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)

Enzymatic activity, hydrolyzing DNA treated with beta-isopropyl-bis-beta-chloroethylamine (HN2-DNA), HN2-DNA exposed at 50 degrees for 1 h, and DNA treated with acid, to acid-soluble fragments was found in extracts from cells of M. lysodeikticus. The endonucleolytic component ofthe indicated activity manifests chromatographic properties on DEAE- and CM-cellulose, close to those for UV-endonuclease. Activity is manifested by UV-irradiated DNA, proflavin, and cyanide. Two electrophoretically homogeneous fractions of UV-endonuclease (after chromatography on DEAE- and CM-cellulose), with molecular weights about 13,000 and 15,000 daltons, exhibit endonucleolytic activity with respect to HN2-DNA, exposed at 50 degrees for 1 h, and with respect to "acid" DNA, treated for 6 min at 70 degrees in citrate buffer, pH 3.5. The activity with respect to the latter substrate is competitively suppressed by UV-irradiated DNA. The most probable substrate of UV-endonuclease, in addition to cyclobutane dimers, is the depurinized region of DNA.
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PMID:The presence of an endonuclease acting on UV-irradiated and depurinized DNA in cells of Micrococcus lysodeikticus. 112 5

Endonuclease V of bacteriophage T4 binds to UV-irradiated deoxyribonucleic acid (DNA) but not to unirradiated DNA. We have developed an assay to detect this binding, based on the retention of enzyme--DNA complexes on nitrocellulose filters. The amount of complex retained, ascertained by using radioactive DNA, is a measure of T4 endonuclease V activity. The assay is simple, rapid, and specific, which makes it useful for detecting T4 endonuclease V activity both in crude lysates and in purified preparations. We have used it to monitor enzyme activity during purification and to study binding of the enzyme to DNA under conditions that minimize the ability of the enzyme to nick DNA. From our data we conclude that (1) T4 endonuclease V binds to UV-irradiated DNA but not to DNA that has been previously incised by the endonuclease, (2) equilibrium between the free and complexed form of the enzyme is attained under our reaction conditions, (3) dissociation of enzyme--DNA complexes is retarded by sodium cyanide, and (4) retention of enzyme--DNA complexes on nitrocellulose filters is enhanced by high concentrations of saline--citrate.
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PMID:Binding of T4 endonuclease V to deoxyribonucleic acid irradiated with ultraviolet light. 624 30

Ninety-two and 33 methicillin-resistant Staphylococcus aureus (MRSA) strains were isolated in Japan and China respectively. They were categorised as ofloxacin-susceptible (MIC < 12.5 mg/L), moderately (MIC 12.5-25 mg/L) or highly (MIC > or = 50 mg/L) ofloxacin-resistant. 4-Quinolone concentrations required to inhibit purified DNA gyrase from the moderately and highly quinolone-resistant MRSA were at least 20 times higher than those required to inhibit the equivalent enzyme from quinolone-susceptible strains. Reconstitution assays demonstrated that the 4-quinolone-resistant MRSA had a mutation in subunit A of DNA gyrase. A portion of the gyrA gene from amino acids codons 40-115 was sequenced. Four moderately resistant and seven highly resistant MRSA contained a Ser-->Leu substitution at amino acid 84; one moderately and one highly resistant MRSA and one moderately resistant methicillin-susceptible S. aureus (MSSA) strain contained a Glu-->Lys substitution at amino acid 88. Eight MRSA, including one quinolone-susceptible strain and one MSSA contained a silent mutation at amino acid 86. Uptake of ofloxacin in moderately resistant strains was almost the same in the presence or absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), whereas in highly resistant strains, uptake increased when CCCP was added. Restriction fragment length analysis of the norA gene with the restriction endonuclease SfcI showed a mutation of nucleotide position 1085 in all MRSA strains tested except for one highly quinolone-resistant strain. Thus the mechanisms of 4-quinolone-resistance in these MRSA isolates involved alterations in both DNA gyrase and antimicrobial uptake and efflux.
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PMID:Mechanisms of 4-quinolone resistance in quinolone-resistant and methicillin-resistant Staphylococcus aureus isolates from Japan and China. 788 4

