Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.22.56 (caspase-3)
 35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The addition of tumor necrosis factor (TNF)-alpha into the cultured porcine kidney LLC-PK1 cells caused apoptosis concomitantly with caspase-3 activation and the inductions of an endogenous Bcl-2 protein. An SDS-polyacrylamide electrophoretic analysis revealed that a 37-kDa protein in a nuclear fraction was increased during TNF-alpha-induced apoptosis. Partial amino acid sequence of the protein was A-L-T-G-H-L-E-E-V, perfectly matching that of annexin I. Immunocytochemistry revealed that annexin I migrated to the nucleus and/or peri-nucleus region upon exposure to TNF-alpha. Overexpression of Bcl-2 proteins inhibited the nuclear localization of annexin I during TNF-alpha-induced apoptosis. Antisense oligodeoxynucleotides complementary to annexin I-inhibited TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling) staining in TNF-alpha-treated cells, suggesting that annexin I expression is a possible prerequisite for the induction of apoptosis by the cytokine. Thus, it is first time to show that annexin I is regulated by an anti-apoptotic Bcl-2 protein in TNF-alpha-induced renal apoptotic events.
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PMID:Overexpression of Bcl-2 inhibits nuclear localization of annexin I during tumor necrosis factor-alpha-mediated apoptosis in porcine renal LLC-PK1 cells. 1554 40

The objective of this study was to investigate the alteration of the protein profile in cells after sonication and to identify the key proteins involved in the process of cell apoptosis. Walker 256 carinosarcoma cells were exposed to focused ultrasound (US) at the intensity of 2.0, 7.0, 10.2, 14.2 and 17.0 W/cm2 (I(spta)) for 10 min in vitro and the morphologic and functional changes of the cells were detected by hematoxylin & eosin staining and flow cytometry, with double staining of fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI). The protein compositions in the cells after sonication were detected by 2-D SDS polyacrylamide gel electrophoresis. Our results showed that apoptosis of Walker 256 carinosarcoma cells could be induced by US. The percentage of early apoptosis and secondary necrosis increased with increasing intensity of US irradiation. Comparing with the protein patterns of cells before sonication, it was found that around 420 new protein spots were present in the gel after sonication. Among them, Hsp60 and Bcl-2 like protein 13 were found to be involved in the process of cell apoptosis and US-induced apoptosis of the cells was probably performed through the pathway of promoting the activation of caspase-3.
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PMID:The alteration of protein profile of Walker 256 carinosarcoma cells during the apoptotic process induced by ultrasound. 1565 39

The transforming growth factor-beta (TGF-beta) 1 is a mediator of extracellular matrix (ECM) gene expression in mesangial cells and the development of diabetic glomerulopathy. Here, we investigate the effects of TGF-beta1 on laminin gamma1 and fibronectin polypeptide expression and cell survival in mouse mesangial cells (MES-13). TGF-beta1 (10 ng/ml) stimulates laminin-gamma1 and fibronectin expression approximately two-fold in a time-dependent manner (0-48 h). TGF-beta1 treatment also retards laminin-gamma1 mobility on SDS-gels, and tunicamycin, an inhibitor of the N-linked glycosylation, blocks the mobility shift. TGF-beta1 increases the binding of laminin gamma1 to WGA-agarose and the binding is abolished by tunicamycin suggesting that laminin gamma1 is modified by N-linked glycosylation. TGF-beta1 also elevates fibronectin glycosylation but its mobility is not altered. The degradation of laminin gamma1 and fibronectin proteins is reduced by their glycosylation. In addition, TGF-beta1 enhances mesangial cell viability and metabolic activities initially (0-24 h); however, eventually leads to cell death (24-48 h). TGF-beta1 elevates pro-apoptotic caspase-3 activity and decrease cell cycle progression factor cyclin D1 expression, which parallels cell death. These results indicate that TGF-beta1 plays an important role in ECM expression, protein glycosylation and demise of mesangial cells in the diabetic glomerular mesangium.
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PMID:Transforming growth factor-beta1 regulation of laminin gamma1 and fibronectin expression and survival of mouse mesangial cells. 1618 Jan 2

