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Sample records for allopurinol xanthine oxidase

  1. Relationship between plasma oxipurinol concentrations and xanthine oxidase activity in volunteers dosed with allopurinol.

    PubMed Central

    Day, R O; Miners, J; Birkett, D J; Graham, G G; Whitehead, A

    1988-01-01

    1. 1-methyl xanthine (1-MX) is metabolized exclusively to 1-methyl uric acid (1-MU) by the enzyme xanthine oxidase. 2. The ratio of 1-MU to 1-MX in the urine, following a dose of 50 mg of 1-MX infused intravenously over 20 min, was used to measure the inhibition of xanthine oxidase induced by different doses of allopurinol. 3. Normal volunteers (n = 8) were given allopurinol 50, 100, 300 and 600 mg daily for 1 week each, in random order and 1 week separated each treatment. Inhibition of xanthine oxidase was assessed twice, on the last 2 days of each treatment week. 4. Steady-state oxipurinol concentrations increased linearly with increasing dose of allopurinol. 5. There was a hyperbolic relationship between the 1-MU/1-MX ratio and plasma oxipurinol concentrations, with an initial steep decline in the ratio which plateaued when plasma oxipurinol was around 4-6 mg l-1. This reduction in the ratio was quickly reversible upon cessation of allopurinol. 6. The 50% and 90% effective inhibitory oxipurinol concentrations, in relation to the 1-MU-/1-MX ratio were 1.4 +/- 0.46 and 4.08 +/- 2.03 mg l-1 respectively. 7. The concentration of oxipurinol required for almost complete inhibition of the enzyme was substantially less than those often observed in clinical practice. PMID:3190993

  2. Therapeutic Effects of Xanthine Oxidase Inhibitors: Renaissance Half a Century after the Discovery of Allopurinol

    PubMed Central

    PACHER, PÁL; NIVOROZHKIN, ALEX; SZABÓ, CSABA

    2008-01-01

    The prototypical xanthine oxidase (XO) inhibitor allopurinol, has been the cornerstone of the clinical management of gout and conditions associated with hyperuricemia for several decades. More recent data indicate that XO also plays an important role in various forms of ischemic and other types of tissue and vascular injuries, inflammatory diseases, and chronic heart failure. Allopurinol and its active metabolite oxypurinol showed considerable promise in the treatment of these conditions both in experimental animals and in small-scale human clinical trials. Although some of the beneficial effects of these compounds may be unrelated to the inhibition of the XO, the encouraging findings rekindled significant interest in the development of additional, novel series of XO inhibitors for various therapeutic indications. Here we present a critical overview of the effects of XO inhibitors in various pathophysiological conditions and also review the various emerging therapeutic strategies offered by this approach. PMID:16507884

  3. Antidepressant-like effects of the xanthine oxidase enzyme inhibitor allopurinol in rats. A comparison with fluoxetine.

    PubMed

    Gürbüz Özgür, Börte; Aksu, Hatice; Birincioğlu, Mustafa; Dost, Turhan

    2015-11-01

    Allopurinol is a xanthine oxidase enzyme inhibitor that is widely used for the treatment of hyperuricemia and gout. The activity of tryptophan 2,3-dioxygenase, which metabolizes tryptophan (TRP), is decreased by xanthine oxidase inhibitors, causing TRP levels in the body to be increased. Increases in TRP levels in the brain might have antidepressant effects. The purpose of this study is to evaluate the antidepressant effects of allopurinol compared to those of fluoxetine, which is a proven antidepressant. Thirty-two Wistar albino male rats were divided into four groups (control, 10mg/kg fluoxetine, 50mg/kg allopurinol, 50mg/kg allopurinol+10 mg/kg fluoxetine; n=8 per group), and forced swimming tests were performed before and after 14days of drug administration. Serotonin, 5-hydroxyindolacetic acid and uric acid levels were measured in blood samples after the final treatment. When allopurinol and fluoxetine were administered separately, a decrease in the duration of immobility and an increased duration of swimming were observed in the forced swimming test. The results showed similar antidepressant efficacies between allopurinol and fluoxetine. However, we found no statistically significant difference in the antidepressant effect of the combined therapy versus single drug therapy.

  4. Hydroxychavicol: a potent xanthine oxidase inhibitor obtained from the leaves of betel, Piper betle.

    PubMed

    Murata, Kazuya; Nakao, Kikuyo; Hirata, Noriko; Namba, Kensuke; Nomi, Takao; Kitamura, Yoshihisa; Moriyama, Kenzo; Shintani, Takahiro; Iinuma, Munekazu; Matsuda, Hideaki

    2009-07-01

    The screening of Piperaceous plants for xanthine oxidase inhibitory activity revealed that the extract of the leaves of Piper betle possesses potent activity. Activity-guided purification led us to obtain hydroxychavicol as an active principle. Hydroxychavicol is a more potent xanthine oxidase inhibitor than allopurinol, which is clinically used for the treatment of hyperuricemia.

  5. Synthesis and Biological Evaluation of Novel Aryl-2H-pyrazole Derivatives as Potent Non-purine Xanthine Oxidase Inhibitors.

    PubMed

    Sun, Zhi-Gang; Zhou, Xiao-Jing; Zhu, Ming-Li; Ding, Wen-Ze; Li, Zhen; Zhu, Hai-Liang

    2015-01-01

    A series of aryl-2H-pyrazole derivatives were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro as potent xanthine oxidase inhibitors. Among them, 2 aryl-2H-pyrazole derivatives showed significant inhibitory activities against xanthine oxidase. Compound 19 emerged as the most potent xanthine oxidase inhibitor (IC50=9.8 µM) in comparison with allopurinol (IC50=9.5 µM). The docking study revealed that compound 19 might have strong interactions with the active site of xanthine oxidase. This compound is thus a new candidate for further development for the treatment of gout.

  6. Xanthine oxidase inhibitors from Garcinia esculenta twigs.

    PubMed

    Zhu, Lun-Lun; Fu, Wen-Wei; Watanabe, Shimpei; Shao, Yi-Nuo; Tan, Hong-Sheng; Zhang, Hong; Tan, Chang-Heng; Xiu, Yan-Feng; Norimoto, Hisayoshi; Xu, Hong-Xi

    2014-12-01

    The EtOAc-soluble portion of the 80 % (v/v) EtOH extract from the twigs of Garcinia esculenta exhibited strong xanthine oxidase inhibition in vitro. Bioassay-guided purification led to the isolation of 1,3,6,7-tetrahydroxyxanthone (3) and griffipavixanthone (8) as the main xanthine oxidase inhibitors, along with six additional compounds (1, 2, 4-7), including two new compounds (1 and 2). This enzyme inhibition was dose dependent with an IC50 value of approximately 1.2 µM for 3 and 6.3 µM for 8. The inhibitory activity of 3 was stronger than the control allopurinol (IC50 value: 5.3 µM). To our knowledge, compound 8 is the first bixanthone that demonstrated potent XO inhibitory activity in vitro. The structures of the new compounds were established by spectroscopic analysis, and the optical properties and absolute stereochemistry of racemic (±) esculentin A (2) were further determined by the calculation of the DP4 probability and analysis of its MTPA ester derivatives.

  7. In vitro xanthine oxidase inhibitory and in vivo hypouricemic activity of herbal coded formulation (Gouticin).

    PubMed

    Akram, Muhammad; Usmanghani, Khan; Ahmed, Iqbal; Azhar, Iqbal; Hamid, Abdul

    2014-05-01

    Currently, natural products have been used in treating gouty arthritis and are recognized as xanthine oxidase inhibitors. Current study was designed to evaluate in vitro xanthine oxidase inhibitory potential of Gouticin and its ingredients extracts and in vivo hypouricemic activity of gouticin tablet 500 mg twice daily. Ethanol extracts of Gouticin and its ingredients were evaluated in vitro, at 200, 100, 50, 25 μ g/ml concentrations for xanthine oxidase inhibitory activity. IC(50) values of Gouticin and its ingredients were estimated. Further, in vivo therapeutic effect of Gouticin was investigated in comparison with allopathic medicine (Allopurinol) to treat gout. Total patients were 200 that were divided into test and control group. Herbal coded medicine (Gouticin) was given to test group and allopathic medicine allopurinol was administered to control group. In vitro, Gouticin has the highest percent inhibition at 96% followed by Allopurinol with 93% inhibition. In vivo study, mean serum uric acid level of patients was 4.62 mg/dl and 5.21mg/dl by use of Gouticin and Allopurinol at end of therapy. The study showed that herbal coded formulation gouticin and its ingredients are potential sources of natural xanthine oxidase inhibitors. Gouticin 500 mg twice daily is more effective than the allopurinol 300mg once daily in the management of gout.

  8. Simulation of the solid state and the first and second hydration shell of the xanthine oxidase inhibitor allopurinol: Structures obtained using DFT and MP2 methods

    NASA Astrophysics Data System (ADS)

    Álvarez-Ros, M. C.; Alcolea Palafox, M.

    2016-05-01

    Allopurinol (AL) was investigated in the isolated state, solid state, and in the hydrated form through an exhaustive quantum-chemical analysis. Full geometry optimization and energy calculations of the 11 most stable tautomers in the isolated state were performed. The most stable one corresponds to AL-15 at all the levels of computation. Several weak bands of the experimental spectrum in Ar-matrix were tentatively assigned to tautomer AL-25. The crystal unit cell of the solid state was simulated by a heptamer form. Comparisons with the experimental values confirm that only tautomer AL-15 appears in the solid state. The hydration of the two most stable tautomers: AL-15 and AL-25 was carried out. Solvent effects were considered using the Tomasi's polarized continuum model (PCM) and by the explicit model (EM) including a variable number of water molecules surrounding the base to simulate the first and second hydration shells. In this second hydration shell the calculated difference in the free energy between both tautomers is so small, that both tautomers can coexist in bull water. Some of the most important conclusions on the effects of the hydration on the molecular structure were presented. The deformation and interaction energies were corrected for basis set superposition error (BSSE) using the counterpoise (CP) procedure.

  9. Xanthine oxidase and xanthine dehydrogenase from an estivating land snail.

    PubMed

    Hermes-Lima, M; Storey, K B

    1995-01-01

    During arousal from estivation in land snails, Otala lactea, active metabolic functions are restored within minutes and oxygen consumption increases dramatically. During the transition from the hypoxic conditions of estivation to normoxia it is possible that xanthine oxidase (XO) in hepatopancreas contributes to the observed lipid peroxidation. Using a fluorometric assay that is based on the oxidation of pterin, the activities and some properties of XO and XO+XDH (sum of XO and xanthine dehydrogenase activities) were measured in hepatopancreas extracts. Km values for pterin for XO and XO+XDH were 9 and 6 microM, respectively, and the Km of XDH for methylene blue was 5 microM. Both XO+XDH and XO activities were inhibited by allopurinol (I50 = 2 microM), pre-incubation at 40 degrees C, and by 5 min H2O2 pre-exposure. Inclusion of azide in the reaction promoted a rise of approximately 70-fold in the inactivation power of H2O2 due to inhibition of high endogenous catalase activity. The I50 for H2O2 of XO+XDH and XO activities in the presence of azide was 0.04 and 0.11 mM, respectively. Unlike the situation for mammalian XO, a previous reduction of O. lactea XO (by pterin) was not necessary to make the enzyme susceptible to H2O2 effects. Interestingly, methylene blue partially prevented both heat- and H2O2-induced inactivation of XO+XDH activity. These data indicate that the formation of an enzyme-methylene blue complex induces protection against heat and oxidative damage at the FAD-active site. Both XO and XO+XDH activites were significantly higher in snails after 35 days of estivation compared with active snails 24 h after arousal from dormancy. The ratio of XO/(XO+XDH) activities was also slightly increased in estivating O. lactea (from 0.07 to 0.09; P < 0.025). XO activity was 0.03 nmol.min-1.mg protein-1 in estivating snails. Compared with hepatopancreas catalase, XO activity is probably too low to contribute significantly to the net generation of oxyradicals, and

  10. 9-Benzoyl 9-deazaguanines as potent xanthine oxidase inhibitors.

    PubMed

    Rodrigues, Marili V N; Barbosa, Alexandre F; da Silva, Júlia F; dos Santos, Deborah A; Vanzolini, Kenia L; de Moraes, Marcela C; Corrêa, Arlene G; Cass, Quezia B

    2016-01-15

    A novel potent xanthine oxidase inhibitor, 3-nitrobenzoyl 9-deazaguanine (LSPN451), was selected from a series of 10 synthetic derivatives. The enzymatic assays were carried out using an on-flow bidimensional liquid chromatography (2D LC) system, which allowed the screening¸ the measurement of the kinetic inhibition constant and the characterization of the inhibition mode. This compound showed a non-competitive inhibition mechanism with more affinity for the enzyme-substrate complex than for the free enzyme, and inhibition constant of 55.1±9.80 nM, about thirty times more potent than allopurinol. Further details of synthesis and enzymatic studies are presented herein.

  11. Xanthine dehydrogenase to xanthine oxidase conversion in ischemic rat intestine

    SciTech Connect

    McKelvey, T.G.; Engerson, T.D.; Elmore, C.R.; Jones, H.P. )

    1990-02-26

    The ischemic conversion of the NADH-producing xanthine dehydrogenase (XDH) to an oxidase form, that produces both superoxide radical and hydrogen peroxide, has been proposed as an important step in initiating oxygen radical-mediated ischemia-reperfusion injury. It has also been reported that two forms of converted oxidase are produced in ischemic rat liver; a reversible xanthine oxidase produced through sulfhydryl oxidation, that can be reconverted to XDH by incubation with 10mM dithiothreitol (Dtt) at 37{degrees}C, and a Dtt-irreversible oxidase produced via proteolysis. The authors report that increased oxidase in the ischemic rat intestine results from significant increases in both the Dtt-reversible and Dtt-irreversible forms of xanthine oxidase. Total oxidase activity (Irreversible + Dtt-reversible) was 19% of the total enzyme activity (XDH + XO) in control ileum and distal jejunum, increased to 26% after 1 hour of ischemia at 37{degrees}C, and significantly to 36% after 1.5 hours. After 3 hours 73% of the activity was in the oxidase form. Irreversible oxidase comprised 15% of the total activity in control intestine, significantly increased to 25% after 2 hours, and further to 42% after 3 hours. Dtt-reversible oxidase was 3% of the total activity in controls, increased to 13% after 1.5 hours, and significantly to 29% after 2 hours.

  12. Genistein effect on xanthine oxidase activity.

    PubMed

    Sumbayev, V V

    2001-01-01

    Genistein was defined to be an allosteric xanthine oxidase inhibitor in the concentrations 0.1-4.0 microM and xanthine oxidase activator with superoxide scavenging activity in the concentrations 5.0 microM and higher. But the most effective allosteric binding with the highest affinity was observed in the genistein concentrations 0.1-1.0 microM. Intraperitoneum injections of genistein (500 micrograms/kg) during three days with the interval 24 hours decrease xanthine oxidase activity in the liver, lung and brain of the Vistar rats.

  13. Endogenous xanthine oxidase-derived O sub 2 metabolites inhibit surfactant metabolism

    SciTech Connect

    Baker, R.R.; Panus, P.C.; Holm, B.A.; Engstrom, P.C.; Freeman, B.A.; Matalon, S. )

    1990-10-01

    The ability of xanthine oxidase (XO)-derived, partially reduced O2 species (PROS) to inhibit surfactant production was examined in freshly isolated alveolar type II (ATII) pneumocytes from New Zealand White rabbits. (Methyl-3H)choline chloride and (1-14C)palmitate incorporation into phosphatidylcholine (PC) decreased in a dose-dependent manner, whereas peak media hydrogen peroxide (H2O2) concentration increased, when 1, 5, or 10 mU/ml XO were added to cell suspensions containing 500 microM xanthine. Addition of 100 microM allopurinol inhibited H2O2 production and abolished the decrease in choline and palmitate incorporation into PC. ATII cells incubated with 500 microM xanthine alone incorporated choline and palmitate at 90 and 80% of control levels, respectively. However, 100 microM allopurinol restored precursor incorporation to control values. To identify a possible intracellular source of PROS, ATII cell xanthine dehydrogenase (XDH) and XO activities were measured. Both total activity (XDH + XO; 45 +/- 7 microU/mg protein) and the percentage activity in the oxidase form (%XO; 30 +/- 4%) remained unchanged in ATII cells incubated in media only (control) for 2 h. In contrast, incubation of ATII cells with 500 microM xanthine resulted in a 50% loss of XDH + XO activity and a 21% increase in %XO within 10 min. After 2 h there was no measurable XDH + XO activity in xanthine-treated cells. Total XDH + XO activity in cells incubated with 500 microM xanthine and 100 microM allopurinol was less than 6% of control values throughout the incubation.

  14. Fluorescence quenching study of quercetin interaction with bovine milk xanthine oxidase

    NASA Astrophysics Data System (ADS)

    Rasoulzadeh, Farzaneh; Jabary, Hamideh Nadjarpour; Naseri, Abdolhossein; Rashidi, Mohammad-Reza

    2009-02-01

    Quercetin is a natural flavonoid with many important therapeutic properties. The interaction of this polyphenolic compound bovine milk xanthine oxidase as one of its major target proteins was studied using fluorescence quenching method for the first time. It was found that the fluorescence quenching of xanthine oxidase occurs through a static mechanism. The results revealed the presence of a single binding site on xanthine oxidase with the binding constant value equals to 1.153 × 10 4 l mol -1 at 310 K and pH 7.4. The thermodynamic parameters were also calculated at different temperatures. The enthalpy and entropy changes were found as -10.661 kJ mol -1 and +43.321 J mol -1 K -1 indicating that both hydrogen binding and hydrophobic are involved in the interaction of this polyphenolic natural compound with xanthine oxidase. The results may provide a ground for further studies with different flavonoids to find a safe alternative for allopurinol, the only xanthine oxidase inhibitor with clinical application.

  15. Thermostable Xanthine Oxidase Activity from Bacillus pumilus RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization.

    PubMed

    Sharma, Nirmal Kant; Thakur, Shikha; Thakur, Neerja; Savitri; Bhalla, Tek Chand

    2016-03-01

    Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051 U/mg dcw (dry cell weight) to 0.209 U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t1/2 at 70 and 80 °C were 5 and 1 h, respectively. Activity of this enzyme was strongly inhibited by Hg(2+), Ag(+) and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.

  16. [Pyonephrosis due to xanthine stones in a bitch treated with allopurinol].

    PubMed

    Maier, R; Lutter, F X; Lohss-Baumgärtner, E

    2014-01-01

    A 2-year-old, neutered, crossbreed bitch was presented as an emergency with painful abdomen, fever and vomiting. The cause of the acute abdomen was a pyonephrosis of the left kidney, caused by four xanthine stones, which had blocked the ureter. After surgical removal of the heavily altered left kidney, the bitch recovered rapidly. Because of a leishmaniasis the bitch had been treated with allopurinol over an extended period, the xanthine stone formation is likely to have resulted from allopurinol usage. Because there were additionally small concrements in the right kidney, the medication was stopped. Subsequently, the dog has received a low purine diet, and the leishmaniasis titer and renal function have been monitored regularly.

  17. Development of a high analytical performance-xanthine biosensor based on layered double hydroxides modified-electrode and investigation of the inhibitory effect by allopurinol.

    PubMed

    Shan, Dan; Wang, Yanna; Zhu, Mingjuan; Xue, Huaiguo; Cosnier, Serge; Wang, Chengyin

    2009-01-01

    The determination of xanthine has considerable importance in clinical and food quality control. Therefore, in this present work, we developed a novel xanthine biosensor based on immobilization of xanthine oxidase (XnOx) by attractive materials layered double hydroxides (LDHs). Amperometric detection of xanthine was evaluated by holding the modified electrode at 0.55V (versus saturated calomel electrode (SCE)). Due to the special properties of LDHs, such as chemical inertia, mechanical and thermal stability, anionic exchange ability, high porosity and swelling properties, XnOx/LDHs-modified electrode exhibited a developed analytical performance. The biosensor provided a linear response to xanthine over a concentration range of 1 x 10(-6)M to 2 x 10(-4)M with a sensitivity of 220 mAM(-1)cm(-2) and a detection limit of 1x10(-7)M based on S/N=3. In addition, the immobilized XnOx layers have been characterized using atomic force microscopy under both air atmosphere and liquid environment, which exhibited the interesting swelling phenomenon of LDHs. The investigation of inhibition of XnOx by allopurinol was carried out using this XnOx/LDHs-modified electrode. The experimental results indicated that inhibitory effect could be achieved by allopurinol with a quasi-reversible competitive type.

  18. IRON REGULATES XANTHINE OXIDASE ACTIVITY IN THE LUNG

    EPA Science Inventory

    The iron chelator deferoxamine has been reported to inhibit both xanthine oxidase (XO) and xanthine dehydrogenase activity, but the relationship of this effect to the availability of iron in the cellular and tissue environment remains unexplored. XO and total xanthine oxidoreduct...

  19. Effects of xanthine oxidase inhibitors on renal function and blood pressure in hypertensive patients with hyperuricemia.

    PubMed

    Kohagura, Kentaro; Tana, Takeshi; Higa, Akira; Yamazato, Masanobu; Ishida, Akio; Nagahama, Kazufumi; Sakima, Atsushi; Iseki, Kunitoshi; Ohya, Yusuke

    2016-08-01

    Hyperuricemia may promote the progression of hypertension and renal dysfunction. However, the effects of hyperuricemia treatment on blood pressure and renal function in adult hypertensive patients with hyperuricemia remain unclear. A total of 137 hypertensive patients with hyperuricemia (96 men and 41 women; mean age of 67 years) who recently started taking xanthine oxidase inhibitors (allopurinol or febuxostat) as outpatients were recruited. Serum uric acid level, estimated glomerular filtration rate (eGFR, ml min(-1) per 1.73 m(2)) and blood pressure (mm Hg) were retrospectively compared immediately before and shortly after starting treatment with xanthine oxidase inhibitors. The mean blood pressure and the eGFR immediately before starting treatment were 128/71 mm Hg and 44.6 ml min(-1) per 1.73 m(2), respectively. Although the eGFR decreased from 46.6 to 44.6 ml min(-1) per 1.73 m(2) before starting treatment with xanthine oxidase inhibitors, it increased to 46.2 ml min(-1) per 1.73 m(2) (P=0.001, compared with immediately before treatment) without any significant changes in blood pressure after the administration of xanthine oxidase inhibitors. Multiple regression analysis revealed that the increase in eGFR after starting xanthine oxidase inhibitor treatment positively correlated with the changes in systolic blood pressure and negatively correlated with the changes in uric acid levels and the use of renin-angiotensin system inhibitors. These results suggest that xanthine oxidase inhibitors may delay the progression of renal dysfunction in adult hypertensive patients with hyperuricemia.

  20. Effects of Xanthine Oxidase Inhibition in Hyperuricemic Heart Failure Patients: The EXACT-HF Study

    PubMed Central

    Givertz, Michael M.; Anstrom, Kevin J.; Redfield, Margaret M.; Deswal, Anita; Haddad, Haissam; Butler, Javed; Tang, W.H. Wilson; Dunlap, Mark E.; LeWinter, Martin M.; Mann, Douglas L.; Felker, G. Michael; O’Connor, Christopher M.; Goldsmith, Steven R.; Ofili, Elizabeth O.; Saltzberg, Mitchell T.; Margulies, Kenneth B.; Cappola, Thomas P.; Konstam, Marvin A.; Semigran, Marc J.; McNulty, Steven E.; Lee, Kerry L.; Shah, Monica R.; Hernandez, Adrian F.

    2015-01-01

    Background Oxidative stress may contribute to heart failure (HF) progression. Inhibiting xanthine oxidase in hyperuricemic HF patients may improve outcomes. Methods and Results We randomized 253 patients with symptomatic HF, left ventricular ejection fraction (LVEF) ≤40%, and serum uric acid levels ≥9.5 mg/dL to receive allopurinol (target dose, 600 mg daily) or placebo in a double-blind, multicenter trial. The primary composite endpoint at 24 weeks was based on survival, worsening HF, and patient global assessment. Secondary endpoints included change in quality of life, submaximal exercise capacity, and LVEF. Uric acid levels were significantly reduced with allopurinol compared to placebo (treatment difference, −4.2 [−4.9, −3.5] mg/dL and −3.5 [−4.2, −2.7] mg/dL at 12 and 24 weeks, respectively, both P<0.0001). At 24 weeks, there was no significant difference in clinical status between the allopurinol- and placebo-treated patients (worsened 45% vs. 46%, unchanged 42% vs. 34%, improved 13% vs. 19%, respectively; P=0.68). At 12 and 24 weeks, there was no significant difference in change in Kansas City Cardiomyopathy Questionnaire scores or 6-minute walk distances between the 2 groups. At 24 weeks, LVEF did not change in either group or between groups. Rash occurred more frequently with allopurinol (10% vs. 2%, P=0.01), but there was no difference in serious adverse event rates between the groups (20% vs. 15%, P=0.36). Conclusions In high-risk HF patients with reduced ejection fraction and elevated uric acid levels, xanthine oxidase inhibition with allopurinol failed to improve clinical status, exercise capacity, quality of life, or LVEF at 24 weeks. PMID:25986447

  1. Monochloramine produces reactive oxygen species in liver by converting xanthine dehydrogenase into xanthine oxidase

    SciTech Connect

    Sakuma, Satoru Miyoshi, Emi; Sadatoku, Namiko; Fujita, Junko; Negoro, Miki; Arakawa, Yukio; Fujimoto, Yohko

    2009-09-15

    In the present study, we assessed the influence of monochloramine (NH{sub 2}Cl) on the conversion of xanthine dehydrogenase (XD) into xanthine oxidase (XO) in rat liver in vitro. When incubated with the partially purified cytosolic fraction from rat liver, NH{sub 2}Cl (2.5-20 {mu}M) dose-dependently enhanced XO activity concomitant with a decrease in XD activity, implying that NH{sub 2}Cl can convert XD into the reactive oxygen species (ROS) producing form XO. The NH{sub 2}Cl (5 {mu}M)-induced XD/XO interconversion in the rat liver cytosol was completely inhibited when added in combination with an inhibitor of NH{sub 2}Cl methionine (25 {mu}M). A sulfhydryl reducing agent, dithiothreitol at concentrations of 0.1, 1 and 5 mM also dose-dependently reversed the NH{sub 2}Cl (5 {mu}M)-induced XD/XO interconversion. These imply that NH{sub 2}Cl itself acts on the XD/XO interconversion, and that this conversion occurs at the cysteine residues in XD. Furthermore, using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate, it was found that NH{sub 2}Cl could increase ROS generation in the cytoplasm of rat primary hepatocyte cultures, and that this increase might be reversed by an XO inhibitor, allopurinol. These results suggest that NH{sub 2}Cl has the potential to convert XD into XO in the liver, which in turn may induce the ROS generation in this region.

  2. Xanthine oxidase inhibiting effects of noni (Morinda citrifolia) fruit juice.

    PubMed

    Palu, Afa; Deng, Shixin; West, Brett; Jensen, Jarakae

    2009-12-01

    Morinda citrifolia L. (noni), family Rubiaceae, has been used in Polynesia for over 2000 years for its reputed health benefits, one of which is its therapeutic effects on gout (langa e hokotanga hui). However, its healing mechanism has not been elucidated. This study showed that in an in vitro bioassay that Tahitian Noni Juice (TNJ) inhibited xanthine oxidase (XO) concentration dependently. Concentrations of 1, 5 and 10 mg/mL of TNJ inhibited XO by 11%, 113% and 148%, respectively, with an IC50 of 3.8 mg compared with an IC50 of 2.4 microm for allopurinol. Noni fruit juice concentrate (NFJC) also inhibited XO concentration dependently. Concentrations of 1 and 5 mg/mL NFJC inhibited XO in vitro by 184% and 159%, respectively. A 0.1 mg/mL methanol extract (NFJME) from the fractionation of noni fruit puree inhibited XO by 64%. It was elucidated that the noni fruit juice inhibitory effect on XO enzymes is the mechanism by which noni ameliorates gout and gout-like diseases. Further, the results also support the traditional usage of noni in the treatment of gout.

  3. Allopurinol

    MedlinePlus

    ... the body caused by certain cancer medications, and kidney stones. Allopurinol is in a class of medications called ... of uric acid may cause gout attacks or kidney stones. Allopurinol is used to prevent gout attacks, not ...

  4. Aldehyde-induced xanthine oxidase activity in raw milk.

    PubMed

    Steffensen, Charlotte L; Andersen, Henrik J; Nielsen, Jacob H

    2002-12-04

    In the present study, the aldehyde-induced pro-oxidative activity of xanthine oxidase was followed in an accelerated raw milk system using spin-trap electron spin resonance (ESR) spectroscopy. The aldehydes acetaldehyde, propanal, hexanal, trans-2-hexenal, trans-2-heptenal, trans-2-nonenal, and 3-methyl-2-butenal were all found to initiate radical reactions when added to milk. Formation of superoxide through aldehyde-induced xanthine oxidase activity is suggested as the initial reaction, as all tested aldehydes were shown to trigger superoxide formation in an ultrahigh temperature (UHT) milk model system with added xanthine oxidase. It was found that addition of aldehydes to milk initially increased the ascorbyl radical concentration with a subsequent decay due to ascorbate depletion, which renders the formation of superoxide in milk with added aldehyde. The present study shows for the first time potential acceleration of oxidative events in milk through aldehyde-induced xanthine oxidase activity.

  5. Purification of xanthine dehydrogenase and sulfite oxidase from chicken liver.

    PubMed

    Ratnam, K; Brody, M S; Hille, R

    1996-05-01

    Xanthine dehydrogenase and sulfite oxidase from chicken liver are oxomolybdenum enzymes which catalyze the oxidation of xanthine to uric acid and sulfite to sulfate, respectively. Independent purification protocols have been previously described for both enzymes. Here we describe a procedure by which xanthine dehydrogenase and sulfite oxidase are purified simultaneously from the same batch of fresh chicken liver. Also, unlike the protocols described earlier, this procedure avoids the use of acetone extraction as well as a heat step, thus minimizing damage to the molybdenum centers of the enzymes.

  6. The effects of allopurinol, uric acid, and inosine administration on xanthine oxidoreductase activity and uric acid concentrations in broilers.

    PubMed

    Settle, T; Carro, M D; Falkenstein, E; Radke, W; Klandorf, H

    2012-11-01

    The purpose of these studies was to determine the effects of uric acid (UA) and inosine administration on xanthine oxidoreductase activity in broilers. In experiment one, 25 broilers were assigned to 5 treatment groups: control, AL (25 mg of allopurinol/kg of body mass), AR (AL for 2 wk followed by allopurinol withdrawal over wk 3), UAF (AL plus 6.25 g of UA sodium salt/kg of feed), and UAI (AL plus 120 mg of UA sodium salt injected daily). The UA administration had no effect on plasma concentration of UA (P > 0.05), and all allopurinol-treated birds had lower (P < 0.05) UA levels than controls. The UA concentrations were restored in both plasma and kidney of AR birds at wk 3, but liver UA concentrations remained lower. Whereas xanthine oxidoreductase (XOR) activity in the liver (LXOR) was reduced (P < 0.05) by allopurinol treatment, XOR activity in the kidney (KXOR) was not affected (P = 0.05). In experiment two, 3 groups of 5 birds each were fed 0 (control), 0.6 M inosine/kg of feed (INO), or INO plus 50 mg of allopurinol/kg of body mass (INOAL). The INOAL birds showed lower total LXOR activity, but KXOR activity was not affected. Both INO and INOAL birds had higher plasma and kidney UA concentrations than controls. The results suggest that regulation of UA production is tissue dependent.

  7. Synthesis and evaluation of xanthine oxidase inhibitory and antioxidant activities of 2-arylbenzo[b]furan derivatives based on salvianolic acid C.

    PubMed

    Tang, Hong-Jin; Zhang, Xiao-Wei; Yang, Lin; Li, Wei; Li, Jia-Huang; Wang, Jin-Xin; Chen, Jun

    2016-11-29

    Xanthine oxidase (XO) is the key enzyme in humans which is related to a variety of diseases such as gout, hyperuricemia and cardiovascular diseases. In this work, a series of 2-arylbenzo[b]furan derivatives were synthesized based on salvianolic acid C, and they were evaluated for xanthine oxidase inhibitory and antioxidant activities. Compounds 5b, 6a, 6e and 6f showed potent xanthine oxidase inhibitory activities with IC50 values ranging from 3.99 to 6.36 μM, which were comparable with that of allopurinol. Lineweaver-Burk plots analysis revealed that the representative derivative 6e could bind to either xanthine oxidase or the xanthine oxidase-xanthine complex, which exhibited a mixed-type competitive mechanism. A DPPH radical scavenging assay showed most of the hydroxyl-functionalized 2-arylbenzo[b]furan derivatives possessed the potent antioxidant activity, which was further validated on LPS-stimulated RAW 264.7 macrophages model. The structure-activity relationships were preliminary analyzed and indicated that the structural skeleton of 2-arylbenzo[b]furan and phenolic hydroxyl groups played an important role in maintaining xanthine oxidase inhibitory effect and antioxidant property for the series of derivatives. Meanwhile, molecular docking studies were performed to further confirm the structure-activity relationships and investigate the proposed binding mechanisms of compounds 5d, 6d and 10d binding to the protein.

  8. Amperometric biosensor based on prussian blue and nafion modified screen-printed electrode for screening of potential xanthine oxidase inhibitors from medicinal plants.

    PubMed

    El Harrad, Loubna; Amine, Aziz

    2016-04-01

    A simple and sensitive amperometric biosensor was developed for the screening of potential xanthine oxidase inhibitors from medicinal plants. This biosensor was prepared by immobilization of xanthine oxidase on the surface of prussian blue modified screen-printed electrodes using nafion and glutaraldehyde. The developed biosensor showed a linear amperometric response at an applied potential of +0.05 V toward the detection of hypoxanthine from 5 μM to 45 μM with a detection limit of 0.4 μM (S/N=3) and its sensitivity was found to be 600 mA M(-1) cm(-2). In addition, the biosensor exhibited a good storage stability. The inhibition of xanthine oxidase by allopurinol was studied under the optimized conditions. The linear range of allopurinol concentration is obtained up to 2.5 μM with an estimated 50% of inhibitionI50=1.8 μM. The developed biosensor was successfully applied to the screening of xanthine oxidase inhibitors from 13 medicinal plants belonging to different families. Indeed, Moroccan people traditionally use these plants as infusion for the treatment of gout and its related symptoms. For this purpose, water extracts obtained from the infusion of these plants were used for the experiments. In this work, 13 extracts were assayed and several of them demonstrated xanthine oxidase inhibitory effect, with an inhibition greater than 50% compared to spectrophotometry measurements that only few extracts showed an inhibition greater than 50%.

  9. Effects of allopurinol on uric acid concentrations, xanthine oxidoreductase activity and oxidative stress in broiler chickens.

    PubMed

    Carro, M D; Falkenstein, E; Radke, W J; Klandorf, H

    2010-01-01

    The purpose of this study was to determine the effects of allopurinol (AL) on xanthine oxidoreductase (XOR) activity and uric acid (UA) levels in chickens. Thirty 5-week-old broilers were divided into three groups and fed 0 (control), 25 (AL25) or 50 (AL50) mg AL per kg of body mass for 5 weeks. Chicks were weighed twice weekly and leukocyte oxidative activity (LOA) and plasma purine levels were determined weekly in five birds per group. Chicks were sacrificed after 2 or 5 weeks, and samples from tissues were taken for analysis of XOR activity. Plasma UA concentrations were lower (P<0.001) and xanthine and hypoxanthine concentrations were greater (P<0.001) in AL25 and AL50 birds compared to controls, whereas no differences (P=0.904) were detected in allantoin concentrations. By week 5, body mass was reduced (P<0.001) to 84.0 and 65.1% of that in controls for AL25 and AL50 broilers, respectively, and LOA was 4.1 times greater (P<0.05) in AL25 compared to control birds. Liver XOR activity was increased by 1.1 and 1.2 times in AL25 and AL50 birds, but there was no change (P>0.05) in XOR activity in the pancreas and intestine. These results suggest that AL effect on XOR activity is tissue dependent.

  10. Xanthine oxidase and the fetal cardiovascular defence to hypoxia in late gestation ovine pregnancy

    PubMed Central

    Kane, Andrew D; Hansell, Jeremy A; Herrera, Emilio A; Allison, Beth J; Niu, Youguo; Brain, Kirsty L; Kaandorp, Joepe J; Derks, Jan B; Giussani, Dino A

    2014-01-01

    Hypoxia is a common challenge to the fetus, promoting a physiological defence to redistribute blood flow towards the brain and away from peripheral circulations. During acute hypoxia, reactive oxygen species (ROS) interact with nitric oxide (NO) to provide an oxidant tone. This contributes to the mechanisms redistributing the fetal cardiac output, although the source of ROS is unknown. Here, we investigated whether ROS derived from xanthine oxidase (XO) contribute to the fetal peripheral vasoconstrictor response to hypoxia via interaction with NO-dependent mechanisms. Pregnant ewes and their fetuses were surgically prepared for long-term recording at 118 days of gestation (term approximately 145 days). After 5 days of recovery, mothers were infused i.v. for 30 min with either vehicle (n = 11), low dose (30 mg kg−1, n = 5) or high dose (150 mg kg−1, n = 9) allopurinol, or high dose allopurinol with fetal NO blockade (n = 6). Following allopurinol treatment, fetal hypoxia was induced by reducing maternal inspired O2 such that fetal basal decreased approximately by 50% for 30 min. Allopurinol inhibited the increase in fetal plasma uric acid and suppressed the fetal femoral vasoconstrictor, glycaemic and lactate acidaemic responses during hypoxia (all P < 0.05), effects that were restored to control levels with fetal NO blockade. The data provide evidence for the activation of fetal XO in vivo during hypoxia and for XO-derived ROS in contributing to the fetal peripheral vasoconstriction, part of the fetal defence to hypoxia. The data are of significance to the understanding of the physiological control of the fetal cardiovascular system during hypoxic stress. The findings are also of clinical relevance in the context of obstetric trials in which allopurinol is being administered to pregnant women when the fetus shows signs of hypoxic distress. PMID:24247986

  11. An amperometric biosensor for xanthine determination prepared from xanthine oxidase immobilized in polypyrrole film.

    PubMed

    Arslan, Fatma; Yaşar, Ahmet; Kiliç, Esma

    2006-01-01

    In order to prepare a biosensor for the determination of xanthine, electropolymerization of pyrrole on Pt surface was carried out with an electrochemical cell containing pyrrole, ferrocene (as a electron mediator) and tetrabutylamonium tetrafluoroborat in acetonitrile by cyclic voltammetry between 0.0 and 0.9V (vs SCE) at a scan rate of 50mV/s upon Pt electrode. Xanthine oxidase was immobilized by a glutaraldehyde/bovine serum albumin (BSA) crosslinking procedure on to polypyrrole film after the electropolymerization processes. The response of the biosensor against xanthine was measured after 3-4 min following the application of a constant potential of + 0.7 V (vs SCE). The resulting biosensor exhibits excellent electrocatalysis for the xanthine. The amperometric determination is based on the electrochemical detection of H202, which is generated in enzymatic reaction of xanthine. The effect of various experimental conditions was examined for the determination of the analytical performance. The sensor responds to xanthine with a detection limit of 1.0 x 10(-6)M. The response current increases linearly with xanthine concentration up to 4.0 x 10(-4) M. The sensor remains relatively stable for 45 days.

  12. Xanthine oxidase-generated hydrogen peroxide is a consequence, not a mediator of cell death.

    PubMed

    Czupryna, Julie; Tsourkas, Andrew

    2012-03-01

    Oxidative stress has been associated with a wide range of diseases including atherosclerosis, cancer and Alzheimer's disease. When present in excessive concentrations, reactive oxygen species (ROS) can cause deleterious effects. This has led to the notion that the anticancer effects of various chemotherapeutics may be mediated, at least in part, by an increase in ROS. To investigate the role of xanthine oxidase (XO), a source of hydrogen peroxide, in cell death, MCF7, HeLa and 293T cells were treated with various cell-death-inducing drugs in the presence and absence of allopurinol, a specific inhibitor of XO. In the absence of allopurinol, each drug led to a time- and concentration-dependent increase in percent DNA fragmentation and ROS levels, regardless of the mechanism of cell death incurred, i.e. caspase dependent and caspase independent. By contrast, pretreatment with allopurinol led to dramatically lower ROS levels in all cases, suggesting that XO is a major contributor to oxidative stress. However, allopurinol did not exhibit a protective effect against cell death. Similarly, the administration of siRNA against XO also did not exhibit a protective effect against cell death. The level of oxidative stress was recorded using the ROS sensor CM-H(2) DCFDA and a ratiometric bioluminescent assay that takes advantage of the increased sensitivity of Firefly luciferase to hydrogen peroxide, compared with a stable variant of Renilla luciferase (RLuc), RLuc8. Overall, these findings suggest that XO-generated hydrogen peroxide, and perhaps hydrogen peroxide in general, is a consequence, but not a mediator of cell death.

  13. Discovery and biological evaluation of some (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety as potent xanthine oxidase inhibitors.

    PubMed

    Zhang, Ting-Jian; Li, Song-Ye; Yuan, Wei-Yan; Wu, Qing-Xia; Wang, Lin; Yang, Su; Sun, Qi; Meng, Fan-Hao

    2017-02-15

    A series of (1H-1,2,3-triazol-4-yl)methoxybenzaldehyde derivatives containing an anthraquinone moiety were synthesized and identified as novel xanthine oxidase inhibitors. Among them, the most promising compounds 1h and 1k were obtained with IC50 values of 0.6μM and 0.8μM, respectively, which were more than 10-fold potent compared with allopurinol. The Lineweaver-Burk plot revealed that compound 1h acted as a mixed-type xanthine oxidase inhibitor. SAR analysis showed that the benzaldehyde moiety played a more important role than the anthraquinone moiety for inhibition potency. The basis of significant inhibition of xanthine oxidase by 1h was rationalized by molecular modeling studies.

  14. Acrylonitrile-induced gastric toxicity in rats: The role of xanthine oxidase

    PubMed Central

    Al-Abbasi, Fahad A.

    2012-01-01

    Summary Background Acrylonitrile (ACN) is an extensively produced aliphatic nitrile. The gastrointestinal tract is an important target organ for ACN toxicity. The objective of the present study was to investigate the role of xanthine oxidase (XO) in ACN-induced gastric toxicity in rats. Material/Methods We assessed the effect of ACN on oxidative stress parameters as xanthine oxidase (XO) and total xanthine dehydrogenase (XD)/ XO activity, superoxide anion (O2·−) production, reduced glutathione (GSH) levels and lipid peroxidation in gastric tissues. Results A single oral dose of ACN (25 mg/kg) caused a significant enhancement in XO activity. ACN also caused a significant depletion of GSH levels, enhanced O2·− production and increased lipid peroxidation in the time-course experiment. In the dose-response experiment, ACN accelerated the conversion of XD to XO, with a significant depletion of gastric GSH in a dose-related manner. A strong negative correlation existed between the levels of GSH and the percentage enhancement in XO activity (r =−0.997). (O2·−) production and malondialdehyde (MDA) formation were significantly elevated in a dose-related manner. Pretreatment with allopurinol (50 mg/kg) significantly protected against ACN-induced rise in XO activity, depletion of GSH, and elevated production of (O2·−). However, pretreatment with diethyl maleate (DEM; 100 mg/kg) significantly aggravated the ACN-induced GSH depletion and rise in XO activity. Furthermore, DEM significantly enhanced (O2·−) and MDA production. Conclusions The present study indicates that enhancement of XO activity could be implicated in ACN-induced gastric damage in rats. PMID:22648241

  15. Xanthine oxidase inhibition attenuates ischemic-reperfusion lung injury

    SciTech Connect

    Lynch, M.J.; Grum, C.M.; Gallagher, K.P.; Bolling, S.F.; Deeb, G.M.; Morganroth, M.L.

    1988-05-01

    Ischemic-reperfusion lung injury is a factor potentially limiting the usefulness of distant organ procurement for heart-lung transplantation. Toxic oxygen metabolites are considered a major etiologic factor in reperfusion injury. Although oxygen-free radicals may be generated by many mechanisms, we investigated the role of xanthine oxidase in this injury process by using lodoxamide, a xanthine oxidase inhibitor, to inhibit ischemic-reperfusion injury in an isolated rat lung model. Isolated rat lungs were perfused with physiologic salt solution (PSS) osmotically stabilized with Ficoll until circulating blood elements were nondetectable in the pulmonary venous effluent. Lungs were rendered ischemic by interrupting ventilation and perfusion for 2 hr at 37/sup 0/C. After the ischemic interval, the lungs were reperfused with whole blood and lung injury was determined by measuring the accumulation of /sup 125/I-bovine serum albumin in lung parenchyma and alveolar lavage fluid as well as by gravimetric measurements. Lung effluent was collected immediately pre- and postischemia for analysis of uric acid by high-pressure liquid chromatography. Lodoxamide (1 mM) caused significant attenuation of postischemic lung injury. Uric acid levels in the lung effluent confirmed inhibition of xanthine oxidase. Protection from injury was not complete, however, implying that additional mechanisms may contribute to ischemic-reperfusion injury in the lung.

  16. Enhancement of hypoxic liver damage by ethanol. Involvement of xanthine oxidase and the role of glycolysis.

    PubMed

    Younes, M; Strubelt, O

    1987-09-15

    Using isolated hemoglobin-free perfused rat livers we investigated the hepatotoxic effects of hypoxia, ethanol or the combination of both. Hypoxia only (90 min) led to a weak toxicity as evidenced by the efflux of the enzymes glutamate-pyruvate-transaminase (GPT) and sorbitol dehydrogenase (SDH). This toxic effect was slightly higher in livers treated with ethanol (3 g/l) under normoxic conditions. Ethanol added under hypoxic conditions, however, showed a strong hepatotoxic effect. Under hypoxic conditions, lactate + pyruvate production was increased fivefold over control, indicating that glycolysis was more effectively undergone as main source of energy. Addition of ethanol suppressed this effect, indicating that ethanol inhibited glycolysis. These results indicate that ethanol potentiates hypoxic liver damage by inhibiting the main metabolic pathway yielding ATP under low oxygen tension resulting in a severe energy deficit. Allopurinol (100 mg/l) inhibited the toxic effects seen with ethanol + hypoxia. Also, the inhibitory action of ethanol on glycolysis was antagonized. Our results are consistent with the following model: hypoxia converts NAD-dependent xanthine dehydrogenase (XD) into the oxygen-dependent xanthine oxidase (XO). Due to hypoxia and ethanol, purine metabolites and acetaldehyde accumulate and are metabolized via XO. This process leads to the production of oxygen radicals which most probably mediate both the inhibition of glycolysis and the direct toxic effects towards liver cells.

  17. Quantitation of rat liver xanthine oxidase by radioimmunoassay. A mechanism for sex-specific differences

    SciTech Connect

    Decker, D.E.; Levinson, D.J.

    1982-03-01

    To further delineate the mechanism responsible for the differences in xanthine oxidase activity in male and female Sprague-Dawley rats, a sensitive and specific radioimmunoassay (RIA) was developed for the measurement of hepatic xanthine oxidase. The RIA could detect as little as 5 mg of liver enzyme. Specificity of the RIA was confirmed by 1) Ouchterlony double immuno-diffusion in which a single precipitin band exhibited xanthine oxidase activity, when crude liver homogenate and an enzyme-specific stain were used; 2) parallelism between purified 125I-labeled xanthine oxidase and serial dilutions of crude liver homogenate; 3) a linear correlation between xanthine oxidase activity and the level of enzyme protein; and 4) a single protein band coincident with purified xanthine oxidase, when an immunoprecipitate prepared from antisera and crude liver homogenate was analyzed on sodium dodecyl sulfate (SDS) polyacrylamide gels. Whether xanthine oxidase activity was assayed in the absence of nicotinamide adenine dinucleotide (NAD+) (oxidase form) or in the presence of NAD+ (dehydrogenase), male values were consistently higher, and both forms of the enzyme correlated significantly with each other. When purified to homogeneity, neither form of the enzyme was appreciably affected by 17 beta-estradiol or testosterone propionate. When the RIA was employed, levels of hepatic xanthine oxidase were significantly greater in male than in female rats. We concluded from these data that increased xanthine oxidase activity in the male corresponds to a greater quantitative complement of xanthine oxidase protein. Furthermore, lower xanthine oxidase activity in the female cannot be explained by immunologically cross-reactive material without enzyme activity nor by a direct sex-steroid enzyme interaction.

  18. Why do most human liver cytosol preparations lack xanthine oxidase activity?

    PubMed

    Barr, John T; Choughule, Kanika V; Nepal, Sahadev; Wong, Timothy; Chaudhry, Amarjit S; Joswig-Jones, Carolyn A; Zientek, Michael; Strom, Stephen C; Schuetz, Erin G; Thummel, Kenneth E; Jones, Jeffrey P

    2014-04-01

    When investigating the potential for xanthine oxidase (XO)-mediated metabolism of a new chemical entity in vitro, selective chemical inhibition experiments are typically used. Most commonly, these inhibition experiments are performed using the inhibitor allopurinol (AP) and commercially prepared human liver cytosol (HLC) as the enzyme source. For reasons detailed herein, it is also a common practice to perfuse livers with solutions containing AP prior to liver harvest. The exposure to AP in HLC preparations could obviously pose a problem for measuring in vitro XO activity. To investigate this potential problem, an HPLC-MS/MS assay was developed to determine whether AP and its primary metabolite, oxypurinol, are retained within the cytosol for livers that were treated with AP during liver harvest. Differences in enzymatic activity for XO and aldehyde oxidase (AO) in human cytosol that can be ascribed to AP exposure were also evaluated. The results confirmed the presence of residual AP (some) and oxypurinol (all) human liver cytosol preparations that had been perfused with an AP-containing solution. In every case where oxypurinol was detected, XO activity was not observed. In contrast, the presence of AP and oxypurinol did not appear to have an impact on AO activity. Pooled HLC that was purchased from a commercial source also contained residual oxypurinol and did not show any XO activity. In the future, it is recommended that each HLC batch is screened for oxypurinol and/or XO activity prior to testing for XO-mediated metabolism of a new chemical entity.

  19. Sensitive electrochemical measurement of hydroxyl radical generation induced by the xanthine-xanthine oxidase system.

    PubMed

    Tatsumi, Hirosuke; Tsuchiya, Yui; Sakamoto, Koichi

    2014-12-15

    A sensitive electrochemical measurement system for hydroxyl radical (OH) was developed using enzyme-catalyzed signal amplification. In the presence of 2,6-xylenol as a trapping agent, glucose as a substrate, and pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) as a catalyst, the amperometric signal of the trapping adduct 2,6-dimethylhydroquinone (DMHQ) produced by the hydroxylation of 2,6-xylenol was able to be amplified and detected sensitively. The limit of detection (signal/noise [S/N]=3) for DMHQ was 1 nM. There was no significant interference from urate and other oxidizable compounds in the reaction mixture at the applied potential of 0V versus Ag/AgCl. This method was employed to observe the OH generation induced by the xanthine-xanthine oxidase (XO) system. The reaction rates of the DMHQ production induced from the xanthine-XO system in the presence and absence of various Fe(III) complexes and proteins were compared. Those with a free coordination site on the Fe atom effectively enhanced the OH generation.

  20. Bioactive compounds of inhibiting xanthine oxidase from Selaginella labordei.

    PubMed

    Tan, Wen-Jie; Xu, Jia-Cheng; Li, Li; Chen, Ke-Li

    2009-01-01

    Four flavone compounds were isolated from the effective fractions inhibiting xanthine oxidase (XOD) of the medicinal plant Selaginella labordei with anti-virus activity, and the structures were elucidated as 4'-methylether robustaflavone (1), robustaflavone (2), eriodictyol (3) and amentoflavone (4). The 50% inhibitory concentration (IC(50)) of the three compounds of inhibiting XOD were 61.0, 0.199, 16.0 and 32.0 mg L(-1), respectively. All of these compounds were isolated from the species for the first time, and eriodictyol was found from Selaginellaceae for the first time. Among these compounds, robustaflavone has been reported as an effective compound against the hepatitis B virus.

  1. Design, synthesis and molecular modeling of aloe-emodin derivatives as potent xanthine oxidase inhibitors.

    PubMed

    Shi, Da-Hua; Huang, Wei; Li, Chao; Liu, Yu-Wei; Wang, Shi-Fan

    2014-03-21

    A series of aloe-emodin derivatives were synthesized and evaluated as xanthine oxidase inhibitors. Among them, four aloe-emodin derivatives showed significant inhibitory activities against xanthine oxidase. The compound 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carbaldehyde (A1) possessed the best xanthine oxidase inhibitory activity with IC50 of 2.79 μM. Lineweaver-Burk plot analysis revealed that A1 acted as a mixed-type inhibitor for xanthine oxidase. The docking study revealed that the molecule A1 had strong interactions with the active site of xanthine oxidase and this result was in agreement with kinetic study. Consequently, compound A1 is a new-type candidate for further development for the treatment of gout.

  2. Phospholipid alterations in cardiac sarcoplasmic reticulum induced by xanthine oxidase: contamination of commercial preparations of xanthine oxidase by phospholipase A/sub 2/

    SciTech Connect

    Gamache, D.A.; Kornberg, L.J.; Bartolf, M.; Franson, R.C.

    1986-05-01

    Incubation of cardiac sarcoplasmic reticulum with xanthine oxidase alone at pH 7.0 resulted in a loss of lipid phosphorus that was potentiated by the addition of xanthine. Using autoclaved E.coli with 1-/sup 14/C-oleate in the 2-acyl position of membrane phospholipids, the authors demonstrate that many, but not all, commercial preparations of xanthine oxidase contain significant phospholipase A/sub 2/ (PLA/sub 2/) activity (64.3-545.6 nmols/min/mg). The PLA/sub 2/ was maximally active in the neutral-alkaline pH range, was Ca/sup 2 +/-dependent, and was unaffected by the addition of xanthine. PLA/sub 2/ activity was totally inhibited by 1mM EDTA whereas radical production by optimal concentrations of xanthine/xanthine oxidase (X/XO) was unaffected by EDTA. Chromatographically purified xanthine oxidase (Sigma Grade III) contained high levels of PLA/sub 2/ activity (64.3 nmols/min/mg) compared to endogenous levels of neutral-active, Ca/sup 2 +/-dependent PLA/sub 2/ measured in various tissue homogenates (less than or equal to 0.5 nmols/ min/mg). Because X/XO mixtures are used extensively to study oxygen free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular PLA/sub 2/ may have influenced previously published reports, and such studies should be interpreted cautiously.

  3. Effects of Greek legume plant extracts on xanthine oxidase, catalase and superoxide dismutase activities.

    PubMed

    Spanou, Chrysoula I; Veskoukis, Aristidis S; Stagos, Dimitrios; Liadaki, Kalliopi; Aligiannis, Nectarios; Angelis, Apostolos; Skaltsounis, Alexios-Leandros; Anastasiadi, Maria; Haroutounian, Serkos A; Kouretas, Dimitrios

    2012-03-01

    Legumes are considered to have beneficial health implications, which have been attributed to their phytochemical content. Polyphenols are considered the most important phytochemical compounds extensively studied for their antioxidant properties. The aim of the present study was to examine the effects of potent antioxidant legume plant extracts on xanthine oxidase (XO), catalase (CAT) and superoxide dismutase (SOD) activities. XO exerts a dual role, as it is the major contributor of free radicals during exercise while it generates uric acid, the most potent antioxidant molecule in plasma. CAT and SOD are two of the main enzymes of the antioxidant defence of tissues. We demonstrate that the majority of the extracts inhibited XO activity, but they had no effect on CAT inhibition and SOD induction when used at low concentrations. These results imply that the tested extracts may be considered as possible source of novel XO inhibitors. However, we have shown that allopurinol administration, a known XO inhibitor, before exercise reduces performance and induces oxidative stress in rats. Considering the fact that the extracts examined had an inhibitory effect on XO activity, possibly posing a restriction in their characterization as antioxidants, phytochemical antioxidant administration before exercise should probably be reconsidered.

  4. Longevity and aging. Role of free radicals and xanthine oxidase. A review.

    PubMed

    Labat-Robert, J; Robert, L

    2014-04-01

    Longevity and aging are differently regulated. Longevity has an important part of genetic determinants, aging is essentially post-genetic. Among the genes involved in longevity determination, sirtuins, activated also by calorie restriction and some others as the TOR pathway, attracted special interest after the insulin–IGF pathway first shown to regulate longevity in model organisms. For most of these genes, postponement of life-threatening diseases is the basis of their action which never exceeds about 35% of all determinants, in humans. Among the post-genetic mechanisms responsible for age-related decline of function, free radicals attracted early interest as well as the Maillard reaction, generating also free radicals. Most attempts to remediate to free radical damage failed however, although different scavenger mechanisms and protective substances are present in the organism. Synthetic protectors were also tested without success. The only example of a successful treatment of a free radical mediated pathology is the case of xanthine oxidase, involved in cardiovascular pathology, essentially during the ischemia-reperfusion process. Its inhibition by allopurinol is currently used to fight this deadly syndrome.

  5. Reexamining Michaelis-Menten enzyme kinetics for xanthine oxidase.

    PubMed

    Bassingthwaighte, James B; Chinn, Tamara M

    2013-03-01

    Abbreviated expressions for enzyme kinetic expressions, such as the Michaelis-Menten (M-M) equations, are based on the premise that enzyme concentrations are low compared with those of the substrate and product. When one does progress experiments, where the solute is consumed during conversion to form a series of products, the idealized conditions are violated. Here, we analyzed data of xanthine oxidase in vitro from Escribano et al. (Biochem J 254: 829, 1988) on two conversions in series, hypoxanthine to xanthine to uric acid. Analyses were done using four models: standard irreversible M-M reactions (model 1), Escribano et al.'s M-M forward reaction expressions with product inhibition (model 2), fully reversible M-M equations (model 3), and standard differential equations allowing forward and backward reactions with mass balance accounting for binding (model 4). The results showed that the need for invoking product inhibition vanishes with more complete analyses. The reactions were not quite irreversible, so the backward reaction had a small effect. Even though the enzyme concentration was only 1-2% of the initial substrate concentrations, accounting for the fraction of solutes bound to the enzyme did influence the parameter estimates, but in this case, the M-M model overestimated Michaelis constant values by only about one-third. This article also presents the research and models in a reproducible and publicly available form.

  6. Substrate specificity of guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase for methyl- and nitrobenzaldehydes.

    PubMed

    Veskoukis, Aristidis S; Kouretas, Demetrios; Panoutsopoulos, Georgios I

    2006-01-01

    Both aldehyde oxidase and xanthine oxidase catalyze the oxidation of a wide range of N-heterocycles and aldehydes. These enzymes are important in the oxidation of N-heterocyclic xenobiotics, whereas their role in the oxidation of xenobiotic aldehydes is usually ignored. The present investigation describes the interaction of methyl- and nitrosubstituted benzaldehydes, in the ortho-, meta- and parapositions, with guinea pig liver aldehyde oxidase and bovine milk xanthine oxidase. The kinetic constants showed that most substituted benzaldehydes are excellent substrates of aldehyde oxidase with lower affinities for xanthine oxidase. Low Km values for aldehyde oxidase were observed with most benzaldehydes tested, with 3-nitrobenzaldehyde having the lowest Km value and 3-methylbenzaldehyde being the best substrate in terms of substrate efficiency (Ks). Additionally, low Km values for xanthine oxidase were found with most benzaldehydes tested. However, all benzaldehydes also had low Vmax values, which made them poor substrates of xanthine oxidase. It is therefore possible that aldehyde oxidase may be critical in the oxidation of xenobiotic and endobiotic derived aldehydes and its role in such reactions should not be ignored.

  7. Electrochemistry of xanthine oxidase and its interaction with nitric oxide.

    PubMed

    Zhou, Hui; Xu, Yi; Chen, Ting; Suzuki, Iwao; Li, Genxi

    2006-02-01

    With the help of nanocrystalline TiO2, the direct electrochemistry of xanthine oxidase (XOD) was achieved and two pairs of redox waves were observed. The interaction between XOD and nitric oxide (NO) was also investigated. The experimental results reveal that NO can be reduced at a XOD-nano TiO2 film modified electrode. When the NO concentration was low, the reduced product, HNO, would inactivate the protein. However, when the NO concentration was high, HNO would continue to react with NO to form N2O2- and N3O3-, which would not inhibit XOD, and thus the amount of active protein did not decrease any further.

  8. Organ distribution and molecular forms of human xanthine dehydrogenase/xanthine oxidase protein.

    PubMed

    Sarnesto, A; Linder, N; Raivio, K O

    1996-01-01

    Xanthine dehydrogenase/xanthine oxidase (XDH/XO) is a major cytoplasmic source of superoxide radicals and hydrogen peroxide, and it is considered important in the pathogenesis of ischemia-reperfusion damage. Because little is known about the enzyme in human tissues, the aims of this study were to purify human XDH/XO and to produce Ab for detection of the protein in Western blots and for quantification by ELISA. We purified human milk XDH/XO, produced Ab for Western blotting and ELISA of the protein, and evaluated the molecular forms and activity-protein relationships in human tissues. The molecular size of the purified protein under nondenaturing conditions was approximately 300 kd. On SDS-PAGE, it was fragmented into four main bands of 143, 125, 87, and 59 kd. Ab recognized bands of similar size in Western blots of the purified preparation and human milk. In fresh liver homogenates treated with anti-proteases, the three largest bands were observed; in the intestine, only the two largest were observed. Serum, brain, heart, and skeletal muscle were negative, whereas some lung and kidney samples showed one faint band of 143 kd. Trypsin treatment of the enzyme converted the large molecular-weight bands into smaller bands, as did incubation of a liver homogenate without anti-proteases. XDH/XO protein concentrations (ng/mg total protein) were 146 +/- 70 in liver and 556 +/- 320 in intestine and less than 5 ng/ml in serum. The relationship of activity to protein (2.7-3.0 mumol/min/mg XDH/XO protein) was constant in liver and intestine during development. We conclude that 1) human XDH/XO has molecular size and subunit structure similar to other mammalian enzymes; 2) the polypeptide chain is unstable, also in the intact cell, despite retained activity; and 3) the amount of inactive XDH/XO in human liver and intestine is apparently small.

  9. Protection against acetaminophen-induced liver injury by allopurinol is dependent on aldehyde oxidase-mediated liver preconditioning

    SciTech Connect

    Williams, C. David; McGill, Mitchell R.; Lebofsky, Margitta; Bajt, Mary Lynn; Jaeschke, Hartmut

    2014-02-01

    Acetaminophen (APAP) overdose causes severe and occasionally fatal liver injury. Numerous drugs that attenuate APAP toxicity have been described. However these compounds frequently protect by cytochrome P450 inhibition, thereby preventing the initiating step of toxicity. We have previously shown that pretreatment with allopurinol can effectively protect against APAP toxicity, but the mechanism remains unclear. In the current study, C3HeB/FeJ mice were administered allopurinol 18 h or 1 h prior to an APAP overdose. Administration of allopurinol 18 h prior to APAP overdose resulted in an 88% reduction in liver injury (serum ALT) 6 h after APAP; however, 1 h pretreatment offered no protection. APAP-cysteine adducts and glutathione depletion kinetics were similar with or without allopurinol pretreatment. The phosphorylation and mitochondrial translocation of c-jun-N-terminal-kinase (JNK) have been implicated in the progression of APAP toxicity. In our study we showed equivalent early JNK activation (2 h) however late JNK activation (6 h) was attenuated in allopurinol treated mice, which suggests that later JNK activation is more critical for the toxicity. Additional mice were administered oxypurinol (primary metabolite of allopurinol) 18 h or 1 h pre-APAP, but neither treatment protected. This finding implicated an aldehyde oxidase (AO)-mediated metabolism of allopurinol, so mice were treated with hydralazine to inhibit AO prior to allopurinol/APAP administration, which eliminated the protective effects of allopurinol. We evaluated potential targets of AO-mediated preconditioning and found increased hepatic metallothionein 18 h post-allopurinol. These data show metabolism of allopurinol occurring independent of P450 isoenzymes preconditions the liver and renders the animal less susceptible to an APAP overdose. - Highlights: • 18 h allopurinol pretreatment protects against acetaminophen-induced liver injury. • 1 h allopurinol pretreatment does not protect from APAP

  10. Effect of polymerization on antioxidant and xanthine oxidase inhibitory potential of sea buckthorn (H. rhamnoides) proanthocyanidins.

    PubMed

    Arimboor, Ranjith; Arumughan, C

    2012-10-01

    Inhibitory potential of sea buckthorn (Hippophae rhamnoides L) seed proanthocyanidins against oxidative stress and xanthine oxidase activity was evaluated. Composition of antioxidant proanthocyanidins was profiled by analyzing the cleavage products obtained by the acid catalyzed hydrolysis in the presence of phloroglucinol. Catechin, epicatechin, gallocatechin, and epigallocatechin were found as the extension and terminal subunits of proanthocyanidins with an average degree of polymerization (ADP) of 14.7. Seed proanthocyanidins showed considerably high antioxidant and xanthine oxidase inhibitory potentials. Antioxidant and xanthine oxidase inhibitory capacity evaluation of proanthocyanidin fractions with varying ADP showed that proanthocyanidins with lower molecular size were more effective as superoxide anion (ADP ≤ 4.2) and hydroxyl radical (ADP ≤ 5.9) scavengers and xanthine oxidase (ADP ≤ 3.1) inhibitors. ADP of the studied proanthocyanidin fractions did not show significant influence on their DPPH and ABTS radical scavenging and ferric reduction capacities.

  11. Xanthine oxidase is one of the major sources of superoxide anion radicals in blood after reperfusion in rats with forebrain ischemia/reperfusion.

    PubMed

    Ono, Takeru; Tsuruta, Ryosuke; Fujita, Motoki; Aki, Hiromi Shinagawa; Kutsuna, Satoshi; Kawamura, Yoshikatsu; Wakatsuki, Jun; Aoki, Tetsuya; Kobayashi, Chihiro; Kasaoka, Shunji; Maruyama, Ikuro; Yuasa, Makoto; Maekawa, Tsuyoshi

    2009-12-11

    We recently reported that excessive superoxide anion radical (O(2)(-)) was generated in the jugular vein during reperfusion in rats with forebrain ischemia/reperfusion using a novel electrochemical sensor and excessive O(2)(-) generation was associated with oxidative stress, early inflammation, and endothelial injury. However, the source of O(2)(-) was still unclear. Therefore, we used allopurinol, a potent inhibitor of xanthine oxidase (XO), to clarify the source of O(2)(-) generated in rats with forebrain ischemia/reperfusion. The increased O(2)(-) current and the quantified partial value of electricity (Q), which was calculated by the integration of the current, were significantly attenuated after reperfusion by pretreatment with allopurinol. Malondialdehyde (MDA) in the brain and plasma, high-mobility group box 1 (HMGB1) in plasma, and intercellular adhesion molecule-1 (ICAM-1) in the brain and plasma were significantly attenuated in rats pretreated with allopurinol with dose-dependency in comparison to those in control rats. There were significant correlations between total Q and MDA, HMGB, or ICAM-1 in the brain and plasma. Allopurinol pretreatment suppressed O(2)(-) generation in the brain-perfused blood in the jugular vein, and oxidative stress, early inflammation, and endothelial injury in the acute phase of forebrain ischemia/reperfusion. Thus, XO is one of the major sources of O(2)(-)- in blood after reperfusion in rats with forebrain ischemia/reperfusion.

  12. Substrate Orientation and Catalysis at the Molybdenum Site in Xanthine Oxidase CRYSTAL STRUCTURES IN COMPLEX WITH XANTHINE AND LUMAZINE

    SciTech Connect

    Pauff, James M.; Cao, Hongnan; Hille, Russ

    2010-01-12

    Xanthine oxidoreductase is a ubiquitous cytoplasmic protein that catalyzes the final two steps in purine catabolism. We have previously investigated the catalytic mechanism of the enzyme by rapid reaction kinetics and x-ray crystallography using the poor substrate 2-hydroxy-6-methylpurine, focusing our attention on the orientation of substrate in the active site and the role of Arg-880 in catalysis. Here we report additional crystal structures of as-isolated, functional xanthine oxidase in the course of reaction with the pterin substrate lumazine at 2.2 {angstrom} resolution and of the nonfunctional desulfo form of the enzyme in complex with xanthine at 2.6 {angstrom} resolution. In both cases the orientation of substrate is such that the pyrimidine subnucleus is oriented opposite to that seen with the slow substrate 2-hydroxy-6-methylpurine. The mechanistic implications as to how the ensemble of active site functional groups in the active site work to accelerate reaction rate are discussed.

  13. A kinetic study of hypoxanthine oxidation by milk xanthine oxidase.

    PubMed Central

    Escribano, J; Garcia-Canovas, F; Garcia-Carmona, F

    1988-01-01

    The course of the reaction sequence hypoxanthine----xanthine----uric acid catalysed by xanthine:oxygen oxidoreductase from milk was investigated on the basis of u.v. spectra taken during the course of hypoxanthine and xanthine oxidations. It was found that xanthine accumulated in the reaction mixture when hypoxanthine was used as a substrate. The time course of the concentrations of hypoxanthine, xanthine intermediate and uric acid product was simulated numerically. The mathematical model takes into account the competition of substrate, intermediate and product and the accumulation of the intermediate at the enzyme. This type of analysis permits the kinetic parameters of the enzyme for hypoxanthine and xanthine to be obtained. PMID:3196295

  14. Allopurinol as a therapeutic option in cardiovascular disease.

    PubMed

    Okafor, Osita N; Farrington, K; Gorog, Diana A

    2017-04-01

    Epidemiological studies indicate that hyperuricaemia is an independent risk factor for cardiovascular disease. Alongside uric acid formation, increased xanthine oxidase activity also results in the formation of oxidative free radicals and superoxide particles. Oxidative stress significantly contributes to the development of cardiovascular disease, including endothelial cell dysfunction, atherosclerosis, vascular calcification and impaired myocardial energetics. Allopurinol, a competitive xanthine oxidase inhibitor, in addition to reducing serum uric acid levels, can act as a free radical scavenger. Although traditionally used for the management of gout, there has been renewed interest in the role of allopurinol in the management of cardiovascular disease. In this review, we summarise the role of the xanthine oxidase pathway in the generation of oxidative stress and evaluate the current body of evidence assessing the clinical effects of allopurinol in patients with cardiovascular disease. A number of small clinical studies have shown a beneficial effect of allopurinol in reducing ischemia-reperfusion injury in the setting of bypass surgery and coronary angioplasty. Additionally, studies in heart failure indicate a potential favourable effect of allopurinol on endothelial dysfunction, LV function and haemodynamic indices, particularly in those with raised serum uric acid levels. Whilst this cheap and readily available pharmacological option may offer a very cost effective therapeutic option, large-scale prospective studies are required to better delineate its role in reducing hard clinical end-points.

  15. Effect of dietary protein and iron on the fractional turnover rate of rat liver xanthine oxidase

    SciTech Connect

    Cherry, D.M.; Amy, N.K.

    1987-12-01

    Rat liver xanthine oxidase activity is regulated in response to dietary protein and iron. To investigate whether the change in activity was mediated by a change in the rate of protein degradation, we measured the fractional turnover rate using the double-isotope technique with (/sup 3/H)- and (/sup 14/C)leucine and calculated the apparent half-life of xanthine oxidase in rats fed diets containing either 20 or 5% casein with either 35 or 5 mg iron/kg diet. Under control conditions, xanthine oxidase had an apparent half-life of 4.8 d and approximately 65% of the enzyme subunits were active. Rats fed diets with low dietary protein had lower xanthine oxidase activity, but the enzyme had a slower fractional turnover rate, resulting in an apparent half-life of 6.4 d, and only 15-20% of the enzyme was active. The apparent half-life of xanthine oxidase increased to 7.5 d in rats fed diets with low dietary iron, but dietary iron did not affect the specific activity of the enzyme or the percentage of active subunits. These results suggest that the loss of enzyme activity is not due to loss of enzyme protein by increased degradation, but rather to inactivation of the enzyme.

  16. Time dependent inhibition of xanthine oxidase in irradiated solutions of folic acid, aminopterin and methotrexate

    SciTech Connect

    Robinson, K.; Pilot, T.F.; Meany, J.E. )

    1990-01-01

    The xanthine oxidase catalyzed oxidation of hypoxanthine was followed by monitoring the formation of uric acid at 290 nm. Inhibition of xanthine oxidase occurs in aqueous solutions of folic acid methotrexate and aminopterin. These compounds are known to dissociate upon exposure to ultraviolet light resulting in the formation of their respective 6-formylpteridine derivatives. The relative rates of dissociation were monitored spectrophotometrically by determining the absorbance of their 2,4-dinitrophenylhydrazine derivatives at 500 nm. When aqueous solutions of folic acid, aminopterin and methotrexate were exposed to uv light, a direct correlation was observed between the concentrations of the 6-formylpteridine derivatives existing in solution and the ability of these solutions to inhibit xanthine oxidase. The relative potency of the respective photolysis products were estimated.

  17. Arterial endothelial barrier dysfunction: actions of homocysteine and the hypoxanthine-xanthine oxidase free radical generating system.

