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)

Neuronal death, secondary to endogenous agents such as glutamate, may involve changes in cytoplasmic calcium. Besides its well recognized role as a second messenger mediating cellular response, calcium is necessary for the activation of endonuclease(s), resulting in DNA fragmentation and cell death. Therefore, we investigated the relationship between changes in cytoplasmic calcium, DNA fragmentation and neuronal death, using PC12 and NCB-20 cell lines. The calcium ionophore, A23187, caused a dose-dependent increase in cytoplasmic calcium, loss of cell viability, increased lactate dehydrogenase (LDH)-release, and DNA fragmentation. DNA fragments, typical of internucleosomal digestion of genomic DNA, characteristic of endonuclease(s) activation, were consistently detected in the incubating medium. Release of DNA fragments into the medium was seen with A23187 in concentrations as low as 10 nM, and within an hour of treatment. Furthermore, calcium added to preparations of PC12 nuclei also produced DNA fragmentation, although, less pronounced than when intact cells were treated with A23187. The findings indicate that A23187-induced neuronal death involves the activation of endonuclease(s). The role of cytoplasmic calcium in this process is supported by evidence that A23187 selectively mobilizes cytoplasmic calcium, and that calcium can directly activate endonuclease(s) in nuclear preparations.
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PMID:Neuronal death, cytoplasmic calcium and internucleosomal DNA fragmentation: evidence for DNA fragments being released from cells. 838 11

The effect of the endonuclease inhibitor aurintricarboxylic acid (ATA) versus NMDA-mediated delayed cell death was examined in an ex vivo chick retinal preparation. Transient exposure to 100 microM NMDA for 60 min followed by a 24-h recovery period resulted in a sevenfold increase in lactate dehydrogenase (LDH) release into the medium. ATA at 100 microM significantly reduced NMDA-mediated LDH release by 60%. In clarifying the mechanism of protection versus NMDA, ATA was found to inhibit several acute NMDA-mediated effects: ATA attenuated NMDA-mediated GABA release in a dose-dependent manner (IC50 = 29.5 microM), prevented NMDA-stimulated cyclic GMP formation, and blocked NMDA-mediated 22Na+ influx. These acute inhibitory effects of ATA were overcome by increasing the NMDA concentration, which suggested a competitive interaction between NMDA and ATA. In a binding assay using membranes prepared from adult rat forebrain, ATA displaced the competitive NMDA receptor ligand [3H]CGS 19755 with an IC50 of 26.9 microM. Maximal displacement was 88% with 100 microM ATA. These studies demonstrate that ATA protected neurons from NMDA-mediated cell death upstream of endonuclease inhibition, i.e., by antagonizing NMDA receptor activity in a manner consistent with competitive antagonism.
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PMID:Aurintricarboxylic acid prevents NMDA-mediated excitotoxicity: evidence for its action as an NMDA receptor antagonist. 839 May 67

Aurintricarboxylic acid (ATA), an inhibitor of Ca(2+)-dependent endonuclease activity, is often used to implicate a role for increased intracellular calcium in mechanistic toxicology studies. We report here on the ability of ATA to inhibit the activity of several NAD(H)/NADP(H)-requiring enzymes (purified or cellular homogenates), including lactic dehydrogenase, alcohol dehydrogenase, cytochrome c reductase, ethoxycoumarin o-dealkylase, isocitric dehydrogenase, glutathione reductase and glucose-6-phosphate dehydrogenase. These results were compared with the ability of ATA to inhibit micrococcal nuclease and rat liver Ca(2+)-dependent endonuclease activity in similar incubations. With the exception of alcohol dehydrogenase, ATA was a potent inhibitor of each of the purified enzymes, with IC50s ranging from 0.5 to 82 microM. In cell homogenates, however, ATA was from 10 to 100-fold less potent at inhibiting these enzymes. When exogenous protein was added to purified enzyme incubations, the effect of ATA was similarly diminished. Our results demonstrate that ATA inhibits a wide range of NAD(H)/NADP(H)-requiring enzymes in in vitro incubations using purified enzymes, but that the inhibitory effects are markedly reduced in incubations which more closely resemble a cellular milieu.
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PMID:Inhibition of NAD(H)/NADP(H)--requiring enzymes by aurintricarboxylic acid. 855 68

