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:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oxidants are generated in vivo by multiple mechanisms, including stimulation of leukocytes, hyperoxia, metabolism of arachidonic acid, and the activation of various oxidases. When the biochemical defences to the oxidants are inadequate, injury of tissues results. This injury was observed in rabbits and rhesus monkeys when pulmonary inflammation was induced with phorbol esters or formylated peptide given intrabronchially. We have recently investigated metabolic changes in various cells exposed to oxidants that are generated from stimulated leukocytes, including H2O2, O2, and HOCl. The target cells used were P388D1 murine macrophage-like tumour cells, human peripheral lymphocytes, GM 1380 human fibroblasts and rabbit alveolar macrophages. The oxidants used were H2O2 and PMA stimulated PMNs or neutroplasts. Lysis could only be prevented when catalase was added within the first 30-40 min of H2O2 exposure indicating that early metabolic changes determined the fate of the cell. Within seconds after the addition of H2O2 to P388D1 cells activation of the hexose monophosphate shunt (HMPS) was observed indicative of increased glutathione cycle activity. At the same time DNA strand breaks (determined by an alkaline unwinding technique) were detected. They resulted in the activation of the DNA repair enzyme poly-ADP-ribose polymerase (pADP-RP) within minutes after the addition of H2O2. At the same time ATP and NAD (the substrate of pADP-RP) concentrations dropped and nicotinamide accumulated extracellularly. 10-15 min after oxidant exposure free intracellular Ca++ concentrations determined by Quin 2 fluorescence started to increase due to release from intracellular stores.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Oxidant and protease injury of the lung. 369 17

Recent studies indicate that arsenic may generate reactive oxygen species to exert its toxicity. However, the mechanism is still unclear. In this study, we demonstrate that arsenite is able to induce apoptosis in a concentration- and time-dependent manner; however, arsenate is unable to do so. An increase of intracellular peroxide levels was accompanied with arsenite-induced apoptosis, as demonstrated by flow cytometry using DCFH-DA. N-Acetyl-L-cysteine (a thiol-containing antioxidant), diphenylene iodonium (an inhibitor of NADPH oxidase), 4,5-dihydro-1,3-benzene disulfonic acid (a selective scavenger of O2-), and catalase significantly inhibit arsenite-induced apoptosis and intracellular fluorescence intensity. In contrast, allopurinol (an inhibitor of xanthine oxidase), indomethacin (an inhibitor of cyclooxygenase), superoxide dismutase, or PDTC had no effect on arsenite-induced cell death. Activation of CPP32 activity, PARP (a DNA repair enzyme) degradation, and release of cytochrome c from mitochondria to the cytosol are involved in arsenite-induced apoptosis, and Bcl-2 antagonize arsenite-induced apoptosis by a mechanism that interferes in the activity of CPP32. These results lead to a working hypothesis that arsenite-induced apoptosis is triggered by the generation of hydrogen peroxide through activation of flavoprotein-dependent superoxide-producing enzymes (such as NADPH oxidase), and hydrogen peroxide might play a role as a mediator to induce apoptosis through release of cytochrome c to cytosol, activation of CPP32 protease, and PARP degradation.
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PMID:Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. 976 29