    PubMed

    Berman, R S; Martin, W

    1993-04-01

    1. Endothelial barrier function was assessed by use of an in vitro model in which transfer of trypan blue-labelled albumin was measured across monolayers of bovine aortic endothelial cells grown on polycarbonate membranes. 2. Addition of either hypoxanthine (0.2 mM) or xanthine oxidase (20 mu ml-1) alone during a 90 min incubation did not affect albumin transfer across endothelial cell monolayers, but a combination of both increased transfer. 3. The increase in albumin transfer induced by hypoxanthine and xanthine oxidase was abolished by catalase (3 u ml-1), reduced by allopurinol (4 mM), but unaffected by superoxide dismutase (6000 u ml-1), the hydroxyl radical scavengers, mannitol (15 mM), dimethylthiourea (10 mM) and N-(2-mercaptopropionyl)-glycine (1 mM), the iron chelator, deferoxamine (0.5 mM), ferric chloride (50 microM), an inhibitor of nitric oxide synthase, NG-nitro-L-arginine (30 microM), or the antioxidant, dithiothreitol (3 mM). 4. Hydrogen peroxide (0.1-30 mM) itself increased albumin transfer across endothelial cell monolayers, exhibiting a biphasic concentration-response curve. The increase induced by 0.1 mM hydrogen peroxide was abolished in the presence of 0.3 u ml-1 catalase whilst that induced by 10 mM hydrogen peroxide was abolished by 3000 u ml-1 catalase. 5. Homocysteine (0.5-1.5 mM) did not affect albumin transfer across endothelial monolayers when added alone, but when added in combination with copper sulphate (50 microM), which catalyses its oxidation, a significant increase in albumin transfer was observed. 6. The increase in albumin transfer induced by the combination of homocysteine (1.5 mM) and copper sulphate was abolished by catalase (1 u ml-1), but was unaffected by superoxide dismutase (6000 u ml-1), mannitol (15 mM), dimethylthiourea (1 mM) or deferoxamine (0.5 mM).7. The data suggest that the endothelial barrier dysfunction induced by the combination of hypoxanthine and xanthine oxidase is mediated solely by the action of

  18. Hypouricaemic action of mangiferin results from metabolite norathyriol via inhibiting xanthine oxidase activity.

    PubMed

    Niu, Yanfen; Liu, Jia; Liu, Hai-Yang; Gao, Li-Hui; Feng, Guo-Hua; Liu, Xu; Li, Ling

    2016-09-01

    Context Mangiferin has been reported to possess a potential hypouricaemic effect. However, the pharmacokinetic studies in rats showed that its oral bioavailability was only 1.2%, suggesting that mangiferin metabolites might exert the action. Objective The hypouricaemic effect and the xanthine oxidase inhibition of mangiferin and norathyriol, a mangiferin metabolite, were investigated. Inhibition of norathyriol analogues (compounds 3-9) toward xanthine oxidase was also evaluated. Materials and methods For a dose-dependent study, mangiferin (1.5-6.0 mg/kg) and norathyriol (0.92-3.7 mg/kg) were administered intragastrically to mice twice daily for five times. For a time-course study, mice received mangiferin and norathyriol both at a single dose of 7.1 μmol/kg. In vitro, inhibition of test compounds (2.4-2.4 mM) against xanthine oxidase activity was evaluated by the spectrophotometrical method. The inhibition type was identified from Lineweaver-Burk plots. Results Norathyriol (0.92, 1.85 and 3.7 mg/kg) dose dependently decreased the serum urate levels by 27.0, 33.6 and 37.4%, respectively. The action was more potent than that of mangiferin at the low dose, but was equivalent at the higher doses. Additionally, the hypouricaemic action of them exhibited a time dependence. In vitro, norathyriol markedly inhibited the xanthine oxidase activities, with the IC50 value of 44.6 μM, but mangiferin did not. The kinetic studies showed that norathyriol was an uncompetitive inhibitor by Lineweaver-Burk plots. The structure-activity relationships exhibited that three hydroxyl groups in norathyriol at the C-1, C-3 and C-6 positions were essential for maintaining xanthine oxidase inhibition. Discussion and conclusion Norathyriol was responsible for the hypouricaemic effect of mangiferin via inhibiting xanthine oxidase activity.

  19. In vitro anti-inflammatory and xanthine oxidase inhibitory activity of Tephrosia purpurea shoot extract.

    PubMed

    Nile, Shivraj H; Khobragade, Chandrahasy N

    2011-10-01

    The methanolic extract of Tephrosia purpurea (Leguminosae) shoots was evaluated in-vitro for its anti-inflammatory and xanthine oxidase inhibitory activity. Anti-inflammatory activity was measured by the Diene-conjugate, HET-CAM and beta-glucuronidase methods. The enzyme inhibitory activity was tested against isolated cow milk xanthine oxidase. The average anti-inflammatory activity of T. purpurea shoot extract in the concentration range of 1-2 microg/mL in the reacting system revealed significant anti-inflammatory activities, which, as recorded by the Diene-conjugate, HET-CAM and beta-glucuronidase assay methods, were 45.4, 10.5, and 70.5%, respectively. Screening of the xanthine oxidase inhibitory activity of the extract in terms of kinetic parameters revealed a mixed type of inhibition, wherein the Km and Vmax values in the presence of 25 to 100 microg/mL shoot extract was 0.20 mM/mL and 0.035, 0.026, 0.023 and 0.020 microg/min, while, for the positive control, the Km and Vmax values were 0.21 mM/mL and 0.043 microg/min, respectively. These findings suggest that T. purpurea shoot extract may possess constituents with good medicinal properties that could be exploited to treat the diseases associated with oxidative stress, xanthine oxidase enzyme activity and inflammation.

  20. Plasma from hemorrhaged mice activates CREB and increases cytokine expression in lung mononuclear cells through a xanthine oxidase-dependent mechanism.

    PubMed

    Shenkar, R; Abraham, E

    1996-02-01

    Hemorrhage rapidly increases plasma xanthine oxidase levels as well as the expression of proinflammatory and immunoregulatory cytokines in the lungs. To determine the role of circulating xanthine oxidase (XO), as well as other plasma factors, in affecting pulmonary cytokine expression, we conducted studies in which plasma from hemorrhaged mice was transferred into unhemorrhaged recipient mice. Administration of posthemorrhage plasma to recipient mice increased the levels of mRNA for interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta 1 (TGF-beta 1) in lung mononuclear cells. No enhancement of mRNA levels for these cytokines was found in the lungs of mice given allopurinol-treated posthemorrhage plasma or fed a tungsten-enriched, XO-depleting diet prior to transfer of posthemorrhage plasma. Among the nuclear transcriptional regulatory factors examined, only the cyclic AMP response-element binding protein (CREB) was activated in nuclear extracts from lung mononuclear cells of mice that were given posthemorrhage plasma. No activation of nuclear factor-kappa B (NF-kappa B), nuclear factor interleukin 6 (NF-IL6), activating protein-1 (AP-1), or serum protein-1 (SP-1) was found. These results suggest that the mechanism for hemorrhage-induced increases in pulmonary cytokine expression is by activation of the enhancer CREB through a tissue XO-dependent pathway initiated by plasma-borne mediators.

  1. In vitro evaluation of selected benzimidazole derivatives as an antioxidant and xanthine oxidase inhibitors.

    PubMed

    Nile, Shivraj H; Kumar, Brajesh; Park, Se W

    2013-09-01

    2-Aryl-1-arylmethyl-1H-benzimidazole derivatives having different side chains on the structure were examined in vitro for their antioxidant abilities by 2,2-diphenyl-1-picryl hydrazine radical scavenging activity, reducing ability, OH radical scavenging activity, inhibition of polyphenol oxidase and xanthine oxidase. Overall, with few exceptions, all the 2-aryl-1-arylmethyl-1H-benzimidazoles showed moderate biological activity with all parameters examined. The 2-aryl-1-arylmethyl-1H-benzimidazoles were found to be reactive toward 2,2-diphenyl-1-picryl hydrazine radical and had considerable reducing ability, with significant xanthine oxidase inhibition. With few exceptions, all the compounds under study were found to possess moderate-to-poor OH radical scavenging activity and inhibited polyphenol oxidase significantly. These findings suggest that these 2-aryl-1-arylmethyl-1H-benzimidazoles can be considered as potential antioxidant and xanthine oxidase inhibitory agents, those might be further, explored for the design of lead antioxidant and antigout drug candidates using in vivo trials.

  2. [Establishment of double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers].

    PubMed

    Xie, Tao; Qin, Zhi-Zhen; Zhou, Rui; Zhao, Ying; Du, Guan-hua

    2015-04-01

    A double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers was established. In the reaction system of xanthine oxidase, WST-1 works as the probe for the ultra oxygen anion generation, and product uric acid works as xanthine oxidase activity indicator. By using SpectraMax M5 continuous spectrum enzyme sign reflectoscope reflector, the changes of these indicators' concentration were observed and the influence factors of this reaction system to establish the high throughput screening model were studied. And the model is confirmed by positive drugs. In the reaction system, the final volume of reaction system is 50 μL and the concentrations of xanthine oxidase is 4 mU x mL(-1), xanthine 250 μmol x L(-1) and WST-1 100 μmol x L(-1), separately. The Z'-factor of model for xanthine oxidase inhibitors is 0.537 4, S/N is 47.519 9; the Z'-factor of model for superoxide anion scavengers is 0.507 4, S/N is 5.388 9. This model for xanthine oxidase inhibitors and superoxide anion scavengers has more common characteristics of the good stability, the fewer reagent types and quantity, the good repeatability, and so on. And it can be widely applied in high-throughput screening research.

  3. Granulomatous reaction to red tattoo pigment treated with allopurinol.

    PubMed

    Godinho, Mariana Marteleto; Aguinaga, Felipe; Grynszpan, Rachel; Lima, Victor Maselli; Azulay, David Rubem; Cuzzi, Tullia; Ramos-E-Silva, Marcia; Manela-Azulay, Mônica

    2015-09-01

    Granulomatous reactions to tattoo ink are most commonly associated with mercury sulfide, a component of red pigments. Treatment options show limited results. Allopurinol, an inhibitor of xanthine oxidase, has been reported as a successful alternative treatment to granulomatous disorders, such as sarcoidosis and granulomatous reactions to fillers and tattoos. We report a case of granulomatous reaction to red tattoo pigment treated with allopurinol for 6 months. Good clinical improvement could be noticed during this time. Two months after we stopped the treatment, the lesion recurred. Allopurinol emerges as an important drug for the management of granulomatous reactions caused by tattoo pigments. Based on the significant clinical improvement noticed during its use, we recommend new studies to elucidate all the potential benefits of the use of allopurinol for the treatment of granulomatous reactions to tattoo ink.

  4. Inhibition of xanthine oxidase by the aldehyde oxidase inhibitor raloxifene: implications for identifying molybdopterin nitrite reductases.

    PubMed

    Weidert, E R; Schoenborn, S O; Cantu-Medellin, N; Choughule, K V; Jones, J P; Kelley, E E

    2014-02-15

    Sources of nitric oxide alternative to nitric oxide synthases are gaining significant traction as crucial mediators of vessel function under hypoxic inflammatory conditions. For example, capacity to catalyze the one electron reduction of nitrite (NO2-) to ·NO has been reported for hemoglobin, myoglobin and molybdopterin-containing enzymes including xanthine oxidoreductase (XOR) and aldehyde oxidase (AO). For XOR and AO, use of selective inhibition strategies is therefore crucial when attempting to assign relative contributions to nitrite-mediated ·NO formation in cells and tissue. To this end, XOR inhibition has been accomplished with application of classic pyrazolopyrimidine-based inhibitors allo/oxypurinol or the newly FDA-approved XOR-specific inhibitor, Uloric® (febuxostat). Likewise, raloxifene, an estrogen receptor antagonist, has been identified as a potent (Ki=1.0 nM) inhibitor of AO. Herein, we characterize the inhibition kinetics of raloxifene for XOR and describe the resultant effects on inhibiting XO-catalyzed ·NO formation. Exposure of purified XO to raloxifene (PBS, pH 7.4) resulted in a dose-dependent (12.5-100 μM) inhibition of xanthine oxidation to uric acid. Dixon plot analysis revealed a competitive inhibition process with a Ki=13 μM. This inhibitory process was more effective under acidic pH; similar to values encountered under hypoxic/inflammatory conditions. In addition, raloxifene also inhibited anoxic XO-catalyzed reduction of NO2- to NO (EC50=64 μM). In contrast to having no effect on XO-catalyzed uric acid production, the AO inhibitor menadione demonstrated potent inhibition of XO-catalyzed NO2- reduction (EC50=60 nM); somewhat similar to the XO-specific inhibitor, febuxostat (EC50=4 nM). Importantly, febuxostat was found to be a very poor inhibitor of human AO (EC50=613 μM) suggesting its usefulness for validating XO-dependent contributions to NO2- reduction in biological systems. Combined, these data indicate care should be taken

  5. Inhibitory effects of cardols and related compounds on superoxide anion generation by xanthine oxidase.

    PubMed

    Masuoka, Noriyoshi; Nihei, Ken-ichi; Maeta, Ayami; Yamagiwa, Yoshiro; Kubo, Isao

    2015-01-01

    5-Pentadecatrienylresorcinol, isolated from cashew nuts and commonly known as cardol (C₁₅:₃), prevented the generation of superoxide radicals catalysed by xanthine oxidase without the inhibition of uric acid formation. The inhibition kinetics did not follow the Michelis-Menten equation, but instead followed the Hill equation. Cardol (C₁₀:₀) also inhibited superoxide anion generation, but resorcinol and cardol (C₅:₀) did not inhibit superoxide anion generation. The related compounds 3,5-dihydroxyphenyl alkanoates and alkyl 2,4-dihydroxybenzoates, had more than a C9 chain, cooperatively inhibited but alkyl 3,5-dihydroxybenzoates, regardless of their alkyl chain length, did not inhibit the superoxide anion generation. These results suggested that specific inhibitors for superoxide anion generation catalysed by xanthine oxidase consisted of an electron-rich resorcinol group and an alkyl chain having longer than C9 chain.

  6. Effect of luteolin on xanthine oxidase: inhibition kinetics and interaction mechanism merging with docking simulation.

    PubMed

    Yan, Jiakai; Zhang, Guowen; Hu, Yuting; Ma, Yadi

    2013-12-15

    Xanthine oxidase (XO) catalyses hypoxanthine and xanthine to uric acid in human metabolism. Overproduction of uric acid will lead to hyperuricemia and finally cause gout and other diseases. Luteolin is one of the major components of celery and green peppers, its inhibitory activity on XO and their interaction mechanism were evaluated by multispectroscopic methods, coupled with molecular simulation. It was found that luteolin reversibly inhibited XO in a competitive manner with inhibition constant (Ki) value of (2.38±0.05)×10(-6) mol l(-1). Luteolin could bind to XO at a single binding site and the binding was driven mainly by hydrophobic interactions. Analysis of synchronous fluorescence and circular dichroism spectra demonstrated that the microenvironment and secondary structure of XO were altered upon interaction with luteolin. The molecular docking results revealed luteolin actually interacted with the primary amino acid residues located within the active site pocket of XO.

  7. Novel insights into the inhibitory mechanism of kaempferol on xanthine oxidase.

    PubMed

    Wang, Yajie; Zhang, Guowen; Pan, Junhui; Gong, Deming

    2015-01-21

    Xanthine oxidase (XO), a key enzyme in purine catabolism, is widely distributed in human tissues. It can catalyze xanthine to generate uric acid and cause hyperuricemia and gout. Inhibition kinetics assay showed that kaempferol inhibited XO activity reversibly in a competitive manner. Strong fluorescence quenching and conformational changes of XO were found due to the formation of a kaempferol-XO complex, which was driven mainly by hydrophobic forces. The molecular docking further revealed that kaempferol inserted into the hydrophobic cavity of XO to interact with some amino acid residues. The main inhibition mechanism of kaempferol on XO activity may be due to the insertion of kaempferol into the active site of XO occupying the catalytic center of the enzyme to avoid the entrance of the substrate and inducing conformational changes of XO. In addition, luteolin exhibited a stronger synergistic effect with kaempferol than did morin at the lower concentration.

  8. Inhibitory effects of DA-9601 on ethanol-induced gastrohemorrhagic lesions and gastric xanthine oxidase activity in rats.

    PubMed

    Huh, Keun; Kwon, Tae Hyup; Shin, Uk Sup; Kim, Won Bae; Ahn, Byoung Ok; Oh, Tae Young; Kim, Jung-Ae

    2003-10-01

    The exposure of gastric mucosa to ethanol produces pathological changes such as inflammatory process, hemorrhagic erosions, even acute ulcers. The gastric mucosal lesions accompanied by a significant decrease of gastric blood flow and increase of reactive oxygen species (ROS) implicate a role of xanthine oxidase in ethanol-induced gastric hemorrhagic erosions. DA-9601, a novel antipeptic formulation of extracts of Artemisia asiatica Nakai, was studied for its inhibitory effect on gastric xanthine oxidase activity and type conversion of the enzyme that has a profound role in free radical generation. Intubation of absolute ethanol (4 g/kg) significantly induced gastrohemorrhagic lesions and lipid peroxidation in the rat stomach. Oral administration of DA-9601 at 40 mg/kg body weight significantly reduced ethanol-induced gastric mucosal hemorrhagic lesions and lipid peroxidation, which was proportional to the inhibitory effect of DA-9601 on alcohol-induced xanthine oxidase-type conversion and enzyme activity. The results suggest that alcohol-induced gastric mucosal damage may be, in part, due to the increased activity of xanthine oxidase and type conversion rate of the enzyme and that the preventive effect of DA-9601 on gastrohemorrhagic lesions would result from its inhibitory action against xanthine oxidase and oxidative stress in alcohol-treated rats.

  9. Effects of allopurinol and oxipurinol on purine synthesis in cultured human cells

    PubMed Central

    Kelley, William N.; Wyngaarden, James B.

    1970-01-01

    In the present study we have examined the effects of allopurinol and oxipurinol on the de novo synthesis of purines in cultured human fibroblasts. Allopurinol inhibits de novo purine synthesis in the absence of xanthine oxidase. Inhibition at lower concentrations of the drug requires the presence of hypoxanthine-guanine phosphoribosyltransferase as it does in vivo. Although this suggests that the inhibitory effect of allopurinol at least at the lower concentrations tested is a consequence of its conversion to the ribonucleotide form in human cells, the nucleotide derivative could not be demonstrated. Several possible indirect consequences of such a conversion were also sought. There was no evidence that allopurinol was further utilized in the synthesis of nucleic acids in these cultured human cells and no effect of either allopurinol or oxipurinol on the long-term survival of human cells in vitro could be demonstrated. At higher concentrations, both allopurinol and oxipurinol inhibit the early steps of de novo purine synthesis in the absence of either xanthine oxidase or hypoxanthine-guanine phosphoribosyltransferase. This indicates that at higher drug concentrations, inhibition is occurring by some mechanism other than those previously postulated. PMID:5415686

  10. Gastric mucosal injury in the rat. Role of iron and xanthine oxidase

    SciTech Connect

    Smith, S.M.; Grisham, M.B.; Manci, E.A.; Granger, D.N.; Kvietys, P.R.

    1987-04-01

    Recent studies have implicated oxygen free radicals in ischemia-reperfusion injury to the gastric mucosa. The aims of the present study were to test the hypothesis that the enzyme xanthine oxidase is the source of the oxygen radicals in the ischemic stomach and determine the importance of the iron-catalyzed Haber-Weiss reaction in generating the cytotoxic oxygen radicals. Gastric mucosal clearance of /sup 51/Cr-labeled red blood cells was measured during a 30-min control period, a 30-min ischemic period (hemorrhage to 25 mmHg arterial pressure), and a 60-80-min reperfusion period (reinfusion of shed blood). In untreated (control) rats, a dramatic rise (100-fold) in the leakage of /sup 51/Cr-labeled red blood cells into the gastric lumen was observed only during the reperfusion period. After the reperfusion period, gastric mucosal damage was further assessed using gross lesion area and histology. Rats were placed on a sodium tungstate diet (to inactivate xanthine oxidase), or treated with either deferoxamine (an iron chelating agent) or superoxide dismutase (a superoxide scavenger). All three interventions substantially reduced /sup 51/Cr-labeled red blood cell clearance and gross lesion area relative to untreated rats. However, tissue injury assessed histologically was similar in both treated and untreated animals. The results of this study support the hypothesis that oxygen free radicals mediate the hemorrhagic shock-induced extravasation of red blood cells. The data also indicate that xanthine oxidase is the source of the oxy-radicals and that the iron-catalyzed Haber-Weiss reaction is largely responsible for hydroxyl radical generation in this model.

  11. Allopurinol and dimethylthiourea reduce brain infarction following middle cerebral artery occlusion in rats.

    PubMed

    Martz, D; Rayos, G; Schielke, G P; Betz, A L

    1989-04-01

    Free radicals have been shown to play an important role in ischemia-reperfusion injury in several organ systems; however, the role of free radicals in central nervous system ischemia has been less well studied. Many potential free radical-generating systems exist. The primary products of these reactions, superoxide and hydrogen peroxide, may combine to produce hydroxyl radicals. Of the many potential sources of free radical generation, the enzyme xanthine oxidase has been shown to be important in ischemia in noncerebral tissue. We investigated the effect of the hydroxyl radical scavenger dimethylthiourea and the xanthine oxidase inhibitor allopurinol on infarct volume in a model of continuous partial ischemia. Male Sprague-Dawley rats were treated with dimethylthiourea or allopurinol before middle cerebral artery occlusion. Infarct volume was measured by triphenyltetrazolium chloride staining of brains removed 3 or 24 hours after occlusion. Stroke volume was reduced by 30% after dimethylthiourea treatment and by 32-35% after allopurinol treatment. At 24 hours after stroke, cortical tissue was more effectively protected than caudate tissue with both agents. Pretreatment with dimethylthiourea and allopurinol also significantly reduced cerebral edema formation and improved blood-brain barrier function as measured by fluorescein uptake. Our results imply that hydroxyl radicals are important in tissue injury secondary to partial cerebral ischemia and that xanthine oxidase may be the primary source of these radicals.

  12. Synthesis, characterization and xanthine oxidase inhibition of Cu(II)-chrysin complex.

    PubMed

    Lin, Suyun; Zeng, Li; Zhang, Guowen; Liao, Yijing; Gong, Deming

    2017-05-05

    Xanthine oxidase (XO) is a key enzyme catalyzing hypoxanthine to xanthine and then uric acid causing hyperuricemia. A Cu(II) complex of chrysin was synthesized and characterized by UV-vis absorption, Fourier transform infrared, nuclear magnetic resonance ((1)H NMR) and mass spectroscopy studies. The interaction of Cu(II)-complex with XO was investigated by spectroscopic methods and molecular simulation. The Cu(II)-chrysin complex exhibited a better inhibitory ability (IC50=0.82±0.034μM) against XO than its corresponding ligands chrysin and Cu(2+) in a mix-competitive manner. The binding affinity of Cu(II)-chrysin complex with XO was much higher than that of chrysin. The hydrogen bonds and van der Waals forces played main roles in the binding. Analysis of circular dichroism spectra indicated that the complex induced the conformational change of XO. The molecular simulation found that the Cu(II)-chrysin complex inserted into the active cavity of XO with Cu acting as a bridge, occupying the catalytic center of the enzyme to avoid entry of the substrate xanthine, leading to the inhibition of XO. This study may provide new insights into the inhibition mechanism of the Cu(II)-chrysin complex as a promising XO inhibitor and its potential application for the treatment of hyperuricemia.

  13. Posttranslational ruling of xanthine oxidase activity in bovine milk by its substrates

    SciTech Connect

    Silanikove, Nissim Shapiro, Fira; Leitner, Gabriel

    2007-11-23

    The aims of this study were to test the hypothesis that the substrates of xanthine oxidase (XO), xanthine and hypoxanthine, are consumed while the milk is stored in the gland between milkings, and to explore how XO activity responds to bacteria commonly associated with subclinical infections in the mammary gland. Freshly secreted milk was obtained following complete evacuation of the gland and induction of milk ejection with oxytocin. In bacteria-free fresh milk xanthine and hypoxanthine were converted to uric acid within 30 min (T{sub 1/2} {approx} 10 min), which in turn provides electrons for formation of hydrogen peroxide and endows the alveolar lumen with passive protection against invading bacteria. On the other hand, the longer residence time of milk in the cistern compartment was not associated with oxidative stress as a result of XO idleness caused by exhaustion of its physiological fuels. The specific response of XO to bacteria species and the resulting bacteria-dependent nitrosative stress further demonstrates that it is part of the gland immune system.

  14. In vitro and in vivo studies on adlay-derived seed extracts: phenolic profiles, antioxidant activities, serum uric acid suppression, and xanthine oxidase inhibitory effects.

    PubMed

    Zhao, Mouming; Zhu, Dashuai; Sun-Waterhouse, Dongxiao; Su, Guowan; Lin, Lianzhu; Wang, Xiao; Dong, Yi

    2014-08-06

    This study aimed to explore the potential of polished adlay, brown adlay, adlay bran, and adlay hull to prevent and treat hyperuricemia. Brown adlay extract effectively decreased the serum uric acid levels of oxonate-induced hyperuricemic rats. Free and bound phenolic extracts from these materials contained significant amounts of phenolics, with free phenolics dominated by chlorogenic acid and p-coumaric acid while bound phenolics dominated by p-coumaric acid and ferulic acid. Free and bound phenolics of adlay bran exhibited significant xanthine oxidase inhibition activities, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities, oxygen radical absorbance capacities, and superoxide radical scavenging activities. Adlay bran phenolics could be effective xanthine oxidase inhibitors and radical scavengers. p-Coumaric acid is a xanthine oxidase inhibitor with strong superoxide radical scavenging activity. However, ferulic acid is a xanthine oxidase inhibitor with weak superoxide radical scavenging activity. Chlorogenic acid is a superoxide radical scavenger with weak xanthine oxidase inhibitory activity.

  15. A new glycosidic antioxidant from Ranunculus muricatus L. (Ranunculaceae) exhibited lipoxygenasae and xanthine oxidase inhibition properties.

    PubMed

    Raziq, Naila; Saeed, Muhammad; Ali, Muhammad Shaiq; Zafar, Salman; Shahid, Muhammad; Lateef, Mehreen

    2017-06-01

    Phytochemical investigation of Ranunculus muricatus L. (Ranunculaceae) led to the isolation of a new metabolite named as ranuncoside from the ethyl acetate fraction of the plant. Structure of the novel compound was elucidated through detailed spectroscopic analyses, using UV, IR, (1)H, (13)C NMR and 2D NMR in combination with EIMS and HR EI-MS techniques. The compound was evaluated for antioxidant activity using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging assay. Its inhibitory potential was tested against lipoxygenase and xanthine oxidase enzymes. Ranuncoside potently scavenged the DPPH free radicals (IC50 = 56.7 ± 0.43 μM) and strongly inhibited the activities of lipoxygenase (IC50 = 63.9 ± 0.17 μM) and xanthine oxidase (IC50 = 43.3 ± 0.22 μM).

  16. Absorption of enzymatically active sup 125 I-labeled bovine milk xanthine oxidase fed to rabbits

    SciTech Connect

    Rzucidlo, S.J. ); Zikakis, J.P. )

    1990-05-01

    Rabbits fed a regular laboratory diet supplemented with a high-fat milk containing xanthine oxidase (XO) were studied to determine the presence of active XO in the blood. A pilot feeding study, where rabbits consumed a high-fat diet containing xanthine oxidase, showed a correlation between dairy food consumption and XO activity in the blood. Antibody to dietary XO was also found. In a second study, rabbits were fed ad libitum the high-fat milk and blood serum samples were tested weekly for XO activity. No elevation in serum XO activity was found. A third study showed that serum XO activity was increased when rabbits were force fed the high-fat milk. The final study consisted of force feeding {sup 125}I-labeled XO to one rabbit to ascertain whether the observed increase in serum XO was due to dietary or endogenous XO. Isoelectric focusing of sera collected from the test rabbit strongly suggested that at least a portion of the serum XO contained the radioactive label. This is the first direct evidence showing the uptake of dietary active XO from the gut.

  17. Antioxidant properties and xanthine oxidase inhibitory effects of Tamus communis L. root extracts.

    PubMed

    Boumerfeg, Sabah; Baghiani, Abderrahmane; Messaoudi, Dalila; Khennouf, Seddik; Arrar, Lekhmici

    2009-02-01

    This study was conducted to search for xanthine oxidase (XO) inhibitors from the root extracts of Tamus communis L. traditionally used in folk medicine in Algeria. Root extracts with different solvents were screened for purified milk xanthine oxidase inhibition. The root extracts (methanol, chloroform and ethyl acetate) and proteins, obtained in distilled water, inhibited bovine, sheep and human milk XO from three species in a concentration-dependent manner, with an additional superoxide scavenging capacity, which reached its highest level with ethyl acetate extract (IC(50) = 0.15, 0.04 and 0.09 g/L) for bovine XO, sheep XO and human XO, respectively. The antioxidant potential was confirmed with the non-enzymatic method, total radical-trapping antioxidant parameter (TRAP) assay, which showed that the Tamus communis L. extracts have a potential antioxidant activity in the same order obtained by using the reduction of cytochrome c, an enzymatic method, in which the antioxidant activity followed a decreasing order: ethyl acetate extract > chloroform extract > protein.

  18. Allopurinol reduces antigen-specific and polyclonal activation of human T cells

    PubMed Central

    Pérez-Mazliah, Damián; Albareda, María C.; Alvarez, María G.; Lococo, Bruno; Bertocchi, Graciela L.; Petti, Marcos; Viotti, Rodolfo J.; Laucella, Susana A.

    2012-01-01

    Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza (Flu) virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases. PMID:23049532

  19. The effect of allopurinol administration on mitochondrial respiration and gene expression of xanthine oxidoreductase, inducible nitric oxide synthase, and inflammatory cytokines in selected tissues of broiler chickens.

    PubMed

    Settle, T; Falkenstein, E; Klandorf, H

    2015-10-01

    Birds have a remarkable longevity for their body size despite an increased body temperature, higher metabolic rate, and increased blood glucose concentrations compared to most mammals. As the end-product of purine degradation, uric acid (UA) is generated in the xanthine/hypoxanthine reactions catalyzed by xanthine oxidoreductase (XOR). In the first study, Cobb × Cobb broilers (n = 12; 4 weeks old) were separated into 2 treatments (n = 6); control (CON) and allopurinol (AL) 35 mg/kg BW (ALLO). The purpose of this study was to assess mitochondrial function in broiler chickens in response to potential oxidative stress generated from the administration of AL for 1 wk. There was a significant reduction in state 3 respiration (P = 0.01) and state 4 respiration (P = 0.007) in AL-treated birds compared to the controls. The purpose of the second study was to assess the effect of AL on gene expression of inflammatory cytokines interferon-γ (IFN)-γ, IL-1β, IL-6, and IL-12p35, as well as inducible nitric oxide synthase and XOR in liver tissue. Cobb × Cobb broilers were separated into two groups at 4 wk age (n = 10); CON and ALLO. After 1 wk AL treatment, half of the birds in each group (CON 1 and ALLO 1) were euthanized while the remaining birds continued on AL treatment for an additional week (CON 2 and ALLO 2). A significant increase in gene expression of XOR, IFN-γ, IL-1β, and IL-12p35 in ALLO 2 birds as compared to birds in CON 2 was detected. Liver UA content was significantly decreased in both ALLO 1(P = 0.003) and ALLO 2 (P = 0.012) birds when compared to CON 1 and CON 2, respectively. The AL reduced liver UA concentrations and increased expression of inflammatory cytokines. Additional studies are needed to determine if AL causes a direct effect on mitochondria or if mitochondrial dysfunction observed in liver mitochondria was due indirectly through increased oxidative stress or increased inflammation.

  20. Synthesis and bioevaluation of 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acids as potent xanthine oxidase inhibitors.

    PubMed

    Guan, Qi; Cheng, Zengjin; Ma, Xiaoxue; Wang, Lijie; Feng, Dongjie; Cui, Yuanhang; Bao, Kai; Wu, Lan; Zhang, Weige

    2014-10-06

    A series of 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acid derivatives (8a-f, 9a-m) were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro. Structure-activity relationship analyses have also been presented. Most of the target compounds exhibited potency levels in the nanomolar range. Compound 9e emerged as the most potent xanthine oxidase inhibitor (IC50 = 5.5 nM) in comparison to febuxostat (IC50 = 18.6 nM). Steady-state kinetics measurements with the bovine milk enzyme indicated a mixed type inhibition with Ki and Ki' values of 0.9 and 2.3 nM, respectively. A molecular modeling study on compounds 9e was performed to gain an insight into its binding mode with xanthine oxidase, and to provide the basis for further structure-guided design of new non-purine xanthine oxidase inhibitors related with 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acid scaffold.

  1. Reactive oxygen species derived from xanthine oxidase interrupt dimerization of breast cancer resistance protein, resulting in suppression of uric acid excretion to the intestinal lumen.

    PubMed

    Ogura, Jiro; Kuwayama, Kaori; Sasaki, Shunichi; Kaneko, Chihiro; Koizumi, Takahiro; Yabe, Keisuke; Tsujimoto, Takashi; Takeno, Reiko; Takaya, Atsushi; Kobayashi, Masaki; Yamaguchi, Hiroaki; Iseki, Ken

    2015-09-01

    The prevalence of hyperuricemia/gout increases with aging. However, the effect of aging on function for excretion of uric acid to out of the body has not been clarified. We found that ileal uric acid clearance in middle-aged rats (11-12 months) was decreased compared with that in young rats (2 months). In middle-aged rats, xanthine oxidase (XO) activity in the ileum was significantly higher than that in young rats. Inosine-induced reactive oxygen species (ROS), which are derived from XO, also decreased ileal uric acid clearance. ROS derived from XO decreased the active homodimer level of breast cancer resistance protein (BCRP), which is a uric acid efflux transporter, in the ileum. Pre-administration of allopurinol recovered the BCRP homodimer level, resulting in the recovering ileal uric acid clearance. Moreover, we investigated the effects of ROS derived from XO on BCRP homodimer level directly in Caco-2 cells using hypoxanthine. Treatment with hypoxanthine decreased BCRP homodimer level. Treatment with hypoxanthine induced mitochondrial dysfunction, suggesting that the decreasing BCRP homodimer level might be caused by mitochondrial dysfunction. In conclusion, ROS derived from XO decrease BCRP homodimer level, resulting in suppression of function for uric acid excretion to the ileal lumen. ROS derived from XO may cause the suppression of function of the ileum for the excretion of uric acid with aging. The results of our study provide a new insight into the causes of increasing hyperuricemia/gout prevalence with aging.

  2. [Allopurinol and its role in the treatment of sarcopenia].

    PubMed

    Ferrando, Beatriz; Olaso-Gonzalez, Gloria; Sebastia, Vicente; Viosca, Enrique; Gomez-Cabrera, Mari Carmen; Viña, Jose

    2014-01-01

    Xanthine oxidase (XO) is an enzyme that catalyzes the oxidation of hypoxanthine to xanthine and uric acid and plays an important role in purine catabolism. The purine analogue, allopurinol, is a well-known inhibitor of XO widely used in the clinical management of gout and conditions associated with hyperuricemia. More recent data indicate that allopurinol reduces oxidative stress and improves vascular function in several cardiometabolic diseases, prolongs exercise time in angina, and improves the efficiency of cardiac contractility in heart failure. XO also plays an important role in free radical generation during skeletal muscle contraction and thus, it has been related to the muscle damage associated to exhaustive exercise. Several research groups have shown the protective effect of allopurinol in the prevention of this type of damage. Based on this background, a critical overview is presented on the possible role of allopurinol in the treatment of sarcopenia, a geriatric syndrome characterized by progressive and generalized loss of skeletal muscle mass and strength with a risk of adverse outcomes, such as physical disability, poor quality of life and death.

  3. Levels and interactions of plasma xanthine oxidase, catalase and liver function parameters in Nigerian children with Plasmodium falciparum infection.

    PubMed

    Iwalokun, B A; Bamiro, S B; Ogunledun, A

    2006-12-01

    Elevated plasma levels of xanthine oxidase and liver function parameters have been associated with inflammatory events in several human diseases. While xanthine oxidase provides in vitro protection against malaria, its pathophysiological functions in vivo and interactions with liver function parameters remain unclear. This study examined the interactions and plasma levels of xanthine oxidase (XO) and uric acid (UA), catalase (CAT) and liver function parameters GOT, GPT and bilirubin in asymptomatic (n=20), uncomplicated (n=32), and severe (n=18) falciparum malaria children aged 3-13 years. Compared to age-matched control (n=16), significant (p<0.05) elevation in xanthine oxidase by 100-550%, uric acid by 15.4-153.8%, GOT and GPT by 22.1-102.2%, and total bilirubin by 2.3-86% according to parasitaemia (geometric mean parasite density (GMPD)=850-87100 parasites/microL) was observed in the malarial children. Further comparison with control revealed higher CAT level (16.2+/-0.5 vs 14.6+/-0.4 U/L; p<0.05) lacking significant (p>0.05) correlation with XO, but lower CAT level (13.4-5.4 U/L) with improved correlations (r=-0.53 to -0.91; p<0.05) with XO among the asymptomatic and symptomatic malaria children studied. 75% of control, 45% of asymptomatic, 21.9% of uncomplicated, and none of severe malaria children had Hb level>11.0 g/dL. Multivariate analyses further revealed significant (p<0.05) correlations between liver function parameters and xanthine oxidase (r=0.57-0.64) only in the severe malaria group. We conclude that elevated levels of XO and liver enzymes are biochemical features of Plasmodium falciparum parasitaemia in Nigerian children, with both parameters interacting differently to modulate the catalase response in asymptomatic and symptomatic falciparum malaria.

  4. Role of xanthine oxidase in the potentiation of doxorubicin-induced cardiotoxicity by mitomycin C.

    PubMed

    Gustafson, D L; Swanson, J D; Pritsos, C A

    1991-09-01

    Clinical evidence has suggested that mitomycin C (MMC) potentiates doxorubicin (DOX) induced cardiotoxicity. In this study a mouse model was used to examine the effect of DOX on the ability of cardiac tissue to bioactivate MMC to generate oxygen radicals. Cardiac damage was assessed by measuring serum CPK-MB isoenzyme levels and thiobarbituric acid reactive substances (TBARS) in the cardiac tissue. The exposure of animals to DOX or DOX and MMC over a three week period led to an increase in serum CPK-MB isoenzyme levels as well as TBARS. Treatment with DOX led to an increase in MMC-dependent, NADH-dependent, cyanide insensitive oxygen consumption, compared to control animals, thereby suggesting increased MMC-dependent oxygen radical generation. Levels of xanthine oxidase (XO; EC 1.1.3.22) and NADPH:cytochrome C reductase, two enzymes known to bioactivate MMC with subsequent oxygen radical generation, were measured in cardiac tissue with a 4.5 x increase in XO activity seen in DOX treated animals vs controls and no change in NADPH:cytochrome C reductase activity. Cardiac levels of xanthine dehydrogenase (XDH; EC 1.1.1.204) activity in DOX treated animals decreased while the XO/XDH ratio increased, suggesting a conversion of XDH to XO following DOX treatment.

  5. Honey as an apitherapic product: its inhibitory effect on urease and xanthine oxidase.

    PubMed

    Sahin, Huseyin

    2016-01-01

    The aim of this study was to evaluate new natural inhibitor sources for the enzymes urease and xanthine oxidase (XO). Chestnut, oak and polyfloral honey extracts were used to determine inhibition effects of both enzymes. In addition to investigate inhibition, the antioxidant capacities of these honeys were determined using total phenolic content (TPC), ferric reducing antioxidant power (FRAP), and DPPH radical scavenging activity assays. Due to their high phenolic content, chestnut and oak honeys are found to be a powerful source for inhibition of both enzymes. Especially, oak honeys were efficient for urease inhibition with 0.012-0.021 g/mL IC50 values, and also chestnut honeys were powerful for XO inhibition with 0.028-0.039 g/mL IC50 values. Regular daily consumption of these honeys can prevent gastric ulcers deriving from Helicobacter pylori and pathological disorders mediated by reactive oxygen species.

  6. The effects of molybate, tungstate and lxd on aldehyde oxidase and xanthine dehydrogenase in Drosophila melanogaster.

    PubMed

    Bentley, M M; Williamson, J H; Oliver, M J

    1981-01-01

    The effects of dietary sodium molybdate and sodium tungstate on eye color and aldehyde oxidase and xanthine dehydrogenase activities have been determined in Drosophila melanogaster. Dietary sodium tungstate administration has been used as a screening procedure to identify two new lxd alleles. Tungstate administration results in increased frequencies of "brown-eyed" flies in lxd stocks and a coordinate decrease in AO and XDH activities in all genotypes tested. The two new lxd alleles affect AO and XDH in a qualitatively but not quantitatively similar fashion to the original lxd allele. AO and XDH activity and AO-CRM levels appear much more sensitive to mutational perturbations of this gene-enzyme than do XDH-CRM levels in the genotypes tested.

  7. The renal phenotype of allopurinol-treated HPRT-deficient mouse

    PubMed Central

    Zarattini, Paola; Clai, Milan; Corbelli, Alessandro; Carraro, Michele; Marchetti, Marialaura; Ronda, Luca; Paredi, Gianluca; Rastaldi, Maria Pia; Percudani, Riccardo

    2017-01-01

    Excess of uric acid is mainly treated with xanthine oxidase (XO) inhibitors, also called uricostatics because they block the conversion of hypoxanthine and xanthine into urate. Normally, accumulation of upstream metabolites is prevented by the hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme. The recycling pathway, however, is impaired in the presence of HPRT deficiency, as observed in Lesch-Nyhan disease. To gain insights into the consequences of purine accumulation with HPRT deficiency, we investigated the effects of the XO inhibitor allopurinol in Hprt-lacking (HPRT-/-) mice. Allopurinol was administered in the drinking water of E12-E14 pregnant mothers at dosages of 150 or 75 μg/ml, and mice sacrificed after weaning. The drug was well tolerated by wild-type animals and heterozygous HPRT+/- mice. Instead, a profound alteration of the renal function was observed in the HPRT-/- model. Increased hypoxanthine and xanthine concentrations were found in the blood. The kidneys showed a yellowish appearance, diffuse interstitial nephritis, with dilated tubules, inflammatory and fibrotic changes of the interstitium. There were numerous xanthine tubular crystals, as determined by HPLC analysis. Oil red O staining demonstrated lipid accumulation in the same location of xanthine deposits. mRNA analysis showed increased expression of adipogenesis-related molecules as well as profibrotic and proinflammatory pathways. Immunostaining showed numerous monocyte-macrophages and overexpression of alpha-smooth muscle actin in the tubulointerstitium. In vitro, addition of xanthine to tubular cells caused diffuse oil red O positivity and modification of the cell phenotype, with loss of epithelial features and appearance of mesenchymal characteristics, similarly to what was observed in vivo. Our results indicate that in the absence of HPRT, blockade of XO by allopurinol causes rapidly developing renal failure due to xanthine deposition within the mouse kidney. Xanthine seems

  8. Dose-dependent effects of allopurinol on human foreskin fibroblast cells and human umbilical vein endothelial cells under hypoxia.

    PubMed

    Sun, Yu; George, Jacob; Rocha, Sonia

    2015-01-01

    Allopurinol, an inhibitor of xanthine oxidase, has been used in clinical trials of patients with cardiovascular and chronic kidney disease. These are two pathologies with extensive links to hypoxia and activation of the transcription factor hypoxia inducible factor (HIF) family. Here we analysed the effects of allopurinol treatment in two different cellular models, and their response to hypoxia. We explored the dose-dependent effect of allopurinol on Human Foreskin Fibroblasts (HFF) and Human Umbilical Vein Endothelial Cells (HUVEC) under hypoxia and normoxia. Under normoxia and hypoxia, high dose allopurinol reduced the accumulation of HIF-1α protein in HFF and HUVEC cells. Allopurinol had only marginal effects on HIF-1α mRNA level in both cellular systems. Interestingly, allopurinol effects over the HIF system were independent of prolyl-hydroxylase activity. Finally, allopurinol treatment reduced angiogenesis traits in HUVEC cells in an in vitro model. Taken together these results indicate that high doses of allopurinol inhibits the HIF system and pro-angiogenic traits in cells.

  9. Deciphering the inhibitory mechanism of genistein on xanthine oxidase in vitro.

    PubMed

    Lin, Suyun; Zhang, Guowen; Pan, Junhui; Gong, Deming

    2015-12-01

    Genistein (Gen), widely distributed in soybean, is proved to be important in homeostasis in the human body. Herein, the inhibitory mechanism of Gen against xanthine oxidase (XO) was studied through multispectroscopic methods and molecular simulation. The inhibition kinetics showed that Gen competitively inhibited XO with an inhibition constant of (1.39 ± 0.11) μM by competing with xanthine for binding to the active site of XO. Fluorescence titration study suggested that the fluorescence quenching mechanism of XO was static, resulting from the formation of a Gen-XO complex at one fold site. The calculated thermodynamic parameters revealed that the interaction process was driven mainly by hydrophobic interactions and hydrogen bonds with affinity of (5.24 ± 0.02) × 10(4) Lmol(-1). Conformational analyses demonstrated that the microenvironment and the secondary structure of XO were changed upon binding of Gen. The molecular docking displayed that Gen bound to the active cavity of XO by interacting with the surrounding amino acid residues (Leu648, Phe649, Glu802, Ser876, Glu879, Arg880, Phe914, Phe1009, Thr1010 and Phe1013). Thus, the inhibition may be attributed to the insertion of Gen into the active site of XO occupying the catalytic center of the enzyme to avoid entry of the substrate and inducing conformational changes of XO (more compact), which was further unfavorable for forming the active cavity and further reduced the landing and oxidation of substrate. This study may offer novel insights into the inhibition mechanism of Gen on XO.

  10. A comparative study on the archives of xanthine oxidase and aldehyde oxidase in different fish species from two rivers in the Western Niger-Delta.

    PubMed

    Isamah, Gabriel K; Asagba, Samuel O

    2004-02-01

    Glycaemia, a classical indicator of stress, xanthine oxidase and aldehyde oxidase which are involved in phase I detoxication were investigated in two different fish species from two rivers with different pollution levels in the Western Niger-Delta. Four sampling zones covering the entire lengths of Warri and Ethiope Rivers respectively were used in this study. For each species of fish five were obtained from a sampling zone in a river. Blood glucose was significantly higher (P < 0.001) in M. electricus from Warri River (82.13 +/- 5.50 mg cm(-3)) compared to the same species from Ethiope River (36.47 +/- 1.49 mg cm(-3)). With the same parameter a similar profile was observed for C. gariepinus; Warri River (56.92 +/- 10.31 mg cm(-3)); Ethiope River (37.65 +/- 0.90 mg cm(-3)) which was also significant (P < 0.01). The activity of xanthine oxidase in M. electricus from Warri River (255.80 +/- 41 it mol cm(-3)) was significantly higher (P < 0.001) compared to the value obtained for the same species (108 +/- 22.36 micro mol cm(-3)) from Ethiope River. Also the activity of xanthine oxidase in C. gariepinus from Warri River (197 +/- 34.65 micro mol cm(-3)) was significantly higher (P < 0.001) when matched with the value obtained for the same species (78.40 +/- 26.84 micro mol cm(-3)) from Ethiope River. That blood glucose level was related to xanthine oxidase activity in the two fish species from Warri River was supported by the high positive correlation between these two parameters (M. electricus. r = 1: C. gariepinus, r = 0.71). The activity of aldehyde oxidase in C. gariepinus from Warri River (143.80 +/- 28.45 micro mol cm(-3)) was significantly higher (P < 0.001) compared to the value obtained for the same species (61.20 +/- 15.21 micro mol cm(-3)) from Ethiope River. A similar profile in aldehyde oxidase activity observed for M. electricus; Warri River (130 +/- 28.39 micro mol cm(-3)); Ethiope River (89 +/- 19.70 micro mol cm(-3)) but an inferior statistical

  11. QM/MM studies of xanthine oxidase: variations of cofactor, substrate, and active-site Glu802.

    PubMed

    Metz, Sebastian; Thiel, Walter

    2010-01-28

    In continuation of our previous QM/MM study on the reductive half-reaction of wild-type xanthine oxidase, we consider the effects of variations in the cofactor, the substrate, and the active-site Glu802 residue on the reaction mechanism. Replacement of the sulfido ligand in the natural cofactor by an oxo ligand leads to a substantial increase in the computed barriers, consistent with the experimentally observed inactivity of this modified cofactor, whereas the selenido form is predicted to have lower barriers and hence higher activity. For the substrate 2-oxo-6-methylpurine, the calculated pathways for three different tautomers show great similarity to those found previously for xanthine, contrary to claims in the literature that the mechanisms for these two substrates are different. Compared with the wild-type enzyme, the conversion of xanthine to uric acid follows a somewhat different pathway in the Glu802 --> Gln mutant which exhibits a lower overall activity, in agreement with recently published kinetic data. The present results confirm the basic stepwise reaction mechanism and the orientation of the substrate that has been proposed in our previous QM/MM work on aldehyde oxidoreductase and xanthine oxidase.

  12. Freeze-Quench Magnetic Circular Dichroism Spectroscopic Study of the "Very Rapid" Intermediate in Xanthine Oxidase.

    PubMed

    Jones, Robert M.; Inscore, Frank E.; Hille, Russ; Kirk, Martin L.

    1999-11-01

    Freeze-quench magnetic circular dichroism spectroscopy (MCD) has been used to trap and study the excited-state electronic structure of the Mo(V) active site in a xanthine oxidase intermediate generated with substoichiometric concentrations of the slow substrate 2-hydroxy-6-methylpurine. EPR spectroscopy has shown that the intermediate observed in the MCD experiment is the "very rapid" intermediate, which lies on the main catalytic pathway. The low-energy (< approximately 30 000 cm(-1)) C-term MCD of this intermediate is remarkably similar to that of the model compound LMoO(bdt) (L = hydrotris(3,5-dimethyl-1-pyrazolyl)borate; bdt = 1,2-benzenedithiolate), and the MCD bands have been assigned as dithiolate S(ip) --> Mo d(xy) and S(op) --> Mo d(xz,yz) LMCT transitions. These transitions result from a coordination geometry of the intermediate where the Mo=O bond is oriented cis to the ene-1,2-dithiolate of the pyranopterin. Since X-ray crystallography has indicated that a terminal sulfido ligand is oriented cis to the ene-1,2-dithiolate in oxidized xanthine oxidase related Desulfovibrio gigas aldehyde oxidoreductase, we have suggested that a conformational change occurs upon substrate binding. The substrate-mediated conformational change is extremely significant with respect to electron-transfer regeneration of the active site, as covalent interactions between the redox-active Mo d(xy) orbital and the S(ip) orbitals of the ene-1,2-dithiolate are maximized when the oxo ligand is oriented cis to the dithiolate plane. This underlies the importance of the ene-1,2-dithiolate portion of the pyranopterin in providing an efficient superexchange pathway for electron transfer. The results of this study indicate that electron-transfer regeneration of the active site may be gated by the orientation of the Mo=O bond relative to the ene-1,2-dithiolate chelate. Poor overlap between the Mo d(xy) orbital and the S(ip) orbitals of the dithiolate in the oxidized enzyme geometry may

  13. Preparation of monoclonal antibodies to xanthine oxidase and other proteins of bovine milk-fat-globule membrane.

    PubMed Central

    Mather, I H; Nace, C S; Johnson, V G; Goldsby, R A

    1980-01-01

    Nine hybridomas secreting monoclonal antibody to proteins of bovine milk-fat-globule membrane were isolated. All nine cell lines continued to secrete monoclonal antibody after serial transfer in culture and after passage as solid tumours in Balb/cJ mice. Four of the cell lines secreted monoclonal antibody specific for xanthine oxidase, one of the major proteins of milk-fat-globule membrane. PMID:6894088

  14. Cardiac contractility in Antarctic teleost is modulated by nitrite through xanthine oxidase and cytochrome p-450 nitrite reductase.

    PubMed

    Garofalo, Filippo; Amelio, Daniela; Gattuso, Alfonsina; Cerra, Maria Carmela; Pellegrino, Daniela

    2015-09-15

    In mammalian and non-mammalian vertebrates, nitrite anion, the largest pool of intravascular and tissue nitric oxide storage, represents a key player of many biological processes, including cardiac modulation. As shown by our studies on Antarctic teleosts, nitrite-dependent cardiac regulation is of great relevance also in cold-blooded vertebrates. This study analysed the influence elicited by nitrite on the performance of the perfused beating heart of two Antarctic stenotherm teleosts, the haemoglobinless Chionodraco hamatus (icefish) and the red-blooded Trematomus bernacchii. Since haemoglobin is crucial in nitric oxide homeostasis, the icefish, a naturally occurring genetic knockout for this protein, provides exclusive opportunities to investigate nitric oxide/nitrite signaling. In vivo, nitrite conversion to nitric oxide requires the nitrite reductase activity of xanthine oxidase and cytochrome P-450, thus the involvement of these enzymes was also evaluated. We showed that, in C. hamatus and T. bernacchii, nitrite influenced cardiac performance by inducing a concentration-dependent positive inotropic effect which was unaffected by nitric oxide scavenging by PTIO in C. hamatus, while it was abolished in T. bernacchii. Specific inhibition of xanthine oxidase and cytochrome P-450 revealed, in the two teleosts, that the nitrite-dependent inotropism required the nitrite reductase activity of both enzymes. We also found that xanthine oxidase is more expressed in C. hamatus than in T. bernacchii, while the opposite was observed concerning cytochrome P-450. Results suggested that in the heart of C. hamatus and T. bernacchii, nitrite is an integral physiological source of nitric oxide with important signaling properties, which require the nitrite reductase activity of xanthine oxidase and cytochrome P-450.

  15. A tungsten supplemented diet attenuates bacterial translocation in chronic portal hypertensive and cholestatic rats: role of xanthine dehydrogenase and xanthine oxidase

    PubMed Central

    Schimpl, G; Pabst, M; Feierl, G; Kuesz, A; Ozbey, H; Takahashi, S; Hollwarth, M

    1999-01-01

    BACKGROUND—Bacterial translocation (BT) plays a major role in the pathophysiological process of spontaneous infections in portal hypertension (PH) and cholestatic jaundice. The major mechanisms promoting BT in experimental animal models are the disruption of the intestinal ecological equilibrium and disruption of the intestinal mucosal barrier. The enzymes xanthine dehydrogenase (XD) and xanthine oxidase (XO) are often implicated as a significant source of oxidants which have a major impact on the impairment of intestinal barrier function.
AIM—To investigate the incidence of BT in rats with PH and obstructive jaundice, and to evaluate the impact of XD and XO.
METHODS—Animals were subjected to sham laparotomy (SL), PH by calibrated stenosis of the portal vein, and common bile duct ligation (CBDL). They were fed either a standard pellet diet or a tungsten supplemented molybdenum-free diet. Four weeks after the operative procedure, intestinal colonisation and BT to portal vein, vena cava, mesenteric lymph nodes, liver, and spleen were determined. Intestinal XD and XO activity were measured enzymatically and histochemically.
RESULTS—Significant (p<0.01) intestinal bacterial overgrowth was present in all PH and CBDL groups compared with the SL group. In normally fed animals after SL, BT occurred in 12%. In PH and after CBDL, the rate of BT increased significantly (p<0.05) to 28% and 54% respectively. In the jejunum of normally fed animals subjected to PH or CBDL, a significant increase in XO was observed (p<0.01). Animals fed a tungsten supplemented diet showed a significant attenuation of BT to 14% in PH and 22% after CBDL (p<0.05). Tungsten treatment completely suppressed jejunal XD and XO activities.
CONCLUSIONS—Significant intestinal bacterial overgrowth, BT, and XD to XO conversion occurred in PH and after CBDL. XD and XO inactivation by a tungsten supplemented molybdenum-free diet significantly reduced the incidence of BT without affecting

  16. Hydroxylated chalcones with dual properties: xanthine oxidase inhibitors and radical scavengers

    PubMed Central

    Hofmann, Emily; Webster, Jonathan; Do, Thuy; Kline, Reid; Snider, Lindsey; Hauser, Quintin; Higginbottom, Grace; Campbell, Austin; Ma, Lili; Paula, Stefan

    2016-01-01

    In this study, we evaluated the abilities of a series of chalcones to inhibit the activity of the enzyme xanthine oxidase (XO) and to scavenge radicals. 20 mono- and polyhydroxylated chalcone derivatives were synthesized by Claisen-Schmidt condensation reactions and then tested for inhibitory potency against XO, a known generator of reactive oxygen species (ROS). In parallel, the ability of the synthesized chalcones to scavenge a stable radical was determined. Structure-activity relationship analysis in conjunction with molecular docking indicated that the most active XO inhibitors carried a minimum of three hydroxyl groups. Moreover, the most effective radical scavengers had two neighboring hydroxyl groups on at least one of the two phenyl rings. Since it has been proposed previously that XO inhibition and radical scavenging could be useful properties for reduction of ROS-levels in tissue, we determined the chalcones’ effects to rescue neurons subjected to ROS-induced stress created by the addition of β-amyloid peptide. Best protection was provided by chalcones that combined good inhibitory potency with high radical scavenging ability in a single molecule, an observation that points to a potential therapeutic value of this compound class. PMID:26762836

  17. Characterization and Thermodynamic Relationship of Three Polymorphs of a Xanthine Oxidase Inhibitor, Febuxostat.

    PubMed

    Patel, Jinish; Jagia, Moksh; Bansal, Arvind Kumar; Patel, Sarsvatkumar

    2015-11-01

    Febuxostat (FXT), a xanthine oxidase inhibitor, is an interesting and unique molecule, which exhibits extensive polymorphism, with over 15 polymorphic forms reported to date. The primary purpose of the study was to characterize the three polymorphic forms with respect to their thermodynamic quantities and establish thermodynamic relationship between them. The polymorphs were characterized by thermal and powder X-ray diffraction methods. Three different methods were used to calculate the transition temperatures (Ttr) and thereby their thermodynamic relationships. Although the first and second method used calorimetric data (melting point and heat of fusion), the third method employed the use of configurational free energy phase diagram. The onset melting points of three polymorphic forms were found to be 482.89 ± 0.37 K for form I, 476.30 ± 1.21 K for form II, and 474.19 ± 0.11 K for form III. Moreover, the powder X-ray diffraction patterns for each form were also unique. The polymorphic pair of form I and II and of form I and III was found to be enantiotropic, whereas pair of form II and III was monotropic. Besides the relative thermodynamic aspects (free energy differences, enthalpy, entropy contributions) using different methods, the pharmaceutical implications and phase transformation aspects have also been covered.

  18. Design and synthesis of aza-flavones as a new class of xanthine oxidase inhibitors.

    PubMed

    Dhiman, Rajni; Sharma, Sahil; Singh, Gagandip; Nepali, Kunal; Singh Bedi, Preet Mohinder

    2013-01-01

    In an attempt to develop non-purine-based xanthine oxidase (XO) inhibitors, keeping in view the complications reported with the use of purine-based XO inhibitors, the flavone framework (a class possessing XO inhibitory potential) was used as lead structure for further optimization. By means of structure-based classical bioisosterism, quinolone was used as an isoster for chromone (a bicyclic unit present in flavones), owing to the bioactive potential and drug-like properties of quinolones. This type of replacement does not alter the shape and structural features required for XO inhibition, and also provides some additional interaction sites, without the loss of hydrogen bonding and hydrophobic and arene-arene interactions. In the present study, a series of 2-aryl/heteroaryl-4-quinolones (aza analogs of flavones) was rationally designed, synthesized and evaluated for in vitro XO inhibitory activity. Some notions about structure-activity relationships are presented indicating the influence of the nature of the 2-aryl ring on the inhibitory activity. Important interactions of the most active compound 3l (IC(50)  = 6.24 µM) with the amino acid residues of the active site of XO were figured out by molecular modeling.

  19. Xanthine oxidase inhibitory activity and hypouricemic effect of aspalathin from unfermented rooibos.

    PubMed

    Kondo, Makoto; Hirano, Yoshiaki; Nishio, Masahiro; Furuya, Yutaka; Nakamura, Hiromichi; Watanabe, Tsuyoshi

    2013-12-01

    Rooibos is rich in flavonoids such as aspalathin, which is a unique C-glycosyl dihydrochalcone, that is used as a traditional herbal tea. This study was designed to evaluate the in vitro xanthine oxidase (XOD) inhibitory activity of the aspalathin-rich fraction (ARF) and purified aspalathin from rooibos. The hypouricemic effects of the ARF and aspalathin on hyperuricemic mice were also assessed. The ARF was prepared from aqueous extract of unfermented rooibos leaves and stems, and it was collected by column chromatography; the aspalathin content in this fraction was 21.4%. The ARF and aspalathin inhibited XOD in a dose-dependent manner. The concentrations of the ARF and aspalathin required to inhibit XOD at 50% (IC50 ) were 20.4 μg/mL (4.4 μg/mL aspalathin equivalents) and 4.5 μg/mL, respectively. Lineweaver-Burk plot analysis indicated that aspalathin was a competitive inhibitor of XOD, and the inhibition constant (Ki) was 3.1 μM. In hyperuricemic mice induced by inosine-5'-monophosphate, treatment with the ARF and aspalathin significantly suppressed the increased plasma uric acid level in a dose-dependent manner. The suppressed plasma uric acid level in mice could be attributed to the XOD inhibitory activity of the ARF and aspalathin. Further study is required to determine the effect of aspalathin or its metabolites on XOD activity in vivo.

  20. Flavonoid glycosides isolated from unique legume plant extracts as novel inhibitors of xanthine oxidase.

    PubMed

    Spanou, Chrysoula; Veskoukis, Aristidis S; Kerasioti, Thalia; Kontou, Maria; Angelis, Apostolos; Aligiannis, Nektarios; Skaltsounis, Alexios-Leandros; Kouretas, Dimitrios

    2012-01-01

    Legumes and the polyphenolic compounds present in them have gained a lot of interest due to their beneficial health implications. Dietary polyphenolic compounds, especially flavonoids, exert antioxidant properties and are potent inhibitors of xanthine oxidase (XO) activity. XO is the main contributor of free radicals during exercise but it is also involved in pathogenesis of several diseases such as vascular disorders, cancer and gout. In order to discover new natural, dietary XO inhibitors, some polyphenolic fractions and pure compounds isolated from two legume plant extracts were tested for their effects on XO activity. The fractions isolated from both Vicia faba and Lotus edulis plant extracts were potent inhibitors of XO with IC(50) values range from 40-135 µg/mL and 55-260 µg/mL, respectively. All the pure polyphenolic compounds inhibited XO and their K(i) values ranged from 13-767 µM. Ten of the compounds followed the non competitive inhibitory model whereas one of them was a competitive inhibitor. These findings indicate that flavonoid isolates from legume plant extracts are novel, natural XO inhibitors. Their mode of action is under investigation in order to examine their potential in drug design for diseases related to overwhelming XO action.

  1. Hydroxylated chalcones with dual properties: Xanthine oxidase inhibitors and radical scavengers.

    PubMed

    Hofmann, Emily; Webster, Jonathan; Do, Thuy; Kline, Reid; Snider, Lindsey; Hauser, Quintin; Higginbottom, Grace; Campbell, Austin; Ma, Lili; Paula, Stefan

    2016-02-15

    In this study, we evaluated the abilities of a series of chalcones to inhibit the activity of the enzyme xanthine oxidase (XO) and to scavenge radicals. 20 mono- and polyhydroxylated chalcone derivatives were synthesized by Claisen-Schmidt condensation reactions and then tested for inhibitory potency against XO, a known generator of reactive oxygen species (ROS). In parallel, the ability of the synthesized chalcones to scavenge a stable radical was determined. Structure-activity relationship analysis in conjunction with molecular docking indicated that the most active XO inhibitors carried a minimum of three hydroxyl groups. Moreover, the most effective radical scavengers had two neighboring hydroxyl groups on at least one of the two phenyl rings. Since it has been proposed previously that XO inhibition and radical scavenging could be useful properties for reduction of ROS-levels in tissue, we determined the chalcones' effects to rescue neurons subjected to ROS-induced stress created by the addition of β-amyloid peptide. Best protection was provided by chalcones that combined good inhibitory potency with high radical scavenging ability in a single molecule, an observation that points to a potential therapeutic value of this compound class.

  2. Flavonoid Glycosides Isolated from Unique Legume Plant Extracts as Novel Inhibitors of Xanthine Oxidase

    PubMed Central

    Spanou, Chrysoula; Veskoukis, Aristidis S.; Kerasioti, Thalia; Kontou, Maria; Angelis, Apostolos; Aligiannis, Nektarios; Skaltsounis, Alexios-Leandros; Kouretas, Dimitrios

    2012-01-01

    Legumes and the polyphenolic compounds present in them have gained a lot of interest due to their beneficial health implications. Dietary polyphenolic compounds, especially flavonoids, exert antioxidant properties and are potent inhibitors of xanthine oxidase (XO) activity. XO is the main contributor of free radicals during exercise but it is also involved in pathogenesis of several diseases such as vascular disorders, cancer and gout. In order to discover new natural, dietary XO inhibitors, some polyphenolic fractions and pure compounds isolated from two legume plant extracts were tested for their effects on XO activity. The fractions isolated from both Vicia faba and Lotus edulis plant extracts were potent inhibitors of XO with IC50 values range from 40–135 µg/mL and 55–260 µg/mL, respectively. All the pure polyphenolic compounds inhibited XO and their Ki values ranged from 13–767 µM. Ten of the compounds followed the non competitive inhibitory model whereas one of them was a competitive inhibitor. These findings indicate that flavonoid isolates from legume plant extracts are novel, natural XO inhibitors. Their mode of action is under investigation in order to examine their potential in drug design for diseases related to overwhelming XO action. PMID:22396752

  3. Role of host xanthine oxidase in infection due to enteropathogenic and Shiga-toxigenic Escherichia coli

    PubMed Central

    Crane, John K

    2013-01-01

    Xanthine oxidase (XO) has been recognized as an important host defense enzyme for decades. In our recent study in Infection and Immunity, we found that enteropathogenic and Shiga-toxigenic E. coli (EPEC and STEC) were far more resistant to killing by the XO pathway than laboratory E. coli strains used in the past. Although XO plus hypoxanthine substrate rarely generated enough H2O2 to kill EPEC and STEC, the pathogens were able to sense the H2O2 and react to it with an increase in expression of virulence factors, most notably Shiga toxin (Stx). H2O2 produced by XO also triggered a chloride secretory response in T84 cell monolayers studied in the Ussing chamber. Adding exogenous XO plus its substrate in vivo did not decrease the number of STEC bacteria recovered from ligated intestinal loops, but instead appeared to worsen the infection and increased the amount of Stx2 toxin produced. XO plus hypoxanthine also increases the ability of Stx2 to translocate across intestinal monolayers. With regard to EPEC and STEC, the role of XO appears more complex and subtle than what has been reported in the past, since XO also plays a role in host-pathogen signaling, in regulating virulence in pathogens, in Stx production and in toxin translocation. Uric acid produced by XO may also be in itself an immune modulator in the intestinal tract. PMID:23811846

  4. Role of host xanthine oxidase in infection due to enteropathogenic and Shiga-toxigenic Escherichia coli.

    PubMed

    Crane, John K

    2013-01-01

    Xanthine oxidase (XO) has been recognized as an important host defense enzyme for decades. In our recent study in Infection and Immunity, we found that enteropathogenic and Shiga-toxigenic E. coli (EPEC and STEC) were far more resistant to killing by the XO pathway than laboratory E. coli strains used in the past. Although XO plus hypoxanthine substrate rarely generated enough H 2O 2 to kill EPEC and STEC, the pathogens were able to sense the H2O2 and react to it with an increase in expression of virulence factors, most notably Shiga toxin (Stx). H 2O 2 produced by XO also triggered a chloride secretory response in T84 cell monolayers studied in the Ussing chamber. Adding exogenous XO plus its substrate in vivo did not decrease the number of STEC bacteria recovered from ligated intestinal loops, but instead appeared to worsen the infection and increased the amount of Stx2 toxin produced. XO plus hypoxanthine also increases the ability of Stx2 to translocate across intestinal monolayers. With regard to EPEC and STEC, the role of XO appears more complex and subtle than what has been reported in the past, since XO also plays a role in host-pathogen signaling, in regulating virulence in pathogens, in Stx production and in toxin translocation. Uric acid produced by XO may also be in itself an immune modulator in the intestinal tract.

  5. Quick identification of xanthine oxidase inhibitor and antioxidant from Erycibe obtusifolia by a drug discovery platform composed of multiple mass spectrometric platforms and thin-layer chromatography bioautography.

    PubMed

    Chen, Zhiyong; Tao, Hongxun; Liao, Liping; Zhang, Zijia; Wang, Zhengtao

    2014-08-01

    As a final step of the purine metabolism process, xanthine oxidase catalyzes the oxidation of hypoxanthine and xanthine into uric acid. Our research has demonstrated that Erycibe obtusifolia has xanthine oxidase inhibitory properties. The purpose of this paper is to describe a new strategy based on a combination of multiple mass spectrometric platforms and thin-layer chromatography bioautography for effectively screening the xanthine oxidase inhibitory and antioxidant properties of E. obtusifolia. This strategy was accomplished through the following steps. (i) Separate the extract of E. obtusifolia into fractions by an autopurification system controlled by liquid chromatography with mass spectrometry. (ii) Determine the active fractions of E. obtusifolia by thin-layer chromatography bioautography. (iii) Identify the structure of the main active compounds with the information provided by direct analysis in real time mass spectrometry. (iv) Calculate the IC50 value of each compound against xanthine oxidase using high-performance liquid chromatography. Using the caulis of E. obtusifolia as the experimental material, seven target peaks were screened out as xanthine oxidase inhibitors or antioxidants. Our screening strategy allows for rapid analysis of small molecules with almost no sample preparation and can be completed within a week, making it a useful assay to identify unstable compounds and provide the empirical foundation for E. obtusifolia as a natural remedy for gout and oxidative-stress-related diseases.

  6. Kinetic study on the inhibition of xanthine oxidase by extracts from two selected Algerian plants traditionally used for the treatment of inflammatory diseases.

    PubMed

    Berboucha, Meriem; Ayouni, Karima; Atmani, Dina; Atmani, Djebbar; Benboubetra, Mustapha

    2010-08-01

    In order to further understand and assess the validity of herbal medicine, we investigated the potential inhibitory effect of various extracts from Fraxinus angustifolia and Pistacia lentiscus, two plants used traditionally in Algeria against several inflammatory diseases such as rheumatism, arthritis, and gout, on purified bovine milk xanthine oxidase (XO) activity. The total phenolic contents of the leaves and bark of F. angustifolia and the leaves and seeds of P. lentiscus were estimated. P. lentiscus aqueous fractions from hexane and chloroform extractions and F. angustifolia aqueous fraction from ethyl acetate extraction inhibited XO activity by 72.74 +/- 2.63% (50% inhibitory concentration [IC(50)] = 27.52 microg/mL), 68.97 +/- 3.89% (IC(50) = 42.46 microg/mL) and 53.92 +/- 3.17% (IC(50) = 58.84 mmicroug/mL), respectively, at 100 microg/mL, compared to that of reference drug, allopurinol (98.18% [IC(50) = 6.34 microg/mL]). Moreover, at a concentration of 50 microg/mL, both P. lentiscus extracts showed inhibition rates higher than 50%. F. angustifolia leaf extracts showed only mild inhibition. Lineweaver-Burk analysis showed that the inhibitory activity exerted by F. angustifolia bark aqueous extract and P. lentiscus aqueous extracts is of mixed type, whereas the leaf extracts from F. angustifolia inhibited XO noncompetitively. Positive correlations were established between XO inhibition and total phenols (r = 0.89) and flavonoids (r = 0.93) for P. lentiscus and with total phenols (r = 0.72) and tannins (r = 0.54) for F. angustifolia. Our findings suggest that the therapeutic use of these plants may be due to the observed XO inhibition, thereby supporting their use in traditional folk medicine against inflammatory-related diseases, in particular, gout.

  7. IFN-γ regulates xanthine oxidase-mediated iNOS-independent oxidative stress in maneb- and paraquat-treated rat polymorphonuclear leukocytes.

    PubMed

    Singh, Deepali; Kumar, Vinod; Singh, Chetna

    2017-03-01

    Maneb (MB) and paraquat (PQ) provoke oxidative stress-mediated cell damage. Role of xanthine oxidase (XO) in oxidative stress and its association with nitric oxide (NO)/NO synthase (NOS) have been widely reported. While inducible NOS (iNOS) is implicated in MB+PQ-induced toxicity in rat polymorphonuclear leukocytes (PMNs), role of XO and its alliance with iNOS have not yet been established. The study investigated the role of XO in MB+PQ-induced oxidative stress in rat PMNs and its regulation by iNOS and inflammatory cytokines. MB+PQ-augmented reactive oxygen species (ROS), superoxide, nitro-tyrosine, lipid peroxidation (LPO), and nitrite levels along with the catalytic activity of iNOS, superoxide dismutase (SOD), and XO. XO inhibitor, allopurinol (AP), alleviated MB+PQ-induced changes except nitrite content and iNOS activity. Conversely, an iNOS inhibitor, aminoguanidine, mitigated MB+PQ-induced LPO, nitrite, iNOS, and nitro-tyrosine levels; however, no change was observed in ROS, SOD, and XO. Nuclear factor-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), tumor necrosis factor-alpha (TNF-α) inhibitor, pentoxyfylline, and an anti-inflammatory agent, dexamethasone, attenuated MB+PQ-induced increase in XO, superoxide, and ROS with parallel reduction in the expression of interferon-gamma (IFN-γ), TNF-α, and interleukin-1β (IL-1β) in rat PMNs. Exogenous IFN-γ, TNF-α, and IL-1β enhanced superoxide, ROS, and XO in the PMNs of control and MB+PQ-treated rats; however, IFN- γ was found to be the most potent inducer. Moreover, AP ameliorated cytokine-induced free radical generation and restored XO activity towards normalcy. The results thus demonstrate that XO mediates oxidative stress in MB+PQ-treated rat PMNs via iNOS-independent but cytokine (predominantly IFN-γ)-dependent mechanism.

  8. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress.

    PubMed

    McNally, J Scott; Davis, Michael E; Giddens, Don P; Saha, Aniket; Hwang, Jinah; Dikalov, Sergey; Jo, Hanjoong; Harrison, David G

    2003-12-01

    Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

  9. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

    NASA Technical Reports Server (NTRS)

    McNally, J. Scott; Davis, Michael E.; Giddens, Don P.; Saha, Aniket; Hwang, Jinah; Dikalov, Sergey; Jo, Hanjoong; Harrison, David G.