The role of endonuclease and poly(ADP-ribose) polymerase activation in various types of cell injuries and death to rabbit renal proximal tubule suspensions was examined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 microM), the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 1 microM), the calcium ionophore ionomycin (5 microM), or the oxidant t-butyl hydroperoxide (TBHP, 0.5 mM) in the absence or presence of the endonuclease inhibitor aurintricarboxylic acid or the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. Lactate dehydrogenase (LDH) release was used as a marker of cell death and analysis of genomic DNA for internucleosomal cleavage was used as a marker of endonuclease activation. Aurintricarboxylic acid and 3-aminobenzamide had no effect on the proximal tubule LDH release produced by 1 h exposure to antimycin A, FCCP, or ionomycin, or 2 h exposure to TBHP. Furthermore, there was no evidence of DNA fragmentation with any compound prior to or after cell death began. As a positive control, proximal tubules exposed to digitonin in the absence of metabolic substrates resulted in the chelator-inhibitable fragmentation of DNA, indicating that the endonuclease is present in proximal tubules. These results show that endonuclease activation did not occur prior to or after cell death began. Furthermore, these results suggest that endonuclease and poly(ADP-ribose) polymerase activation do not play a role in this model of acute renal proximal tubule cell injury and death induced by agents that cause oxidative stress, mitochondrial dysfunction, or increases in cytosolic free calcium.
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PMID:Absence of endonuclease activation during acute cell death in renal proximal tubules. 839 29

Terminally differentiated PC12 cells are a useful neuron-like model for studying programmed cell death in response to nerve growth factor (NGF) deprivation. This in vitro model was used to investigate the mechanism by which cyanide-induced histotoxic hypoxia produces neuronal degeneration. Treatment of undifferentiated PC12 cells with 0.1 mM KCN for 24 h did not produce cell death. In contrast, treatment of differentiated PC12 cell cultures with 0.1 mM KCN for 24 h increased cell death by 43% when compared with control cultures, as measured by trypan blue dye exclusion and lactate dehydrogenase release assays. The Ca2+/Mg(2+)-dependent endonuclease inhibitor aurintricarboxylic acid and the transcriptional inhibitor actinomycin D partially attenuated hypoxic toxicity, suggesting roles for endonuclease activation and transcription in this model of neuronal death. Extracted DNA from cyanide-treated neurons demonstrated cleavage into oligonucleosomal fragments on gel electrophoresis. Transmission electron microscopic analysis showed morphological changes consistent with apoptotic cell death, including membrane blebbing and convolution, as well as chromatin condensation and margination to the nuclear membrane. Addition of either ascorbate or catalase to the cultures partially attenuated the loss of cell viability induced by cyanide, and decreased the incidence of apoptotic cells after treatment, based on the in situ detection of DNA strand breaks. The ability of cyanide to elevate intracellular oxidant species was determined by microfluorescence in differentiated PC12 cells loaded with the oxidant-sensitive dye 2',7'-dichlorofluorescin. Exposure of cells to 0.1 mM KCN produced a rapid generation of oxidants that was blocked approximately 50% by ascorbate or catalase. These observations indicate that cyanide induces apoptosis in terminally differentiated, and not undifferentiated, PC12 cells, and that antioxidants significantly reduce the incidence of cyanide-induced apoptosis.
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PMID:Cyanide-induced apoptosis and oxidative stress in differentiated PC12 cells. 875 10

Cyanide is a mitochondrial poison and its toxicity is mediated through histotoxic hypoxia. Although cyanide is regarded as a neurotoxin, its other toxic manifestations are also well documented. Cyanide triggers all those events which can lead to DNA damage, but its genotoxic potential has not been established yet. The present investigation addresses the DNA damage induced by cyanide in rat thymocytes in vitro. Cell viability (eosin Y exclusion and LDH leakage) along with DNA strand breaks were measured in thymocytes exposed to 1.25-10 mM KCN for various time intervals. Cleavage into oligonucleosomal fragments of extracted DNA from cyanide treated thymocytes were visualized on gel electrophoresis. Cyanide produced both time and dose dependent DNA fragmentation accompanied by cytotoxicity. The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was minimal in Ca2+ free medium. The DNA fragmentation was attenuated by Zn2+ (modulator of Ca2+/Mg2+-dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). Cyanide induced DNA damage was further observed in baby hamster kidney cells (BHK-21), where unlike thymocytes, internucleosomal DNA fragmentation was not observed. Thymocytes were more sensitive to cyanide as compared to BHK-21 cells.
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PMID:Cyanide induced DNA fragmentation in mammalian cell cultures. 935 39

