In Silico and 3D QSAR Studies of Natural Based Derivatives as Xanthine Oxidase Inhibitors.

BACKGROUND: A large number of disorders and their symptoms emerge from deficiency or overproduction of specific metabolites has drawn the attention for the discovery of new therapeutic agents for the treatment of disorders. Various approaches such as computational drug design have provided the new methodology for the selection and evaluation of target protein and the lead compound mechanistically. For instance, the overproduction of xanthine oxidase causes the accumulation of uric acid which can prompt gout. OBJECTIVE: In the present study we critically discussed the various techniques such as 3-D QSAR and molecular docking for the study of the natural based xanthine oxidase inhibitors with their mechanistic insight into the interaction of xanthine oxidase and various natural leads. CONCLUSION: The computational studies of deferent natural compounds were discussed as a result the flavonoids, anthraquinones, xanthones shown the remarkable inhibitory potential for xanthine oxidase inhibition moreover the flavonoids such as hesperidin and rutin were found as promising candidates for further exploration.

Bisphenol A promotes hyperuricemia via activating xanthine oxidase.

The prevalence of hyperuricemia has increased rapidly over the past decades. Bisphenol A (BPA) is an environmental endocrine disruptor. We investigated the effects of BPA on uric acid metabolism and its potential mechanisms. Experiments were performed in different animal models, cell cultures, and humans. In 3 different animal models, BPA exposure increased serum and hepatic uric acid with enhanced activity of xanthine oxidase (XO) in liver, whereas the excretion of uric acid was unchanged. Both in vivo and in vitro, BPA-induced uric acid production was decreased after treatment with allopurinol, which is a XO inhibitor. XO led to the accumulation of uric acid after xanthine was added, with the enzyme-catalyzed reaction, which was enhanced by BPA. Altered secondary structures of XO were found by circular dichroism analysis in the conditions of different BPA concentrations. Molecular docking portrayed Asp360 and Lys422 of XO to be the preferred binding sites for BPA. Mutation of both sites significantly blocked the effect of BPA on XO activity. In humans, patients with hyperuricemia exhibited higher levels of serum BPA than subjects without hyperuricemia. These findings demonstrate BPA promotes hyperuricemia by increasing hepatic uric acid synthesis via the activation of XO, probably through direct binding.-Ma, L., Hu, J., Li, J., Yang, Y., Zhang, L., Zou, L., Gao, R., Peng, C., Wang, Y., Luo, T., Xiang, X., Qing, H., Xiao, X., Wu, C., Wang, Z., He, J. C., Li, Q., Yang, S. Bisphenol A promotes hyperuricemia via activating xanthine oxidase.

Crucial involvement of xanthine oxidase in the intracellular signalling networks associated with human myeloid cell function.

Xanthine oxidase (XOD) is an enzyme which plays a central role in purine catabolism by converting hypoxanthine into xanthine and then further into uric acid. Here we report that XOD is activated in THP-1 human myeloid cells in response to pro-inflammatory and growth factor stimulation. This effect occurred following stimulation of THP-1 cells with ligands of plasma membrane associated TLRs 2 and 4, endosomal TLRs 7 and 8 as well as stem cell growth factor (SCF). Hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) transcription complexes were found to be responsible for XOD upregulation. Importantly, the mammalian target of rapamycin (mTOR), a major myeloid cell translation regulator, was also found to be essential for XOD activation. Specific inhibition of XOD by allopurinol and sodium tungstate led to an increase in intracellular AMP levels triggering downregulation of mTOR activation by phosphorylation of its T2446 residue. Taken together, our results demonstrate for the first time that XOD is not only activated by pro-inflammatory stimuli or SCF but also plays an important role in maintaining mTOR-dependent translational control during the biological responses of human myeloid cells.

Xanthine oxidase inhibition by febuxostat attenuates experimental atherosclerosis in mice.

