Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin-agarose or a trypsin-agarose column. In both cases, SDS-PAGE showed two bands of M(r) 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K(i)s for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6 x 10(-7) and 1.3 x 10(-5)M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.
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PMID:A novel trypsin inhibitor from Peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis. 1259 Sep 27

Enediyne antibiotics have been reported to be the most potent cytotoxic antitumor agents. The pathway by which these compounds cleave DNA and induce apoptosis of tumor cells may be different from the caspase-mediated pathways that initiate typical apoptosis. In this report, we studied the apoptosis induced by lidamycin (LDM), a member of the enediyne antibiotic family, and compared the characteristics of LDM-induced apoptosis with those of typical apoptosis induced by mitomycin C or etoposide. Chromatin condensation occurred very rapidly and appeared as speckles in human hepatoma BEL-7402 and breast carcinoma MCF-7 cells after treatment with 1 microM LDM. In addition, co-staining the cells with the mitochondria-specific dye Mitosensor and the DNA-specific dye Hoechst 33342 enabled the visualization of mitochondria in normal control and LDM-treated cells but not in mitomycin C-treated cells. Neither the caspase inhibitor VAD-fmk nor the caspase-3 inhibitor DEVD-fmk was able to inhibit the DNA ladder patterns caused by LDM in BEL-7042 or MCF-7 cells. Smaller fragments of histone H1 cleaved by LDM were detected by SDS-PAGE, indicating that the site of LDM action is the internucleosomal structure. Although caspase-9, caspase-3/7, and caspase-6 activities were increased in BEL-7402 cells, and caspase-7 activity was increased in MCF-7 cells after treatment with 1 microM LDM, this occurred much later, indicating that chromatin condensation reached the maximal level rapidly while caspase activities still remained low. Taken together, these results demonstrate that LDM induced rapid DNA cleavage and chromatin condensation independently of caspase activities; this may contribute to its highly potent cytotoxicity toward tumor cells.
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PMID:Non-caspase-mediated apoptosis contributes to the potent cytotoxicity of the enediyne antibiotic lidamycin toward human tumor cells. 1278 28

The melanin-free ink of the cephalopod Sepia officinalis is shown to contain a heat labile proteinaceous component toxic to a variety of cell lines, including PC12 cells. Gel filtration chromatography indicated that the toxic component was concentrated in those fractions eluted at a molecular weight higher than 100 kDa and exhibiting the highest tyrosinase activity. SDS-PAGE analysis of the active fractions displayed a single major band migrating at an approximate molecular weight of 100 kDa, identical with that of the single tyrosinase band in the melanin-free ink. These data unambiguously demonstrated the identity of the toxic component with tyrosinase. Treatment of purified Sepia as well as of mushroom tyrosinase with an immobilized version of proteinase K resulted in a parallel loss of tyrosinase activity and cytotoxicity. Sepia apotyrosinase was ineffective in inducing cytotoxicity in PC12 cells. Purified Sepia tyrosinase was found to induce a significant increase in caspase 3 activity in PC12 cells, leading eventually to an irreversible apoptotic process. Overall, these results disclose a hitherto unrecognized property of tyrosinase that may lead to a reappraisal of its biological significance beyond that of a mere pigment producing enzyme.
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PMID:Toxicity of melanin-free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase. 1290 67

Oligonucleosomal fragmentation of nuclear DNA is the late-stage apoptosis hallmark. In apoptotic mammalian cells the fragmentation is catalyzed by DFF40/CAD DNase primarily activated by caspase 3 through the site-specific proteolytic cleavage of DFF45/ICAD. A deletion in the casp3 gene of human breast adenocarcinoma MCF-7 results in lack of procaspase 3 in these cells. The absence of caspase 3 in MCF-7 leads to disability to activate oligonucleosomal DNA fragmentation in TNF-alpha induced cell death. In this study, sodium palmitate was used as an apoptotic stimulus for MCF-7. It has been shown that palmitate but not TNF-alpha induces both apoptotic changes in nuclei and oligonucleosomal DNA fragmentation in casp3-mutated MCF-7. Activation and accumulation of 40-50 kD DFF40-like DNases in nuclei of palmitate-treated apoptotic MCF-7 were detected by SDS-DNA-PAGE assay. Microsomal fraction of apoptotic MCF-7 does not contain any detectable DNases, but activates 40-50 kD nucleases when incubated with human placental chromatin. Furthermore, microsomes of apoptotic MCF-7 induce oligonucleosomal fragmentation of chromatin in a cell-free system. Both the activation of DNases and chromatin fragmentation are suppressed in the presence of the caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome-associated caspase 7 is suggested to play an essential role in the induction of oligonucleosomal DNA fragmentation in casp3-deficient MCF-7 cells.
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PMID:Oligonucleosomal DNA fragmentation in MCF-7 cells undergoing palmitate-induced apoptosis. 1475 30