Electrophoretic mobility shift assay (EMSA) or gel shift assay is one of the most powerful methods for studying protein-DNA interactions. Typically, 32P-labeled DNA probes containing the sequence bound by the protein of interest are used in EMSA (rEMSA). Although rEMSA is sensitive and practicable, it relies on the handling of hazardous radioisotopes, and does not easily allow quantification. We developed a non-radioactive procedure using fluorescence (Cyano dye Cy5) labeled oligodeoxynucleotide duplexes as specific probes (fEMSA) and an automatic DNA sequencer for analysis. Testing different DNA-binding proteins (restriction endonuclease EcoRII, transcription factor NFkappaB and it's subunit p50) the results in fEMSA and rEMSA are similar in regard to quality, reproducibility, and sensitivity. fEMSA allows a semiquantitative screening of large amounts of samples for specific DNA binding activities and is, therefore, a high throughput technology for semiquantitative analysis of DNA-protein interaction.
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PMID:A fluorescence based non-radioactive electrophoretic mobility shift assay. 1072 39

Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxidase causing histotoxic hypoxia. It is primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induced apoptosis in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN) produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was attenuated by Zn2+ (modulator of Ca2+ dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). In a continuation of this work, in the present study we have shown that the cytotoxicity and DNA fragmentation induced by 5 mM KCN was preceded by loss of mitochondrial integrity (MTT assay and rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive oxygen species (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragmentation (Hoechst 33342 staining), a phenomenon that characterises the 'apoptotic' type of cell death. The in vitro toxic insult of KCN was challenged by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5-3 h) of various pharmacological agents viz., Trolox (antioxidant), EGTA (Ca2+ modulator) and aurintricarboxylic acid (ATA; Ca2+/Mg2+ dependent endonuclease inhibitor). In addition, Quercetin (antioxidant) was tested as simultaneous treatment alone and was found to be ineffective. On the basis of various biochemical indices and DNA fragmentation (quantitative and qualitative), simultaneous treatment of Trolox was found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in oxidative stress-mediated cell injury which is an early event preceding DNA damage. Both EGTA and ATA could not prevent this damage. Trolox also increased the LD(50) of KCN in mice 2.5-fold as compared to 1.8- and 1.6-fold for EGTA and ATA, respectively.
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PMID:Pharmacological interventions of cyanide-induced cytotoxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo. 1127 22

Over the past two decades, the incidence of infections due to Candida glabrata, a yeast with intrinsic low susceptibility to azole antifungals, has increased markedly. Respiratory deficiency due to mutations in mitochondrial DNA (mtDNA) associated with resistance to azoles frequently occurs in vitro in this species. In order to specify the relationships between respiration and azole susceptibility, the effects of respiratory chain inhibitors on a wild-type isolate of C. glabrata were evaluated. Respiration of blastoconidia was immediately blocked after extemporaneous addition of potassium cyanide, whereas a 4-h preincubation was required for sodium azide. Antifungal susceptibility determined by a disk diffusion method on Casitone agar containing sodium azide showed a significant decrease in the susceptibility to azoles. Biweekly subculturing on Casitone agar supplemented with sodium azide was therefore performed. This resulted after 40 passages in the isolation of a respiration-deficient mutant, as suggested by its lack of growth on glycerol-containing agar. This respiratory deficiency was confirmed by flow cytometric analysis of blastoconidia stained with rhodamine 123 and by oxygraphy. Moreover, transmission electron microscopy and restriction endonuclease analysis of the mtDNA of mutant cells demonstrated the mitochondrial origin of the respiratory deficiency. Finally, this mutant exhibited cross-resistance to all the azoles tested. In conclusion, blockage of respiration in C. glabrata induces decreased susceptibility to azoles, culminating in azole resistance due to the deletion of mtDNA. This mechanism could explain the induction of petite mutations by azole antifungals which have been demonstrated to act directly on the mitochondrial respiratory chain.
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PMID:Relationships between respiration and susceptibility to azole antifungals in Candida glabrata. 1260 11

A DNAzyme, synthetically modified with both primary amines and imidazoles, is found to act as a M2+ -independent AP lyase-endonuclease. In the course of the cleavage reaction, this DNAzyme forms a covalent Schiff base intermediate with an abasic site on a complementary oligodeoxyribonucleotide. This intermediate, which is inferred from NaCNBH3 trapping as well as cyanide inhibition, does not evidently accumulate because the second step, dehydrophosphorylative elimination, is fast compared to Schiff base formation. The 5'-product that remains linked to the catalyst hydrolyzes slowly to regenerate free catalyst. The use of duly modified DNAzymes to perform Schiff base catalysis demonstrates the value of modified nucleotides for enhancing the catalytic repertoire of nucleic acids. This work suggests that DNAzymes will be capable of catalyzing aldol condensation reactions.
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PMID:Covalent Schiff base catalysis and turnover by a DNAzyme: a M2+ -independent AP-endonuclease mimic. 1505 4


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