Atherosclerosis is a chronic inflammatory disease due to lipid deposition in the arterial wall. Multiple mechanisms participate in the inflammatory process, including oxidative stress. Xanthine oxidase (XO) is a major source of reactive oxygen species (ROS) and has been linked to the pathogenesis of atherosclerosis, but the underlying mechanisms remain unclear. Here, we show enhanced XO expression in macrophages in the atherosclerotic plaque and in aortic endothelial cells in ApoE(-/-) mice, and that febuxostat, a highly potent XO inhibitor, suppressed plaque formation, reduced arterial ROS levels and improved endothelial dysfunction in ApoE(-/-) mice without affecting plasma cholesterol levels. In vitro, febuxostat inhibited cholesterol crystal-induced ROS formation and inflammatory cytokine release in murine macrophages. These results demonstrate that in the atherosclerotic plaque, XO-mediated ROS formation is pro-inflammatory and XO-inhibition by febuxostat is a potential therapy for atherosclerosis.

Autophagy induced by cathepsin S inhibition induces early ROS production, oxidative DNA damage, and cell death via xanthine oxidase.

Cathepsin S plays multiple roles in MHC class II antigen presentation, extracellular matrix degradation, angiogenesis, and tumorogenesis. Our previous study revealed that targeting cathepsin S could induce cellular cytotoxicity and reduce cell viability. For the current study, we further investigated the molecular mechanism responsible for targeting cathepsin S-induced cell death and its association with autophagy. Distinct from regulation of the classic autophagy pathway by reactive oxygen species (ROS), we demonstrated that autophagy is the genuine regulator of early ROS production. The molecular silencing of autophagy-dependent ATG genes (ATG5, ATG7, and LC3) and the pharmacologic inhibition of autophagy with 3-MA and wortmannin reduced ROS production significantly. In addition, xanthine oxidase (XO), which is upregulated by autophagy, is required for early ROS production, oxidative DNA damage, and consequent cell death. Autophagy inhibition suppresses the upregulation of XO, which is induced by cathepsin S inhibition, resulting in reduced ROS generation, DNA damage, and cell death. Collectively, our study reveals a noncanonical molecular pathway in which, after the inhibition of cathepsin S, autophagy induces early ROS production for oxidative DNA damage and cell death through XO.

Nicorandil, a K(atp) channel opener, alleviates chronic renal injury by targeting podocytes and macrophages.

Nicorandil exhibits a protective effect in the vascular system, which is thought to be due to vasodilatation from opening ATP-dependent potassium channels and donation of nitric oxide. Recently, nicorandil was shown to be renoprotective in models of acute kidney injury and glomerulonephritis. However, the specific mechanisms of renoprotection are unclear. We evaluated the effect of nicorandil on the rat remnant kidney model of chronic kidney disease. Blood pressure was unchanged by a 10-wk course of nicorandil, while albuminuria was significantly reduced. Glomerular injury and tubulointerstitial injury were also ameliorated by nicorandil. Oxidative stress, as noted by renal nitrotyrosine level and urine 8-hydroxy-2'-deoxyguanosine, were elevated in this model and was significantly reduced by nicorandil treatment. Treatment was associated with maintenance of the mitochondrial antioxidant, manganese SOD, in podocytes and with suppression of xanthine oxidase expression in infiltrating macrophages. Interestingly, these two cell types express sulfonylurea receptor 2 (SUR2), a binding site of nicorandil in the ATP-dependent K channel. Consistently, we found that stimulating SUR2 with nicorandil prevented angiotensin II-mediated upregulation of xanthine oxidase in the cultured macrophage, while xanthine oxidase expression was rather induced by blocking SUR2 with glibenclamide. In conclusion, nicorandil reduces albuminuria and ameliorates renal injury by blocking oxidative stress in chronic kidney disease.

Noninvasive delayed limb ischemic preconditioning attenuates myocardial ischemia-reperfusion injury in rats by a mitochondrial K(ATP) channel-dependent mechanism.

