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)

FIV is a lentivirus infection of cats which induces an immunodeficiency syndrome associated with early qualitative defects in antigen-specific T cell function and with late quantitative defects in CD4+ T lymphocytes. We have observed that peripheral blood mononuclear cells (PBMC) from FIV-infected cats have impaired survival in culture. The mechanism of this in vitro dysfunction and depletion is not known. We have proposed that inappropriate induction of programmed cell death (apoptosis) could account for these in vitro defects. Here, we report that PBMC from FIV-infected cats, with impaired T cell blastogenesis and impaired survival in vitro, undergo an active cell death upon culture, which has the morphological and biochemical characteristics of programmed cell death (PCD). Apoptosis occurred in all six asymptomatic FIV-infected cats, and in none of the nine uninfected cats, which were studied. Changes in cell morphology under both light and electron microscopy, and fragmentation of genomic DNA were characteristic for apoptosing cells. Cell death was spontaneous and occurred in the absence of any stimuli, and culture with the T cell mitogen, concanavalin A (Con A), did not significantly enhance cell death. Activation-induced cell death was inhibited, in a dose-dependent manner, by addition to the incubation medium of zinc, which has been shown to inhibit the action of endonuclease responsible for the characteristic fragmentation of DNA. Since apoptosis has recently been implicated in AIDS pathogenesis, FIV infection may prove useful to study this aspect of retroviral, in particular HIV, infection.
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PMID:Programmed cell death (apoptosis) as a mechanism of cell death in peripheral blood mononuclear cells from cats infected with feline immunodeficiency virus (FIV). 839 41

Cell death occurring by apoptosis has become widely recognized as an integral component of the life cycle of many cell types. Apoptosis can be induced in many tissues by a wide variety of endogenous signals, including glucocorticoids. However, even though there are glucocorticoid receptors present in almost all cells, only certain lymphoid cells are susceptible to glucocorticoid-induced apoptosis. The basis for this selective regulation of programmed cell death is unknown. Internucleosomal chromatin degradation is an integral characteristic of apoptosis, common to all cells undergoing this form of programmed cell death. Thus, we have developed an in vitro assay that recapitulates apoptotic DNA degradation in isolated nuclei and have identified a nuclease activity with internucleosomal specificity that is present in nuclear extracts of thymocytes undergoing glucocorticoid-induced apoptosis. We have now extended these studies to analyze the molecular properties of the crude enzyme and to further elucidate the mechanism of its regulation during the tissue-specific induction of apoptosis. In vitro, optimal internucleosomal cleavage activity was detected in the presence of millimolar concentrations of calcium and magnesium or manganese. Nuclease activity was inhibited by three agents previously shown to block apoptosis (zinc, aurintricarboxylic acid, and sodium), suggesting that the nuclease we detected in apoptotic thymocytes is responsible for the DNA degradation associated with apoptosis. Size-fractionation analysis of thymocyte nuclear extract under native conditions revealed a protein with an apparent molecular mass of 22.7 kilodaltons and a sedimentation coefficient of 3.5S. Interestingly, when extracts from control thymocytes, inactive in internucleosomal cleavage activity, were subjected to gel filtration or sucrose density gradient fractionation, internucleosomal cleavage activity was detected. The physical characteristics of this endonuclease activity were identical to those found in thymocyte extract from glucocorticoid-treated rats. Repressed or latent internucleosomal cleavage activity was also detected in hepatocyte nuclear extracts, but this activity was not activated by glucocorticoid treatment. These data suggest that glucocorticoid-induced apoptosis involves cell-specific activation of a latent endonuclease, rather than nuclease induction.
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PMID:Mechanism of tissue-specific induction of internucleosomal deoxyribonucleic acid cleavage activity and apoptosis by glucocorticoids. 839 69

Apoptosis, or programmed cell death, is a physiological process whereby a target cell dies in response to a specific signal. A prominent model system used to study this process is the glucocorticoid-mediated killing of immature thymocytes. Following glucocorticoid treatment, apoptotic thymocytes undergo a series of distinct morphological alteration including cellular shrinkage, blebbing of the cytoplasmic membrane, and chromatin condensation. The chromatin condensation that occurs during apoptosis is associated with a characteristic endonuclease activity that degrades the genome at internucleosomal sites. To study this characteristic endonuclease activity further, nuclear extracts were prepared from thymocytes of glucocorticoid-treated chicks and nuclease activity present in the protein extracts was analyzed using chicken red blood cell nuclei as a substrate. Using this in vitro assay system, it was demonstrated that the avian endonuclease activity degrades chromatin at internucleosomal sites and can be inhibited by EDTA and zinc ions. Current efforts are focused on purifying the avian apoptotic endonuclease and further characterizing this nuclease activity.
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PMID:Programmed cell death in avian thymocytes: role of the apoptotic endonuclease. 839 96