    2003-01-01

    Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress.

  10. Synthesis, crystal structures, fluorescence and xanthine oxidase inhibitory activity of pyrazole-based 1,3,4-oxadiazole derivatives

    NASA Astrophysics Data System (ADS)

    Qi, De-Qiang; Yu, Chuan-Ming; You, Jin-Zong; Yang, Guang-Hui; Wang, Xue-Jie; Zhang, Yi-Ping

    2015-11-01

    A series of pyrazole-based 1,3,4-oxadiazole derivatives were rationally designed and synthesized in good yields by following a convenient route. All the newly synthesized molecules were fully characterized by IR, 1H NMR and elemental analysis. Eight compounds were structurally determined by single crystal X-ray diffraction analysis. The fluorescence properties of all the compounds were investigated in dimethyl sulfoxide media. In addition, these newly synthesized compounds were evaluated for in vitro inhibitory activity against commercial enzyme xanthine oxidase (XO) by measuring the formation of uric acid from xanthine. Among the compounds synthesized and tested, 3d and 3e were found to be moderate inhibitory activity against commercial XO with IC50 = 72.4 μM and 75.6 μM. The studies gave a new insight in further optimization of pyrazole-based 1,3,4-oxadiazole derivatives with excellent fluorescence properties and XO inhibitory activity.

  11. Xanthine oxidase and lens oxidative stress markers in diabetic and senile cataract patients.

    PubMed

    Miric, Dijana J; Kisic, Bojana B; Zoric, Lepsa D; Mitic, Radoslav V; Miric, Bratislav M; Dragojevic, Ilija M

    2013-01-01

    Xanthine oxidase (XOD) is a prooxidant enzyme possibly implicated in diabetic lens injury and genesis of senile cataract (SC). We evaluated the impact of diabetes on XOD activity and its relationships with lens oxidative stress markers in patients operated on for SC. Serum and lens XOD activities, lens malondialdehyde (MDA), conjugated dienes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and reduced glutathione (GSH) levels were measured in 62 non-diabetic and 29 diabetic patients operated on for SC. Lens XOD, SOD, GPx and GSH levels were gradually declining, while MDA and serum XOD were increasing with patient's age. Lens XOD activity was positively correlated with conjugated dienes concentration (rho=0.316; p=0.003) while being inversely correlated with age (rho=-0.371; p<0.001), indicating that low ocular expression of XOD could be related to lower intensity of oxidative stress and delayed occurrence of SC. When samples were adjusted for confounding factors, serum XOD (p<0.001), lens XOD (p=0.003) and conjugated dienes (p=0.002) were significantly higher in diabetic than in non-diabetic group. Lens SOD and GPx were moderately increased while MDA and GSH were unchanged in diabetic, compared with non-diabetic SC group. Blood HbA1C concentration was positively correlated with lens XOD (rho=0.346; p<0.001) as well as serum XOD activity (rho=0.485; p<0.001). These results suggest that poor glycemic control may upregulate systemic and ocular XOD activities contributing to lens oxidative stress and possibly to earlier onset of cataract.

  12. Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta.

    PubMed

    Soucy, Kevin G; Lim, Hyun Kyo; Attarzadeh, David O; Santhanam, Lakshmi; Kim, Jae Hyung; Bhunia, Anil K; Sevinc, Baris; Ryoo, Sungwoo; Vazquez, Marcelo E; Nyhan, Daniel; Shoukas, Artin A; Berkowitz, Dan E

    2010-05-01

    Radiation exposure is associated with the development of various cardiovascular diseases. Although irradiation is known to cause elevated oxidant stress and chronic inflammation, both of which are detrimental to vascular function, the molecular mechanisms remain incompletely understood. We previously demonstrated that radiation causes endothelial dysfunction and increased vascular stiffness by xanthine oxidase (XO) activation. In this study, we investigated whether dietary inhibition of XO protects against radiation-induced vascular injury. We exposed 4-mo-old rats to a single dose of 0 or 5 Gy gamma radiation. These rats received normal drinking water or water containing 1 mM oxypurinol, an XO inhibitor. We measured XO activity and superoxide production in rat aorta and demonstrated that both were significantly elevated 2 wk after radiation exposure. However, oxypurinol treatment in irradiated rats prevented aortic XO activation and superoxide elevation. We next investigated endothelial function through fluorescent measurement of nitric oxide (NO) and vascular tension dose responses. Radiation reduced endothelium-dependent NO production in rat aorta. Similarly, endothelium-dependent vasorelaxation in the aorta of irradiated rats was significantly attenuated compared with the control group. Dietary XO inhibition maintained NO production at control levels and prevented the development of endothelial dysfunction. Furthermore, pulse wave velocity, a measure of vascular stiffness, increased by 1 day postirradiation and remained elevated 2 wk after irradiation, despite unchanged blood pressures. In oxypurinol-treated rats, pulse wave velocities remained unchanged from baseline throughout the experiment, signifying preserved vascular health. These findings demonstrate that XO inhibition can offer protection from radiation-induced endothelial dysfunction and cardiovascular complications.

  13. Xanthine oxidase inhibitory activity of the methanolic extracts of selected Jordanian medicinal plants

    PubMed Central

    Hudaib, Mohammad M.; Tawaha, Khaled A.; Mohammad, Mohammad K.; Assaf, Areej M.; Issa, Ala Y.; Alali, Feras Q.; Aburjai, Talal A.; Bustanji, Yasser K.

    2011-01-01

    Background: The search for novel xanthine oxidase (XO) inhibitors with a higher therapeutic activity and fewer side effects are desired not only to treat gout but also to combat various other diseases associated with the XO activity. At present, the potential of developing successful natural products for the management of XO-related diseases is still largely unexplored. In the present study, we have screened the methanolic extracts of various Jordanian medicinal plants for their XO inhibitory activities using an optimized protocol. Materials and Methods: The methanolic extracts of 23 medicinal plants, belonging to 12 families, were tested in vitro, at 200 μg/ml concentrations, for their XO inhibitory potential. The dose-dependent inhibition profiles of the most active plants were further evaluated by estimating the IC50 values of their corresponding extracts. Results: Six plants were found most active (% inhibition more than 39%). These plants are Salvia spinosa L. (IC50 = 53.7 μg/ml), Anthemis palestina Boiss. (168.0 μg/ml), Chrysanthemum coronarium L. (199.5 μg/ml), Achillea biebersteinii Afansiev (360.0 μg/ml), Rosmarinus officinalis L. (650.0 μg/ml), and Ginkgo biloba L. (595.8 μg/ml). Moreover, four more plants, namely Lavandula angustifolia Mill. (28.7% inhibition), Helianthemum ledifolium (L.) Mill. (28.4%), Majorana syriaca (L.) Kostel. (25.1%), and Mentha spicata L. (22.5%) showed a XO inhibitory activity in the range of 22–30%. Conclusion: The study showed that many of the tested plant species are potential sources of natural XO inhibitors that can be developed, upon further investigation, into successful herbal drugs for treatment of gout and other XO-related disorders. PMID:22262935

  14. Xanthine Oxidase Activity in Type 2 Diabetes Mellitus Patients with and without Diabetic Peripheral Neuropathy

    PubMed Central

    Filipovic-Danic, Snezana; Miric, Marko B.; Puhalo-Sladoje, Dragana

    2016-01-01

    This study investigated the relationship between serum xanthine oxidase (XOD) activity and the occurrence of diabetic peripheral neuropathy (DPN) in type 2 diabetes mellitus (T2DM) patients. Serum XOD activity, ischemia-modified albumin (IMA), uric acid (UA), albumin, glycated hemoglobin (HbA1c), advanced glycation end products (AGE), total free thiols, atherogenic index of plasma (AIP), and body mass index (BMI) were measured in 80 T2DM patients (29 with and 51 without DPN), and 30 nondiabetic control subjects. Duration of diabetes, hypertension, medication, and microalbuminuria was recorded. Serum XOD activities in controls, non-DPN, and DPN were 5.7 ± 2.4 U/L, 20.3 ± 8.6 U/L, and 27.5 ± 10.6 U/L (p < 0.01), respectively. XOD activity was directly correlated to IMA, UA, BMI, HbA1c, and AGE, while inversely correlated to serum total free thiols. A multivariable logistic regression model, which included duration of diabetes, hypertension, AIP, HbA1c, UA, and XOD activity, revealed HbA1c [OR = 1.03 (1.00–1.05); p = 0.034] and XOD activity [OR = 1.07 (1.00–1.14); p = 0.036] as independent predictors of DPN. Serum XOD activity was well correlated to several other risk factors. These results indicate the role of XOD in the development of DPN among T2DM patients. PMID:27965983

  15. Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase: The Sequential Hydroxylation of Hypoxanthine to Uric Acid

    SciTech Connect

    Cao, Hongnan; Pauff, James M.; Hille, Russ

    2010-11-29

    Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp{sup 2}-hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 {angstrom} resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6 {angstrom} resolution have been determined, showing in each case two alternate orientations of substrate in the two active sites of the crystallographic asymmetric unit. One orientation is such that it is expected to yield hydroxylation at C-2 of substrate, yielding xanthine. The other suggests hydroxylation at C-8 to give 6,8-dihydroxypurine, a putative product not previously thought to be generated by the enzyme. Kinetic experiments demonstrate that >98% of hypoxanthine is hydroxylated at C-2 rather than C-8, indicating that the second crystallographically observed orientation is significantly less catalytically effective than the former. Theoretical calculations suggest that enzyme selectivity for the C-2 over C-8 of hypoxanthine is largely due to differences in the intrinsic reactivity of the two sites. For the orientation of hypoxanthine with C-2 proximal to the molybdenum center, the disposition of substrate in the active site is such that Arg880 and Glu802, previous shown to be catalytically important for the conversion of xanthine to uric acid, play similar roles in hydroxylation at C-2 as at C-8. Contrary to the literature, we find that 6,8-dihydroxypurine is effectively converted to uric acid by xanthine oxidase.

  16. Phytochemical Composition, Antioxidant and Xanthine Oxidase Inhibitory Activities of Amaranthus cruentus L. and Amaranthus hybridus L. Extracts

    PubMed Central

    Nana, Fernand W.; Hilou, Adama; Millogo, Jeanne F.; Nacoulma, Odile G.

    2012-01-01

    This paper describes a preliminary assessment of the nutraceutical value of Amaranthus cruentus (A. cruentus) and Amaranthus hybridus (A. hybridus), two food plant species found in Burkina Faso. Hydroacetonic (HAE), methanolic (ME), and aqueous extracts (AE) from the aerial parts were screened for in vitro antioxidant and xanthine oxidase inhibitory activities. Phytochemical analyses revealed the presence of polyphenols, tannins, flavonoids, steroids, terpenoids, saponins and betalains. Hydroacetonic extracts have shown the most diversity for secondary metabolites. The TLC analyses of flavonoids from HAE extracts showed the presence of rutin and other unidentified compounds. The phenolic compound contents of the HAE, ME and AE extracts were determined using the Folin–Ciocalteu method and ranged from 7.55 to 10.18 mg Gallic acid equivalent GAE/100 mg. Tannins, flavonoids, and flavonols ranged from 2.83 to 10.17 mg tannic acid equivalent (TAE)/100 mg, 0.37 to 7.06 mg quercetin equivalent (QE) /100 mg, and 0.09 to 1.31 mg QE/100 mg, respectively. The betacyanin contents were 40.42 and 6.35 mg Amaranthin Equivalent/100 g aerial parts (dry weight) in A. cruentus and A. hybridus, respectively. Free-radical scavenging activity expressed as IC50 (DPPH method) and iron reducing power (FRAP method) ranged from 56 to 423 µg/mL and from 2.26 to 2.56 mmol AAE/g, respectively. Xanthine oxidase inhibitory activities of extracts of A. cruentus and A. hybridus were 3.18% and 38.22%, respectively. The A. hybridus extract showed the best antioxidant and xanthine oxidase inhibition activities. The results indicated that the phytochemical contents of the two species justify their traditional uses as nutraceutical food plants. PMID:24281664

  17. An oxidative coupling product of luteolin with cysteine ester and its enhanced inhibitory activity for xanthine oxidase.

    PubMed

    Masuda, Toshiya; Nojima, Shoko; Miura, Yukari; Honda, Sari; Masuda, Akiko

    2015-08-15

    Oxidative coupling reactions of several flavonoids with a cysteine ester (a radicalic and nucleophilic biochemical) were carried out and the abilities of the coupling products against xanthine oxidase (XO) were screened. One of the products, derived from luteolin, showed a notable inhibitory effect. A potent XO inhibitory compound was isolated from the complex mixture of the product of the coupling of luteolin and cysteine ethyl ester, and its structure was determined by NMR and MS analysis. The compound has a unique 1,4-thiazine ring unit on the luteolin B-ring and is inhibited XO 4.5 times more strongly than it did luteolin.

  18. Quantitative electron spin resonance (ESR) analysis of antioxidative properties using the acetaldehyde/xanthine oxidase system

    NASA Astrophysics Data System (ADS)

    Souchard, J.-P.; Nepveu, F.

    1998-05-01

    We present a method for the quantitative ESR analysis of the antioxidant properties of drugs using the acetaldhehyde/xanthine oxidase (AC/XOD) superoxide generating system and 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) as spin trap. In stoichiometric conditions (AC/XOD, 60 mM/0.018 U), the resulting paramagnetic DMPO adduct disappeared with superoxide dismutase and remained when catalase or DMSO were used. That adduct was dependent only on superoxide and resulted from the trapping of a carboxyl radical by DMPO (aN = 15.2 G, aH = 18.9 G). Similar results were obtained using 4-pyridyl-l-oxide-N-t-butyl nitrone (POBN) as spin trap. The ESR signal of the DMPO-CO2- adduct was very stable and allowed quantitative analysis of the antioxidative activity of redox molecules from an IC{50} value representing the concentration causing 50% inhibition of its intensity. Among the tested compounds, manganese(II), complexes were the most effective, 25 times as active as ascorbic acid or (+)catechin and 500-fold more antioxidative than Trolox^R. Nous présentons une méthode d'analyse quantitative de l'activité antioxydante de composés d'intérêt pharmaceutique basée sur le système acétaldéhyde/xanthine oxydase (AC/XOD), l'utilisation de la RPE et du piégeage de spin avec le 5,5-diméthyl-l-pyrroline-N-oxyde (DMPO). Dans les conditions stoechiométriques {AC/XOD, 60 mM/0,018 U/ml}, l'adduit radicalaire résultant de ce système disparaît en présence de superoxyde dismutase et persiste en présence de catalase ou de DMSO. Cet adduit ne dépend que de la présence de l'anion superoxyde et provient du piégeage d'un radical carboxyle CO2- sur le DMPO (aN = 15.2 G, aH = 18.9 G). Des résultats similaires ont été obtenus avec le piégeur de spin 4-pyridyl-l-oxyde-N-t-butyl nitrone (POBN). Le signal RPE de l'adduit DMPO-CO2- est très stable et permet la quantification de l'activité antioxydante de pharmacophores redox par la détermination de la CI{50}, concentration qui

  19. Noncompetitive and irreversible inhibition of xanthine oxidase by benzimidazole analogues acting at the functional flavin adenine dinucleotide cofactor.

    PubMed

    Skibo, E B

    1986-07-29

    Benzimidazole derivatives possessing a leaving group in the 2 alpha-position and either 4,7-dione, 4,7-diol, or 4,7-dimethoxy substituents were examined as inhibitors of buttermilk xanthine oxidase. The quinone and hydroquinone derivatives are not inhibitors of xanthine-oxygen reductase activity, even though the latter is a powerful alkylating agent. The methoxylated hydroquinones are linear noncompetitive inhibitors, the best of which is the 2 alpha-bromo analogue (Ki = 46 microM). During xanthine-oxygen reductase activity, the 2 alpha-bromo analogue irreversibly traps the reduced enzyme. Formation of a C(4a) adduct of the reduced functional FAD cofactor is postulated on the basis of UV-visible spectral evidence and reconstitution of the enzyme after removal of the altered FAD. A probable sequence of events is reversible binding at or near the reduced cofactor followed by adduct formation. It is concluded that potent tight binding inhibitors could be designed that act at the FAD cofactor rather than the purine active site.

  20. Measurement of xanthine oxidase inhibition activity of phenolics and flavonoids with a modified cupric reducing antioxidant capacity (CUPRAC) method.

    PubMed

    Ozyürek, Mustafa; Bektaşoğlu, Burcu; Güçlü, Kubilay; Apak, Reşat

    2009-03-16

    Various dietary polyphenolics have been found to show an inhibitory effect on xanthine oxidase (XO) which mediates oxidative stress-originated diseases because of its ability to generate reactive oxygen species (ROS), including superoxide anion radical (O(2)(-)) and hydrogen peroxide. XO activity has usually been determined by following the rate of uric acid formation from xanthine-xanthine oxidase (X-XO) system using the classical XO activity assay (UV-method) at 295nm. Since some polyphenolics have strong absorption from the UV to visible region, XO-inhibitory activity of polyphenolics was alternatively determined without interference by directly measuring the formation of uric acid and hydrogen peroxide using the modified CUPRAC (cupric reducing antioxidant capacity) spectrophotometric method at 450nm. The CUPRAC absorbance of the incubation solution due to the reduction of Cu(II)-neocuproine reagent by the products of the X-XO system decreased in the presence of polyphenolics, the difference being proportional to the XO inhibition ability of the tested compound. The structure-activity relationship revealed that the flavones and flavonols with a 7-hydroxyl group such as apigenin, luteolin, kaempferol, quercetin, and myricetin inhibited XO-inhibitory activity at low concentrations (IC(50) values from 1.46 to 1.90microM), while the flavan-3-ols and naringin were less inhibitory. The findings of the developed method for quercetin and catechin in the presence of catalase were statistically alike with those of HPLC. In addition to polyphenolics, five kinds of herbs were evaluated for their XO-inhibitory activity using the developed method. The proposed spectrophotometric method was practical, low-cost, rapid, and could reliably assay uric acid and hydrogen peroxide in the presence of polyphenols (flavonoids, simple phenolic acids and hydroxycinnamic acids), and less open to interferences by UV-absorbing substances.

  1. Synthesis, screening and docking of fused pyrano[3,2-d]pyrimidine derivatives as xanthine oxidase inhibitor.

    PubMed

    Kaur, Manroopraj; Kaur, Amandeep; Mankotia, Suhani; Singh, Harbinder; Singh, Arshdeep; Singh, Jatinder Vir; Gupta, Manish Kumar; Sharma, Sahil; Nepali, Kunal; Bedi, Preet Mohinder Singh

    2017-05-05

    In view of developing effective xanthine oxidase (XO) enzyme inhibitors, a series of 100 pyrano[3,2-d]pyrimidine derivatives was synthesized and evaluated for its in vitro XO enzyme inhibition. Structure activity relationship has also been established. Among all the synthesized compounds, 4d, 8d and 9d were found to be the most potent enzyme inhibitors with IC50 values of 8μM, 8.5μM and 7μM, respectively. Compound 9d was further investigated in enzyme kinetic studies and the Lineweaver-Burk plot revealed that the compound 9d was mixed type inhibitor. Molecular properties of the most potent compounds 4d, 8d and 9d, have also been calculated. Docking study was performed to investigate the recognition pattern between xanthine oxidase and the most potent XO inhibitor, 9d. The study suggests that 9d may block the activity of XO sufficiently enough to prevent the substrate from binding to its active site.

  2. Allopurinol Use during Pregnancy - Outcome of 31 Prospectively Ascertained Cases and a Phenotype Possibly Indicative for Teratogenicity

    PubMed Central

    Hoeltzenbein, Maria; Stieler, Katja; Panse, Mary; Wacker, Evelin; Schaefer, Christof

    2013-01-01

    Allopurinol is a purine analogue that inhibits xanthine oxidase. It is mainly used for the treatment of hyperuricemia in patients with gout or tumor lysis syndrome. Experience with allopurinol in pregnancy is scarce. In 2011, Kozenko et al. reported on a child with multiple malformations after maternal treatment with allopurinol throughout pregnancy. Possible teratogenicity of allopurinol was proposed due to the similarity of the pattern of malformations in children with mycophenolate embryopathy. A possible common mechanism of both drugs, i.e. disruption of purine synthesis, was discussed. We report on the outcome of 31 prospectively ascertained pregnancies with allopurinol exposure at least during first trimester. Pregnancy outcomes were 2 spontaneous abortions, 2 elective terminations of pregnancy and 27 live born children. The overall rate of major malformations (3.7%) and of spontaneous abortions (cumulative incidence 11%, 95%-CI 3–40) were both within the normal range. However, there was one child with severe malformations including microphthalmia, cleft lip and palate, renal hypoplasia, low-set ears, hearing deficit, bilateral cryptorchidism, and micropenis. The striking similarity of the anomalies in this child and the case described by Kozenko et al. might be considered as a signal for teratogenicity. Thus, we would recommend caution with allopurinol treatment in the first trimester, until further data are available. PMID:23840514

  3. Pallidifloside D from Smilax riparia enhanced allopurinol effects in hyperuricemia mice.

    PubMed

    Hou, Pi-Yong; Mi, Chao; He, Yi; Zhang, Jun; Wang, Shu-Qing; Yu, Fei; Anderson, Samantha; Zhang, Yan-Wen; Wu, Xiao-Hui

    2015-09-01

    Pallidifloside D, a saponin glycoside constituent from the total saponins of Smilax riparia, had been proved to be effective in hyperuricemic control. Allopurinol is a commonly used medication to treat hyperuricemia and its complications. In this study, we evaluated whether Pallidifloside D could enhance allopurinol's effects by decreasing the serum uric acid level in a hyperuricemic mouse model induced by potassium oxonate. We found that, compared with allopurinol alone, the combination of allopurinol and Pallidifloside D significantly decreased the serum uric acid level and increased the urine uric acid level (both P<0.05), leading to the normalized serum and urine uric acid concentrations. Data on serum, urine creatinine and BUN supported these observations. Our results showed that the synergistic effects of allopurinol combined with Pallidifloside D were linked to the inhibition of both serum and hepatic xanthine oxidase (XOD), the down-regulation of renal mURAT1 and mGLUT9, and the up-regulation of mOAT1. Our data may have a potential value in clinical practice in the treatment of gout and other hyperuricemic conditions.

  4. Allopurinol reduces severity of delayed neurologic sequelae in experimental carbon monoxide toxicity in rats.

    PubMed

    Dong, Guangtao; Ren, Ming; Wang, Xiujie; Jiang, Hongquan; Yin, Xiang; Wang, Shuyu; Wang, Xudong; Feng, Honglin

    2015-05-01

    Approximately half of those who survive severe carbon monoxide (CO) poisoning develop delayed neurologic sequelae. Growing evidence supports the crucial role of free radicals in delayed brain injury associated with CO toxicity. Xanthine oxidase (XO) has been reported to play a pivotal role in the generation of reactive oxygen species (ROS) in CO poisoning. A recent report indicates that allopurinol both attenuated oxidative stress and possessed anti-inflammatory properties in an animal model of acute liver failure. In this study, we aimed to explore the potential of allopurinol to reduce the severity of delayed neurologic sequelae. The rats were first exposed to 1000 ppm CO for 40 min and then to 3000 ppm CO for another 20 min. Following CO poisoning, the rats were injected with allopurinol (50 mg/kg, i.p.) six times. Results showed that allopurinol significantly reduced neuronal death and suppressed expression of pro-inflammatory factors, including tumor necrosis factor-α, intercellular adhesion molecule-1, ionized calcium-binding adapter molecule 1, and degraded myelin basic protein. Furthermore, behavioral studies revealed an improved performance in the Morris water maze test. Our findings indicated that allopurinol may have protective effects against delayed neurologic sequelae caused by CO toxicity.

  5. Pleiotrophin-induced endothelial cell migration is regulated by xanthine oxidase-mediated generation of reactive oxygen species.

    PubMed

    Tsirmoula, Sotiria; Lamprou, Margarita; Hatziapostolou, Maria; Kieffer, Nelly; Papadimitriou, Evangelia

    2015-03-01

    Pleiotrophin (PTN) is a heparin-binding growth factor that induces cell migration through binding to its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and integrin alpha v beta 3 (ανβ3). In the present work, we studied the effect of PTN on the generation of reactive oxygen species (ROS) in human endothelial cells and the involvement of ROS in PTN-induced cell migration. Exogenous PTN significantly increased ROS levels in a concentration and time-dependent manner in both human endothelial and prostate cancer cells, while knockdown of endogenous PTN expression in prostate cancer cells significantly down-regulated ROS production. Suppression of RPTPβ/ζ through genetic and pharmacological approaches, or inhibition of c-src kinase activity abolished PTN-induced ROS generation. A synthetic peptide that blocks PTN-ανβ3 interaction abolished PTN-induced ROS generation, suggesting that ανβ3 is also involved. The latter was confirmed in CHO cells that do not express β3 or over-express wild-type β3 or mutant β3Y773F/Y785F. PTN increased ROS generation in cells expressing wild-type β3 but not in cells not expressing or expressing mutant β3. Phosphoinositide 3-kinase (PI3K) or Erk1/2 inhibition suppressed PTN-induced ROS production, suggesting that ROS production lays down-stream of PI3K or Erk1/2 activation by PTN. Finally, ROS scavenging and xanthine oxidase inhibition completely abolished both PTN-induced ROS generation and cell migration, while NADPH oxidase inhibition had no effect. Collectively, these data suggest that xanthine oxidase-mediated ROS production is required for PTN-induced cell migration through the cell membrane functional complex of ανβ3 and RPTPβ/ζ and activation of c-src, PI3K and ERK1/2 kinases.

  6. Mechanism of Xanthine Oxidase Catalyzed Biotransformation of HMX Under Anaerobic Conditions

    DTIC Science & Technology

    2003-01-01

    xanthine. The nitrite mediated inhibition of HMX biotransformation was determined by assaying the XO against HMX in the presence of increasing NaNO2 ...Anal. Chem. 338 (1990) 41–45. [2] C.A. Myler, W. Sisk, in: G.S. Sayler, R. Fox, J.W. Blackburn (Eds.), Environmental Biotechnology for Waste Treatment

  7. Olea europaea leaf (Ph.Eur.) extract as well as several of its isolated phenolics inhibit the gout-related enzyme xanthine oxidase.

    PubMed

    Flemmig, J; Kuchta, K; Arnhold, J; Rauwald, H W

    2011-05-15

    In Mediterranean folk medicine Olea europaea L. leaf (Ph.Eur.) preparations are used as a common remedy for gout. In this in vitro study kinetic measurements were performed on both an 80% ethanolic (v/v) Olea europaea leaf dry extract (OLE) as well as on nine of its typical phenolic constituents in order to investigate its possible inhibitory effects on xanthine oxidase (XO), an enzyme well known to contribute significantly to this pathological process. Dixon and Lineweaver-Burk plot analysis were used to determine K(i) values and the inhibition mode for the isolated phenolics, which were analysed by RP-HPLC for standardisation of OLE. The standardised OLE as well as some of the tested phenolics significantly inhibited the activity of XO. Among these, the flavone aglycone apigenin exhibited by far the strongest effect on XO with a K(i) value of 0.52 μM. In comparison, the known synthetic XO inhibitor allopurinol, used as a reference standard, showed a K(i) of 7.3 μM. Although the phenolic secoiridoid oleuropein, the main ingredient of the extract (24.8%), had a considerable higher K(i) value of 53.0 μM, it still displayed a significant inhibition of XO. Furthermore, caffeic acid (K(i) of 11.5 μM; 1.89% of the extract), luteolin-7-O-β-D-glucoside (K(i) of 15.0 μM; 0.86%) and luteolin (K(i) of 2.9 μM; 0.086%) also contributed significantly to the XO inhibiting effect of OLE. For oleuropein, a competitive mode of inhibition was found, while all other active substances displayed a mixed mode of inhibition. Tyrosol, hydroxytyrosol, verbascoside, and apigenin-7-O-β-D-glucoside, which makes up for 0.3% of the extract, were inactive in all tested concentrations. Regarding the pharmacological in vitro effect of apigenin-7-O-β-D-glucoside, it has to be considered that it is transformed into the active apigenin aglycone in the mammalian body, thus also contributing substantially to the anti-gout activity of olive leaves. For the first time, this study provides a

  8. Xanthine urolithiasis in a dachshund.

    PubMed

    Flegel, T; Freistadt, R; Haider, W

    1998-10-10

    Calculi were located in the kidneys, the ureters and the bladder of a two-year-old male dachshund. The yellow-greenish calculi developed as a result of impaired transformation of xanthine to uric acid resulting in an increased concentration of xanthine in the urine. The cause of the impaired catabolism of xanthine was probably a disorder of the xanthine oxidase enzyme, which catalyses the transformation of xanthine to uric acid.

  9. X-ray crystal structure of arsenite-inhibited xanthine oxidase: μ-sulfido,μ-oxo double bridge between molybdenum and arsenic in the active site.

    PubMed

    Cao, Hongnan; Hall, James; Hille, Russ

    2011-08-17

    Xanthine oxidoreductase is a molybdenum-containing enzyme that catalyzes the hydroxylation reaction of sp(2)-hybridized carbon centers of a variety of substrates, including purines, aldehydes, and other heterocyclic compounds. The complex of arsenite-inhibited xanthine oxidase has been characterized previously by UV-vis, electron paramagnetic resonance, and X-ray absorption spectroscopy (XAS), and the catalytically essential sulfido ligand of the square-pyrimidal molybdenum center has been suggested to be involved in arsenite binding through either a μ-sulfido,μ-oxo double bridge or a single μ-sulfido bridge. However, this is contrary to the crystallographically observed single μ-oxo bridge between molybdenum and arsenic in the desulfo form of aldehyde oxidoreductase from Desulfovibrio gigas (an enzyme closely related to xanthine oxidase), whose molybdenum center has an oxo ligand replacing the catalytically essential sulfur, as seen in the functional form of xanthine oxidase. Here we use X-ray crystallography to characterize the molybdenum center of arsenite-inhibited xanthine oxidase and solve the structures of the oxidized and reduced inhibition complexes at 1.82 and 2.11 Å resolution, respectively. We observe μ-sulfido,μ-oxo double bridges between molybdenum and arsenic in the active sites of both complexes. Arsenic is four-coordinate with a distorted trigonal-pyramidal geometry in the oxidized complex and three-coordinate with a distorted trigonal-planar geometry in the reduced complex. The doubly bridged binding mode is in agreement with previous XAS data indicating that the catalytically essential sulfur is also essential for the high affinity of reduced xanthine oxidoreductase for arsenite.

  10. X-ray Crystal Structure of Arsenite-Inhibited Xanthine Oxidase:[mu]-Sulfido,[mu]-Oxo Double Bridge between Molybdenum and Arsenic in the Active Site

    SciTech Connect

    Cao, Hongnan; Hall, James; Hille, Russ

    2012-10-23

    Xanthine oxidoreductase is a molybdenum-containing enzyme that catalyzes the hydroxylation reaction of sp{sup 2}-hybridized carbon centers of a variety of substrates, including purines, aldehydes, and other heterocyclic compounds. The complex of arsenite-inhibited xanthine oxidase has been characterized previously by UV-vis, electron paramagnetic resonance, and X-ray absorption spectroscopy (XAS), and the catalytically essential sulfido ligand of the square-pyrimidal molybdenum center has been suggested to be involved in arsenite binding through either a {mu}-sulfido,{mu}-oxo double bridge or a single {mu}-sulfido bridge. However, this is contrary to the crystallographically observed single {mu}-oxo bridge between molybdenum and arsenic in the desulfo form of aldehyde oxidoreductase from Desulfovibrio gigas (an enzyme closely related to xanthine oxidase), whose molybdenum center has an oxo ligand replacing the catalytically essential sulfur, as seen in the functional form of xanthine oxidase. Here we use X-ray crystallography to characterize the molybdenum center of arsenite-inhibited xanthine oxidase and solve the structures of the oxidized and reduced inhibition complexes at 1.82 and 2.11 {angstrom} resolution, respectively. We observe {mu}-sulfido,{mu}-oxo double bridges between molybdenum and arsenic in the active sites of both complexes. Arsenic is four-coordinate with a distorted trigonal-pyramidal geometry in the oxidized complex and three-coordinate with a distorted trigonal-planar geometry in the reduced complex. The doubly bridged binding mode is in agreement with previous XAS data indicating that the catalytically essential sulfur is also essential for the high affinity of reduced xanthine oxidoreductase for arsenite.

  11. Discovery of xanthine oxidase inhibitors from a complex mixture using an online, restricted-access material coupled with column-switching liquid chromatography with a diode-array detection system.

    PubMed

    Li, De-qiang; Zhao, Jing; Li, Shao-ping; Zhang, Qing-wen

    2014-03-01

    To find potential lead compounds for antigout drug discovery, an automated online, restricted-access material coupled with column-switching liquid chromatography with a diode-array detection (RAM-LC-DAD) system was developed for screening of xanthine oxidase (XO) inhibitors and their affinity rankings in complex mixtures. The system was first evaluated by analyzing a mixture of six compounds with known inhibition of XO. Nonspecific binding to the denatured XO was investigated and used as the control for screening. Subsequently, the newly developed system was applied to screening of a natural product, Oroxylum indicum extract, and four compounds which could specifically interact with XO were found and identified as oroxin B, oroxin A, baicalin, and baicalein. The results were verified by a competitive binding test using the known competitive inhibitor allopurinol and were further validated by an inhibition assay in vitro. The online RAM-LC-DAD system developed was shown to be a simple and effective strategy for the rapid screening of bioactive compounds from a complex mixture.

  12. Enhanced activity of the free radical producing enzyme xanthine oxidase in hypoxic rat liver. Regulation and pathophysiologic significance.

    PubMed Central

    Brass, C A; Narciso, J; Gollan, J L

    1991-01-01

    It has been widely proposed that conversion of xanthine dehydrogenase (XDH) to its free radical-producing form, xanthine oxidase (XOD), underlies ischemic/reperfusion injury, although the relationship of this conversion to hypoxia and its physiologic control have not been defined. This study details the time course and control of this enzymatic interconversion. In a functionally intact, isolated perfused rat liver model, mean % XOD activity increased as a function of both the duration (25 to 45% in 3 h) and degree (r = 0.97) of hypoxia. This process was markedly accelerated in ischemic liver by an overnight fast (45 vs. 30% at 2 h), and by imposing a short period of in vivo ischemia (cardiopulmonary arrest 72%). Moreover, only under these conditions was there a significant rise in the XOD activity due to the conformationally altered XDH molecule (XODc, 18%), as well as concomitant morphologic injury. Neither circulating white blood cells nor thrombosis appeared to contribute to the effects of in vivo ischemia on enzyme conversion. Thus, it is apparent that conversion to the free radical-producing state, with high levels of XOD activity and concurrent cellular injury, can be achieved during a relatively short period of hypoxia under certain well-defined physiologic conditions, in a time course consistent with its purported role in modulating reperfusion injury. These data also suggest that the premorbid condition of organ donors (e.g., nutritional status and relative state of hypoxia) is important in achieving optimal organ preservation. Images PMID:1991828

  13. The Treatment of Gout and Disorders of Uric Acid Metabolism with Allopurinol

    PubMed Central

    Ogryzlo, M. A.; Urowitz, M. B.; Weber, H. M.; Houpt, J. B.

    1966-01-01

    Allopurinol (4-hydroxypyrazolo (3,4-d)-pyrimidine) is a potent xanthine oxidase inhibitor which inhibits the oxidation of naturally occurring oxypurines, thus decreasing uric acid formation. The clinical and metabolic effects of this agent were studied in 80 subjects with primary and secondary gout and other disorders of uric acid metabolism. Allopurinol has been universally successful in lowering the serum uric acid concentration and uric acid excretion to normal levels, while not significantly affecting the clearance of urate or other aspects of renal function. Oxypurine excretion increased concomitantly with the fall in urine uric acid. The agent is particularly valuable in the management of problems of gout with azotemia, acute uric acid nephropathy and uric acid urolithiasis. The minor side effects, clinical indications and theoretical complications are discussed. PMID:5923471

  14. Coupling of [33S]sulphur to molybdenum(V) in different reduced forms of xanthine oxidase.

    PubMed Central

    Malthouse, J P; George, G N; Lowe, D J; Bray, R C

    1981-01-01

    Different reduced forms of xanthine oxidase, labelled specifically in the cyanide-labile site with 33S, were prepared and examined by electron paramagnetic resonance. Coupling of this isotope to molybdenum(V) was quantified with the help of computer simulations and found to differ markedly from one reduced form to another. The xanthine Very Rapid signal shows strong, highly anisotropic, coupling with A(33S)av. 1.27 mT. For this signal, axes of the g- and A(33S)-tensors are rotated relative to one another. One axis of the A-tensor is in the plane of gxx ang gyy, but rotated by 40 degrees relative to the gxx axis, whereas the direction of weakest coupling to sulphur deviates by 10 degrees from the gzz axis. In contrast with this signal, only rather weaker coupling was observed in different types of Rapid signal [A(33S)av. 0.3--0.4 mT], and in the Inhibited signal coupling was weaker still [A(33S)av. 0.1--0.2 mT]. Clearly, there must be substantial differences in the structures of the molybdenum centre in the different signal-giving species, with the sulphur atom perhaps in an equatorial type of ligand position in the Very Rapid species but in a more axial one in the other species. Structures are discussed in relation to the mechanism of action of the enzyme and the nature of the proton-accepting group that participates in turnover. PMID:6280672

  15. Screening inhibitors of xanthine oxidase from natural products using enzyme immobilized magnetic beads by high-performance liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Wang, Ting; Li, Dapeng; Yu, Boyang; Qi, Jin

    2017-03-06

    In this study, high-performance liquid chromatography coupled with tandem mass spectrometry was used to assess the results of bioactive compound screening from natural products using immobilized enzyme magnetic beads. We compared three commercial magnetic beads with modified amino, carboxy and N-hydroxysuccinimide groups, respectively. Amino magnetic beads performed best for immobilization and were selected for further experiments. Xanthine oxidase was immobilized on amino magnetic beads and applied to screen potential inhibitors in fresh Zingiber officinale Roscoe, extracts of Scutellaria baicalensis Georgi and Pueraria lobata Ohwi. In total, 12 potential xanthine oxidase ligands were identified from fresh Zingiber root and Scutellaria root extracts, of which eight were characterized and the concentration required for 50% inhibition was determined. Preliminary structure-function relationships were discussed based on these results. A convenient and effective method was therefore developed for the identification of active compounds from complex natural product mixtures. This article is protected by copyright. All rights reserved.

  16. In vitro antioxidant, lipoxygenase and xanthine oxidase inhibitory activities of fractions from Cienfuegosia digitata Cav., Sida alba L. and Sida acuta Burn f. (Malvaceae).

    PubMed

    Konaté, K; Souza, A; Coulibaly, A Y; Meda, N T R; Kiendrebeogo, M; Lamien-Meda, A; Millogo-Rasolodimby, J; Lamidi, M; Nacoulma, O G

    2010-11-15

    In this study polyphenol content, antioxidant activity, lipoxygenase (LOX) and Xanthine Oxidase (XO) inhibitory effects of n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of aqueous acetone extracts from S. alba L., S. acuta Burn f and Cienfuegosia digitata Cav. were investigated. The total phenolics, flavonoids, flavonols and total tannins were determined by spectrophotometric methods using Folin-ciocalteu, AlCl3 reagents and tannic acid, respectively. The antioxidant potential was evaluated using three methods: inhibition of free radical 2,2-diphenyl-1-picrylhydramzyl (DPPH), ABTS radical cation decolorization assay and Iron (III) to iron (II) reduction activity (FRAP). For enzymatic activity, lipoxygenase and xanthine oxidase inhibitory activities were used. This study shows a relationship between polyphenol contents, antioxidant and enzymatic activities. Present results showed that ethyl acetate and dichloromethane fractions elicit the highest polyphenol content, antioxidant and enzymatic activities.

  17. Bioactive compounds, antioxidant, xanthine oxidase inhibitory, tyrosinase inhibitory and anti-inflammatory activities of selected agro-industrial by-products.

    PubMed

    Oskoueian, Ehsan; Abdullah, Norhani; Hendra, Rudi; Karimi, Ehsan

    2011-01-01

    Evaluation of abundantly available agro-industrial by-products for their bioactive compounds and biological activities is beneficial in particular for the food and pharmaceutical industries. In this study, rapeseed meal, cottonseed meal and soybean meal were investigated for the presence of bioactive compounds and antioxidant, anti-inflammatory, xanthine oxidase and tyrosinase inhibitory activities. Methanolic extracts of rapeseed meal showed significantly (P < 0.01) higher phenolics and flavonoids contents; and significantly (P < 0.01) higher DPPH and nitric oxide free radical scavenging activities when compared to that of cottonseed meal and soybean meal extracts. Ferric thiocyanate and thiobarbituric acid tests results showed rapeseed meal with the highest antioxidant activity (P < 0.01) followed by BHT, cotton seed meal and soybean meal. Rapeseed meal extract in xanthine oxidase and tyrosinase inhibitory assays showed the lowest IC(50) values followed by cottonseed and soybean meals. Anti-inflammatory assay using IFN-γ/LPS stimulated RAW 264.7 cells indicated rapeseed meal is a potent source of anti-inflammatory agent. Correlation analysis showed that phenolics and flavonoids were highly correlated to both antioxidant and anti-inflammatory activities. Rapeseed meal was found to be promising as a natural source of bioactive compounds with high antioxidant, anti-inflammatory, xanthine oxidase and tyrosinase inhibitory activities in contrast to cotton and soybean meals.

  18. Substrate orientation and specificity in xanthine oxidase: crystal structures of the enzyme in complex with indole-3-acetaldehyde and guanine.

    PubMed

    Cao, Hongnan; Hall, James; Hille, Russ

    2014-01-28

    Xanthine oxidase is a molybdenum-containing hydroxylase that catalyzes the hydroxylation of sp(2)-hybridized carbon centers in a variety of aromatic heterocycles as well as aldehydes. Crystal structures of the oxidase form of the bovine enzyme in complex with a poor substrate indole-3-acetaldehyde and the nonsubstrate guanine have been determined, both at a resolution of 1.6 Å. In each structure, a specific and unambiguous orientation of the substrate in the active site is observed in which the hydroxylatable site is oriented away from the active site molybdenum center. The orientation seen with indole-3-acetaldehyde has the substrate positioned with the indole ring rather than the exocyclic aldehyde nearest the molybdenum center, indicating that the substrate must rotate some 30° in the enzyme active site to permit hydroxylation of the aldehyde group (as observed experimentally), accounting for the reduced reactivity of the enzyme toward this substrate. The principal product of hydroxylation of indole-3-acetaldehyde by the bovine enzyme is confirmed to be indole-3-carboxylic acid based on its characteristic UV-vis spectrum, and the kinetics of enzyme reduction are reported. With guanine, the dominant orientation seen crystallographically has the C-8 position that might be hydroxylated pointed away from the active site molybdenum center, in a configuration resembling that seen previously with hypoxanthine (a substrate that is effectively hydroxylated at position 2). The ∼180° reorientation required to permit reaction is sterically prohibited, indicating that substrate (mis)orientation in the active site is a major factor precluding formation of the highly mutagenic 8-hydroxyguanine.

  19. Bioassay-Guided Isolation and Identification of Xanthine Oxidase Inhibitory Constituents from the Leaves of Perilla frutescens.

    PubMed

    Huo, Li-Na; Wang, Wei; Zhang, Chun-Yu; Shi, Hai-Bo; Liu, Yang; Liu, Xiao-Hong; Guo, Bing-Hua; Zhao, Dong-Mei; Gao, Hua

    2015-09-25

    Activity-directed fractionation and purification processes were employed to identify xanthine oxidase (XO) inhibitory compounds from the leaves of Perilla frutescens. The total extract was evaluated in vitro on XO inhibitory activity and in vivo in an experimental model with potassium oxonate-induced hyperuricemia in mice which was used to evaluate anti-hyperuricemic activity. The crude extract showed expressive urate-lowering activity results. Solvent partitioning of the total extract followed by macroporous resin column chromatography of the n-butanol extract yielded four extracts and eluted parts. Among them, only the 70% ethanol eluted part of the n-butanol extract showed strong activity and therefore was subjected to separation and purification using various chromatographic techniques. Five compounds showing potent activity were identified by comparing their spectral data with literature values to be caffeic acid, vinyl caffeate, rosmarinic acid, methyl rosmarinate, and apigenin. These results indicate that pending further study, these compounds could be used as novel natural product agents for the treatment of hyperuricemia.

  20. Xanthine oxidase inhibition for the treatment of cardiovascular disease: an updated systematic review and meta‐analysis

    PubMed Central

    Dierckx, Riet; Mohee, Kevin; Clark, Andrew L.; Cleland, John G.

    2016-01-01

    Abstract Background Previous studies have shown that xanthine oxidase inhibitors (XOI) might improve outcome for patients with cardiovascular disease. However, more evidence is required. Methods and results We published a meta‐analysis of trials conducted before 2014 examining the effects of XOI on mortality in patients with cardiovascular disease. At least two further trials (N = 323 patients) have since been published. Accordingly, we repeated our analysis after a further search for randomized controlled trials of XOI in PubMed/MEDLINE, EMBASE, and Cochrane Databases. We identified eight relevant trials with 1031 patients. The average age of the patients was 61 years and 68% were men (one study did not report gender). There were 57 deaths in these eight trials, 26 in those assigned to XOI, and 31 in those assigned to the control. The updated meta‐analysis could not confirm a reduction in mortality for patients assigned to XOI compared with placebo (odds ratio 0.84) but 95% confidence intervals were wide (0.48–1.47). Conclusions This updated meta‐analysis does not suggest that XOI exert a large reduction in mortality but also cannot exclude the possibility of substantial harm or benefit. PMID:28217311

  1. Effects of aqueous soybean, mistletoe and red clover extracts on activities of adenosine deaminase and xanthine oxidase enzyme.

    PubMed

    Namuslu, M; Kocaoglu, H; Celik, H T; Avci, A; Devrim, E; Genc, Y; Gocmen, E; Erguder, I B; Durak, I

    2014-01-01

    Soybean (Glycine max), mistletoe (Viscum album) and red clover (Trifolium pratence) have been argued to have anti-cancer effects. In the present study it was aimed to investigate possible effects of these plant extracts on the activities of DNA turn-over enzymes, namely adenosine deaminase (ADA) and xanthine oxidase (XO) in cancerous and non-cancerous gastric and colon tissues. For this aim, 6 cancerous and 6 non-cancerous adjacent human gastric tissues, and 7 cancerous and 7 non-cancerous adjacent colon tissues were obtained by surgical operations. Our results suggest that aqueous soybean, mistletoe and red clover extracts may exhibit anti-tumoral activity by depleting hypoxanthine concentration in the cancer cells through XO activation, which may lead to lowered salvage pathway activity necessary for the cancer cells to proliferate in the cancerous colon tissue. Some foods like soybean, mistletoe and red clover may provide nutritional support to medical cancer therapy through inhibiting and/or activating key enzymes in cancer metabolism (Tab. 4, Ref. 33).

  2. ESR studies on reaction of saccharide with the free radicals generated from the xanthine oxidase/hypoxanthine system containing iron.

    PubMed

    Luo, G M; Qi, D H; Zheng, Y G; Mu, Y; Yan, G L; Yang, T S; Shen, J C

    2001-03-09

    The free radicals generated from the iron containing system of xanthine oxidase and hypoxanthine (Fe-XO/HX) were directly detected by using spin trapping. It was found that not only superoxide anion (O(2)*-) and hydroxyl radical (OH*), but also alkyl or alkoxyl radicals (R*) were formed when saccharides such as glucose, fructose and sucrose were added into the Fe-XO/HX system. The generated amount of R* was dependent on the kind and concentration of saccharides added into the Fe-XO/HX system and no R* were detected in the absence of saccharides, indicating that there is an interaction between the saccharide molecules and the free radicals generated from the Fe-XO/HX system and saccharide molecules are essential for generating R* in the Fe-XO/HX system. It is expected that the toxicity of R* would be greater than of hydrophilic O(2)*- and OH* because they are liposoluble and their lives are longer and the active sites of biomolecules are closely related with lipophilic phase, thus they can damage cells more seriously than O(2)*- and OH*. The R* generated from the saccharide containing Fe-XO/HX can be effectively scavenged by selenium containing abzyme (Se-abzyme), indicating Se-abzyme is a promising antioxidant.

  3. Pharmacological Basis for Use of Selaginella moellendorffii in Gouty Arthritis: Antihyperuricemic, Anti-Inflammatory, and Xanthine Oxidase Inhibition

    PubMed Central

    Zhao, Ping; Chen, Ke-li; Zhang, Guo-li

    2017-01-01

    This study was aimed at evaluating the effects of Selaginella moellendorffii Hieron. (SM) on gouty arthritis and getting an insight of the possible mechanisms. HPLC method was developed for chemical analysis. The paw oedema, the neutrophil accumulation, inflammatory mediators, lipid peroxidation, and histopathological changes of the joints were analyzed in gouty arthritis rat model, and the kidney injury and serum urate were detected in hyperuricemic mice. Pharmacokinetic result demonstrated that the main apigenin glycosides might be quantitatively transformed into apigenin in the mammalian body. Among these compounds, the apigenin exhibited the strongest effect on xanthine oxidase (XOD). SM aqueous extract has proved to be active in reducing hyperuricemia in dose-dependent manner, and the levels of blood urea nitrogen (BUN) and creatinine (Cr) in high dose group were decreased significantly as compared with hyperuricemic control group (P < 0.01). The high dose of SM extract could significantly prevent the paw swelling, reduce gouty joint inflammatory features, reduce the release of IL-1β and TNF-α, lower malondialdehyde (MDA) and myeloperoxidase (MPO) levels, and increase superoxide dismutase (SOD) level (P < 0.01). For the first time, this study provides a rational basis for the traditional use of SM aqueous extract against gout in folk medicine. PMID:28250791

  4. Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

    PubMed

    López-Cruz, Roberto I; Pérez-Milicua, Myrna Barjau; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal-Vertiz, Jaime A; de la Rosa, Alejandro; Vázquez-Medina, José P; Zenteno-Savín, Tania

    2014-05-01

    Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas.

  5. Antioxidant activity of minimally processed red chicory (Cichorium intybus L.) evaluated in xanthine oxidase-, myeloperoxidase-, and diaphorase-catalyzed reactions.

    PubMed

    Lavelli, Vera

    2008-08-27

    Minimally processed red chicory products (Cichorium intybus L. var. silvestre) were studied for their polyphenol content and antioxidant activity evaluated by using the synthetic 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl radical and three model reactions catalyzed by relevant enzymatic sources of reactive oxygen species, namely, xanthine oxidase, myeloperoxidase, and diaphorase. Products were analyzed at the time of production and after storage at 4 degrees C within either a gas permeable film or a gas barrier film. The antioxidant activity and contents of hydroxycinnamic acids and flavonoids decreased by less than 20% during storage of the minimally processed red chicory products. Total phenolics were significantly correlated with the antioxidant activity evaluated with both the synthetic radical and the enzyme-catalyzed reactions. On a molar basis, red chicory phenolics were as efficient as the reference compound Trolox in scavenging the synthetic radical. However, red chicory phenolics had a much higher inhibitory activity than Trolox in the model enzymatic systems.

  6. Competitive binding experiments can reduce the false positive results of affinity-based ultrafiltration-HPLC: A case study for identification of potent xanthine oxidase inhibitors from Perilla frutescens extract.

    PubMed

    Wang, Zhiqiang; Kwon, Shin Hwa; Hwang, Seung Hwan; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

    2017-03-24

    The purpose of this study was to assess the possibility of using competitive binding experiments with ultrafiltration-HPLC analysis to identify potent xanthine oxidase (XO) inhibitors from the Perilla frutescens extract as an attempt to reduce the number of false positive results. To isolate the enzyme-ligand complex from unbound compounds, the P. frutescens extract was either incubated in the absence of XO, in the presence of XO, or with the active site blocked XO before the ultrafiltration was performed. Allopurinaol was used as the XO active site blocker. The unbound compounds were subjected to HPLC analysis. The degree of total binding (TBD) and degree of specific binding (SBD) of each compound were calculated using the peak areas. TBD represents the binding affinities of compounds from the P. frutescens extract for the XO binding site. SBD represents the XO competitive binding between allopurinol and ligands from the extract samples. Two criteria were applied to select putative targets that could help avoid false positives. These include TBD>30% and SBD>10%. Using that approach, kaempferol-3-O-rutinoside, rosmarinic acid, methyl-rosmarinic acid, apigenin, and 4',5,7-trimethoxyflavone were identified, from total 11 compounds, as potent XO inhibitors. Finally, apigenin, 4',5,7-trimethoxyflavone, and luteolin were XO inhibitors verified through an XO inhibition assay and structural simulation of the complex. These results showed that the newly developed strategy has the advantage that the number of targets identified via ultrafiltration-HPLC can be narrowed from many false positives. However, not all false positives can be eliminated with this approach. Some potent inhibitors might also be excluded with the use of this method. The limitations of this method are also discussed herein.

  7. Phlomis mauritanica extracts reduce the xanthine oxidase activity, scavenge the superoxide anions, and inhibit the aflatoxin B1-, sodium azide-, and 4-nitrophenyldiamine-induced mutagenicity in bacteria.

    PubMed

    Limem, Ilef; Bouhlel, Ines; Bouchemi, Meriem; Kilani, Soumaya; Boubaker, Jihed; Ben-Sghaier, Mohamed; Skandrani, Ines; Behouri, Wissem; Neffati, Aicha; Ghedira, Kamel; Chekir-Ghedira, Leila

    2010-06-01

    Four extracts were prepared from the leaves of Phlomis mauritanica: lyophilized infusion, total oligomer flavonoids, methanol, and ethyl acetate extracts. The antimutagenic properties of these extracts were investigated by assessing the inhibition of the mutagenic effects of direct-acting mutagens such as sodium azide and 4-nitrophenylenediamine and indirect-acting mutagens like aflatoxin B1 (AFB1) using the Ames assay. The four extracts prepared from P. mauritanica strongly inhibit the mutagenicity induced by AFB1 in both Salmonella typhimurium TA 100 and TA 98 assay systems. Lyophilized infusion and methanol extracts at the dose of 250 microg per plate reduced AFB1 mutagenicity by 93% and 91%, respectively, in S. typhymurium strain TA 100. We examined also the antioxidant effect of these extracts by the enzymatic xanthine/xanthine oxidase assay. Result indicated that total oligomer flavonoids and ethyl acetate and methanol extracts were potent inhibitors of xanthine oxidase activity. In contrast, lyophilized infusion, total oligomer flavonoids, and methanol extracts exhibited a high degree of superoxide anion scavenging. Our findings emphasize the potential of P. mauritanica extracts to prevent mutations and oxidant effects. Furthermore, the results presented here could be an additional argument to support the use of this species as a medicinal and dietary plant.

  8. High-performance liquid chromatography coupled with post-column dual-bioactivity assay for simultaneous screening of xanthine oxidase inhibitors and free radical scavengers from complex mixture.

    PubMed

    Li, D Q; Zhao, J; Li, S P

    2014-06-06

    Xanthine oxidase (XO) can catalyze hypoxanthine and xanthine to generate uric acid and reactive oxygen species (ROS), including superoxide anion radical (O₂(•-)) and hydrogen peroxide. XO inhibitors and free radical scavengers are beneficial to the treatment of gout and many related diseases. In the present study, an on-line high-performance liquid chromatography (HPLC) coupled with post-column dual-bioactivity assay was established and successfully applied to simultaneously screening of XO inhibitors and free radical scavengers from a complex mixture, Oroxylum indicum extract. The integrated system of HPLC separation, bioactivity screening and mass spectrometry identification was proved to be simple and effective for rapid and sensitive screening of individual bioactive compounds in complex mixtures.

  9. Low-interferences Determination of the Antioxidant Capacity in Fruits Juices Based on Xanthine Oxidase and Mediated Amperometric Measurements in the Reduction Mode.

    PubMed

    Bucur, Madalina-Petruta; Radulescu, Maria-Cristina; Bucur, Bogdan; Radu, Gabriel Lucian

    2016-01-01

    A low-interferences enzymatic sensor for evaluating the antioxidant capacity was developed. Xanthine oxidase was used to produce superoxide radicals that spontaneously dismutate to hydrogen peroxide. Low xanthine concentrations were used to minimize the rapid dismutation of the superoxide radical before its fast reaction with antioxidants. The sensor operates in the reduction mode, and evaluations with low interferences of the antioxidant capacity are based on the detection of remaining hydrogen peroxide using Prussian blue electrodes at low potentials. The linear calibration graph is between 2 - 10 μM ascorbic acid. No interferences were observed from easily oxidisable substances including uric acid, which is produced in the enzymatic reaction or other substances usually found in foods. The method was used to evaluate the antioxidant capacity in different real juice samples.

  10. HZE ⁵⁶Fe-ion irradiation induces endothelial dysfunction in rat aorta: role of xanthine oxidase.

    PubMed

    Soucy, Kevin G; Lim, Hyun Kyo; Kim, Jae Hyung; Oh, Young; Attarzadeh, David O; Sevinc, Baris; Kuo, Maggie M; Shoukas, Artin A; Vazquez, Marcelo E; Berkowitz, Dan E

    2011-10-01

    Ionizing radiation has been implicated in the development of significant cardiovascular complications. Since radiation exposure is associated with space exploration, astronauts are potentially at increased risk of accelerated cardiovascular disease. This study investigated the effect of high atomic number, high-energy (HZE) iron-ion radiation on vascular and endothelial function as a model of space radiation. Rats were exposed to a single whole-body dose of iron-ion radiation at doses of 0, 0.5 or 1 Gy. In vivo aortic stiffness and ex vivo aortic tension responses were measured 6 and 8 months after exposure as indicators of chronic vascular injury. Rats exposed to 1 Gy iron ions demonstrated significantly increased aortic stiffness, as measured by pulse wave velocity. Aortic rings from irradiated rats exhibited impaired endothelial-dependent relaxation consistent with endothelial dysfunction. Acute xanthine oxidase (XO) inhibition or reactive oxygen species (ROS) scavenging restored endothelial-dependent responses to normal. In addition, XO activity was significantly elevated in rat aorta 4 months after whole-body irradiation. Furthermore, XO inhibition, initiated immediately after radiation exposure and continued until euthanasia, completely inhibited radiation-dependent XO activation. ROS production was elevated after 1 Gy irradiation while production of nitric oxide (NO) was significantly impaired. XO inhibition restored NO and ROS production. Finally, dietary XO inhibition preserved normal endothelial function and vascular stiffness after radiation exposure. These results demonstrate that radiation induced XO-dependent ROS production and nitroso-redox imbalance, leading to chronic vascular dysfunction. As a result, XO is a potential target for radioprotection. Enhancing the understanding of vascular radiation injury could lead to the development of effective methods to ameliorate radiation-induced vascular damage.

  11. Conformational alterations induced by novel green 16-E2-16 gemini surfactant in xanthine oxidase: Biophysical insights from tensiometry, spectroscopy, microscopy and molecular modeling.

    PubMed

    Akram, Mohd; Bhat, Imtiyaz Ahmad; Bhat, Waseem Feeroze; Kabir-ud-Din

    2015-01-01

    Herein we report the interaction of a biodegradable gemini surfactant, ethane-1,2-diyl bis(N,N-dimethyl-N-hexadecylammoniumacetoxy) dichloride (16-E2-16) with bovine milk xanthine oxidase (XO), employing tensiometry, fluorescence spectroscopy, UV spectroscopy, far-UV circular dichroism spectroscopy (CD), Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and computational molecular modeling. Surface tension results depict substantial changes in the micellar as well as interfacial parameters (CMC, ΠCMC, γCMC, Γmax, Amin, ΔGmic° and ΔGads°) of 16-E2-16 gemini surfactant upon XO combination, deciphering the interaction of XO with the gemini surfactant. Fluorescence measurements reveal that 16-E2-16 gemini surfactant causes quenching in the xanthine oxidase (XO) fluorescence spectra via static procedure and the values of various evaluated binding parameters (KSV, Kb, kq, ΔGb° and n) describe that 16-E2-16 effectively binds to XO. Three dimensional fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, F1F3 ratio, UV, CD, FTIR, SEM and TEM results delineate changes in the secondary structure of xanthine oxidase. Molecular docking results provide complement to the steady-state fluorescence findings and support the view that quenching occurs due to non-polar environment experienced by aromatic residues of the enzyme. The results of this study can help scientists to tune the conformation of an enzyme (XO) with biocompatible amphiphilic microstructures, which will help to unfold further understanding in the treatment modes of various diseases like gout, hyperuricemia, liver and brain necrosis.

  12. Assessment of Antioxidant and Phenolic Compound Concentrations as well as Xanthine Oxidase and Tyrosinase Inhibitory Properties of Different Extracts of Pleurotus citrinopileatus Fruiting Bodies

    PubMed Central

    Alam, Nuhu; Yoon, Ki Nam; Lee, Kyung Rim; Kim, Hye Young; Shin, Pyung Gyun; Cheong, Jong Chun; Yoo, Young Bok; Shim, Mi Ja; Lee, Min Woong

    2011-01-01

    Cellular damage caused by reactive oxygen species has been implicated in several diseases, thus establishing a significant role for antioxidants in maintaining human health. Acetone, methanol, and hot water extracts of Pleurotus citrinopileatus were evaluated for their antioxidant activities against β-carotene-linoleic acid and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, reducing power, ferrous ion-chelating abilities, and xanthine oxidase inhibitory activities. In addition, the tyrosinase inhibitory effects and phenolic compound contents of the extracts were also analyzed. Methanol and acetone extracts of P. citrinopileatus showed stronger inhibition of β-carotene-linoleic acid compared to the hot water extract. Methanol extract (8 mg/mL) showed a significantly high reducing power of 2.92 compared to the other extracts. The hot water extract was more effective than the acetone and methanole extracts for scavenging DPPH radicals. The strongest chelating effect (92.72%) was obtained with 1.0 mg/mL of acetone extract. High performance liquid chromatography analysis detected eight phenolic compounds, including gallic acid, protocatechuic acid, chlorogenic acid, ferulic acid, naringenin, hesperetin, formononetin, and biochanin-A, in an acetonitrile and hydrochloric acid (5 : 1) solvent extract. Xanthine oxidase and tyrosinase inhibitory activities of the acetone, methanol, and hot water extracts increased with increasing concentration. This study suggests that fruiting bodies of P. citrinopileatus can potentially be used as a readily accessible source of natural antioxidants. PMID:22783067

  13. Aldehyde oxidase-catalysed oxidation of methotrexate in the liver of guinea-pig, rabbit and man.

    PubMed

    Jordan, C G; Rashidi, M R; Laljee, H; Clarke, S E; Brown, J E; Beedham, C

    1999-04-01

    Although 7-hydroxymethotrexate is a major metabolite of methotrexate during high-dose therapy, negligible methotrexate-oxidizing activity has been found in-vitro in the liver in man. The goals of this study were to determine the role of aldehyde oxidase in the metabolism of methotrexate to 7-hydroxymethotrexate in the liver and to study the effects of inhibitors and other substrates on the metabolism of methotrexate. Methotrexate, (+/-)-methotrexate and (-)-methotrexate were incubated with partially purified aldehyde oxidase from the liver of rabbit, guinea-pig and man and the products analysed by HPLC. Rabbit liver aldehyde oxidase was used for purposes of comparison. In-vitro aldehyde oxidase from the liver of man catalyses the oxidation of methotrexate to 7-hydroxymethotrexate, but the turnover is low. However, formation of 7-hydroxy-methotrexate from all forms of methotrexate by the liver in guinea-pig and man was significantly inhibited in the presence of 100 microM menadione and chlorpromazine, potent inhibitors of aldehyde oxidase. Allopurinol (100 microM) had a negligible inhibitory effect on liver aldehyde oxidase from guinea-pig and man. Allopurinol is a xanthine oxidase inhibitor. The production of 7-hydroxymethotrexate was enhanced in the presence of allopurinol. Although aldehyde oxidase is also responsible for some of this conversion, it is also possible that the closely related xanthine oxidase is responsible for the formation of 7-hydroxymethotrexate. By employing potent selective inhibitors of aldehyde oxidase, menadione and chlorpromazine, we have demonstrated for the first time that liver aldehyde oxidase from man is minimally involved in methotrexate oxidation.

  14. Tandem orientation of duplicated xanthine dehydrogenase genes from Arabidopsis thaliana: differential gene expression and enzyme activities.

    PubMed

    Hesberg, Christine; Hänsch, Robert; Mendel, Ralf R; Bittner, Florian

    2004-04-02

    Xanthine dehydrogenase from the plant Arabidopsis thaliana was analyzed on molecular and biochemical levels. Whereas most other organisms appear to own only one gene for xanthine dehydrogenase A. thaliana possesses two genes in tandem orientation spaced by 704 base pairs. The cDNAs as well as the proteins AtXDH1 and AtXDH2 share an overall identity of 93% and show high homologies to xanthine dehydrogenases from other organisms. Whereas AtXDH2 mRNA is expressed constitutively, alterations of AtXDH1 transcript levels were observed at various stresses like drought, salinity, cold, and natural senescence, but also after abscisic acid treatment. Transcript alteration did not mandatorily result in changes of xanthine dehydrogenase activities. Whereas salt treatment had no effect on xanthine dehydrogenase activities, cold stress caused a decrease, but desiccation and senescence caused a strong increase of activities in leaves. Because AtXDH1 presumably is the more important isoenzyme in A. thaliana it was expressed in Pichia pastoris, purified, and used for biochemical studies. AtXDH1 protein is a homodimer of about 300 kDa consisting of identical subunits of 150 kDa. Like xanthine dehydrogenases from other organisms AtXDH1 uses hypoxanthine and xanthine as main substrates and is strongly inhibited by allopurinol. AtXDH1 could be activated by the purified molybdenum cofactor sulfurase ABA3 that converts inactive desulfo-into active sulfoenzymes. Finally it was found that AtXDH1 is a strict dehydrogenase and not an oxidase, but is able to produce superoxide radicals indicating that besides purine catabolism it might also be involved in response to various stresses that require reactive oxygen species.

  15. Molecular Docking Analysis of Selected Clinacanthus nutans Constituents as Xanthine Oxidase, Nitric Oxide Synthase, Human Neutrophil Elastase, Matrix Metalloproteinase 2, Matrix Metalloproteinase 9 and Squalene Synthase Inhibitors

    PubMed Central

    Narayanaswamy, Radhakrishnan; Isha, Azizul; Wai, Lam Kok; Ismail, Intan Safinar

    2016-01-01

    Background: Clinacanthus nutans (Burm. f.) Lindau has gained popularity among Malaysians as a traditional plant for anti-inflammatory activity. Objective: This prompted us to carry out the present study on a selected 11 constituents of C. nutans which are clinacoside A–C, cycloclinacoside A1, shaftoside, vitexin, orientin, isovitexin, isoorientin, lupeol and β-sitosterol. Materials and Methods: Selected 11 constituents of C. nutans were evaluated on the docking behavior of xanthine oxidase (XO), nitric oxide synthase (NOS), human neutrophil elastase (HNE), matrix metalloproteinase (MMP 2 and 9), and squalene synthase (SQS) using Discovery Studio Version 3.1. Also, molecular physicochemical, bioactivity, absorption, distribution, metabolism, excretion, and toxicity (ADMET), and toxicity prediction by computer assisted technology analyzes were also carried out. Results: The molecular physicochemical analysis revealed that four ligands, namely clinacoside A–C and cycloclinacoside A1 showed nil violations and complied with Lipinski's rule of five. As for the analysis of bioactivity, all the 11 selected constituents of C. nutans exhibited active score (>0) toward enzyme inhibitors descriptor. ADMET analysis showed that the ligands except orientin and isoorientin were predicted to have Cytochrome P4502D6 inhibition effect. Docking studies and binding free energy calculations revealed that clinacoside B exhibited the least binding energy for the target enzymes except for XO and SQS. Isovitexin and isoorientin showed the potentials in the docking and binding with all of the six targeted enzymes, whereas vitexin and orientin docked and bound with only NOS and HNE. Conclusion: This present study has paved a new insight in understanding these 11 C. nutans ligands as potential inhibitors against XO, NOS, HNE, MMP 2, MMP 9, and SQS. SUMMARY Isovitexin and isoorientin (Clinacanthus nutans constituent) showed potentials in the docking and binding with all of the six targeted

  16. The role of aldehyde oxidase in ethanol-induced hepatic lipid peroxidation in the rat.

    PubMed Central

    Shaw, S; Jayatilleke, E

    1990-01-01

    Hepatic lipid peroxidation has been implicated in the pathogenesis of alcohol-induced liver injury, but the mechanism(s) by which ethanol metabolism or resultant free radicals initiate lipid peroxidation is not fully defined. The role of the molybdenum-containing enzymes aldehyde oxidase and xanthine oxidase in the generation of such free radicals was investigated by measuring alkane production (lipoperoxidation products) in isolated rat hepatocytes during ethanol metabolism. Inhibition of aldehyde oxidase and xanthine oxidase (by feeding tungstate at 100 mg/day per kg) decreased alkane production (80-95%), whereas allopurinol (20 mg/kg by mouth), a marked inhibitor of xanthine oxidase, inhibited alkane production by only 35-50%. Addition of acetaldehyde (0-100 microM) (in the presence of 50 microM-4-methylpyrazole) increased alkane production in a dose-dependent manner (Km of aldehyde oxidase for acetaldehyde 1 mM); menadione, an inhibitor of aldehyde oxidase, virtually inhibited alkane production. Desferrioxamine (5-10 microM) completely abolished alkane production induced by both ethanol and acetaldehyde, indicating the importance of catalytic iron. Thus free radicals generated during the metabolism of acetaldehyde by aldehyde oxidase may be a fundamental mechanism in the initiation of alcohol-induced liver injury. PMID:2363695

  17. Effects of allopurinol and deferoxamine on reperfusion injury of the brain in newborn piglets after neonatal hypoxia-ischemia.

    PubMed

    Peeters-Scholte, Cacha; Braun, Kees; Koster, Johanna; Kops, Nicole; Blomgren, Klas; Buonocore, Giuseppe; van Buul-Offers, Sylvia; Hagberg, Henrik; Nicolay, Klaas; van Bel, Frank; Groenendaal, Floris

    2003-10-01

    The hypothesis was tested that treatment with allopurinol, a xanthine oxidase inhibitor, or deferoxamine, a chelator of nonprotein-bound iron, preserved cerebral energy metabolism, attenuated development of edema, and improved histologic outcome in the newborn piglet at 24 h after hypoxia-ischemia. Thirty-two newborn piglets were subjected to 1 h of hypoxia-ischemia by occluding both carotid arteries and reducing the fraction of inspired oxygen; five newborn piglets served as sham-operated controls. The depth of hypoxia-ischemia was controlled by phosphorous magnetic resonance spectroscopy. Upon reperfusion and reoxygenation, piglets received vehicle (n= 12), allopurinol (30 mg/kg/d, n = 10), or deferoxamine (12.5 mg/kg/d, n = 10). The cerebral energy status was determined with phosphorous magnetic resonance spectroscopy. The presence of vasogenic edema was assessed by T2-weighted magnetic resonance imaging. Brain cell injury was assessed with caspase-3 activity, histology, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end (TUNEL)-labeling. At 24 h after hypoxia-ischemia, the phosphocreatine/inorganic phosphate ratios were significantly decreased in vehicle-treated, but not in allopurinol- or deferoxamine-treated piglets. Water T2 values were significantly increased at 24 h after hypoxia-ischemia in cerebral cortex, thalamus, and striatum of vehicle-treated piglets, but not in allopurinol- and deferoxamine-treated piglets. No differences in caspase-3 activity, histologic outcome, or TUNEL-labeling were demonstrated between the three treatment groups. We suggest that allopurinol and deferoxamine may have an additional value in the treatment of perinatal hypoxia-ischemia with other neuroprotective agents or in combination with hypothermia.

  18. Induction of xanthine oxidase activity, endoplasmic reticulum stress and caspase activation by sodium metabisulfite in rat liver and their attenuation by Ghrelin.

    PubMed

    Ercan, Sevim; Kencebay, Ceren; Basaranlar, Goksun; Derin, Narin; Aslan, Mutay

    2015-02-01

    Sodium metabisulfite is used as a preservative in many food preparations but can oxidize to sulfite radicals initiating molecular oxidation. Ghrelin is a peptide hormone primarily produced in the stomach and has anti-inflammatory and anti-oxidant effects on gastrointestinal and cardiovascular systems. This study was performed to elucidate the effect of ghrelin on sulfite-induced endoplasmic reticulum (ER) stress and caspase activation in rat peripheral organs. Xanthine oxidase (XO), xanthine dehydrogenase (XDH) enzyme activities, ER stress markers [phosphorylated PKR-like ER kinase (pPERK); C/EBP-homologous protein (CHOP)], caspase-3, -8, -9 activities, nuclear factor kappa-B (NF-κB) levels were determined in liver, heart and kidney of rats treated with sodium metabisulfite and/or ghrelin for 5 weeks. Sodium metabisulfite treatment significantly elevated XO activity, induced expression of GRP78, CHOP and increased caspase-3, -8 and -9 activities in liver but had no significant effect in heart and kidney. Ghrelin treatment decreased XO activity to baseline levels and attenuated ER stress and caspase activation in liver tissue of sodium metabisulfite treated rats. In conclusion, metabolism of sodium metabisulfite in liver tissue increased XO activity, induced ER stress and caused caspase activation which was attenuated by ghrelin treatment. Ghrelin's hepatoprotective effect could be through modulation of XO activity.

  19. Discovery of xanthine oxidase inhibitors and/or α-glucosidase inhibitors by carboxyalkyl derivatization based on the flavonoid of apigenin.