Anthrax toxin protective antigen (PA) binds cell surface receptors (e.g. ANTXR1,2), forms heptameric pores, and translocates lethal factor (LF) or oedema factor (OF) into the cytoplasm of mammalian cells. In the current study, we sought to determine how receptor levels influence these events, by examining PA heptamer stability and related processes in macrophages that overexpress ANTXR1 (RAW 264.7ANTXR1). In these experiments, PA-oligomers demonstrated an extended half-life in RAW 264.7ANTXR1 macrophages, with SDS-resistant heptamers detected up to 10 h following treatment, while levels of PA-oligomers declined within 3 h in control cells. RAW 264.7ANTXR1 macrophages were also more sensitive to lethal toxin, a combination of PA and LF. Surprisingly, we found that PA alone was cytotoxic to RAW 264.7ANTXR1 cells. Further analysis found that PA cytotoxicity required direct interaction with ANTXR1, oligomerization, channel formation, endosomal acidification, and was independent of the ANTXR1 cytoplasmic tail. PA intoxication of RAW 264.7ANTXR1 macrophages resulted in caspase-3 activation, with corresponding DNA fragmentation and proteolytic cleavage of poly-ADP-ribose polymerase, as well as activation of Bid, suggesting cell death occurred via apoptosis. Overall, results from the current study suggest that receptor levels dictate the extent of PA oligomer stability, and shifts in this normal process can lead to cell death via apoptosis in the absence of toxin catalytic subunits.
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PMID:Cytotoxic activity of Bacillus anthracis protective antigen observed in a macrophage cell line overexpressing ANTXR1. 1688 31

The role of proteinases of the histiophagous ciliate Philasterides dicentrarchi, purified by affinity chromatography in bacitracin-Sepharose, on apoptosis (programmed cell death) of turbot pronephric leucocytes (PL) was investigated. The results showed that more than 90% of proteinases purified by bacitracin-Sepharose were cysteine proteinases, which lacked significant caspase-3-like activity and generated three main gelatinolytic bands of molecular weights 36, 45 and 77 kDa as determined by gelatine-SDS-PAGE and immunoblot. Viability of PL cells after 24 h stimulation with P. dicentrarchi cysteine proteinases did not differ from that of non-stimulated cells. Apoptosis was confirmed by: (i) caspase activity, (ii) DNA fragmentation, and (iii) nucleus fragmentation. The caspase-3-like activity in PL incubated for 4h in the presence of 125, 250 and 500 microg/ml of proteinases increased in a dose-dependent fashion. The PL DNA was fragmented following 24-h exposure to P. dicentrarchi cysteine proteinases and characteristic DNA ladders consisting of multimers of approximately 180-200 pb were produced. Morphological changes, such as chromatin condensation and nucleus fragmentation, were observed under fluorescence microscopy after DAPI staining of the PL cells incubated with cysteine proteinase-incubated for 24 h. The results suggest that the pathogenic scuticociliate P. dicentrarchi may induce host leucocyte programmed cell death via the production of cysteine proteinases, as a mechanism of pathogenesis and evasion of the turbot innate immune response.
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PMID:Scuticociliate proteinases may modulate turbot immune response by inducing apoptosis in pronephric leucocytes. 1704 29