A major event in apoptosis is the digestion of chromatin into oligonucleosomal fragments. However, the enzymes responsible for the DNA degradation have not been well characterized. Here we report the purification of an endonuclease from human spleen cell nuclei that is likely to be responsible for DNA digestion in apoptosis. Enzyme activity was measured by a sensitive fluorometric assay, which assesses the conversion of plasmid DNA from a supercoiled to an open form. The endonuclease was extracted from isolated nuclei with NaCl between 100 and 350 mM and was further purified by chromatography on columns of phosphocellulose, Superdex 75, and chelating Sepharose (Zn2+ form). By gel filtration, the apparent molecular mass was 22-26 kDa; on SDS-polyacrylamide gel electrophoresis, the purified enzyme showed a single 27-kDa band. The enzyme required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity. It was inhibited by Zn2+ (100% inhibition at 50 microM) and by high (> 10 mM) concentrations of Ca2+. Aurintricarboxylic acid, spermine, p-(hydroxymercuri)benzoate, and N-ethylmaleimide were also endonuclease inhibitors. No inhibition was observed with iodoacetamide, G-actin, or nucleoside 3',5'-bisphosphates. An optimum pH of 8.0 was found. When added to human CCRF-CEM lymphoblast nuclei, that do not contain the endonuclease, the purified splenic enzyme digested the chromatin into the mono- and oligonucleosomal fragments that are characteristic of apoptosis. On the basis of this result, and the observation that the activators and inhibitors of the purified endonuclease closely parallel those that affect apoptosis, it seems likely that this enzyme is involved in the apoptotic degradation of DNA in human lymphocytes.
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PMID:Ca2+/Mg(2+)-dependent endonuclease from human spleen: purification, properties, and role in apoptosis. 839 24

Exposing a macrophage-like murine cell line to copper-oxidised low-density lipoprotein led to DNA fragmentation which was inhibited by the putative Ca2+/Mg2+ endonuclease inhibitor, zinc sulphate. DNA fragmentation preceded loss of membrane impermeability. These results suggest that apoptosis may be a mechanism of macrophage foam cell death in atherosclerotic lesions in the arterial wall.
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PMID:Fragmentation of DNA in P388D1 macrophages exposed to oxidised low-density lipoprotein. 840 60

Spontaneous mutants that were resistant to zinc were isolated from Alcaligenes eutrophus CH34 containing either the native plasmid pMOL28 or a derivative derepressed for its self-transfer, pMOL50. With the cured plasmid-free derivative of CH34, strain AE104, such mutants were not detected. The mutations, which were shown to be located in the plasmid, increased the level of the nickel and cobalt resistance determined by the cnr locus. The chromate resistance closely linked to the cnr locus was not affected by these mutations. In the Znr mutants, the resistance to zinc and nickel was constitutively expressed. Uptake studies showed that the zinc resistance in a Znr mutant resulted from reduced accumulation of zinc ions in comparison with that in the plasmid-free strain. Reduced accumulation of zinc was also observed to a lesser degree in the parental strain induced with nickel, suggesting that zinc interferes with the Ni2+ and Co2+ efflux system. A 12.2-kb EcoRI-XbaI restriction endonuclease fragment containing the cnr locus was cloned from plasmid pMOL28 harboring the mutation and shortened to an 8.5-kb EcoRI-PstI-PstI fragment conferring resistance to zinc, nickel, and cobalt. The 12.2-kb EcoRI-XbaI fragment was also reduced to a 9.7-kb BamHI fragment still encoding weak resistance to nickel and cobalt but not to zinc. Complementation studies demonstrated the recessivity of the cnr mutations with a Znr phenotype. Such mutations thus allow positive selection of mutants affected in the expression of the cnr operon.
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PMID:A new type of Alcaligenes eutrophus CH34 zinc resistance generated by mutations affecting regulation of the cnr cobalt-nickel resistance system. 842 50

Apoptosis is a major form of cell death, characterized morphologically by chromatin condensation and biochemically by endonuclease cleavage of DNA into oligonucleosomal fragments. Recently, we reported that zinc arrested dexamethasone-induced apoptosis in thymocytes at an early stage, as characterized morphologically by condensation of heterochromatin into clumps abutting the nuclear membrane. In this study, we show that zinc completely inhibits endonuclease cleavage of DNA into oligonucleosomal fragments but does not prevent the cleavage of DNA into high molecular weight fragments. These results indicate that the formation of these high molecular weight fragments, which correlates with the very early morphological features of apoptosis, is a critical event in glucocorticoid-induced apoptosis. The formation of these high molecular weight fragments, despite the inhibition by zinc of the endonuclease cleavage of DNA, suggests that key enzyme(s), other than the Ca2+/Mg(2+)-dependent endonuclease, are involved at the earliest stages of induction of apoptosis.
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PMID:Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation. 842 79

