Expression of green fluorescent protein as a marker for effects of antileishmanial compounds in vitro.
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Transgenic Leishmania infantum promastigotes, which constitutively express green fluorescent protein (GFP) in their cytoplasm, were used to monitor the effects of antileishmanial compounds in real time. The GFP-based assay provided a reliable measure of drug-induced inhibitory effects on protein expression, resulting in a dynamic picture of the responses of leishmanial promastigotes to the compounds tested. (+info)
Spinal cord metabolism during thoracic aortic cross-clamping in pigs with special reference to the effect of allopurinol.
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OBJECTIVE: investigate the metabolic response of the spinal cord and the effect of allopurinol following cross clamping of the descending thoracic aorta in a porcine model. DESIGN: experimental animal study. MATERIALS: twelve domestic swine. Six pigs were pre-treated with allopurinol, while six pigs served as controls. METHODS: measurement of extracellular concentrations of glucose, pyruvate, lactate, glycerol and glutamate using microdialysis in the lumbar spinal cord. Measurement of lumbar spinal blood flow using laser Doppler technique. RESULTS: for all animals there was a significant decrease in concentrations of glucose and pyruvate together with a significant increase in the lactate-pyruvate ratio during aortic cross clamping. There was also a significant increase in glycerol concentrations 60 min after cross clamping, and a significant decrease in glutamate concentrations after 50 min. No differences in concentrations of glucose, pyruvate, lactate and glutamate or the glutamate-pyruvate ratio were observed between animals used as controls and those treated with allopurinol. The laser Doppler flux decreased to 40% of pre cross-clamp level, returning to normal values at declamping. CONCLUSION: the changes in energy-related metabolites reflect a considerable ischaemia in the spinal cord tissue but there was no convincing effect of allopurinol on the lumbar spinal cord metabolism during thoracic aortic cross clamping in this model. (+info)
Use of anti-(tumour necrosis factor-alpha) antibody or 3-deaza-adenosine as additives to promote protection by University of Wisconsin solution in ischaemia/reperfusion injury.
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Experimental interventions that reduce ischaemia/reperfusion (I/R) lung injury can be used to improve the properties of preservation solutions. We attempted to increase the attenuation of I/R injury by University of Wisconsin solution (UW) by adding an antibody against tumour necrosis factor-alpha (TNF-alpha), to neutralize TNF-alpha, and/or by adding 3-deaza-adenosine (c3-Ado), to inhibit leucocyte adhesion and the biosynthesis of ICAM-1 (intercellular cell-adhesion molecule 1). We examined I/R injury using an isolated rat lung model. Six different solutions were perfused individually, followed by evaluation of I/R injury: (1) 0.9% NaCl (normal saline; NS), (2) NS+anti-TNF-alpha antibody, (3) UW alone, (4) UW+anti-TNF-alpha, (5) UW+c3-Ado and (6) UW+anti-TNF-alpha+c3-Ado. Haemodynamic changes, lung weight gain, capillary filtration coefficient, TNF-alpha levels and lung pathology were analysed in order to evaluate I/R injury. Compared with lungs perfused with NS, lungs treated with NS+anti-TNF-alpha showed less I/R injury. The addition of anti-TNF-alpha and/or c3-Ado to UW reduced I/R injury compared with unmodified UW. Among the six solutions tested, UW containing anti-TNF-alpha antibody reduced I/R injury to the greatest extent. We conclude that addition of anti-TNF-alpha antibody or c3-Ado protects against I/R lung injury when using UW. Further investigation of the improved properties of modified UWs would be beneficial with regard to lung transplantation research. (+info)
Apoptotic degeneration of germ cells in cryptorchid testis is associated with an increased level of reactive oxygen species, and may be prevented by antioxidant treatment. The objective of this study was to investigate whether xanthine oxidase inhibitors could confer such protection. Unilateral cryptorchidism was surgically induced in the immature rats, which were then left untreated or treated with xanthine oxidase inhibitors, and the testes were evaluated 7 days after the operation. In the control group, the weight of the cryptorchid testis was decreased to 47% of that of the contralateral scrotal testis. However, administration of a xanthine oxidase inhibitor allopurinol (> or = 1 mg/kg/day) significantly attenuated weight reduction of the cryptorchid testis (68-77% of the contralateral scrotal testis, P < 0.01 versus control). Another highly specific xanthine oxidase inhibitor, BOF-4272, also attenuated cryptorchidism-induced testis regression. The effects of allopurinol were associated with decreased apoptosis in germ cells as evaluated by in-situ staining of fragmented DNA. In testicular cells cultured at 37 degrees C, either allopurinol or BOF-4272 prevented DNA cleavage characteristic of apoptosis. In conclusion, xanthine oxidase inhibitors can inhibit germ cell apoptosis induced by experimental cryptorchidism, and may be considered for treatment of male infertility associated with heat stress. (+info)
Effect of allopurinol on mortality and hospitalisations in chronic heart failure: a retrospective cohort study.