    PubMed

    Su, Zhuo-Ran; Fan, Shi-Yong; Shi, Wei-Guo; Zhong, Bo-Hua

    2015-07-15

    Three series of apigenin derivatives have been prepared by coupling the carboxyl alkyl group to 4'-, 5- or 7-hydroxyl groups of apigenin respectively. Preliminary biological evaluation in vitro revealed that xanthine oxidase inhibitory activity was improved by modifications at 4'-position and decreased by similar modifications at 5-, 7-positions while α-glucosidase inhibitory activity was maintained by modifications at 5-, 7-positions but lost by modifications at 4'-position. Administration (ip) of 7e markedly lowered serum uric acid levels in potassium oxonate induced hyperuricemic mouse model and administration (p.o.) of 11d or 11e effectively suppressed the elevation of serum glucose in the oral sucrose tolerance test in mice, while apigenin were not significantly effective in both tests.

  20. Inhibitory Effects of Tart Cherry (Prunus cerasus) Juice on Xanthine Oxidoreductase Activity and its Hypouricemic and Antioxidant Effects on Rats.

    PubMed

    Haidari, F; Mohammad Shahi, M; Keshavarz, S A; Rashidi, M R

    2009-03-01

    The aim of this study was to investigate the effect of tart cherry juice on serum uric acid levels, hepatic xanthine oxidoreductase activity and two non-invasive biomarkers of oxidative stress (total antioxidant capacity and malondialdehyde concentration), in normal and hyperuricemic rats. Tart cherry juice (5 ml/kg) was given by oral gavage to rats for 2 weeks. Allopurinol (5 mg/kg) was used as a positive control and was also given by oral gavage. Data showed that tart cherry juice treatment did not cause any significant reduction in the serum uric acid levels in normal rats, but significantly reduced (P<0.05) the serum uric acid levels of hyperuricemic rats in a time-dependent manner. Tart cherry juice treatment also inhibited hepatic xanthine oxidase/dehydrogenase activity. Moreover, a significant increase (P<0.05) in serum total antioxidant capacity was observed in tart cherry juice treated-rats in both normal and hyperuricemic groups. The oral administration of tart cherry juice also led to a significant reduction (P<0.05) in MDA concentration in the hyperuricemic rats. Although the hypouricemic effect of allopurinol, as a putative inhibitor of xanthine oxidoreductase, was much higher than that of tart cherry, it could not significantly change anti-oxidative parameters. These features of tart cherry make it an attractive candidate for the prophylactic treatment of hyperuricaemia, particularly if it is to be taken on a long-term basis. Further investigations to define its clinical efficacy would be highly desirable.

  1. Organic compounds present in airborne particles stimulate superoxide production and DNA fragmentation: role of NOX and xanthine oxidase in animal tissues.

    PubMed

    Busso, Iván Tavera; Silva, Guillermo Benjamín; Carreras, Hebe Alejandra

    2016-08-01

    Suspended particulate matter trigger the production of reactive oxygen species. However, most of the studies dealing with oxidative damage of airborne particles focus on the effects of individual compounds and not real mixtures. In order to study the enzymatic superoxide production resulting from the exposition to a complex mixture, we derived organic extracts from airborne particles collected daily in an urban area and exposed kidney, liver, and heart mammal tissues. After that, we measured DNA damage employing the comet assay. We observed that in every tissue, NADPH oxidase and xanthine oxidase were involved in O2 (-) production when they were exposed to the organic extracts, as the lucigenin's chemiluminescence decays when enzymes were inhibited. The same trend was observed with the percentage of cells with comets, since DNA damage was higher when they were exposed to same experimental conditions. Our data allow us to hypothesize that these enzymes play an important role in the oxidative stress produced by PAHs and that there is a mechanism involving them in the O2 (-)generation.

  2. Xanthine oxidoreductase and its inhibitors: relevance for gout.

    PubMed

    Day, Richard O; Kamel, Bishoy; Kannangara, Diluk R W; Williams, Kenneth M; Graham, Garry G

    2016-12-01

    Xanthine oxidoreductase (XOR) is the rate-limiting enzyme in purine catabolism and converts hypoxanthine to xanthine, and xanthine into uric acid. When concentrations of uric acid exceed its biochemical saturation point, crystals of uric acid, in the form of monosodium urate, emerge and can predispose an individual to gout, the commonest form of inflammatory arthritis in men aged over 40 years. XOR inhibitors are primarily used in the treatment of gout, reducing the formation of uric acid and thereby, preventing the formation of monosodium urate crystals. Allopurinol is established as first-line therapy for gout; a newer alternative, febuxostat, is used in patients unable to tolerate allopurinol. This review provides an overview of gout, a detailed analysis of the structure and function of XOR, discussion on the pharmacokinetics and pharmacodynamics of XOR inhibitors-allopurinol and febuxostat, and the relevance of XOR in common comorbidities of gout.

  3. Hepatic xanthine oxidase activity and purine nucleosides levels as physiological mediators to analyze a subcutaneous treatment with (PhSe)2 in mice infected by Toxoplasma gondii.

    PubMed

    Doleski, Pedro H; Leal, Daniela B R; Machado, Vanessa S; Bottari, Nathieli B; Casali, Emerson A; Moritz, Cesar E J; Camillo, Giovana; Vogel, Fernanda F; Stefani, Lenita M; da Silva, Aleksandro Schafer

    2017-03-01

    The aim of this study was to evaluate the levels of purine nucleosides and xanthine oxidase (XO) activity in the liver of mice chronically infected by Toxoplasma gondii and treated with diphenyl diselenide (PhSe)2. For this experiment, forty Swiss mice were used. Twenty animals were orally infected by approximately 50 bradizoites of a cystogenic ME-49 strain of T. gondii, and the same number of uninfected mice was used as a control group. Ten infected and ten uninfected mice were subcutaneously treated twice (days 1 and 20 post-infection (PI)) with 5 μmol kg(-1) of (PhSe)2. On day 30 PI, liver samples were collected to measure the levels of hypoxanthine (HYPO), xanthine (XAN), uric acid (UA), and XO activity. Infected animals showed increased (P < 0.05) levels of hepatic XAN and UA, as well as XO activity compared to uninfected animals. The use of (PhSe)2 in healthy mice increased the levels of all nucleosides, but decreased XO activity compared to healthy untreated animals. The group of infected and treated animals showed increased XAN and UA levels, and XO activity compared to the healthy control group, however infected and treated mice showed a decrease in the XO activity compared to the infected untreated group. We conclude that chronic infection caused by T. gondii can induce hepatic changes, such as increased UA levels and XO activity, that can increase the pro-oxidative profile. The (PhSe)2 treatment of healthy animals altered the levels of nucleosides, possibly due to low XO activity that decreased nucleoside degradation. Finally, (PhSe)2 treatment decreased XO activity in the infected group and increased nucleoside levels; however it was unable to reduce the UA levels found during the infection.

  4. Allopurinol induced erythroderma.

    PubMed

    Sharma, Geeta; Govil, Dinesh Chandra

    2013-01-01

    Allopurinol, a widely prescribed urate lowering agent is responsible for various adverse drug reactions, including erythroderma. A 45-year-old male patient was admitted with the complaints of fever, redness and scaling all over the body after 3-4 weeks of allopurinol treatment for asymptomatic hyperuricemia. Elevated liver enzymes were detected in his blood analysis. Skin biopsy was consistent with drug induced erythroderma. Allopurinol was stopped and steroids were started. Patient improved over a period of 2 weeks.

  5. Quantitative analysis of phenolic metabolites from different parts of Angelica keiskei by HPLC-ESI MS/MS and their xanthine oxidase inhibition.

    PubMed

    Kim, Dae Wook; Curtis-Long, Marcus J; Yuk, Heung Joo; Wang, Yan; Song, Yeong Hun; Jeong, Seong Hun; Park, Ki Hun

    2014-06-15

    Angelica keiskei is used as popular functional food stuff. However, quantitative analysis of this plant's metabolites has not yet been disclosed. The principal phenolic compounds (1-16) within A. keiskei were isolated, enabling us to quantify the metabolites within different parts of the plant. The specific quantification of metabolites (1-16) was accomplished by multiple reaction monitoring (MRM) using a quadruple tandem mass spectrometer. The limit of detection and limit of quantitation were calculated as 0.4-44 μg/kg and 1.5-148 μg/kg, respectively. Abundance and composition of these metabolites varied significantly across different parts of plant. For example, the abundance of chalcones (12-16) decreased as follows: root bark (10.51 mg/g)>stems (8.52 mg/g)>leaves (2.63 mg/g)>root cores (1.44 mg/g). The chalcones were found to be responsible for the xanthine oxidase (XO) inhibition shown by this plant. The most potent inhibitor, xanthoangelol inhibited XO with an IC50 of 8.5 μM. Chalcones (12-16) exhibited mixed-type inhibition characteristics.

  6. Nitric oxide retards xanthine oxidase-mediated superoxide anion generation in Phalaenopsis flower: an implication of NO in the senescence and oxidative stress regulation.

    PubMed

    Tewari, Rajesh Kumar; Kumar, Praveen; Kim, Soohyun; Hahn, Eun-Joo; Paek, Kee-Yoeup

    2009-02-01

    Senescence is a developmentally regulated and highly ordered sequence of events. Senescence leads to abscission of plant organs and eventually leads to death of a plant or part of it. Present study revealed that Phalaenopsis flower undergo senescence due to over activation of O(2) (.-)generating xanthine oxidase (XO), which consequently increases the concentrations of O(2) (.-) leading to enhanced oxidative damage and disturbed cellular redox environment as indicated by increased lipid peroxidation and DHA/AsA + DHA ratio, respectively. While activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and non-specific peroxidase (POD) were enhanced in sepals and petals of old flower, activities of catalase (CAT) and glutathione reductase (GR) were decreased. Exogenous application of nitric oxide (NO) retarded H(2)O(2)-induced senescence of Phalaenopsis flower by downregulating activity of XO and concentrations of O(2) (.-), H(2)O(2) and malondialdehyde (MDA, an index of lipid peroxidation). Exogenous application of NO also downregulated SOD activity and upregulated antioxidant enzymes involved in the detoxification of H(2)O(2) (CAT and APX), and in the regulation of redox couples viz, monodehydroascorbate reductase (MDHAR) and GR, together with the modulation in non-protein thiol status and DHA/AsA + DHA ratio.

  7. The effect of pH on the exchangeability with deuterium of protons coupled to molybdenum(V) in the active and the desulpho forms of xanthine oxidase.

    PubMed Central

    Malthouse, J P; Bray, R C

    1983-01-01

    The effect of pH variation on the exchangeability with deuterium of protons strongly coupled to Mo(V) in the active and desulpho forms of xanthine oxidase was studied by e.p.r. and rapid freezing, in extension of the work of Gutteridge, Tanner & Bray [Biochem. J. (1978) 175, 887-897]. Above neutrality, exchange rates increased with increasing pH. Detailed studies were made on the desulpho enzyme under a variety of conditions, and exchange rate constants at 22 degrees C ranged from 0.16s -1 at pH 6.6 to 1.6s -1 at pH 11.3. The mechanism of proton exchange in the enzyme is discussed. The interpretation by the above workers that the strongly coupled proton of the active enzyme is on sulphur and that of the desulpho enzyme is on oxygen remains valid (and is in agreement with other work), as do their proposals for the structures of the protonated and deprotonated species. However, pK values cannot be calculated from the exchange data. It is likely that the relatively low rates of exchange observed are due to the difference of structure between the protonated and the deprotonated forms. In the case of the desulpho enzyme, an exchange mechanism, which involves the proton exchanging both as such and along with oxygen in the form of a hydroxyl ion, is discussed. PMID:6312970

  8. Activities of cytochrome P450 1A2, N-acetyltransferase 2, xanthine oxidase, and cytochrome P450 2D6 are unaltered in children with cystic fibrosis.

    PubMed

    Kennedy, Mary Jayne; Scripture, Charity D; Kashuba, Angela D M; Scott, Christy S; Gaedigk, Andrea; Kearns, Gregory L

    2004-03-01

    The activities of hepatic cytochrome P450 (CYP) 1A2, N-acetyltransferase 2 (NAT-2), xanthine oxidase (XO), and CYP2D6 were evaluated in 12 young children (aged 3-8 years) with mild cystic fibrosis (CF) and 12 age-matched healthy control subjects by use of standard caffeine and dextromethorphan phenotyping methods. Subjects were given 4 oz of Coca-Cola (approximately 35 mg caffeine) (The Coca-Cola Company, Atlanta, Ga) and a single 0.5-mg/kg dose of dextromethorphan. Urine was collected for 8 hours after biomarker administration, and enzyme activity was assessed by use of previously validated caffeine and dextromethorphan molar ratios. CYP2D6 genotyping was also performed in 10 of 12 subjects with CF and 11 of 12 control subjects. There were no significant differences in the urinary molar ratios for any of the enzyme systems evaluated. These data suggest that CF does not alter the activities of CYP1A2, NAT-2, XO, and CYP2D6. Altered biotransformation of drugs in this patient population is likely enzyme- and isoform-specific and thus is apparent for only selected compounds that are substrates for enzymes other than CYP1A2, NAT-2, XO, and CYP2D6.

  9. Caffeine affects the biological responses of human hematopoietic cells of myeloid lineage via downregulation of the mTOR pathway and xanthine oxidase activity

    PubMed Central

    Abooali, Maryam; Yasinska, Inna M.; Casely-Hayford, Maxwell A.; Berger, Steffen M.; Fasler-Kan, Elizaveta; Sumbayev, Vadim V.

    2015-01-01

    Correction of human myeloid cell function is crucial for the prevention of inflammatory and allergic reactions as well as leukaemia progression. Caffeine, a naturally occurring food component, is known to display anti-inflammatory effects which have previously been ascribed largely to its inhibitory actions on phosphodiesterase. However, more recent studies suggest an additional role in affecting the activity of the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways, although detailed molecular events underlying its mode of action have not been elucidated. Here, we report the cellular uptake of caffeine, without metabolisation, by healthy and malignant hematopoietic myeloid cells including monocytes, basophils and primary acute myeloid leukaemia mononuclear blasts. Unmodified caffeine downregulated mTOR signalling, which affected glycolysis and the release of pro-inflammatory/pro-angiogenic cytokines as well as other inflammatory mediators. In monocytes, the effects of caffeine were potentiated by its ability to inhibit xanthine oxidase, an enzyme which plays a central role in human purine catabolism by generating uric acid. In basophils, caffeine also increased intracellular cyclic adenosine monophosphate (cAMP) levels which further enhanced its inhibitory action on mTOR. These results demonstrate an important mode of pharmacological action of caffeine with potentially wide-ranging therapeutic impact for treating non-infectious disorders of the human immune system, where it could be applied directly to inflammatory cells. PMID:26384306

  10. Identification of Pyrogallol in the Ethyl Acetate-Soluble Part of Coffee as the Main Contributor to Its Xanthine Oxidase Inhibitory Activity.

    PubMed

    Honda, Sari; Masuda, Toshiya

    2016-10-10

    In this study, ethyl acetate-soluble parts of hot-water extracts from roasted coffee beans were found to demonstrate potent xanthine oxidase (XO) inhibition. The XO inhibitory activities and chlorogenic lactone contents (chlorogenic lactones have previously been identified as XO inhibitors in roast coffee) were measured for ethyl acetate-soluble parts prepared from coffee beans roasted to three different degrees. Although chlorogenic lactone contents decreased with higher degrees of roasting, the XO inhibitory activity did not decrease. These data led us to investigate new potent inhibitors present in these ethyl acetate-soluble extracts. Repeated assay-guided purifications afforded a highly potent XO inhibitor, which was eluted before chlorogenic lactones via medium-pressure chromatography using an octadecylsilica gel column. The obtained inhibitor was identified as pyrogallol (1,2,3-trihydroxybenzene), which had an IC50 of 0.73 μmol L(-1), much stronger than that of other related polyphenolic compounds. Quantitative analysis of pyrogallol and chlorogenic lactones revealed that pyrogallol (at concentrations of 33.9 ± 4.2 nmol mL(-1) in light roast coffee and 39.4 ± 3.9 nmol mL(-1) in dark roast coffee) was the main XO inhibitor in hot-water extracts of roasted coffee beans (i.e., drinking coffee).

  11. Flavonoid content, free radical scavenging and increase in xanthine oxidase inhibitory activity in Galgeun-tang following fermentation with Lactobacillus plantarum.

    PubMed

    Kim, Dong-Seon; Um, Young Ran; Ma, Jin Yeul

    2014-11-01

    Galgeun-tang (GT) prior to and following fermentation with Lactobacillus plantarum was analyzed to determine the total polyphenol and flavonoid contents and the antioxidant activity. GT, fermented GT (FGT) and their three solvent-partitioned fractions, which were prepared by successive partitioning with ethyl acetate (EtOAc), butanol (BuOH) and water, were evaluated for total polyphenol and flavonoid contents, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and xanthine oxidase (XO) inhibitory activity. Following fermentation, the total polyphenol content only increased slightly; however, the flavonoid content increased by 24.3%. The radical scavenging activity increased from 22.4 to 27.5% and the XO inhibitory activity increased from 20.2 to 62.4% at 500 µg/ml. The EtOAc fraction among the solvent‑partitioned fractions demonstrated the highest total polyphenol and flavonoid contents, radical scavenging activities and XO inhibitory activity, and the quantity also markedly increased following fermentation.

  12. Mechanism-based pharmacokinetic-pharmacodynamic modeling of salvianolic acid A effects on plasma xanthine oxidase activity and uric acid levels in acute myocardial infarction rats.

    PubMed

    Wang, Haidong; Li, Xi; Zhang, Wenting; Liu, Yao; Wang, Shijun; Liu, Xiaoquan; He, Hua

    2017-03-01

    1. Salvianolic acid A (SalA) was found to attenuate plasma uric acid (UA) concentration and xanthine oxidase (XO) activity in acute myocardial infraction (AMI) rats, which was characterized with developed mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) model. 2. AMI was induced in rats by coronary artery ligation. Surviving AMI rats received a single intravenous dose of 5 mg/kg of SalA and normal saline. The plasma SalA concentrations were determined by HPLC-MS/MS method. The plasma UA concentrations were determined by HPLC method and plasma XO activity were measured spectrophotometrically. An integrated mathematical model characterized the relationship between plasma UA and SalA. 3. Pharmacokinetics was described using two-compartment model for SalA with linear metabolic process. In post-AMI rats, XO activity and UA concentrations were increased, while SalA dosing palliated this increase. These effects were well captured by using two series of transduction models, simulating the delay of inhibition on XO driven by SalA and UA elevation resulted from the multiple factors, respectively. 4. The effect was well described by the developed PK-PD model, indicating that SalA can exert cardiovascular protective effects by decreasing elevated plasma UA levels induced by AMI.

  13. Xanthine Oxidase Activity Is Associated with Risk Factors for Cardiovascular Disease and Inflammatory and Oxidative Status Markers in Metabolic Syndrome: Effects of a Single Exercise Session

    PubMed Central

    Feoli, Ana Maria Pandolfo; Macagnan, Fabrício Edler; Piovesan, Carla Haas; Bodanese, Luiz Carlos; Siqueira, Ionara Rodrigues

    2014-01-01

    Objective. The main goal of the present study was to investigate the xanthine oxidase (XO) activity in metabolic syndrome in subjects submitted to a single exercise session. We also investigated parameters of oxidative and inflammatory status. Materials/Methods. A case-control study (9 healthy and 8 MS volunteers) was performed to measure XO, superoxide dismutase (SOD), glutathione peroxidase activities, lipid peroxidation, high-sensitivity C-reactive protein (hsCRP) content, glucose levels, and lipid profile. Body mass indices, abdominal circumference, systolic and diastolic blood pressure, and TG levels were also determined. The exercise session consisted of 3 minutes of stretching, 3 minutes of warm-up, 30 minutes at a constant dynamic workload at a moderate intensity, and 3 minutes at a low speed. The blood samples were collected before and 15 minutes after the exercise session. Results. Serum XO activity was higher in MS group compared to control group. SOD activity was lower in MS subjects. XO activity was correlated with SOD, abdominal circumference, body mass indices, and hsCRP. The single exercise session reduced the SOD activity in the control group. Conclusions. Our data support the association between oxidative stress and risk factors for cardiovascular diseases and suggest XO is present in the pathogenesis of metabolic syndrome. PMID:24967004

  14. Caffeine affects the biological responses of human hematopoietic cells of myeloid lineage via downregulation of the mTOR pathway and xanthine oxidase activity.

    PubMed

    Gibbs, Bernhard F; Gonçalves Silva, Isabel; Prokhorov, Alexandr; Abooali, Maryam; Yasinska, Inna M; Casely-Hayford, Maxwell A; Berger, Steffen M; Fasler-Kan, Elizaveta; Sumbayev, Vadim V

    2015-10-06

    Correction of human myeloid cell function is crucial for the prevention of inflammatory and allergic reactions as well as leukaemia progression. Caffeine, a naturally occurring food component, is known to display anti-inflammatory effects which have previously been ascribed largely to its inhibitory actions on phosphodiesterase. However, more recent studies suggest an additional role in affecting the activity of the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways, although detailed molecular events underlying its mode of action have not been elucidated. Here, we report the cellular uptake of caffeine, without metabolisation, by healthy and malignant hematopoietic myeloid cells including monocytes, basophils and primary acute myeloid leukaemia mononuclear blasts. Unmodified caffeine downregulated mTOR signalling, which affected glycolysis and the release of pro-inflammatory/pro-angiogenic cytokines as well as other inflammatory mediators. In monocytes, the effects of caffeine were potentiated by its ability to inhibit xanthine oxidase, an enzyme which plays a central role in human purine catabolism by generating uric acid. In basophils, caffeine also increased intracellular cyclic adenosine monophosphate (cAMP) levels which further enhanced its inhibitory action on mTOR. These results demonstrate an important mode of pharmacological action of caffeine with potentially wide-ranging therapeutic impact for treating non-infectious disorders of the human immune system, where it could be applied directly to inflammatory cells.

  15. Adenosine deaminase, 5'-nucleotidase, xanthine oxidase, superoxide dismutase, and catalase activities in gastric juices from patients with gastric cancer, ulcer, and atrophic gastritis.

    PubMed

    Durak, I; Ormeci, N; Akyol, O; Canbolat, O; Kavutçu, M; Bülbül, M

    1994-04-01

    Adenosine deaminase (ADA), 5'-Nucleotidase (5NT), Xanthine oxidase (XO), Cu-Zn Superoxide dismutase (SOD) and Catalase (CAT) activities were determined in gastric juices from patients with gastric cancer, ulcer, gastritis and from healthy subjects. Enzyme activities were given as units per ml gastric juice and units per mg protein in gastric juice. ADA, 5NT and XO activities were found lower and protein concentrations were found higher in the cancer group than controls. There was however no significant difference between Cu-Zn SOD activities of the cancer and control groups. In all groups including control one, we could not find catalase activities in most of the samples. On the other hand, ADA, 5NT activities and protein concentrations in the gastric juice were lower in the gastritis group than control group. In the ulcer group, we found higher Cu-Zn SOD and XO activities and lower 5NT activity and protein concentrations compared with control values. In an attempt to establish statistical correlations between mean enzyme activities, pH and protein concentrations in the gastric juices of the groups, we found noticeable intra and inter-correlations, which indicated possible relations between DNA and free radical metabolizing enzymes.

  16. Acute kidney injury observed during phase 1 clinical trials of a novel xanthine oxidase/URAT1 dual inhibitor PF-06743649.

    PubMed

    Dua, Pinky; Gurrell, Rachel; Kirby, Simon; Sudworth, Maria; Loudon, Peter T

    2016-08-01

    The objective of these clinical studies was to assess the safety and urate lowering activity of a novel urate transporter 1 (URAT1)/ xanthine oxidase (XO) inhibitor PF-06743649 in healthy subjects and gout patients. Escalating doses of PF-06743649 or placebo were given to healthy young subjects, healthy elderly subjects and gout patients. Serum uric acid (sUA) and urinary pharmacodynamic markers were assayed, and safety was assessed by collection of adverse events and assessment of safety labs, ECGs and vital signs. Administration of PF-06743649 led to rapid decrease in sUA in all cohorts; in gout patients, a change from baseline of 69 % was observed for the 40 mg dose. Urinary and serum biomarkers were consistent with inhibition of both URAT1 and XO. Although dosing was otherwise well tolerated, two subjects experienced serious adverse events of acute kidney injury. Both subjects exhibited increased serum creatinine and blood urea nitrogen in the first 3 days post first dose and were hospitalised. One subject exhibited oliguria for the first 24 h. Both subjects made a complete recovery with minimal intervention. PF-06743649 was effective at rapidly lowering sUA, but further development was terminated for an identified renal safety risk.

  17. Enzymatic oxidation of phthalazine with guinea pig liver aldehyde oxidase and liver slices: inhibition by isovanillin.

    PubMed

    Panoutsopoulos, Georgios I; Beedham, Christine

    2004-01-01

    The enzymes aldehyde oxidase and xanthine oxidase catalyze the oxidation of a wide range of N-heterocycles and aldehydes. These enzymes are widely known for their role in the metabolism of N-heterocyclic xenobiotics where they provide a protective barrier by aiding in the detoxification of ingested nitrogen-containing heterocycles. Isovanillin has been shown to inhibit the metabolism of aromatic aldehydes by aldehyde oxidase, but its inhibition towards the heterocyclic compounds has not been studied. The present investigation examines the oxidation of phthalazine in the absence and in the presence of the inhibitor isovanillin by partially purified aldehyde oxidase from guinea pig liver. In addition, the interaction of phthalazine with freshly prepared guinea pig liver slices, both in the absence and presence of specific inhibitors of several liver oxidizing enzymes, was investigated. ldehyde oxidase rapidly converted phthalazine into 1-phthalazinone, which was completely inhibited in the presence of isovanillin (a specific inhibitor of aldehyde oxidase). In freshly prepared liver slices, phthalazine was also rapidly converted to 1-phthalazinone. The formation of 1-phthalazinone was completely inhibited by isovanillin, whereas disulfiram (a specific inhibitor of aldehyde dehydrogenase) only inhibited 1-phthalazinone formation by 24% and allopurinol (a specific inhibitor of xanthine oxidase) had little effect. Therefore, isovanillin has been proved as an inhibitor of the metabolism of heterocyclic substrates, such as phthalazine, by guinea pig liver aldehyde oxidase, since it had not been tested before. Thus it would appear from the inhibitor results that aldehyde oxidase is the predominant enzyme in the oxidation of phthalazine to 1-phthalazinone in freshly prepared guinea pig liver slices, whereas xanthine oxidase only contributes to a small extent and aldehyde dehydrogenase does not take any part.

  18. In vitro xanthine oxidase and albumin denaturation inhibition assay of Barringtonia racemosa L. and total phenolic content analysis for potential anti-inflammatory use in gouty arthritis

    PubMed Central

    Osman, Nurul Izzati; Sidik, Norrizah Jaafar; Awal, Asmah; Adam, Nurul Athirah Mohamad; Rezali, Nur Inani

    2016-01-01

    Aim: This study was conducted to evaluate the in vitro anti-inflammatory activities and total phenolic content (TPC) of methanolic extracts of infloresence axes, endosperms, leaves, and pericarps of Barringtonia racemosa L. Methods: The anti-inflammatory study was conducted by assessing the potential through xanthine oxidase (XO) and albumin denaturation inhibition assays. Meanwhile, the TPC in the extracts were assessed by Folin-Ciocalteu assay. Results: In the XO inhibition assay, the infloresence axes extract was found to exert the highest inhibition capacity at 0.1% (w/v) with 59.54 ± 0.001% inhibition followed by leaves (58.82 ± 0.001%), pericarps (57.99 ± 0.003%), and endosperms (57.20 ± 0.003%) extracts. Similarly in the albumin denaturation inhibition assay, the infloresence axes extract had shown the greatest inhibition capacity with 70.58 ± 0.004% inhibition followed by endosperms (66.80 ± 0.024%), leaves (65.29 ± 0.006%), and pericarps extracts (43.33 ± 0.002%). Meanwhile, for TPC analysis, leaves extract was found to have the highest phenolic content (53.94 ± 0.000 mg gallic acid equivalent [GAE]/g DW) followed by infloresence axes (31.54 ± 0.001 mg GAE/g DW), endosperms (22.63 ± 0.001 mg GAE/g DW), and the least was found in pericarps (15.54 ± 0.001 mg GAE/g DW). Conclusion: The results indeed verified the in vitro anti-inflammatory activities of B. racemosa and supported its potential to be used in alleviating gouty arthritis and XO-related diseases. PMID:27757263

  19. Bioactive compounds from Carissa opaca roots and xanthine oxidase and alpha-amylase inhibitory activities of their methanolic extract and its fractions in different solvents

    PubMed Central

    Saeed, Ramsha; Ahmed, Dildar

    2015-01-01

    Background: Carissa opaca is known for its many ethnomedicinal uses. There was a need to study its bioactivities and identify its phytochemicals. Objective: The objective was to isolate and identify phytochemicals from roots of C. opaca and to evaluate xanthine oxidase (XO) and alpha-amylase inhibitory activities of their methanolic extract and its fractions. Materials and Methods: Methanolic extract of finely divided powder of roots of C. opaca was obtained by cold maceration, followed by its fractionation to obtain hexane, chloroform, ethyl acetate, n-butanolic, and aqueous fractions. Phytochemicals screening was done by standard protocols. XO and alpha-amylase inhibitory activities of the methanolic extract and its fractions were studied. The most active ethyl acetate fraction was subjected to the column and thin layer chromatography to isolate its compounds, which were identified by gas chromatography-mass spectrometry and high-performance liquid chromatography comparison. Results: Methanolic extract displayed significant activity against both the enzymes with IC50 of 156.0 mg/mL and 5.6 mg/mL for XO and alpha-amylase, respectively. Ethyl acetate fraction showed highest activity against both the enzymes with IC50 of 129 mg/mL and 4.9 mg/mL for XO and alpha-amylase, respectively. Chloroform fraction had IC50 of 154.2 mg/mL and 5.5 mg/mL for XO and alpha-amylase, respectively. Aqueous fraction exhibited significant efficacy against alpha-amylase (IC50 5.0 mg/mL). Hexane fraction showed good activity against alpha-amylase in a dose-dependent manner but exhibited opposite trend against XO. The compounds isolated from ethyl acetate fraction included limonene, vanillin, lupeol, rutin, quercetin, b-sitosterol, Vitamin E, 2-hydroxyacetophenone, naphthalenone, 2,3,3-trimethyl-2-(3-methylbuta-1,3-dienyl)-6-methylenecyclohexanone, and 2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester. Conclusions: Moderately polar phytochemicals of C. opaca roots possess exploitable

  20. Pitfalls, prevention, and treatment of hyperuricemia during tumor lysis syndrome in the era of rasburicase (recombinant urate oxidase)

    PubMed Central

    Pession, Andrea; Melchionda, Fraia; Castellini, Claudia

    2008-01-01

    Along with hydration and urinary alkalinization, allopurinol has been the standard agent for the management of hyperuricemia in patients with a high tumor burden at risk of tumor lysis syndrome; however, this agent often fails to prevent and treat this complication effectively. Rasburicase (recombinant urate oxidase) has been shown to be effective in reducing uric acid and preventing uric acid accumulation in patients with hematologic malignancies with hyperuricemia or at high risk of developing it. Rasburicase acts at the end of the purine catabolic pathway and, unlike allopurinol, does not induce accumulation of xanthine or hypoxanthine. Its rapid onset of action and the ability to lower pre-existing elevated uric acid levels are the advantages of rasburicase over allopurinol. Rasburicase represents an effective alternative to allopurinol to promptly reduce uric acid levels, improve patient’s electrolyte status, and reverse renal insufficiency. The drug, initially studied in pediatric patients with acute lymphoblastic leukemia and aggressive non-Hodgkin lymphoma, seems to show comparable benefit in adults with similar lymphoid malignancies or at high risk of tumor lysis syndrome. Current and future trials will evaluate alternative doses and different schedules of rasburicase to maintain its efficacy while reducing its cost. The review provides a comprehensive and detailed review of pathogenesis, laboratory, and clinical presentation of TLS together with clinical studies already performed both in pediatric and adult patients. PMID:19707436

  1. Allopurinol hypersensitivity reactions: desensitization strategies and new therapeutic alternative molecules.

    PubMed

    Calogiuri, Gianfranco; Nettis, Eustachio; Di Leo, Elisabetta; Foti, Caterina; Ferrannini, Antonio; Butani, Lavjay

    2013-02-01

    Allopurinol, an analog of hypoxanthine has been worldwide used for the treatment of hyperuricemia and gout for over 40 years. Unfortunately some patients assuming this medication have developed hypersensitivity reactions ranging from mild cutaneous eruption to more severe clinical manifestations such as allopurinol hypersensitivity syndrome or Steven-Johnson syndrome and lethal toxic epidermal necrolysis. Various strategies of slow desensitization have been elaborated to reintroduce allopurinol in a part of these patients, mainly patients affected by mild skin reactions as fixed drug eruption or exanthema. However, several new uricosuric therapies have been recently introduced. Actually drugs as recombinant urate oxidase and febuxostat are under post-marketing surveillance to control potential adverse effects related to their immunogenicity even.

  2. Multicentre, prospective, randomised, open-label, blinded end point trial of the efficacy of allopurinol therapy in improving cardiovascular outcomes in patients with ischaemic heart disease: protocol of the ALL-HEART study

    PubMed Central

    Ford, Ian; Walker, Andrew; Hawkey, Chris; Begg, Alan; Avery, Anthony; Taggar, Jaspal; Wei, Li; Struthers, Allan D; MacDonald, Thomas M

    2016-01-01

    Introduction Ischaemic heart disease (IHD) is one of the most common causes of death in the UK and treatment of patients with IHD costs the National Health System (NHS) billions of pounds each year. Allopurinol is a xanthine oxidase inhibitor used to prevent gout that also has several positive effects on the cardiovascular system. The ALL-HEART study aims to determine whether allopurinol improves cardiovascular outcomes in patients with IHD. Methods and analysis The ALL-HEART study is a multicentre, controlled, prospective, randomised, open-label blinded end point (PROBE) trial of allopurinol (up to 600 mg daily) versus no treatment in a 1:1 ratio, added to usual care, in 5215 patients aged 60 years and over with IHD. Patients are followed up by electronic record linkage and annual questionnaires for an average of 4 years. The primary outcome is the composite of non-fatal myocardial infarction, non-fatal stroke or cardiovascular death. Secondary outcomes include all-cause mortality, quality of life and cost-effectiveness of allopurinol. The study will end when 631 adjudicated primary outcomes have occurred. The study is powered at 80% to detect a 20% reduction in the primary end point for the intervention. Patient recruitment to the ALL-HEART study started in February 2014. Ethics and dissemination The study received ethical approval from the East of Scotland Research Ethics Service (EoSRES) REC 2 (13/ES/0104). The study is event-driven and results are expected after 2019. Results will be reported in peer-reviewed journals and at scientific meetings. Results will also be disseminated to guideline committees, NHS organisations and patient groups. Trial registration number 32017426, pre-results. PMID:27609859

  3. Comparative Study of Renal Protective Effects of Allopurinol and N-Acetyl-Cysteine on Contrast Induced Nephropathy in Patients Undergoing Cardiac Catheterization

    PubMed Central

    Bhawani, Goru; Kumari, Neera; Murthy, Kasturi SN; Lalwani, Vinod; Raju, CH Narasimha

    2014-01-01

    Objectives : To evaluate the difference in the renal protective effects of allopurinol and n-acetyl cysteine along with saline hydration in patients of contrast induced nephropathy (CIN) post cardiac interventions. Background: CIN remains a common complication of cardiac procedures. Radio contrast agents can cause a reduction in renal function that may be related to oxidative stress underlining various patho- physiologies. Conflicting evidence suggests that administration of allopurinol, a xanthine oxidase inhibitor can prevent CIN. Materials and Methods: This is a study of 500 patients undergoing angiography and coronary revascularisation in patients showing significant coronary block. The angiography positive patients (275) were prospectively randomised to different treatment protocol to study for their reno-protective effect. The patients received either of the three drugs saline hydration (SH, 1ml/kg/hr), n-acetylcysteine (SH+NAC, 600 mg bd) or Allopurinol (SH+ALLP, 300 mg/day) 12 hours before and after administration of radio contrast agent. Levels of serum creatinine and blood urea of the 275 patients recorded at 24 hour interval were noted post angioplasty over a course of 5 days in patients receiving either omnipaque (125) or visipaque (150) contrast media. All the 500 patients were also assessed for development of any kind of adverse drug effects/reactions with the two contrast media. Results: CIN occurred in 56 of 500 the patients (10.6%) who underwent angiography and 49 of 275 patients (17.8%) who underwent angioplasty. In the omnipaque group CIN occurred in 16/40, 8/40, nil/45 in patients receiving SH, NAC plus SH and SH plus ALLP respectively. In the visipaque group CIN occurred in 15/50, 10/50, nil/50 in the three treatments groups respectively. Allopurinol maintained a consistent fall in the serum creatinine & blood urea levels from the baseline values from the end of the 1st day (p < .01 & .001) in both the category. Visipaque proved to be better

  4. Cisplatin-induced renal toxicity via tumor necrosis factor-α, interleukin 6, tumor suppressor P53, DNA damage, xanthine oxidase, histological changes, oxidative stress and nitric oxide in rats: protective effect of ginseng.

    PubMed

    Yousef, Mokhtar I; Hussien, Hend M

    2015-04-01

    Cisplatin is an effective chemotherapeutic agent successfully used in the treatment of a wide range of solid tumors, while its usage is limited due to its nephrotoxicity. The present study was undertaken to examine the effectiveness of ginseng to ameliorate the renal nephrotoxicity, damage in kidney genomic DNA, tumor necrosis factor-α, interleukin 6, tumor suppressor P53, histological changes and oxidative stress induced by cisplatin in rats. Cisplatin caused renal damage, including DNA fragmentation, upregulates gene expression of tumor suppressor protein p53 and tumor necrosis factor-α and IL-6. Cisplatin increased the levels of kidney TBARS, xanthine oxidase, nitric oxide, serum urea and creatinine. Cisplatin decreased the activities of antioxidant enzymes (GST, GPX, CAT and SOD), ATPase and the levels of GSH. A microscopic examination showed that cisplatin caused kidney damage including vacuolization, severe necrosis and degenerative changes. Ginseng co-treatment with cisplatin reduced its renal damage, oxidative stress, DNA fragmentation and induced DNA repair processes. Also, ginseng diminished p53 activation and improved renal cell apoptosis and nephrotoxicity. It can be concluded that, the protective effects of ginseng against cisplatin induced-renal damage was associated with the attenuation of oxidative stress and the preservation of antioxidant enzymes.

  5. Allopurinol-mediated lignocellulose-derived microbial inhibitor tolerance by Clostridium beijerinckii during acetone-butanol-ethanol (ABE) fermentation.

    PubMed

    Ujor, Victor; Agu, Chidozie Victor; Gopalan, Venkat; Ezeji, Thaddeus Chukwuemeka

    2015-04-01

    In addition to glucans, xylans, and arabinans, lignocellulosic biomass hydrolysates contain significant levels of nonsugar components that are toxic to the microbes that are typically used to convert biomass to biofuels and chemicals. To enhance the tolerance of acetone-butanol-ethanol (ABE)-generating Clostridium beijerinckii NCIMB 8052 to these lignocellulose-derived microbial inhibitory compounds (LDMICs; e.g., furfural), we have been examining different metabolic perturbation strategies to increase the cellular reductant pools and thereby facilitate detoxification of LDMICs. As part of these efforts, we evaluated the effect of allopurinol, an inhibitor of NAD(P)H-generating xanthine dehydrogenase (XDH), on C. beijerinckii grown in furfural-supplemented medium and found that it unexpectedly increased the rate of detoxification of furfural by 1.4-fold and promoted growth, butanol, and ABE production by 1.2-, 2.5-, and 2-fold, respectively. Since NAD(P)H/NAD(P)(+) levels in C. beijerinckii were largely unchanged upon allopurinol treatment, we postulated and validated a possible basis in DNA repair to account for the solventogenic gains with allopurinol. Following the observation that supplementation of allopurinol in the C. beijerinckii growth media mitigates the toxic effects of nalidixic acid, a DNA-damaging antibiotic, we found that allopurinol elicited 2.4- and 6.7-fold increase in the messenger RNA (mRNA) levels of xanthine and hypoxanthine phosphoribosyltransferases, key purine-salvage enzymes. Consistent with this finding, addition of inosine (a precursor of hypoxanthine) and xanthine led to 1.4- and 1.7-fold increase in butanol production in furfural-challenged cultures of C. beijerinckii. Taken together, our results provide a purine salvage-based rationale for the unanticipated effect of allopurinol in improving furfural tolerance of the ABE-fermenting C. beijerinckii.

  6. Allopurinol therapy and cataractogenesis in humans

    SciTech Connect

    Lerman, S.; Megaw, J.M.; Gardner, K.

    1982-08-01

    Long-term ingestion of allopurinol, an antihyperuricemic agent used to treat gout, may be related to the development of lens opacities in relatively young patients (second to fifth decades of life). Cataracts obtained from three patients taking allopurinol were subjected to high-resolution phosphorescence spectroscopy. The characteristic allopurinol triplet was demonstrated in all three cataracts. Identical spectra were obtained for normal human lenses incubated in media containing 10(-3)M allopurinol and exposed to 1.2 mW/cm2 ultraviolet radiation for 16 hours; control lenses (irradiated without allopurinol) showed no allopurinol triplets. Similar data were obtained for lenses from rats given one dose of allopurinol and exposed to ultraviolet radiation overnight. These data provide evidence that allopurinol can be photobound in rat and human lenses and suggest its cataractogenic potential.

  7. Xanthine Oxidoreductase in Drug Metabolism: Beyond a Role as a 
Detoxifying Enzyme

    PubMed Central

    Battelli, Maria Giulia; Polito, Letizia; Bortolotti, Massimo; Bolognesi, Andrea

    2016-01-01

    The enzyme xanthine oxidoreductase (XOR) catalyzes the last two steps of purine catabolism in the highest uricotelic primates. XOR is an enzyme with dehydrogenase activity that, in mammals, may be converted into oxidase activity under a variety of pathophysiologic conditions. XOR activity is highly regulated at the transcriptional and post-translational levels and may generate reactive oxygen and nitrogen species, which trigger different consequences, ranging from cytotoxicity to inflammation. The low specificity for substrates allows XOR to metabolize a number of endogenous metabolites and a variety of exogenous compounds, including drugs. The present review focuses on the role of XOR as a drug-metabolizing enzyme, specifically for drugs with anticancer, antimicrobial, antiviral, immunosuppressive or vasodilator activities, as well as drugs acting on metabolism or inducing XOR expression. XOR has an activating role that is essential to the pharmacological action of quinone drugs, cyadox, antiviral nucleoside analogues, allopurinol, nitrate and nitrite. XOR activity has a degradation function toward thiopurine nucleotides, pyrazinoic acid, methylxanthines and tolbutamide, whose half-life may be prolonged by the use of XOR inhibitors. In conclusion, to avoid potential drug interaction risks, such as a toxic excess of drug bioavailability or a loss of drug efficacy, caution is suggested in the use of XOR inhibitors, as in the case of hyperuricemic patients affected by gout or tumor lysis syndrome, when it is necessary to simultaneously administer therapeutic substances that are activated or degraded by the drug-metabolizing activity of XOR. PMID:27458036

  8. Allopurinol and kidney function: An update.

    PubMed

    Stamp, Lisa K; Chapman, Peter T; Palmer, Suetonia C

    2016-01-01

    Allopurinol is the most commonly used urate lowering therapy in the management of gout. Despite the fact that it has been available for over 40 years there is ongoing debate about optimal allopurinol dosing in gout patients with chronic kidney disease. Given that gout is common in patients with renal impairment, clinicians need to be aware of the relationships between serum urate and kidney function as well as the effects of allopurinol on kidney function and vice versa. The use of allopurinol in patients on dialysis is an understudied area. Dialysis reduces plasma oxypurinol concentrations, therefore the dose and time of administration in relationship to dialysis need to be carefully considered. Recently, it has been suggested that there may be a role for allopurinol in patients with chronic kidney disease without gout. Observational studies have reported an association between serum urate and chronic kidney disease and end stage renal failure. The effect of urate lowering therapy with allopurinol on progression of kidney disease has been examined in small studies with varying results. Larger clinical trials are currently underway. This review will examine the relationships between allopurinol and kidney function in adults with and without renal disease and address allopurinol dosing in gout patients with impaired kidney function.

  9. NADPH oxidase-derived reactive oxygen species contribute to impaired cutaneous microvascular function in chronic kidney disease.

    PubMed

    DuPont, Jennifer J; Ramick, Meghan G; Farquhar, William B; Townsend, Raymond R; Edwards, David G

    2014-06-15

    Oxidative stress promotes vascular dysfunction in chronic kidney disease (CKD). We utilized the cutaneous circulation to test the hypothesis that reactive oxygen species derived from NADPH oxidase and xanthine oxidase impair nitric oxide (NO)-dependent cutaneous vasodilation in CKD. Twenty subjects, 10 stage 3 and 4 patients with CKD (61 ± 4 yr; 5 men/5 women; eGFR: 39 ± 4 ml·min(-1)·1.73 m(-2)) and 10 healthy controls (55 ± 2 yr; 4 men/6 women; eGFR: >60 ml·min(-1)·1.73 m(-2)) were instrumented with 4 intradermal microdialysis fibers for the delivery of 1) Ringer solution (Control), 2) 10 μM tempol (scavenge superoxide), 3) 100 μM apocynin (NAD(P)H oxidase inhibition), and 4) 10 μM allopurinol (xanthine oxidase inhibition). Skin blood flow was measured via laser-Doppler flowmetry during standardized local heating (42°C). N(g)-nitro-l-arginine methyl ester (L-NAME; 10 mM) was infused to quantify the NO-dependent portion of the response. Cutaneous vascular conductance (CVC) was calculated as a percentage of the maximum CVC achieved during sodium nitroprusside infusion at 43°C. Cutaneous vasodilation was attenuated in patients with CKD (77 ± 3 vs. 88 ± 3%, P = 0.01), but augmented with tempol and apocynin (tempol: 88 ± 2 (P = 0.03), apocynin: 91 ± 2% (P = 0.001). The NO-dependent portion of the response was reduced in patients with CKD (41 ± 4 vs. 58 ± 2%, P = 0.04), but improved with tempol and apocynin (tempol: 58 ± 3 (P = 0.03), apocynin: 58 ± 4% (P = 0.03). Inhibition of xanthine oxidase did not alter cutaneous vasodilation in either group (P > 0.05). These data suggest that NAD(P)H oxidase is a source of reactive oxygen species and contributes to microvascular dysfunction in patients with CKD.

  10. Pharmacokinetics and Metabolism of Allopurinol Riboside,

    DTIC Science & Technology

    1991-05-01

    095 Pharmacokinetics and metabolism of allopurinol riboside T"ere are no safe and effective oral drugs to treat leishmaniasis and Chagas ’ disease. Thc...leishmaniasis and Chagas ’ disease. 2 .3 typically require muLltiple parenteral doses. are e\\pen- [hese diseases occur in millions of people on a world- sive...40 ° C until analysis. Urine samples and perchloric allopurinol riboside were quantitated (in 85 determina acid extracts of plasma were analyzed for

  11. Pathways of nitrogen assimilation in cowpea nodules studied using /sup 15/N/sub 2/ and allopurinol. [Vigna unguiculata L. Walp. cv Vita

    SciTech Connect

    Atkins, C.A.; Storer, P.J.; Pate, J.S.

    1988-01-01

    In the presence of 0.5 millimolar allopurinol (4-hydroxypyrazolo (3,4-d)pyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.2.3.2), intact attached nodules of cowpea (vigna unguiculata L. Walp. cv Vita 3) formed (/sup 15/N)xanthine from /sup 15/N/sub 2/ at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de novo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.87.99.2) activity. Negligible /sup 15/N -labeling of asparagine from /sup 15/N/sub 2/ was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery.

  12. Hyperuricemia-Related Diseases and Xanthine Oxidoreductase (XOR) Inhibitors: An Overview

    PubMed Central

    Chen, Changyi; Lü, Jian-Ming; Yao, Qizhi

    2016-01-01

    Uric acid is the final oxidation product of purine metabolism in humans. Xanthine oxidoreductase (XOR) catalyzes oxidative hydroxylation of hypoxanthine to xanthine to uric acid, accompanying the production of reactive oxygen species (ROS). Uric acid usually forms ions and salts known as urates and acid urates in serum. Clinically, overproduction or under-excretion of uric acid results in the elevated level of serum uric acid (SUA), termed hyperuricemia, which has long been established as the major etiologic factor in gout. Accordingly, urate-lowering drugs such as allopurinol, an XOR-inhibitor, are extensively used for the treatment of gout. In recent years, the prevalence of hyperuricemia has significantly increased and more clinical investigations have confirmed that hyperuricemia is an independent risk factor for cardiovascular disease, hypertension, diabetes, and many other diseases. Urate-lowering therapy may also play a critical role in the management of these diseases. However, current XOR-inhibitor drugs such as allopurinol and febuxostat may have significant adverse effects. Therefore, there has been great effort to develop new XOR-inhibitor drugs with less or no toxicity for the long-term treatment or prevention of these hyperuricemia-related diseases. In this review, we discuss the mechanism of uric acid homeostasis and alterations, updated prevalence, therapeutic outcomes, and molecular pathophysiology of hyperuricemia-related diseases. We also summarize current discoveries in the development of new XOR inhibitors. PMID:27423335

  13. Effect of Systemic Antioxidant Allopurinol Therapy on Skin Flap Survival

    PubMed Central

    Rasti Ardakani, Mehdi; Al-Dam, Ahmed; Rashad, Ashkan; Shayesteh Moghadam, Ali

    2017-01-01

    BACKGROUND It has been reported that systemic administration of allopurinol improves cell survival. This study was aimed to evaluate effects of allopurinol on skin flaps in dogs. METHODS Twenty dogs underwent one skin flap surgery with a 2-week interval. The first procedure was performed according to the standard protocols. The second phase was started by a 1-week pretreatment with allopurinol. Length of the necrotic zone was measured and recorded daily. At each phase, flaps were removed and sent for histopathological study after 1 week observation. RESULTS Mean length of the necrotic zone in allopurinol treated skin flaps has been significantly less than normal flaps over all 7 days of observation (p<0.0001). Histopathology study showed less inflammation and more normal tissue structure in the allopurinol treated skin flaps. CONCLUSION It was demonstrated that systemic administration of allopurinol significantly improved skin flap survival. PMID:28289614

  14. Predicting allopurinol response in patients with gout

    PubMed Central

    Duffull, Stephen B.; Merriman, Tony R.; Dalbeth, Nicola; Barclay, Murray L.; Stamp, Lisa K.

    2015-01-01

    Aims The primary aim of this research was to predict the allopurinol maintenance doses required to achieve the target plasma urate of ≤0.36 mmol l−1. Methods A population analysis was conducted in nonmem using oxypurinol and urate plasma concentrations from 133 gout patients. Maintenance dose predictions to achieve the recommended plasma urate target were generated. Results The urate response was best described by a direct effects model. Renal function, diuretic use and body size were found to be significant covariates. Dose requirements increased approximately 2‐fold over a 3‐fold range of total body weight and were 1.25–2 fold higher in those taking diuretics. Renal function had only a modest impact on dose requirements. Conclusions Contrary to current guidelines, the model predicted that allopurinol dose requirements were determined primarily by differences in body size and diuretic use. A revised guide to the likely allopurinol doses to achieve the target plasma urate concentration is proposed. PMID:26451524

  15. Effects of Parsley (Petroselinum crispum) and its Flavonol Constituents, Kaempferol and Quercetin, on Serum Uric Acid Levels, Biomarkers of Oxidative Stress and Liver Xanthine Oxidoreductase Aactivity inOxonate-Induced Hyperuricemic Rats.

    PubMed

    Haidari, Fatemeh; Keshavarz, Seid Ali; Mohammad Shahi, Majid; Mahboob, Soltan-Ali; Rashidi, Mohammad-Reza

    2011-01-01

    Increased serum uric acid is known to be a major risk related to the development of several oxidative stress diseases. The aim of this study was to investigate the effect of parsley, quercetin and kaempferol on serum uric acid levels, liver xanthine oxidoreductase activity and two non-invasive biomarkers of oxidative stress (total antioxidant capacity and malondialdehyde concentration) in normal and oxonate-induced hyperuricemic rats. A total of 60 male Wistar rats were randomly divided into ten equal groups; including 5 normal groups (vehicle, parsley, quercetin, kaempferol and allopurinol) and 5 hyperuricemic groups (vehicle, parsley, quercetin, kaempferol and allopurinol). Parsley (5 g/Kg), quercetin (5 mg/Kg), kaempferol (5 mg/Kg) and allopurinol (5 mg/Kg) were administrated to the corresponding groups by oral gavage once a day for 2 weeks. The results showed that parsley and its flavonol did not cause any significant reduction in the serum uric acid levels in normal rats, but significantly reduced the serum uric acid levels of hyperuricemic rats in a time-dependent manner. All treatments significantly inhibited liver xanthine oxidoreductase activity. Parsley, kaempferol and quercetin treatment led also to a significant increase in total antioxidant capacity and decrease in malondialdehyde concentration in hyperuricemic rats. Although the hypouricemic effect of allopurinol was much higher than that of parsley and its flavonol constituents, it could not significantly change oxidative stress biomarkers. These features of parsley and its flavonols make them as a possible alternative for allopurinol, or at least in combination therapy to minimize the side effects of allopurinol to treat hyperuricemia and oxidative stress diseases.

  16. Allopurinol and Cardiovascular Outcomes in Adults With Hypertension.

    PubMed

    MacIsaac, Rachael L; Salatzki, Janek; Higgins, Peter; Walters, Matthew R; Padmanabhan, Sandosh; Dominiczak, Anna F; Touyz, Rhian M; Dawson, Jesse

    2016-03-01

    Allopurinol lowers blood pressure in adolescents and has other vasoprotective effects. Whether similar benefits occur in older individuals remains unclear. We hypothesized that allopurinol is associated with improved cardiovascular outcomes in older adults with hypertension. Data from the United Kingdom Clinical Research Practice Datalink were used. Multivariate Cox-proportional hazard models were applied to estimate hazard ratios for stroke and cardiac events (defined as myocardial infarction or acute coronary syndrome) associated with allopurinol use over a 10-year period in adults aged >65 years with hypertension. A propensity-matched design was used to reduce potential for confounding. Allopurinol exposure was a time-dependent variable and was defined as any exposure and then as high (≥300 mg daily) or low-dose exposure. A total of 2032 allopurinol-exposed patients and 2032 matched nonexposed patients were studied. Allopurinol use was associated with a significantly lower risk of both stroke (hazard ratio, 0.50; 95% confidence interval, 0.32-0.80) and cardiac events (hazard ratio, 0.61; 95% confidence interval, 0.43-0.87) than nonexposed control patients. In exposed patients, high-dose treatment with allopurinol (n=1052) was associated with a significantly lower risk of both stroke (hazard ratio, 0.58; 95% confidence interval, 0.36-0.94) and cardiac events (hazard ratio, 0.65; 95% confidence interval, 0.46-0.93) than low-dose treatment (n=980). Allopurinol use is associated with lower rates of stroke and cardiac events in older adults with hypertension, particularly at higher doses. Prospective clinical trials are needed to evaluate whether allopurinol improves cardiovascular outcomes in adults with hypertension.

  17. [Allopurinol hypersensitivity syndrome. A report of two cases].

    PubMed

    Rodríguez-Arámbula, Adriana; Arenas-Velázquez, Elsa; Castanedo-Cázares, Juan Pablo; Hernández-Blanco, Diana; Oros-Ovalle, Cuauhtémoc; Torres-Álvarez, Bertha

    2016-01-01

    Patients in treatment with allopurinol are in risk of having life threatening adverse reactions particularly at the beginning of the treatment. Two percent of the patients prescribed with this drug have associated severe cutaneous adverse reactions. We present two cases of allopurinol hypersensitivity syndrome in mexican patients in which asymptomatic hyperuricemia was the indication to its use. The general physician and the specialist must be alert of this syndrome that causes elevate morbidity and mortality.

  18. Xanthine dehydrogenase-1 silencing in Aedes aegypti mosquitoes promotes a blood feeding-induced adulticidal activity.

    PubMed

    Isoe, Jun; Petchampai, Natthida; Isoe, Yurika E; Co, Katrina; Mazzalupo, Stacy; Scaraffia, Patricia Y

    2017-02-08

    Aedesaegypti has 2 genes encoding xanthine dehydrogenase (XDH). We analyzed XDH1 and XDH2 gene expression by real-time quantitative PCR in tissues from sugar- and blood-fed females. Differential XDH1 and XDH2 gene expression was observed in tissues dissected throughout a time course. We next exposed females to blood meals supplemented with allopurinol, a well-characterized XDH inhibitor. We also tested the effects of injecting double-stranded RNA (dsRNA) against XDH1, XDH2, or both. Disruption of XDH by allopurinol or XDH1 by RNA interference significantly affected mosquito survival, causing a disruption in blood digestion, excretion, oviposition, and reproduction. XDH1-deficient mosquitoes showed a persistence of serine proteases in the midgut at 48 h after blood feeding and a reduction in the uptake of vitellogenin by the ovaries. Surprisingly, analysis of the fat body from dsRNA-XDH1-injected mosquitoes fell into 2 groups: one group was characterized by a reduction of the XDH1 transcript, whereas the other group was characterized by an up-regulation of several transcripts including XDH1, glutamine synthetase, alanine aminotransferase, catalase, superoxide dismutase, ornithine decarboxylase, glutamate receptor, and ammonia transporter. Our data demonstrate that XDH1 plays an essential role and that XDH1 has the potential to be used as a metabolic target for Ae.aegypti vector control.-Isoe, J., Petchampai, N., Isoe, Y. E., Co, K., Mazzalupo, S., Scaraffia, P. Y. Xanthine dehydrogenase-1 silencing in Aedes aegypti mosquitoes promotes a blood feeding-induced adulticidal activity.

  19. Reexamining Michaelis-Menten Enzyme Kinetics for Xanthine Oxidase

    ERIC Educational Resources Information Center

    Bassingthwaighte, James B.; Chinn, Tamara M.

    2013-01-01

    Abbreviated expressions for enzyme kinetic expressions, such as the Michaelis-Menten (M-M) equations, are based on the premise that enzyme concentrations are low compared with those of the substrate and product. When one does progress experiments, where the solute is consumed during conversion to form a series of products, the idealized conditions…

  20. Allopurinol use in a New Zealand population: prevalence and adherence.

    PubMed

    Horsburgh, Simon; Norris, Pauline; Becket, Gordon; Arroll, Bruce; Crampton, Peter; Cumming, Jacqueline; Keown, Shirley; Herbison, Peter

    2014-07-01

    Allopurinol is effective for the control of gout and its long-term complications when taken consistently. There is evidence that adherence to allopurinol therapy varies across population groups. This may exacerbate differences in the burden of gout on population groups and needs to be accurately assessed. The aim of this study was to describe the prevalence of allopurinol use in a region of New Zealand using community pharmacy dispensing data and to examine the levels of suboptimal adherence in various population groups. Data from all community pharmacy dispensing databases in a New Zealand region were collected for a year covering 2005/2006 giving a near complete picture of dispensings to area residents. Prevalence of allopurinol use in the region by age, sex, ethnicity and socioeconomic position was calculated. Adherence was assessed using the medication possession ratio (MPR), with a MPR of 0.80 indicative of suboptimal adherence. Multiple logistic regression was used to explore variations in suboptimal adherence across population groups. A total of 953 people received allopurinol in the study year (prevalence 3%). Prevalence was higher in males (6%) than in females (1%) and Māori (5%) than non-Māori (3%). The overall MPR during the study was 0.88, with 161 (22%) of patients using allopurinol having suboptimal adherence. Non-Māori were 54% less likely to have suboptimal allopurinol adherence compared to Māori (95% CI 0.30-0.72, p = 0.001). These findings are consistent with those from other studies nationally and internationally and point to the important role for health professionals in improving patient adherence to an effective gout treatment.

  1. Protein Conformational Gating of Enzymatic Activity in Xanthine Oxidoreductase

    SciTech Connect

    Ishikita, Hiroshi; Eger, Bryan T.; Okamoto, Ken; Nishino, Takeshi; Pai, Emil F.

    2012-05-24

    In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E{sub sq/hq}) is {approx}170 mV, a striking difference. The former greatly prefers NAD{sup +} as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD{sup +}), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 {angstrom} resolution crystal structures for XDH, XO, the NAD{sup +}- and NADH-complexed XDH, E{sub sq/hq} were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E{sub sq/hq} difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD{sup +} binding at XDH. Instead, the positive charge of the NAD{sup +} ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD{sup +} molecule all contribute to altering E{sub sq/hq} upon NAD{sup +} binding to XDH.

  2. Allopurinol hypersensitivity: investigating the cause and minimizing the risk.

    PubMed

    Stamp, Lisa K; Day, Richard O; Yun, James

    2016-04-01

    Allopurinol is the most commonly prescribed urate-lowering therapy for the management of gout. Serious adverse reactions associated with allopurinol, while rare, are feared owing to the high mortality. Such reactions can manifest as a rash combined with eosinophilia, leukocytosis, fever, hepatitis and progressive kidney failure. Risk factors for allopurinol-related severe adverse reactions include the recent introduction of allopurinol, the presence of the HLA-B(*)58:01 allele, and factors that influence the drug concentration. The interactions between allopurinol, its metabolite, oxypurinol, and T cells have been studied, and evidence exists that the presence of the HLA-B(*)58:01 allele and a high concentration of oxypurinol function synergistically to increase the number of potentially immunogenic-peptide-oxypurinol-HLA-B(*)58:01 complexes on the cell surface, thereby increasing the risk of T-cell sensitization and a subsequent adverse reaction. This Review will discuss the above issues and place this in the clinical context of reducing the risk of serious adverse reactions.

  3. Acute renal and hepatic failure associated with allopurinol treatment.

    PubMed

    Fagugli, R M; Gentile, G; Ferrara, G; Brugnano, R

    2008-12-01

    Hyperuricemia is present in about 5% of the population, and allopurinol is frequently used to treat it. The use of this drug can be associated with a number of side effects, indicating allergic reactions, such as skin rash, reversible after its withdrawal. In some cases more severe hypersensitivity reactions may be seen, such as erythema multiforme exudativum, or Steven-Johnson Syndrome (SJS). Reversible clinical hepatotoxicity, as well as acute renal failure, may also develop after allopurinol therapy. We describe here the case of a 74-year-old woman with chronic renal failure who was admitted to hospital after 1 week of sore throat and fever, presenting mucous membrane lesions, widespread blistering of the skin, evolving to flaccid vesicles and bullae, and extensive epidermal detachment associated with acute renal failure and cholestatic jaundice. A diagnosis of allopurinol-induced toxic epidermal necrolysis (TEN) was established. Allopurinol was discontinued, and intensive care management was required: the patient was successfully treated by using intravenous immunoglobulin (IVIg), standard hemodialysis, and albumin dialysis (Molecular Adsorbents Recirculating System - MARS, Teraklin AG, Rostock, Germany). Allopurinol-induced TEN is extremely rare, however, the survival rate is extremely low. Clinicians should be aware of this potentially severe adverse effect. This report emphasizes the importance of an aggressive pharmacological and dialysis treatment in the case of TEN.

  4. Kinetic mechanism of chicken liver xanthine dehydrogenase.

    PubMed Central

    Bruguera, P; Lopez-Cabrera, A; Canela, E I

    1988-01-01

    The kinetic behaviour of chicken-liver xanthine dehydrogenase (xanthine/NAD+ oxidoreductase; EC 1.2.1.37) has been studied. Steady-state results, obtained from a wide range of concentrations of substrates and products, were fitted by rational functions of degree 1:1, 1:2, 2:2 and 3:3 with respect to substrates, and 0:1, 1:1, 0:2 and 1:2 with regard to products, using a non-linear regression program which guarantees the fit. The goodness of fit was improved using a computer program that combines model discrimination, parameter refinement and sequential experimental design. The AIC and F tests were also used for model discrimination. For comparative purposes, the xanthine/oxygen oxidoreductase reaction was also studied. From the functions which give the maximum improvement, the complete rate equation was deduced. The significance of the terms was stated by the above methods. It was concluded that xanthine dehydrogenase requires a minimum mechanism of degree 1:1 for xanthine, 2:2 for NAD+, 1:1 for uric acid and 1:2 for NADH in the xanthine/NAD+ oxidoreductase reaction. These are the minimum degrees required but a rate equation of higher degree is not excluded. PMID:3422556

  5. Allopurinol in the treatment of acquired reactive perforating collagenosis*

    PubMed Central

    Tilz, Hemma; Becker, Jürgen Christian; Legat, Franz; Schettini, Antonio Pedro Mendes; Inzinger, Martin; Massone, Cesare

    2013-01-01

    Acquired reactive perforating collagenosis is a perforating dermatosis usually associated with different systemic diseases, mainly diabetes mellitus and/or chronic renal insufficiency. Different therapies have been tried but treatment is not standardized yet and remains a challenge. In the last few years, allopurinol has been reported as a good therapeutic option for acquired reactive perforating collagenosis. We describe the case of a 73-year-old man affected by acquired reactive perforating collagenosis associated with diabetes type 1 and chronic renal failure with secondary hyperparathyroidism. The patient was successfully treated with allopurinol 100mg once/day p.o.. PMID:23539010

  6. Case report of extensive metabolism by aldehyde oxidase in humans: pharmacokinetics and metabolite profile of FK3453 in rats, dogs, and humans.

    PubMed

    Akabane, Takafumi; Tanaka, Kohichiro; Irie, Megumi; Terashita, Shigeyuki; Teramura, Toshio

    2011-05-01

    We describe the preclinical and clinical pharmacokinetic profiles of FK3453 [6-(2-amino-4-phenylpyrimidin-5-yl)-2-isopropylpyridazin-3(2H)-one] and the mechanism responsible for poor oral exposure of FK3453 in humans. FK3453 showed favourable profiles in preclinical pharmacokinetic studies, including satisfactory absolute bioavailability and total body clearance in animals (30.5%-41.4%, 54.7%-68.2%, and 71.3%-93.4% and 10.8-17.6, 1.9-17.1, and 5.0 mL/min/kg in male rats, female rats, and dogs, respectively), and good metabolic stability in liver microsomes (42.3, 14.5, and 1.1 mL/min/kg in male rats, dogs, and humans, respectively). However, despite these promising preclinical findings, plasma concentrations of FK3453 in humans were extremely low, with the oxidative metabolite of the aminopyrimidine moiety (M4) identified as a major metabolite. Given that aldehyde oxidase (AO) and xanthine oxidase (XO) were presumed to be the enzymes responsible for M4 formation, we investigated the mechanism of M4 formation using human liver subcellular fractions. M4 was detected in the incubation mixture with S9 and cytosol but not with microsomes, and M4 formation was inhibited by AO inhibitors (menadione, isovanillin) but not by cytochrome P-450 inhibitor (1-aminobenzotiazole) or XO inhibitor (allopurinol). These results suggest M4 formation is catalyzed by AO, and therefore, its poor exposure in humans was attributed to extensive AO metabolism.

  7. Construction of novel xanthine biosensor by using polymeric mediator/MWCNT nanocomposite layer for fish freshness detection.

    PubMed

    Dervisevic, Muamer; Custiuc, Esma; Çevik, Emre; Şenel, Mehmet

    2015-08-15

    A novel nanocomposite host matrix for enzyme immobilization of xanthine oxidase was developed by incorporating MWCNT in poly(GMA-co-VFc) copolymer film. In the food industry fish is a product with a very low commercial life, and a high variability as well elevated level of xanthine is an important biomarker as a sign of spoilage. The fabricated process was characterized by scanning electron microscopy (SEM), and the electrochemical behaviors of the biosensor were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The prepared enzyme electrodes exhibited maximum response at pH 7.0 and 45°C +0.35 V and reached 95% of steady-state current in about ∼ 4 s and its sensitivity was 16 mAM(-1). Linear ranges (2-28 μM, 28-46 and 46-86 μM), analytical performance and a low detection limit 0.12 μM obtained from the xanthine biosensor gives reliable results in measuring xanthine concentration in the fish meat. All the results indicating that the resulting biosensor exhibited a good response to xanthine that was related to the addition of MWCNT in the polymeric mediator film which played an important role in the biosensor performance. In addition, the biosensor exhibited high good storage stability and satisfactory anti-interference ability.

  8. Xanthine oxidoreductase is required for genotoxic stress-induced NKG2D ligand expression and gemcitabine-mediated antitumor activity

    PubMed Central

    Xu, Xiulong; Rao, Geetha; Li, Yi

    2016-01-01

    MICA/B (the major histocompatibility antigen-related chain A and B) and Rae I are stress-inducible ligands for the immune-receptor NKG2D. Mechanisms by which genotoxic stress and DNA damage induce the expression of NKG2D ligands remain incompletely understood. Here, we report that inhibition of xanthine oxidoreductase (XOR) activity by allopurinol or inhibition of XOR expression by gene knockdown abrogated genotoxic stress-induced expression of MICA/B and Rae I in three tumor cell lines. XOR knockdown also blocked gemcitabine-mediated antitumor activity in an orthotopic syngeneic mouse model of breast cancer. As a rate-limiting enzyme in the purine catabolic pathway, XOR generates two end-products, uric acid and reactive oxygen species (ROS). ROS scavenging had an insignificant effect on genotoxic drug-induced MICA/B expression but modestly inhibited radiation-induced MICA/B expression. Exogenous uric acid (in the form of monosodium urate) induced MICA/B expression by activating the MAP kinase pathway. Allopurinol blocked genotoxic stress-induced MAP kinase activation. Our study provides mechanistic insights into genotoxic stress-induced activation of the MAP kinase pathway and suggests that XOR is required for genotoxic stress-induced NKG2D ligand expression and gemcitabine-mediated antitumor activity. PMID:27494876

  9. A case of allopurinol-induced fixed drug eruption confirmed with a lymphocyte transformation test.

    PubMed

    Kim, Min-Hye; Shim, Eun-Jin; Jung, Jae-Woo; Sohn, Seong-Wook; Kang, Hye-Ryun

    2012-09-01

    Allopurinol is one of the causative drugs that induce fixed drug eruption (FDE). The lymphocyte transformation test (LTT) is a safe and reliable diagnostic procedure for drug allergy, but is reported to be rarely positive in patients with FDE. In the current case, we performed an LTT and successfully confirmed allopurinol as the offending drug. This case report suggests that an LTT should be an optional diagnostic tool for FDE or delayed reaction due to allopurinol.

  10. Xanthine biosensor based on the direct oxidation of xanthine at an electrogenerated oligomer film.

    PubMed

    Mu, Shaolin; Shi, Qiaofang

    2013-09-15

    Poly(o-aminophenol-co-pyrogallol) (PAP) was first synthesized via the electrochemical copolymerization of o-aminophenol and pyrogallol in the acidic solution, using a reduced graphene oxide/glassy carbon (RGO/GC) electrode as a working electrode. Reduced graphene oxide played an important role in increasing PAP amount deposited on the RGO/GC electrode compared to that on the bare GC electrode, which is due to that RGO has the large specific surface area. The results from the spectra of IR, (1)H NMR and ESR and the measurement of molecular weight demonstrated that PAP is an oligomer with the free radicals and exhibited good redox activity in a wide pH range from pH<1-9.0 and can effectively catalyze xanthine oxidation due to the presence of the free radicals and the reversible redox groups in the copolymer chain. On the basis of the direct oxidation of xanthine on PAP, the PAP/RGO/GC electrode was used as a xanthine biosensor. The biosensor showed a linear range from 1.0 to 120μM xanthine at pH 6.0 with a correction coefficient of 0.9965 and fast response to xanthine oxidation. The peak potential of xanthine oxidation shifted from 0.814 to 0.668V as pH increased from 5.0 to 7.5.

  11. NADPH Oxidases in Vascular Pathology

    PubMed Central

    Konior, Anna; Schramm, Agata; Czesnikiewicz-Guzik, Marta

    2014-01-01

    Abstract Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of “kindling radicals,” which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814. PMID:24180474

  12. The First Mammalian Aldehyde Oxidase Crystal Structure

    PubMed Central

    Coelho, Catarina; Mahro, Martin; Trincão, José; Carvalho, Alexandra T. P.; Ramos, Maria João; Terao, Mineko; Garattini, Enrico; Leimkühler, Silke; Romão, Maria João

    2012-01-01

    Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity. PMID:23019336

  13. Lesinurad in combination with allopurinol: results of a phase 2, randomised, double-blind study in patients with gout with an inadequate response to allopurinol

    PubMed Central

    Perez-Ruiz, Fernando; Sundy, John S; Miner, Jeffrey N; Cravets, Matthew; Storgard, Chris

    2016-01-01

    Objectives To assess the efficacy and tolerability of lesinurad, an oral selective uric acid reabsorption inhibitor, in combination with allopurinol versus allopurinol alone in patients with gout and an inadequate response to allopurinol. Methods Patients (N=227) with an inadequate response to allopurinol, defined as serum urate (sUA) ≥6 mg/dL on ≥2 occasions ≥2 weeks apart despite ≥6 weeks of allopurinol, were randomised 2:1 to 4 weeks of double-blind treatment with lesinurad (200, 400 or 600 mg/day) or matching placebo in combination with their prestudy allopurinol dose (200–600 mg/day). Colchicine prophylaxis for gout flares was required. The primary end point was percent reduction from baseline sUA levels at 4 weeks. A pharmacokinetic substudy was also conducted. Safety was assessed throughout. Results Patients (n=208) received ≥1 dose of blinded medication. Lesinurad 200, 400 and 600 mg in combination with allopurinol produced significant mean percent reductions from baseline sUA of 16%, 22% and 30%, respectively, versus a mean 3% increase with placebo (p<0.0001, all doses vs placebo). Similar results were observed in patients with mild or moderate renal insufficiency (estimated creatinine clearance 30 to <90 mL/min). The incidence of ≥1 treatment-emergent adverse event was 46%, 48% and 54% with lesinurad 200, 400 and 600 mg, respectively, and 46% with placebo (most frequent, gout flares, arthralgia, headache and nasopharyngitis), with no deaths or serious adverse events. Conclusions Lesinurad achieves clinically relevant and statistically significant reductions in sUA in combination with allopurinol in patients who warrant additional therapy on allopurinol alone. Trial registration number NCT01001338. PMID:26742777

  14. Xanthine oxidoreductase in atherosclerosis pathogenesis: not only oxidative stress.

    PubMed

    Battelli, Maria Giulia; Polito, Letizia; Bolognesi, Andrea

    2014-12-01

    Endothelial xanthine oxidoreductase (XOR) together with NAD(P)H oxidase and nitric oxide (NO) synthase plays a physiologic role in inflammatory signalling, the regulation of NO production and vascular function. The oxidative stress generated by these enzymes may induce endothelial dysfunction, leading to atherosclerosis, cardiovascular diseases and metabolic syndrome. XOR activity creates both oxidant and anti-oxidant products that are implicated in the development of hypertension, smoking vascular injury, dyslipidemia and diabetes, which are the main risk factors of atherosclerosis. In particular, uric acid may have a protective as well as a detrimental role in vascular alterations, thus justifying the multi-directional effects of XOR inhibition. Moreover, XOR products are associated with cell differentiation, leading to adipogenesis and foam cell formation, as well as to the production of monocyte chemoattractant protein-1 from arterial smooth muscle cells, after proliferation and migration. The role of XOR in adipogenesis is also connected with insulin resistance and obesity, two main features of type 2 diabetes.