We previously demonstrated in rats that noninvasive delayed limb ischemic preconditioning (LIPC) induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb per day for three days confers the same cardioprotective effect as local ischemic preconditioning of the heart, but the mechanism has not been studied in depth. The aim of this project was to test the hypothesis that delayed LIPC enhances myocardial antioxidative ability during ischemia-reperfusion by a mitochondrial K(ATP) channel (mito K(ATP))-dependent mechanism. Rats were randomized to five groups: ischemia-reperfusion (IR)-control group, myocardial ischemic preconditioning (MIPC) group, LIPC group, IR-5HD group and LIPC-5HD group. The MIPC group underwent local ischemic preconditioning induced by three cycles of 5-min occlusion and 5-min reperfusion of the left anterior descending coronary arteries. The LIPC and LIPC-5HD groups underwent LIPC induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb using a modified blood pressure aerocyst per day for three days. All rats were subjected to myocardial ischemia-reperfusion injury. The IR-5HD and LIPC-5HD groups received the mito K(ATP) channel blocker 5-hydroxydecanoate Na (5-HD) before and during the myocardial ischemia-reperfusion injury. Compared with the IR-control group, both the LIPC and MIPC groups showed an amelioration of ventricular arrhythmia, reduced myocardial infarct size, increased activities of total superoxide dismutase, manganese-superoxide dismutase (Mn-SOD) and glutathione peroxidase, increased expression of Mn-SOD mRNA and decreased xanthine oxidase activity and malondialdehyde concentration. These beneficial effects of LIPC were prevented by 5-HD. In conclusion, delayed LIPC offers similar cardioprotection as local IPC. These results support the hypothesis that the activation of mito K(ATP) channels enhances myocardial antioxidative ability during ischemia-reperfusion, thereby contributing, at least in part, to the anti-arrhythmic and anti-infarct effects of delayed LIPC.

New evidence for the role of TNF-alpha in liver ischaemic/reperfusion injury.

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) plays a key role in causing ischaemia/reperfusion (I/R) injury. I/R also causes activation of xanthine oxidase and dehydrogenase (XDH + XO) system that, via generated free radicals, causes organ damage. We investigated the effect of ischaemia, reperfusion and non-ischaemic prolonged perfusion (NIP) on TNF-alpha and XDH + XO production in an isolated perfused rat liver model. MATERIALS AND METHODS: Rat livers underwent 150 min NIP (control group) or two hours of ischaemia followed by reperfusion (I/R group). TNF-alpha (TNF-alpha mRNA and protein level), XDH + XO production and bile secretion were determined in tissue and effluent at baseline, at 120 min of ischaemia, after 30 min of reperfusion (I/R group) and after 120 and 150 min of prolonged perfusion (control). RESULTS: Unexpectedly, neither ischaemia nor reperfusion had any effect on TNF-alpha production. TNF-alpha in effluent was 11 +/- 4.8 pg mL(-1) at baseline, 7 +/- 3.2 pg mL(-1) at the end of ischaemia, and 13 +/- 5.3 pg mL(-1) after 30 min of reperfusion. NIP, however, caused a significant increase of TNF-alpha synthesis and release. TNF-alpha effluent level after 120 and 150 min of perfusion was 392 +/- 78.7 pg mL(-1) and 408 +/- 64.3 pg mL(-1), respectively. TNF-alpha mRNA in tissue was also significantly elevated compared to baseline levels (1.31 +/- 0.2 P < 0.001 and 1.38 P < 0.002, respectively). Decrease of liver function (expressed by bile secretion) during I/R and NIP was accompanied by significant XDH + XO elevation. CONCLUSION: This is the first evidence that NIP, and not I/R, is the decisive trigger for TNF-alpha production. This study leads to a better understanding of pathogenesis of liver I/R and perfusion damage.

Mechanism of in vitro pancreatic cancer cell growth inhibition by melanoma differentiation-associated gene-7/interleukin-24 and perillyl alcohol.

The death rate for pancreatic cancer approximates the number of new cases each year, and when diagnosed, current therapeutic regimens provide little benefit in extending patient survival. These dire statistics necessitate the development of enhanced single or combinatorial therapies to decrease the pathogenesis of this invariably fatal disease. Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a potent cancer gene therapeutic because of its broad-spectrum cancer-specific apoptosis-inducing properties as well as its multipronged indirect antitumor activities. However, pancreatic cancer cells show inherent resistance to mda-7/IL-24 that is caused by a block of translation of mda-7/IL-24 mRNA in these tumor cells. We now reveal that a dietary agent perillyl alcohol (POH) in combination with Ad.mda-7 efficiently reverses the mda-7/IL-24 "protein translational block" by inducing reactive oxygen species, thereby resulting in mda-7/IL-24 protein production, growth suppression, and apoptosis. Pharmacologic inhibitor and small interfering RNA studies identify xanthine oxidase as a major source of superoxide radical production causing these toxic effects. Because both POH and Ad.mda-7 are being evaluated in clinical trials, combining a dietary agent and a virally delivered therapeutic cytokine provides an innovative approach for potentially treating human pancreatic cancer.