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

Hypoxia is considered to result in a necrotic form of cell injury. We have recently demonstrated a role of endonuclease activation, generally considered a feature of apoptosis, to be almost entirely responsible for DNA damage in hypoxic injury to renal tubular epithelial cells. The role of reactive oxygen metabolites in endonuclease-induced DNA damage and cell death in chemical hypoxic injury has not been previously examined. LLC-PK1 cells exposed to chemical hypoxia with antimycin A resulted in enhanced generation of intracellular reactive oxygen species as measured by oxidation of a sensitive fluorescent probe, 2',7'-dichlorofluorescin diacetate. Superoxide dismutase, a scavenger of superoxide radical, significantly reduced the fluorescence induced by antimycin A and provided significant protection against chemical hypoxia-induced DNA strand breaks (as measured by the alkaline unwinding assay). Pyruvate, a scavenger of hydrogen peroxide, provided significant protection against chemical hypoxia-induced DNA strand breaks and DNA fragmentation (as measured by agarose gel electrophoresis). The interaction between superoxide anion and hydrogen peroxide in the presence of a metal catalyst leads to generation of other oxidant species such as hydroxyl radical. Hydroxyl radical scavengers, dimethylthiourea, salicylate, and sodium benzoate, and two metal chelators, deferoxamine and 1,10-phenanthroline, also provided marked protection against DNA strand breaks and DNA fragmentation. These scavengers of reactive oxygen metabolites and metal chelators provided significant protection against cell death as measured by trypan blue exclusion and lactate dehydrogenase release. Taken together, these data indicate that reactive oxygen species play an important role in the endonuclease activation and consequent DNA damage, as well as cell death in chemical hypoxic injury to renal tubular epithelial cells.
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PMID:Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells. 876 Feb 63

This study has demonstrated the toxicity to human monocyte-macrophages of low-density lipoprotein (LDL) which had been artificially oxidized using copper sulphate. The assays of cell damage used were tritiated adenine release, neutral red staining, lactate dehydrogenase leakage, and MTT dye reduction. Toxicity was concentration- and time-dependent. Exposure to native LDL under the same conditions did not result in toxicity. Transmission electron microscopy of cells exposed to oxidized LDL showed characteristic changes of apoptosis, including chromatin condensation and a decrease in cell volume. There was extensive loss of cell surface protrusions and evidence of the phagocytosis of apoptotic cells by neighbouring monocyte-macrophages. Apoptotic features preceded the increased membrane permeability revealed by the release of radioactivity from cells preloaded with tritiated adenine and by lactate dehydrogenase leakage. DNA fragmentation was indicated by nick end-labelling using the terminal transferase enzyme (TUNEL). The number of TUNEL-positive cells was markedly greater in cells exposed to oxidized LDL, compared with those incubated as no-additions controls. Inhibition of de novo protein synthesis with cycloheximide and of Ca2+/Mg(2+)-activated endonuclease activity with aurintricarboxylic acid or zinc ion did not inhibit the toxicity produced by oxidized LDL.
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PMID:Apoptosis in human monocyte-macrophages exposed to oxidized low density lipoprotein. 877 86

Acetaminophen (N-acetyl-p-aminophenol [APAP]) hepatotoxicity is a process characterized by Ca2+ deregulation. Cellular functions utilizing Ca2+ as a second messenger molecule affect both cytosolic and nuclear signal transduction. Many studies have independently shown Ca2+-related effects on target molecules in response to toxic doses of APAP; however, the primary Ca2+ target resulting in liver necrosis has not been determined. We hypothesize that Ca2+-dependent DNA damage is a critical event in liver necrosis caused by alkylating hepatotoxins. In this study, Ca2+-dependent endonuclease activity was determined from DNA single-strand lesions measured by fluorometric analysis of DNA unwinding. The status of cytosolic Ca2+ was determined by measuring Ca2+-dependent activation of glycogen phosphorylase a. Primary cultures of mouse hepatocytes exposed to a toxic concentration of APAP showed twofold and greater increases in glycogen phosphorylase a stimulation at 6 hours, which was reversible with Ca2+-chelating agents. Cell death was preceded by a large decline in intact, double-stranded DNA. Following toxic administration of APAP, the percentage of total double-stranded DNA was significantly reduced by 2 hours. At 6 and 24 hours, genomic integrity was compromised by 26% and 37%, respectively, compared with untreated controls. Hepatotoxic effects of APAP-mediated Ca2+ deregulation were confirmed in both primary mouse hepatocytes and the human hepatoblastoma HepG2 cell line by lactate dehydrogenase (LDH) release and tetrazolium reduction using the 3-4,5-dimethylthiazole-2-yl-2,5-diphenyltetrazolium bromide thiazol blue(MTT) assay. The Ca2+ chelator, ethylene glycol-bis (beta-aminoethyl ether) N',N',N', N'-tetraacetic acid (EGTA), blocked APAP-induced phosphorylase a activation and necrotic cell death, but failed to inhibit phosphorylase a activation by the adenosine 3',5'-cyclic monophosphate (cAMP) analogue, dibutyryl cAMP, indicating little or no contribution of the cAMP pathway to phosphorylase a stimulation during APAP-induced necrotic death. Results with these in vitro models of liver injury are interpreted as supporting the hypothesis that increased Ca2+ availability plays a major role in the progression of APAP-dependent cellular necrosis, and that the nucleus is a critical target for APAP hepatotoxicity.
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PMID:Calcium-dependent DNA damage and adenosine 3',5'-cyclic monophosphate-independent glycogen phosphorylase activation in an in vitro model of acetaminophen-induced liver injury. 918 64