  15. A critical reappraisal of allopurinol dosing, safety, and efficacy for hyperuricemia in gout.

    PubMed

    Chao, Jeannie; Terkeltaub, Robert

    2009-04-01

    Allopurinol, the first-line drug for serum urate-lowering therapy in gout, is approved by the US Food and Drug Administration for a dose up to 800 mg/d and is available as a low-cost generic drug. However, the vast majority of allopurinol prescriptions are for doses < or = 300 mg/d, which often fails to adequately treat hyperuricemia in gout. This situation has been promoted by longstanding, non-evidence-based guidelines for allopurinol use calibrated to renal function (and oxypurinol levels) and designed, without proof of efficacy, to avoid allopurinol hypersensitivity syndrome. Severe allopurinol hypersensitivity reactions are not necessarily dose-dependent and do not always correlate with serum oxypurinol levels. Limiting allopurinol dosing to < or = 300 mg/d suboptimally controls hyperuricemia and fails to adequately prevent hypersensitivity reactions. However, the long-term safety of elevating allopurinol dosages in chronic kidney disease requires further study. The emergence of novel urate-lowering therapeutic options, such as febuxostat and uricase, makes timely this review of current allopurinol dosing guidelines, safety, and efficacy in gout hyperuricemia therapy, including patients with chronic kidney disease.

  16. Electronic spectra and structures of some biologically important xanthines

    NASA Astrophysics Data System (ADS)

    Shukla, M. K.; Mishra, P. C.

    1994-08-01

    Electronic absorption and fluorescence spectra of aqueous solutions of xanthine, caffeine, theophylline and theobromine have been studied at different pH. The observed spectra have been interpreted in terms of neutral and ionic forms of the molecules with the help of molecular orbital calculations. At neutral and acidic pH, the spectra can be assigned to the corresponding most stable neutral forms, with the exception that the fluorescence of xanthine at acidic pH appears to originate from the lowest singlet excited state of a cation of the molecule. At alkaline pH, xanthine and theophylline exist mainly as their monoanions. In xanthine and theophylline at alkaline pH, fluorescence originates from the lowest singlet excited state of the corresponding anion. However, in caffeine and theobromine, even at alkaline pH, fluorescence belongs to the neutral species. On the whole, the properties of xanthine are quite different from those of the methyl xanthines.

  17. Formation and Detoxification of Reactive Oxygen Species

    ERIC Educational Resources Information Center

    Kuciel, Radoslawa; Mazurkiewicz, Aleksandra

    2004-01-01

    A model of reactive oxygen species metabolism is proposed as a laboratory exercise for students. The superoxide ion in this model is generated during the reaction of oxidation of xanthine, catalyzed by xanthine oxidase. The effect of catalase, superoxide dismutase, and allopurinol on superoxide ion generation and removal in this system is also…

  18. Low-dose allopurinol plus azathioprine/cyclosporin/prednisolone, a novel immunosuppressive regimen.

    PubMed

    Chocair, P; Duley, J; Simmonds, H A; Cameron, J S; Ianhez, L; Arap, S; Sabbaga, E

    1993-07-10

    Early rejection can still complicate renal transplantation even with cyclosporin. We added low-dose allopurinol (25 mg on alternative days) to "triple" immunosuppression with cyclosporin, prednisolone, and azathioprine for twelve recipients of cadaver renal grafts. The controls were fifteen patients on triple therapy alone. Only one rejection episode occurred among the allopurinol-treated patients, whereas eleven controls had rejections (seven with more than one episode). Allopurinol may be toxic when combined with azathioprine, yet the bone marrow tolerated the new regimen well. As expected, reduction of the azathioprine dose was necessary in the treated group.

  19. Xanthines Studied via Femtosecond Fluorescence Spectroscopy.

    PubMed

    Changenet-Barret, Pascale; Kovács, Lajos; Markovitsi, Dimitra; Gustavsson, Thomas

    2016-12-03

    Xanthines represent a wide class of compounds closely related to the DNA bases adenine and guanine. Ubiquitous in the human body, they are capable of replacing natural bases in double helices and give rise to four-stranded structures. Although the use of their fluorescence for analytical purposes was proposed, their fluorescence properties have not been properly characterized so far. The present paper reports the first fluorescence study of xanthine solutions relying on femtosecond spectroscopy. Initially, we focus on 3-methylxanthine, showing that this compound exhibits non-exponential fluorescence decays with no significant dependence on the emission wavelength. The fluorescence quantum yield (3 × 10(-4)) and average decay time (0.9 ps) are slightly larger than those found for the DNA bases. Subsequently, we compare the dynamical fluorescence properties of seven mono-, di- and tri-methylated derivatives. Both the fluorescence decays and fluorescence anisotropies vary only weakly with the site and the degree of methylation. These findings are in line with theoretical predictions suggesting the involvement of several conical intersections in the relaxation of the lowest singlet excited state.

  20. Allopurinol initiation and change in blood pressure in older adults with hypertension.

    PubMed

    Beattie, Catherine J; Fulton, Rachael L; Higgins, Peter; Padmanabhan, Sandosh; McCallum, Linsay; Walters, Matthew R; Dominiczak, Anna F; Touyz, Rhian M; Dawson, Jesse

    2014-11-01

    Hypertension is a key risk factor for cardiovascular disease, and new treatments are needed. Uric acid reduction lowers blood pressure (BP) in adolescents, suggesting a direct pathophysiological role in the development of hypertension. Whether the same relationship is present in older adults is unknown. We explored change in BP after allopurinol initiation using data from the UK Clinical Practice Research Datalink. Data were extracted for patients with hypertension aged >65 years who were prescribed allopurinol with pretreatment and during treatment BP readings. Data from comparable controls were extracted. The change in BP in patients with stable BP medication was the primary outcome and was compared between groups. Regression analysis was used to adjust for potential confounding factors, and a propensity-matched sample was generated. Three hundred sixty-five patients who received allopurinol and 6678 controls were included. BP fell in the allopurinol group compared with controls (between-group difference in systolic and diastolic BP: 2.1 mm Hg; 95% confidence interval, -0.6 to 4.8; and 1.7 mm Hg; 95% confidence interval, 0.4-3.1, respectively). Allopurinol use was independently associated with a fall in both systolic and diastolic BP on regression analysis (P<0.001). Results were consistent in the propensity-matched sample. There was a trend toward greater fall in BP in the high-dose allopurinol group, but change in BP was not related to baseline uric acid level. Allopurinol use is associated with a small fall in BP in adults. Further studies of the effect of high-dose allopurinol in adults with hypertension are needed.

  1. Enhanced vasodilator activity of nitrite in hypertension: critical role for erythrocytic xanthine oxidoreductase and translational potential.

    PubMed

    Ghosh, Suborno M; Kapil, Vikas; Fuentes-Calvo, Isabel; Bubb, Kristen J; Pearl, Vanessa; Milsom, Alexandra B; Khambata, Rayomand; Maleki-Toyserkani, Sheiva; Yousuf, Mubeen; Benjamin, Nigel; Webb, Andrew J; Caulfield, Mark J; Hobbs, Adrian J; Ahluwalia, Amrita

    2013-05-01

    Elevation of circulating nitrite (NO2(-)) levels causes vasodilatation and lowers blood pressure in healthy volunteers. Whether these effects and the underpinning mechanisms persist in hypertension is unknown. Therefore, we investigated the consequences of systemic nitrite elevation in spontaneously hypertensive rats and conducted proof-of-principle studies in patients. Nitrite caused dose-dependent blood pressure-lowering that was profoundly enhanced in spontaneously hypertensive rats versus normotensive Wistar Kyoto controls. This effect was virtually abolished by the xanthine oxidoreductase (XOR) inhibitor, allopurinol, and associated with hypertension-specific XOR-dependent nitrite reductase activity localized to the erythrocyte but not the blood vessel wall. To determine whether these pathways translate to human hypertension, we investigated the effects of elevation of circulating nitrite levels in 15 drug naïve grade 1 hypertensives. To elevate nitrite, we used a dose of dietary nitrate (≈ 3.5 mmol) that elevated nitrite levels ≈ 1.5-fold (P<0.01); a rise shown previously to exert no significant blood pressure-lowering effects in normotensives. This dose caused substantial reductions in systolic (≈ 12 mm Hg) and diastolic blood pressures (P<0.001) and pulse wave velocity (P<0.05); effects associated with elevations in erythrocytic XOR expression and XOR-dependent nitrite reductase activity. Our observations demonstrate the improved efficacy of inorganic nitrate and nitrite in hypertension as a consequence of increased erythrocytic XOR nitrite reductase activity and support the concept of dietary nitrate supplementation as an effective, but simple and inexpensive, antihypertensive strategy.

  2. Immunological comparison of sulfite oxidase

    SciTech Connect

    Pollock, V.; Barber, M.J. )

    1991-03-11

    Polyclonal antibodies (rabbit), elicited against FPLC-purified chicken and rat liver sulfite oxidase (SO), have been examined for inhibition and binding to purified chicken (C), rat (R), bovine (B), alligator (A) and shark (S) liver enzymes. Anti-CSO IgG cross-reacted with all five enzymes, with varying affinities, in the order CSO=ASO{gt}RSO{gt}BSO{gt}SSO. Anti-ROS IgG also cross-reacted with all five enzymes in the order RSO{gt}CSO=ASO{gt}BSO{gt}SSO. Anti-CSO IgG inhibited sulfite:cyt. c reductase (S:CR), sulfite:ferricyanide reductase (S:FR) and sulfite:dichlorophenolindophenol reductase (S:DR) activities of CSO to different extents (S:CR{gt}S:FR=S:DR). Similar differential inhibition was found for anti-ROS IgG and RSO S:CR, S:FR and S:DR activities. Anti-CSO IgG inhibited S:CR activities in the order CSO=ASO{much gt}SSO{gt}BSO. RSO was uninhibited. For anti-RSO IgG the inhibition order was RSO{gt}SSO{gt}BSO{gt}ASO. CSO was uninhibited. Anti-CSO and RSO IgGs partially inhibited Chlorella nitrate reductase (NR). Minor cross-reactivity was found for xanthine oxidase. Common antigenic determinants for all five SO's and NR are indicated.

  3. 5-fluorocytosine susceptibility of pathogenic fungi in the presence of allopurinol: potential for improving the therapeutic index of 5-fluorocytosine.

    PubMed Central

    Kerkering, T M; Schwartz, P M; Espinel-Ingroff, A; Turek, P J; Diasio, R B

    1983-01-01

    The minimal inhibitory concentration of 5-fluorocytosine in 18 pathogenic fungal isolates was not altered by either allopurinol (100 microM) or oxypurinol (100 microM). Since allopurinol at this level clinically has been demonstrated to interfere with 5-fluorouracil anabolism, thereby reducing toxicity owing to 5-fluorouracil, allopurinol may be useful in counteracting the 5-fluorouracil-induced myelotoxicity observed in patients being treated with 5-fluorocytosine without interfering with the antifungal activity of 5-fluorocytosine. PMID:6639003

  4. Effects of allopurinol on plasma uric acid levels in normouricaemic and hyperuricaemic green iguanas (Iguana iguana).

    PubMed

    Hernandez-Divers, S J; Martinez-Jimenez, D; Bush, S; Latimer, K S; Zwart, P; Kroeze, E J B Veldhuis

    2008-01-26

    A two-phase cross-over therapeutic study was performed with 19 green iguanas (Iguana iguana) maintained within a preferred optimum temperature range of 26 to 37 degrees C. During phase 1, they were fed a normal vegetarian diet and medicated orally with either allopurinol or a placebo control once a day for seven days. Uric acid concentrations, total protein, packed-cell volumes (pcv) and bodyweights were recorded from each lizard before and after treatment to determine the effects of allopurinol. In phase 2, after a 10-day washout period, the iguanas were fed a high protein diet to induce hyperuricaemia. Normo- and hyperuricaemic iguanas that received 24.2 (3.2) mg/kg allopurinol had significantly lower mean (sd) uric acid concentrations (100.3 [53.1] micromol/l) than the controls (159.3 [100.3] micromol/l). There were no detectable interactions between the doses of allopurinol or placebo, and the iguanas' diet, weight, pcv or total protein. The allopurinol was well tolerated, and there was no significant clinical, gross or histological evidence of hepatic or renal toxicity in the iguanas that received the drug. However, in the kidneys of the hyperuricaemic iguanas that did not receive allopurinol there were proliferative changes in the glomeruli and degeneration of tubular epithelia. Allopurinol given orally at 25 mg/kg daily is able to reduce plasma uric acid levels by 41 to 45 per cent, and is therefore recommended for the treatment of hyperuricaemia in the green iguana.

  5. Studies on bi-layer osmotic pump tablets of water-insoluble allopurinol with large dose: in vitro and in vivo.

    PubMed

    Wang, Xun; Nie, Shu-fang; Li, Wei; Luan, Lin; Pan, Weisan

    2007-09-01

    Controlled release bi-layer osmotic pump tablets (BOPT) of water-insoluble allopurinol with large dose (150 mg/BOPT) were successfully prepared merely with sodium chloride as osmotic promoting agent and polyethylene oxide (PEO) as suspending agent. Formulations of the two kinds of agents were investigated in order to discuss their effects on the release behavior of BOPT, and then the optimal formulation was evaluated. The pharmacokinetics studies of allopurinol and its active metabolite oxypurinol in two-preparation and two-period crossover design relative to the equivalent dose of commercially common allopurinol tablets were evaluated in six Beagle dogs. And the pharmacokinetics results showed that allopurinol BOPT were able to provide a slow release of allopurinol, and oxypurinol were bioequivalent between allopurinol BOPT and common allopurinol tablets. A good in vitro-in vivo correlation of allopurinol was also proved. In conclusion, water-insoluble drugs with large dose can be designed to BOPT for efficacy and safety use.

  6. Involvement of Xanthine Oxidoreductase-related Oxidative Stress in a Dermatophagoides farinae-induced Asthma Model of NC/Nga Mice.

    PubMed

    Setiawan, Heri; Nagaoka, Kenjiro; Kubo, Masayuki; Fujikura, Yoshihisa; Ogino, Keiki

    2016-06-01

    Oxidative stress is widely known to play a role in asthma. However, the contribution of xanthine oxidoreductase (XOR) as a source of the superoxide anion radical (O2-) in oxidative stress associated with asthma has not yet been examined in detail. Here we investigated pathophysiological changes in XOR in an experimental model of asthma induced by the house dust mite Dermatophagoides farinae (Df). In the lungs of Df-treated mice, the production of O2 - from XOR increased and the nitrite concentrations decreased, whereas the protein expression of XOR remained unchanged. Moreover, the protein expression levels of XOR and the hydrogen peroxide (H2O2) concentrations in bronchoalveolar lavage fluid (BALF) were higher in the Df-treated mice than in saline-treated mice. Immunohistochemically, although XOR was highly localized in the bronchial epithelial cells of the saline-treated mice, immunostaining for XOR was absent in the bronchial epithelium of Df-treated mice. These results suggest that oxidative stress is up-regulated by increases in the conversion of the dehydrogenase form (xanthine dehydrogenase; XDH) of XOR to the oxidase form (xanthine oxidase; XOD).

  7. The possible antianginal effect of allopurinol in vasopressin-induced ischemic model in rats

    PubMed Central

    Al-Zahrani, Yahya A.; Al-Harthi, Sameer E.; Khan, Lateef M.; El-Bassossy, Hani M.; Edris, Sherif M.; A. Sattar, Mai A. Alim

    2015-01-01

    The anti-anginal effects of allopurinol were assessed in experimental model rats of angina and their effects were evaluated with amlodipine. In the vasopressin-induced angina model, oral administration of allopurinol in dose of 10 mg/kg revealed remarkably analogous effects in comparison with amlodipine such as dose-dependent suppression of vasopressin-triggered time, duration and severity of ST depression. In addition, allopurinol produced dose dependent suppression of plasma Malondialdehyde (MDA) level, systolic blood pressure, cardiac contractility and cardiac oxygen consumption; while in contrast, amlodipine minimally suppressed the elevation of plasma MDA level. Endothelial NO synthase (eNOS) expression, serum nitrate were strikingly increased, however lipid profile was significantly reduced. Seemingly, allopurinol was found to be more potent than amlodipine – a calcium channel antagonist. To conclude, it was explicitly observed and verified that on the ischemic electrocardiography (ECG) changes in angina pectoris model in rats, allopurinol exerts a significant protective effects, reminiscent of enhancement of vascular oxidative stress, function of endothelial cells, improved coronary blood flow in addition to the potential enhancement in myocardial stress. Moreover, our findings were in conformity with several human studies. PMID:26594114

  8. Accelerated tissue aging and increased oxidative stress in broiler chickens fed allopurinol.

    PubMed

    Klandorf, H; Rathore, D S; Iqbal, M; Shi, X; Van Dyke, K

    2001-06-01

    Uric acid has been hypothesized as being one of the more important antioxidants in limiting the accumulation of glycosylated endproducts in birds. Study 1 was designed to quantitatively manipulate the plasma concentrations of uric acid using hemin and allopurinol while study 2 determined their effects on skin pentosidine, the shear force value of Pectoralis major muscle, plasma glucose, body weight and chemiluminescence monitored oxidative stress in broiler chickens. Hemin was hypothesized to raise uric acid concentrations thereby lowering oxidative stress whereas allopurinol was hypothesized to lower uric acid concentrations and raise measures of oxidative stress. In study 1 feeding allopurinol (10 mg/kg body weight) to 8-week-old broiler chicks (n=50) for 10 days decreased plasma uric acid by 57%. However, hemin (10 mg/kg body weight) increased uric acid concentrations 20%. In study 2, 12-week-old broiler chicks (n=90) were randomly assigned to either an ad libitum (AL) diet or a diet restricted (DR) group. Each group was further divided into three treatments (control, allopurinol or hemin fed). Unexpectedly, hemin did not significantly effect uric acid concentrations but increased (P<0.05) measures of chemiluminescence dependent oxidative stress in both the DR and AL birds probably due to the ability of iron to generate oxygen radicals. Allopurinol lowered concentrations of uric acid and increased (P<0.05) the oxidative stress in the AL birds at week 22, reduced (P<0.05) body weight in both the AL and DR fed birds at 16 and 22 weeks of age, and markedly increased (P<0.001) shear force values of the pectoralis major muscle. Skin pentosidine levels increased (P<0.05) in AL birds fed allopurinol or hemin fed birds, but not in the diet restricted birds at 22 weeks. The significance of these studies is that concentrations of plasma uric acid can be related to measures of oxidative stress, which can be linked to tissue aging.

  9. [The uric acid-lowering action of benzbromarone effervescent granules and allopurinol. Comparative studies (author's transl)].

    PubMed

    Mertz, D P

    1978-10-20

    The daily administration of 25 mg effervescent granules is about equipotent with a daily dose of 200 mg allopurinol, giving due attention to the caution necessary for uricosuric therapy. The daily administration of 50 mg benzbromarone effervescent granules has a slight but significantly greater uric acid-lowering effect than treatment with 300 mg allopurinol per day. The tolerance of benzbromarone in the active form named was good in all cases. Side effects were not observed. Since benzbromarone as effervescent granules is taken with plenty of fluid this takes care of an increased fluid intake and introduces a motivation for doing so.

  10. Chronic allopurinol treatment during the last trimester of pregnancy in sows: effects on low and normal birth weight offspring.

    PubMed

    Gieling, Elise T; Antonides, Alexandra; Fink-Gremmels, Johanna; Ter Haar, Kim; Kuller, Wikke I; Meijer, Ellen; Nordquist, Rebecca E; Stouten, Jacomijn M; Zeinstra, Elly; van der Staay, Franz Josef

    2014-01-01

    Low-birth-weight (LBW) children are born with several risk factors for disease, morbidity and neonatal mortality, even if carried to term. Placental insufficiency leading to hypoxemia and reduced nutritional supply is the main cause for LBW. Brain damage and poor neurological outcome can be the consequence. LBW after being carried to term gives better chances for survival, but these children are still at risk for poor health and the development of cognitive impairments. Preventive therapies are not yet available. We studied the risk/efficacy of chronic prenatal treatment with the anti-oxidative drug allopurinol, as putative preventive treatment in piglets. LBW piglets served as a natural model for LBW. A cognitive holeboard test was applied to study the learning and memory abilities of these allopurinol treated piglets after weaning. Preliminary analysis of the plasma concentrations in sows and their piglets suggested that a daily dose of 15 mg.kg(-1) resulted in effective plasma concentration of allopurinol in piglets. No adverse effects of chronic allopurinol treatment were found on farrowing, birth weight, open field behavior, learning abilities, relative brain, hippocampus and spleen weights. LBW piglets showed increased anxiety levels in an open field test, but cognitive performance was not affected by allopurinol treatment. LBW animals treated with allopurinol showed the largest postnatal compensatory body weight gain. In contrast to a previous study, no differences in learning abilities were found between LBW and normal-birth-weight piglets. This discrepancy might be attributable to experimental differences. Our results indicate that chronic prenatal allopurinol treatment during the third trimester of pregnancy is safe, as no adverse side effects were observed. Compensatory weight gain of treated piglets is a positive indication for the chronic prenatal use of allopurinol in these animals. Further studies are needed to assess the possible preventive effects

  11. Allopurinol Therapy in Gout Patients Does Not Associate with Beneficial Cardiovascular Outcomes: A Population-Based Matched-Cohort Study

    PubMed Central

    Kok, Victor C.; Horng, Jorng-Tzong; Chang, Wan-Shan; Hong, Ya-Fang; Chang, Tzu-Hao

    2014-01-01

    Introduction Previous studies have shown an association between gout and/or hyperuricemia and a subsequent increase in cardiovascular disease (CVD) outcomes. Allopurinol reduces vascular oxidative stress, ameliorates inflammatory state, improves endothelial function, and prevents atherosclerosis progression. Accordingly, we tested the hypothesis that a positive association between allopurinol therapy in gout patients and future cardiovascular outcomes is present using a population-based matched-cohort study design. Methods Patients aged ≥40 years with newly diagnosed gout having no pre-existing severe form of CVD were separated into allopurinol (n = 2483) and non-allopurinol (n = 2483) groups after matching for age, gender, index date, diabetes mellitus, hypertension, hyperlipidemia, and atrial fibrillation. The two groups were also balanced in terms of uric acid nephrolithiasis, acute kidney injury, hepatitis, and Charlson comorbidity index. Results With a median follow-up time of 5.25 years, the allopurinol group had a modest increase in cardiovascular risk [relative risk, 1.20; 95% confidence interval (CI), 1.08–1.34]. A Cox proportional hazard model adjusted for chronic kidney disease, uremia, and gastric ulcer gave a hazard ratio (HR) for cardiovascular outcomes of 1.25 (95% CI, 1.10–1.41) in gout patients receiving allopurinol compared with the non-allopurinol group. In further analysis of patients receiving urate-lowering therapy, the uricosuric agent group (n = 1713) had an adjusted HR of 0.83 (0.73–0.95) for cardiovascular events compared with the allopurinol group. Conclusions The current population-based matched-cohort study did not support the association between allopurinol therapy in gout patients with normal risk for cardiovascular sequels and beneficial future cardiovascular outcomes. Several important risk factors for cardiovascular disease, such as smoking, alcohol consumption, body mass index, blood pressure were not

  12. Role of xanthine oxidoreductase in the anti-thrombotic effects of nitrite in rats in vivo.

    PubMed

    Kramkowski, K; Leszczynska, A; Przyborowski, K; Kaminski, T; Rykaczewska, U; Sitek, B; Zakrzewska, A; Proniewski, B; Smolenski, R T; Chabielska, E; Buczko, W; Chlopicki, S

    2016-01-01

    The mechanisms underlying nitrite-induced effects on thrombosis and hemostasis in vivo are not clear. The goal of the work described here was to investigate the role of xanthine oxidoreductase (XOR) in the anti-platelet and anti-thrombotic activities of nitrite in rats in vivo. Arterial thrombosis was induced electrically in rats with renovascular hypertension by partial ligation of the left renal artery. Sodium nitrite (NaNO2, 0.17 mmol/kg twice daily for 3 days, p.o) was administered with or without one of the XOR-inhibitors: allopurinol (ALLO) and febuxostat (FEB) (100 and 5 mg/kg, p.o., for 3 days). Nitrite treatment (0.17 mmol/kg), which was associated with a significant increase in NOHb, nitrite/nitrate plasma concentration, resulted in a substantial decrease in thrombus weight (TW) (0.48 ± 0.03 mg vs. vehicle [VEH] 0.88 ± 0.08 mg, p < 0.001) without a significant hypotensive effect. The anti-thrombotic effect of nitrite was partially reversed by FEB (TW = 0.63 ± 0.06 mg, p < 0.05 vs. nitrites), but not by ALLO (TW = 0.43 ± 0.02 mg). In turn, profound anti-platelet effect of nitrite measured ex vivo using collagen-induced whole-blood platelet aggregation (70.5 ± 7.1% vs. VEH 100 ± 4.5%, p < 0.05) and dynamic thromboxaneB2 generation was fully reversed by both XOR-inhibitors. In addition, nitrite decreased plasminogen activator inhibitor-1 concentration (0.47 ± 0.13 ng/ml vs. VEH 0.62 ± 0.04 ng/ml, p < 0.05) and FEB/ALLO reversed this effect. In vitro the anti-platelet effect of nitrite (1 mM) was reversed by FEB (0.1 mM) under hypoxia (0.5%O2) and normoxia (20%O2). Nitrite treatment had no effect on coagulation parameters. In conclusion, the nitrite-induced anti-platelet effect in rats in vivo is mediated by XOR, but XOR does not fully account for the anti-thrombotic effects of nitrite.

  13. Xanthine urolithiasis in a cat: a case report and evaluation of a candidate gene for xanthine dehydrogenase.

    PubMed

    Tsuchida, Shuichi; Kagi, Akiko; Koyama, Hidekazu; Tagawa, Masahiro

    2007-12-01

    Xanthine urolithiasis was found in a 4-year-old spayed female Himalayan cat with a 10-month history of intermittent haematuria and dysuria. Ultrasonographs indicated the existence of several calculi in the bladder that were undetectable by survey radiographic examination. Four bladder stones were removed by cystotomy. The stones were spherical brownish-yellow and their surface was smooth and glossy. Quantitative mineral analysis showed a representative urolith to be composed of more than 95% xanthine. Ultrasonographic examination of the bladder 4.5 months postoperatively indicated the recurrence of urolithiasis. Analysis of purine concentration in urine and blood showed that the cat excreted excessive amounts of xanthine. In order to test the hypothesis that xanthinuria was caused by a homozygote of the inherited mutant allele of a gene responsible for deficiency of enzyme activity in purine degradation pathway, the allele composition of xanthine dehydrogenase (XDH) gene (one of the candidate genes for hereditary xanthinuria) was evaluated. The cat with xanthinuria was a heterozygote of the polymorphism. A single nucleotide polymorphism analysis of the cat XDH gene strongly indicated that the XDH gene of the patient cat was composed of two kinds of alleles and ruled out the hypothesis that the cat inherited the same recessive XDH allele suggesting no activity from a single ancestor.

  14. Xanthine derivatives in the heart: blessed or cursed?

    PubMed

    Szentmiklósi, A J; Cseppentō, A; Gesztelyi, R; Zsuga, J; Körtvély, A; Harmati, G; Nánási, P P

    2011-01-01

    Methylxanthines, such as theophylline, have been used to treat cardiorespiratory disorders, whereas caffeine is the most widely consumed psychoactive agent in various soft drinks. Because of the worldwide use of these drugs and the recently synthesized xanthine derivatives, an intensive research on the cardiac actions of these substances is under progress. This review focuses on the molecular mechanisms involved in the actions of xanthine derivatives with special reference to their adenosine receptor antagonistic properties. The main basic and human studies on the action of xanthines on impulse initiation and conduction, as well as the electrophysiological and mechanical activity of the working myocardium will be overviewed. The potential beneficial and harmful actions of the methylxanthines will be discussed in light of the recent experimental and clinical findings. The pharmacological features and clinical observations with adenosine receptor subtype-specific xanthine antagonists are also the subject of this paper. Based on the adenosine receptor-antagonistic activity of these compounds, it can be raised that xanthine derivatives might inhibit the cardioprotective action of endogenous adenosine on various subtypes (A(1), A(2A), A(2B) and A(3)) of adenosine receptors. Adenosine is an important endogenous substance with crucial role in the regulation of cardiac function under physiological and pathological conditions (preconditioning, postconditioning, ischemia/reperfusion injury). Recent clinical studies show that acute administration of caffeine or theophylline can inhibit various types of preconditioning in human subjects. There are no human studies, however, for the cardiovascular actions of long-term administration of these drugs. Upregulation of adenosine receptors and increased effectiveness of adenosine receptor-related cardiovascular functions have been observed after long-lasting treatment with methylxanthines. In addition, there are data indicating that

  15. Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X-ray Absorption Spectroscopy.

    PubMed

    Reschke, Stefan; Mebs, Stefan; Sigfridsson-Clauss, Kajsa G V; Kositzki, Ramona; Leimkühler, Silke; Haumann, Michael

    2017-02-20

    Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5-10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo(VI) ion with each one MPT, Mo═O, Mo-O(-), and Mo═S ligand, and a weak Mo-O(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (pK ∼ 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (pK ∼ 8.8) is an inhibitor at alkaline pH. A similar acidic pK for the WT and Q179A variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo-OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation.

  16. A Randomized Controlled Trial of Allopurinol in Patients With Peripheral Arterial Disease

    PubMed Central

    Robertson, Alan J.; Struthers, Allan D.

    2016-01-01

    Background Patients with peripheral arterial disease (PAD) are limited by intermittent claudication in the distance they can walk. Allopurinol has been shown in coronary arterial disease to prolong exercise before angina occurs, likely by prevention of oxygen wastage in tissues and reduction of harmful oxidative stress. Methods In this study we evaluated whether allopurinol could prolong the time to development of leg pain in participants with PAD. In a double-blind, randomized controlled clinical trial participants were randomized to receive either allopurinol 300 mg twice daily or placebo for 6 months. The primary outcome was change in exercise capacity on treadmill testing at 6 months. Secondary outcomes were 6-minute walking distance, Walking Impairment Questionnaire, SF-36 questionnaire, flow-mediated dilatation, and oxidized low-density lipoprotein. Outcome measures were repeated midstudy and at the end of study. The mean age of the 50 participants was 68.4 ± 1.2 years with 39 of 50 (78%) male. Results Five participants withdrew during the study (2 active, 3 placebo). There was a significant reduction in uric acid levels in those who received active treatment of 52.1% (P < 0.001), but no significant change in either the pain-free or the maximum walking distance. Other measures of exercise capacity, blood vessel function, and the participants' own assessment of their health and walking ability also did not change during the course of the study. Conclusions Although allopurinol has been shown to be of benefit in a number of other diseases, in this study there was no evidence of any improvement after treatment in patients with PAD. PMID:26277090

  17. Xanthine nephrolithiasis in a galician blond beef calf.

    PubMed

    Miranda, Marta; Rigueira, Lucas; Suárez, María L; Carbajales, Paloma; Moure, Pablo; Fidalgo, Luis E; Failde, Daniel; Vázquez, Sonia

    2010-07-01

    A six-month-old female Galician Blond beef calf presented signs of apathy, anorexia and weight loss. The analysis of a blood sample confirmed renal failure. Bilateral nephrolithiasis was diagnosed at necropsy. Quantitative analysis revealed the nephroliths to be composed of 100 per cent xanthine. In cattle, xanthinuria has only been described in the Japanese Black breed, but never before in other breeds. Clinical history suggested a naturally occurring xanthinuria.

  18. Selective bronchodilators from 1-(5'-oxohexyl)xanthines.

    PubMed

    Miyamoto, K I; Sakai, R; Yamamoto, Y; Konno, K; Sanae, F; Hasegawa, T; Takagi, K

    1992-11-01

    A series of twenty one 1-(5'-oxohexyl)xanthines substituted with alkyl chains at the N3 and N7 positions of the xanthine nucleus were prepared and their relaxant activity in guinea-pig isolated tracheal muscle and positive chronotropic activity in isolated right atrium of guinea-pig were compared. The tracheal relaxant activities were markedly increased with alkyl chain length at the N3 position, but decreased by the N7 alkylation. The positive chronotropic activities in the right atrium were increased by introduction of an n-propyl group at the N3 position but decreased by substitution of longer alkyl chains, and the action on the heart was diminished by N7 substitution. The activities of compounds on cAMP-phosphodiesterase (PDE) and binding of [3H]8-cyclopentyl-1,3-dipropylxanthine were measured in the homogenate of tracheal muscle and the membrane preparation of cerebral cortex, respectively. No relationship among tracheal muscle relaxant activity, cAMP-PDE inhibitory activity and adenosine antagonism of these xanthines was observed, and other action mechanisms should be considered for their relaxant activities. This study indicated that N3 alkylation is important for the selectivity for tracheal muscle, while the introduction of long alkyl chains such as n-butyl and n-pentyl groups at the N3 and N7 positions diminished the potency for the right atrium in guinea-pigs. 3-n-Pentyl- and 7-methyl-3-n-pentyl-1-(5'-oxohexyl)xanthines showed much higher bronchoselectivity than oxpentifylline and theophylline.

  19. Mechanism of allopurinol-mediated increase in enzyme activity in man

    PubMed Central

    Beardmore, Thomas D.; Cashman, Jay S.; Kelley, William N.

    1972-01-01

    Allopurinol therapy in man interferes with pyrimidine biosynthesis de novo by inhibition of one or both of the two enzymes, orotate phosphoribosyltransferase (OPRT) and orotidylic decarboxylase (ODC), responsible for the conversion of orotic acid to uridine-5′-monophosphate. Inhibition of this pathway in vivo is followed in 1-3 wk by an increase in the activity of both of these enzymes in erythrocytes and of ODC in circulating leukocytes. This drug-mediated increase in enzyme activity in erythrocytes could not be attributed to enzyme stabilization or induction in vivo but appeared to be due to enzyme “activation.” “Activation” of the OPRT enzyme was directly demonstrated in erythrocytes studied in vitro after incubation with oxipurinol, and to a lesser extent, with allopurinol. No evidence for “activation” of the ODC enzyme was demonstrated in vitro. This response to allopurinol therapy provides an excellent model for examining the mechanism of increased enzyme activity in response to drug administration. PMID:5032526

  20. Purification and partial characterisation of camel milk xanthine oxidoreductase.

    PubMed

    Baghiani, A; Harrison, R; Benboubetra, M

    2003-12-01

    Xanthine oxidoreductase (XOR) was purified in the presence of dithiothrietol from camel milk with yields of up to 22.2mg/l that were comparable to those obtained from bovine and human milk sources. On SDS-PAGE, the freshly purified camel milk XOR had a protein flavin (A280/A450) ratio of 5.3 +/- 0.4 and appeared homogenous with a single major band of approximately Mr 145.3 KDa. Surprisingly, in all the batches (n = 8) purified camel milk XOR showed no detectable activity towards xanthine or NADH. The molybdenum content of camel XOR was comparable to human and goat milk enzymes. After resulphuration, camel milk XOR gave a specific activity of 1.1 nmol/min/mg and 13.0 nmol/min/mg enzyme towards pterin (fluorimetric assay) and xanthine (spectrophotometric assay) respectively. This activity was markedly lower than that of human, bovine and goat enzymes obtained under the same conditions. These findings suggest that the molybdo-form of camel enzyme is totally under desulpho inactive form. It is possible that camel neonates are equipped with an enzymic system that reactivates XOR in their gut and consequently generates antibacterial reactive oxygen species.

  1. Xanthine Oxidoreductase-Derived Reactive Species: Physiological and Pathological Effects

    PubMed Central

    Bortolotti, Massimo

    2016-01-01

    Xanthine oxidoreductase (XOR) is the enzyme that catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid and is widely distributed among species. In addition to this housekeeping function, mammalian XOR is a physiological source of superoxide ion, hydrogen peroxide, and nitric oxide, which can function as second messengers in the activation of various pathways. This review intends to address the physiological and pathological roles of XOR-derived oxidant molecules. The cytocidal action of XOR products has been claimed in relation to tissue damage, in particular damage induced by hypoxia and ischemia. Attempts to exploit this activity to eliminate unwanted cells via the construction of conjugates have also been reported. Moreover, different aspects of XOR activity related to phlogosis, endothelial activation, leukocyte activation, and vascular tone regulation, have been taken into consideration. Finally, the positive and negative outcomes concerning cancer pathology have been analyzed because XOR products may induce mutagenesis, cell proliferation, and tumor progression, but they are also associated with apoptosis and cell differentiation. In conclusion, XOR activity generates free radicals and other oxidant reactive species that may result in either harmful or beneficial outcomes. PMID:26823950

  2. Effects of alkyl substituents of xanthine on phosphodiesterase isoenzymes.

    PubMed

    Miyamoto, K; Sakai, R; Kurita, M; Ohmae, S; Sanae, F; Sawanishi, H; Hasegawa, T; Takagi, K

    1995-03-01

    The structure-activity relationships of a series of alkylxanthine derivatives were investigated. The partition coefficient of alkylxanthines enlarged with an elongation of the alkyl chain at the 1-, 3-, or 7-position of xanthine. There was a mild correlation between the apparent partition coefficient and the tracheal relaxant activity or the inhibitory activity on phosphodiesterase (PDE) IV isoenzyme, while the tracheal relaxant activity closely correlated with the PDE IV inhibitory activity. Regarding substituents at different positions, the alkylation at the 3-position increased the inhibitory activity on every PDE isoenzyme. The alkylation at the 1-position potentiated the inhibitory activity on PDE IV with the alkyl chain length, but decreased the activities on other PDE isoenzymes. The alkylation at the 7-position was characteristic in its decrease in inhibitory activity on PDE III. These results suggested that the potency of the inhibitory activity of xanthine derivatives on PDE isoenzymes is not dependent simply upon their hydrophobicity but upon change in the affinity for the active sites on PDE isoenzymes by the introduction of the alkyl group at particular positions of the xanthine skeleton.

  3. Effects of alkyl substitutions of xanthine skeleton on bronchodilation.

    PubMed

    Sakai, R; Konno, K; Yamamoto, Y; Sanae, F; Takagi, K; Hasegawa, T; Iwasaki, N; Kakiuchi, M; Kato, H; Miyamoto, K

    1992-10-30

    Structure-activity relationships in a series of 1,3,7-trialkyl-xanthine were studied with guinea pigs. Relaxant actions in the tracheal muscle were increased with alkyl chain length at the 1- and 3-positions of the xanthine skeleton, but decreased by alkylation at the 7-position. Positive chronotropic actions in the right atrium were potentiated with 3-alkyl chain length but tended to decrease with 1-alkylation and diminish by 7-substitution. Consequently, while the 1- and 3-substitutions were equally important for the tracheal smooth muscle relaxation, the substitution at the 1-position was more important than the 3-substitution for bronchoselectivity. The 7-alkylation may be significant to cancel heart stimulation. There were good correlations between the smooth muscle relaxant action and the cyclic AMP-PDE inhibitory activity in 3-substituents and the affinity for adenosine (A1) receptors in 1-, 3-, and 7-substituents. This suggests that not only the cyclic AMP-PDE inhibitory activity but also the adenosine antagonistic activity is important in the bronchodilatory effects of alkylxanthines. Among these xanthine derivatives, 1-butyl-3-propylxanthine and its 7-methylated derivative showed high bronchoselectivity in the in vitro and in vivo experiments compared to theophylline and enprofylline and may be new candidates for bronchodilator.

  4. Purification and characterization of a novel caffeine oxidase from Alcaligenes species.

    PubMed

    Mohapatra, B R; Harris, N; Nordin, R; Mazumder, A

    2006-09-18

    Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.

  5. Allopurinol Reduces the Lethality Associated with Acute Renal Failure Induced by Crotalus durissus terrificus Snake Venom: Comparison with Probenecid

    PubMed Central

    Frezzatti, Rodrigo; Silveira, Paulo Flavio

    2011-01-01

    Background Acute renal failure is one of the most serious complications of envenoming resulting from Crotalus durissus terrificus bites. This study evaluated the relevance of hyperuricemia and oxidative stress and the effects of allopurinol and probenecid in renal dysfunction caused by direct nephrotoxicity of C. d. terrificus venom. Methodology/Principal Findings Hematocrit, protein, renal function and redox status were assessed in mice. High ratio of oxidized/reduced glutathione and hyperuricemia induced by C. d. terrificus venom were ameliorated by both, allopurinol or probenecid, but only allopurinol significantly reduced the lethality caused by C. d. terrificus venom. The effectiveness of probenecid is compromised probably because it promoted hypercreatinemia and hypocreatinuria and worsed the urinary hypo-osmolality in envenomed mice. In turn, the highest effectiveness of allopurinol might be due to its ability to diminish the intracellular formation of uric acid. Conclusions/Significance Data provide consistent evidences linking uric acid with the acute renal failure induced by C. d. terrificus venom, as well as that this envenoming in mice constitutes an attractive animal model suitable for studying the hyperuricemia and that the allopurinol deserves to be clinically evaluated as an approach complementary to anti-snake venom serotherapy. PMID:21909449

  6. [Forms of xanthine oxidoreductase in the tissues of Japanese quail].

    PubMed

    Jankela, J; Baranovská, M; Antalíková, J

    1993-01-01

    The Japanese quail tissues--liver, kidney and pancreas were analysed for the presence of forms of xanthine oxidoreductase utilised cofactors NAD+, molecular oxygen or artificial acceptor--methylene blue, as well as for the validity of correlation between enzymatic activity and diet protein content. Four groups of animals with the experimental diets, the formulae of which are given in Tab. I, and control group with a commercial mash were fed for ten days. For enzyme preparation, the rough purification of cytoplasmic fraction with subsequent dialysis was used. The xanthine oxidoreductase utilised NAD+ (XOR-NAD) was detected in all examined tissues (Fig. 1), whereby the correlation of enzymatic activity with diet protein content was shown only in liver, according our previous findings (Jankela; 1978; Baranovská and Gazo, 1990). The values in liver and pancreas of animals fed a commercial mash were somewhat out of the range of linearity, probably because of the presence of nonprotein substances in mash, which affected the XOR activity in these organs (Jankela, 1992). The XOR utilised O2 (Fig. 2) was only detected in liver and kidney with certain activity in animals fed free protein diet. The percentage of this enzyme form was below 18% of the total activity (Fig. 5). The xanthine oxidoreductase utilised methylene blue (XOR-MM) was detected in liver, kidney and pancreas (Fig. 3). The correlation of enzymatic activity with diet protein content was linear in liver and kidney. The percentage of XOR-MM activity was very high, it amounted to 55% of the total activity (Fig. 4).(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Effect of allopurinol, sulphasalazine, and vitamin C on aspirin induced gastroduodenal injury in human volunteers.

    PubMed Central

    McAlindon, M E; Muller, A F; Filipowicz, B; Hawkey, C J

    1996-01-01

    BACKGROUND--The mechanisms of aspirin induced gastroduodenal injury are not fully understood. Aspirin induces the release of reactive oxygen metabolites in animal models, which may contribute to mucosal injury. AIMS--To investigate the effects of aspirin administered with placebo or antioxidants on gastric mucosal reactive oxygen metabolite release and gastroduodenal injury in human volunteers. SUBJECTS--Fourteen healthy volunteers participated in the study (seven male; mean age 27 years, range 20-40). METHODS--In a double blind, randomised, crossover study, volunteers received aspirin 900 mg twice daily and either placebo, allopurinol 100 mg twice daily, sulphasalazine 1 g twice daily or vitamin C 1 g twice daily for three days. Injury was assessed endoscopically and by quantifying mucosal reactive oxygen metabolite release by measuring chemiluminescence before and after each treatment. The effect on prostanoids was determined by measuring ex vivo antral prostaglandin E2 (PGE2) synthesis and serum thromboxane B2 (TXB2). RESULTS--No drug reduced any parameter of gastric injury but vitamin C reduced duodenal injury assessed by Lanza score (p < 0.005). Chemiluminescence increased after aspirin both with placebo (p < 0.05) and vitamin C (p < 0.05). Post-treatment chemiluminescence was lower in subjects taking allopurinol (p < 0.05) or sulphasalazine (p < 0.005) than in those taking placebo with aspirin. CONCLUSIONS--In this study, aspirin induced gastric injury was associated with reactive oxygen metabolite release. This was reduced by sulphasalazine and allopurinol, although macroscopic injury was not affected. Vitamin C, however, was shown to have a previously unrecognised protective effect against aspirin induced duodenal injury. PMID:8707080

  8. Albumin microparticles as the carriers for allopurinol and applicable for the treatment of ischemic stroke

    NASA Astrophysics Data System (ADS)

    Aganyants, Hovsep Alexandr; Nikohosyan, Gayane; Danielyan, Kristine Edgar

    2016-11-01

    Albumin nanoparticles are already used for the treatment of the cancer. In our current work, it is presented the technique for the preparation of small-size 1- to 5-micron particles coated with the allopurinol. We propose that this combination of the compounds might be useful for the ischemic stroke treatment as the agent preventing formation of the brain edema, reactive oxygen species, and initiation of cells regeneration. Glutaraldehyde was used for the polymerization of albumin. Determination of the particle size was performed by the light as well as phase contrast microscopies and analyzed by Pixcavator 6.0 and Image Tool programs. Modification and establishment of iodine-based method served as the base for quantification of bound with the particles and free allopurinol. As a consequence of the experiments, the best formulation of glutaraldehyde ratio and albumin quantity as well as conditions for the formation of the smallest sized spheroid-shaped particles were found for the further in vivo application.

  9. The Impact of Single Nucleotide Polymorphisms on Human Aldehyde OxidaseS

    PubMed Central

    Hartmann, Tobias; Terao, Mineko; Garattini, Enrico; Teutloff, Christian; Alfaro, Joshua F.; Jones, Jeffrey P.; Leimkühler, Silke

    2012-01-01

    Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95% and a yield of 50 μg/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs. PMID:22279051

  10. Gypenosides Inhibits Xanthine Oxidoreductase and Ameliorates Urate Excretion in Hyperuricemic Rats Induced by High Cholesterol and High Fat Food (Lipid Emulsion)

    PubMed Central

    Pang, Minxia; Fang, Yingying; Chen, Suhong; Zhu, Xuexin; Shan, Chaowen; Su, Jie; Yu, Jingjing; Li, Bo; Yang, Yao; Chen, Bo; Liang, Kailun; Hu, Huiming; Lv, Guiyuan

    2017-01-01

    Background The aim of this study was to study the effects of gypenosides (GPS) on lowering uric acid (UA) levels in hyperuricemic rats induced by lipid emulsion (LE) and the related mechanisms. GPS are natural saponins extracted from Gynostemma pentaphyllum. Material/Methods Forty-eight male SD rats were randomly divided into six groups: normal, model, two positive controls, and two GPS treated groups (two different doses of GPS). The normal group rats were fed a basic diet, and the other rats were orally pretreated with LE. Urine and blood were collected at regular intervals. Full automatic biochemical analyzer was used to detect the concentration levels of serum UA (SUA), serum creatinine (SCr), BUN, and urine UA (UUA), and urine creatinine (UCr) and fractional excretion of UA (FEUA). ELISA kits were used to detect enzymes activities: xanthine oxidase (XOD), adenosime deaminase (ADA), guanine deaminase (GDA), and xanthine dehydrogenase (XDH). Immunohistochemistry was used to observe kidney changes and protein (URAT1, GLUT9, and OAT1) expression levels. RT-PCR was used to detect the relevant mRNA expression levels. Results Treatment with GPS significantly reduced the SUA, prevented abnormal weight loss caused by LE, and improved kidney pathomorphology. Treatment with GPS also decreased the levels of XOD, ADA, and XDH expression, increased the kidney index and FEUA, downregulated URAT1 and GLUT9 expression and upregulated OAT1 expression in the kidney. Conclusions GPS may be an effective treatment for hyperuricemia via a decrease in xanthine oxidoreductase through the XOD/XDH system; and via an increase in urate excretion through regulating URAT1, GLUT9, and OAT1 transporters. PMID:28258276

  11. Increase in thyroid stimulating hormone levels in patients with gout treated with inhibitors of xanthine oxidoreductase.

    PubMed

    Perez-Ruiz, Fernando; Chinchilla, Sandra Pamela; Atxotegi, Joana; Urionagüena, Irati; Herrero-Beites, Ana Maria; Aniel-Quiroga, Maria Angeles

    2015-11-01

    Increase in thyroid stimulating hormone (TSH) levels over the upper normal limit has been reported in a small percentage of patients treated with febuxostat in clinical trials, but a mechanistic explanation is not yet available. In an observational parallel longitudinal cohort study, we evaluated changes in TSH levels in patients with gout at baseline and during urate-lowering treatment with febuxostat. Patients to be started on allopurinol who had a measurement of TSH in the 6-month period prior to baseline evaluation were used for comparison. TSH levels and change in TSH levels at 12-month follow-up were compared between groups. Patients with abnormal TSH levels or previous thyroid disease or on amiodarone were not included for analysis. Eighty-eight patients treated with febuxostat and 87 with allopurinol were available for comparisons. Patients to be treated with febuxostat had higher urate levels and TSH levels, more severe gout, and poorer renal function, but were similar regarding other characteristics. A similar rise in TSH levels was observed in both groups (0.4 and 0.5 µUI/mL for febuxostat and allopurinol, respectively); at 12-mo, 7/88 (7.9 %) of patients on febuxostat and 4/87 (3.4 %) of patients on allopurinol showed TSH levels over 0.5 µUI/mL. Doses prescribed (corrected for estimated glomerular filtration rate in the case if patients on allopurinol) and baseline TSH levels were determinants of TSH levels at 12-month follow-up. No impact on free T4 (fT4) levels was observed. Febuxostat, but also allopurinol, increased TSH levels in a dose-dependent way, thus suggesting rather a class effect than a drug effect, but with no apparent impact on either clinical or fT4 levels.

  12. Allopurinol reduces oxidative stress in the ovine fetal cardiovascular system after repeated episodes of ischemia-reperfusion.

    PubMed

    Derks, Jan B; Oudijk, Martijn A; Torrance, Helen L; Rademaker, Carin M A; Benders, Manon J; Rosen, Karl G; Cindrova-Davies, Tereza; Thakor, Avnesh S; Visser, Gerard H A; Burton, Graham J; van Bel, Frank; Giussani, Dino A

    2010-11-01

    In complicated labor, neonatal outcome may depend not only on the extent of fetal asphyxia and acidosis but also on the effects on the fetal cardiovascular system of reactive oxygen species (ROS) generated during the ischemia-reperfusion (I/R) associated with repeated compressions of the umbilical cord. This study tested the hypothesis that maternal treatment with clinical doses of the antioxidant allopurinol in the setting of fetal asphyxia would reduce oxidative stress in the fetal cardiovascular system. The hypothesis was tested in chronically instrumented fetal sheep in late gestation by investigating the effects of maternal treatment with therapeutic doses of allopurinol or vehicle on the fetal cardiovascular system during and after episodes of I/R. The latter were produced by repeated, measured compressions of the umbilical cord. The data show that maternal treatment with allopurinol helped maintain umbilical blood flow and it reduced fetal cardiac oxidative stress after I/R of the type associated with clinically relevant acidemia and repetitive fetal heart rate decelerations. The data support the hypothesis tested and suggest that maternal treatment with allopurinol may offer plausible clinical intervention in the management of perinatal asphyxia in complicated labor.

  13. Quercetin and allopurinol reduce liver thioredoxin-interacting protein to alleviate inflammation and lipid accumulation in diabetic rats

    PubMed Central

    Wang, Wei; Wang, Chuang; Ding, Xiao-Qin; Pan, Ying; Gu, Ting-Ting; Wang, Ming-Xing; Liu, Yang-Liu; Wang, Fu-Meng; Wang, Shui-Juan; Kong, Ling-Dong

    2013-01-01

    Background and Purpose Thioredoxin-interacting protein (TXNIP), a regulator of cellular oxidative stress, has been associated with activation of NOD-like receptor 3 (NLRP3) inflammasome, inflammation and lipid metabolism, suggesting it has a role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) in diabetes. In this study we investigated whether TXNIP is involved in type 1 diabetes-associated NAFLD and whether antioxidants, quercetin and allopurinol, alleviate NAFLD by targeting TXNIP. Experimental Approach Diabetes was induced in male Sprague-Dawley rats by a single i.p. injection of 55 mg·kg−1 streptozotocin. Quercetin and allopurinol were given p.o. to diabetic rats for 7 weeks. Hepatic function, oxidative stress, inflammation and lipid levels were determined. Rat BRL-3A and human HepG2 cells were exposed to high glucose (30 mM) in the presence and absence of antioxidants, TXNIP siRNA transfection or caspase-1 inhibitor, Ac-YVAD-CMK. Key Results Quercetin and allopurinol significantly inhibited the TXNIP overexpression, activation of NLRP3 inflammasome, down-regulation of PPARα and up-regulation of sterol regulatory element binding protein-1c (SREBP-1c), SREBP-2, fatty acid synthase and liver X receptor α, as well as elevation of ROS and IL-1β in diabetic rat liver. These effects were confirmed in hepatocytes in vitro and it was further shown that TXNIP down-regulation contributed to the suppression of NLRP3 inflammasome activation, inflammation and changes in PPARα and SREBPs. Conclusions and Implications Inhibition of hepatic TXNIP by quercetin and allopurinol contributes to the reduction in liver inflammation and lipid accumulation under hyperglycaemic conditions. The targeting of hepatic TXNIP by quercetin and allopurinol may have therapeutic implications for prevention of type 1 diabetes-associated NAFLD. PMID:23647015

  14. Proteinuria reduction after treatment with miltefosine and allopurinol in dogs naturally infected with leishmaniasis

    PubMed Central

    Proverbio, Daniela; Spada, Eva; de Giorgi, Giada Bagnagatti; Perego, and Roberta

    2016-01-01

    Aim: The aim of this study was to evaluate changes in proteinuria in dogs naturally infected with visceral leishmaniasis, following treatment with miltefosine (MLF) and allopurinol. Materials and Methods: Medical records of 40 dogs with leishmaniasis, treated with 2 mg/kg MLF every 24 h PO and 10 mg/kg allopurinol every 12 h for 28 days were reviewed. 20 dogs were included in the study, and clinical staging was performed following guidelines of the Canine leishmaniasis (CanL) Working Group, and dogs were categorized for proteinuria according to the International Renal Interest Society (IRIS) staging system. Clinical score, indirect fluorescent antibody test titer, serum total protein, gamma globulin (IgG), serum creatinine and urea concentration, and urine protein creatinine ratio (UP/C) were recorded at the time of diagnosis before the start of therapy (D0) and at the end of 28 days of therapy (D28). Results: Following the CanL Working Group staging, all 20 dogs were classified as the clinical Stage C (Clinical disease) before and after the cycle of treatment. Before the cycle of therapy, dogs were categorized according to the IRIS staging system, as: 9/20 non-proteinuric (NP), 7/20 borderline proteinuric (BP), and 4/20 proteinuric (P). After treatment, 12/20 dogs were NP, 7/20 were BP, and 1/20 was P. There was a significant change in UP/C values before and after one cycle of treatment with MLF. In detail, after 28 days of therapy, 2 of 9 NP dogs became BP, 3 of the 7 BP dogs became NP, and 2 of the 4 P dogs became NP. Conclusion: This study showed a significant decrease in UP/C values occurred after one cycle of treatment with MLF and allopurinol in dogs naturally affected with CanL. This suggests that MLF does not increase proteinuria, and the use of MLF could be considered for the management of dogs with leishmaniasis, particularly in those with impaired renal function at the time of diagnosis. PMID:27651682

  15. 1-Methylxanthine derived from theophylline as an in vivo biochemical probe of allopurinol effect.

    PubMed

    Birkett, D J; Miners, J O; Day, R O

    1991-08-01

    The urinary 1-methyluric acid (1MU) to 1-methylxanthine (1MX) ratio has been assessed as a biochemical index of oxipurinol effect in vivo in man. Dosing with theophylline was used to produce 1MX as an intermediate metabolite in six healthy volunteers. A sigmoid Emax model was fitted to the data and gave a mean plasma oxipurinol IC50 of 3.0 +/- 1.1 mg l-1, a mean exponent n of 3.4 +/- 2.1 and a mean IC90 of 8.5 +/- 5.9 mg l-1. There was marked interindividual variability in the steepness of the plasma oxipurinol concentration response relationship, and in the plasma oxipurinol IC90 values. The study has confirmed the feasibility of using single doses of allopurinol to construct individual plasma oxipurinol concentration-response curves.

  16. Gene silencing in phlebotomine sand flies: Xanthine dehydrogenase knock down by dsRNA microinjections.

    PubMed

    Sant'Anna, Mauricio R; Alexander, Bruce; Bates, Paul A; Dillon, Rod J

    2008-06-01

    Lutzomyia longipalpis are vectors of medically important visceral leishmaniasis in South America. Blood-fed adult females digest large amounts of protein, and xanthine dehydrogenase is thought to be a key enzyme involved in protein catabolism through the production of urate. Large amounts of heme are also released during digestion with potentially damaging consequences, as heme can generate oxygen radicals that damage lipids, proteins and nucleic acids. However, urate is an antioxidant that may prevent such oxidative damage produced by heme. We investigated xanthine dehydrogenase by developing the RNAi technique for sand flies and used this technique to knock down the Lu. longipalpis xanthine dehydrogenase gene to evaluate its role in survival of adult females after blood feeding. The gene sequence of Lu. longipalpis xanthine dehydrogenase is described together with expression in different life cycle stages and RNAi knock down. Semi-quantitative RT-PCR of xanthine dehydrogenase expression showed a significant increase in expression after bloodmeal ingestion. Microinjection of dsRNA via the thorax of 1-day-old adult female sand flies resulted in approximately 40% reduction of xanthine dehydrogenase gene expression in comparison to flies injected with a control dsRNA. A significant reduction of urate in the whole body and excretions of Lu. longipalpis was observed after dsRNA xanthine dehydrogenase microinjection and feeding 96h later on rabbit blood. Sand flies injected with XDH dsRNA also exhibit significantly reduced life span in comparison with the mock-injected group when fed on sucrose or when rabbit blood fed, showing that urate could be indeed an important free radical scavenger in Lu. longipalpis. The demonstration of xanthine dehydrogenase knock down by dsRNA microinjection, low mortality of microinjected insects and the successful bloodfeeding of injected insects demonstrated the utility of RNAi as a tool for functional analysis of genes in phlebotomine

  17. Caffeine and other xanthines as cytochemical blockers and removers of heterocyclic DNA intercalators from chromatin.

    PubMed

    Lyles, Mark B; Cameron, Ivan L

    2002-01-01

    Caffeine (CAF) and other xanthines non-covalently bind with the cationic fluorescent dye acridine orange (AO) and with other heterocyclic mutagens and carcinogens that are known to intercalate into double-stranded DNA (dsDNA). Fluorescence microscopy and spectrofluorometry studies were employed to test the ability of caffeine and certain other methyl substituted xanthines, with different binding affinities for AO, to inhibit and to reverse the intercalation of AO and other heterocyclic agents from intercalation with the DNA of nuclear chromatin of air-dried cells. Results indicated that xanthines with binding affinity for AO greater than 150 m(-1) block the AO molecule in a concentration dependent manner and comply with mass action kinetics. Thus CAF and other xanthines can be used to either inhibit intercalation of AO into nuclear DNA or to remove AO once intercalated into nuclear DNA. The interactions between other planar heterocyclics, xanthines, and nuclear chromatin dsDNA were also found to be non-covalent. Studies are needed to determine the ability of CAF and other xanthines to block and/or remove polyaromatic hydrocarbon (PAH) intercalators from the DNA of living cells.

  18. Treatment of calcium nephrolithiasis in the patient with hyperuricosuria.

    PubMed

    Arowojolu, Omotayo; Goldfarb, David S

    2014-12-01

    Nearly one-third of patients with calcium stones have hyperuricosuria. In vitro studies and clinical trials have investigated the relationship between uric acid and calcium stones, but the association between hyperuricosuria and calcium stone formation in patients is still being debated. Uric acid appears to cause salting out of calcium oxalate in human urine. However, the importance of this in vitro phenomenon to the proposed association is not supported in cross-sectional observational studies. A small placebo-controlled randomized clinical trial showed that allopurinol decreased the rate of recurrent calcium oxalate calculi in patients with hyperuricosuria and normocalciuria. An assessment of the effect of combination therapy of allopurinol with indapamide showed no additive effect. Allopurinol may have antioxidant effects that are responsible for its reducing calcium stone formation, which are independent of xanthine oxidase inhibition. In addition, a newer xanthine oxidoreductase inhibitor, febuxostat, may also be effective in the prevention of calcium stones, as it reduces urinary uric acid excretion.

  19. Effect of N-acetylcysteine, allopurinol and vitamin E on jaundice-induced brain oxidative stress in rats.

    PubMed

    Karageorgos, Nikolaos; Patsoukis, Nikolaos; Chroni, Elisabeth; Konstantinou, Dimitris; Assimakopoulos, Stelios F; Georgiou, Christos

    2006-09-21

    We examined the possible protective effect of certain antioxidants (N-acetylcysteine, allopurinol and vitamin E) against the oxidative stress of brain tissue induced by experimental obstructive jaundice in rats. Thirty-six male Wistar rats were randomly divided into six groups; group I control, group II sham operated, group III bile duct ligated and groups IV, V, and VI in which the rats, after bile duct ligation, were given every day an intraperitoneal injection with N-acetylcysteine, allopurinol and Vit-E respectively. All rats were sacrificed on the tenth day by exsanguination and the oxidative state in samples from cortex, midbrain and cerebellum was assessed by measuring the thiol redox state and lipid peroxidation quantified by MDA measurements. The main finding was that all three antioxidants decrease lipid peroxidation in the three brain areas. Cysteine levels increased and protein thiol levels were reserved only in the group treated with N-acetylcysteine, whereas oxidized glutathione increased dramatically in the group treated with allopurinol, suggesting that each antioxidant agent had a certain influence profile on the different antioxidant defense systems. The observed effects of the antioxidants in this experimental model could also provide insight into some aspects of jaundice-induced hepatic encephalopathy in humans.

  20. NADPH oxidases, reactive oxygen species, and hypertension: clinical implications and therapeutic possibilities.

    PubMed

    Paravicini, Tamara M; Touyz, Rhian M

    2008-02-01

    Reactive oxygen species (ROS) influence many physiological processes including host defense, hormone biosynthesis, fertilization, and cellular signaling. Increased ROS production (termed "oxidative stress") has been implicated in various pathologies, including hypertension, atherosclerosis, diabetes, and chronic kidney disease. A major source for vascular and renal ROS is a family of nonphagocytic NAD(P)H oxidases, including the prototypic Nox2 homolog-based NAD(P)H oxidase, as well as other NAD(P)H oxidases, such as Nox1 and Nox4. Other possible sources include mitochondrial electron transport enzymes, xanthine oxidase, cyclooxygenase, lipoxygenase, and uncoupled nitric oxide synthase. NAD(P)H oxidase-derived ROS plays a physiological role in the regulation of endothelial function and vascular tone and a pathophysiological role in endothelial dysfunction, inflammation, hypertrophy, apoptosis, migration, fibrosis, angiogenesis, and rarefaction, important processes underlying cardiovascular and renal remodeling in hypertension and diabetes. These findings have evoked considerable interest because of the possibilities that therapies against nonphagocytic NAD(P)H oxidase to decrease ROS generation and/or strategies to increase nitric oxide (NO) availability and antioxidants may be useful in minimizing vascular injury and renal dysfunction and thereby prevent or regress target organ damage associated with hypertension and diabetes. Here we highlight current developments in the field of reactive oxygen species and cardiovascular disease, focusing specifically on the recently identified novel Nox family of NAD(P)H oxidases in hypertension. We also discuss the potential role of targeting ROS as a therapeutic possibility in the management of hypertension and cardiovascular disease.

  1. Selective tracheal relaxation and phosphodiesterase-IV inhibition by xanthine derivatives.

    PubMed

    Miyamoto, K; Kurita, M; Ohmae, S; Sakai, R; Sanae, F; Takagi, K

    1994-05-17

    The effects of substitutions in the xanthine nucleus on tracheal relaxant activity, atrium chronotropic activity, adenosine A1 affinity, and inhibitory activities on cyclic AMP-phosphodiesterase isoenzymes in guinea pigs were studied. Substitution with a long alkyl chain at the N1-position of xanthine nucleus increased the tracheal relaxant activity without leading to positive chronotropic action, and long alkyl chains at the N3-position increased both activities. N7-substitutions with n-propyl and 2'-oxopropyl groups, such as in denbufylline, increased bronchoselectivity. N7-substitution decreased the adenosine A1 affinity, but substitution at either the N1- or N3-position increased it. The bronchorelaxant activity of xanthine derivatives was closely correlated with their inhibition of phosphodiesterase-IV, but not with their adenosine A1 affinity; the positive chronotropic effects were related to their inhibition of phosphodiesterase-III. This study confirms that the bronchorelaxation of xanthine derivatives is mediated by inhibition of the isoenzyme phosphodiesterase-IV. The results of structure-activity analysis suggest that substitutions at the N1- and N7-positions should be tried in the development of xanthine derivatives that are selective bronchodilators and phosphodiesterase-IV inhibitors.

  2. Succinate oxidase in Neurospora.

    PubMed

    West, D J; Woodward, D O

    1973-02-01

    Two kinetically distinct states of succinate oxidase have been detected in the mitochondria of Neruospora crassa. One state has a K(m) for succinate of 4.1 x 10(-3)m, and the other has a K(m) for succinate of 3.5 x 10(-4)m. The high K(m) state was found in freshly extracted mitochondria from either 20- or 72-hr mycelium. However, the succinate oxidase activity in mitochondria from 20-hr mycelium rapidly deteriorated in vitro, leaving a stable residual activity with the lower K(m) for succinate. Adenosine triphosphate (ATP) plus Mg(2+) stabilized the high K(m) state in these preparations. The high K(m) state of succinate oxidase was further characterized by a two- to threefold increase in activity over the pH range 6.6 to 8.0 and by classical competitive inhibition by fumarate and malonate. By contrast, the low K(m) state of succinate oxidase showed a relatively flat response to pH over the range 6.6 to 8.0 and a nonclassical pattern of inhibition by fumarate and malonate, as shown by nonlinear plots of reciprocal velocity versus reciprocal substrate concentration in the presence of inhibitor or reciprocal velocity versus inhibitor concentration at fixed substrate concentrations. The relationship of mycelial age to the in vitro stability of succinate oxidase is considered with reference to probable changes in the relative pool sizes of extra- and intramitochondrial ATP in response to changes in the rate of glycolysis.

  3. Ocular signs, diagnosis and long-term treatment with allopurinol in a cat with leishmaniasis.

    PubMed

    Richter; Schaarschmidt-Kiener; Krudewig

    2014-06-01

    A case of leishmaniasis with predominantly ocular signs in a cat living in Switzerland and it's treatment is reported. The cat was imported from Spain 4 years earlier and was initially presented with chronic uveitis. Laboratory test results were negative for feline immunodeficiency virus (FIV), feline infectious peritonitis (FIP), feline leukaemia virus (FeLV) and Toxoplasma gondii, as well as for Bartonella haenselae and Leishmania spp. Twenty-one months later the cat was presented again because of development of keratitis and granulomatous blepharitis. Blood cell count revealed severe Pancytopenia; Cytology of fine needle aspirates of granulomatous lesions on both upper eyelids and of a corneal smear revealed intracytoplasmatic microorganisms. A preliminary diagnosis of leishmaniasis was supported by positive polymerase chain reaction from bone marrow and eyelid samples for Leishmania infantum DNA and by a high serum antibody titer for Leishmania spp. Treatment with Allopurinol (10 mg/kg, BID) orally led to rapid improvement of ocular signs, general condition and blood cell count with complete remission of lid and corneal lesions within 2 months of treatment.

  4. The Effects of Lowering Uric Acid Levels Using Allopurinol on Components of Metabolic Syndrome

    PubMed Central

    Heimbach, Esther J.; Bowden, Rodney G.; Griggs, Jackson O.; Beaujean, A. Alexander; Doyle, Eva I.; Doyle, Robert D.

    2012-01-01

    Background Researchers have reported an independent direct relationship between lipid levels and hyperuricemia with MetS. The purpose of this study was to determine the relationship between serum uric acid levels and lipids among patients on allopurinol. Methods A retrospective secondary data analysis was conducted on 66 adult patients from a family health clinic in Central Texas. Medical records used were recorded during a nine year period (2002 - 2010) ascertaining the relationship between uric acid and lipids. Results Spearman correlations revealed a weak correlation between uric acid and total cholesterol, a weak correlation between uric acid and triglycerides and LDL-C. A weak inverse correlation was discovered between uric acid and HDL-C. A moderate correlation was discovered when all lipid variables combined were compared to uric acid. Conclusions We discovered LDL-C and triglycerides to be significant predictors of uric acid with weak correlations. Additionally, weak correlations existed between uric acid and total cholesterol and HDL-C with an inverse relationship discovered with HDL-C. These findings support the literature suggesting that uric acid is more likely to be associated with total cholesterol and triglycerides. In addition, new discoveries serve as an indication that LDL-C may also be associated with uric acids levels. The mechanism by which uric acid may regulate lipids is elusive but suggestions have included suppression of lipid peroxidase and decreases in critical lipase activity.

  5. Antioxidant effect of naturally occurring xanthines on the oxidative damage of DNA bases

    NASA Astrophysics Data System (ADS)

    Vieira, A. J. S. C.; Telo, J. P.; Pereira, H. F.; Patrocínio, P. F.; Dias, R. M. B.

    1999-01-01

    The repair of the oxidised radicals of adenine and guanosine by several naturally occurring xanthines was studied. Each pair of DNA purine/xanthine was made to react with the sulphate radical and the decrease of the concentration of both compounds was measured by HPLC as a function of irradiation time. The results show that xanthine efficiently prevents the oxidation of the two DNA purines. Theophyline and paraxanthine repair the oxidised radical of adenine but not the one from guanosine. Theobromine and caffeine do not show any protecting effect. An order of the oxidation potentials of all the purines studied is proposed. La réparation des radicaux oxydés de l'adénine et de la guanosine par des xanthines naturelles a été étudiée en soumettant chaque paire base de l'ADN/xanthine à l'oxydation par le radical sulfate et en mesurant par HPLC la disparition des deux composés en fonction du temps d'irradiation. Les résultats montrent que la xanthine joue un rôle protecteur efficace contre l'oxydation des deux purines de l'ADN. La théophyline et la paraxanthine réparent le radical oxydé de l'adénine mais pas celui de la guanosine. La théobromine et la cafeíne n'ont pas d'effet protecteur. Un ordre de potentiels d'oxydation des purines étudiées est proposé.

  6. Electron-paramagnetic-resonance parameters of molybdenum(V) in sulphite oxidase from chicken liver.

    PubMed Central

    Lamy, M T; Gutteridge, S; Bary, R C

    1980-01-01

    A study has been made of e.p.r. signals due to Mo(V) in reduced sulphite oxidase (EC 1.8.3.1) from chicken liver. Reduction by SO3(2-), or photochemically in the presence of a deazaflavin derivative, produces spectra indistinguishable from one another. Three types of spectra from the enzyme were distingusihed and shown to correspond to single chemical species, since they could be simulated at both 9 and 35 GHz by using the same parameters. These were the low-pH form of the enzyme, with gav. 1.9805, the high-pH form, with gav. 1.9681 and a phosphate complex, with gav. 1.9741. The low-H form shows interaction with a single exchangeable proton, with A(1H)av. (hyperfine coupling constant) = 0.98 mT, probably in the form of an MoOH group. Parameters of the signals are compared with those for signals from xanthine oxidase and nitrate reductase. The signal from the phosphate complex of sulphite oxidase in unique among anion complexes of Mo-containing enzymes in showing no hyperfine coupling to protons. There is no evidence for additional weakly coupled protons or nitrogen nuclei in the sulphite oxidase signals. The possibility is considered that the enzymic mechanism involves abstraction of a proton and two electrons from HSO3- by a Mo = O group in the enzyme. PMID:6249254

  7. Effect of antiperoxidative drugs on gastric damage induced by ethanol in rats

    SciTech Connect

    Mizui, T.; Sato, H.; Hirose, F.; Doteuchi, M.