Pseudomonas aeruginosa possesses an extensive armament of genes involved in oxidative stress defense, including katB-ankB, ahpB, and ahpC-ahpF. Transcription of these genes was regulated in response to H(2)O(2), paraquat, or organic peroxides. Expression of katB-lacZ and the observed KatB catalase levels in P. aeruginosa PAO1 were induced up to 250-fold after exposure to oxidative stress-generating compounds. Also, ahpB-lacZ and ahpC-lacZ expression was 90- and 3-fold higher, respectively, upon exposure to paraquat. The dose- and time-response curves revealed that 1 microM paraquat was sufficient for half-maximal activation of each reporter fusion within 5 min of exposure. Expression of these genes was not observed in a DeltaoxyR mutant, indicating that OxyR was essential for this response. The transcriptional start sites of katB-ankB, ahpB, and ahpC-ahpF were mapped, putative OxyR-binding sites were identified upstream of the -35 promoter elements, and direct binding of purified OxyR protein to these target promoters was demonstrated. The oxyR mutant was hypersusceptible to oxidative stress-generating agents, including H(2)O(2) and paraquat, in spite of total KatA catalase activity being comparable to that of the wild type. The oxyR phenotype was fully complemented by a plasmid containing the oxyR gene, while any of the katB, ahpB, or ahpCF genes alone resulted in only marginal complementation. Increased katB-lacZ expression and higher KatB catalase levels were detected in a DeltaahpCF background compared to wild-type bacteria, suggesting a compensatory function for KatB in the absence of AhpCF. In P. aeruginosa, oxyR is located upstream of recG, encoding a putative DNA repair enzyme. oxyR-lacZ and recG-lacZ reporter activities and oxyR-recG mRNA analysis showed that oxyR and recG are organized in an operon and expressed constitutively with regard to oxidative stress from a single promoter upstream of oxyR. Mutants affected in recG but not oxyR were dramatically impaired in DNA damage repair as measured by sensitivity to UV irradiation. In conclusion, we present evidence that the oxyR-recG locus is essential for oxidative stress defense and for DNA repair.
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PMID:Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF. 1091 87

To test the hypothesis that asbestos-mediated cell injury is mediated through an oxidant-dependent mitochondrial pathway, isolated mesothelial cells were examined for mitochondrial DNA damage as determined by quantitative PCR. Mitochondrial DNA damage occurred at fourfold lower concentrations of crocidolite asbestos compared with concentrations required for nuclear DNA damage. DNA damage by asbestos was preceded by oxidant stress as shown by confocal scanning laser microscopy using MitoTracker Green FM and the oxidant probe Redox Sensor Red CC-1. These events were associated with dose-related decreases in steady-state mRNA levels of cytochrome c oxidase, subunit 3 (COIII), and NADH dehydrogenase 5. Subsequently, dose-dependent decreases in formazan production, an indication of mitochondrial dysfunction, increased mRNA expression of pro- and antiapoptotic genes, and increased numbers of apoptotic cells were observed in asbestos-exposed mesothelial cells. The possible contribution of mitochondrial-derived pathways to asbestos-induced apoptosis was confirmed by its significant reduction after pretreatment of cells with a caspase-9 inhibitor. Apoptosis was decreased in the presence of catalase. Last, use of HeLa cells transfected with a mitochondrial transport sequence targeting the human DNA repair enzyme 8-oxoguanine DNA glycosylase to mitochondria demonstrated that asbestos-induced apoptosis was ameliorated with increased cell survival. Studies collectively indicate that mitochondria are initial targets of asbestos-induced DNA damage and apoptosis via an oxidant-related mechanism.
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PMID:Asbestos induces mitochondrial DNA damage and dysfunction linked to the development of apoptosis. 1290 82

Many studies suggest green tea is a cancer chemopreventive agent. This effect has been attributed to its major constituent (-)-epigallocatechin-3-gallate (EGCG). EGCG is also observed to have cytotoxic anticancer effects, especially when used in combination with certain chemotherapeutic agents. The biochemical actions of EGCG in chemoprevention and anticancer effects have been studied; however, the mechanisms of action are not clearly understood. We show here by expression genomics the effects of EGCG (25 micromol/L) in the Ha-ras gene transformed human bronchial epithelial 21BES cells. We found induction of temporal changes in gene expression and the coalescence of specific genetic pathways by EGCG. In this experimental system, hydrogen peroxide (H2O2) was produced. By treating cells with EGCG in the presence or absence of catalase, we further distinguished gene expression changes that are mediated by H2O2 from those that are H2O2 independent. Many genes and cellular pathways, including genes of the transforming growth factor-beta signaling pathway, were H2O2 dependent because the effects were abolished by catalase. Gene expression changes that were not affected by catalase included those of the bone morphogenetic protein signaling pathway, peptidylprolyl isomerase (cyclophilin)-like 2, alkylated DNA repair enzyme alkB, polyhomeotic-like 2, and homeobox D1. We show further that EGCG and H2O2 differentially transactivated the bone morphogenetic protein and the transforming growth factor-beta response element promoter reporters, respectively, thus confirming results from DNA microarray analysis. The elucidation of gene expression changes between H2O2-dependent and H2O2-independent responses helps us better understand the cancer chemopreventive and anticancer actions of EGCG.
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PMID:Gene expression changes induced by green tea polyphenol (-)-epigallocatechin-3-gallate in human bronchial epithelial 21BES cells analyzed by DNA microarray. 1536 3

