Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The process of cell death caused by influenza virus infection in cultured MDCK and HeLa cells was analysed. This infection gave rise to nuclear fragmentation and chromatin condensation accompanied by chromosomal DNA fragmentation into oligonucleosomes. Chromosomal DNA fragmentation progressed concomitantly with cell lysis of MDCK cells and HeLa cells, producing high and low yields of virus particles, respectively, indicating that the extent of cell lysis was not proportional to the virus production. The endonuclease inhibitor zinc blocked DNA fragmentation in MDCK cells. Cycloheximide inhibited DNA fragmentation as well as cell lysis. Inhibition occurred when the drug was added to the medium within 2 h after infection but not efficiently at 4 h or later. Infection induced the Fas Ag gene, which encodes a possible apoptosis-mediating molecule, in the early infectious stage followed by the expression of Fas Ag on the cell surface. These results suggested that influenza virus infection causes apoptotic death of cultured cells, and their fate might be determined at an early stage of the infection by induction of an apoptotic gene.
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PMID:Induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells. 750 71

Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death.
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PMID:Cell nucleus and DNA fragmentation are not required for apoptosis. 752 18

Activation of a triplet of nuclear proteins (NP42-50) was observed in human Jurkat T cell line following treatment with an antibody to CD95 (Fas/Apo-1), a cell surface molecule involved in apoptotic cell death. The nuclease activity, corresponding to a triplet of proteins observed at approximately 42, 45, and 50 kDa in size, was extractable, heat-stable, and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing deoxyribonucleic acids (SDS-PAGE-DNA) assay. The NP42-50 activity requires the presence of Mg2+/Ca2+ and is insensitive to inactivation by heating at 80 degrees C for 5 min. Zinc effectively inhibited the enzymatic activity of NP42-50 on SDS-PAGE-DNA and also protected Jurkat cells from the CD95-mediated apoptosis in cell cultures. The nuclease activation, however, was not a unique pathway for the CD95-mediated cell death. The apoptosis induced by arabinofuranosyl cytosine, a chemotherapeutic agent, also activated the NP42-50 nuclease activity in Jurkat cells, suggesting that a similar cascade of subsequent events in apoptosis may occur in most instances although many different signals can initiate apoptotic cell death in various cell types. The nuclease identified by this study appears to be distinguishable from DNase I or DNase II by its molecular characteristics and its enzymatic requirements. The NP42-50, with respect to the nuclease activity closely associated with apoptotic cell death, may serve as a candidate for the endonuclease(s) involved in the cleavage of DNA into fragments during apoptosis.
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PMID:A triplet of nuclease proteins (NP42-50) is activated in human Jurkat cells undergoing apoptosis. 755 79

We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction.
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PMID:Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification. 857 Jun 10

Autolytic DNA breakdown, detected as smears in electrophoretic gels, is a late event in necrosis. On the other hand, internucleosomal DNA cleavage, visualized as ladders, is thought to be a hallmark of apoptosis. We now report that this specific form of DNA fragmentation also occurs during necrosis and is an early event but appears to be triggered by proteolytic mechanisms significantly different from those documented in apoptosis. Treatment of MDCK cells with a mitochondrial uncoupler and a Ca2+ ionophore led to ATP depletion, necrotic morphology, and progressive fragmentation of DNA in an internucleosomal or ladder pattern. DNA breakdown was immediately preceded by increased permeability of the plasma membrane to macromolecules. Provision of glycine along with the noxious agents did not modify the extent of ATP depletion, but prevented plasma membrane damage. This was accompanied by complete inhibition of DNA fragmentation. Internucleosomal DNA cleavage was observed also during necrosis after rapid permeabilization of plasma membranes by detergents or streptolysin-O in hepatocytes, thymocytes, and P19, Jurkat, and MDCK cells. DNA fragmentation associated with necrosis was Ca2+/Mg2+ dependent, was suppressed by endonuclease inhibitors, and was abolished by serine protease inhibitors but not by inhibitors of interleukin-1 beta converting enzyme (ICE)-related proteases or caspases. Moreover, unlike apoptosis, it was not accompanied by caspase-mediated proteolysis. On the other hand, the cleavage-site-directed chymotryptic inhibitor N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK) suppressed DNA fragmentation not only in necrotic cells but also during Fas-mediated apoptosis, without inhibiting caspase-related proteolysis. The results suggest a novel pathway of endonuclease activation during necrosis not involving the participation of caspases. In addition, they indicate that techniques based on double-strand DNA breaks may not reliably differentiate between apoptosis and necrosis.
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PMID:Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death. Involvement of serine but not cysteine proteases. 935 45