The rapid cold-hardening (RCH) response increases the cold tolerance of insects by protecting against non-freezing, cold-shock injury. Apoptosis, or programmed cell death, plays important roles in development and the elimination of sub-lethally damaged cells. Our objectives were to determine whether apoptosis plays a role in cold-shock injury and, if so, whether the RCH response protects against cold-induced apoptosis in Drosophila melanogaster. The present study confirmed that RCH increased the cold tolerance of the adults at the organismal level. No flies in the cold-shocked group survived direct exposure to 7 degrees C for 2 h, whereas significantly more flies in the RCH group survived exposure to 7 degrees C for 2 h after a 2-h exposure to 5 degrees C. We used a TUNEL assay to detect and quantify apoptotic cell death in five groups of flies including control, cold-shocked, RCH, heat-shocked (37.5 degrees C, 30 min), and frozen (20 degrees C, 24 h) and found that apoptosis was induced by cold shock, heat shock, and freezing. The RCH treatment significantly improved cell viability by 38% compared to the cold-shocked group. Cold shock-induced DNA fragmentation shown by electrophoresis provided further evidence for apoptosis. SDS-PAGE analysis revealed an RCH-specific protein band with molecular mass of approximately 150 kDa. Western-blotting revealed three proteins that play key roles in the apoptotic pathway: caspase-9-like (apoptotic initiator), caspase-3-like (apoptotic executioner) and Bcl-2 (anti-apoptotic protein). Consequently, the results of this study support the hypothesis that the RCH response protects against cold-shock-induced apoptosis.
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PMID:Rapid cold-hardening protects Drosophila melanogaster from cold-induced apoptosis. 1724 39

Modulation of surface properties of biomembranes by any ligand leading to permeabilization, fusion, rupture, etc. is a fundamental requirement for many biological processes. In this work, we present the interaction of piroxicam, a long acting Non-Steroidal Anti-Inflammatory Drug (NSAID) with isolated mitochondria, membrane mimetic systems, intact cells and a mitochondrial protein cytochrome c. Dye permeabilization study on isolated mitochondria indicates that piroxicam can permeabilize mitochondrial membrane. Direct imaging by Scanning Electron Microscope (SEM) shows that piroxicam induces changes in mitochondrial membrane morphology leading to fusion and rupture. Transmission Electron Microscope (TEM) imaging of piroxicam treated DMPC vesicles and mixed micelles formed from CTAB and SDS show that causing membrane fusion is a general property of piroxicam at physiological pH. In intact cells viz., V79 Chinese Hamster lung fibroblast, piroxicam is capable of releasing cytochrome c from mitochondria into the cytosol in a dose dependent manner along with the enhancement of downstream proapoptotic event viz., increase in caspase-3 activity. We have also shown that piroxicam can reduce cytochrome c within a time frame relevant to its lifetime in blood plasma. UV-visible spectroscopy has been used to study the reaction mechanism and kinetics in detail, allowing us to propose and validate a Michaelis-Menten like reaction scheme. CD spectroscopy shows that small but significant changes occur in the structure of cytochrome c when reduced by piroxicam.
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PMID:Interaction of piroxicam with mitochondrial membrane and cytochrome c. 1730 18

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, in the cytosolic fraction of the cerebral cortex of newborn piglets. The present study examines the mechanism of caspase-9 activation during hypoxia and tests the hypothesis that the ATP and cytochrome c-dependent activation of caspase-9 increases in the cytosol of the cerebral cortex of newborn piglets. Newborn piglets were divided into normoxic (Nx, n=4), and hypoxic (Hx, n=4) groups. Anesthetized, ventilated animals were exposed to an FiO(2) of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Cytosolic fraction was isolated and passed through a G25-Sephadex column to remove endogenous ATP and cytochrome c. Fractions were collected and protein determined by UV spectrophotometry at 280 nm. Eluted high-molecular weight samples from normoxic and hypoxic animals were divided into four subgroups: subgroup 1 (control), incubated without added ATP and cytochrome c; subgroup 2, incubated with added ATP; subgroup 3, incubated with added cytochrome c; and subgroup 4, incubated with added ATP and cytochrome c. The incubation was carried out at 37 degrees C for 30 min. Following incubation, the protein was separated by 12% SDS-PAGE and active caspase-9 was detected using specific active caspase-9 antibody. Protein bands were detected by enhanced chemiluminescence. Protein density was determined by imaging densitometry and expressed as absorbance (OD x mm(2)). ATP (mumol/g brain) level was 4.7 +/- 0.18 in normoxic, as compared to 1.53 +/- 0.16 in hypoxic (p < 0.05 vs. Nx). PCr (mumol/g brain) level was 4.03 +/- 0.11 in the normoxic and 1.1 +/- 0.3 in the hypoxic brain (p < 0.05 vs. Nx). In the normoxic preparations, active caspase-9 density increased by 9, 4 and 20% in the presence of ATP, cytochrome c and ATP+cytochrome c, respectively. In the hypoxic preparations, active caspase-9 density increased by 30, 45 and 60% in the presence of ATP, cytochrome c and ATP+cytochrome c, respectively. These results show that incubation with ATP, cytochrome c and ATP+cytochrome c result in a significantly increased activation of caspase-9 in the hypoxic group (p < 0.05). We conclude that the ATP and cytochrome c dependent activation of caspase-9 is increased during hypoxia. We propose that the ATP and cytochrome c sites of apoptotic protease activating factor I that mediate caspase-9 activation are modified during hypoxia.
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PMID:ATP and cytochrome c-dependent activation of caspase-9 during hypoxia in the cerebral cortex of newborn piglets. 1797 8