Redox-independent activation of NF-kappaB by Pseudomonas aeruginosa pyocyanin in a cystic fibrosis airway epithelial cell line.

The roles of the Pseudomonas aeruginosa-derived pigment pyocyanin (PYO) as an oxidant and activator of the proinflammatory transcription factor NF-kappaB were tested in a cystic fibrosis (CF) airway epithelial cell line, CF15. 100 microm PYO on its own had no effect or only small effects to activate NF-kappaB (<1.5-fold), but PYO synergized with the TLR5 agonist flagellin. Flagellin activated NF-kappaB 4-20-fold, and PYO increased these activations >2.5-fold. PYO could have synergized with flagellin to activate NF-kappaB by redox cycling with NADPH, generating superoxide (O(2)*), hydrogen peroxide (H(2)O(2)), and hydroxyl radical (HO*). Cytosol-targeted, redox-sensitive roGFP1 and imaging microscopy showed that 1-100 microm PYO oxidized CF15 cytosol redox potential (Psi(cyto)) from -325 mV (control) to -285 mV. O(2)* (derived from KO(2)*. or xanthine + xanthine oxidase) or H(2)O(2) oxidized Psi(cyto) dose-dependently but did not activate NF-kappaB, even in the presence of flagellin, and 400 microm H(2)O(2) inhibited NF-kappaB. Overexpressing intracellular catalase decreased effects of PYO and H(2)O(2) on Psi(cyto) but did not affect flagellin + PYO-activated NF-kappaB. Catalase also reversed the inhibitory effects of H(2)O(2) on NF-kappaB. The HO* scavenger DMSO did not alter the effects of PYO on Psi(cyto) and NF-kappaB. The synergistic NF-kappaB activation was calcium-independent. Thus, in the presence of flagellin, PYO activated NF-kappaB through a redox- and calcium-independent effect.

A comparison of reactive oxygen species metabolism in the rat aorta and vena cava: focus on xanthine oxidase.

Reactive oxygen species (ROS) are important mediators in vascular biology. Venous function, although relevant to cardiovascular disease, is still understudied. We compared aspects of ROS metabolism between a major artery (the aorta) and a major vein (the vena cava, VC) of the rat, with the hypothesis that venous ROS metabolism would be overall increased compared with its arterial counterpart. Superoxide and hydrogen peroxide (H2O2) release in basal conditions was higher in VC compared with aorta. The antioxidant capacity for H2O2 was also higher in VC than in aorta. Exogenous superoxide induced a higher contraction in VC compared with aorta. Protein expression of three major ROS metabolizing enzymes, xanthine oxidase (XO), CuZn-SOD, and catalase, was higher in VC compared with aorta. Because XO seemed a likely source of the higher VC ROS levels, we examined it further and found higher mRNA expression and activity of XO in VC compared with aorta. We also investigated the impact of XO inhibition by allopurinol on aorta and VC functional responses to norepinephrine, ANG II, ET-1, and ACh. Maximal ET-1-mediated contraction was decreased by allopurinol in VC but not in the aorta. Our results suggest that there are overall differences in ROS metabolism between aorta and VC, with the latter operating normally at a higher set point, releasing but also being able to handle, higher ROS levels. We propose XO to be an important source for these differences. The result of this particular comparison may be reflective of a general arteriovenous contrast.

A role for IOP1 in mammalian cytosolic iron-sulfur protein biogenesis.