    1987-08-10

    Lesion formation due to oral administration of absolute ethanol could be prevented by parenteral pretreatment with antiperoxidative drugs such as butylated hydroxytoluene (BHT), quercetin and quinacrine. Also effective were allopurinol and oxypurinol, inhibitors of xanthine oxidase, but not superoxide dismutase (SOD) and hydroxyl radical scavengers, such as sodium benzoate and dimethyl sulfoxide (DMSO). BHT, quercetin, quinacrine and sulfhydryl compounds such as reduced glutathione and cysteamine which offer gastroprotection in vivo against ethanol inhibited lipid peroxidation induced in vitro by ferrous ion in porcine gastric mucosal homogenate, but SOD, sodium benzoate, DMSO, allopurinol and oxypurinol did not. These results suggest the possibility that an active species, probably derived from free iron mobilized by the xanthine oxidase system, other than oxygen radicals such as hydroxyl formation in the gastric mucosa after absolute ethanol administration. 38 references, 1 figure, 4 tables.

  8. N-acetyl cysteine versus allopurinol in the prevention of contrast nephropathy in patients with chronic kidney disease: A randomized controlled trial

    PubMed Central

    Sadineni, R.; Karthik, K. R.; Swarnalatha, G.; Das, U.; Taduri, G.

    2017-01-01

    Contrast media administration can lead to acute deterioration in renal function particularly in patients with pre-existing chronic kidney disease. This prospective, randomized controlled open-label parallel group study was undertaken at Nizam's Institute of Medical Sciences, Hyderabad, from June to December 2015. A total of 95 patients were included, of which 35 received n-acetylcysteine (NAC) + normal saline (NS), 30 patients received allopurinol (ALL) + NS, and 30 patients received placebo. In our study, the overall incidence of CIN was 24%. Incidence of CIN in NAC + NS, ALL + NS, and placebo group were 20%, 16%, and 36%, respectively. The major finding of this study was there was no significant difference between NAC and allopurinol in the prevention of contrast nephropathy. However, only allopurinol was superior to placebo. In our study, hyperuricemia and baseline serum creatinine were the only risk factors associated with CIN. PMID:28356658

  9. A comparative study of efficacy and safety of febuxostat and allopurinol in pyrazinamide-induced hyperuricemic tubercular patients

    PubMed Central

    Pichholiya, Meenu; Yadav, Arvind Kumar; Luhadia, S. K.; Tahashildar, Jameela; Aseri, M. L.

    2016-01-01

    Objectives: To compare the efficacy and safety of febuxostat and allopurinol in pyrazinamide (PZA)-induced hyperuricemia in patients taking antitubercular therapy (ATT). Methods: This randomized controlled study was conducted at a tertiary care teaching institute of Rajasthan in all the sputum-positive tubercular patients aged between 18 and 65 years of either sex. Serum uric acid level was monitored at 0th, 2nd, 4th, 6th, and 8th week of ATT. Patients whose uric acid level was found to be increased at 2nd week were finally recruited in the study. Ninety patients who developed hyperuricemia due to ATT were divided randomly into three groups (Group A - febuxostat, Group B - allopurinol, and Group C - control) of thirty patients each. Mean serum uric acid levels were calculated at all the weeks in all the groups, and serum uric acid levels were compared by applying student's t-test and ANOVA. Results: Mean serum uric acid level decreased from 10.698 mg/dl (at 2nd week) to 7.846 mg/dl (at 8th week) in Group A and from 11.34 mg/dl (at 2nd week) to 7.280 mg/dl (at 8th week) in Group B. Numbers of adverse events encountered across both the treatment groups were same with both the drugs. Conclusion: Allopurinol and febuxostat were equally efficacious in lowering PZA induced raised serum uric acid level in tubercular patients, and it was possible to continue ATT without withdrawing PZA. PMID:27721537

  10. Safety and efficacy of oral febuxostat for treatment of HLA-B*5801-negative gout: a randomized, open-label, multicentre, allopurinol-controlled study

    PubMed Central

    Yu, K-H; Lai, J-H; Hsu, P-N; Chen, D-Y; Chen, C-J; Lin, H-Y

    2016-01-01

    Objectives: This phase IIIB study compared the efficacy and safety of febuxostat and allopurinol in gout patients with or without tophi who were HLA-B*5801 negative. Method: Eligible patients were randomized to a febuxostat group (80 mg QD) or an allopurinol group (300 mg QD). Following an initial 2-week washout period, over the next 12 weeks we made five measurements of serum urate levels along with assessments of adverse events (AEs). Results: Forty-three out of 152 screened subjects (28.3%) were ineligible either because of the presence of the HLA-B*5801 allele or for various other reasons. The febuxostat group (n = 54) and the allopurinol group (n = 55) had no significant differences in demographic or baseline characteristics. From week 2 to week 12, the febuxostat group had a significantly lower serum urate level than the allopurinol group (p ≤ 0.001 for all comparisons) and significantly more patients with serum urate levels less than 6.0 mg/dL. The serum urate levels of the febuxostat group declined by more than 40% from week 2 to week 12 and this decrease was greater than that in the allopurinol group (~30%). The two groups were similar in terms of AEs. Conclusions: Febuxostat was more effective than allopurinol in reducing the serum urate levels of Han Chinese patients with gout or tophaceous gout who were HLA-B*5801 negative, without causing any serious skin reactions. Febuxostat should be considered for treatment of Han Chinese patients with gout who are HLA-B*5801 negative. PMID:26771445

  11. Spectroscopic Signatures and Structural Motifs in Isolated and Hydrated Xanthine: a Computational Study

    NASA Astrophysics Data System (ADS)

    Singh, Vipin Bahadur

    2016-06-01

    The conformational landscapes of xanthine and its hydrated complex have been investigated by MP2 and DFT methods. The ground state geometry optimization yield five lowest energy conformers of xanth1-(H2O)1 complex at the MP2/6-311++G(d,p) level of theory for the first time. We investigated the low-lying excited states of bare xanthine by means of coupled cluster singles and approximate doubles (CC2) and TDDFT methods and a satisfactory interpretation of the electronic absorption spectra1 is obtained. The difference between the S0-S1 transition energy due to the most stable and the second most stable stable conformation of xanthine was found to be 859 wn. One striking feature is the coexistence of the blue and red shift of the vertical excitation energy of the optically bright state S1 of xanthine upon forming complex with a water at C2=O and C6=O carbonyl sites, respectively. The lowest singlet ππ* excited-state of the xanth1-(H2O)1 complex involving C2=O carbonyl are strongly blue shifted which is in agreement with the result of R2PI spectra of singly hydrated xanthine. While for the most stable and the second most stable xanth1-(H2O)1 complexes involving C6=O carbonyl, the lowest singlet ππ* excited-state is red shifted. The effect of hydration on S1 excited state due to bulk water environment was mimicked by a combination of polarizable continuum solvent model (PCM) and conductor like screening model (COSMO), which also shows a blue shift in accordance with the result of electronic absorption spectra in aqueous solution. This hypsochromic shift, is expected to be the result of the changes in the π-electron delocalization extent of molecule because of hydrogen bond formation. The optimized structure of xanthine dimer, computed the first time by MP2 and DFT methods. The binding energy of this dimer linked by double N-H…O=C hydrogen bonds was found to be 88 kj/mole at the MP2/6-311++G(d,p) level of theory. Computed IR spectra is found in remarkable agreement

  12. CSF xanthine, homovanillic acid, and their ratio as biomarkers of Parkinson's disease.

    PubMed

    LeWitt, Peter; Schultz, Lonni; Auinger, Peggy; Lu, Mei

    2011-08-23

    Diminished nigrostriatal dopaminergic neurotransmission is a biochemical hallmark of Parkinson's disease. Despite this, a reliable trait biomarker of sporadic Parkinson's disease has not emerged from measurements of cerebrospinal fluid dopamine metabolites. Previous studies have highlighted strong neurochemical relationships between dopamine and various purine compounds. In this study, we analyzed cerebrospinal fluid concentrations of homovanillic acid (the major catabolite of dopamine) and the purine compound xanthine for a comparison of 217 unmedicated Parkinson's disease subjects and 26 healthy controls. These compounds were highly correlated for both the Parkinson's disease subjects (r=0.68) and for controls (r=0.73; both groups, p<0.001). While neither homovanillic acid nor xanthine concentrations differentiated Parkinson's disease from controls, their ratio did. For controls, the mean [xanthine]/[homovanillic acid] quotient was 13.1±5.5 as compared to the Parkinson's disease value of 17.4±6.7 at an initial lumbar CSF collection (p=0.0017), and 19.7±8.7 (p<0.001) at a second CSF collection up to 24 months later. The [xanthine]/[homovanillic acid] ratio in the Parkinson's disease subjects differed as a function of disease severity, as measured by the sum of Unified Parkinson's Disease Rating Scale Activities of Daily Living and Motor Exam ratings. The [xanthine]/[homovanillic acid] ratio also increased between the first and second CSF collections, suggesting that this quotient provides both a state and trait biomarker of Parkinson's disease. These observations add to other neurochemical evidence that links purine metabolism to Parkinson's disease.

  13. Superoxide Free Radicals Are Produced in Glyoxysomes 1

    PubMed Central

    Sandalio, Luisa M.; Fernández, Victor M.; Rupérez, Francisco L.; Del Río, Luis A.

    1988-01-01

    The production of superoxide free radicals in pellet and supernatant fractions of glyoxysomes, specialized plant peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons, was investigated. Upon inhibition of the endogenous superoxide dismutase, xanthine, and hypoxanthine induced in glyoxysomal supernatants the generation of O2− radicals and this was inhibited by allopurinol. In glyoxysomal pellets, NADH stimulated the generation of superoxide radicals. Superoxide production by purines was due to xanthine oxidase, which was found predominantly in the matrix of glyoxysomes. The generation of O2− radicals in glyoxysomes by endogenous metabolites suggests new active oxygen-related roles for glyoxysomes, and for peroxisomes in general, in cellular metabolism. PMID:16666081

  14. Enzymatic antioxidant defence mechanism in rat intestinal tissue is changed after ischemia–reperfusion. Effects of an allopurinol plus antioxidant combination

    PubMed Central

    Kaçmaz, Murat; Öztürk, H. Serdar; Karaayvaz, Muammer; Güven, Cengiz; Durak, Ílker

    1999-01-01

    Objectives To establish the antioxidant status of rat intestinal tissues after ischemia–reperfusion and to determine if pretreatment with an allopurinol and antioxidant vitamin combination gives any protection against mucosal injury. Experimental animals Twenty rats were divided into 4 groups of 5 animals each. Methods Group 1 (control) rats were not subjected to ischemia–reperfusion and received no allopurinol plus vitamin combination; group 2 rats received vitamins C (200 mg/kg) and E (100 mg/kg) and allopurinol (50 mg/kg) combination daily for 3 days preoperatively; group 3 rats were subjected to ischemia–reperfusion only; and group 4 rats were subjected to ischemia–reperfusion and received the vitamin and allopurinol combination. Main outcome measures Activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) enzymes, the level of thiobarbituric acid-reagent substances (TBARS) and histologic grading of tissue samples. Results SOD and GSH-Px activities were decreased, but the CAT activity and TBARS level increased. Pretreatment of the rats with the allopurinol-vitamin C-vitamin E combination did not have any significant effect on the enzyme activities. However, it resulted in important reductions in the TBARS tissue levels. Histologic investigation revealed significant mucosal injury in group 3 rats compared with group 4 rats (mean [and standard deviation] for grading, 4.6 [0.5] versus 1.8 [0.4]). Conclusions The enzymatic antioxidant defence system was significantly changed after ischemia–reperfusion and intestinal tissue was exposed to increased oxidant stress, the results of which were peroxidation of some cellular structures and increased concentrations of oxidative products. Although antioxidant treatment did not drastically affect the enzyme activities or afford complete protection of cellular structures against deformation, it apparently could eliminate oxygen radicals and prevent peroxidative reactions. PMID

  15. Discovery of piperonal-converting oxidase involved in the metabolism of a botanical aromatic aldehyde

    PubMed Central

    Doi, Shiori; Hashimoto, Yoshiteru; Tomita, Chiaki; Kumano, Takuto; Kobayashi, Michihiko

    2016-01-01

    Piperonal-catabolizing microorganisms were isolated from soil, the one (strain CT39-3) exhibiting the highest activity being identified as Burkholderia sp. The piperonal-converting enzyme involved in the initial step of piperonal metabolism was purified from strain CT39-3. Gene cloning of the enzyme and a homology search revealed that the enzyme belongs to the xanthine oxidase family, which comprises molybdoenzymes containing a molybdopterin cytosine dinucleotide cofactor. We found that the piperonal-converting enzyme acts on piperonal in the presence of O2, leading to formation of piperonylic acid and H2O2. The growth of strain CT39-3 was inhibited by higher concentrations of piperonal in the culture medium. Together with this finding, the broad substrate specificity of this enzyme for various aldehydes suggests that it would play an important role in the defense mechanism against antimicrobial compounds derived from plant species. PMID:27905507

  16. Development, optimization and biological evaluation of chitosan scaffold formulations of new xanthine derivatives for treatment of type-2 diabetes mellitus.

    PubMed

    Lupascu, Florentina Geanina; Dash, Mamoni; Samal, Sangram Keshari; Dubruel, Peter; Lupusoru, Catalina Elena; Lupusoru, Raoul-Vasile; Dragostin, Oana; Profire, Lenuta

    2015-09-18

    New xanthine derivatives as antidiabetic agents were synthesized and new chitosan formulations have been developed in order to improve their biological and pharmacokinetic profile. Their physicochemical properties in terms of particle size, morphology, swelling degree, crystalline state, the loading efficiency as well as in vitro release and biodegradation rate were evaluated. According to the results the optimized formulations have a high drug loading efficiency (more than 70%), small particle size, a good release profile in the simulated biological fluids (the percentage of cumulative release being more than 55%) and improved biodegradation rate in reference with chitosan microparticles. The presence of xanthine derivatives (6, 7) in chitosan microparticles was demonstrated by means of FTIR analysis. The X-ray diffraction (XRD) proved that xanthine derivatives present a crystalline state. The biological evaluation assays confirmed the antioxidant and antidiabetic effects of the xanthine derivatives (6, 7) and their chitosan formulations (CS-6, CS-7). Xanthine derivative 6 showed a high antiradical scavenging effect (DPPH remaining=41.78%). It also reduced the glucose blood level with 59.30% and recorded level of glycosylated hemoglobin was 4.53%. The effect of its chitosan formulation (CS-6) on the level of blood glucose (114.5mg/dl) was even more intense than the one recorded by pioglitazone (148.5mg/dl) when used as standard antidiabetic drug. These results demonstrated the potential application of xanthine derivative 6 and its chitosan formulation (CS-6) in the treatment of the diabetes mellitus syndrome.

  17. Interactions of xanthines with activated carbon. I. Kinetics of the adsorption process

    NASA Astrophysics Data System (ADS)

    Navarrete Casas, R.; García Rodriguez, A.; Rey Bueno, F.; Espínola Lara, A.; Valenzuela Calahorro, C.; Navarrete Guijosa, A.

    2006-06-01

    Because of their pharmaceutical and industrial applications, we have studied the adsorption of xanthine derivates (caffeine and theophylline) by activated carbon. To this end, we examined kinetic, equilibrium and thermodynamic aspects of the process. This paper reports the kinetics results. The experimental results indicate that the process was first order in C and the overall process was assumed to involve a single, reversible adsorption-desorption process obeying a kinetic law postulated by us.

  18. Methods for the synthesis of aza(deaza)xanthines as a basis of biologically active compounds

    NASA Astrophysics Data System (ADS)

    Babkov, D. A.; Geisman, A. N.; Khandazhinskaya, A. L.; Novikov, M. S.

    2016-03-01

    The review covers methods for the synthesis of aza(deaza)xanthines, i.e., fused pyrrolo-, pyrazolo- and triazolopyrimidine heterocyclic systems, which are common core structures of various biologically active compounds. The extensive range of modern synthetic approaches is organized according to target structures and starting building blocks. The presented material is intended to benefit broad audience of specialists in the fields of organic, medicinal and pharmaceutical chemistry. The bibliography includes 195 references.

  19. Polyphenols decreased liver NADPH oxidase activity, increased muscle mitochondrial biogenesis and decreased gastrocnemius age-dependent autophagy in aged rats.

    PubMed

    Laurent, Caroline; Chabi, Beatrice; Fouret, Gilles; Py, Guillaume; Sairafi, Badie; Elong, Cecile; Gaillet, Sylvie; Cristol, Jean Paul; Coudray, Charles; Feillet-Coudray, Christine

    2012-09-01

    This study explored major systems of reactive oxygen species (ROS) production and their consequences on oxidative stress, mitochondriogenesis and muscle metabolism in aged rats, and evaluated the efficiency of 30-day oral supplementation with a moderate dose of a red grape polyphenol extract (RGPE) on these parameters. In the liver of aged rats, NADPH oxidase activity was increased and mitochondrial respiratory chain complex activities were altered, while xanthine oxidase activity remained unchanged. In muscles, only mitochondrial activity was modified with aging. The oral intake of RGPE decreased liver NADPH oxidase activity in the aged rats without affecting global oxidative stress, suggesting that NADPH oxidase was probably not the dominant detrimental source of production of O(2)·(-) in the liver. Interestingly, RGPE supplementation increased mitochondrial biogenesis and improved antioxidant status in the gastrocnemius of aged rats, while it had no significant effect in soleus. RGPE supplementation also decreased age-dependent autophagy in gastrocnemius of aged rats. These results extended existing findings on the beneficial effects of RGPE on mitochondriogenesis and muscle metabolism in aged rats.

  20. Lysyl oxidase in colorectal cancer.

    PubMed

    Cox, Thomas R; Erler, Janine T

    2013-11-15

    Colorectal cancer is the third most prevalent form of cancer worldwide and fourth-leading cause of cancer-related mortality, leading to ~600,000 deaths annually, predominantly affecting the developed world. Lysyl oxidase is a secreted, extracellular matrix-modifying enzyme previously suggested to act as a tumor suppressor in colorectal cancer. However, emerging evidence has rapidly implicated lysyl oxidase in promoting metastasis of solid tumors and in particular colorectal cancer at multiple stages, affecting tumor cell proliferation, invasion, and angiogenesis. This emerging research has stimulated significant interest in lysyl oxidase as a strong candidate for developing and deploying inhibitors as functional efficacious cancer therapeutics. In this review, we discuss the rapidly expanding body of knowledge concerning lysyl oxidase in solid tumor progression, highlighting recent advancements in the field of colorectal cancer.

  1. Respiratory burst oxidase of fertilization.

    PubMed Central

    Heinecke, J W; Shapiro, B M

    1989-01-01

    Partially reduced oxygen species are toxic, yet sea urchin eggs synthesize H2O2 in a "respiratory burst" at fertilization, as an extracellular oxidant to crosslink their protective surface envelopes. To study the biochemical mechanism for H2O2 production, we have isolated an NADPH-specific oxidase fraction from homogenates of unfertilized Strongylocentrotus purpuratus eggs that produces H2O2 when stimulated with Ca2+ and MgATP2-. Concentrations of free Ca2+ previously implicated in regulation of egg activation modulate the activity of the oxidase. Inhibitors were used to test the relevance of this oxidase to the respiratory burst of fertilization. Procaine, two phenothiazines, and N-ethylmaleimide (but not iodoacetamide) inhibited H2O2 production by the oxidase fraction and oxygen consumption by activated eggs. The ATP requirement suggested that protein kinase activity might regulate the respiratory burst of fertilization; consonant with this hypothesis, H-7 and staurosporine were inhibitory. The respiratory burst oxidase of fertilization is an NADPH:O2 oxidoreductase that appears to be regulated by a protein kinase; although it bears a remarkable resemblance to the neutrophil oxidase, unlike the latter it does not form O2- as its initial product. PMID:2537493

  2. Bronchodilator activity of xanthine derivatives substituted with functional groups at the 1- or 7-position.

    PubMed

    Miyamoto, K; Yamamoto, Y; Kurita, M; Sakai, R; Konno, K; Sanae, F; Ohshima, T; Takagi, K; Hasegawa, T; Iwasaki, N

    1993-05-14

    Xanthine derivatives with several functional groups at the 1- or 7-position were synthesized, and their pharmacological activities in guinea pigs were studied. In general, the in vitro tracheal relaxant action and positive chronotropic action of 3-propylxanthines were increased by substitutions with nonpolar functional groups at the 1-position, but decreased by any substitution at the 7-position. On the other hand, because positive chronotropic actions of substituents with allyl, aminoalkyl, alkoxyalkyl, and normal alkyl groups were much less than tracheal muscle became very high with substitutions of 3'-butenyl, (dimethylamino)ethyl, 2'-ethoxyethyl, 3'-methoxypropyl, and n-propyl groups at the 1-position and of 2'-ethoxyethyl, 2'-oxopropyl, and n-propyl groups at the 7-position, compared with theophylline and the corresponding unsubstituted xanthines, 3-propylxanthine and 1-methyl-3-propylxanthine. When compounds were intraduodenally administered to the guinea pig, 1-(2'-ethoxyethyl)-, 1-(3'-methoxypropyl)-, 1-(3'-butenyl)-, and 1-[(dimethylamino)-ethyl]-3-propylxanthines, 1-methyl-7-(2'-oxopropyl)-3-propylxanthine, and denbufylline (1,3-di-n-butyl-7-(2'-oxopropyl)xanthine) effectively inhibited the acetylcholine-induced bronchospasm without heart stimulation or central nervous system-stimulation at the effective dosage range. Particularly, the bronchodilatory effect of 1-(2'-ethoxyethyl)-3-propylxanthine was much stronger and more continuous than those of theophylline and pentoxifylline. On the other hand, there were certain relationships among the in vitro tracheal relaxant activities of these compounds, their affinities for adenosine (A1) receptors in the brain membrane, and their inhibition of cyclic AMP-phosphodiesterase (PDE) in the tracheal muscle. The affinity for A2 receptors of these compounds was very low or negligible. This suggests that both the action on A1 receptors or interaction with adenosine and the cyclic AMP-PDE inhibitory activity contribute

  3. Cost-Effectiveness Analysis of HLA-B*5801 Testing in Preventing Allopurinol-Induced SJS/TEN in Thai Population

    PubMed Central

    Saokaew, Surasak; Tassaneeyakul, Wichittra; Maenthaisong, Ratree; Chaiyakunapruk, Nathorn

    2014-01-01

    Background Stevens-Johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN), caused by allopurinol therapy, are strongly associated with the human leukocyte antigen (HLA), HLA-B*5801. Identification of HLA-B*5801 genotype before prescribing allopurinol offers the possibility of avoiding allopurinol-induced SJS/TEN. As there is a paucity of evidence about economic value of such testing, this study aims to determine the cost-effectiveness of HLA-B*5801 testing compared with usual care (no genetic testing) before allopurinol administration in Thailand. Methods and Finding A decision analytical and Markov model was used to estimate life time costs and outcomes represented as quality adjusted life years (QALYs) gained. The model was populated with relevant information of the association between gene and allopurinol-induced SJS/TEN, test characteristics, costs, and epidemiologic data for Thailand from a societal perspective. Input data were obtained from the literature and a retrospective database analysis. The results were expressed as incremental cost per QALY gained. A base-case analysis was performed for patients at age 30. A series of sensitivity analyses including scenario, one-way, and probabilistic sensitivity analyses were constructed to explore the robustness of the findings. Based on a hypothetical cohort of 1,000 patients, the incremental total cost was 923,919 THB (USD 29,804) and incremental QALY was 5.89 with an ICER of 156,937.04 THB (USD 5,062) per QALY gained. The cost of gout management, incidence of SJS/TEN, case fatality rate of SJS/TEN, and cost of genetic testing are considered very influential parameters on the cost-effectiveness value of HLA-B*5801 testing. Conclusions The genetic testing for HLA-B*5801 before allopurinol administration is considered a highly potential cost-effective intervention in Thailand. The findings are sensitive to a number of factors. In addition to cost-effectiveness findings, consideration of other factors including

  4. Hyperuricemia induced by the uricosuric drug probenecid in rats.

    PubMed

    Shinosaki, T; Yonetani, Y

    1991-04-01

    Stimulation of uric acid production by the well-known uricosuric drug probenecid was studied using potassium oxonate-treated rats and eviscerated rats subjected to functional hepatectomy. In oxonate-treated rats, probenecid was hyperuricosuric, increasing the glomerular-filtered amounts of uric acid and causing marked hyperuricemia. This could be completely blocked by combination dosing with allopurinol, an inhibitor of xanthine oxidase. In eviscerated rats subjected to functional hepatectomy, probenecid also increased plasma uric acid and urinary uric acid excretion, but when given together with allopurinol, the increase of plasma uric acid was abolished with a remarkable increase of plasma hypoxanthine and xanthine. When probenecid was given by combination dosing with propranolol, a beta adrenoceptor antagonist, the hyperuricemia was also completely blocked. Thus, probenecid is concluded to stimulate uric acid production, probably via some interaction with endogenous catecholamine, resulting in hyperuricemia in rats, although it is a practical hypouricemic drug in humans.

  5. The management of gout

    PubMed Central

    Rheumatology, Andrew Finch; Rheumatologist, Paul Kubler

    2016-01-01

    SUMMARY Gout is a common inflammatory arthritis that is increasing in prevalence. It is caused by the deposition of urate crystals. Non-steroidal anti-inflammatory drugs, colchicine and corticosteroids are options for the management of acute gout. They are equally efficacious and comorbidities guide the best choice. Allopurinol is an effective treatment for reducing concentrations of uric acid. Renal function guides the starting dose of allopurinol and the baseline serum uric acid concentration guides the maintenance dose. Febuxostat is another xanthine oxidase inhibitor. It is clinically equivalent to allopurinol. Uricosuric drugs, such as probenecid, increase uric acid excretion. New drugs in this class will soon become available and are likely to have a role in the treatment of patients who do not respond to other drugs. PMID:27756973

  6. Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase.

    PubMed

    McDonald, Allison E; Amirsadeghi, Sasan; Vanlerberghe, Greg C

    2003-12-01

    The mitochondrial alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) are two similar members of the membrane-bound diiron carboxylate group of proteins. AOX is a ubiquinol oxidase present in all higher plants, as well as some algae, fungi, and protists. It may serve to dampen reactive oxygen species generation by the respiratory electron transport chain. PTOX is a plastoquinol oxidase in plants and some algae. It is required in carotenoid biosynthesis and may represent the elusive oxidase in chlororespiration. Recently, prokaryotic orthologues of both AOX and PTOX proteins have appeared in sequence databases. These include PTOX orthologues present in four different cyanobacteria as well as an AOX orthologue in an alpha-proteobacterium. We used PCR, RT-PCR and northern analyses to confirm the presence and expression of the PTOX gene in Anabaena variabilis PCC 7120. An extensive phylogeny of newly found prokaryotic and eukaryotic AOX and PTOX proteins supports the idea that AOX and PTOX represent two distinct groups of proteins that diverged prior to the endosymbiotic events that gave rise to the eukaryotic organelles. Using multiple sequence alignment, we identified residues conserved in all AOX and PTOX proteins. We also provide a scheme to readily distinguish PTOX from AOX proteins based upon differences in amino acid sequence in motifs around the conserved iron-binding residues. Given the presence of PTOX in cyanobacteria, we suggest that this acronym now stand for plastoquinol terminal oxidase. Our results have implications for the photosynthetic and respiratory metabolism of these prokaryotes, as well as for the origin and evolution of eukaryotic AOX and PTOX proteins.

  7. [Alternative oxidase in industrial fungi].

    PubMed

    Gu, Shuai; Liu, Qiang; He, Hao; Li, Shuang

    2015-01-01

    Filamentous fungi have been used in industrial fermentation extensively. Based on non-phosphorylating electron transport process, alternative respiration pathway (ARP) acts as an energy overflow, which can balance carbon metabolism and electron transport, allow the continuance of tricarboxylic acid cycle without the formation of ATP, and permit the turnover of carbon skeletons. Alternative respiration pathway also plays an important role in the stress response of fungi and the physiological function of conditioned pathogen. Alternative oxidase (AOX) is the terminal oxidase responsible for the activity of alternative respiration pathway, which exists widely in higher plants, parts of fungi and algae. Owing to the property that alternative oxidase (AOX) is sensitive to salicylhydroxamic acid (SHAM) and insensitive to conventional inhibitors of cytochrome respiration, alternative respiration pathway by AOX is also named as cyanide-resistant respiration (CRR). In recent years, the study of the alternative respiration pathway and alternative oxidase has been a hot topic in the area involving cellular respiration metabolism. In this review we summarized the latest research advances about the functions of alternative respiration pathway and alternative oxidase in industrial fungi.

  8. Determination of Xanthine in the Presence of Hypoxanthine by Adsorptive Stripping Voltammetry at the Mercury Film Electrode

    PubMed Central

    Farias, Percio Augusto Mardini; Castro, Arnaldo Aguiar

    2014-01-01

    A stripping method for the determination of xanthine in the presence of hypoxanthine at the submicromolar concentration levels is described. The method is based on controlled adsorptive accumulation at the thin-film mercury electrode followed by a fast linear scan voltammetric measurement of the surface species. Optimum experimental conditions were found to be the use of 1.0 × 10−3 mol L−1 NaOH solution as supporting electrolyte, an accumulation potential of 0.00 V for xanthine and −0.50 V for hypoxanthine–copper, and a linear scan rate of 200 mV second−1. The response of xanthine is linear over the concentration ranges of 20–140 ppb. For an accumulation time of 30 minutes, the detection limit was found to be 36 ppt (2.3 × 10−10 mol L−1). Adequate conditions for measuring the xanthine in the presence of hypoxanthine, copper and other metals, uric acid, and other nitrogenated bases were also investigated. The utility of the method is demonstrated by the presence of xanthine associated with hypoxanthine, uric acid, nitrogenated bases, ATP, and ssDNA. PMID:24940040

  9. Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase

    PubMed Central

    Clinch, Keith; Crump, Douglas R.; Evans, Gary B.; Hazleton, Keith Z.; Mason, Jennifer M.; Schramm, Vern L.

    2013-01-01

    The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C- nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme. PMID:23810424

  10. Xanthine Dehydrogenase (XDH) cross-reacting material in mutants of Drosophila melanogaster deficient in XDH activity.

    PubMed

    Browder, L W; Tucker, L; Wilkes, J

    1982-02-01

    Rocket immunoelectrophoresis was used to estimate xanthine dehydrogenase cross-reacting material (XDH-CRM) in strains containing the cin and cin mutant genes, which are deficient in XDH enzymatic activity. CRM levels were determined as percentages of CRM in the Oregon-R wild-type strain. The mutant strains contain 72 and 76% of Oregon-R CRM, respectively. CRM levels in strains containing the XDH-deficient mutant genes lxd and mal are 93 and 105%, respectively. The high levels of CRM in these four mutant strains indicate that the primary effects of the mutant genes are on the function of XDH protein rather than its accumulation.

  11. Expression of alternative oxidase in tomato

    SciTech Connect

    Kakefuda, M.; McIntosh, L. )

    1990-05-01

    Tomato fruit ripening is characterized by an increase in ethylene biosynthesis, a burst in respiration (i.e. the climacteric), fruit softening and pigmentation. As whole tomatoes ripened from mature green to red, there was an increase in the alternative oxidase capacity. Aging pink tomato slices for 24 and 48 hrs also showed an increase of alternative oxidase and cytochrome oxidase capacities. Monoclonal antibodies prepared to the Sauromatum guttatum alternative oxidase were used to follow the appearance of alternative oxidase in tomato fruits. There is a corresponding increase in a 36kDa protein with an increase in alternative oxidase capacity. Effects of ethylene and norbornadiene on alternative oxidase capacity were also studied. We are using an alternative oxidase cDNA clone from potato to study the expression of mRNA in ripening and wounded tomatoes to determine if the gene is transcriptionally regulated.

  12. Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine.

    PubMed

    Doré, Andrew S; Robertson, Nathan; Errey, James C; Ng, Irene; Hollenstein, Kaspar; Tehan, Ben; Hurrell, Edward; Bennett, Kirstie; Congreve, Miles; Magnani, Francesca; Tate, Christopher G; Weir, Malcolm; Marshall, Fiona H

    2011-09-07

    Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A(2A) receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A(2A) selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.

  13. Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase.

    PubMed

    Coelho, Catarina; Foti, Alessandro; Hartmann, Tobias; Santos-Silva, Teresa; Leimkühler, Silke; Romão, Maria João

    2015-10-01

    Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-Å resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-Å resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

  14. Metabolism of purine alkaloids and xanthine in leaves of maté (Ilex paraguariensis).

    PubMed

    Yin, Yuling; Katahira, Riko; Ashihara, Hiroshi

    2015-05-01

    Accumulation and metabolism of purine alkaloids in leaves of maté (Ilex paraguariensis) were investigated. In winter, leaves accumulated caffeine but not theobromine, indicating that caffeine is the end product of purine alkaloid synthesis in maté. To elucidate the purine alkaloid metabolism in maté leaves, the metabolic fate of [8-(14)C]theobromine, [8-(14)C]theophylline, [8-(14)C]caffeine and [8-(14)C] xanthine was investigated in the leaf disks of young and mature leaves. In young maté leaves, significant amounts of theobromine and theophylline were utilized for caffeine biosynthesis, but the conversion was not observed in mature leaves. A small amount of theophylline was converted to theobromine. Practically no caffeine catabolism was detected in maté leaves during a 24 h-incubation. Catabolism of theobromine and theophylline via 3-methylxanthine was observed mainly in mature leaves. Xanthine was catabolised extensively via ureides in both young and mature leaves, but limited amounts are also utilized for the synthesis of theobromine, theophylline and caffeine. Possible pathways for the metabolism of purine alkaloids in maté leaves are discussed.

  15. Effect of ethanol on metabolism of purine bases (hypoxanthine, xanthine, and uric acid).

    PubMed

    Yamamoto, Tetsuya; Moriwaki, Yuji; Takahashi, Sumio

    2005-06-01

    There are many factors that contribute to hyperuricemia, including obesity, insulin resistance, alcohol consumption, diuretic use, hypertension, renal insufficiency, genetic makeup, etc. Of these, alcohol (ethanol) is the most important. Ethanol enhances adenine nucleotide degradation and increases lactic acid level in blood, leading to hyperuricemia. In beer, purines also contribute to an increase in plasma uric acid. Although rare, dehydration and ketoacidosis (due to ethanol ingestion) are associated with the ethanol-induced increase in serum uric acid levels. Ethanol also increases the plasma concentrations and urinary excretion of hypoxanthine and xanthine via the acceleration of adenine nucleotide degradation and a possible weak inhibition of xanthine dehydrogenase activity. Since many factors such as the ALDH2*1 gene and ADH2*2 gene, daily drinking habits, exercise, and dehydration enhance the increase in plasma concentration of uric acid induced by ethanol, it is important to pay attention to these factors, as well as ingested ethanol volume, type of alcoholic beverage, and the administration of anti-hyperuricemic agents, to prevent and treat ethanol-induced hyperuricemia.

  16. The effect of dimerization on the excited state behavior of methylated xanthine derivatives: a computational study.

    PubMed

    Nachtigallová, Dana; Aquino, Adelia J A; Horn, Shawn; Lischka, Hans

    2013-08-01

    The behavior of monomers and dimers of methylated xanthine derivatives in their excited states is investigated by means of the ADC(2), CASSCF, and CASPT2 methods. The results of the calculations of stationary points in the ground and excited states, minima on the S0/S1 crossing seams and the relaxation pathways are used to provide the interpretation of experimental observations of the monomer xanthine derivatives. The effect of dimerization on the excited state properties is studied for various relative orientations of the monomers in the dimer complexes in comparison with the relevant monomer species. A significant stabilization in the excited state minima of dimers is observed. These can act as trapping sites. Various types of conical intersections, with both localized and delocalized characters of wavefunctions, have been found, mainly energetically above the lowest bright excited state in the FC region. In addition, structures with the bonds formed between the two monomers were also found on the crossing seams. The possibility of ultrafast relaxation via these conical intersections is discussed.

  17. [Alternative oxidase - never ending story].

    PubMed

    Szal, Bożena; Rychter, Anna M

    2016-01-01

    Investigations of plant cyanide resistant respiration lead to the discovery in mitochondrial respiratory chain of the second terminal oxidase, alternative oxidase (AOX). AOX transfers electrons from reduced ubiquinone to oxygen omitting two coupling places thus lowering energetic efficiency of respiration. The presence of AOX was shown in all plants and also in some fungi, mollusca and protista. In termogenic plants the activity of AOX is connected with heat production. In other organisms AOX activity is important for maintaining metabolic homeostasis (carbon metabolism, cell redox state and energy demand) and ROS homeostasis. In this article structure of plant AOX protein and the regulation on molecular levels was described. Possible role of AOX as stress marker was pointed and the possibility of using AOX in human gene therapy was discussed.

  18. Development of the pteridine pathway in the zebrafish, Danio rerio.

    PubMed

    Ziegler, I; McDonald, T; Hesslinger, C; Pelletier, I; Boyle, P; McDonaldo, T

    2000-06-23

    In the zebrafish, the peripheral neurons and the pigment cells are derived from the neural crest and share the pteridine pathway, which leads either to the cofactor tetrahydrobiopterin or to xanthophore pigments. The components of the pteridine pattern were identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin, isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP cyclohydrolase I activity during the first 24-h postfertilization, followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and sepiapterin reductase, suggest an early supply of tetrahydrobiopterin for neurotransmitter synthesis in the neurons and for tyrosine supply in the melanophores. At 48-h postfertilization, sepiapterin formation branches off the de novo pathway of tetrahydrobiopterin synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves as a precursor for the formation of 7-oxobiopterin, which may be further catabolized to isoxanthopterin and 2,4,7-trioxopteridine. Neither 7, 8-dihydrobiopterin nor biopterin is a substrate for xanthine oxidoreductase. In contrast, both of these compounds are oxidized at C-7 by a xanthine oxidase variant form, which is inactivated by KCN, but is insensitive to allopurinol. The oxidase and the dehydrogenase form of xanthine oxidoreductase as well as the xanthine oxidase variant have specific developmental patterns. It follows that GTP cyclohydrolase I, the formation of sepiapterin, and the xanthine oxidoreductase family control the pteridine pathway in the zebrafish.

  19. Lysyl oxidase in cancer research.

    PubMed

    Perryman, Lara; Erler, Janine T

    2014-01-01

    Metastasis is the main reason for cancer-associated deaths and therapies are desperately needed to target the progression of cancer. Lysyl oxidase (LOX) plays a pivotal role in cancer progression, including metastasis, and is therefore is an attractive therapeutic target. In this review we will breakdown the process of cancer progression and the various roles that LOX plays has in the advancement of cancer. We will highlight why LOX is an exciting therapeutic target for the future.

  20. Apparent affinity of some 8-phenyl-substituted xanthines at adenosine receptors in guinea-pig aorta and atria.

    PubMed Central

    Collis, M. G.; Jacobson, K. A.; Tomkins, D. M.

    1987-01-01

    1 Some 8-phenyl-substituted, 1,3 dipropyl xanthines have previously been demonstrated to have a 20-400 fold greater affinity for A1 binding sites in rat CNS membranes than for A2 adenosine receptors in intact CNS cells from guinea-pigs. In the present study these compounds (1,3, dipropyl-8-phenylxanthine: DPPX; 1,3 dipropyl-8-(2 amino-4-chlorophenyl) xanthine: PACPX; 8-(4-(2-amino-ethyl)amino) carbonyl methyl oxyphenyl)-1,3-dipropylxanthine: XAC; and D-Lys-XAC) together with two that have not been reported to exhibit A1-receptor selectively (8-(p-sulphophenyl)theophylline: 8-PST; 8-(4-carboxy methyl oxyphenyl)-1,3-dipropylxanthine: XCC) have been evaluated as antagonists of the effects of 2-chloroadenosine in two isolated cardiovascular tissues. 2 The isolated tissues used were guinea-pig atria (bradycardic response) and aorta (relaxation), which are thought to possess A1 and A2 adenosine receptors, respectively. 3 All the xanthines antagonized responses evoked by 2-chloroadenosine in both tissues but did not affect responses evoked by acetylcholine (atria) or sodium nitrite (aorta). 4 The xanthines, 8-PST, XAC, D-Lys XAC, XCC and DPPX appeared to be competitive antagonists of the effects of 2-chloroadenosine, as Schild plot slopes did not differ significantly from unity. The 1,3-dipropyl substituted compounds had pA2 values from 6.5 to 7.4 and were more potent than the 1,3 dimethyl substituted 8-PST (pA2 4.9 to 5). 5 For individual xanthines, there was no difference between pA2 values obtained in the atria and in the aorta.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3664093

  1. Molybdenum(VI) salts convert the xanthine oxidoreductase apoprotein into the active enzyme in mouse L929 fibroblastic cells.

    PubMed Central

    Falciani, F; Terao, M; Goldwurm, S; Ronchi, A; Gatti, A; Minoia, C; Li Calzi, M; Salmona, M; Cazzaniga, G; Garattini, E

    1994-01-01

    The mouse L929 fibroblastic cell line presents low, but detectable, levels of the mRNA encoding xanthine oxidoreductase under basal conditions, and it responds to type I and type II interferons by inducing the expression of the transcript [Falciani, Ghezzi, Terao, Cazzaniga, and Garattini (1992) Biochem. J. 285, 1001-1008]. This cell line, however, does not show any detectable amount of xanthine oxidoreductase enzymic activity, either before or after treatment with the cytokines. Molybdenum(VI) salts, in the millimolar range, are capable of activating xanthine oxidoreductase in L929 cells both under basal conditions and after treatment with interferon-alpha. The increase is observed in mouse L929 as well as in clones derived from it, but not in many other human and mouse cell lines. The induction observed in L929 cells is post-translational in nature and it is insensitive to cycloheximide, indicating that the molybdenum ion converts a pool of inactive xanthine oxidoreductase apoenzyme into its holoenzymic form. When grown in the absence of sodium molybdate, the L929 cell line has undetectable intracellular levels of the molybdenum cofactor, since the cell extracts are unable to complement the nitrate reductase defect of the nit-1 mutant of Neurospora crassa. L929 cells grown in the presence of millimolar concentrations of sodium molybdate, however, become competent to complement the nit-1 defect. L929 cells accumulate molybdenum ion inside the intracellular compartment as efficiently as TEnd cells, a mouse endothelial cell line that expresses xanthine oxidoreductase activity both under basal conditions and after treatment with interferon-gamma, suggesting that L929 cells have a defect in one or more of the metabolic steps leading to the synthesis of the molybdenum cofactor. Images Figure 1 Figure 2 Figure 4 PMID:8129733

  2. The terminal oxidases of Paracoccus denitrificans.

    PubMed

    de Gier, J W; Lübben, M; Reijnders, W N; Tipker, C A; Slotboom, D J; van Spanning, R J; Stouthamer, A H; van der Oost, J

    1994-07-01

    Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding subunit I of the aa3-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (delta ctaDI, delta ctaDII), indicating that they are isoforms of subunit I of the aa3-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. This protohaem-containing oxidase, called cytochrome bb3, is the only quinol oxidase expressed under the conditions used. In a triple oxidase mutant (delta ctaDI, delta ctaDII, cyoB::KmR) an alternative cytochrome c oxidase has been characterized; this cbb3-type oxidase has been partially purified. Both cytochrome aa3 and cytochrome bb3 are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb3 has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.

  3. Acyclic Immucillin Phosphonates. Second-Generation Inhibitors of Plasmodium falciparum Hypoxanthine- Guanine-Xanthine Phosphoribosyltransferase

    SciTech Connect

    Hazelton, Keith Z.; Ho, Meng-Chaio; Cassera, Maria B.; Clinch, Keith; Crump, Douglas R.; Rosario Jr., Irving; Merino, Emilio F.; Almo, Steve C.; Tyler, Peter C.; Schramm, Vern L.

    2012-06-22

    We found that Plasmodium falciparum is the primary cause of deaths from malaria. It is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. We present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.

  4. The Reductive Half-reaction of Xanthine Dehydrogenase from Rhodobacter capsulatus

    PubMed Central

    Hall, James; Reschke, Stefan; Cao, Hongnan; Leimkühler, Silke; Hille, Russ

    2014-01-01

    The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu232 in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu232 being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu232 of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme. PMID:25258317

  5. Expression and purification of a functional uric acid-xanthine transporter (UapA).

    PubMed

    Leung, James; Karachaliou, Mayia; Alves, Claudia; Diallinas, George; Byrne, Bernadette

    2010-07-01

    The Nucleobase-Ascorbate Transporters (NATs) family includes carriers with fundamental functions in uptake of key cellular metabolites, such as uric acid or vitamin C. The best studied example of a NAT transporter is the uric acid-xanthine permease (UapA) from the model ascomycete Aspergillus nidulans. Detailed genetic and biochemical analyses have revealed much about the mechanism of action of this protein; however, the difficulties associated with handling eukaryotic membrane proteins have limited efforts to elucidate the precise structure-function relationships of UapA by structural analysis. In this manuscript, we describe the heterologous overexpression of functional UapA as a fusion with GFP in different strains of Saccharomyces cerevisiae. The UapA-GFP construct expressed to 2.3 mg/L in a pep4Delta deletion strain lacking a key vacuolar endopeptidase and 3.8 mg/L in an npi1-1 mutant strain with defective Rsp5 ubiquitin ligase activity. Epifluorescence microscopy revealed that the UapA-GFP was predominately localized to the plasma membrane in both strains, although a higher intensity of fluorescence was observed for the npi1-1 mutant strain plasma membrane. In agreement with these observations, the npi1-1 mutant strain demonstrated a approximately 5-fold increase in uptake of [(3)H]-xanthine compared to the pep4Delta deletion strain. Despite yielding the best results for functional expression, in-gel fluorescence of the UapA-GFP expressed in the npi1-1 mutant strain revealed that the protein was subject to significant proteolytic degradation. Large scale expression of the protein using the pep4Delta deletion strain followed by purification produced mg quantities of pure, monodispersed protein suitable for further structural and functional studies. In addition, this work has generated a yeast cell based system for performing reverse genetics and other targeted approaches, in order to further understand the mechanism of action of this important model protein.

  6. Allopurinol ameliorates thioacetamide-induced acute liver failure by regulating cellular redox-sensitive transcription factors in rats.

    PubMed

    Demirel, Ulvi; Yalniz, Mehmet; Aygün, Cem; Orhan, Cemal; Tuzcu, Mehmet; Sahin, Kazim; Ozercan, Ibrahim Hanifi; Bahçecioğlu, Ibrahim Halil

    2012-08-01

    Oxidative stress plays important role in the development of acute liver failure. In this study, we investigated effects of allopurinol (AP) upon thioacetamide (TAA)-induced liver injury and the potential mechanisms leading to amelioration in inflammation with AP treatment. Acute liver failure was induced by intraperitoneal administration of TAA (300 mg/kg/day for 2 days). Thirty-five rats were divided into five groups as control (group 1), TAA (group 2), TAA + 25AP (group 3), TAA + 50 AP (group 4), and TAA + 100AP (group 5). The number of animals in each group was seven. At the end of the study, histopathological, biochemical, and western blot analysis were done. TAA treatment significantly increased serum levels of aminotransferases, liver malondialdehyde (MDA), nuclear factor-kappa B (NF-қB ), activator protein-1 (AP-1), tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) levels, and the necro-inflammation scores. Nevertheless, nuclear factor E2-related factor-2 and heme oxygenase-1 (HO-1) expressions in the liver were decreased by TAA. AP treatment significantly lowered the serum levels of aminotransferases (P < 0.01) and liver MDA, NF-κB, AP-1, TNF-α, COX-2, and IL-6 expressions (P < 0.05). Moreover, AP restored the liver Nrf2 and HO-1 expressions and improved the necro-inflammation scores significantly. AP improves oxidative stress-induced liver damage by regulating cellular redox-sensitive transcriptor factors and expression of pro-inflammatory and antioxidant defense mechanisms. AP probably exerts these beneficiary features by its free radical scavenging ability in a dose-dependent manner.

  7. Monoamine Oxidase Inhibitors: Clinical Review

    PubMed Central

    Remick, Ronald A.; Froese, Colleen

    1990-01-01

    Monoamine oxidase inhibitors (MAOIs) are effective antidepressant agents. They are increasingly and effectively used in a number of other psychiatric and non-psychiatric medical syndromes. Their potential for serious toxicity (i.e., hypertensive reaction) is far less than original reports suggest, and newer reversible substrate-specific MAOIs may offer even less toxicity. The author reviews the pharmacology, mechanism of action, clinical indications, and dosing strategies of MAOIs. The common MAOI side-effects (hypotension, weight gain, sexual dysfunction, insomnia, daytime sedation, myoclonus, and hypertensive episodes) are described and management techniques suggested. Recent clinical developments involving MAOIs are outlined. PMID:21233984

  8. Use of allopurinol with low-dose 6-mercaptopurine in inflammatory bowel disease to achieve optimal active metabolite levels: A review of four cases and the literature

    PubMed Central

    Witte, Todd N; Ginsberg, Allen L

    2008-01-01

    BACKGROUND: At least one-third of patients with inflammatory bowel disease do not respond or are intolerant to therapy with 6-mercaptopurine (6-MP). A subgroup fails to attain optimal levels of 6-thioguanine nucleotide (6-TGN) and instead shunts to 6-methylmercaptopurine nucleotide (6-MMPN). PATIENTS AND METHODS: A retrospective chart review was conducted, and four patients are described who had been previously unable to achieve optimal 6-TGN metabolite levels until allopurinol was added to their treatment. RESULTS: All four patients achieved optimal 6-TGN levels and undetectable 6-MMPN with a mean 6-MP dose of 0.49 mg/kg. Three achieved steroid-free clinical remission. Two of those three patients had normalization of liver enzymes; one patient had baseline normal liver enzymes despite an initial 6-MMPN level of 27,369 pmol/8×108 red blood cells. Two patients experienced reversible leukopenia. CONCLUSIONS: Combination allopurinol and low-dose 6-MP is an effective means to achieve optimal metabolite levels and steroid-free clinical remission in previously refractory patients. Caution is advised. PMID:18299738

  9. Mitochondrial cytochrome c oxidase deficiency.

    PubMed

    Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique; Bouchereau, Juliette; Schiff, Manuel; El-Khoury, Riyad; Tzagoloff, Alexander; Rustin, Pierre

    2016-03-01

    As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance of studying different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy.

  10. Mitochondrial Cytochrome c Oxidase Deficiency

    PubMed Central

    Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique; Bouchereau, Juliette; Schiff, Manuel; El-Khoury, Riyad; Tzagoloff, Alexander; Rustin, Pierre

    2016-01-01

    As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance to study different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy. PMID:26846578

  11. Miracle Fruit (Synsepalum dulcificum) Exhibits as a Novel Anti-Hyperuricaemia Agent.

    PubMed

    Shi, Yeu-Ching; Lin, Kai-Sian; Jhai, Yi-Fen; Lee, Bao-Hong; Han, Yifan; Cui, Zhibin; Hsu, Wei-Hsuan; Wu, She-Ching

    2016-01-26

    Miracle fruit (Synsepalum dulcificum) belongs to the Sapotaceae family. It can change flavors on taste buds, transforming acidic tastes to sweet. We evaluated various miracle fruit extracts, including water, butanol, ethyl acetate (EA), and hexane fractions, to determine its antioxidant effects. These extracts isolated from miracle fruit exerted potential for reduction of uric acid and inhibited xanthine oxidase activity in vitro and in monosodiumurate (MSU)-treated RAW264.7 macrophages. Moreover, we also found that the butanol extracts of miracle fruit attenuated oxonic acid potassium salt-induced hyperuricaemia in ICR mice by lowering serum uric acid levels and activating hepatic xanthine oxidase. These effects were equal to those of allopurinol, suggesting that the butanol extract of miracle fruit could be developed as a novel anti-hyperuricaemia agent or health food.

  12. Gout - a guide for the general and acute physicians.

    PubMed

    Abhishek, Abhishek; Roddy, Edward; Doherty, Michael

    2017-02-01

    Gout is the most prevalent inflammatory arthritis and affects 2.5% of the general population in the UK. It is also the only arthritis that has the potential to be cured with safe, inexpensive and well tolerated urate-lowering treatments, which reduce serum uric acid by either inhibiting xanthine oxidase - eg allopurinol, febuxostat - or by increasing the renal excretion of uric acid. Of these, xanthine oxidase inhibitors are used first line and are effective in 'curing' gout in the vast majority of patients. Gout can be diagnosed on clinical grounds in those with typical podagra. However, in those with involvement of other joints, joint aspiration is recommended to demonstrate monosodium urate crystals and exclude other causes of acute arthritis, such as septic arthritis. However, a clinical diagnosis of gout can be made if joint aspiration is not feasible. This review summarises the current understanding of the pathophysiology, clinical presentation, investigations and treatment of gout.

  13. Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor

    PubMed Central

    Hirano, Kazufumi; Chen, Woei Shin; Chueng, Adeline L.W.; Dunne, Angela A.; Seredenina, Tamara; Filippova, Aleksandra; Ramachandran, Sumitra; Bridges, Angela; Chaudry, Laiq; Pettman, Gary; Allan, Craig; Duncan, Sarah; Lee, Kiew Ching; Lim, Jean; Ma, May Thu; Ong, Agnes B.; Ye, Nicole Y.; Nasir, Shabina; Mulyanidewi, Sri; Aw, Chiu Cheong; Oon, Pamela P.; Liao, Shihua; Li, Dizheng; Johns, Douglas G.; Miller, Neil D.; Davies, Ceri H.; Browne, Edward R.; Matsuoka, Yasuji; Chen, Deborah W.; Jaquet, Vincent

    2015-01-01

    Abstract Aims: The NADPH oxidase (NOX) family of enzymes catalyzes the formation of reactive oxygen species (ROS). NOX enzymes not only have a key role in a variety of physiological processes but also contribute to oxidative stress in certain disease states. To date, while numerous small molecule inhibitors have been reported (in particular for NOX2), none have demonstrated inhibitory activity in vivo. As such, there is a need for the identification of improved NOX inhibitors to enable further evaluation of the biological functions of NOX enzymes in vivo as well as the therapeutic potential of NOX inhibition. In this study, both the in vitro and in vivo pharmacological profiles of GSK2795039, a novel NOX2 inhibitor, were characterized in comparison with other published NOX inhibitors. Results: GSK2795039 inhibited both the formation of ROS and the utilization of the enzyme substrates, NADPH and oxygen, in a variety of semirecombinant cell-free and cell-based NOX2 assays. It inhibited NOX2 in an NADPH competitive manner and was selective over other NOX isoforms, xanthine oxidase, and endothelial nitric oxide synthase enzymes. Following systemic administration in mice, GSK2795039 abolished the production of ROS by activated NOX2 enzyme in a paw inflammation model. Furthermore, GSK2795039 showed activity in a murine model of acute pancreatitis, reducing the levels of serum amylase triggered by systemic injection of cerulein. Innovation and Conclusions: GSK2795039 is a novel NOX2 inhibitor that is the first small molecule to demonstrate inhibition of the NOX2 enzyme in vivo. Antioxid. Redox Signal. 23, 358–374. PMID:26135714

  14. Identification of Crucial Amino Acids in Mouse Aldehyde Oxidase 3 That Determine Substrate Specificity

    PubMed Central

    Mahro, Martin; Brás, Natércia F.; Cerqueira, Nuno M. F. S. A.; Teutloff, Christian; Coelho, Catarina; Romão, Maria João; Leimkühler, Silke

    2013-01-01

    In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis. PMID:24358164

  15. A Novel Reaction Mediated by Human Aldehyde Oxidase: Amide Hydrolysis of GDC-0834

    PubMed Central

    Wong, Susan; Kirkpatrick, Donald S.; Liu, Lichuan; Khojasteh, S. Cyrus; Hop, Cornelis E. C. A.; Barr, John T.; Jones, Jeffrey P.; Halladay, Jason S.

    2015-01-01

    GDC-0834, a Bruton’s tyrosine kinase inhibitor investigated as a potential treatment of rheumatoid arthritis, was previously reported to be extensively metabolized by amide hydrolysis such that no measurable levels of this compound were detected in human circulation after oral administration. In vitro studies in human liver cytosol determined that GDC-0834 (R)-N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo- 4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b] thiophene-2-carboxamide) was rapidly hydrolyzed with a CLint of 0.511 ml/min per milligram of protein. Aldehyde oxidase (AO) and carboxylesterase (CES) were putatively identified as the enzymes responsible after cytosolic fractionation and mass spectrometry-proteomics analysis of the enzymatically active fractions. Results were confirmed by a series of kinetic experiments with inhibitors of AO, CES, and xanthine oxidase (XO), which implicated AO and CES, but not XO, as mediating GDC-0834 amide hydrolysis. Further supporting the interaction between GDC-0834 and AO, GDC-0834 was shown to be a potent reversible inhibitor of six known AO substrates with IC50 values ranging from 0.86 to 1.87 μM. Additionally, in silico modeling studies suggest that GDC-0834 is capable of binding in the active site of AO with the amide bond of GDC-0834 near the molybdenum cofactor (MoCo), orientated in such a way to enable potential nucleophilic attack on the carbonyl of the amide bond by the hydroxyl of MoCo. Together, the in vitro and in silico results suggest the involvement of AO in the amide hydrolysis of GDC-0834. PMID:25845827

  16. A novel ring oxidation of 4- or 5-substituted 2H-oxazole to corresponding 2-oxazolone catalyzed by cytosolic aldehyde oxidase.

    PubMed

    Arora, Vinod K; Philip, Thomas; Huang, Stella; Shu, Yue-Zhong

    2012-09-01

    The ring oxidation of 2H-oxazole, or C2-unsubstituted oxazole, to 2-oxazolone, a cyclic carbamate, was observed on various 4- or 5-substituted oxazoles. Using 5-(3-bromophenyl)oxazole as a model compound, its 2-oxazolone metabolite M1 was fully characterized by liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance. The reaction mainly occurred in the liver cytosolic fraction without the requirement of cytochrome P450 enzymes and cofactor NADPH. Investigations into the mechanism of formation of 2-oxazolone using various chemical inhibitors indicated that the reaction was primarily catalyzed by aldehyde oxidase and not by xanthine oxidase. In addition, cytosol incubation of 5-(3-bromophenyl)oxazole in the medium containing H₂¹⁸O led to the ¹⁸O incorporation into M1, substantiating the reaction mechanism of a typical molybdenum hydroxylase. The rank order of liver cytosols for the 2-oxazolone formation was mouse > monkey ≫ rat and human liver cytosol, whereas M1 was not formed in dog liver cytosol. Because the reaction was observed with a number of 4- or 5-substituted 2H-oxazoles in mouse liver cytosols, 2H-oxazoles represent a new substrate chemotype for ring oxidation catalyzed by aldehyde oxidase.

  17. Genetic Heterogeneity within Electrophoretic "Alleles" of Xanthine Dehydrogenase in DROSOPHILA PSEUDOOBSCURA

    PubMed Central

    Singh, R. S.; Lewontin, R. C.; Felton, A. A.

    1976-01-01

    An experimental plan for an exhaustive determination of genic variation at structural gene loci is presented. In the initial steps of this program, 146 isochromosomal lines from 12 geographic populations of D. pseudoobscura were examined for allelic variation of xanthine dehydrogenase by the serial use of 4 different electrophoretic conditions and a heat stability test. The 5 criteria revealed a total of 37 allelic classes out of the 146 genomes examined where only 6 had been previously revealed by the usual method of gel electrophoresis. This immense increase in genic variation also showed previously unsuspected population differences between the main part of the species distribution and the isolated population of Bogotá, Colombia, in conformity with the known partial reproductive isolation of the Bogotá population. The average heterozygosity at the Xdh locus is at least 72% in natural populations. This result, together with the very large number of alleles segregating and the pattern of allelic frequencies, has implications for theories of genetic polymorphism which are discussed. PMID:1001881

  18. Study on inclusion complex of cyclodextrin with methyl xanthine derivatives by fluorimetry

    NASA Astrophysics Data System (ADS)

    Wei, Yan-Li; Ding, Li-Hua; Dong, Chuan; Niu, Wei-Ping; Shuang, Shao-Min

    2003-10-01

    The inclusion complexes of β-cyclodextrin (β-CD) and HP-β-cyclodextrin (HP-β-CD) with caffeine, theophylline and theobromine were investigated by fluorimetry. Various factors affecting the formation of inclusion complexes were discussed in detail including forming time, pH effect and temperature. The results indicate that inclusion process was affected seriously by laying time and pH. The forming time of β-CD inclusion complexes is much longer than that of HP-β-CD. The optimum pH range is about 7-12 for caffeine, 8-10 for TP, 10.5-12 for TB. The intensities of their fluorescence increase with the decreasing of temperature. Their maximum excitation wavelengths are all in the range of 280-290 nm. The emission wavelength of caffeine and theophylline are both in the range of 340-360 nm, and that of theobromine is about 325 nm. The fluorescence signals are intensified with the increasing concentration of CD. The stoichiometry of the inclusion complexes of CD with these three methyl xanthine derivatives are all 1:1 and the formation constant are all calculated.

  19. Xanthine microsensor based on polypyrrole molecularly imprinted film modified carbon fiber microelectrodes.

    PubMed

    Liu, Bin; Wang, Xiao-Li; Lian, Hui-Ting; Sun, Xiang-Ying

    2013-09-15

    A molecularly imprinted polymers (MIPs) microsensor was presented as a carbon fiber microelectrode (CFME) coating for specifically recognizing xanthine (Xan). The polymeric film was obtained based on the imprinted procedure of electropolymerization of pyrrole in the presence of the template molecule Xan by cyclic voltammetry, and template was removed by magnetic stirring. Under the optimum conditions, a satisfactory molecularly binding selectivity of Xan was obtained from the MIPs microsensor with an imprinting factor (IF) of 6.63 and a linear response to concentration in certain ranges. The ranges are from 4.0 × 10⁻⁶ to 6.0 × 10⁻⁵ M and from 8.0 × 10⁻⁵ to 2.0 × 10⁻³ M with a detection limit of 2.5 × 10⁻⁷ M. Meanwhile, good stability (relative standard deviation [RSD] = 3.2%, n = 10) and reproducibility (RSD = 2.0%, n = 10) were observed, and recoveries ranging from 96.9 to 102.5% were calculated when applied to Xan determination in real blood serum samples.

  20. Catalysis and Structure of Zebrafish Urate Oxidase Provide Insights into the Origin of Hyperuricemia in Hominoids

    PubMed Central

    Marchetti, Marialaura; Liuzzi, Anastasia; Fermi, Beatrice; Corsini, Romina; Folli, Claudia; Speranzini, Valentina; Gandolfi, Francesco; Bettati, Stefano; Ronda, Luca; Cendron, Laura; Berni, Rodolfo; Zanotti, Giuseppe; Percudani, Riccardo

    2016-01-01

    Urate oxidase (Uox) catalyses the first reaction of oxidative uricolysis, a three-step enzymatic pathway that allows some animals to eliminate purine nitrogen through a water-soluble compound. Inactivation of the pathway in hominoids leads to elevated levels of sparingly soluble urate and puts humans at risk of hyperuricemia and gout. The uricolytic activities lost during evolution can be replaced by enzyme therapy. Here we report on the functional and structural characterization of Uox from zebrafish and the effects on the enzyme of the missense mutation (F216S) that preceded Uox pseudogenization in hominoids. Using a kinetic assay based on the enzymatic suppression of the spectroscopic interference of the Uox reaction product, we found that the F216S mutant has the same turnover number of the wild-type enzyme but a much-reduced affinity for the urate substrate and xanthine inhibitor. Our results indicate that the last functioning Uox in hominoid evolution had an increased Michaelis constant, possibly near to upper end of the normal range of urate in the human serum (~300 μM). Changes in the renal handling of urate during primate evolution can explain the genetic modification of uricolytic activities in the hominoid lineage without the need of assuming fixation of deleterious mutations. PMID:27922051

  1. Dietary inhibitors of monoamine oxidase A.

    PubMed

    Dixon Clarke, Sarah E; Ramsay, Rona R

    2011-07-01

    Inhibition of monoamine oxidase is one way to treat depression and anxiety. The information now available on the pharmacokinetics of flavonoids and of the components of tobacco prompted an exploration of whether a healthy diet (with or without smoking) provides active compounds in amounts sufficient to partially inhibit monoamine oxidase. A literature search was used to identify dietary monoamine oxidase inhibitors, the levels of these compounds in foods, the pharmacokinetics of the absorption and distribution, and tissue levels observed. An estimated daily intake and the expected tissue concentrations were compared with the measured efficacies of the compounds as inhibitors of monoamine oxidases. Norharman, harman and quercetin dietary presence, pharmacokinetics, and tissue levels were consistent with significant levels reaching neuronal monoamine oxidase from the diet or smoking; 1,2,3,4-tetrahydroisoquinoline, eugenol, 1-piperoylpiperidine, and coumarin were not. Quercetin was equipotent with norharman as a monoamine oxidase A inhibitor and its metabolite, isorhamnetin, also inhibits. Total quercetin was the highest of the compounds in the sample diet. Although bioavailability was variable depending on the source, a healthy diet contains amounts of quercetin that might give sufficient amounts in brain to induce, by monoamine oxidase A inhibition, a small decrease in neurotransmitter breakdown.

  2. The relaxant and spasmogenic effects of some xanthine derivatives acting on guinea-pig isolated trachealis muscle.

    PubMed Central

    Small, R. C.; Boyle, J. P.; Cortijo, J.; Curtis-Prior, P. B.; Davies, J. M.; Foster, R. W.; Hofer, P.

    1988-01-01

    1. Caffeine (10 mM)-induced relaxation of guinea-pig isolated trachealis was attenuated and converted to a small spasmogenic response on cooling to 22 degrees C. The relaxant response was restored on rewarming to 37 degrees C and was abolished by indomethacin (2.8 microM). Cooling to 22 degrees C in the presence of indomethacin revealed spasmogenic responses to caffeine which were abolished on rewarming to 37 degrees C. 2. Trachealis treated with indomethacin (2.8 microM) was repeatedly dosed with acetylcholine (ACh, 10 microM). Caffeine (1 or 10 mM), added as each ACh-induced spasm reached equilibrium, transiently augmented but then suppressed the spasm. On cooling from 37 degrees C to 12 degrees C, the increment in spasm evoked by caffeine increased relative to the spasm evoked by ACh. 3. Trachealis treated with indomethacin (2.8 microM) was repeatedly dosed with caffeine (10 mM). At 37 degrees C caffeine had little effect but it caused spasm when the tissue was cooled to 32 degrees C. Spasm amplitude increased as cooling progressed to 12 degrees C. Similar results were obtained with caffeine (1 mM). 4. At 37 degrees C, caffeine, enprofylline, 1,3,7,9-tetramethylxanthinium (TMX), theobromine, theophylline, xanthine and forskolin each caused concentration-dependent suppression of tracheal tone. Among the xanthine derivatives the rank order of potency was enprofylline greater than theophylline greater than caffeine greater than theobromine greater than xanthine greater than TMX. 5. In trachealis treated with indomethacin (2.8 microM) and maintained at 12 degrees C, the xanthines each caused concentration-dependent spasm. The rank order of potency was theobromine greater than or equal to theophylline greater than or equal to caffeine greater than or equal to enprofylline greater than xanthine greater than TMX. Forskolin was devoid of spasmogenic activity. 6. Trachealis treated with indomethacin (2.8 microM) and maintained at 12 degrees C, was repeatedly dosed with

  3. Structural insights into sulfite oxidase deficiency.

    PubMed

    Karakas, Erkan; Wilson, Heather L; Graf, Tyler N; Xiang, Song; Jaramillo-Busquets, Sandra; Rajagopalan, K V; Kisker, Caroline

    2005-09-30

    Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

  4. Selective cytotoxicity of an oxygen-radical-generating enzyme conjugated to a monoclonal antibody.

    PubMed Central

    Battelli, M G; Abbondanza, A; Tazzari, P L; Dinota, A; Rizzi, S; Grassi, G; Gobbi, M; Stirpe, F

    1988-01-01

    The monoclonal antibody 8A, which recognizes a human plasma cell-associated antigen, was covalently linked to xanthine oxidase in a conjugate maintaining both immunological and enzymatic properties. A significant degree of target cell lysis was obtained at an enzyme concentration that was ineffective on non-target cells and on myeloid staminal cells (CFU-GM). The cytotoxic activity was abolished by an excess of antibody, by allopurinol and by superoxide dismutase and catalase. A possible use of the conjugate for bone marrow purging in multiple myeloma patients is suggested. PMID:3262464

  5. Prokaryotic origins for the mitochondrial alternative oxidase and plastid terminal oxidase nuclear genes.

    PubMed

    Finnegan, Patrick M; Umbach, Ann L; Wilce, Jackie A

    2003-12-18

    The mitochondrial alternative oxidase is a diiron carboxylate quinol oxidase (Dox) found in plants and some fungi and protists, but not animals. The plastid terminal oxidase is distantly related to alternative oxidase and is most likely also a Dox protein. Database searches revealed that the alpha-proteobacterium Novosphingobium aromaticivorans and the cyanobacteria Nostoc sp. PCC7120, Synechococcus sp. WH8102 and Prochlorococcus marinus subsp. pastoris CCMP1378 each possess a Dox homolog. Each prokaryotic protein conforms to the current structural models of the Dox active site and phylogenetic analyses suggest that the eukaryotic Dox genes arose from an ancestral prokaryotic gene.

  6. Activation of polyphenol oxidase of chloroplasts.

    PubMed

    Tolbert, N E

    1973-02-01

    Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or -18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles x mg(-1) chlorophyll x hr(-1). Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes.Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30

  7. Direct Activation of β-Cell KATP Channels with a Novel Xanthine Derivative

    PubMed Central

    Raphemot, Rene; Swale, Daniel R.; Dadi, Prasanna K.; Jacobson, David A.; Cooper, Paige; Wojtovich, Andrew P.; Banerjee, Sreedatta; Nichols, Colin G.

    2014-01-01

    ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet β-cell membrane potential, calcium influx, and insulin secretion, and consequently, represent important drug targets for metabolic disorders of glucose homeostasis. The KATP channel opener diazoxide is used clinically to treat intractable hypoglycemia caused by excessive insulin secretion, but its use is limited by off-target effects due to lack of potency and selectivity. Some progress has been made in developing improved Kir6.2/SUR1 agonists from existing chemical scaffolds and compound screening, but there are surprisingly few distinct chemotypes that are specific for SUR1-containing KATP channels. Here we report the serendipitous discovery in a high-throughput screen of a novel activator of Kir6.2/SUR1: VU0071063 [7-(4-(tert-butyl)benzyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione]. The xanthine derivative rapidly and dose-dependently activates Kir6.2/SUR1 with a half-effective concentration (EC50) of approximately 7 μM, is more efficacious than diazoxide at low micromolar concentrations, directly activates the channel in excised membrane patches, and is selective for SUR1- over SUR2A-containing Kir6.1 or Kir6.2 channels, as well as Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and voltage-gated potassium channel 2.1. Finally, we show that VU0071063 activates native Kir6.2/SUR1 channels, thereby inhibiting glucose-stimulated calcium entry in isolated mouse pancreatic β cells. VU0071063 represents a novel tool/compound for investigating β-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry. PMID:24646456

  8. Decreased xanthine oxidoreductase is a predictor of poor prognosis in early‐stage gastric cancer

    PubMed Central

    Linder, N; Haglund, C; Lundin, M; Nordling, S; Ristimäki, A; Kokkola, A; Mrena, J; Wiksten, J‐P; Lundin, J

    2006-01-01

    Background Xanthine oxidoreductase (XOR) is a key enzyme in the degradation of DNA, RNA and high‐energy phosphates. About half of the patients with breast cancer have a decrease in XOR expression. Patients with breast cancer with unfavourable prognosis are independently identified by the loss of XOR. Aim To assess the clinical relevance of XOR expression in gastric cancer. Methods XOR levels were studied by immunohistochemistry in tissue microarray specimens of 337 patients with gastric cancer and the relation between XOR expression and a series of clinicopathological variables, as well as disease‐specific survival, was assessed. Results XOR was moderately decreased in 41% and was undetectable in another 14% of the tumours compared with the corresponding normal tissue. Decreased XOR was associated with advanced stage, deep tumour penetration, diffusely spread tumour location, positive lymph node status, large tumour size, non‐curative disease, cellular aneuploidy, high S‐phase fraction and high cyclooxygenase‐2 expression, but not with p53 expression or Borrmann classification. Down regulation of XOR was associated with unfavourable outcome, and the cumulative 5‐year gastric cancer‐specific survival in patients with strong XOR expression was 47%, compared with 22% in those with moderate to negative expression (p<0.001). This was also true in patients with stage I–II (p = 0.01) and lymph node‐negative (p = 0.02) disease, as well as in patients with smaller (⩽5 cm) tumours (p = 0.02). Conclusion XOR expression in gastric cancer may be a new marker for a more aggressive gastric cancer biology, similar to that previously reported for breast cancer. PMID:16935971

  9. Azide inhibition of urate oxidase.

    PubMed

    Gabison, Laure; Colloc'h, Nathalie; Prangé, Thierry

    2014-07-01

    The inhibition of urate oxidase (UOX) by azide was investigated by X-ray diffraction techniques and compared with cyanide inhibition. Two well characterized sites for reagents are present in the enzyme: the dioxygen site and the substrate-binding site. To examine the selectivity of these sites towards azide inhibition, several crystallization conditions were developed. UOX was co-crystallized with azide (N3) in the presence or absence of either uric acid (UA, the natural substrate) or 8-azaxanthine (8AZA, a competitive inhibitor). In a second set of experiments, previously grown orthorhombic crystals of the UOX-UA or UOX-8AZA complexes were soaked in sodium azide solutions. In a third set of experiments, orthorhombic crystals of UOX with the exchangeable ligand 8-nitroxanthine (8NXN) were soaked in a solution containing uric acid and azide simultaneously (competitive soaking). In all assays, the soaking periods were either short (a few hours) or long (one or two months). These different experimental conditions showed that one or other of the sites, or the two sites together, could be inhibited. This also demonstrated that azide not only competes with dioxygen as cyanide does but also competes with the substrate for its enzymatic site. A model in agreement with experimental data would be an azide in equilibrium between two sites, kinetically in favour of the dioxygen site and thermodynamically in favour of the substrate-binding site.

