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:1.17.3.2 (xanthine oxidase)
8,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Light-emitting chemical reactions (chemiluminescence, CL) and biological reactions (bioluminescence, BL) have a diverse range of analytical applications but relatively few have been adopted by routine clinical laboratories. Advantages of CL and BL assays include sensitivity (attomole and sub-attomole detection limits), speed (signal generated in a few seconds and in some cases stable for several hours), nonhazardous reagents, and simple procedures. The most promising clinical applications are in immunoassay, protein blotting, and DNA probe assays. Chemiluminescent molecules exploited as labels include luminol, isoluminol, acridinium esters, thioesters and sulfonamides, and phenanthridinium esters. Separation and nonseparation assays have been devised, based on isoluminol and acridinium ester labels. The combination of the amplification properties of an enzyme and a CL or BL detection reaction provides a highly sensitive analytical system. Since 1983, CL and BL methods have been developed for many enzyme labels, e.g., alkaline phosphatase, glucose-6-phosphate dehydrogenase, horseradish peroxidase, Renilla luciferase, and xanthine oxidase. Currently, the most successful enzyme assays are the enhanced CL method for a peroxidase label involving a mixture of luminol, hydrogen peroxide, and an enhancer (e.g., p-iodophenol) and the direct CL method for alkaline phosphatase, with an adamantyl 1,2-dioxetane phenyl phosphate as substrate. Both systems are very sensitive (the detection limit for alkaline phosphatase when using the dioxetane reagent is 0.001 amol) and produce long-lived light emission (greater than 30 min), which is ideal for membrane applications in which light emission is detected with photographic film or a charge-coupled device camera.
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PMID:Chemiluminescent and bioluminescent techniques. 189 71

The potential role of oxidative stress conditions in the induction of heat shock proteins was studied in human umbilical vein endothelial cells. We compared the effects of temperature (43 to 45 degrees C), exposure to hydrogen peroxide (H2O2) and oxygen metabolites generated by the enzyme system hypoxanthine-xanthine oxidase (O2- plus H2O2), as well as exposure to 95% O2, on the expression of the major 70-kD heat shock proteins (hsp70). Northern blot analysis indicated that: (1) heat shock induced a rapid and marked increase in hsp70 mRNA levels that reached a maximum during recovery from a 30-min exposure to 45 degrees C; (2) treatment with a 5-mM H2O2 bolus or 50 mU/ml xanthine oxidase also increased hsp70 mRNA levels but to a lesser extent than heat shock (about 10 and 25 times less, respectively); (3) no change was detected after a 5-day exposure to 95% O2. Nuclear run on transcription data and kinetics of mRNA decay in the presence of actinomycin D indicated that the observed increase in hsp70 mRNA levels in both heat-shocked and H2O2-treated cells was mainly due to a transcriptional induction. The kinetics of hsp70 synthesis correlated with the accumulation of hsp70 mRNA. Two-dimensional gel electrophoresis and immunologic analysis of these heat shock proteins revealed a series of at least five distinct hsp70 isoforms induced in heat-shocked cells, whereas only a specific subset of these proteins, mainly one acidic isoform, was induced in very low amounts in response to H2O2 treatment. These results clearly indicate that the endothelial cell responses to oxidative stress and heat shock differ in both qualitative and quantitative terms in respect to hsp70 induction. They also suggest that the intensity of this response to oxidative stress conditions may vary depending on the nature of the oxidative challenge.
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PMID:Differential expression of hsp70 stress proteins in human endothelial cells exposed to heat shock and hydrogen peroxide. 191 Aug 12