The biogenesis of cytosolic iron-sulfur (Fe-S) proteins in mammalian cells is poorly understood. In Saccharomyces cerevisiae, there is a pathway dedicated to cytosolic Fe-S protein maturation that involves several essential proteins. One of these is Nar1, which intriguingly is homologous to iron-only hydrogenases, ancient enzymes that catalyze the formation of hydrogen gas in anaerobic bacteria. There are two orthologues of Nar1 in mammalian cells, iron-only hydrogenase-like protein 1 (IOP1) and IOP2 (also known as nuclear prelamin A recognition factor). We examined IOP1 for a potential role in mammalian cytosolic Fe-S protein biogenesis. We found that knockdown of IOP1 in both HeLa and Hep3B cells decreases the activity of cytosolic aconitase, an Fe-S protein, but not that of mitochondrial aconitase. Knockdown of IOP2, in contrast, had no effect on either. The decrease in aconitase activity upon IOP1 knockdown is rescued by expression of a small interference RNA-resistant version of IOP1. Upon loss of its Fe-S cluster, cytosolic aconitase is known to be converted to iron regulatory protein 1, and consistent with this, we found that IOP1 knockdown increases transferrin receptor 1 mRNA levels and decreases ferritin heavy chain protein levels. IOP1 knockdown also leads to a decrease in activity of xanthine oxidase, a distinct cytosolic Fe-S protein. Taken together, these results provide evidence that IOP1 is involved in mammalian cytosolic Fe-S protein maturation.

The effects of ginkgo biloba extract on tissue adenosine deaminase, xanthine oxidase, myeloperoxidase, malondialdehyde, and nitric oxide in cisplatin-induced nephrotoxicity.

This study was carried out to determine if Ginkgo Biloba Extract (GBE or Egb 761) exerts a beneficial effect against cisplatin-induced renal failure in rats. Sprague Dawley rats were divided into four groups. The first group (control) received orally 1 mL/kg/day of 0.9% saline by an oral carrier vehicle on days 1 to 10. The second group was injected with 7 mg/kg cisplatin intraperitoneally (i.p.) on the fourth day, once only. The third group (vit E+cisplatin) was administered 10 mg/kg/day i.p. vit E on 1 to 10 days with one dose of i.p. cisplatin (7 mg/kg) injection on the fourth day. The fourth group (GBE+cisplatin) was given GBE orally at 100 mg/mL/kg started on the first day up to the tenth day with one dose of cisplatin (7 mg/kg) injection on the fourth day. Cisplatin was found to lead a statistically significant increase in plasma BUN and creatinine levels, as well as urine micro total protein (MTP) levels, leading to acute renal failure (ARF) in rats. Renal xanthine oxidase (XO) activities increased in all groups (statistically significant in cisplatin + GBE-treated rats; P < 0.001). Adenosine deaminase (AD) activities were increased in cisplatin-treated rats, and decreased in cisplatin+GBE-treated (P < 0.041) and cisplatin+vit E-treated (P < 0.005) rats, compared to controls. Malondialdehyde (MDA), nitric oxide (NO) levels and myeloperoxidase (MPO) activities were increased in the kidney tissue of cisplatin-treated rats. Vit E improved plasma creatinine and urine MTP levels, together with tissue MDA, NO levels, and MPO activities. But GBE had no statistically significant effect on those parameters. These results indicate that increased XO, AD and MPO activities, as well as MDA and NO levels play a critical role in cisplatin nephrotoxicity. GBE has been shown to protect against cisplatin-induced nephrotoxicity.

Salix caprea inhibits skin carcinogenesis in murine skin: inhibition of oxidative stress, ornithine decarboxylase activity and DNA synthesis.