Thapsigargin, a specific inhibitor of the endoplasmic reticular Ca(2+)-ATPase, has been used previously to mobilize calcium release from intracellular calcium stores. We now show that thapsigargin (1-10 microM) induces apoptosis in a neuroblastoma cell line (SH-SY5Y) and in fetal rat cerebrocortical cultures. Cell death measured by lactate dehydrogenase release was observed 24-48 hours after treatment with thapsigargin. In both cases, DNA extracts from thapsigargin treated cells showed laddering, typical of endonuclease-mediated internucleosomal cleavages. The presence of DNA fragments was also confirmed by an ELISA designed for detecting nucleosomes in apoptotic cells. Cycloheximide reduced the extent of DNA fragmentation and injury in thapsigargin-treated cells. Dantrolene, an inhibitor of calcium release from intracellular stores partially abolished the effect of thapsigargin, suggesting that the initial Ca2+ rise may be the signalling event in this apoptotic cell death pathway. We propose that thapsigargin-induced cell death in cultured neuronal cells may be a useful system to study the molecular and genetic events involved in apoptosis.
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PMID:Thapsigargin induces apoptosis in SH-SY5Y neuroblastoma cells and cerebrocortical cultures. 931 98

The homogenate from Caco-2 cells of day 13 or 15 after subculturing had high omega-hydroxylation activity of docosahexaenoic acid (22:6(n-3)) or arachidonic acid (20:4(n-6)). Activity, maximal at pH 8.0, was inhibited in the presence of CO or metyrapone and in the absence of NADPH. Omega-hydroxylation activity of lauric acid in the homogenate was not detected. Apparent Michaelis constant (Km) values for 22:6(n-3) and 20:4(n-6) were found to be 4 and 7 microM. Omega-hydroxylation activity considerably increased with growth up to day 13 and then decreased until day 20 even though alkaline phosphatase (ALP) and leucine-aminopeptidase (LAP) activity increased with growth to day 20. Metyrapone in cultures caused omega-hydroxylation, ALP and LAP activity to decrease, while sodium butyrate dose-dependently increased that of omega-hydroxylation, ALP and an endogenous endonuclease and decreased lactate dehydrogenase (LDH) activity in culture medium. The omega-hydroxylation system thus appears quite likely to be associated with cytochrome P450, differentiation and/or apoptosis rather than cytotoxic cell death of Caco-2 cells. Substrate specificity, however, differed from that of human cytochrome P450 4A11.
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PMID:Docosahexaenoic/arachidonic acid omega-hydroxylation system and differentiation in the human colonic adenocarcinoma cell line, Caco-2. 946 91

We compared neurotoxicity of piperine and low K+ on cultured cerebellar granule neurons. As considered from lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide reduction, both piperine and shifting from high K+ (25 mM) to low K+ (5.4 mM) were cytotoxic to cerebellar granule neurons. Protein synthesis inhibitors, cycloheximide and anisomycin, and an endonuclease inhibitor, aurintricarboxylic acid, were protective against low K+-induced neuronal death whereas they were ineffective against that induced by piperine. D-alpha-tocopherol, trolox, and a spin trap 3,3,5,5-tetramethyl-1-pyrroline-1-oxide were protective against piperine neurotoxicity whereas they had no effect on that induced by low K+. These results suggest that piperine and low K+ may exert neurotoxic effects on cerebellar granule neurons through different mechanisms. Death of cerebellar granule neurons induced by piperine may be mediated by non-apoptotic mechanisms and may involve membrane lipid peroxidation and/or free radical generation.
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PMID:Death of cerebellar granule neurons induced by piperine is distinct from that induced by low potassium medium. 948 73


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