Isolated nuclei from mammalian cells contain a Ca(2+)-dependent endonuclease [1]. The produced DNA fragmentation is a necessary step in the sequence of events resulting in apoptosis [2]. We report here that zinc inhibits the DNA fragmentation in dependence of the free Ca2+ concentrations, suggesting that a balance between zinc and calcium might regulate the Ca(2+)-dependent endonuclease. Incubation of nuclei with different free calcium concentrations combined with cadmium shows a stronger inhibition of the DNA fragmentation than zinc. Cadmium inhibits the endonuclease in a calcium-independent way. Surprisingly cadmium alone is able to stimulate the endonuclease, thus to replace Ca2+.
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PMID:Cadmium and zinc mediated changes of the Ca(2+)-dependent endonuclease in apoptosis. 843 10

A 26-kDa endonuclease has been purified to homogeneity from zinc-sufficient Euglena gracilis. The protein binds to single-stranded DNA with a higher affinity than to double-stranded DNA, but it exhibits nucleolytic activity toward both. Thus, it converts supercoiled plasmid pBR322 DNA into the linear form, a property characteristic of endonucleases, and it continues to act on the linearized DNA until it is completely degraded. It also hydrolyzes heat-denatured, single-stranded calf thymus DNA. Moreover, at amounts below 1 microgram, it enhances RNA synthesis by RNA polymerase II, a characteristic observed with other DNases. Its addition to an in vitro transcription assay increases RNA synthesis up to 3-fold. The nuclease requires two metal components to carry out its enzymatic activities. It hydrolyzes DNA only in the presence of millimolar amounts of magnesium or micromolar quantities of other activating metal ions, such as manganese, zinc, or cobalt. However, even when optimal concentrations of Mg2+ are present, micromolar amounts of the metal-chelating agents OP and HQSA completely inhibit pBR322 digestion. Transcription enhancement is also inhibited completely by both chelators at concentrations that do not affect the intrinsic polymerase II activity. By atomic absorption spectrometry, the enzyme contains 1 g-atom of Zn/mol, which is the likely target of chelator action. The nuclease protein can also be isolated from zinc-deficient E. gracilis, but remarkably it then contains 1 mol of Cu/g-atom and no zinc.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A Euglena gracilis zinc endonuclease. 844 31

Certain anti-cancer agents are known to induce apoptosis in human tumour cells. However, these agents are intrinsically cytotoxic against cells of normal tissue origin, including myelocytes and immunocytes. Here we show that a naturally occurring flavone of citrus origin, tangeretin (5,6,7,8,4'-pentamethoxyflavone), induces apoptosis in human promyelocytic leukaemia HL-60 cells, whereas the flavone showed no cytotoxicity against human peripheral blood mononuclear cells (PBMCs). The growth of HL-60 cells in vitro assessed by [3H]thymidine incorporation or tetrazolium crystal formation was strongly suppressed in the presence of tangeretin; the IC50 values range between 0.062 and 0.173 microM. Apoptosis of HL-60 cells, assessed by cell morphology and DNA fragmentation, was demonstrated in the presence of > 2.7 microM tangeretin. Flow cytometric analysis of tangeretin-treated HL-60 cells also demonstrated apoptotic cells with low DNA content and showed a decrease of G1 cells and a concomitant increase of S and/or G2/M cells. Apoptosis was evident after 24 h of incubation with tangeretin, and the tangeretin effect as assessed by DNA fragmentation or growth inhibition was significantly attenuated in the presence of Zn2+, which is known to inhibit Ca(2+)-dependent endonuclease activity. Ca2+ and Mg2+, in contrast, promoted the effect of tangeretin. Cycloheximide significantly decreased the tangeretin effect on HL-60 cell growth, suggesting that protein synthesis is required for flavonoid-induced apoptosis. Tangeretin showed no cytotoxicity against either HL-60 cells or mitogen-activated PBMCs even at high concentration (27 microM) as determined by a dye exclusion test. Moreover, the flavonoid was less effective on growth of human T-lymphocytic leukaemia MOLT-4 cells or on blastogenesis of PBMCs. These results suggest that tangeretin inhibits growth of HL-60 cells in vitro, partially through induction of apoptosis, without causing serious side-effects on immune cells.
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PMID:Citrus flavone tangeretin inhibits leukaemic HL-60 cell growth partially through induction of apoptosis with less cytotoxicity on normal lymphocytes. 851 48


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