Quantitative proteome analysis of cisplatin-induced apoptosis in total Jurkat T cell lysates was performed in order to identify modified proteins. Proteins were labeled in cell culture with stable isotopes of arginines, and fractionated by SDS-PAGE. Subsequently, tryptic peptides were analyzed by nano-LC coupled offline to MALDI-TOF/TOF-MS as an alternative to commonly used online LC-ESI-MS. As a result, 26 proteins were found with a relative abundance higher than 1.5, thereof 19 already known and seven unknown to be involved in apoptosis (adenine phosphoribosyltransferase, microsomal signal peptidase 25 kDa subunit, phosphomevalonate kinase, probable rRNA processing protein EBP2, RNA-binding protein 4, transmembrane protein 33, and tetratricopeptide repeat domain 9C). Immunoblotting of core-binding factor beta and elongation factor 2 revealed similar quantitative changes as detected by the SILAC-based proteomics approach. Strikingly, 8 of 26 identified apoptosis-modified proteins contained at least one RNA-binding motif. Three caspase cleavage sites of the 54 kDa nuclear RNA-binding protein (p54nrb) were mapped at DQLD(231) (downward arrow)D, DQVD(286) (downward arrow)R, and MMPD(422) (downward arrow)G by applying caspase-3 to the in vitro translated protein and mutation analysis. The determined caspase cleavage sites were located C-terminal to the two RNA-binding motifs and one (DQLD(231) (downward arrow)D) within the NOPS domain of p54nrb. Concisely, quantitative protein data generated by offline LC-MALDI-MS were shown to be particularly accurate. Furthermore, only regulated peptides were selected in a result-dependent manner for MS/MS analyses and revealed novel apoptosis-modified proteins.
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PMID:Quantitative proteome analysis of cisplatin-induced apoptotic Jurkat T cells by stable isotope labeling with amino acids in cell culture, SDS-PAGE, and LC-MALDI-TOF/TOF MS. 1798 30

In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by exogenous C(2)-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 micromol/L C(2)-ceramide in vitro. Flow cytometer was used to detect the mitochondrial membrane potential (DeltaPhi(m)). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C(2)-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived activator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3 for 24 h. The results showed that DeltaPhi(m) began to decrease from 6 h after 25 and 50 micromol/L C(2)-ceramide treatment (P<0.05) and cyclosporin A (CsA) could inhibit the collapse of DeltaPhi(m) through regulating mitochondrial membrane permeability transition pore. There was no effect of C(2)-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 micromol/L C(2)-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P<0.05). Also there was expression of cleaved caspase-3 with C(2)-ceramide treatment. After the treatment with caspase inhibitor, C(2)-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C(2)-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the release of Smac was caspase-dependent.
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PMID:Ceramide induces release of mitochondrial proapoptotic proteins in caspase-dependent and -independent manner in HT-29 cells. 1817 93


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