  10. Azide inhibition of urate oxidase

    PubMed Central

    Gabison, Laure; Colloc’h, Nathalie; Prangé, Thierry

    2014-01-01

    The inhibition of urate oxidase (UOX) by azide was investigated by X-ray diffraction techniques and compared with cyanide inhibition. Two well characterized sites for reagents are present in the enzyme: the dioxygen site and the substrate-binding site. To examine the selectivity of these sites towards azide inhibition, several crystallization conditions were developed. UOX was co-crystallized with azide (N3) in the presence or absence of either uric acid (UA, the natural substrate) or 8-azaxanthine (8AZA, a competitive inhibitor). In a second set of experiments, previously grown orthorhombic crystals of the UOX–UA or UOX–8AZA complexes were soaked in sodium azide solutions. In a third set of experiments, orthorhombic crystals of UOX with the exchangeable ligand 8-nitroxanthine (8NXN) were soaked in a solution containing uric acid and azide simultaneously (competitive soaking). In all assays, the soaking periods were either short (a few hours) or long (one or two months). These different experimental conditions showed that one or other of the sites, or the two sites together, could be inhibited. This also demonstrated that azide not only competes with dioxygen as cyanide does but also competes with the substrate for its enzymatic site. A model in agreement with experimental data would be an azide in equilibrium between two sites, kinetically in favour of the dioxygen site and thermodynamically in favour of the substrate-binding site. PMID:25005084

  11. Investigation of solvent polarity effect on molecular structure and vibrational spectrum of xanthine with the aid of quantum chemical computations.

    PubMed

    Polat, Turgay; Yıldırım, Gurcan

    2014-04-05

    The main scope of this study is to determine the effects of 8 solvents on the geometric structure and vibrational spectra of the title compound, xanthine, by means of the DFT/B3LYP level of theory in the combination with the polarizable conductor continuum model (CPCM) for the first time. After determination of the most-steady state (favored structure) of the xanthine molecule, the role of the solvent polarity on the SCF energy (for the molecule stability), atomic charges (for charge distribution) and dipole moments (for molecular charge transfer) belonging to tautomer is discussed in detail. The results obtained indicate not only the presence of the hydrogen bonding and strong intra-molecular charge transfer (ICT) in the compound but the increment of the molecule stability with the solvent polarity, as well. Moreover, it is noted that the optimized geometric parameters and the theoretical vibrational frequencies are in good agreement with the available experimental results found in the literature. In fact, the correlations between the experimental and theoretical findings for the molecular structures improve with the enhancement of the solvent polarity. At the same time, the dimer forms of the xanthine compound are simulated to describe the effect of intermolecular hydrogen bonding on the molecular geometry and vibrational frequencies. It is found that the CO and NH stretching vibrations shift regularly to lower frequency value with higher IR intensity as the dielectric medium enhances systematically due to the intermolecular NH⋯O hydrogen bonds. Theoretical vibrational spectra are also assigned based on the potential energy distribution (PED) using the VEDA 4 program.

  12. GPCR Ligand Dendrimer (GLiDe) Conjugates: Adenosine Receptor Interactions of a Series of Multivalent Xanthine Antagonists

    PubMed Central

    Kecskés, Angela; Tosh, Dilip K.; Wei, Qiang; Gao, Zhan-Guo; Jacobson, Kenneth A.

    2011-01-01

    Previously, G protein–coupled receptor (GPCR) agonists were tethered from polyamidoamine (PAMAM) dendrimers to provide high receptor affinity and selectivity. Here we prepared GPCR Ligand Dendrimer (GLiDe) conjugates from a potent adenosine receptor (AR) antagonist; such agents are of interest for treating Parkinson’s disease, asthma, and other conditions. Xanthine amine congener (XAC) was appended with an alkyne group on an extended C8 substituent for coupling by Cu(I)-catalyzed click chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. These conjugates also contained triazole-linked PEG groups (8 or 22 moieties per 64 terminal positions) for increasing water-solubility and optionally prosthetic groups for spectroscopic characterization and affinity labeling. Human AR binding affinity increased progressively with the degree of xanthine substitution to reach Ki values in the nM range. The order of affinity of each conjugate was hA2AAR > hA3AR > hA1AR, while the corresponding monomer was ranked hA2AAR > hA1AR ≥ hA3AR. The antagonist activity of the most potent conjugate 14 (34 xanthines per dendrimer) was examined at the Gi-coupled A1AR. Conjugate 14 at 100 nM right-shifted the AR agonist concentration-response curve in a cyclic AMP functional assay in a parallel manner, but at 10 nM (lower than its Ki value) it significantly suppressed the maximal agonist effect in calcium mobilization. This is the first systematic probing of a potent AR antagonist tethered on a dendrimer and its activity as a function of variable loading. PMID:21539392

  13. Stabilizing the heterologously expressed uric acid-xanthine transporter UapA from the lower eukaryote Aspergillus nidulans.

    PubMed

    Leung, James; Cameron, Alexander D; Diallinas, George; Byrne, Bernadette

    2013-02-01

    Despite detailed genetic and mutagenic analysis and a recent high-resolution structure of a bacterial member of the nucleobase-ascorbate transporter (NAT) family, understanding of the mechanism of action of eukaryotic NATs is limited. Preliminary studies successfully expressed and purified wild-type UapA to high homogeneity; however, the protein was extremely unstable, degrading almost completely after 48 h at 4°C. In an attempt to increase UapA stability we generated a number of single point mutants (E356D, E356Q, N409A, N409D, Q408E and G411V) previously shown to have reduced or no transport activity, but correct targeting to the membrane. The mutant UapA constructs expressed well as GFP fusions in Saccharomyces cerevisiae and exhibited similar fluorescent size exclusion chromatography (FSEC) profiles to the wild-type protein, following solubilization in 1% DDM, LDAO or OM + 1 mM xanthine. In order to assess the relative stabilities of the mutants, solubilized fractions prepared in 1% DDM + 1 mM xanthine were heated at 45°C for 10 min prior to FSEC. The Q408E and G411V mutants gave markedly better profiles than either wild-type or the other mutants. Further FSEC analysis following solubilization of the mutants in 1% NG ± xanthine confirmed that G411V is more stable than the other mutants, but showed that Q408E is unstable under these conditions. G411V and an N-terminally truncated construct G411VΔ1-11 were submitted to large-scale expression and purification. Long-term stability analysis revealed that G411VΔ1-11 was the most stable construct and the most suited to downstream structural studies.

  14. Mechanisms of lung neutrophil activation after hemorrhage or endotoxemia: roles of reactive oxygen intermediates, NF-kappa B, and cyclic AMP response element binding protein.

    PubMed

    Shenkar, R; Abraham, E

    1999-07-15

    Acute inflammatory lung injury occurs frequently in the setting of severe infection or blood loss. Accumulation of activated neutrophils in the lungs and increased pulmonary proinflammatory cytokine levels are major characteristics of acute lung injury. In the present experiments, we examined mechanisms leading to neutrophil accumulation and activation in the lungs after endotoxemia or hemorrhage. Levels of IL-1 beta, TNF-alpha, and macrophage inflammatory protein-2 mRNA were increased in lung neutrophils from endotoxemic or hemorrhaged mice compared with those present in lung neutrophils from control mice or in peripheral blood neutrophils from endotoxemic, hemorrhaged, or control mice. The transcriptional regulatory factors NF-kappa B and cAMP response element binding protein were activated in lung but not blood neutrophils after hemorrhage or endotoxemia. Xanthine oxidase inhibition, achieved by feeding allopurinol or tungsten-containing diets, did not affect neutrophil trafficking to the lungs after hemorrhage or endotoxemia. Xanthine oxidase inhibition did prevent hemorrhage- but not endotoxemia-induced increases in proinflammatory cytokine expression among lung neutrophils. Hemorrhage- or endotoxemia-associated activation of NF-kappa B in lung neutrophils was not affected by inhibition of xanthine oxidase. cAMP response element binding protein activation was increased after hemorrhage, but not endotoxemia, in mice fed xanthine oxidase-inhibiting diets. Our results indicate that xanthine oxidase modulates cAMP response element binding protein activation and proinflammatory cytokine expression in lung neutrophils after hemorrhage, but not endotoxemia. These findings suggest that the mechanisms leading to acute inflammatory lung injury after hemorrhage differ from those associated with endotoxemia.

  15. Selective inhibition of human acetylcholinesterase by xanthine derivatives: in vitro inhibition and molecular modeling investigations.

    PubMed

    Mohamed, Tarek; Osman, Wesseem; Tin, Gary; Rao, Praveen P N

    2013-08-01

    The commonly used beverage and psychostimulant caffeine is known to inhibit human acetylcholinesterase enzyme. This pharmacological activity of caffeine is partly responsible for its cognition enhancing properties. However, the exact mechanisms of its binding to human cholinesterases (acetyl and butyrylcholinesterase; hAChE and hBuChE) are not well known. In this study, we investigated the cholinesterase inhibition by the xanthine derivatives caffeine, pentoxifylline, and propentofylline. Among them, propentofylline was the most potent AChE inhibitor (hAChE IC₅₀=6.40 μM). The hAChE inhibitory potency was of the order: caffeine (hAChE IC₅₀=7.25 μM)50 μM) relative to the reference agent donepezil (hBuChE IC₅₀=13.60 μM). Molecular modeling investigations indicate that caffeine binds primarily in the catalytic site (Ser203, Glu334 and His447) region of hAChE whereas pentoxifylline and propentofylline are able to bind to both the catalytic site and peripheral anionic site due to their increased bulk/size, thereby exhibiting superior AChE inhibition relative to caffeine. In contrast, their lack of hBuChE inhibition is due to a larger binding site and lack of key aromatic amino acids. In summary, our study has important implications in the development of novel caffeine derivatives as selective AChE inhibitors with potential application as cognitive enhancers and to treat various forms of dementia.

  16. Cinnamaldehyde and allopurinol reduce fructose-induced cardiac inflammation and fibrosis by attenuating CD36-mediated TLR4/6-IRAK4/1 signaling to suppress NLRP3 inflammasome activation.

    PubMed

    Kang, Lin-Lin; Zhang, Dong-Mei; Ma, Chun-Hua; Zhang, Jian-Hua; Jia, Ke-Ke; Liu, Jia-Hui; Wang, Rong; Kong, Ling-Dong

    2016-06-08

    Fructose consumption induces metabolic syndrome to increase cardiovascular disease risk. Cinnamaldehyde and allopurinol possess anti-oxidative and anti-inflammatory activity to relieve heart injury in metabolic syndrome. But the mechanisms of fructose-induced cardiac injury, and cardioprotective effects of cinnamaldehyde and allopurinol are not completely understood. In this study, fructose-fed rats displayed metabolic syndrome with elevated serum ox-LDL, cardiac oxidative stress, inflammation and fibrosis. Scavenger receptor CD36, Toll-like receptor 4 (TLR4), TLR6, IL-1R-associated kinase 4/1 (IRAK4/1), nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome, interleukin-1β, transforming growth factor-β (TGF-β), drosophila mothers against DPP homolog (Smad) 2/3 phosphorylation and Smad4 were increased in animal and H9c2 cell models. These pathological processes were further evaluated in ox-LDL or fructose-exposed H9c2 cells pretreated with ROS scavenger and CD36 specific inhibitor, or IRAK1/4 inhibitor, and transfected with CD36, NLRP3, or IRAK4/1 siRNA, demonstrating that NLPR3 inflammasome activation through CD36-mediated TLR4/6-IRAK4/1 signaling may promote cardiac inflammation and fibrosis. Cinnamaldehyde and allopurinol reduced cardiac oxidative stress to suppress NLPR3 inflammasome activation and TGF-β/Smads signaling by inhibiting CD36-mediated TLR4/6-IRAK4/1 signaling under fructose induction. These results suggest that the blockage of CD36-mediated TLR4/6-IRAK4/1 signaling to suppress NLRP3 inflammasome activation by cinnamaldehyde and allopurinol may protect against fructose-induced cardiac inflammation and fibrosis.

  17. Cinnamaldehyde and allopurinol reduce fructose-induced cardiac inflammation and fibrosis by attenuating CD36-mediated TLR4/6-IRAK4/1 signaling to suppress NLRP3 inflammasome activation

    PubMed Central

    Kang, Lin-Lin; Zhang, Dong-Mei; Ma, Chun-Hua; Zhang, Jian-Hua; Jia, Ke-Ke; Liu, Jia-Hui; Wang, Rong; Kong, Ling-Dong

    2016-01-01

    Fructose consumption induces metabolic syndrome to increase cardiovascular disease risk. Cinnamaldehyde and allopurinol possess anti-oxidative and anti-inflammatory activity to relieve heart injury in metabolic syndrome. But the mechanisms of fructose-induced cardiac injury, and cardioprotective effects of cinnamaldehyde and allopurinol are not completely understood. In this study, fructose-fed rats displayed metabolic syndrome with elevated serum ox-LDL, cardiac oxidative stress, inflammation and fibrosis. Scavenger receptor CD36, Toll-like receptor 4 (TLR4), TLR6, IL-1R-associated kinase 4/1 (IRAK4/1), nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome, interleukin-1β, transforming growth factor-β (TGF-β), drosophila mothers against DPP homolog (Smad) 2/3 phosphorylation and Smad4 were increased in animal and H9c2 cell models. These pathological processes were further evaluated in ox-LDL or fructose-exposed H9c2 cells pretreated with ROS scavenger and CD36 specific inhibitor, or IRAK1/4 inhibitor, and transfected with CD36, NLRP3, or IRAK4/1 siRNA, demonstrating that NLPR3 inflammasome activation through CD36-mediated TLR4/6-IRAK4/1 signaling may promote cardiac inflammation and fibrosis. Cinnamaldehyde and allopurinol reduced cardiac oxidative stress to suppress NLPR3 inflammasome activation and TGF-β/Smads signaling by inhibiting CD36-mediated TLR4/6-IRAK4/1 signaling under fructose induction. These results suggest that the blockage of CD36-mediated TLR4/6-IRAK4/1 signaling to suppress NLRP3 inflammasome activation by cinnamaldehyde and allopurinol may protect against fructose-induced cardiac inflammation and fibrosis. PMID:27270216

  18. Effect of naphthalene on cytochrome oxidase activity

    SciTech Connect

    Harmon, H.J.

    1988-01-01

    Previous reports have demonstrated that naphthalene inhibits oxygen consumption in Daphnia magna tissue culture cells, and intact mitochondria and submitochondrial particles. These studies were extended to algal mitochondrial respiration as well as photosynthetic activity. The authors were able to demonstrate the specific site of apparent respiratory inhibition to be coenzyme Q (ubiquinone, UQ) and later to demonstrate the molecular basis of this inhibition at ubiquinone. The authors previously could not demonstrate an effect of naphthalene on cytochrome oxidase activity. However, the observation that naphthalene can stimulate respiration in algae prompted the reinvestigation of the effect of naphthalene on the kinetics of cytochrome oxidase. Cytochrome oxidase is a multi-subunit membranous protein responsible for the oxidation of cytochrome c and the reduction of molecular oxygen to water. Because of the complicated nature and mechanism of this enzyme, the potential exists for multiple and possibly opposite effects of naphthalene on its function.

  19. Validation of a Rapid, Robust, Inexpensive Screening Method for Detecting the HLA-B*58:01 Allele in the Prevention of Allopurinol-Induced Severe Cutaneous Adverse Reactions.

    PubMed

    Nguyen, Dinh Van; Vida, Christopher; Chu, Hieu Chi; Fulton, Richard; Li, Jamma; Fernando, Suran L

    2017-01-01

    The HLA B*58:01 allele has been worldwide reported as a pharmacogenetic susceptibility to allopurinol-induced severe cutaneous adverse reactions (SCARs). To prevent these life-threatening conditions, the American College of Rheumatology hingly recommended that the HLA-B*58:01 be screened prior to the initiation of allopurinol therapy. Therefore, we developed a rapid, robust, inexpensive screening method using SYBR® Green real time PCR to detect the HLA-B*58:01 allele. A total of 119 samples were tested. The assay has a sensitivity of 100% (95% CI: 69.15%-100%), a specificity of 100% (95% CI: 96.67%-100%), a positive predictive value of 100% (95% CI: 69.15%-100%) and a negative predictive value of 100% (95% CI: 96.67%-100%). HLA-B*58:01 genotyping results showed 100% agreement with those obtained from Luminex SSO/SBT/SSP. The lowest limit of detection of this method is 0.8 ng/μL of DNA. The unit cost of the test is only $3.8 USD. This novel screening test using SYBR® real time PCR would be appropriate to identify individuals with the HLA-B*58:01 allele for the prevention of allopurinol-induced SCARs.

  20. Potential Pharmacologic Treatments for Cystinuria and for Calcium Stones Associated with Hyperuricosuria

    SciTech Connect

    Goldfarb, David S.

    2012-03-14

    Two new potential pharmacologic therapies for recurrent stone disease are described. The role of hyperuricosuria in promoting calcium stones is controversial with only some but not all epidemiologic studies demonstrating associations between increasing urinary uric acid excretion and calcium stone disease. The relationship is supported by the ability of uric acid to 'salt out' (or reduce the solubility of) calcium oxalate in vitro. A randomized, controlled trial of allopurinol in patients with hyperuricosuria and normocalciuria was also effective in preventing recurrent stones. Febuxostat, a nonpurine inhibitor of xanthine oxidase (also known as xanthine dehydrogenase or xanthine oxidoreductase) may have advantages over allopurinol and is being tested in a similar protocol, with the eventual goal of determining whether urate-lowering therapy prevents recurrent calcium stones. Treatments for cystinuria have advanced little in the past 30 years. Atomic force microscopy has been used recently to demonstrate that effective inhibition of cystine crystal growth is accomplished at low concentrations of L-cystine methyl ester and L-cystine dimethyl ester, structural analogs of cystine that provide steric inhibition of crystal growth. In vitro, L-cystine dimethyl ester had a significant inhibitory effect on crystal growth. The drug's safety and effectiveness will be tested in an Slc3a1 knockout mouse that serves as an animal model of cystinuria.

  1. Purification of the Alpha Glycerophosphate Oxidase From Trypanosomes.

    DTIC Science & Technology

    1992-08-01

    is the purifica- tion of the glycerphosphate oxidase from the terminal oxidase in bloodstream trypanosomes. African trypanosomiasis remains one of the...oxidase from the terminal oxidase in bloodstream trypanosomes. African trypanosomiasis remains one of the major diseases in the world today, affecting...interest as a possible target for drug chemotherapy . At present only suramin and organic arsenicals remain as the mainstay of chemotherapy , despite their

  2. An oxidase road to platelet adhesion.

    PubMed

    Krause, Diane S

    2016-03-17

    Platelet adhesion to collagen via collagen receptors is an important part of thrombosis. In this issue of Blood, Matsuura et al identify collagen receptors as previously unrecognized targets of the extracellular enzyme lysyl oxidase (LOX), the level of which is increased in myeloproliferative neoplasms (MPNs) and other conditions associated with pathological thromboses.

  3. Polyphenol oxidase activity in annual forage clovers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO)-mediated phenol reactions in red clover (Trifolium pratense L.) bind forage protein and reduce proteolysis, producing beneficial effects on forage protein degradability, silage fermentation, and soil-N cycling. We evaluated PPO activity in seven previously untested annual c...

  4. A colorimetric assay for cytokinin oxidase.

    PubMed

    Libreros-Minotta, C A; Tipton, P A

    1995-11-01

    A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal and p-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.

  5. Exploiting algal NADPH oxidase for biophotovoltaic energy.

    PubMed

    Anderson, Alexander; Laohavisit, Anuphon; Blaby, Ian K; Bombelli, Paolo; Howe, Christopher J; Merchant, Sabeeha S; Davies, Julia M; Smith, Alison G

    2016-01-01

    Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.

  6. Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition.

    PubMed Central

    Koenig, K; Andreesen, J R

    1990-01-01

    The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with [185W]tungstate. This labeling was used as a reporter to purify both labile proteins. The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps. Both radioactive proteins were separated and purified to homogeneity. Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively. With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis. Molybdate was absolutely required for incorporation of 185W, formation of cross-reacting material, and enzymatic activity. The latter parameters showed a perfect correlation. This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase. The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000. The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme. The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined. The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Fu1 was catalyzed by two different dehydrogenases. Images PMID:2170335

  7. Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism.

    PubMed

    Yang, Ting; Peleli, Maria; Zollbrecht, Christa; Giulietti, Alessia; Terrando, Niccolo; Lundberg, Jon O; Weitzberg, Eddie; Carlström, Mattias

    2015-06-01

    Oxidative stress contributes to the pathogenesis of many disorders, including diabetes and cardiovascular disease. Immune cells are major sources of superoxide (O2(∙-)) as part of the innate host defense system, but exaggerated and sustained O2(∙-) generation may lead to progressive inflammation and organ injuries. Previous studies have proven organ-protective effects of inorganic nitrite, a precursor of nitric oxide (NO), in conditions manifested by oxidative stress and inflammation. However, the mechanisms are still not clear. This study aimed at investigating the potential role of nitrite in modulating NADPH oxidase (NOX) activity in immune cells. Mice peritoneal macrophages or human monocytes were activated by lipopolysaccharide (LPS), with or without coincubation with nitrite. O2(∙-) and peroxynitrite (ONOO(-)) formation were detected by lucigenin-based chemiluminescence and fluorescence techniques, respectively. The intracellular NO production was measured by DAF-FM DA fluorescence. NOX isoforms and inducible NO synthase (iNOS) expression were detected by qPCR. LPS increased both O2(∙-) and ONOO(-) production in macrophages, which was significantly reduced by nitrite (10µmol/L). Mechanistically, the effects of nitrite are (1) linked to increased NO generation, (2) similar to that observed with the NO donor DETA-NONOate, and (3) can be abolished by the NO scavenger carboxy-PTIO or by the xanthine oxidase (XO) inhibitor febuxostat. Nox2 expression was increased in activated macrophages, but was not influenced by nitrite. However, nitrite attenuated LPS-induced upregulation of iNOS expression. Similar to that observed in mice macrophages, nitrite also reduced O2(∙-) generation in LPS-activated human monocytes. In conclusion, XO-mediated reduction of nitrite attenuates NOX activity in activated macrophages, which may modulate the inflammatory response.

  8. Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

    PubMed Central

    Yin, DeLu (Tyler); Urresti, Saioa; Lafond, Mickael; Johnston, Esther M.; Derikvand, Fatemeh; Ciano, Luisa; Berrin, Jean-Guy; Henrissat, Bernard; Walton, Paul H.; Davies, Gideon J.; Brumer, Harry

    2015-01-01

    Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure–function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications. PMID:26680532

  9. Inhibition of rat fat cell lipolysis by monoamine oxidase and semicarbazide-sensitive amine oxidase substrates.

    PubMed

    Visentin, Virgile; Prévot, Danielle; Marti, Luc; Carpéné, Christian

    2003-04-18

    It has been demonstrated that amine oxidase substrates stimulate glucose transport in cardiomyocytes and adipocytes, promote adipogenesis in pre-adipose cell lines and lower blood glucose in diabetic rats. These insulin-like effects are dependent on amine oxidation by semicarbazide-sensitive amine oxidase or by monoamine oxidase. The present study aimed to investigate whether amine oxidase substrates also exhibit another insulin-like property, the inhibition of lipolysis. We therefore tested the influence of tyramine and benzylamine on lipolytic activity in rat adipocytes. These amines did not modify basal lipolysis but dose-dependently counteracted the stimulation induced by lipolytic agents. The response to 10 nM isoprenaline was totally inhibited by tyramine 1 mM. The blockade produced by inhibition of amine oxidase activity or by 1 mM glutathione suggested that the generation of oxidative species, which occurs during amine oxidation, was involved in tyramine antilipolytic effect. Among the products resulting from amine oxidation, only hydrogen peroxide was antilipolytic in a manner that was potentiated by vanadate, as for tyramine or benzylamine. Antilipolytic responses to tyramine and to insulin were sensitive to wortmannin. These data suggest that inhibition of lipolysis is a novel insulin-like effect of amine oxidase substrates which is mediated by hydrogen peroxide generated during amine oxidation.

  10. The Overexpression of NALP3 Inflammasome in Knee Osteoarthritis Is Associated with Synovial Membrane Prolidase and NADPH Oxidase 2.

    PubMed

    Clavijo-Cornejo, Denise; Martínez-Flores, Karina; Silva-Luna, Karina; Martínez-Nava, Gabriela Angélica; Fernández-Torres, Javier; Zamudio-Cuevas, Yessica; Guadalupe Santamaría-Olmedo, Mónica; Granados-Montiel, Julio; Pineda, Carlos; López-Reyes, Alberto

    2016-01-01

    Osteoarthritis is characterized by the presence of proinflammatory cytokines and reactive oxygen species. We aimed to clarify the role of prooxidant enzyme content at the synovial membrane level and how it correlates with the inflammatory process in patients with knee osteoarthritis (KOA). In synovial membranes from KOA patients and control group, we analyzed the protein content of prooxidant enzymes such as Nox2, xanthine oxidase (XO), and prolidase as well as the proinflammatory NALP3. Results show that protein content of prolidase and Nox2 increased 4.8- and 8.4-fold, respectively, and XO showed an increasing trend, while the NALP3 inflammasome increased 5.4-fold with respect to control group. Levels of prolidase and XO had a positive correlation between the levels of NALP3 and Nox2. By principal component analysis the protein expression pattern by study groups was evaluated. Three clusters were identified; protein expression patterns were higher for clusters two (prolidase) and three (XO and Nox2) between KOA patients and controls. Data suggest that prooxidant enzymes increase in synovial membrane of KOA patients and may contribute to the inflammatory state and degradation of the articular cartilage.

  11. The Overexpression of NALP3 Inflammasome in Knee Osteoarthritis Is Associated with Synovial Membrane Prolidase and NADPH Oxidase 2

    PubMed Central

    Clavijo-Cornejo, Denise; Martínez-Flores, Karina; Silva-Luna, Karina; Fernández-Torres, Javier; Zamudio-Cuevas, Yessica; Guadalupe Santamaría-Olmedo, Mónica

    2016-01-01

    Osteoarthritis is characterized by the presence of proinflammatory cytokines and reactive oxygen species. We aimed to clarify the role of prooxidant enzyme content at the synovial membrane level and how it correlates with the inflammatory process in patients with knee osteoarthritis (KOA). In synovial membranes from KOA patients and control group, we analyzed the protein content of prooxidant enzymes such as Nox2, xanthine oxidase (XO), and prolidase as well as the proinflammatory NALP3. Results show that protein content of prolidase and Nox2 increased 4.8- and 8.4-fold, respectively, and XO showed an increasing trend, while the NALP3 inflammasome increased 5.4-fold with respect to control group. Levels of prolidase and XO had a positive correlation between the levels of NALP3 and Nox2. By principal component analysis the protein expression pattern by study groups was evaluated. Three clusters were identified; protein expression patterns were higher for clusters two (prolidase) and three (XO and Nox2) between KOA patients and controls. Data suggest that prooxidant enzymes increase in synovial membrane of KOA patients and may contribute to the inflammatory state and degradation of the articular cartilage. PMID:27777643

  12. The reductive half-reaction of xanthine dehydrogenase from Rhodobacter capsulatus: the role of Glu232 in catalysis.

    PubMed

    Hall, James; Reschke, Stefan; Cao, Hongnan; Leimkühler, Silke; Hille, Russ

    2014-11-14

    The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme.

  13. HPTLC Analysis, Antioxidant and Antigout Activity of Indian Plants

    PubMed Central

    Nile, Shivraj Hariram; Park, Se Won

    2014-01-01

    The HPTLC analysis, antioxidant, and antigout activity of Asparagus racemosus, Withania somnifera, Vitex negundo, Plumbago zeylanica, Butea monosperma and Tephrosia purpurea extracts were investigated. The chemical fingerprinting were carried out by high performance thin layer chromatography (HPTLC), antioxidant activity by ABTS, DPPH, FRAP radical scavenging assays, and antiogout activity by cow milk xanthine oxidase. The HPTLC fingerprint qualitatively revealed predominant amount of flavonoids. The TEAC values ranged from 45.80 to 140 µM trolox/100 g dry weight for ABTS, from 85 to 430 µM trolox/ 100 g dw DPPH, and 185 to 560 µM trolox/100 g dw for FRAP respectively. Plants used in this study was found to inhibit the toxicity, as seen from the decreased LPO and increased GSH, SOD and CAT levels. The total phenolic and flavonoid content ranged from 10.21 to 28.17 and 5.80 to 10.1 mg of gallic acid equivalents (GAE)/100 gdw respectively. The plant extracts demonstrated significant xanthine oxidase inhibitory activity at 100 g/mL and revealed an inhibition greater than 50 % and IC50 values below the standard. This effect was almost similar to the activity of allopurinol (Standard drug) against xanthine oxidase (90.2 ± 0.4 %). These plant root extract will be subjected for further extensive studies to isolate and identify their active constituents which are useful for against inflammation and gout. PMID:25237348

  14. HPTLC Analysis, Antioxidant and Antigout Activity of Indian Plants.

    PubMed

    Nile, Shivraj Hariram; Park, Se Won

    2014-01-01

    The HPTLC analysis, antioxidant, and antigout activity of Asparagus racemosus, Withania somnifera, Vitex negundo, Plumbago zeylanica, Butea monosperma and Tephrosia purpurea extracts were investigated. The chemical fingerprinting were carried out by high performance thin layer chromatography (HPTLC), antioxidant activity by ABTS, DPPH, FRAP radical scavenging assays, and antiogout activity by cow milk xanthine oxidase. The HPTLC fingerprint qualitatively revealed predominant amount of flavonoids. The TEAC values ranged from 45.80 to 140 µM trolox/100 g dry weight for ABTS, from 85 to 430 µM trolox/ 100 g dw DPPH, and 185 to 560 µM trolox/100 g dw for FRAP respectively. Plants used in this study was found to inhibit the toxicity, as seen from the decreased LPO and increased GSH, SOD and CAT levels. The total phenolic and flavonoid content ranged from 10.21 to 28.17 and 5.80 to 10.1 mg of gallic acid equivalents (GAE)/100 gdw respectively. The plant extracts demonstrated significant xanthine oxidase inhibitory activity at 100 g/mL and revealed an inhibition greater than 50 % and IC50 values below the standard. This effect was almost similar to the activity of allopurinol (Standard drug) against xanthine oxidase (90.2 ± 0.4 %). These plant root extract will be subjected for further extensive studies to isolate and identify their active constituents which are useful for against inflammation and gout.

  15. Maternal fructose drives placental uric acid production leading to adverse fetal outcomes

    PubMed Central

    Asghar, Zeenat A.; Thompson, Alysha; Chi, Maggie; Cusumano, Andrew; Scheaffer, Suzanne; Al-Hammadi, Noor; Saben, Jessica L.; Moley, Kelle H.

    2016-01-01

    Maternal metabolic diseases increase offspring risk for low birth weight and cardiometabolic diseases in adulthood. Excess fructose consumption may confer metabolic risks for both women and their offspring. However, the direct consequences of fructose intake per se are unknown. We assessed the impact of a maternal high-fructose diet on the fetal-placental unit in mice in the absence of metabolic syndrome and determined the association between maternal serum fructose and placental uric acid levels in humans. In mice, maternal fructose consumption led to placental inefficiency, fetal growth restriction, elevated fetal serum glucose and triglyceride levels. In the placenta, fructose induced de novo uric acid synthesis by activating the activities of the enzymes AMP deaminase and xanthine oxidase. Moreover, the placentas had increased lipids and altered expression of genes that control oxidative stress. Treatment of mothers with the xanthine oxidase inhibitor allopurinol reduced placental uric acid levels, prevented placental inefficiency, and improved fetal weights and serum triglycerides. Finally, in 18 women delivering at term, maternal serum fructose levels significantly correlated with placental uric acid levels. These findings suggest that in mice, excess maternal fructose consumption impairs placental function via a xanthine oxidase/uric acid-dependent mechanism, and similar effects may occur in humans. PMID:27125896

  16. Maternal fructose drives placental uric acid production leading to adverse fetal outcomes.

    PubMed

    Asghar, Zeenat A; Thompson, Alysha; Chi, Maggie; Cusumano, Andrew; Scheaffer, Suzanne; Al-Hammadi, Noor; Saben, Jessica L; Moley, Kelle H

    2016-04-29

    Maternal metabolic diseases increase offspring risk for low birth weight and cardiometabolic diseases in adulthood. Excess fructose consumption may confer metabolic risks for both women and their offspring. However, the direct consequences of fructose intake per se are unknown. We assessed the impact of a maternal high-fructose diet on the fetal-placental unit in mice in the absence of metabolic syndrome and determined the association between maternal serum fructose and placental uric acid levels in humans. In mice, maternal fructose consumption led to placental inefficiency, fetal growth restriction, elevated fetal serum glucose and triglyceride levels. In the placenta, fructose induced de novo uric acid synthesis by activating the activities of the enzymes AMP deaminase and xanthine oxidase. Moreover, the placentas had increased lipids and altered expression of genes that control oxidative stress. Treatment of mothers with the xanthine oxidase inhibitor allopurinol reduced placental uric acid levels, prevented placental inefficiency, and improved fetal weights and serum triglycerides. Finally, in 18 women delivering at term, maternal serum fructose levels significantly correlated with placental uric acid levels. These findings suggest that in mice, excess maternal fructose consumption impairs placental function via a xanthine oxidase/uric acid-dependent mechanism, and similar effects may occur in humans.

  17. Natural Compounds as Modulators of NADPH Oxidases

    PubMed Central

    2013-01-01

    Reactive oxygen species (ROS) are cellular signals generated ubiquitously by all mammalian cells, but their relative unbalance triggers also diseases through intracellular damage to DNA, RNA, proteins, and lipids. NADPH oxidases (NOX) are the only known enzyme family with the sole function to produce ROS. The NOX physiological functions concern host defence, cellular signaling, regulation of gene expression, and cell differentiation. On the other hand, increased NOX activity contributes to a wide range of pathological processes, including cardiovascular diseases, neurodegeneration, organ failure, and cancer. Therefore targeting these enzymatic ROS sources by natural compounds, without affecting the physiological redox state, may be an important tool. This review summarizes the current state of knowledge of the role of NOX enzymes in physiology and pathology and provides an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols. PMID:24381714

  18. Lysyl Oxidase and the Tumor Microenvironment

    PubMed Central

    Wang, Tong-Hong; Hsia, Shih-Min; Shieh, Tzong-Ming

    2016-01-01

    The lysyl oxidase (LOX) family of oxidases contains a group of extracellular copper-dependent enzymes that catalyze the cross-linking of collagen and elastin by oxidation, thus maintaining the rigidity and structural stability of the extracellular matrix (ECM). Aberrant expression or activation of LOX alters the cellular microenvironment, leading to many diseases, including atherosclerosis, tissue fibrosis, and cancer. Recently, a number of studies have shown that LOX is overexpressed in most cancers and that it is involved in the regulation of tumor progression and metastasis. In contrast, a few reports have also indicated the tumor-suppressing role of LOX. In this short review, we discuss recent research on the correlations between LOX and cancer. Further, the role of LOX in tumor microenvironment remodeling, tumorigenesis, and metastasis and the underlying mechanisms have also been elucidated. PMID:28036074

  19. Strain differences in the responsiveness between Sprague-Dawley and Fischer rats to nephropathy induced by FYX-051, a xanthine oxidoreductase inhibitor

    SciTech Connect

    Ashizawa, Naoki . E-mail: L2-26899@fujiyakuhin.co.jp; Shimo, Takeo; Matsumoto, Koji; Oba, Kazuhiko; Nakazawa, Takashi; Nagata, Osamu

    2006-12-15

    To determine a rat strain appropriate for carcinogenicity testing of FYX-051, a xanthine oxidoreductase inhibitor, we performed a 4-week oral toxicity study by administering 0.3, 1 and 3 mg/kg, and 1, 3 and 10 mg/kg of FYX-051 to male Sprague-Dawley (SD) and Fischer (F344) rats, respectively. Histopathology revealed that the degree of FYX-051-induced nephropathy was 3-fold stronger in SD rats than in F344 rats. Our previous study demonstrated that the key factor of species differences in FYX-051-induced nephropathy is purine metabolism. This observation led us to examine the involvement of purine metabolism in differences among two strains of rats. However, purine metabolism was proven not to be implicated as an important factor. Subsequently, other factors responsible for the strain differences were examined. FYX-051-induced increases in plasma xanthine concentrations were higher in SD rats than in F344 rats, suggesting more remarkable effects on pharmacodynamics in the former than the latter. Urinary volume was greater in F344 rats administered 10 mg/kg of FYX-051 (6.8 ml/h/kg) than in SD rats administered 3 mg/kg of FYX-051 (5.0 ml/h/kg), implying easier xanthine excretion in the former. Urinary xanthine solubility was 55 mg/dl in F344 rats aged 6 weeks, in contrast to 38 mg/dl in SD rats of the same age. Also, there were no significant differences in exposure levels at the same dose between SD and F344 rats. The outcomes of exposure levels and renal histopathology in both rats suggest the possibility that F344 rats could be exposed to a 3-fold higher amount of drug than SD rats in a carcinogenicity bioassay. The present study, therefore, suggested that strain differences of nephrotoxicity were caused by the combined effects of pharmacodynamics, xanthine excretion capacity, and urinary xanthine solubility. Furthermore, these results indicate that F344 rats would be a suitable strain for the carcinogenicity study of FYX-051.

  20. Lysyl oxidase mediates hypoxic control of metastasis.

    PubMed

    Erler, Janine T; Giaccia, Amato J

    2006-11-01

    Hypoxic cancer cells pose a great challenge to the oncologist because they are especially aggressive, metastatic, and resistant to therapy. Recently, we showed that elevation of the extracellular matrix protein lysyl oxidase (LOX) correlates with metastatic disease and is essential for hypoxia-induced metastasis. In an orthotopic rodent model of breast cancer, a small-molecule or antibody inhibitor of LOX abolished metastasis, offering preclinical validation of this enzyme as a therapeutic target.

  1. Ligand interactions with galactose oxidase: mechanistic insights.

    PubMed Central

    Whittaker, M M; Whittaker, J W

    1993-01-01

    Interactions between galactose oxidase and small molecules have been explored using a combination of optical absorption, circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to detect complex formation and characterize the products. Anions bind directly to the cupric center in both active and inactive galactose oxidase, converting to complexes with optical and EPR spectra that are distinctly different from those of the starting aquo enzyme. Azide binding is coupled to stoichiometric proton uptake by the enzyme, reflecting the generation of a strong base (pKa > 9) in the active site anion adduct. At low temperature, the aquo enzyme converts to a form that exhibits the characteristic optical and EPR spectra of an anion complex, apparently reflecting deprotonation of the coordinated water. Anion binding results in a loss of the optical transition arising from coordinated tyrosine, implying displacement of the axial tyrosine ligand on forming the adduct. Nitric oxide binds to galactose oxidase, forming a specific complex exhibiting an unusual EPR spectrum with all g values below 2. The absence of Cu splitting in this spectrum and the observation that the cupric EPR signal from the active site metal ion is not significantly decreased in the complex suggest a nonmetal interaction site for NO in galactose oxidase. These results have been interpreted in terms of a mechanistic scheme where substrate binding displaces a tyrosinate ligand from the active site cupric ion, generating a base that may serve to deprotonate the coordinated hydroxyl group of the substrate, activating it for oxidation. The protein-NO interactions may probe a nonmetal O2 binding site in this enzyme. PMID:8386015

  2. Imaging Monoamine Oxidase in the Human Brain

    SciTech Connect

    Fowler, J. S.; Volkow, N. D.; Wang, G-J.; Logan, Jean

    1999-11-10

    Positron emission tomography (PET) studies mapping monoamine oxidase in the human brain have been used to measure the turnover rate for MAO B; to determine the minimum effective dose of a new MAO inhibitor drug lazabemide and to document MAO inhibition by cigarette smoke. These studies illustrate the power of PET and radiotracer chemistry to measure normal biochemical processes and to provide information on the effect of drug exposure on specific molecular targets.

  3. Tetrazolium Oxidase Polymorphism in Rainbow Trout

    PubMed Central

    Cederbaum, Stephen D.; Yoshida, Akira

    1972-01-01

    Tetrazolium oxidase from the blood and liver of rainbow trout was found to be genetically polymorphic. The inheritance pattern of the liver enzyme was compatible only with a one locus-two allele hypothesis. The enzymes in the blood while having an electrophoretically identical polymorphism could differ genotypically from that of the liver in a given fish. The significance of these findings to the understanding of the evolution of the salmonid genome is discussed. PMID:4675090

  4. Acrolein activates matrix metalloproteinases by increasing reactive oxygen species in macrophages

    SciTech Connect

    O'Toole, Timothy E. Zheng Yuting; Hellmann, Jason; Conklin, Daniel J.; Barski, Oleg; Bhatnagar, Aruni

    2009-04-15

    Acrolein is a ubiquitous component of environmental pollutants such as automobile exhaust, cigarette, wood, and coal smoke. It is also a natural constituent of several foods and is generated endogenously during inflammation or oxidation of unsaturated lipids. Because increased inflammation and episodic exposure to acrolein-rich pollutants such as traffic emissions or cigarette smoke have been linked to acute myocardial infarction, we examined the effects of acrolein on matrix metalloproteinases (MMPs), which destabilize atherosclerotic plaques. Our studies show that exposure to acrolein resulted in the secretion of MMP-9 from differentiated THP-1 macrophages. Acrolein-treatment of macrophages also led to an increase in reactive oxygen species (ROS), free intracellular calcium ([Ca{sup 2+}]{sub i}), and xanthine oxidase (XO) activity. ROS production was prevented by allopurinol, but not by rotenone or apocynin and by buffering changes in [Ca{sup 2+}]{sub I} with BAPTA-AM. The increase in MMP production was abolished by pre-treatment with the antioxidants Tiron and N-acetyl cysteine (NAC) or with the xanthine oxidase inhibitors allopurinol or oxypurinol. Finally, MMP activity was significantly stimulated in aortic sections from apoE-null mice containing advanced atherosclerotic lesions after exposure to acrolein ex vivo. These observations suggest that acrolein exposure results in MMP secretion from macrophages via a mechanism that involves an increase in [Ca{sup 2+}]{sub I}, leading to xanthine oxidase activation and an increase in ROS production. ROS-dependent activation of MMPs by acrolein could destabilize atherosclerotic lesions during brief episodes of inflammation or pollutant exposure.

  5. Arabidopsis alternative oxidase sustains Escherichia coli respiration.

    PubMed Central

    Kumar, A M; Söll, D

    1992-01-01

    Glutamyl-tRNA reductase, encoded by the hemA gene, is the first enzyme in porphyrin biosynthesis in many organisms. Hemes, important porphyrin derivatives, are essential components of redox enzymes, such as cytochromes. Thus a hemA Escherichia coli strain (SASX41B) is deficient in cytochrome-mediated aerobic respiration. Upon complementation of this strain with an Arabidopsis thaliana cDNA library, we isolated a clone which permitted the SASX41B strain to grow aerobically. The clone encodes the gene for Arabidopsis alternative oxidase, whose deduced amino acid sequence was found to have 71% identity with that of the enzyme from the voodoo lily, Sauromatum guttatum. The Arabidopsis protein is expressed as a 31-kDa protein in E. coli and confers on this organism cyanide-resistant growth, which in turn is sensitive to salicylhydroxamate. This implies that a single polypeptide is sufficient for alternative oxidase activity. Based on these observations we propose that a cyanide-insensitive respiratory pathway operates in the transformed E. coli hemA strain. Introduction of this pathway now opens the way to genetic/molecular biological investigations of alternative oxidase and its cofactor. Images PMID:1438286

  6. NAD(P)H oxidase and renal epithelial ion transport

    PubMed Central

    Schreck, Carlos

    2011-01-01

    A fundamental requirement for cellular vitality is the maintenance of plasma ion concentration within strict ranges. It is the function of the kidney to match urinary excretion of ions with daily ion intake and nonrenal losses to maintain a stable ionic milieu. NADPH oxidase is a source of reactive oxygen species (ROS) within many cell types, including the transporting renal epithelia. The focus of this review is to describe the role of NADPH oxidase-derived ROS toward local renal tubular ion transport in each nephron segment and to discuss how NADPH oxidase-derived ROS signaling within the nephron may mediate ion homeostasis. In each case, we will attempt to identify the various subunits of NADPH oxidase and reactive oxygen species involved and the ion transporters, which these affect. We will first review the role of NADPH oxidase on renal Na+ and K+ transport. Finally, we will review the relationship between tubular H+ efflux and NADPH oxidase activity. PMID:21270341

  7. Exploring non-covalent interactions in guanine- and xanthine-based model DNA quadruplex structures: a comprehensive quantum chemical approach.

    PubMed

    Yurenko, Yevgen P; Novotný, Jan; Sklenář, Vladimir; Marek, Radek

    2014-02-07

    The study aimed to cast light on the structure and internal energetics of guanine- and xanthine-based model DNA quadruplexes and the physico-chemical nature of the non-covalent interactions involved. Several independent approaches were used for this purpose: DFT-D3 calculations, Quantum Theory of Atoms in Molecules, Natural Bond Orbital Analysis, Energy Decomposition Analysis, Compliance Constant Theory, and Non-Covalent Interaction Analysis. The results point to an excellent degree of structural and energetic compatibility between the two types of model quadruplexes. This fact stems from both the structural features (close values of van der Waals volumes, pore radii, geometrical parameters of the H-bonds) and the energetic characteristics (comparable values of the energies of formation). It was established that hydrogen bonding makes the greatest (∼50%) contribution to the internal stability of the DNA quadruplexes, whereas the aromatic base stacking and ion coordination terms are commensurable and account for the rest. Energy decomposition analysis performed for guanine (Gua) and xanthine (Xan) quartets B4 and higher-order structures consisting of two or three stacked quartets indicates that whereas Gua structures benefit from a high degree of H-bond cooperativity, Xan models are characterized by a more favorable and cooperative π-π stacking. The results of electron density topological analysis show that Na(+)/K(+) ion coordination deeply affects the network of non-covalent interactions in Gua models due to the change in the twist angle between the stacked tetrads. For Xan models, ion coordination makes tetrads in stacks more planar without changing the twist angle. Therefore, the presence of the ion seems to be essential for the formation of planar stacks in Xan-based DNA quadruplexes. Detailed study of the nature of ion-base coordination suggests that this interaction has a partially covalent character and cannot be considered as purely electrostatic

  8. Individual variation in hepatic aldehyde oxidase activity.

    PubMed

    Al-Salmy, H S

    2001-04-01

    Aldehyde oxidase (AO) is a molybdo-flavo enzyme expressed predominantly in the liver, lung, and kidney. AO plays a major role in oxidation of aldehydes, as well as oxidation of various N-heterocyclic compounds of pharmacological and toxicological importance including antiviral (famciclovir), antimalarial (quinine), antitumour (methotrexate), and nicotine. The aim of this study was to investigate cytosolic aldehyde oxidase activity in human liver. Cytosolic AO was characterised using both the metabolism of N-[(2-dimethylamino)ethyl] acridine-4-carboxamide (DACA) and benzaldehyde to form DACA-9(10H)-acridone (quantified by HPLC with fluorescence detection) and benzoic acid (quantified spectrophotometrically). Thirteen livers (10 female, 3 male) were examined. The intrinsic clearance (Vmax/Km) of DACA varied 18-fold (0.03-0.50 m/min/mg). Vmax ranged from 0.20-3.10 nmol/ min/mg, and Km ranged from 3.5-14.2 microM. In the same specimens, the intrinsic clearance for benzaldehyde varied 5-fold (0.40-1.8 ml/min/mg). Vmax ranged from 3.60-12.6 nmol/min/mg and Km ranged from 3.6-14.6 microM. Furthermore, there were no differences in AO activity between male and female human livers, nor was there any relationship to age of donor (range 29-73 years), smoking status, or disease status. In conclusion, our results showed that there are variations in AO activity in human liver. These variations in aldehyde oxidase activity might reflect individual variations or they might be due to AO stability during processing and storage.

  9. Nox NADPH Oxidases and the Endoplasmic Reticulum

    PubMed Central

    Araujo, Thaís L.S.; Abrahão, Thalita B.

    2014-01-01

    Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between

  10. Separation of putrescine oxidase and spermidine oxidase in foetal bovine serum with the aid of a specific radioactive assay of spermidine oxidase.

    PubMed Central

    Gahl, W A; Vale, A M; Pitot, H C

    1980-01-01

    1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate. PMID:7406861

  11. Teneligliptin Decreases Uric Acid Levels by Reducing Xanthine Dehydrogenase Expression in White Adipose Tissue of Male Wistar Rats

    PubMed Central

    2016-01-01

    We investigated the effects of teneligliptin on uric acid metabolism in male Wistar rats and 3T3-L1 adipocytes. The rats were fed with a normal chow diet (NCD) or a 60% high-fat diet (HFD) with or without teneligliptin for 4 weeks. The plasma uric acid level was not significantly different between the control and teneligliptin groups under the NCD condition. However, the plasma uric acid level was significantly decreased in the HFD-fed teneligliptin treated rats compared to the HFD-fed control rats. The expression levels of xanthine dehydrogenase (Xdh) mRNA in liver and epididymal adipose tissue of NCD-fed rats were not altered by teneligliptin treatment. On the other hand, Xdh expression was reduced significantly in the epididymal adipose tissue of the HFD-fed teneligliptin treated rats compared with that of HFD-fed control rats, whereas Xdh expression in liver did not change significantly in either group. Furthermore, teneligliptin significantly decreased Xdh expression in 3T3-L1 adipocytes. DPP-4 treatment significantly increased Xdh expression in 3T3-L1 adipocytes. With DPP-4 pretreatment, teneligliptin significantly decreased Xdh mRNA expression compared to the DPP-4-treated 3T3-L1 adipocytes. In conclusion, our studies suggest that teneligliptin reduces uric acid levels by suppressing Xdh expression in epididymal adipose tissue of obese subjects. PMID:27652270

  12. Analysis of xanthine dehydrogenase mRNA levels in mutants affecting the expression of the rosy locus.

    PubMed Central

    Covington, M; Fleenor, D; Devlin, R B

    1984-01-01

    Xanthine dehydrogenase (XDH) mRNA levels were measured in a number of mutants and natural variants affecting XDH gene expression. Two variants, ry+4 and ry+10, contain cis-acting elements which map to a region flanking the 5' end of the XDH gene. Ry+4, which has 2-3 times more XDH protein than a wild type strain, has 3.2 times more XDH mRNA. Ry+10 has 50% of the wild type XDH level and 54% of the wild type XDH mRNA level. Three rosy mutants which map within the structural gene were also examined. Two of these had little if any XDH mRNA, but the third mutant had 1.3 times more XDH mRNA than wild type flies. Another mutant, ry2 , which contains no XDH protein and has a 9KB transposable element inserted into the XDH gene, has normal levels of XDH mRNA transcripts which are also the same size as those found in the wild type strain. Changes in XDH mRNA levels were measured during Drosophila development and found to parallel changes in the amount of XDH protein. In addition, there were no large changes in the size of XDH mRNA during development. Images PMID:6588363

  13. Xanthine and 8-oxoguanine in G-quadruplexes: formation of a G·G·X·O tetrad.

    PubMed

    Cheong, Vee Vee; Heddi, Brahim; Lech, Christopher Jacques; Phan, Anh Tuân

    2015-12-02

    G-quadruplexes are four-stranded structures built from stacked G-tetrads (G·G·G·G), which are planar cyclical assemblies of four guanine bases interacting through Hoogsteen hydrogen bonds. A G-quadruplex containing a single guanine analog substitution, such as 8-oxoguanine (O) or xanthine (X), would suffer from a loss of a Hoogsteen hydrogen bond within a G-tetrad and/or potential steric hindrance. We show that a proper arrangement of O and X bases can reestablish the hydrogen-bond pattern within a G·G·X·O tetrad. Rational incorporation of G·G·X·O tetrads in a (3+1) G-quadruplex demonstrated a similar folding topology and thermal stability to that of the unmodified G-quadruplex. pH titration conducted on X·O-modified G-quadruplexes indicated a protonation-deprotonation equilibrium of X with a pKa ∼6.7. The solution structure of a G-quadruplex containing a G·G·X·O tetrad was determined, displaying the same folding topology in both the protonated and deprotonated states. A G-quadruplex containing a deprotonated X·O pair was shown to exhibit a more electronegative groove compared to that of the unmodified one. These differences are likely to manifest in the electronic properties of G-quadruplexes and may have important implications for drug targeting and DNA-protein interactions.

  14. NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

    PubMed Central

    Röhm, Marc; Grimm, Melissa J.; D'Auria, Anthony C.; Almyroudis, Nikolaos G.

    2014-01-01

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47phox−/−) mice which had resolved in wild-type mice by day 5 but progressed in p47phox−/− mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47phox−/− mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323

  15. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity.

    PubMed

    Peng, Zeyu; Dittmer, Neal T; Lang, Minglin; Brummett, Lisa M; Braun, Caroline L; Davis, Lawrence C; Kanost, Michael R; Gorman, Maureen J

    2015-04-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism.

  16. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity

    PubMed Central

    Peng, Zeyu; Dittmer, Neal T.; Lang, Minglin; Brummett, Lisa M.; Braun, Caroline L.; Davis, Lawrence C.; Kanost, Michael R.; Gorman, Maureen J.

    2015-01-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surpring because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism. PMID:25701385

  17. Combination treatment with 6-mercaptopurine and allopurinol in HepG2 and HEK293 cells – Effects on gene expression levels and thiopurine metabolism

    PubMed Central

    Haglund, Sofie; Vikingsson, Svante; Almer, Sven; Söderman, Jan

    2017-01-01

    Combination treatment with low-dose thiopurine and allopurinol (AP) has successfully been used in patients with inflammatory bowel disease with a so called skewed thiopurine metabolite profile. In red blood cells in vivo, it reduces the concentration of methylated metabolites and increases the concentration of the phosphorylated ones, which is associated with improved therapeutic efficacy. This study aimed to investigate the largely unknown mechanism of AP on thiopurine metabolism in cells with an active thiopurine metabolic pathway using HepG2 and HEK293 cells. Cells were treated with 6-mercaptopurine (6MP) and AP or its metabolite oxypurinol. The expression of genes known to be associated with thiopurine metabolism, and the concentration of thiopurine metabolites were analyzed. Gene expression levels were only affected by AP in the presence of 6MP. The addition of AP to 6MP affected the expression of in total 19 genes in the two cell lines. In both cell lines the expression of the transporter SLC29A2 was reduced by the combined treatment. Six regulated genes in HepG2 cells and 8 regulated genes in HEK293 cells were connected to networks with 18 and 35 genes, respectively, present at known susceptibility loci for inflammatory bowel disease, when analyzed using a protein-protein interaction database. The genes identified as regulated as well as the disease associated interacting genes represent new candidates for further investigation in the context of combination therapy with thiopurines and AP. However, no differences in absolute metabolite concentrations were observed between 6MP+AP or 6MP+oxypurinol vs. 6MP alone in either of the two cell lines. In conclusion; the effect of AP on gene expression levels requires the presence of 6MP, at least in vitro. Previously described AP-effects on metabolite concentrations observed in red blood cells in vivo could not be reproduced in our cell lines in vitro. AP’s effects in relation to thiopurine metabolism are complex

  18. Effect of hexacosanol on the characteristics of novel sustained-release allopurinol solid lipospheres (SLS): factorial design application and product evaluation.

    PubMed

    El-Gibaly, I; Abdel-Ghaffar, S K

    2005-04-27

    This investigation involved the evaluation of the effect of hexacosanol (HC, ceryl alcohol), a new hydrophobic wax modifier (WM) in comparison with conventional modifiers, on the development of sustained-release allopurinol (AP) solid lipospheres (SLS) intended for use in a suspension formulation and other oral dosage forms. Various beeswax (BW)/WM blends (composition ratio 1:1) were thus used to prepare SLS by a modified oil-in-water emulsion meltable disperse-phase (MDP) encapsulation method without using organic solvents and the influence of these blends on the drug encapsulation efficiency (EE), size distribution and the time for 50% of the drug to be released (t50%) was investigated. Results indicated that incorporation of HC in wall matrix of SLS provided the means to enhance the EE of AP and to modulate the rate of drug release into dissolution media (simulated gastric fluid (S.G.F.: pH 1.2) and simulated intestinal fluid (S.I.F.: pH 7.4). The effects of the process variables; HC concentration, dispersant (pluronic F-68: PF-68) concentration and drug:wax ratio were also studied on the properties of AP-loaded SLS by a 2(3) factorial design. The EE values were in the range of 80.8-92.67%. The only significant parameter affecting (P<0.01) the size and size distribution of the SLS formulations was the amount of the PF-68, whereas the factor with the biggest influence (P<0.05) on the drug EE was the initial loading of AP (in terms of the drug:wax ratio). The amount of HC blended with wax and the initial drug loading significantly (P<0.01) affected the t50% values of all of the formulations. The release of AP was more extended (t50% values (S.I.F.; pH 7.4)=9.91-25.36 h, depending on the drug:wax ratio) and surface morphology of SLS was improved with higher HC content (15%, w/w) formulations. The release patterns fitted the Baker-Lonsdale dissolution kinetics for spherical matrices. A significant decrease of plasma uric acid levels (P<0.05) and hepatic impairment

  19. Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii.

    PubMed

    Jurtshuk, P; Mueller, T J; Wong, T Y

    1981-09-14

    A membrane-bound cytochrome oxidase for Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using as ascorbate-TMPD oxidation assay. The oxidase was 'solubilized' from a sonic-type electron-transport particle (R3 fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27-70% (NH4)2SO4. The highly purified cytochrome oxidase has a V of 60-78 microgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN3 and NH2OH; NaNO2 (but not NaNO3) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c4 and cytochrome o; cytochromes a1 and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c4-o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a3 oxidase of mammalian mitochondria.

  20. Molecular Evolution of Cytochrome bd Oxidases across Proteobacterial Genomes

    PubMed Central

    Degli Esposti, Mauro; Rosas-Pérez, Tania; Servín-Garcidueñas, Luis Eduardo; Bolaños, Luis Manuel; Rosenblueth, Monica; Martínez-Romero, Esperanza

    2015-01-01

    This work is aimed to resolve the complex molecular evolution of cytochrome bd ubiquinol oxidase, a nearly ubiquitous bacterial enzyme that is involved in redox balance and bioenergetics. Previous studies have created an unclear picture of bd oxidases phylogenesis without considering the existence of diverse types of bd oxidases. Integrated approaches of genomic and protein analysis focused on proteobacteria have generated a molecular classification of diverse types of bd oxidases, which produces a new scenario for interpreting their evolution. A duplication of the original gene cluster of bd oxidase might have occurred in the ancestors of extant α-proteobacteria of the Rhodospirillales order, such as Acidocella, from which the bd-I type of the oxidase might have diffused to other proteobacterial lineages. In contrast, the Cyanide-Insensitive Oxidase type may have differentiated into recognizable subtypes after another gene cluster duplication. These subtypes are widespread in the genomes of α-, β-, and γ-proteobacteria, with occasional instances of lateral gene transfer. In resolving the evolutionary pattern of proteobacterial bd oxidases, this work sheds new light on the basal taxa of α-proteobacteria from which the γ-proteobacterial lineage probably emerged. PMID:25688108

  1. Detection and characterization of a multicopper oxidase from Nitrosomonas europaea.

    PubMed

    Lawton, Thomas J; Rosenzweig, Amy C

    2011-01-01

    Blue copper oxidase (BCO) is a multicopper oxidase (MCO) found in Nitrosomonas europaea as well as in other ammonia-oxidizing organisms. In this chapter, we detail methods used to detect, isolate, and characterize BCO from N. europaea. A method for identifying and classifying MCOs commonly found in nitrifiers based on primary sequence is also described.

  2. Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.

    PubMed Central

    Lambowitz, A M; Sabourin, J R; Bertrand, H; Nickels, R; McIntosh, L

    1989-01-01

    Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species. Images PMID:2524649

  3. Lysyl oxidase activity in human normal skins and postburn scars.

    PubMed

    Hayakawa, T; Hino, N; Fuyamada, H; Nagatsu, T; Aoyama, H

    1976-09-06

    Lysyl oxidase activity of human normal skins derived from the frontal thighs of 33 subjects showed large variations and the mean value was 11 455 +/- 7 172 (S.D.) cpm/g of wet weight tissue. The age of lesion affected the lysyl oxidase activity in postburn scars. Granulation tissues showed a fairly low activity; however, the activity increased sharply within 2--3 months, and reached a significantly higher value than that of normal skin. The high level of activity continued for up to 2--3 years, then gradually decreased to normal range after 5 years or so. Lysyl oxidase activity was detected only after 4 M urea treatment of tissues. Benzylamine oxidase activity also showed large variations in both normal skins and postburn scars, with mean values of: 0.128 +/- 0.077 (S.D.) and 0.145 +/- 0.090 (S.D.) mmol/g of wet weight/h, respectively. No correlation was observed between lysyl oxidase and benzylamine oxidase activities. The granulation tissues showed significantly high values of benzylamine oxidase activity in contrast to the low values of lysyl oxidase activity.

  4. [A case of Xanthinuria in a patient with marked hypouricemia].

    PubMed

    Martella, Vilma; Sozzo, Efisio; Montagna, Elio; Stefanizzi, Salvatore; Cito, Annarita; Marinello, Enrico; Terzuoli, Lucia; Micheli, Vanna; Napoli, Marcello

    2011-01-01

    Xanthinuria is a rare autosomal recessive disorder associated with a deficiency of xanthine oxidoreductase (XOR), which normally catalyzes the conversion of hypoxanthine to uric acid. The effects of this deficit are an elevated concentration of hypoxanthine and xanthine in the blood and urine, hypouricemia, and hypouricuria. The deficit in XOR can be isolated (type I xanthinuria) or associated with a deficit in aldehyde oxidase (type II xanthinuria) and sulfite oxidase (type III xanthinuria). While the first two variants have a benign course, are often asymptomatic (20%), and clinically indistinguishable, type III xanthinuria is a harmful form that leads to infant death due to neurological damage. The clinical symptoms (kidney stones, CKD, muscle and joint pain, peptic ulcer) are the result of the accumulation of xanthine, which is highly insoluble, in the body fluids. We describe a case of type I xanthinuria in a 52-year-old woman who presented with hypouricemia, hypouricuria and kidney stones. The diagnosis was based on purine catabolite levels in urine and serum measured by 3 nonroutine methods: high-pressure liquid chromatography, mass spectrometry, and magnetic resonance imaging. To identify the type of xanthinuria the allopurinol test was used. We believe that these tests will facilitate the diagnosis of xantinuria especially in asymptomatic patients without the need for a biopsy of the liver or intestines, which is useful only for scientific purposes.

  5. The complex roles of NADPH oxidases in fungal infection

    PubMed Central

    Hogan, Deborah; Wheeler, Robert T.

    2014-01-01

    Summary NADPH oxidases play key roles in immunity and inflammation that go beyond the production of microbicidal reactive oxygen species (ROS). The past decade has brought a new appreciation for the diversity of roles played by ROS in signaling associated with inflammation and immunity. NADPH oxidase activity affects disease outcome during infections by human pathogenic fungi, an important group of emerging and opportunistic pathogens that includes Candida, Aspergillus and Cryptococcus species. Here we review how alternative roles of NADPH oxidase activity impact fungal infection and how ROS signaling affects fungal physiology. Particular attention is paid to roles for NADPH oxidase in immune migration, immunoregulation in pulmonary infection, neutrophil extracellular trap formation, autophagy and inflammasome activity. These recent advances highlight the power and versatility of spatiotemporally controlled redox regulation in the context of infection, and point to a need to understand the molecular consequences of NADPH oxidase activity in the cell. PMID:24905433

  6. The NADH oxidase-Prx system in Amphibacillus xylanus.

    PubMed

    Niimura, Youichi

    2007-01-01

    Amphibacillus NADH oxidase belongs to a growing new family of peroxiredoxin-linked oxidoreductases including alkyl hydroperoxide reductase F (AhpF). Like AhpF it displays extremely high hydroperoxide reductase activity in the presence of a Prx, thus making up the NADH oxidase-Prx system. The NADH oxidase primarily catalyzes the reduction of oxygen by NADH to form H2O2, while the Prx immediately reduces H2O2 (or ROOH) to water (or ROH). Consequently, the NADH oxidase-Prx system catalyzes the reduction of both oxygen and hydrogen peroxide to water with NADH as the preferred electron donor. The NADH oxidase-Prx system is widely distributed in aerobically growing bacteria lacking a respiratory chain and catalase, and plays an important role not only in scavenging hydroperoxides but also in regenerating NAD in these bacteria.

  7. Genotypic Variation in Cytokinin Oxidase from Phaseolus Callus Cultures 1

    PubMed Central

    Kaminek, Miroslav; Armstrong, Donald J.

    1990-01-01

    Genotypic variation in cytokinin oxidase has been detected in enzyme preparations from Phaseolus vulgaris L. cv Great Northern and Phaseolus lunatus L. cv Kingston callus cultures. Although cytokinin oxidase preparations from Great Northern and Kingston callus tissues appear to have very similar substrate specificities, the cytokinin oxidase activities from the two callus tissues were found to differ in a number of other properties. The cytokinin oxidase from P. vulgaris cv Great Northern callus tissue exhibited a pH optimum of 6.5 (bisTris) and had a strong affinity for the lectin concanavalin A. The cytokinin oxidase from P. lunatus cv Kingston callus tissue exhibited a pH optimum of 8.4 (Taps) and did not bind to concanavalin A. The two enzymes also differed in position of elution when chromatographed on DEAE-cellulose. Both cytokinin oxidase activities exhibited enhanced activity and lower pH optima in the presence of copper-imidazole complexes, but the optimum copper-imidazole ratio and the magnitude of enhancement differed for the two activities. In both callus tissues, transient increases in the supply of exogenous cytokinins induced increases in cytokinin oxidase activity. The differences in pH optima and in glycosylation (as evidenced by the observed difference in lectin affinity) of the cytokinin oxidases from Great Northern and Kingston callus tissues suggest that the compartmentation of cytokinin oxidase may differ in the two callus tissues. The possibility that enzyme compartmentation and isozyme variation in cytokinin oxidase may play a role in the regulation of cytokinin degradation in plant tissues is discussed in relation to known differences in the rates of cytokinin degradation in Great Northern and Kingston callus tissues. Images Figure 6 PMID:16667652

  8. Expression of the alternative oxidase complements cytochrome c oxidase deficiency in human cells

    PubMed Central

    Dassa, Emmanuel P; Dufour, Eric; Gonçalves, Sérgio; Paupe, Vincent; Hakkaart, Gertjan A J; Jacobs, Howard T; Rustin, Pierre

    2009-01-01

    Cytochrome c oxidase (COX) deficiency is associated with a wide spectrum of clinical conditions, ranging from early onset devastating encephalomyopathy and cardiomyopathy, to neurological diseases in adulthood and in the elderly. No method of compensating successfully for COX deficiency has been reported so far. In vitro, COX-deficient human cells require additional glucose, pyruvate and uridine for normal growth and are specifically sensitive to oxidative stress. Here, we have tested whether the expression of a mitochondrially targeted, cyanide-resistant, alternative oxidase (AOX) from Ciona intestinalis could alleviate the metabolic abnormalities of COX-deficient human cells either from a patient harbouring a COX15 pathological mutation or rendered deficient by silencing the COX10 gene using shRNA. We demonstrate that the expression of the AOX, well-tolerated by the cells, compensates for both the growth defect and the pronounced oxidant-sensitivity of COX-deficient human cells. PMID:20049701

  9. Alternative oxidase and plastoquinol terminal oxidase in marine prokaryotes of the Sargasso Sea.

    PubMed

    McDonald, Allison E; Vanlerberghe, Greg C

    2005-04-11

    Alternative oxidase (AOX) represents a non-energy conserving branch in mitochondrial electron transport while plastoquinol terminal oxidase (PTOX) represents a potential branch in photosynthetic electron transport. Using a metagenomics dataset, we have uncovered numerous and diverse AOX and PTOX genes from the Sargasso Sea. Sequence similarity, synteny and phylogenetic analyses indicate that the large majority of these genes are from prokaryotes. AOX appears to be widely distributed among marine Eubacteria while PTOX is widespread among strains of cyanobacteria closely related to the high-light adapted Prochlorococcus marinus MED4, as well as Synechococcus. The wide distribution of AOX and PTOX in marine prokaryotes may have important implications for productivity in the world's oceans.

  10. Spectroscopic investigations and hydrogen bond interactions of 8-aza analogues of xanthine, theophylline and caffeine: a theoretical study.

    PubMed

    Karthika, Mylsamy; Kanakaraju, Ramasamy; Senthilkumar, Lakshmipathi

    2013-04-01

    The structure, spectral properties and the hydrogen bond interactions of 8-aza analogues of xanthine, theophylline and caffeine have been studied by using quantum chemical methods. The time-dependent density functional theory (TD-DFT) and the singly excited configuration interaction (CIS) methods are employed to optimize the excited state geometries of isolated 8-azaxanthine, 8-azatheophylline tautomers and 8-azacaffeine in both the gas and solvent phases. The solvent phase calculations are performed using the polarizable continuum model (PCM). The absorption and emission spectra are calculated using the time-dependent density functional theory (TD-DFT) method. The results from the TD-DFT calculations reveal that the excitation spectra are red shifted relative to absorption in aqueous medium. These changes in the transition energies are qualitatively comparable to the experimental data. The examination of molecular orbital reveals that the molecules with a small H→L energy gap possess maximum absorption and emission wavelength. The relative stability and hydrogen bonded interactions of mono and heptahydrated 8-azaxanthine, 8-azatheophylline tautomers and 8-azacaffeine have been studied using the density functional theory (DFT) and Møller Plesset perturbation theory (MP2) implementing the 6-311++G(d,p) basis set. The formation of strong N-H...O bond has resulted in the highest interaction energy among the monohydrates. Hydration does not show any significant impact on the stability of heptahydrated complexes. The atoms in molecule (AIM) and natural bonding orbital (NBO) analyses have been performed to elucidate the nature of the hydrogen bond interactions in these complexes.

  11. Contribution of xanthine oxidoreductase to mammary epithelial and breast cancer cell differentiation in part modulates inhibitor of differentiation-1.

    PubMed

    Fini, Mehdi A; Monks, Jenifer; Farabaugh, Susan M; Wright, Richard M

    2011-09-01

    Loss of xanthine oxidoreductase (XOR) has been linked to aggressive breast cancer in vivo and to breast cancer cell aggressiveness in vitro. In the present study, we hypothesized that the contribution of XOR to the development of the normal mammary gland may underlie its capacity to modulate breast cancer. We contrasted in vitro and in vivo developmental systems by differentiation marker and microarray analyses. Human breast cancer microarray was used for clinical outcome studies. The role of XOR in differentiation and proliferation was examined in human breast cancer cells and in a mouse xenograft model. Our data show that XOR was required for functional differentiation of mammary epithelial cells both in vitro and in vivo. Poor XOR expression was observed in a mouse ErbB2 breast cancer model, and pharmacologic inhibition of XOR increased breast cancer tumor burden in mouse xenograft. mRNA microarray analysis of human breast cancer revealed that low XOR expression was significantly associated with time to tumor relapse. The opposing expression of XOR and inhibitor of differentiation-1 (Id1) during HC11 differentiation and mammary gland development suggested a potential functional relationship. While overexpression of Id1 inhibited HC11 differentiation and XOR expression, XOR itself modulated expression of Id1 in differentiating HC11 cells. Overexpression of XOR both inhibited Id1-induced proliferation and -stimulated differentiation of Heregulin-β1-treated human breast cancer cells. These results show that XOR is an important functional component of differentiation whose diminished expression contributes to breast cancer aggressiveness, and they support XOR as both a breast cancer biomarker and a target for pharmacologic activation in therapeutic management of aggressive breast cancer.

  12. [NADPH oxidases, Nox: new isoenzymes family].

    PubMed

    Chuong Nguyen, Minh Vu; Lardy, Bernard; Paclet, Marie-Hélène; Rousset, Francis; Berthier, Sylvie; Baillet, Athan; Grange, Laurent; Gaudin, Philippe; Morel, Françoise

    2015-01-01

    NADPH oxidases, Nox, are a family of isoenzymes, composed of seven members, whose sole function is to produce reactive oxygen species (ROS). Although Nox catalyze the same enzymatic reaction, they acquired from a common ancestor during evolution, specificities related to their tissue expression, subcellular localization, activation mechanisms and regulation. Their functions could vary depending on the pathophysiological state of the tissues. Indeed, ROS are not only bactericidal weapons in phagocytes but also essential cellular signaling molecules and their overproduction is involved in chronic diseases and diseases of aging. The understanding of the mechanisms involved in the function of Nox and the emergence of Nox inhibitors, require a thorough knowledge of their nature and structure. The objectives of this review are to highlight, in a structure/function approach, the main similar and differentiated properties shared by the human Nox isoenzymes.

  13. Visualization of monoamine oxidase in human brain

    SciTech Connect

    Fowler, J.S.; Volkow, N.D.; Wang, G.J.; Pappas, N.; Shea, C.; MacGregor, R.R.; Logan, J.

    1996-12-31

    Monoamine oxidase is a flavin enzyme which exists in two subtypes, MAO A and MAO B. In human brain MAO B predominates and is largely compartmentalized in cell bodies of serotonergic neurons and glia. Regional distribution of MAO B was determined by positron computed tomography with volunteers after the administration of deuterium substituted [11C]L-deprenyl. The basal ganglia and thalamus exhibited the greatest concentrations of MAO B with intermediate levels in the frontal cortex and cingulate gyrus while lowest levels were observed in the parietal and temporal cortices and cerebellum. We observed that brain MAO B increases with are in health normal subjects, however the increases were generally smaller than those revealed with post-mortem studies.