(+)-Catechin possesses a broad range of pharmacological properties, including antioxidative effect. However, little is reported on the mechanism by which (+)-catechin protects microglia cells from DNA damage by oxidative stress. In this study, TUNEL assay and DNA electrophorysis indicated that (+)-catechin markedly blocked DNA fragmentation and apoptosis of microglia cells by tBHP exposure. A potent antioxidative effect of (+)-catechin was confirmed by comparison with a putative antioxidant agent, N-acetylcysteine at the lower doses. Furthermore, the increased intracellular ROS by tBHP exposure were scavenged by elevated activities of catalase (CAT) and superoxide dismutase (SOD) after (+)-catechin treatment. (+)-Catechin partially inhibited the activation of caspase-3, thereby both cleavage of poly (ADP-ribose) polymerase (PARP) and degradation of inhibitor of caspase-activated DNase (ICAD) were effectively abolished. In addition, the expression of PARP for repair of impaired DNA was significantly increased by (+)-catechin treatment. Taken together, these data suggest that protective effects of (+)-catechin against oxidative DNA damage of microglia cells is exerted by the increased expression of DNA repair enzyme PARP and antioxidant enzyme activities.
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PMID:Elevated levels of DNA repair enzymes and antioxidative enzymes by (+)-catechin in murine microglia cells after oxidative stress. 1675 84

This report describes a novel and efficient method for walking the sequence of a genomic deoxyribonucleic acid (DNA) from a known region to an unknown region based on an oligodeoxynucleotide (oligo) cassette-mediated polymerase chain reaction technique. In this method, genomic DNA is digested by a restriction enzyme that generates a sticky 5'-end, followed by ligation of a one-base excess oligo-adaptor using T4 DNA ligase. The adaptor consists of two complementary oligos that form the same sticky end as the digested genomic DNA fragments, except that the 5'-overhang base overlaps the corresponding 3'-end base of the restriction site. This overhanging terminal base prevents ligation between the adaptors, and the appropriate molar ratio of adaptor to genomic DNA enables specific amplification of the target sequence. T4 DNA ligase catalyzes both the ligation of the phosphorylated overhang base of the adaptor to genomic DNA and the excision of the corresponding 3'-terminal base of the genomic DNA. This sequence-specific exonuclease activity of T4 DNA ligase was confirmed by ligation of an alternative adaptor in which the 5'-terminal base was not consistent with the corresponding 3'-terminal base. Using this technique, the 3'- and 5'-flanking sequences of the catalase gene of the ciliate Paramecium bursaria were determined.
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PMID:One-base excess adaptor ligation method for walking uncloned genomic DNA. 1807 44