Interactions between rat pulmonary artery endothelial cells and hydrogen peroxide or toxic oxygen products from phorbol ester-activated human neutrophils result in endothelial cell killing defined by 51Cr release. It has been shown that this cytotoxic reaction can be blocked by the presence of catalase, iron chelators, or scavengers of the hydroxyl radical. Evidence shows that products from xanthine oxidase of endothelial cells are necessary for the toxic effects of hydrogen peroxide or phorbol ester-activated neutrophils. Addition of xanthine oxidase inhibitors protects against phorbol ester-mediated injury of endothelial cells. Preloading of endothelial cells with superoxide dismutase attenuates injury caused either by hydrogen peroxide or phorbol ester-activated neutrophils. Conversion of xanthine dehydrogenase to xanthine oxidase in endothelial cells occurs during contact of endothelial cells by activated neutrophils. This conversion is not related to oxygen products of neutrophils. Conversion of xanthine dehydrogenase to xanthine oxidase in endothelial cells is also induced by endothelial cell contact with C5a, N'-formyl-methionyl-leucyl-phenylalanine (fMLP), or tumor necrosis factor alpha (TNF alpha). Interaction of hydrogen peroxide with endothelial cells rapidly depletes adenosine triphosphate (ATP) and causes the extracellular appearance of xanthine and hypoxanthine. Agents that protect endothelial cells from the toxic effects of hydrogen peroxide do not prevent falls in cellular ATP caused by hydrogen peroxide, indicating that ATP levels do not necessarily correlate with cytotoxic events. A synergy between hydrogen peroxide and proteases in endothelial cell killing has been demonstrated. TNF alpha causes alterations in endothelial cells, the result of which is increased susceptibility to killing by PMA-activated neutrophils.
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PMID:Mechanisms of endothelial cell killing by H2O2 or products of activated neutrophils. 192 18

Selected immunotherapies (tumor necrosis factor, interleukin-1, interleukin-2, and gamma interferon), chemotherapeutic agents (mitomycin, platinum, doxorubicin [Adriamycin], and bleomycin), and radiation therapy have been described to exert cytotoxicity through the generation of reactive oxygen species, including superoxide and hydrogen peroxide. Tumor necrosis factor, however, has been shown to impart increased resistance in vitro and in vivo against reactive oxygen species stress, including radiation therapy and oxygen toxicity, possibly because of the induction of increased cellular buffering capacities. It is unknown whether the sensitivity of a lung cancer cell to reactive oxygen species therapy is altered by tumor necrosis factor through the induction of free radical scavenging enzymes such as manganese superoxide dismutase. This question was investigated as follows: A549 lung adenocarcinoma cells, exposed for 24 hours to 0, 0.1, 1.0, or 10 micrograms/ml concentrations of tumor necrosis factor, were exposed to hypoxanthine plus xanthine oxidase, a superoxide generating system, for varying intervals. The number of cells surviving 5 days after the stress was determined, and cells exposed to tumor necrosis factor were examined by Northern Blot analysis for induction of the manganese superoxide dismutase gene. The hypoxanthine-xanthine oxidase stress alone caused a time-dependent decrease in survival; however, pretreatment with tumor necrosis factor increased cell survival significantly. Moreover, the cells exposed to tumor necrosis factor had a fivefold increase in the number of manganese superoxide dismutase transcripts. These findings suggest that tumor necrosis factor may confer resistance of lung cancer cells to subsequent reactive oxygen species-based therapies, and the resistance of these cells may be due to increased expression of manganese superoxide dismutase. Clinical treatment failures may result, especially if tumor necrosis factor is given concurrently with other therapies.
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PMID:Tumor necrosis factor-alpha alters response of lung cancer cells to oxidative stress. 196 Sep 95