Ligation of major histocompatability complex class I (MHC-I) molecules expressed on T cells leads to both growth arrest and apoptosis. The aim of the current study was to investigate the intracellular signal pathways that mediate these effects. MHC-I ligation of human Jurkat T cells induced a morphologically distinct form of apoptosis within 6 h. A specific caspase inhibitor, which inhibited Fas-induced apoptosis, did not affect apoptosis induced by MHC-I ligation. Furthermore, MHC-I-induced apoptosis did not involve cleavage and activation of the poly(ADP- ribose) polymerase (PARP) endonuclease or degradation of genomic DNA into the typical fragmentation ladder, both prominent events of Fas-induced apoptosis. These results suggest that MHC-I ligation of Jurkat T cells induce apoptosis through a signal pathway distinct from the Fas molecule. In our search for other signal pathways leading to apoptosis, we found that the regulatory 85-kD subunit of the phosphoinositide-3 kinase (PI-3) kinase was tyrosine phosphorylated after ligation of MHC-I and the PI-3 kinase inhibitor wortmannin selectively blocked MHC-I-, but not Fas-induced, apoptosis. As the c-Jun NH2-terminal kinase (JNK) can be activated by PI-3 kinase activity, and has been shown to be involved in apoptosis of lymphocytes, we examined JNK activation after MHC-I ligation. Strong JNK activity was observed after MHC-I ligation and the activity was completely blocked by wortmannin. Inhibition of JNK activity, by transfecting cells with a dominant-negative JNKK- MKK4 construct, led to a strong reduction of apoptosis after MHC-I ligation. These results suggest a critical engagement of PI-3 kinase-induced JNK activity in apoptosis induced by MHC-I ligation.
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PMID:Ligation of major histocompatability complex (MHC) class I molecules on human T cells induces cell death through PI-3 kinase-induced c-Jun NH2-terminal kinase activity: a novel apoptotic pathway distinct from Fas-induced apoptosis. 939 57

We compared two methods to stain apoptotic cells, one using terminal deoxynucleotidyl transferase (TDT), the other DNA polymerase I, using leukemia cell lines treated with anti-Fas monoclonal antibody (MAb). Both TDT and polymerase I strongly reacted with fragmented nuclei of apoptotic MOLT-16 and Jurkat cells, but only polymerase I strongly reacted with nonfragmented nuclei of early apoptotic cells. Anti-Fas MAb-treated MOLT-4 cells showed morphological changes corresponding to early apoptosis and were strongly positive for polymerase I only. MOLT-16 and Jurkat cells treated with anti-Fas MAb and inhibitors of endonuclease and poly(ADP-ribose) polymerase showed the morphology of early apoptosis but were not strongly stained by TDT. Because DNA polymerase I has nick-translation activity, it is possible that DNA polymerase I reaction is positive in early apoptotic cells by detecting single-strand DNA cleavage, which occurs before extensive oligonucleosomal DNA cleavage and late morphological changes of apoptosis in leukemia cell lines. Although TDT is widely used to stain apoptotic cells, DNA polymerase I may be more applicable in special cases of apoptosis, in which cells undergo single-strand rather than double-strand DNA breaks. However, the procedure has limitations, such as the necessity to use cell smears for comparison with the TDT reaction. (J Histochem Cytochem 46:85-90, 1998)
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PMID:Comparison of two methods of staining apoptotic cells of leukemia cell lines. Terminal deoxynucleotidyl transferase and DNA polymerase I reactions. 940 97

Induction of apoptosis by death receptors such as Fas or tumour necrosis factor (TNF) R1 leads to distinct changes in cell morphology, activation of the caspase protease cascade, and the degradation of nuclear chromatin by activated nucleases. Here, we describe the purification and cDNA cloning of a novel 40 kDa endonuclease from Jurkat cells that is activated by caspases. This protein, designated caspase-activated nuclease (CPAN), is sufficient to degrade naked DNA and to induce apoptotic morphology and DNA fragmentation in naive nuclei. CPAN is highly homologous to a recently described mouse nuclease, CAD [1], and may represent the human homologue. Our data on the human cDNA as well as additional data on the mouse homologue suggest that a 30 amino-acid portion of the recently published mouse sequence [1] is incorrect. We show that the activity of human CPAN is regulated by DFF45 [2], an inhibitor necessary for CPAN expression and stabilization in an inactive state in living cells. Proteolytic cleavage of DFF45 by caspases in vitro leads to dissociation of DFF45 fragments from CPAN and activation of CPAN as an endonuclease. CPAN is a tightly regulated endonuclease with unique characteristics that might represent a distinctive family of endonucleases.
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PMID:CPAN, a human nuclease regulated by the caspase-sensitive inhibitor DFF45. 956 Mar 46