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OBJECTIVE: To examine whether allopurinol is associated with any alteration in mortality and hospitalisations in patients with chronic heart failure (CHF). This hypothesis is based on previous data that a high urate concentration is independently associated with mortality with a risk ratio of 4.23 in CHF. DESIGN: Retrospective cohort study. SETTING: Medicines Monitoring Unit, Ninewells Hospital, Dundee, UK. PATIENTS: 1760 CHF patients divided into four groups: those on no allopurinol, those on long term low dose allopurinol, those on short term low dose allopurinol, and those on long term high dose allopurinol. MAIN OUTCOME MEASURES: Total mortality, cardiovascular mortality, cardiovascular hospitalisations, cardiovascular mortality or hospitalisations. RESULTS: Long term low dose allopurinol was associated with a significant worsening in mortality over those who never received allopurinol (relative risk 2.04, 95% confidence interval (CI) 1.48 to 2.81). This may be because low dose allopurinol is insufficient to negate the adverse effect of a high urate concentration. However, long term high dose (> or = 300 mg/day) allopurinol was associated with a significantly better mortality than longstanding low dose allopurinol (relative risk 0.59, 95% CI 0.37 to 0.95). This may mean that high dose allopurinol can fully negate the adverse effect of urate and return the mortality to normal. CONCLUSIONS: Long term high dose allopurinol may be associated with a better mortality than long term low dose allopurinol in patients with CHF because of a dose related beneficial effect of allopurinol against the well described adverse effect of urate. Further work is required to substantiate or refute this finding. (+info)
Imbalance between xanthine oxidase and nitric oxide synthase signaling pathways underlies mechanoenergetic uncoupling in the failing heart.
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Inhibition of xanthine oxidase (XO) in failing hearts improves cardiac efficiency by an unknown mechanism. We hypothesized that this energetic effect is due to reduced oxidative stress and critically depends on nitric oxide synthase (NOS) activity, reflecting a balance between generation of nitric oxide (NO) and reactive oxygen species. In dogs with pacing-induced heart failure (HF), ascorbate (1000 mg) mimicked the beneficial energetic effects of allopurinol, increasing both contractility and efficiency, suggesting an antioxidant mechanism. Allopurinol had no additive effect beyond that of ascorbate. Crosstalk between XO and NOS signaling was assessed. NOS inhibition with N(G)-monomethyl-L-arginine (L-NMMA; 20 mg/kg) had no effect on basal contractility or efficiency in HF, but prevented the +26.2+/-3.5% and +66.5+/-17% enhancements of contractility and efficiency, respectively, observed with allopurinol alone. Similarly, improvements in contractility and energetics due to ascorbate were also inhibited by L-NMMA. Because of the observed NOS-XO crosstalk, we predicted that in normal hearts NOS inhibition would uncover a depression of energetics caused by XO activity. In normal conscious dogs, L-NMMA increased myocardial oxygen consumption (MVO2) while lowering left ventricular external work, reducing efficiency by 31.1+/-3.8% (P<0.005). Lowered efficiency was reversed by XO inhibition (allopurinol, 200 mg) or by ascorbate without affecting cardiac load or systemic hemodynamics. Single-cell immunofluorescence detected XO protein in cardiac myocytes that was enhanced in HF, consistent with autocrine signaling. These data show that both NOS and XO signaling systems participate in the regulation of myocardial mechanical efficiency and that upregulation of XO relative to NOS contributes to mechanoenergetic uncoupling in heart failure. (+info)
Xanthine oxidase is involved in free radical production in type 1 diabetes: protection by allopurinol.