trans-Resveratrol (3,4',5-trihydroxystilbene; RES), a polyphenol found in particularly high concentrations in red wine, has recently attracted intense interest for its potentially beneficial effects on human health. Here, we report the effects of long-term exposure to micromolar concentrations of RES on antioxidant and DNA repair enzyme activities in a human cell line (MRC-5). RES had either no effect on, or reduced the activities of glutathione peroxidase, catalase and CuZn superoxide dismutase (SOD), in treatments lasting up to 2 weeks. RES failed to induce activities of the DNA base excision repair enzymes apurinic/apyrimidinic endonuclease and DNA polymerase beta. However, it dramatically and progressively induced mitochondrial MnSOD expression and activity. Two weeks exposure to RES increased MnSOD protein level 6-fold and activity 14-fold. Thus, long-term exposure of human cells to RES results in a highly specific upregulation of MnSOD, and this may be an important mechanism by which it elicits its effects in human cells.
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PMID:Molecular mechanisms of oxidative stress resistance induced by resveratrol: Specific and progressive induction of MnSOD. 1816 10

The groundwater arsenicals have brought dreadful misery for the people residing in the endemic regions of West Bengal, India. Arsenic-related anomalies include arsenicosis, hyperkera-tosis, gastric complications, liver fibrosis, peripheral neuropathy, and cancer. Some of these diseases have been frequently associated with overproduction of reactive oxygen species that cause DNA damage and improper functioning of body's antioxidant defense mechanism. Natural polyphenols present in tea serve as excellent antioxidants. In the present study, an attempt has been made to elucidate the role of representative polyphenols and extracts of green and black tea in modulating sodium arsenite (As III)-induced DNA damage in normal human lymphocytes. Comet assay was used to detect the DNA damage. Arsenic-induced oxidative stress was measured with generation of reactive oxygen species, lipid peroxidation, and activity of some antioxidant enzymes. Expression of some repair enzymes such as poly(ADP-ribose) polymerase and DNA polymerase beta was measured to assess the effect of tea on DNA repair. Tea afforded efficient reduction of As III-induced DNA damage in human lymphocytes. Tea also quenched the excessive production of reactive oxygen species by arsenic, reduced the elevated levels of lipid peroxidation, and increased the activity of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase. Furthermore, tea enhanced recovery of DNA damage, which was indicative of repair as confirmed by unscheduled DNA synthesis and pronounced expression of DNA repair enzyme poly(ADP-ribose) polymerase. It is speculated that the antioxidant potential and repair-inducing capacity of tea might help in combating the severe genotoxic effects induced by arsenic in the human population.
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PMID:In vitro mitigation of arsenic toxicity by tea polyphenols in human lymphocytes. 1819 36

Artificial selection in rat has yielded high-capacity runners (HCR) and low-capacity runners (LCR) that differ in intrinsic (untrained) aerobic exercise ability and metabolic disease risk. To gain insight into how oxygen metabolism may have been affected by selection, we compared mitochondrial function, oxidative DNA damage (8-dihydroxy-guanosine; 8dOHG), and antioxidant enzyme activities in soleus muscle (Sol) and gastrocnemius muscle (Gas) of adult and aged LCR vs. HCR rats. In Sol of adult HCR rats, maximal ADP-stimulated respiration was 37% greater, whereas in Gas of adult HCR rats, there was a 23% greater complex IV-driven respiratory capacity and 54% greater leak as a fraction of electron transport capacity (suggesting looser mitochondrial coupling) vs. LCR rats. H(2)O(2) emission per gram of muscle was 24-26% greater for both muscles in adult HCR rats vs. LCR, although H(2)O(2) emission in Gas was 17% lower in HCR, after normalizing for citrate synthase activity (marker of mitochondrial content). Despite greater H(2)O(2) emission, 8dOHG levels were 62-78% lower in HCR rats due to 62-96% higher superoxide dismutase activity in both muscles and 47% higher catalase activity in Sol muscle in adult HCR rats, with no evidence for higher 8 oxoguanine glycosylase (OGG1; DNA repair enzyme) protein expression. We conclude that genetic segregation for high running capacity has generated a molecular network of cellular adaptations, facilitating a superior response to oxidative stress.
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PMID:Lower oxidative DNA damage despite greater ROS production in muscles from rats selectively bred for high running capacity. 2114 74


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