Chemoprevention of free radical-mediated diseases including cancer by natural products is an emerging discipline due to its wider applicability and acceptance. The present study deals with the chemopreventive effect of Salix caprea against phorbol ester-induced oxidative stress and tumor promotion in murine skin. In the present investigation, it was observed that a single application of 12-O-tetradecanoyl-13-phorbol acetate (TPA) (20 nmol/0.2 ml acetone/animal) caused a significant (P < 0.05) depletion of cutaneous antioxidants viz., glutathione, glutathione reductase, glutathione peroxidase, catalase and phase II drug metabolizing enzymes viz., glutathione-S-transferase, quinone reductase. An increase in the hydrogen peroxide generation and protein oxidation (measured in terms of protein carbonyl content) was also observed with a single application of TPA. However, the pretreatment of animals with different doses of Salix caprea (0.5, 1.0 and 1.5 mg/kg/0.2 ml acetone) caused a significant recovery in the TPA-mediated depletion in antioxidant levels. The pretreatment of animals with Salix caprea was observed to inhibit the TPA-mediated depletion in phase II enzymes. It was also observed that Salix caprea reversed the TPA-mediated depletion in the activity of phase II enzymes that is an important characteristic of cancer chemopreventive agents. Phorbol esters are known to induce the tumor promotion by increasing rate of DNA synthesis, ornithine decarboxylase activity (ODC), and xanthine oxidase activity. In the present investigation, it was observed that the pretreatment of animals with Salix caprea caused a significant (P < 0.05) depletion in the TPA-induced DNA synthesis, ODC and xanthine oxidase activity in mice skin. Salix caprea significantly reduced the tumor promotion in mice skin when tested in two-stage chemical carcinogenesis model. It was observed to inhibit significantly P < 0.05) the 7,12-dimethyl benz[a] anthracene (DMBA)-initiated phorbol ester promoted skin carcinogenesis. It was concluded from the results that Salix caprea is an effective antioxidant and chemopreventive agent against phorbol ester-induced tumor promotion.

Upregulation of xanthine oxidase by tobacco smoke condensate in pulmonary endothelial cells.

Tobacco smoking has been causally linked to the development of chronic obstructive pulmonary disease. It has been reported that the reactive oxygen species (ROS)- generating enzyme xanthine dehydrogenase/oxidase (XO) is increased in smoking-related stomach ulcers and that gastric mucosal damage caused by tobacco smoke can be blocked by the XO inhibitor allopurinol. In order to test the hypothesis that tobacco may cause the upregulation of XO in the lung, cultured rat pulmonary microvascular endothelial cells were exposed to tobacco smoke condensate (TSC). TSC at a concentration of 20 microg/mL significantly upregulated XO activity after 24 h of exposure. Longer exposure (1 week) to a lower concentration of TSC (2 microg/mL) also caused an increase in XO activity. Unlike hypoxia, TSC treatment did not alter the phosphorylation of XO. However, TSC treatment increased XO mRNA expression and the XO gene promoter activity. Furthermore, actinomycin D blocked the activation of XO by TSC. In conclusion, our results indicate that tobacco smoke condensate causes upregulation of XO transcription and activity.

Functional regulation of xanthine oxidoreductase expression and localization in the mouse mammary gland: evidence of a role in lipid secretion.

Xanthine oxidoreductase (XOR), a key enzyme of purine metabolism, has been implicated in the secretion of the milk fat droplet in lactating mammary epithelial cells, possibly through structural interactions with other milk fat globule proteins including butyrophilin (Btn) and adipophilin (ADPH). To help determine the mechanism by which XOR is regulated, we examined the expression and localization of XOR in the non-secretory states of late pregnancy and induced involution compared with the state of active secretion. XOR mRNA levels started to increase at mid-pregnancy, turned sharply upwards at the onset of lactation and decreased rapidly with forced involution, indicating transcriptional control of the enzyme level by differentiation and secretory function. During pregnancy and involution the enzyme was diffusely distributed in the cytoplasm, but moved rapidly to the apical membrane of the cells when secretion was activated, where it colocalized with both Btn and ADPH, similar to the situation in the milk fat globule itself. Size-exclusion chromatography of solubilized milk fat globule membrane proteins showed that XOR formed a sulphydryl-bond-dependent complex with Btn and ADPH in the milk fat globule membrane. XOR returned to a diffuse cytoplasmic localization shortly after induced involution, while Btn remained localized to the apical membrane, suggesting that localization of XOR is not dependent on the presence of Btn in the apical membrane. Our findings indicate that the expression and membrane association of XOR in the mammary gland are tightly regulated by secretory activity, and suggest that the apical membrane association of XOR regulates the coupling of lipid droplets to the apical plasma membrane during milk lipid secretion.