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The aim of this work was to study the mechanism of free radical formation in type 1 diabetes and its possible prevention. We have found oxidation of blood glutathione and an increase in plasma lipoperoxide levels in both human type 1 diabetes and experimental diabetes. Peroxide production by mitochondria does not increase in diabetes. On the contrary, the activity of xanthine oxidase, a superoxide-generating enzyme, increases in liver and plasma of diabetic animals. The increase in plasma xanthine oxidase activity may be explained by the increase in the hepatic release of this enzyme, which is not due to nonspecific membrane damage: release of other hepatic enzymes, such as the amino transferases, does not increase in diabetes. Superoxide formation by aortic rings of rabbits increases significantly in diabetes. This is completely inhibited by allopurinol, an inhibitor of xanthine oxidase. Heparin, which releases xanthine oxidase from the vessel wall, also decreases superoxide formation by aortic rings of diabetic animals. Treatment with allopurinol decreases oxidative stress in type 1 diabetic patients: hemoglobin glycation, glutathione oxidation, and the increase in lipid peroxidation are prevented. These results may have clinical significance in the prevention of late-onset vascular complications of diabetes. (+info)
Role of mitochondrial oxidant generation in endothelial cell responses to hypoxia.
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Endothelial cells increase their secretion of the cytokine interleukin-6 (IL-6) during hypoxia, which then acts in an autocrine fashion to increase the permeability of cell monolayers. These responses are attenuated by antioxidants, suggesting that reactive oxygen species (ROS) participate in signaling in hypoxic endothelium. We tested whether mitochondria are responsible for these ROS in human umbilical vein endothelial cells exposed to hypoxia. Oxidation of the probe 2', 7'-dichlorodihydrofluorescein to fluorescent dichlorofluorescein or the probe dihydroethidium was used to assess oxidant signaling, whereas permeability was assessed by using transendothelial electrical resistance. Hypoxia elicited increases in dichlorofluorescein and dihydroethidium fluorescence that were abrogated by the mitochondrial electron transport (ET) inhibitors rotenone (2 micromol/L) and diphenyleneiodonium (5 micromol/L). The same ET inhibitors also attenuated hypoxia-induced increases in nuclear factor-kappaB (NF-kappaB) activation, although they did not abrogate NF-kappaB activation in response to endotoxin (lipopolysaccharide). ET inhibition also abolished the hypoxia-induced increases in IL-6 mRNA expression, hypoxia-stimulated IL-6 secretion into the media, and the hypoxia-induced increases in transendothelial electrical resistance of human umbilical vein endothelial cell monolayers. By contrast, the above responses to hypoxia were not significantly affected by treatment with the NAD(P)H oxidase inhibitor apocynin (30 micromol/L), the xanthine oxidase inhibitor allopurinol (100 micromol/L), or the NO synthase inhibitor N-nitro-L-arginine (100 micromol/L). We conclude that ROS signals originating from the mitochondrial ET chain trigger the increase in NF-kappaB activation, the transcriptional activation of IL-6, the secretion of IL-6 into the cell culture media, and the increases in endothelial permeability observed during hypoxia. (+info)