Single smooth muscle cells were isolated from the basilar artery of the rat by enzymatic dispersion. The membrane properties of the cells were assessed using the patch-electrode voltage-clamp technique, and cell viability was monitored using fluorescein diacetate uptake. Exposure of the cells to oxyhemoglobin (5 microM) resulted in 1) contraction, 2) the appearance of membrane blebs, 3) an increase in the outward potassium currents, 4) a decrease in the membrane resistance, and 5) cell death. In contrast, no effect of oxyhemoglobin on cultured murine neuroblastoma cells was observed. Methemoglobin (100 microM) had no effects on the smooth muscle cells. Catalase (300 units/ml) or dimethyl sulfoxide (0.5%) protected against the effects of oxyhemoglobin; superoxide dismutase (100-1,000 units/ml) provided only partial protection. Exposure of the cells to superoxide anions generated by xanthine (1 mM) plus xanthine oxidase (10 units/l) or to hydrogen peroxide (500 microM) caused an increase in the outward potassium currents without affecting membrane resistance. Generation of hydroxyl radicals by metal ions plus hydrogen peroxide caused the same effects as oxyhemoglobin, that is, an increase in the potassium currents, followed by a decrease in the membrane resistance and cell death. In conclusion, it appears that oxyhemoglobin exerts its effects on vascular smooth muscle cells by the generation of free radicals, chiefly hydroxyl radicals.
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PMID:Free radicals mediate actions of oxyhemoglobin on cerebrovascular smooth muscle cells. 199 46

Reoxygenation injury that occurs when blood circulation is restored to previously ischemic tissues is currently discussed as a pathophysiological entity distinct from the primary anoxic injury that develops during ischemia per se. To test the hypothesis that reoxygenation injury in hepatocytes is caused by a postischemic burst of reactive oxygen species (ROS), including superoxide radicals, O2-., and hydrogen peroxide, H2O2, we performed a cytochemical study exploiting the peroxidase activity within peroxisomes as a sensitive ultrastructural detector of intracellular H2O2 generation. The osmiophilic polymer formed when tissue peroxidase is incubated with 3,3'-diaminobenzidine (DAB) and H2O2 was used as a marker for endogenous H2O2 in rat liver slices in short-term organ culture subjected to a cycle of 60-min ischemic anoxia and 30-min reoxygenation in the presence of DAB without exogenous H2O2. Peroxisomal reaction product was quantitatively evaluated in transmission electron micrographs of systematically sampled hepatocytes. Mean densities of positive peroxisomes per 1,000 micron2 (+/- SE) in liver slices subjected to various treatments were as follows: continuous anoxia (negative control) 0 +/- 0; normoxia + exogenous H2O2 (positive control) 45 +/- 12; normoxia only 26 +/- 2; ischemia-reoxygenation 13 +/- 6; ischemia-reoxygenation + xanthine oxidase inhibitor, oxypurinol 5 +/- 3; ischemia-reoxygenation + peroxidase inhibitor, aminotriazole 7 +/- 3. Endogenous H2O2 can be detected in hepatocytes by electron microscopic cytochemistry and may in part derive from xanthine oxidase, but it is not substantially increased in the postischemic state. We conclude that hepatocytes do not exhibit a postischemic burst of reactive oxygen species that could cause reoxygenation injury.
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PMID:Cytochemical studies of hydrogen peroxide generation in postischemic hepatocytes. 199 89

Using a highly specific assay that minimizes enzyme inactivation in vitro, we found that rabbit myocardial tissue contained low levels of xanthine oxidase (XO) and xanthine dehydrogenase (XD) activity that were effectively inhibited by pretreatment of hearts with allopurinol. In parallel, allopurinol treatment also improved ventricular developed pressure, peak systolic pressure, and coronary flow in isolated hearts subjected to 30 min of normothermic global ischemia and 30 min of reperfusion. Although function was protected by allopurinol treatment, creatine kinase (CK) release was not altered by allopurinol. Inhibition of myocardial XO with allopurinol did not increase myocardial ATP or phosphocreatine. In addition, allopurinol did not scavenge superoxide anion or hydrogen peroxide in vitro. The results support the possibility that relatively low amounts of XO activity, similar to levels reported in human myocardium, may contribute to cardiac ischemia-reperfusion injury.
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PMID:Existence and participation of xanthine oxidase in reperfusion injury of ischemic rabbit myocardium. 200 Sep 75

Conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the toxic reactions of subsequent XO-derived superoxide, hydrogen peroxide and hydroxyl radical, have been suggested to be critical factors in several mechanisms of tissue pathophysiology. In the lung, intracellular XO-derived products may modulate type II pneumocyte surfactant turnover and barrier function, jeopardizing the pulmonary air-blood barrier. We characterized total cellular XDH/XO enzymatic activity in freshly isolated and cultured rat pulmonary type II epithelial cells. Type II cells were isolated and cultured on fibronectin-pretreated dishes, with a plating efficiency after 36 h in culture of 40% or 14% when quantified via cellular protein or DNA, respectively. Over the subsequent 96 h in culture, monolayer DNA was unchanged, whereas protein per cell increased continuously. Alterations in different cellular enzymatic activities were also detected in these cultured cells. In culture, total cellular XDH/XO and catalase activities decreased in a logarithmical fashion with respect to time, whether normalized for cellular protein or DNA. The rate of loss of these enzymes was greatest when normalized for cell protein, but was also significant when the activities were normalized for DNA. When compared to freshly isolated type II cells, catalase and total XDH/XO activities normalized for protein decreased 78% and 72%, respectively, during the first 36 h of culture. After 132 h in culture, XDH/XO and catalase activities normalized for protein decreased 93% and 84%, respectively, when compared to freshly isolated cell values. Total cellular XDH/XO activity in the oxidase form (% XO) was initially 31% in freshly isolated type II cells and increased to 67% during the 132 h culture period. In contrast to the loss of total cellular XDH/XO and catalase, no significant change in lactate dehydrogenase (LDH) activity occurred during culture of the type II cells. In type II cells the conversion of XDH to XO, the cytotoxic potential of XO, and the activity of the hydrogen peroxide scavenger, catalase, is expected to be strongly influenced by in vitro culture. Thus, strong consideration should be made before transposing information obtained from cultured type II cells to in vivo situations.
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PMID:Characterization of cultured alveolar epithelial cell xanthine dehydrogenase/oxidase. 200 13

Free radicals may arise from a number of sources as a result of a variety of cellular mechanisms; they are generated under both normal and pathological circumstances. The xanthine oxidase pathway, the arachidonic acid pathway, invading leucocytes, catecholamine oxidation, and mitochondrial activity can all lead to the production of a variety of reactive oxygen intermediates including superoxide, hydrogen peroxide, and the hydroxyl radical. Whatever their source, free radicals can be extremely toxic to the cell and they are capable of causing major membrane injury by initiating lipid peroxidation or by altering the activity of membrane-bound enzyme systems which control ionic movement. The cell possesses highly efficient protective mechanisms, including antioxidants such as vitamins C and E and the enzymes superoxide dismutase and catalase, all of which are designed to prevent the occurrence of free radical-induced injury under normal conditions. However, during ischaemia and reperfusion, these protective mechanisms may be overwhelmed and severe free radical-mediated injury may occur. Ischaemia may prime the myocardium for free radical-induced injury. The great majority of the evidence that manipulation of free radicals may protect against such injury is, however, circumstantial.
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PMID:Free radicals and the heart. 202 51

From in vitro studies involving multilamellar liposomes or other artificial systems, several groups of workers have deduced that Trolox (a water-soluble analogue of vitamin E) and ascorbate are synergistic antioxidants. Here, we demonstrate that while Trolox and ascorbate individually protect cultured hepatocytes against oxyradicals generated either with xanthine oxidase plus hypoxanthine or with hydrogen peroxide, the two antioxidants do not appear to be synergistic when used in equimolar combinations. Also, in a rat model of hepatic ischemia-reperfusion, we observed that infusion of Trolox or ascorbate (7.5-10 mumol/kg body weight) into the postischemic liver reduced the reperfusion injury by 76 or 67%, respectively. However, when both compounds were used together (each at the same dose as used separately), the organ salvage amounted to only 79%. Therefore, there is no evidence of synergism between Trolox and ascorbate in our in vitro and especially in vivo systems.
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PMID:Trolox and ascorbate: are they synergistic in protecting liver cells in vitro and in vivo? 203 21


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