Purification and characterization of multiple forms of rat liver xanthine oxidoreductase expressed in baculovirus-insect cell system.

cDNA of rat liver xanthine oxidoreductase (XOR), a molybdenum-containing iron-sulfur flavoprotein, was expressed in a baculovirus-insect cell system. The expressed XOR consisted of a heterogeneous mixture of native dimeric, demolybdo-dimeric, and monomeric forms, each of which was separated and purified to homogeneity. All the expressed forms contained flavin, of which the semiquinone form was stable during dithionite titration after dithiothreitol treatment, indicating that the flavin domains of all the expressed molecules have the intact conformations interconvertible between NAD(+)-dependent dehydrogenase (XDH) and O(2)-dependent oxidase (XO) types. The absorption spectrum and metal analyses showed that the monomeric form lacks not only molybdopterin but also one of the iron-sulfur centers. The reductive titration of the monomer with dithionite showed that the monomeric form required only three electrons for complete reduction, and the redox potential of the iron-sulfur center in the monomeric form is a lower value than that of FAD. In contrast to native or demolybdo-dimeric XDHs, the monomer showed a very slow reductive process with NADH under anaerobic conditions, although the conformation around FAD is a dehydrogenase form, suggesting the important role of the iron-sulfur center in the reductive process of FAD with the reduced pyridine nucleotide.

Xanthine oxidoreductase and xanthine oxidase in human cornea.

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.

Burn depth affects dermal interstitial fluid pressure, free radical production, and serum histamine levels in rats.

BACKGROUND: We measured the amount of edema and the free radical production in burn-injured skin and the serum histamine levels, as well as changes in dermal interstitial fluid pressure. METHODS: Thirty-six Wistar rats with 20% total body surface area burns of different depth were resuscitated by lactated Ringer's solution intravenously. The rats were divided into a deep burn (DB) group (n = 12), a superficial dermal burn (SDB) group (n = 12), and a sham burn (Sham) group (n = 12). Dermal interstitial fluid hydrostatic pressure (Pif), total skin water and xanthine oxidase activity, and serum histamine levels were measured until 60 minutes postburn. RESULTS: In the DB group, dermal Pif significantly fell to -35.9 +/- 11.0 and -40.9 +/- 7.0 mm Hg at 10 and 15 minutes postburn, respectively (p < 0.05); it returned to preburn values at 50 minutes postburn. In the SDB group, dermal Pif was slightly negative but did not markedly change. Total skin water was significantly higher than that of the DB and the Sham groups; however, in the SDB group, serum histamine and dermal xanthine oxidase were significantly higher than in the DB group at 15, 30, and 45 minutes postburn (p < 0.05). CONCLUSION: The fluid-resuscitated DB produced a more negative dermal Pif than the SDB, but less dermal edema. In contrast, the SDB appeared to mainly generate dermal edema formation by wound free radical production and serum histamine release. The dermal Pif is one of the factors associated with edema formation immediately after deep burns. However, an increase in vascular permeability associated with oxygen radical production plays a more important role in dermal edema formation than does dermal Pif.

Inducible nitric oxide synthase, but not xanthine oxidase, is highly expressed in interstitial pneumonias and granulomatous diseases of human lung.

We assessed the distribution and expression of inducible nitric oxide synthase (i-NOS), endothelial nitric oxide synthase (e-NOS), and xanthine oxidase (XAO) in usual interstitial pneumonia, desquamative interstitial pneumonia, and granulomatous diseases. The material consisted of biopsy specimens from 5 healthy subjects (nonsmokers), 9 patients with usual interstitial pneumonia, 11 with desquamative interstitial pneumonia, 14 with sarcoidosis, and 8 with extrinsic allergic alveolitis. i-NOS was expressed intensively in inflammatory but not infibrotic lesions. It was expressed most prominently in alveolar macrophages and alveolar epithelium of all disorders and in the granulomas of sarcoidosis and extrinsic allergic alveolitis. In contrast with i-NOS, e-NOS was expressed prominently in control lung tissue samples but also in granulomas of sarcoidosis and extrinsic allergic alveolitis. Reverse transcription-polymerase chain reaction performed on bronchoalveolar lavage fluid samples from patients with sarcoidosis or usual interstitial pneumonia andfrom healthy subjects indicated positivity for XAO, but immunohistochemical analysis in samples from healthy lung and all parenchymal lung disorders showed no immunoreactivity for XAO. i-NOS has an important role in the pathogenesis of interstitial lung diseases, being up-regulated during the inflammatory but not during the fibrotic disease stage.