koh et al. 2008), the phosphorylated cAMP-responsive-element binding protein and Bcl-2, while 10(-8) M VIP, in a VIP antagonist (SN)VIPhyb-sensitive manner, increased ATP level by 38% (p < 0.02) and decreased glycogen level by 32% (p < 0.02). VPAC1 (not VPAC2) receptor was expressed in CE cells. Thus, CE cell VIP/VPAC1 signaling is both anti-inflammatory and protective in the corneal endothelium.
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PMID:VIP down-regulates the inflammatory potential and promotes survival of dying (neural crest-derived) corneal endothelial cells ex vivo: necrosis to apoptosis switch and up-regulation of Bcl-2 and N-cadherin. 1925 Mar 42

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