Programmed cell death (PCD), also known as apoptosis, is a genetically controlled cellular response, manifested in morphologically distinct non-necrotic cellular destruction: cell shrinkage, cytoplasmic "boiling", condensation of chromatin, loss of nuclear membrane followed by DNA fragmentation and cell membrane blebbing, all of which initiate the formation of apoptotic bodies. During the early stages of PCD, cell membrane phospholipid asymmetry is altered, resulting in the dislocation of phosphatidylserine (PS) to the cell surface. During apoptosis, DNA is cut by endonucleases at DNA-linked sites between nucleosomes, producing a number of multimers of nucleosomal DNA units in the cell nuclei. The mechanism of apoptosis and the cellular signals involved in its induction have been studied during thymic prenatal ontogenesis and postnatal development, mainly in immature thymocytes and in the cells of the reticulo-epithelial (RE) network. In thymocytes or resting T lymphocytes, p53 tumor suppressor protein was identified to be a critical mediator of apoptosis in response to DNA damage. The cellular interaction of immature, cortical thymocytes (characterized by a double positive CD4+CD8+TCRlow immunophenotype) with thymic RE cells induces positive selection of T lymphocytes that recognize, but are not activated by self-MHC molecules (tolerance induction). Double positive CD4+CD8+CD3- thymocytes undergo Fas-mediated apoptosis, while CD4+CD8+CD3+ cells use the CD3 mediated pathway of PCD. Two step, apoptotic cell death is mainly restricted to the CD4+CD8+TCRdull thymocyte subpopulation. T-lymphocytes which do not undergo positive selection are killed by apoptosis in response to a number of intrinsic and extrinsic factors, such as chemical toxins, viral infections, X- and UV irradiation, mild hyperthermia, the actions of various hormones, extracellular survival factors, calcium ionophores (such as A23187), various chemotherapeutic drugs (adriamycin, actinomycin D, etc) and antibodies directed to the CD3-TCR (T cell receptor) complex. Immature thymocytes also undergo a second selective process, so-called negative selection, when thymic stromal cells eliminate autoreactive T lymphocytes. This process has been termed clonal deletion and also involves apoptosis. In addition to the two intrathymic T lymphocyte selection mechanisms, Immunocompetent, but autoreactive T lymphocytes which have already reached the periphery are also eliminated by apoptosis. All the divers stimuli of PCD are associated with an increase in the concentration of cytosolic calcium ions (Ca+2), which activate an endogenous endonuclease. This trigger for PCD results in rapid cleavage of DNA, a hallmark of apoptosis. Despite the diversity of the signals that can trigger apoptosis, the changes in cellular morphology characteristic of PCD are very similar. The uniformity of the morphological changes suggests the existence of a predetermined, final and common cell suicide pathway. Apoptosis requires energy in the form of ATP, indicating that PCD, as opposed to necrosis, is an energy dependent, active physiological and pathophysiological phenomenon.
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PMID:Apoptosis in the mammalian thymus during normal histogenesis and under various in vitro and in vivo experimental conditions. 957 34

Characteristic steps in the course of cellular apoptosis are the induction of chromatin condensation and cleavage of the DNA, leading to the formation of oligomers of nucleosomes. Since the H1 histones represent functional elements that are essential for the generation of highly condensed chromatin structures, we analysed the total cellular H1 histones of five leukaemic and three solid human tumour cell lines, comparing the H1 pattern of exponentially growing cells with that of apoptotic cells. For the induction of apoptosis, cell lines were treated with the water-soluble camptothecin derivative, topotecan (a topoisomerase I inhibitor), or with an apoptosis-inducing monoclonal anti-CD95 (Fas/APO-1) antibody. Total histone H1 proteins were isolated by extraction with 5% perchloric acid and were analysed by means of capillary zone electrophoresis (CZE) separation. The identities of the peaks representing different histone H1 subtypes on CZE electropherograms were confirmed by analysis of preparations (recombinant proteins purified from transformed yeast used as internal standards) mixed with each of the subtypes respectively. The progress of topotecan- or anti-CD95-induced cell death was monitored by flow cytometry analysis and also by agarose electrophoresis of fragmented DNA. During early apoptosis of three of these cell lines, we observed the induction of internucleosomal DNA cleavage and, simultaneously, a typical change in the histone H1 protein pattern, leading to an increase in the relative amounts of histone subtypes H1.4 and H1.5. Upon apoptosis induction, these changes were only observed in correlation with the occurrence of DNA fragmentation, thus possibly reflecting a prerequisite for DNA accessibility and/or endonuclease activity.
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PMID:Changes in the protein pattern of H1 histones associated with apoptotic DNA fragmentation. 988 31


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