Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.17.3.2 (xanthine oxidase)
 8,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Here we report a method to determine superoxide scavenging efficiency, using kinetic analysis of cytochrome c reduction and an automated UV/vis microtiter plate reader. Superoxide (O(2)(-&z. rad;)) was generated by xanthine oxidase metabolism of hypoxanthine, and quantified by following reduction of cytochrome c by O(2)(-&z. rad;) as increasing absorbance at 550 nm. Reaction conditions were established that provided a linear increase in O(2)(-&z.rad;) generation for more than 20 min, and good reproducibility over time. The majority of cytochrome c reduction was blocked by superoxide dismutase, indicating cytochrome c reduction derived predominantly from O(2)(-&z.rad;). Although EDTA is commonly included in this assay to eliminate undesirable Fenton side-reactions with H(2)O(2) (a co-product of reactions that use xanthine oxidase to produce O(2)(-&z.rad;)), we found that catalase, but not EDTA, blocked suicide elimination of cytochrome c from the reaction. Finally, we demonstrate the feasibility of evaluating superoxide scavenging abilities on small samples extracted from two types of neuronal cultures, a hypothalamic neuronal cell line (GT1 trk cells) and primary mouse cortical cell cultures. This assay allows rapid, high throughput assessments of superoxide scavenging efficacy for small molecules of interest, as well as for cell or tissue extracts.
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PMID:Rapid microplate assay for superoxide scavenging efficiency. 1078 68

We have studied neurotoxicity induced by pharmacological concentrations of 3-hydroxykynurenine (3-HK), an endogenous toxin implicated in certain neurodegenerative diseases, in cerebellar granule cells, PC12 pheochromocytoma cells, and GT1-7 hypothalamic neurosecretory cells. In all three cell types, the toxicity was induced in a dose-dependent manner by 3-HK at high micromolar concentrations and had features characteristic of apoptosis, including chromatin condensation and internucleosomal DNA cleavage. In cerebellar granule cells, the 3-HK neurotoxicity was unaffected by xanthine oxidase inhibitors but markedly potentiated by superoxide dismutase and its hemelike mimetic, MnTBAP [manganese(III) tetrakis(benzoic acid)porphyrin chloride]. Catalase blocked 3-HK neurotoxicity in the absence and presence of superoxide dismutase or MnTBAP. The formation of H(2)O(2) was demonstrated in PC12 and GT1-7 cells treated with 3-HK, by measuring the increase in the fluorescent product, 2',7'-dichlorofluorescein. In both PC12 and cerebellar granule cells, inhibitors of the neutral amino acid transporter that mediates the uptake of 3-HK failed to block 3-HK toxicity. However, their toxicity was slightly potentiated by the iron chelator, deferoxamine. Taken together, our results suggest that neurotoxicity induced by pharmacological concentrations of 3-HK in these cell types is mediated primarily by H(2)O(2), which is formed most likely by auto-oxidation of 3-HK in extracellular compartments. 3-HK-induced death of PC12 and GT1-7 cells was protected by dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. The protection by dantrolene was associated with a marked increase in the protein level of Bcl-2, a prominent antiapoptotic gene product. Moreover, overexpression of Bcl-2 in GT1-7 cells elicited by gene transfection suppressed 3-HK toxicity. Thus, dantrolene may elicit its neuroprotective effects by mechanisms involving up-regulation of the level and function of Bcl-2 protein.
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PMID:Neuronal apoptosis induced by pharmacological concentrations of 3-hydroxykynurenine: characterization and protection by dantrolene and Bcl-2 overexpression. 1085 50