Recently, a new coronavirus was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with Severe Acute Respiratory Syndrome (SARS) and the causative association with SARS was determined. To reveal further the characteristics of the virus and to provide insight about the molecular mechanism of SARS etiology, a proteomic strategy was utilized to identify the structural proteins of SARS coronavirus (SARS-CoV) isolated from Vero E6 cells infected with the BJ-01 strain of the virus. At first, Western blotting with the convalescent sera from SARS patients demonstrated that there were various structural proteins of SARS-CoV in the cultured supernatant of virus infected-Vero E6 cells and that nucleocaspid (N) protein had a prominent immunogenicity to the convalescent sera from the patients with SARS, while the immune response of spike (S) protein probably binding with membrane (M) glycoprotein was much weaker. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the complex protein constituents, and the strategy of continuous slicing from loading well to the bottom of the gels was utilized to search thoroughly the structural proteins of the virus. The proteins in sliced slots were trypsinized in-gel and identified by mass spectrometry. Three structural proteins named S, N and M proteins of SARS-CoV were uncovered with the sequence coverage of 38.9, 93.1 and 28.1% respectively. Glycosylation modification in S protein was also analyzed and four glycosylation sites were discovered by comparing the mass spectra before and after deglycosylation of the peptides with PNGase F digestion. Matrix-assisted laser desorption/ionization-mass spectrometry determination showed that relative molecular weight of intact N protein is 45 929 Da, which is very close to its theoretically calculated molecular weight 45 935 Da based on the amino acid sequence deduced from the genome with the first amino acid methionine at the N-terminus depleted and second, serine, acetylated, indicating that phosphorylation does not happen at all in the predicted phosphorylation sites within infected cells nor in virus particles. Intriguingly, a series of shorter isoforms of N protein was observed by SDS-PAGE and identified by mass spectrometry characterization. For further confirmation of this phenomenon and its related mechanism, recombinant N protein of SARS-CoV was cleaved in vitro by caspase-3 and -6 respectively. The results demonstrated that these shorter isoforms could be the products from cleavage of caspase-3 rather than that of caspase-6. Further, the relationship between the caspase cleavage and the viral infection to the host cell is discussed.
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PMID:Proteomic analysis on structural proteins of Severe Acute Respiratory Syndrome coronavirus. 1476 Jul 22

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

Oligonucleosomal fragmentation of nuclear DNA is the late stage hallmark of the apoptotic process. In mammalian apoptotic cells fragmentation is catalyzed by DFF40/ CAD DNase. DFF40/CAD primary activated through site-specific proteolytic cleavage by caspase 3. The absence of caspase 3 in MCF-7 leads to lack of oligonucleosomal DNA fragmentation under numerous apoptotic stimuli. In this study it was shown that palmitate induces apoptotic changes of nuclei and oligonucleosomal DNA fragmentation in casp3 deficient MCF-7. Activation and accumulation of 40-50 kDa DFF40 like DNases in nuclei and cytoplasm of palmitate-treated MCF-7 were detected by SDS-DNA-PAGE assay. Microsomes of apoptotic MCF-7 activate 40-50 kDa nucleases when incubated with human placental chromatin and induce oligonucleosomal fragmentation of chromatin in cell free system. Both DNases activation and chromatin fragmentation are suppressed in presence of caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome associated caspase 7 is suggested to play the principal role in induction of oligonucleosomal DNA fragmentation of casp3 defitient MCF-7.
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PMID:Oligonucleosome DNA fragmentation of caspase 3 deficient MCF-7 cells in palmitate-induced apoptosis. 1556 68

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


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