  14. NADPH Oxidases in Lung Health and Disease

    PubMed Central

    Bernard, Karen; Hecker, Louise; Luckhardt, Tracy R.; Cheng, Guangjie

    2014-01-01

    Abstract Significance: The evolution of the lungs and circulatory systems in vertebrates ensured the availability of molecular oxygen (O2; dioxygen) for aerobic cellular metabolism of internal organs in large animals. O2 serves as the physiologic terminal acceptor of mitochondrial electron transfer and of the NADPH oxidase (Nox) family of oxidoreductases to generate primarily water and reactive oxygen species (ROS), respectively. Recent advances: The purposeful generation of ROS by Nox family enzymes suggests important roles in normal physiology and adaptation, most notably in host defense against invading pathogens and in cellular signaling. Critical issues: However, there is emerging evidence that, in the context of chronic stress and/or aging, Nox enzymes contribute to the pathogenesis of a number of lung diseases. Future Directions: Here, we review evolving functions of Nox enzymes in normal lung physiology and emerging pathophysiologic roles in lung disease. Antioxid. Redox Signal. 20, 2838–2853. PMID:24093231

  15. Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

    PubMed

    Cervelli, Manuela; Leonetti, Alessia; Cervoni, Laura; Ohkubo, Shinji; Xhani, Marla; Stano, Pasquale; Federico, Rodolfo; Polticelli, Fabio; Mariottini, Paolo; Agostinelli, Enzo

    2016-10-01

    Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

  16. Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

    PubMed Central

    Hernández-Romero, Diana; Solano, Francisco; Sanchez-Amat, Antonio

    2005-01-01

    Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds. PMID:16269713

  17. Dephenolization of industrial wastewaters catalyzed by polyphenol oxidase

    SciTech Connect

    Atlow, S.C.; Bonadonna-Aparo, L.; Klibanov, A.M.

    1984-01-01

    A new enzymatic method for the removal of phenols from industrial aqueous effluents has been developed. The method uses the enzyme polyphenol oxidase which oxidizes phenols to the corresponding o-quinones; the latter then undergo a nonenzymatic polymerization to form water-insoluble aggregates. Therefore, the enzyme in effect precipitates phenols from water. Polyphenol oxidase has been found to nearly completely dephenolize solutions of phenol in the concentration range from 0.01 to 1.0 g/L. The enzymatic treatment is effective over a wide range of pH and temperature; a crude preparation of polyphenol oxidase (mushroom extract) is as effective as a purified, commercially obtained version. In addition to phenol itself, polyphenol oxidase is capable of precipitating from water a number of substituted phenols (cresols, chlorophenols, naphthol, etc.). Also, even pollutants which are unreactive towards polyphenol oxidase can be enzymatically coprecipitated with phenol. The polyphenol oxidase treatment has been successfully used to dephenolize two different real industrial wastewater samples, from a plant producing triarylphosphates and from a coke plant. The advantage of the polyphenol oxidase dephenolization over the peroxidase-catalyzed one previously elaborated by the authors is that the former enzyme uses molecular oxygen instead of costly hydrogen peroxide (used by peroxidase) as an oxidant.

  18. Characterization of two amine oxidases from Aspergillus carbonarius AIU 205.

    PubMed

    Sugawara, Asami; Matsui, Daisuke; Yamada, Miwa; Asano, Yasuhisa; Isobe, Kimiyasu

    2015-06-01

    We have reported that Aspergillus carbonarius AIU 205, which was isolated by our group, produced three enzymes exhibiting oxidase activity for 4-aminobutanamide (4-ABAD) (J. Biosci. Bioeng., 117, 263-268, 2014). Among three enzymes, characteristics of enzyme I have been revealed, but those of the other two enzymes have not. In this study, we purified enzymes II and III, and compared their characteristics with those of enzyme I. Enzymes II and III also oxidized aliphatic monoamines, aromatic amines, and aliphatic aminoalcohols. In addition, the oxidase activity of both enzymes was strongly inhibited by carbonyl reagents and specific inhibitors for copper-containing amine oxidases. Thus, enzymes II and III were also classified into the copper-containing amine oxidase group (EC 1.4.3.6) along with enzyme I. However, these three enzymes differed from each other in their enzymatic, kinetic, and physicochemical properties. The N-terminal amino acid sequences also differed from each other; that of enzyme I was modified, that of enzyme II was similar to those of peroxisomal copper-containing amine oxidases, and that of enzyme III was similar to those of copper-containing amine oxidases from the genus Aspergillus. Therefore, we concluded that A. carbonarius AIU 205 produced three different types of amine oxidase in the mycelia.

  19. THE PREPARATION AND PROPERTIES OF HIGHLY PURIFIED ASCORBIC ACID OXIDASE

    PubMed Central

    Powers, Wendell H.; Lewis, Stanley; Dawson, Charles R.

    1944-01-01

    1. A method is described for the preparation of a highly purified ascorbic acid oxidase containing 0.24 per cent copper. 2. Using comparable activity measurements, this oxidase is about one and a half times as active on a dry weight basis as the hitherto most highly purified preparation described by Lovett-Janison and Nelson. The latter contained 0.15 per cent copper. 3. The oxidase activity is proportional to the copper content and the proportionality factor is the same as that reported by Lovett-Janison and Nelson. 4. When dialyzed free of salt, the blue concentrated oxidase solutions precipitate a dark green-blue protein which carries the activity. This may be prevented by keeping the concentrated solutions about 0.1 M in Na2HPO4. 5. When highly diluted for activity measurements the oxidase rapidly loses activity (irreversibly) previous to the measurement, unless the dilution is made with a dilute inert protein (gelatin) solution. Therefore activity values obtained using such gelatin-stabilized dilute solutions of the oxidase run considerably higher than values obtained by the Lovett-Janison and Nelson technique. 6. The effect of pH and substrate concentration on the activity of the purified oxidase in the presence and absence of inert protein was studied. PMID:19873382

  20. CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity.

    PubMed

    Su, Jianmei; Bao, Peng; Bai, Tenglong; Deng, Lin; Wu, Hui; Liu, Fan; He, Jin

    2013-01-01

    Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10(-6)±0.21 M·min(-1) and 0.32±0.02 s(-1), respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a

  1. Multilayered polyelectrolyte microcapsules: interaction with the enzyme cytochrome C oxidase.

    PubMed

    Pastorino, Laura; Dellacasa, Elena; Noor, Mohamed R; Soulimane, Tewfik; Bianchini, Paolo; D'Autilia, Francesca; Antipov, Alexei; Diaspro, Alberto; Tofail, Syed A M; Ruggiero, Carmelina

    2014-01-01

    Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components. This work provides a significant input towards the fabrication of an integrated device made of biological components and based on specific biomolecular functions and properties.

  2. [An endogenous inhibitor of monoamine oxidase A (tribulin A) from brain: purification and structure identification].

    PubMed

    Medvedev, A E; Kamyshanskaia, N S; Halket, J; Glover, V; Sandler, A

    1995-05-01

    The endogenous monoamine oxidase inhibitor, tribulin, contains several components which selectively (or nonselectively) inhibit monoamine oxidases A and B. The pig brain tribulin component selectively inhibiting monoamine oxidase A was purified and identified as 4-hydroxyphenylethanol using gas chromatography-mass spectrometry. This compound was also found in the rabbit brain tribulin fraction which selectively inhibits monoamine oxidase A but has no influence on monoamine oxidase B. 4-Hydroxyphenylethanol inhibits monoamine oxidase A in an incompetitive manner with respect to the substrate, serotonin (Ki = 1.4 mM). Possible pathways of 4-hydroxyphenylethanol synthesis and its biological importance as the monoamine oxidase A inhibiting component of tribulin are discussed.

  3. Isolation of oxidase-negative Pseudomonas aeruginosa from sputum culture.

    PubMed

    Hampton, K D; Wasilauskas, B L

    1979-05-01

    Two isolates of Pseudomonas aeruginosa lacking characteristic indophenol oxidase were recovered from a sputum specimen. A discussion of the characteristic biochemical tests and antibiograms along with a possible explanation for this phenomenon is presented.

  4. Polyphenol oxidase produced during encystation of Acanthamoeba castellanii.

    PubMed

    Sykes, D E; Band, R N

    1985-08-01

    Acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. Enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. Encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. Acanthamoeba polyphenol oxidase has a pH optimum of 6.0 and a Km value of 0.21 mM with dihydroxyphenylalanine. The enzyme does not oxidize tyrosine, and it is inhibited by chloride but not by inhibitors of peroxidase. Its synthesis coincides with encystation, and known inhibitors of polyphenol oxidase prevent encystation. Polyphenol oxidase may have a role in making the cyst resistant to mechanical and chemical breakdown.

  5. Beyond brown: polyphenol oxidases as enzymes of plant specialized metabolism.

    PubMed

    Sullivan, Michael L

    2014-01-01

    Most cloned and/or characterized plant polyphenol oxidases (PPOs) have catechol oxidase activity (i.e., they oxidize o-diphenols to o-quinones) and are localized or predicted to be localized to plastids. As a class, they have broad substrate specificity and are associated with browning of produce and other plant materials. Because PPOs are often induced by wounding or pathogen attack, they are most generally believed to play important roles in plant defense responses. However, a few well-characterized PPOs appear to have very specific roles in the biosynthesis of specialized metabolites via both tyrosinase (monophenol oxidase) and catechol oxidase activities. Here we detail a few examples of these and explore the possibility that there may be many more "biosynthetic" PPOs.

  6. Stimulation of cellular XTT reduction by cytochrome oxidase inhibitors.

    PubMed

    Kunimoto, S; Nosaka, C; Takeuchi, T

    1999-06-01

    XTT reducing activity by CHO and L1210 cells was found to be stimulated by the presence of cytochrome oxidase inhibitors such as NaN3 or KCN. Among the other respiratory chain inhibitors, antimycin A (a complex III inhibitor) and chlorpromazine inhibited cellular XTT reduction, and rotenone and malonate showed slight inhibition and no effect, respectively. It is suggested that XTT reduction is coupled with the respiratory chain via cytochrome c, which is located between complexes III and IV (cytochrome oxidase).

  7. The role of human xanthine oxidoreductase (HXOR), anti-HXOR antibodies, and microorganisms in synovial fluid of patients with joint inflammation.

    PubMed

    Al-Muhtaseb, Najah; Al-Kaissi, Elham; Thawaini, Abdul Jalil; Eldeen, Zuhair Muhi; Al-Muhtaseb, Sabah; Al-Saleh, Badiee

    2012-08-01

    This work is to investigate the levels of human xanthine oxidoreductase (HXOR), its antibodies, and microorganisms in synovial fluid of patients with untreated rheumatoid joint diseases. Synovial fluids were collected from sixty-four patients with rheumatoid joint diseases. Sixty-four age-matched individuals were included as control. Xanthine oxidoreductase (XOR) proteins level and anti-XOR antibodies were determined in the blood and synovial fluid, using human XOR as antigen, by enzyme-linked immunosorbent (ELISA) assay. Synovial fluids were cultured for bacteria and fungi. The titers of XOR protein in the synovial fluid of patients with rheumatoid arthritis were 90.43 ± 23.37 μg/ml (mean ± SD, n = 29) and up to 62.42 ± 8.74 μg/ml (mean ± SD, n = 35) in other joint inflammation. Anti-HXOR antibodies titers in patients were 167.72 ± 23.64 μg/ml, n = 64, which was significantly higher in rheumatoid arthritis patients. The results indicated that anti-HXOR antibodies in synovial fluids have a protective role as high concentrations against XOR were detected in inflammatory arthritis. These antibodies play a role in eliminating XOR from synovial fluids. However, immune complex formation could activate complement and participate in propagating the inflammatory cycle. Synovial aspirate ordinary microbial cultures were negative for any bacteria or fungi, but that does not exclude organisms of special culture requirements.

  8. Confirmation of a blocked amino terminus of sulfhydryl oxidase

    SciTech Connect

    Janolino, V.G.; Morrison-Rowe, S.J.; Swaisgood, H.E. )

    1990-09-01

    The isolation of sulfhydryl oxidase from bovine milk in a suitably pure form for sequencing was carried out by transient covalent affinity chromatography of diafiltered whey using cysteinylsuccinamidopropyl-glass as matrix. The glutathione-eluted proteins were separated by SDS-PAGE. By radiolabeling the affinity chromatography-purified enzyme with ({sup 14}C)iodoacetate before subjecting to SDS-PAGE, the sulfhydryl oxidase band was identified, because sulfhydryl oxidase is known to be inactivated by alkylation of one sulfhydryl group per mole. The results confirmed that sulfhydryl oxidase corresponds to the 85 ({plus minus} 5)-kDa band observed on SDS-PAGE. The protein band corresponding to radiolabeled sulfhydryl oxidase was recovered from SDS-PAGE gels by electrophoretic elution and by electroblotting on polyvinylidene difluoride membrane and subjected to gas phase sequencing. Precautions were taken during electrophoretic elution to prevent reactions that result in N-terminal blocking. Both methods of protein recovery yielded negative results when subjected to sequence analysis indicating that the N-terminus of sulfhydryl oxidase is blocked.

  9. Characterization of monomeric substates of ascorbate oxidase.

    PubMed

    Di Venere, Almerinda; Nicolai, Eleonora; Rosato, Nicola; Rossi, Antonello; Finazzi Agrò, Alessandro; Mei, Giampiero

    2011-05-01

    Ascorbate oxidase (AAO) is a large, multidomain, dimeric protein whose folding/unfolding pathway is characterized by a complex, multistep process. Here we used fluorescence correlation spectroscopy to demonstrate the formation of partially folded monomers by pH-induced full dissociation into subunits. Hence, the structural features of monomeric AAO could be studied by fluorescence and CD spectroscopy. We found that the monomers keep their secondary structure, whereas subtle conformational changes in the tertiary structure become apparent. AAO dissociation has also been studied when unfolding the protein by high hydrostatic pressure at different pH values. A strong protein concentration dependence was observed at pH 8, whereas the enzyme was either monomeric or dimeric at pH 10 and 6, respectively. The calculated volume change associated with the unfolding of monomeric AAO, ΔV ∼ -55 mL·mol(-1), is in the range observed for most proteins of the same size. These findings demonstrate that partially folded monomeric species might populate the energy landscape of AAO and that the overall AAO stability is crucially controlled by a few quaternary interactions at the subunits' interface.

  10. The function of ascorbate oxidase in tobacco.

    PubMed

    Pignocchi, Cristina; Fletcher, John M; Wilkinson, Joy E; Barnes, Jeremy D; Foyer, Christine H

    2003-07-01

    The function of the apoplastic enzyme ascorbate oxidase (AO) was investigated in tobacco (Nicotiana tabacum). The abundance of AO mRNA was up-regulated by light. Cytosolic ascorbate peroxidase (APX1) transcripts were also highest in the light. In contrast, L-galactono-gamma-lactone dehydrogenase, stromal APX, and thylakoid APX transcripts remained constant over the day/night cycle. Salicylic acid inhibited growth, increased expression of the pathogenesis-related protein (PR) 1a, and decreased AO transcript abundance. In contrast, the application of auxin enhanced growth and increased AO and PR 1a gene expression. Therefore, AO transcript abundance varied in a manner similar to hormone-mediated changes in plant growth. To study the effects of modified AO expression on growth, transformed tobacco plants expressing AO in the sense and antisense orientations were generated. The resultant large changes in apoplastic AO activity in the transformed tobacco plants had little effect on whole leaf ascorbate (AA) content, but they had dramatic effects on apoplastic AA levels. Enhanced AO activity oxidized the apoplastic AA pool, whereas decreased AO activity increased the amount of AA compared with dehydroascorbate. A relationship was observed between AO activity and plant height and biomass. Native AO transcript levels were no longer subject to light/dark regulation in AO sense and antisense plants. Taken together, these data show that there is an interaction between hormone, redox, and light signals at the level of the apoplast via modulation of ion of AA content.

  11. Monoamine oxidase: Radiotracer chemistry and human studies

    DOE PAGES

    Fowler, Joanna S.; Logan, Jean; Shumay, Elena; ...

    2015-03-01

    Monoamine oxidase (MAO) oxidizes amines from both endogenous and exogenous sources thereby regulating the concentration of neurotransmitter amines such as serot onin, norepinephrine and dopamine as well as many xenobiotics. MAO inhibitor drugs are used in the treatment of Parkinson’s disease and in depression stimulating the development of radiotracer tools to probe the role of MAO in normal human biology and in disease. Over the past 30 since the first radiotracers were developed and the first PET images of MAO in humans were carried out, PET studies of brain MAO in healthy volunteers and in patients have identified different variablesmore » which have contributed to different MAO levels in brain and in peripheral organs. MAO radiotracers and PET have also been used to study the current and developing MAO inhibitor drugs including the selection of doses for clinical trials. In this article, we describe (1) the development of MAO radiotracers; (2) human studies including the relationship of brain MAO levels to genotype, personality, neurological and psychiatric disorders; (3) examples of the use of MAO radiotracers in drug research and development. We will conclude with outstanding needs to improve the radiotracers which are currently used and possible new applications.« less

  12. Monoamine oxidase: Radiotracer chemistry and human studies

    SciTech Connect

    Fowler, Joanna S.; Logan, Jean; Shumay, Elena; Alia-Klein, Nelly; Wang, Gene-Jack; Volkow, Nora D.

    2015-03-01

    Monoamine oxidase (MAO) oxidizes amines from both endogenous and exogenous sources thereby regulating the concentration of neurotransmitter amines such as serot onin, norepinephrine and dopamine as well as many xenobiotics. MAO inhibitor drugs are used in the treatment of Parkinson’s disease and in depression stimulating the development of radiotracer tools to probe the role of MAO in normal human biology and in disease. Over the past 30 since the first radiotracers were developed and the first PET images of MAO in humans were carried out, PET studies of brain MAO in healthy volunteers and in patients have identified different variables which have contributed to different MAO levels in brain and in peripheral organs. MAO radiotracers and PET have also been used to study the current and developing MAO inhibitor drugs including the selection of doses for clinical trials. In this article, we describe (1) the development of MAO radiotracers; (2) human studies including the relationship of brain MAO levels to genotype, personality, neurological and psychiatric disorders; (3) examples of the use of MAO radiotracers in drug research and development. We will conclude with outstanding needs to improve the radiotracers which are currently used and possible new applications.

  13. Modular assembly of yeast cytochrome oxidase.

    PubMed

    McStay, Gavin P; Su, Chen Hsien; Tzagoloff, Alexander

    2013-02-01

    Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high-molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits. We propose a model of COX biogenesis in which Cox1p and the two other mitochondrial gene products, Cox2p and Cox3p, constitute independent assembly modules, each with its own complement of subunits. Unlike their bacterial counterparts, which are composed only of the individual core subunits, the final sequence in which the mitochondrial modules associate to form the holoenzyme may have been conserved during evolution.

  14. Polyphenol oxidase from yacon roots (Smallanthus sonchifolius).

    PubMed

    Neves, Valdir Augusto; da Silva, Maraiza Aparecida

    2007-03-21

    Polyphenol oxidase (E.C. 1.14.18.1) (PPO) extracted from yacon roots (Smallanthus sonchifolius) was partially purified by ammonium sulfate fractionation and separation on Sephadex G-100. The enzyme had a molecular weight of 45 490+/-3500 Da and Km values of 0.23, 1.14, 1.34, and 5.0 mM for the substrates caffeic acid, chlorogenic acid, 4-methylcatechol, and catechol, respectively. When assayed with resorcinol, DL-DOPA, pyrogallol, protocatechuic, p-coumaric, ferulic, and cinnamic acids, catechin, and quercetin, the PPO showed no activity. The optimum pH varied from 5.0 to 6.6, depending on substrate. PPO activity was inhibited by various phenolic and nonphenolic compounds. p-Coumaric and cinnamic acids showed competitive inhibition, with Ki values of 0.017 and 0.011 mM, respectively, using chlorogenic acid as substrate. Heat inactivation from 60 to 90 degrees C showed the enzyme to be relatively stable at 60-70 degrees C, with progressive inactivation when incubated at 80 and 90 degrees C. The Ea (apparent activation energy) for inactivation was 93.69 kJ mol-1. Sucrose, maltose, glucose, fructose, and trehalose at high concentrations appeared to protect yacon PPO against thermal inactivation at 75 and 80 degrees C.

  15. Origin and evolution of lysyl oxidases

    PubMed Central

    Grau-Bové, Xavier; Ruiz-Trillo, Iñaki; Rodriguez-Pascual, Fernando

    2015-01-01

    Lysyl oxidases (LOX) are copper-dependent enzymes that oxidize primary amine substrates to reactive aldehydes. The best-studied role of LOX enzymes is the remodeling of the extracellular matrix (ECM) in animals by cross-linking collagens and elastin, although intracellular functions have been reported as well. Five different LOX enzymes have been identified in mammals, LOX and LOX-like (LOXL) 1 to 4, showing a highly conserved catalytic carboxy terminal domain and more divergence in the rest of the sequence. Here we have surveyed a wide selection of genomes in order to infer the evolutionary history of LOX. We identified LOX proteins not only in animals, but also in many other eukaryotes, as well as in bacteria and archaea – which reveals a pre-metazoan origin for this gene family. LOX genes expanded during metazoan evolution resulting in two superfamilies, LOXL2/L3/L4 and LOX/L1/L5. Considering the current knowledge on the function of mammalian LOX isoforms in ECM remodeling, we propose that LOXL2/L3/L4 members might have preferentially been involved in making cross-linked collagen IV-based basement membrane, whereas the diversification of LOX/L1/L5 forms contributed to chordate/vertebrate-specific ECM innovations, such as elastin and fibronectin. Our work provides a novel view on the evolution of this family of enzymes. PMID:26024311

  16. Leflunomide, a Reversible Monoamine Oxidase Inhibitor.

    PubMed

    Petzer, Jacobus P; Petzer, Anél

    2016-01-01

    A screening study aimed at identifying inhibitors of the enzyme, monoamine oxidase (MAO), among clinically used drugs have indicated that the antirheumatic drug, leflunomide, is an inhibitor of both MAO isoforms. Leflunomide inhibits human MAO-A and MAO-B and exhibits IC50 values of 19.1 μM and 13.7 μM, respectively. The corresponding Ki values are 17.7 μM (MAO-A) and 10.1 μM (MAO-B). Dialyses of mixtures of the MAO enzymes and leflunomide show that inhibition of the MAOs by leflunomide is reversible. The principal metabolite of leflunomide, teriflunomide (A77 1726), in contrast is not an MAO inhibitor. This study concludes that, although leflunomide is only moderately potent as an MAO inhibitor, isoxazole derivatives may represent a general class of MAO inhibitors and this heterocycle may find application in MAO inhibitor design. In this respect, MAO inhibitors are used in the clinic for the treatment of depressive illness and Parkinson's disease, and are under investigation as therapy for certain types of cancer, Alzheimer's disease and age-related impairment of cardiac function.

  17. Molecular "wiring" glucose oxidase in supramolecular architecture.

    PubMed

    Deng, Liu; Liu, Ying; Yang, Guocheng; Shang, Li; Wen, Dan; Wang, Fuan; Xu, Zhiai; Dong, Shaojun

    2007-07-01

    Supramolecular organized multilayers were constructed by multiwalled carbon nanotubes modified with ferrocene-derivatized poly(allylamine) redox polymer and glucose oxidase by electrostatic self-assembly. From the analysis of voltammetric signals and fluorescence results, a linear increment of the coverage of enzyme per bilayer was estimated, which demonstrated that the multilayer is constructed in a spatially ordered manner. The cyclic voltammograms obtained from the indium tin oxide (ITO) electrodes coated by the (Fc-PAH@CNT/GOx)n multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers; that is, the sensitivity is tunable by controlling the number of bilayers associated with ITO electrodes. The incorporation of redox-polymer-functionalized carbon nanotubes (CNT) into enzyme films resulted in a 6-10-fold increase in the glucose electrocatalytic current; the bimolecular rate constant of FADH2 oxidation (wiring efficiency) was increased up to 12-fold. Impedance spectroscopy data have yielded the electron diffusion coefficient (De) of this nanostructure to be over 10(-8) cm2 s(-1), which is typically higher than those systems without CNT by at least a factor of 10, indicating that electron transport in the new supramolecular architecture was enhanced by communication of the redox active site of enzyme, redox polymer, and CNT.

  18. MONOAMINE OXIDASE: RADIOTRACER DEVELOPMENT AND HUMAN STUDIES.

    SciTech Connect

    FOWLER,J.S.; LOGAN,J.; VOLKOW,N.D.; WANG,G.J.; MACGREGOR,R.R.; DING,Y.S.

    2000-09-28

    PET is uniquely capable of providing information on biochemical transformations in the living human body. Although most of the studies of monoamine oxidase (MAO) have focused on measurements in the brain, the role of peripheral MAO as a phase 1 enzyme for the metabolism of drugs and xenobiotics is gaining attention (Strolin Benedetti and Tipton, 1998; Castagnoli et al., 1997.). MAO is well suited for this role because its concentration in organs such as kidneys, liver and digestive organs is high sometimes exceeding that in the brain. Knowledge of the distribution of the MAO subtypes within different organs and different cells is important in determining which substrates (and which drugs and xenobiotics) have access to which MAO subtypes. The highly variable subtype distribution with different species makes human studies even more important. In addition, the deleterious side effects of combining MAO inhibitors with other drugs and with foodstuffs makes it important to know the MAO inhibitory potency of different drugs both in the brain and in peripheral organs (Ulus et al., 2000). Clearly PET can play a role in answering these questions, in drug research and development and in discovering some of the factors which contribute to the highly variable MAO levels in different individuals.

  19. Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans.

    PubMed

    Sugio, Tsuyoshi; Hisazumi, Tomohiro; Kanao, Tadayoshi; Kamimura, Kazuo; Takeuchi, Fumiaki; Negishi, Atsunori

    2006-07-01

    It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.

  20. Effects of feeding endophyte-infected fescue seed to Holstein cows during the dry period on plasma nitric oxide (NO), xanthine oxidase (XO) and haptoglobin (Hp) status in newborn calves.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fescue toxicosis in cattle, caused by ingestion of endophyte-infected fescue (EIF), is associated with decreased feed intake, growth, milk production and reproductive efficiency as well as decreased resistance to heat, transportation and immune stress. Increased inflammatory response to immune chal...

  1. Modeling the effects of estradiol and progesterone on the acute phase proinflammatory axis: Variability in tumor necrosis factor-alpha, nitric oxide, and xanthine oxidase responses to endotoxin challenge in steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The severity of host response in some diseases differs between sexes and this dimorphism has been attributed to the immunomodulating effects of reproductive steroid hormones. In females, susceptibility to disease stress has been associated with reproductive status and attributed to prevailing proge...

  2. Conversion of Escherichia coli pyruvate oxidase to an 'alpha-ketobutyrate oxidase'.

    PubMed Central

    Chang, Y Y; Cronan, J E

    2000-01-01

    Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate ('alpha-ketobutyrate'), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380 had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in K(m) for pyruvate and a 10-fold lower V(max) with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme. PMID:11104678

  3. Identification of the alternative terminal oxidase of higher plant mitochondria

    PubMed Central

    Elthon, Thomas E.; McIntosh, Lee

    1987-01-01

    In addition to cytochrome oxidase, plant mitochondria have a second terminal oxidase called the alternative oxidase. The alternative oxidase is of great interest in that energy is not conserved when electrons flow through it. The potential energy of the system is thus lost as heat, and, in plants with high levels of the alternative oxidase, this results in thermogenesis. We have purified the alternative oxidase from mitochondria of the thermogenic spadix of Sauromatum guttatum and have identified its polypeptide constituents by using polyclonal antibodies. A 166-fold purification was achieved through a combination of cation-exchange (carboxymethyl-Sepharose) and hydrophobic-interaction (phenyl-Sepharose) chromatography. Polyclonal antibodies raised to the CM-Sepharose fractions readily immunoprecipitated alternative oxidase activity and immunoprecipitated four of the proteins that copurify with the activity. These proteins have apparent molecular masses of 37, 36, 35.5, and 35 kDa. Polyclonal antibodies raised individually to the 37-, 36-, and 35.5- plus 35-kDa proteins cross-reacted with all of these proteins, indicating the presence of common antigenic sites. The 37-kDa protein appears to be constitutive in Sauromatum, whereas expression of the 36- and 35-kDa proteins was correlated with presence of alternative pathway activity. The 35.5-kDa protein appears with loss of alternative pathway activity during senescence, indicating that this protein may be a degradation product of the 36-kDa protein. Binding of anti-36-kDa protein antibodies to total mitochondrial protein blots of five plant species indicated that similar proteins were always present when alternative pathway activity was observed. Images PMID:16593898

  4. Cytokinin Oxidase from Phaseolus vulgaris Callus Tissues 1

    PubMed Central

    Chatfield, J. Mark; Armstrong, Donald J.

    1987-01-01

    The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N6-(Δ2-isopentenyl)-adenine-2,8-3H (i6 Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N6-side chain of i6Ade. PMID:16665511

  5. Pentamines as substrate for human spermine oxidase

    PubMed Central

    Takao, Koichi; Shirahata, Akira; Samejima, Keijiro; Casero, Robert A.; Igarashi, Kazuei; Sugita, Yoshiaki

    2013-01-01

    Substrate activities of various linear polyamines to human spermine oxidase (hSMO) were investigated. The activities were evaluated by monitoring the amount of H2O2 released from sample polyamines by hSMO. H2O2 was measured by a HPLC method that analyzed fluorescent dimers derived from the oxidation of homovanillic acid in the presence of horseradish peroxidase. Six triamines were tested and were found not to be hSMO substrates. Of sixteen tetramines tested, spermine (Spm) was the most active substrate, followed by homospermine and N-butylated Spm. Pentamines showed a characteristic pattern of substrate activity. Of thirteen pentamines tested, 3343 showed higher substrate activity than Spm, and 4343 showed similar activity to Spm. The activities of the other pentamines were as follows: 3443, 4443, 4344, 3344, 4334, 4444, and 3334 (in decreasing order). Product amines released from these pentamines by hSMO were then analyzed by HPLC. Triamine was the only observed product, and the amount of triamine was nearly equivalent to that of released H2O2. A marked difference in the pH dependency curves between tetramines and pentamines suggested that hSMO favored reactions with a non-protonated secondary nitrogen at the cleavage site. The Km and Vmax values for Spm and 3343 at pH 7.0 and 9.0 were consistent with the higher substrate activity of 3343 compared to Spm, as well as with the concept of a non-protonated secondary nitrogen at the cleavage site being preferred, and 3343 was well degraded at a physiological pH by hSMO. PMID:23449327

  6. Monoamine oxidase inhibitory components from Cayratia japonica.

    PubMed

    Han, Xiang Hua; Hong, Seong Su; Hwang, Ji Sang; Lee, Myung Koo; Hwang, Bang Yeon; Ro, Jai Seup

    2007-01-01

    Seven flavonoids were isolated from the whole plants and fruits of Cayratia japonica through the activity-guided isolation of a methanol extract using a monoamine oxidase (MAO) inhibition assay as a monitor. The chemical structures of the isolates were assigned as apigenin-7-O-beta-D-glucuronopyranoside (1), apigenin (2), luteolin (3), luteolin-7-O-beta-D-glucopyranoside (4), (+)-dihydroquercetin (taxifolin) (5), (+)-dihydrokaempferol (aromadendrin) (6) and quercetin (7). Among the isolated compounds, flavones such as apigenin (2) and luteolin (3), as well as the flavonol, quercetin (7) showed potent inhibitory effects against the MAO activity with IC50 values of 6.5, 22.6, and 31.6 microM, respectively. However, the flavone glycosides, apigenin-7-O-beta-D-glucuronopyranoside (1) and luteolin-7-O-beta-D-glucopyranoside (4), showed mild MAO inhibition (IC50 values: 81.7 and 118.6 microM, respectively). The flavanonol derivatives, taxifolin (5) and aromadendrin (6), also showed weak inhibition (IC50 values: 154.7 and 153.1 microM, respectively). Furthermore, quercetin (7) had a more potent inhibitory effect on MAO-A (IC50 value: 2.8 microM) than MAO-B (IC50 value: 90.0 microM). Apigenin (2) and luteolin (3) also preferentially inhibited MAO-A (IC50 values: 1.7 and 4.9 microM, respectively) compared with MAO-B (IC50 values: 12.8 and 59.7 microM, respectively).

  7. Cation binding site of cytochrome c oxidase: progress report.

    PubMed

    Vygodina, Tatiana V; Kirichenko, Anna; Konstantinov, Alexander A

    2014-07-01

    Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca(2+)-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the "slow" form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

  8. Crystal Structure of Alcohol Oxidase from Pichia pastoris

    PubMed Central

    Valerius, Oliver; Feussner, Ivo; Ficner, Ralf

    2016-01-01

    FAD-dependent alcohol oxidases (AOX) are key enzymes of methylotrophic organisms that can utilize lower primary alcohols as sole source of carbon and energy. Here we report the crystal structure analysis of the methanol oxidase AOX1 from Pichia pastoris. The crystallographic phase problem was solved by means of Molecular Replacement in combination with initial structure rebuilding using Rosetta model completion and relaxation against an averaged electron density map. The subunit arrangement of the homo-octameric AOX1 differs from that of octameric vanillyl alcohol oxidase and other dimeric or tetrameric alcohol oxidases, due to the insertion of two large protruding loop regions and an additional C-terminal extension in AOX1. In comparison to other alcohol oxidases, the active site cavity of AOX1 is significantly reduced in size, which could explain the observed preference for methanol as substrate. All AOX1 subunits of the structure reported here harbor a modified flavin adenine dinucleotide, which contains an arabityl chain instead of a ribityl chain attached to the isoalloxazine ring. PMID:26905908

  9. NADPH OXIDASE: STRUCTURE AND ACTIVATION MECHANISMS (REVIEW). NOTE I.

    PubMed

    Filip-Ciubotaru, Florina; Manciuc, Carmen; Stoleriu, Gabriela; Foia, Liliana

    2016-01-01

    NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase), with its generically termed NOX isoforms, is the major source of ROS (reactive oxigen species) in biological systems. ROS are small oxygen-derived molecules with an important role in various biological processes (physiological or pathological). If under physiological conditions some processes are beneficial and necessary for life, under pathophysiological conditions they are noxious, harmful. NADPH oxidases are present in phagocytes and in a wide variety of nonphagocytic cells. The enzyme generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling them to molecular oxygen to produce superoxide anion, a reactive free-radical. Structurally, NADPH oxidase is a multicomponent enzyme which includes two integral membrane proteins, glycoprotein gp9 1 Phox and adaptor protein p22(phox), which together form the heterodimeric flavocytochrome b558 that constitutes the core of the enzyme. During the resting state, the multidomain regulatory subunits p40P(phox), p47(phox), p67(Phox) are located in the cytosol organized as a complex. The activation of phagocytic NADPH oxidase occurs through a complex series of protein interactions.

  10. Use of febuxostat in the management of gout in the United Kingdom

    PubMed Central

    Waller, Arabella; Jordan, Kelsey M.

    2016-01-01

    Gout is the most common cause of inflammatory arthritis worldwide. Despite clinical cure being achievable and multiple evidence-based guidelines having been published, the incidence and prevalence continues to increase and the condition remains undertreated. Concerns regarding allopurinol have limited its use in those with renal impairment. Febuxostat, a novel xanthine oxidase inhibitor requiring no dose adjustment in mild−moderate renal impairment was launched in the United Kingdom (UK) in 2010. We review published data on the efficacy, safety and tolerability of febuxostat and provide an opinion on its place in the management of gout in the UK in the context of other published guidelines. One phase II trial, multiple phase III trials [febuxostat versus allopurinol controlled trial (FACT), APEX, CONFIRMS] and two open-label extension trials have demonstrated febuxostat given at the doses commonly used in UK practice (80 mg, 120 mg) to reduce serum urate more effectively than those receiving fixed-dose allopurinol. Overall adverse event rates were comparable across treatment groups aside from gout flare (more common in febuxostat-treated patients) and concerns regarding cardiovascular toxicity are being further evaluated in two large trials. If the outcomes of these are favourable, we would anticipate a marked increase in the use of febuxostat in the UK market. We would advocate the use of febuxostat to target a serum urate < 0.3 mmol/l (5 mg/dl) as a second-line urate-lowering therapy in patients with hyperuricaemia, and clinical gout in those intolerant of allopurinol, or in those in whose renal function precludes optimal dose escalation to achieve target serum urate. We would advise prophylaxis against gouty flare with colchicine, nonsteroidal anti-inflammatory drugs (NSAIDs), or Cyclo-oxygenase-2 selective NSAID (COXIB) after febuxostat initiation. PMID:28255339

  11. Red clover polyphenol oxidase and lipid metabolism.

    PubMed

    Van Ranst, G; Lee, M R F; Fievez, V

    2011-02-01

    Increasing the polyunsaturated fatty acid (PUFA) composition of milk is acknowledged to be of benefit to consumer health. Despite the high PUFA content of forages, milk fat contains only about 3% of PUFA and only about 0.5% of n-3 fatty acids. This is mainly due to intensive lipid metabolism in the rumen (lipolysis and biohydrogenation) and during conservation (lipolysis and oxidation) such as drying (hay) and ensiling (silage). In red clover, polyphenol oxidase (PPO) has been suggested to protect lipids against degradation, both in the silage as well as in the rumen, leading to a higher output of PUFA in ruminant products (meat and milk). PPO mediates the oxidation of phenols and diphenols to quinones, which will readily react with nucleophilic binding sites. Such binding sites can be found on proteins, resulting in the formation of protein-bound phenols. This review summarizes the different methods that have been used to assess PPO activity in red clover, and an overview on the current understanding of PPO activity and activation in red clover. Knowledge on these aspects is of major importance to fully harness PPO's lipid-protecting role. Furthermore, we review the studies that evidence PPO-mediated lipid protection and discuss its possible importance in lab-scale silages and further in an in vitro rumen system. It is demonstrated that high (induction of) PPO activity can lead to lower lipolysis in the silage and lower biohydrogenation in the rumen. There are three hypotheses on its working mechanism: (i) protein-bound phenols could directly bind to enzymes (e.g. lipases) as such inhibiting them; (ii) binding of quinones in and between proteins embedded in a lipid membrane (e.g. in the chloroplast) could lead to encapsulation of the lipids; (iii) direct binding of quinones to nucleophilic sites in polar lipids also could lead to protection. There is no exclusive evidence on which mechanism is most important, although there are strong indications that only lipid

  12. Forage polyphenol oxidase and ruminant livestock nutrition

    PubMed Central

    Lee, Michael R. F.

    2014-01-01

    Polyphenol oxidase (PPO) is predominately associated with the detrimental effect of browning fruit and vegetables, however, interest within PPO containing forage crops (crops to be fed to animals) has grown since the browning reaction was associated with reduced nitrogen (N) losses in silo and the rumen. The reduction in protein breakdown in silo of red clover (high PPO forage) increased the quality of protein, improving N-use efficiency [feed N into product N (e.g., Milk): NUE] when fed to ruminants. A further benefit of red clover silage feeding is a significant reduction in lipolysis (cleaving of glycerol-based lipid) in silo and an increase in the deposition of beneficial C18 polyunsaturated fatty acid (PUFA) in animal products, which has also been linked to PPO activity. PPOs protection of plant protein and glycerol based-PUFA in silo is related to the deactivation of plant proteases and lipases. This deactivation occurs through PPO catalyzing the conversion of diphenols to quinones which bind with cellular nucleophiles such as protein reforming a protein-bound phenol (PBP). If the protein is an enzyme (e.g., protease or lipase) the complexing denatures the enzyme. However, PPO is inactive in the anaerobic rumen and therefore any subsequent protection of plant protein and glycerol based-PUFA in the rumen must be as a result of events that occurred to the forage pre-ingestion. Reduced activity of plant proteases and lipases would have little effect on NUE and glycerol based-PUFA in the rumen due to the greater concentration of rumen microbial proteases and lipases. The mechanism for PPOs protection of plant protein in the rumen is a consequence of complexing plant protein, rather than protease deactivation per se. These complexed proteins reduce protein digestibility in the rumen and subsequently increase undegraded dietary protein flow to the small intestine. The mechanism for protecting glycerol-based PUFA has yet to be fully elucidated but may be associated

  13. Expression of alternative oxidase in Drosophila ameliorates diverse phenotypes due to cytochrome oxidase deficiency.

    PubMed

    Kemppainen, Kia K; Rinne, Juho; Sriram, Ashwin; Lakanmaa, Matti; Zeb, Akbar; Tuomela, Tea; Popplestone, Anna; Singh, Satpal; Sanz, Alberto; Rustin, Pierre; Jacobs, Howard T

    2014-04-15

    Mitochondrial dysfunction is a significant factor in human disease, ranging from systemic disorders of childhood to cardiomyopathy, ischaemia and neurodegeneration. Cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain, is a frequent target. Lower eukaryotes possess alternative respiratory-chain enzymes that provide non-proton-translocating bypasses for respiratory complexes I (single-subunit reduced nicotinamide adenine dinucleotide dehydrogenases, e.g. Ndi1 from yeast) or III + IV [alternative oxidase (AOX)], under conditions of respiratory stress or overload. In previous studies, it was shown that transfer of yeast Ndi1 or Ciona intestinalis AOX to Drosophila was able to overcome the lethality produced by toxins or partial knockdown of complex I or IV. Here, we show that AOX can provide a complete or substantial rescue of a range of phenotypes induced by global or tissue-specific knockdown of different cIV subunits, including integral subunits required for catalysis, as well as peripheral subunits required for multimerization and assembly. AOX was also able to overcome the pupal lethality produced by muscle-specific knockdown of subunit CoVb, although the rescued flies were short lived and had a motility defect. cIV knockdown in neurons was not lethal during development but produced a rapidly progressing locomotor and seizure-sensitivity phenotype, which was substantially alleviated by AOX. Expression of Ndi1 exacerbated the neuronal phenotype produced by cIV knockdown. Ndi1 expressed in place of essential cI subunits produced a distinct residual phenotype of delayed development, bang sensitivity and male sterility. These findings confirm the potential utility of alternative respiratory chain enzymes as tools to combat mitochondrial disease, while indicating important limitations thereof.

  14. Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

    SciTech Connect

    Pan, Heng-Yen; Whittaker, Mei M.; Bouveret, Romaric; Berna, Anne; Bernier, Francois; Whittaker, James W. . E-mail: jim@ebs.ogi.edu

    2007-05-18

    High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an {alpha}-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4 x 10{sup 4} U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.

  15. Purification and two-dimensional crystallization of bacterial cytochrome oxidases.

    PubMed

    Warne, A; Wang, D N; Saraste, M

    1995-12-01

    A novel strategy which employes chromatography on an immobilized metal ion has been developed for the purification of bacterial cytochrome c and quinol oxidases. Many bacterial oxidase complexes appear to have a natural affinity to bind to the chelated copper ion. A combination of three different chromatographic principles (anion exchange, metal-affinity and gel filtration) makes an effective tool chest for the preparation of homogeneous and protein-chemically pure bacterial oxidases. These preparations have been used for two-dimensional crystallization. Until now, crystals have been obtained using the Paracococcus denitrificans and Rhodobacter sphaeroides cytochrome aa3 and the Escherichia coli cytochrome bo. The crystals diffract to approximately 2.5 nm in negative stain and have potential for further structural studies.

  16. Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

    PubMed Central

    Pan, Heng-Yen; Whittaker, Mei M.; Bouveret, Romaric; Berna, Anne; Bernier, François; Whittaker, James W.

    2007-01-01

    High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an α-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4×104 U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions. PMID:17399681

  17. Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha.

    PubMed

    Veenhuis, M; van Dijken, J P; Harder, W

    1976-12-01

    The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes D-aminoacid oxidase and L-alpha-hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.

  18. A xanthine derivative denbufylline inhibits negative inotropic response to verapamil in guinea pig ventricular papillary muscles, independent of its phosphodiesterase inhibitory activity.

    PubMed

    Sanae, F; Ohmae, S; Takagi, K; Miyamoto, K

    1995-11-01

    A phosphodiesterase (PDE) III inhibitor, amrinone, inhibited both the negative inotropic actions of verapamil and nicardipine in guinea pig ventricular papillary muscle; this effect was canceled by the protein kinase A inhibitor H-89. The PDE IV inhibitor 1,3-di-n-butyl-7-(2'-oxopropyl)xanthine (denbufylline), which elicited a negative inotropic action by itself, attenuated the action of verapamil up to 10 microM, without any interaction with nicardipine. The attenuation by denbufylline was not influenced by H-89. This suggests that in the ventricular papillary muscle, denbufylline acts on some verapamil-sensitive site(s) in the membrane and interferes with the calcium channel function without involvement of its PDE inhibitory activity.

  19. A Conserved Steroid Binding Site in Cytochrome c Oxidase

    SciTech Connect

    Qin, Ling; Mills, Denise A.; Buhrow, Leann; Hiser, Carrie; Ferguson-Miller, Shelagh

    2010-09-02

    Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

  20. Functional characterization of gibberellin oxidases from cucumber, Cucumis sativus L.

    PubMed

    Pimenta Lange, Maria João; Liebrandt, Anja; Arnold, Linda; Chmielewska, Sara-Miriam; Felsberger, André; Freier, Eduard; Heuer, Monika; Zur, Doreen; Lange, Theo

    2013-06-01

    Cucurbits have been used widely to elucidate gibberellin (GA) biosynthesis. With the recent availability of the genome sequence for the economically important cucurbit Cucumis sativus, sequence data became available for all genes potentially involved in GA biosynthesis for this species. Sixteen cDNAs were cloned from root and shoot of 3-d to 7-d old seedlings and from mature seeds of C. sativus. Two cDNAs code for GA 7-oxidases (CsGA7ox1, and -2), five for GA 20-oxidases (CsGA20ox1, -2, -3, -4, and -5), four for GA 3-oxidases (CsGA3ox1, -2, -3, and -4), and another five for GA 2-oxidases (CsGA2ox1, -2, -3, -4, and -5). Their enzymatic activities were investigated by heterologous expression of the cDNAs in Escherichia coli and incubation of the cell lysates with (14)C-labelled, D2-labelled, or unlabelled GA-substrates. The two GA 7-oxidases converted GA12-aldehyde to GA12 efficiently. CsGA7ox1 converted GA12 to GA14, to 15α-hydroxyGA12, and further to 15α-hydroxyGA14. CsGA7ox2 converted GA12 to its 12α-hydroxylated analogue GA111. All five GA 20-oxidases converted GA12 to GA9 as a major product, and to GA25 as a minor product. The four GA 3-oxidases oxidized the C19-GA GA9 to GA4 as the only product. In addition, three of them (CsGA3ox2, -3, and -4) converted the C20-GA GA12 to GA14. The GA 2-oxidases CsGA2ox1, -2, -3, and -4 oxidized the C19-GAs GA9 and GA4 to GA34 and GA51, respectively. CsGA2ox2, -3, and -4 converted GA51 and GA34 further to respective GA-catabolites. In addition to C19-GAs, CsGA2ox4 also converted the C20-GA GA12 to GA110. In contrast, CsGA2ox5 oxidized only the C20 GA12 to GA110 as the sole product. As shown for CsGA20ox1 and CsGA3ox1, similar reactions were catalysed with 13-hydroxlyated GAs as substrates. It is likely that these enzymes are also responsible for the biosynthesis of 13-hydroxylated GAs in vivo that occur at low levels in cucumber.

  1. Rational pharmacotherapy (RPT) in goutology: Define the serum uric acid target & treat-to-target patient cohort and review on urate lowering therapy (ULT) applying synthetic drugs.

    PubMed

    Jansen, Tim L

    2015-07-01

    A gout revolution is at hand as can be seen from the number of publications and our recent increase in a better understanding of gout regarding imaging, regarding pathogenetic involvement of crystals, cells and cytokines, as well as regarding new pharmacotherapeutic options. We should now focus on rational pharmacotherapy to significantly improve gout care. With modern combinations of xanthine oxidase inhibition PLUS uricosuric all serum urate concentrations can be targeted. The pharmacotherapeutic literature of synthetic urate lowering treatment is reviewed and a plea is given for rational pharmacotherapy combining different modes of action aiming at the rheumatologically predefined optimal serum urate concentrations instead of a more reluctant approach to just lower a serum urate to any lower level with a fixed dose allopurinol.

  2. The involvement of reactive oxygen species in hypoxic injury to rat liver.

    PubMed

    Younes, M; Strubelt, O

    1988-03-01

    Isolated perfused livers from fasted, but not from fed rats showed hepatotoxic responses when subjected to 30 min of hypoxia followed by 60 min of reoxygenation. Toxicity was evident by a release of glutamate-pyruvate-transaminase, lactate dehydrogenase and glutathione into the perfusate, by a depletion of hepatic glutathione and by an accumulation of calcium in the liver. This indicates, that the liver is resistant to hypoxic injury as long as glycogen is present to maintain anaerobic ATP-synthesis. This is substantiated by the fact that addition of fructose--but not glucose--to the medium resulted in a protection of the liver against hypoxic injury concomitant with its degradation to lactate + pyruvate. Superoxide dismutase, catalase, desferrioxamine and allopurinol prevented hypoxic liver injury suggesting a substantial role of reactive oxygen species formed via the xanthine oxidase reaction in mediating hypoxic liver injury.

  3. Beware the low urine pH--the major cause of the increased prevalence of nephrolithiasis in the patient with type 2 diabetes.

    PubMed

    Bell, D S H

    2012-04-01

    There is an increased prevalence of nephrolithiasis and an increase in the incidence of renal colic in patients with diabetes, obesity, hypertension and insulin resistance because of an increased frequency of uric acid crystallization. Uric acid crystallization occurs in the milieu of an acid urine and is not due to hyperuricosuria as with insulin resistance, urinary uric acid levels are generally decreased because of increased renal tubular reabsorption. However, in the presence of insulin resistance, there is decreased renal tubular generation of ammonia and increased sodium absorption leading to acidification of the urine and uric acid crystallization. The presence of a low urine pH should alert the clinician to the increased risk of nephrolithiasis particularly in the obese, diabetic or hypertensive patient. Prevention of nephrolithiasis can be achieved in susceptible individuals either by alkalizing the urine and/or by further decreasing the uric acid content of the urine with a xanthine oxidase inhibitor such as allopurinol.

  4. Expression of terminal oxidases under nutrient-starved conditions in Shewanella oneidensis: detection of the A-type cytochrome c oxidase

    PubMed Central

    Le Laz, Sébastien; kpebe, Arlette; Bauzan, Marielle; Lignon, Sabrina; Rousset, Marc; Brugna, Myriam

    2016-01-01

    Shewanella species are facultative anaerobic bacteria that colonize redox-stratified habitats where O2 and nutrient concentrations fluctuate. The model species Shewanella oneidensis MR-1 possesses genes coding for three terminal oxidases that can perform O2 respiration: a bd-type quinol oxidase and cytochrome c oxidases of the cbb3-type and the A-type. Whereas the bd- and cbb3-type oxidases are routinely detected, evidence for the expression of the A-type enzyme has so far been lacking. Here, we investigated the effect of nutrient starvation on the expression of these terminal oxidases under different O2 tensions. Our results reveal that the bd-type oxidase plays a significant role under nutrient starvation in aerobic conditions. The expression of the cbb3-type oxidase is also modulated by the nutrient composition of the medium and increases especially under iron-deficiency in exponentially growing cells. Most importantly, under conditions of carbon depletion, high O2 and stationary-growth, we report for the first time the expression of the A-type oxidase in S. oneidensis, indicating that this terminal oxidase is not functionally lost. The physiological role of the A-type oxidase in energy conservation and in the adaptation of S. oneidensis to redox-stratified environments is discussed. PMID:26815910

  5. Peroxisomal polyamine oxidase and NADPH-oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana

    PubMed Central

    Andronis, Efthimios A.; Moschou, Panagiotis N.; Toumi, Imene; Roubelakis-Angelakis, Kalliopi A.

    2014-01-01

    Homeostasis of reactive oxygen species (ROS) in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA) are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd) and spermine to putrescine and Spd, respectively, is catalyzed by two peroxisomal PA oxidases (AtPAO). However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI). Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions (O2•− ), but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX). On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and O2•− . These results suggest that the ratio of O2•− /H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of O2•− by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed. PMID:24765099

  6. Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies.

    PubMed

    Andjelković, Ana; Oliveira, Marcos T; Cannino, Giuseppe; Yalgin, Cagri; Dhandapani, Praveen K; Dufour, Eric; Rustin, Pierre; Szibor, Marten; Jacobs, Howard T

    2015-12-17

    The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression.

  7. Covalent immobilization of ascorbate oxidase onto polycarbonate strip for L-ascorbic acid detection.

    PubMed

    Kannoujia, Dileep Kumar; Kumar, Saroj; Nahar, Pradip

    2012-10-01

    Herein, a simple and rapid method is described for detection of L-ascorbic acid by ascorbate oxidase immobilized onto polycarbonate strip pre-activated by 1-fluoro-2-nitro-4-azidobenzene in photochemical reaction. Covalent attachment of ascorbate oxidase was confirmed by XPS studies. The immobilized-ascorbate oxidase shows higher pH, thermal and storage stability in comparison to free enzyme.

  8. Dopa oxidase activity and ceruloplasmin in the sera of hamsters with melanoma.

    PubMed

    Vachtenheim, J; Pavel, S; Duchon, J

    1981-01-01

    Two simple spectrophotometric assays have been employed for the measurement of dopa oxidase activity and ceruloplasmin polyphenol oxidase activity in the sera from normal hamsters and hamsters bearing melanotic melanoma. Both activities were found to be augmented in tumor animals, the dopa oxidase activity much more prominently. The levels of the enzymes tested increased proportionally to the tumor mass.

  9. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420...

  10. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420...

  11. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420...

  12. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420...

  13. 21 CFR 866.2420 - Oxidase screening test for gonorrhea.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Oxidase screening test for gonorrhea. 866.2420 Section 866.2420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2420...

  14. Amine oxidase activity regulates the development of pulmonary fibrosis.

    PubMed

    Marttila-Ichihara, Fumiko; Elima, Kati; Auvinen, Kaisa; Veres, Tibor Z; Rantakari, Pia; Weston, Christopher; Miyasaka, Masayuki; Adams, David; Jalkanen, Sirpa; Salmi, Marko

    2017-03-01

    In pulmonary fibrosis, an inflammatory reaction and differentiation of myofibroblasts culminate in pathologic deposition of collagen. Amine oxidase copper containing-3 (AOC3) is a cell-surface expressed oxidase that regulates leukocyte extravasation. Here we analyzed the potential role of AOC3 using gene-modified and inhibitor-treated mice in a bleomycin-induced pulmonary fibrosis model. Inflammation and fibrosis of lungs were assessed by histologic, flow cytometric, and quantitative PCR analysis. AOC3-deficient mice showed a 30-50% reduction in fibrosis, collagen synthesis, numbers of myofibroblasts, and accumulation of CD4(+) lymphocytes, NK T cells, macrophages, and type 2 innate lymphoid cells compared with wild-type control mice. AOC3 knock-in mice, which express a catalytically inactive form of AOC3, were also protected from lung fibrosis. In wild-type mice, a small-molecule AOC3 inhibitor treatment reduced leukocyte infiltration, myofibroblast differentiation, and fibrotic injury both in prophylactic and early therapeutic settings by about 50% but was unable to reverse the established fibrosis. AOC3 was also induced in myofibroblasts in human idiopathic pulmonary fibrosis. Thus, the oxidase activity of AOC3 contributes to the development of lung fibrosis mainly by regulating the accumulation of pathogenic leukocyte subtypes, which drive the fibrotic response.-Marttila-Ichihara, F., Elima, K., Auvinen, K., Veres, T. Z., Rantakari, P., Weston, C., Miyasaka, M., Adams, D., Jalkanen, S., Salmi, M. Amine oxidase activity regulates the development of pulmonary fibrosis.

  15. Solubilized cytochrome c oxidase from Paracoccus denitrificans is a monomer.

    PubMed

    Ludwig, B; Grabo, M; Gregor, I; Lustig, A; Regenass, M; Rosenbusch, J P

    1982-05-25

    Cytochrome c oxidase purified from the bacterium Paracoccus denitrificans was analyzed by analytical ultracentrifugation. In the detergent octyltetra/pentaoxyethylene (C8E45), the isolated enzyme exhibits a molecular weight of 79,000 to 84,000. The detergent-solubilized enzyme is thus a monomer which contains one copy of each of the two subunits.

  16. The number of nucleotide binding sites in cytochrome C oxidase.

    PubMed

    Rieger, T; Napiwotzki, J; Hüther, F J; Kadenbach, B

    1995-12-05

    The binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), [35S]ATP alpha S and 8-azido-[gamma-32P]ATP to isolated cytochrome c oxidase of bovine heart and liver and to the two-subunit enzyme of Paracoccus dentrificans was studied by measuring the fluorescence change or bound radioactivity, respectively. With TNP-ATP three binding sites were determined at cytochrome c oxidase from bovine heart and liver, both with two dissociation constants Kd of about 0.2 and 0.9 microM. Trypsin treatment of the enzyme from bovine heart, resulted in one binding site with a Kd of 0.3 microM. The two-subunit enzyme of Paracoccus dentrificans had only one binding site with a Kd of 3.6 microM. The binding of [35S]ATP alpha S to cytochrome c oxidase was studied by equilibrium dialysis. With the enzyme of bovine heart seven and the enzyme of liver six high-affinity binding sites with apparent Kd's of 7.5 and 12 microM, respectively, were obtained. The two-subunit enzyme of Paracoccus denitrificans had one binding site with a Kd of 20 microM. The large number of binding sites at cytochrome c oxidase from bovine heart, mainly at nuclear coded subunits, was verified by photoaffinity labelling with 8-azido-[gamma-32P]ATP.

  17. Purification of cytochrome c oxidase by lysine-affinity chromatography.

    PubMed

    Felsch, J; Kotake, S; Copeland, R A

    1992-02-01

    A method for the purification of cytochrome c oxidase that is based on the affinity of this enzyme for polycations such as poly-L-lysine is described. When detergent extracts of bovine cardiac mitochondria were applied to either a poly-L-lysine-agarose or a lysine-Sepharose column at low ionic strength, cytochrome c oxidase was found to adhere tightly, whereas the bulk of the proteins were eluted by washing with the same buffer. The cytochrome c oxidase was eluted by application of a linear potassium chloride gradient to the columns. The resulting enzyme was identical to that obtained by more traditional purification methods in terms of its subunit composition, optical and resonance Raman spectra, and cytochrome c oxidizing activity. When detergent extracts of spheroplasts from Paracoccus denitrificans were applied to these columns, the cytochrome c oxidase from this organism was also found to adhere tightly. Thus this purification method appears applicable to both prokaryotic and eukaryotic forms of the enzyme. The advantages of this new purification method are that it is less labor intensive than the traditional procedure and less expensive than methods based on cytochrome c-affinity chromatography.

  18. HypC, the anthrone oxidase involved in aflatoxin biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Based on gene disruption and enzyme activity, hypC, an open reading frame in the pksA (aflC)/nor-1 (aflD) intergenic region in the aflatoxin biosynthesis cluster, encodes a 17 kDa oxidase that catalyzes the conversion of norsolorinic acid anthrone to norsolorinic acid....

  19. Reducing peanut allergens by high pressure combined with polyphenol oxidase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO) has been shown to reduce major peanut allergens (Ara h 1 and Ara h 2). Because high pressure (HP) can increase enzyme activity, we postulated that further reduction of peanut allergens can be achieved through HP combined with PPO. Peanut extracts were treated with each of th...

  20. The glucose oxidase-peroxidase assay for glucose

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The glucose oxidase-peroxidase assay for glucose has served as a very specific, sensitive, and repeatable assay for detection of glucose in biological samples. It has been used successfully for analysis of glucose in samples from blood and urine, to analysis of glucose released from starch or glycog...

  1. Platinum Nanoparticles: Efficient and Stable Catechol Oxidase Mimetics.

    PubMed

    Liu, Yi; Wu, Haohao; Chong, Yu; Wamer, Wayne G; Xia, Qingsu; Cai, Lining; Nie, Zhihong; Fu, Peter P; Yin, Jun-Jie

    2015-09-09

    Although enzyme-like nanomaterials have been extensively investigated over the past decade, most research has focused on the peroxidase-like, catalase-like, or SOD-like activity of these nanomaterials. Identifying nanomaterials having oxidase-like activities has received less attention. In this study, we demonstrate that platinum nanoparticles (Pt NPs) exhibit catechol oxidase-like activity, oxidizing polyphenols into the corresponding o-quinones. Four unique approaches are employed to demonstrate the catechol oxidase-like activity exerted by Pt NPs. First, UV-vis spectroscopy is used to monitor the oxidation of polyphenols catalyzed by Pt NPs. Second, the oxidized products of polyphenols are identified by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution mass spectrometry (HRMS) identification. Third, electron spin resonance (ESR) oximetry techniques are used to confirm the O2 consumption during the oxidation reaction. Fourth, the intermediate products of semiquinone radicals formed during the oxidation of polyphenols are determined by ESR using spin stabilization. These results indicate Pt NPs possess catechol oxidase-like activity. Because polyphenols and related bioactive substances have been explored as potent antioxidants that could be useful for the prevention of cancer and cardiovascular diseases, and Pt NPs have been widely used in the chemical industry and medical science, it is essential to understand the potential effects of Pt NPs for altering or influencing the antioxidant activity of polyphenols.

  2. Polyphenol oxidase activity in co-ensiled temperate grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO) and its o-diphenol substrates have been shown to effectively decrease proteolytic activity during the ensiling of forages such as red clover. Orchardgrass and smooth bromegrass both contain high levels of PPO activity, but lack appropriate levels of o-diphenols to adequately...

  3. The proton pump of heme-copper oxidases.

    PubMed

    Papa, S; Capitanio, N; Glaser, P; Villani, G

    1994-05-01

    Proton pumping heme-copper oxidases represent the terminal, energy-transfer enzymes of respiratory chains in prokaryotes and eukaryotes. The CuB-heme a3 (or heme o) binuclear center, associated with the largest subunit I of cytochrome c and quinol oxidases, is directly involved in the coupling between dioxygen reduction and proton pumping. The role of the other subunits is less clear. The following aspects will be covered in this paper: i) the efficiency of coupling in the mitochondrial aa3 cytochrome c oxidase. In particular, the effect of respiratory rate and protonmotive force on the H+/e- stoichiometry and the role of subunit IV; ii) mutational analysis of the aa3 quinol oxidase of Bacillus subtilis addressed to the role of subunit III, subunit IV and specific residues in subunit I; iii) possible models of the protonmotive catalytic cycle at the binuclear center. The observations available suggest that H+/e- coupling is based on the combination of protonmotive redox catalysis at the binuclear center and co-operative proton transfer in the protein.

  4. Targeting NADPH oxidase and phospholipases A2 in Alzheimer's disease.

    PubMed

    Simonyi, Agnes; He, Yan; Sheng, Wenwen; Sun, Albert Y; Wood, W Gibson; Weisman, Gary A; Sun, Grace Y

    2010-06-01

    Alzheimer's disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Abeta) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Abeta. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Abeta in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A(2) (PLA(2)) and secretory PLA(2). In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Abeta NADPH oxidase and PLA(2) can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.

  5. [L-lysine-alpha-oxidase activity of some Trichoderma species].

    PubMed

    Smirnova, I P; Khaduev, S Kh

    1984-01-01

    Trichoderma cultures were tested for their ability to produce L-lysine-alpha-oxidase. The highest enzyme activity was manifested by T. harzianum (MGU), T. longibrachiatum Rifai VKM F-2025 and T. aureoviride Rifai VKM F-2026. The biosynthesis of the enzyme did not depend on the growth of the cultures and did not vary among the species.

  6. Beyond brown: polyphenol oxidases as enzymes of plant specialized metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most cloned and/or characterized plant polyphenol oxidases (PPOs) have catecholase activity (i.e., they oxidize o-diphenols to o-quinones) and are localized or predicted to be localized to plastids. As a class, they have broad substrate specificity and are associated with browning of produce and oth...

  7. Purification of gibberellin sub 53 -oxidase from spinach

    SciTech Connect

    Wilson, T.M.; Zeevaart, J.A.D. )

    1989-04-01

    Spinach is a long-day rosette plants, in which stem growth is mediated by gibberellins. It has been shown that two enzymatic steps, GA{sub 53}-oxidase and GA{sub 19}-oxidase, are controlled by light. To develop an understanding into this light regulation, purification of GA{sub 53}-oxidase has been undertaken. The original assay relied on the HPLC separation of the product and substrate, but was considered too slow for the development of a purification scheme. A TLC system was developed which in conjunction with improvements to the assay conditions was sensitive and gave rapid results. The partial purification of the GA{sub 53}-oxidase is achieved by a high speed centrifugation, 40-55% ammonium sulfate precipitation, an hydroxyapatite column, Sephadex G-100 column and an anion exchange FPLC column, Mono Q HR10/10, yielding 1000-fold purification and 15% recovery. Monoclonal antibodies to the protein will be raised and used to further characterize the enzyme.

  8. Berberine-induced apoptosis in human prostate cancer cells is initiated by reactive oxygen species generation

    SciTech Connect

    Meeran, Syed M.; Katiyar, Suchitra; Katiyar, Santosh K.

    2008-05-15

    Phytochemicals show promise as potential chemopreventive or chemotherapeutic agents against various cancers. Here we report the chemotherapeutic effects of berberine, a phytochemical, on human prostate cancer cells. The treatment of human prostate cancer cells (PC-3) with berberine induced dose-dependent apoptosis but this effect of berberine was not seen in non-neoplastic human prostate epithelial cells (PWR-1E). Berberine-induced apoptosis was associated with the disruption of the mitochondrial membrane potential, release of apoptogenic molecules (cytochrome c and Smac/DIABLO) from mitochondria and cleavage of caspase-9,-3 and PARP proteins. This effect of berberine on prostate cancer cells was initiated by the generation of reactive oxygen species (ROS) irrespective of their androgen responsiveness, and the generation of ROS was through the increased induction of xanthine oxidase. Treatment of cells with allopurinol, an inhibitor of xanthine oxidase, inhibited berberine-induced oxidative stress in cancer cells. Berberine-induced apoptosis was blocked in the presence of antioxidant, N-acetylcysteine, through the prevention of disruption of mitochondrial membrane potential and subsequently release of cytochrome c and Smac/DIABLO. In conclusion, the present study reveals that the berberine-mediated cell death of human prostate cancer cells is regulated by reactive oxygen species, and therefore suggests that berberine may be considered for further studies as a promising therapeutic candidate for prostate cancer.

  9. O2 free radicals: cause of ischemia-reperfusion injury to cardiac Na+-K+-ATPase

    SciTech Connect

    Kim, M.S.; Akera, T.

    1987-02-01

    The role of O2 free radicals in the reduction of sarcolemmal Na+-K+-ATPase, which occurs during reperfusion of ischemic heart, was examined in isolated guinea pig heart using exogenous scavengers of O2 radicals and an inhibitor of xanthine oxidase. Ischemia and reperfusion reduced Na+-K+-ATPase activity and specific (3H)ouabain binding to the enzyme in ventricular muscle homogenates and also markedly lowered sodium pump activity estimated from ouabain-sensitive 86Rb+ uptake by ventricular muscle slices. These effects of ischemia and reperfusion were prevented to various degrees by O2-radical scavengers, such as superoxide dismutase, catalase, dimethyl-sulfoxide, histidine, or vitamin E or by the xanthine oxidase inhibitor, allopurinol. The degree of protection afforded by these agents paralleled that of reduction in enhanced lipid peroxidation of myocardial tissue as estimated from malondialdehyde production. These results strongly suggest that O2 radicals play a crucial role in the injury to sarcolemmal Na+-K+-ATPase during reperfusion of ischemic heart.

  10. Reduction of 2,4-dinitrotoluene by Wistar rat liver microsomal and cytosol fractions.

    PubMed

    Mori, M; Matsuhashi, T; Miyahara, T; Shibata, S; Izima, C; Kozuka, H

    1984-10-01

    2,4-Dinitrotoluene (2,4-DNT) is an important industrial nitroaromatic compound. 2,4-Diaminotoluene (2,4-DAT), one of the urinary metabolites of 2,4-DNT, is carcinogenic when fed to rats. The objectives of these studies were to determine whether 2,4-DAT was formed from 2,4-DNT in rat liver and to clarify the nature of enzymes responsible for reduction of 2,4-DNT to 2,4-DAT. Data obtained from thin-layer and high-pressure liquid chromatography indicated that metabolites produced by microsomal preparations were 2-amino-4-nitrotoluene (2A4NT) and its isomer (4A2NT). This microsomal activity is probably mediated by cytochrome P-450 because the reduction is blocked by carbon monoxide and primary amines [aniline, n-octylamine, and 2,4-dichloro-6-phenylphenoxyethylamine (DPEA)]. In contrast, 2,4-DNT was metabolized via 2A4NT and 4A2NT to 2,4-DAT by cytosolic preparations. The greatest part of the reduction activity was due to cytosolic xanthine oxidase because the reduction was blocked by allopurinol. The results of this investigation suggest that reduction of 2,4-DNT to 2,4-DAT by cytosolic xanthine oxidase may play a role in 2,4-DNT hepatocarcinogenicity.

  11. Structure of the trypanosome cyanide-insensitive alternative oxidase

    PubMed Central

    Shiba, Tomoo; Kido, Yasutoshi; Sakamoto, Kimitoshi; Inaoka, Daniel Ken; Tsuge, Chiaki; Tatsumi, Ryoko; Takahashi, Gen; Balogun, Emmanuel Oluwadare; Nara, Takeshi; Aoki, Takashi; Honma, Teruki; Tanaka, Akiko; Inoue, Masayuki; Matsuoka, Shigeru; Saimoto, Hiroyuki; Moore, Anthony L.; Harada, Shigeharu; Kita, Kiyoshi

    2013-01-01

    In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O2 reduction. PMID:23487766

  12. Expression studies of gibberellin oxidases in developing pumpkin seeds.

    PubMed

    Frisse, Andrea; Pimenta, Maria João; Lange, Theo

    2003-03-01

    Two cDNA clones, 3-ox and 2-ox, have been isolated from developing pumpkin (Cucurbita maxima) embryos that show significant amino acid homology to gibberellin (GA) 3-oxidases and 2-oxidases, respectively. Recombinant fusion protein of clone 3-ox converted GA(12)-aldehyde, GA(12), GA(15), GA(24), GA(25), and GA(9) to GA(14)-aldehyde, GA(14), GA(37), GA(36), GA(13), and GA(4), respectively. Recombinant 2-ox protein oxidized GA(9), GA(4), and GA(1) to GA(51), GA(34), and GA(8), respectively. Previously cloned GA 7-oxidase revealed additional 3beta-hydroxylation activity of GA(12). Transcripts of this gene were identified in endosperm and embryo of the developing seed by quantitative reverse transcriptase-polymerase chain reaction and localized in protoderm, root apical meristem, and quiescent center by in situ hybridization. mRNA of the previously cloned GA 20-oxidase from pumpkin seeds was localized in endosperm and in tissues of protoderm, ground meristem, and cotyledons of the embryo. However, transcripts of the recently cloned GA 20-oxidase from pumpkin seedlings were found all over the embryo, and in tissues of the inner seed coat at the micropylar end. Previously cloned GA 2beta,3beta-hydroxylase mRNA molecules were specifically identified in endosperm tissue. Finally, mRNA molecules of the 3-ox and 2-ox genes were found in the embryo only. 3-ox transcripts were localized in tissues of cotyledons, protoderm, and inner cell layers of the root apical meristem, and 2-ox transcripts were found in all tissues of the embryo except the root tips. These results indicate tissue-specific GA-biosynthetic pathways operating within the developing seed.

  13. Oxidative metabolic pathway of lenvatinib mediated by aldehyde oxidase.

    PubMed

    Inoue, Kazuko; Mizuo, Hitoshi; Kawaguchi, Shinki; Fukuda, Katsuyuki; Kusano, Kazutomi; Yoshimura, Tsutomu

    2014-08-01

    Lenvatinib is a multityrosine kinase inhibitor that inhibits vascular endothelial growth factor receptors, and is being developed as an anticancer drug. P450s are involved in one of the elimination pathways of lenvatinib, and mono-oxidized metabolites, such as N-oxide (M3) and desmethylated metabolite (M2), form in rats, dogs, monkeys, and humans. Meanwhile, two other oxidative metabolites are produced only in monkey and human liver S9 fractions, and their structures have been identified using high-resolution mass spectrometry as a quinolinone form of lenvatinib (M3') and a quinolinone form of desmethylated lenvatinib (M2'). The formation of M3' from lenvatinib occurred independently of NADPH and was effectively inhibited by typical inhibitors of aldehyde oxidase, indicating the involvement of aldehyde oxidase, but not P450s, in this pathway. M2' was a dioxidized metabolite arising from a combination of mono-oxidation and desmethylation and could only be produced from M2 in a NADPH-independent manner; M2' could not be generated from M3 or M3'. These results suggested that M2' is formed from lenvatinib by a unique two-step pathway through M2. Although both lenvatinib and M2 were substrates for aldehyde oxidase, an enzyme kinetic study indicated that M2 was a much more favorable substrate than lenvatinib. No inhibitory activities of lenvatinib, M2', or M3' and no significant inhibitory activities of M2 or M3 on aldehyde oxidase were observed, suggesting a low possibility of drug-drug interactions in combination therapy with substrates of aldehyde oxidase.

  14. Inheritance of polyphenol oxidase activity in wheat breeding lines derived from matings of low polyphenol oxidase parents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO) in grain plays a major role in time-dependent discoloration of wheat (Triticum aestivum L.) products, especially fresh noodles. Breeding wheat cultivars with low or nil PPO activity can reduce the undesirable product darkening. The low PPO line PI 117635 was crossed to two...

  15. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

    PubMed

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-03-29

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo.

  16. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

    PubMed Central

    Molitor, Christian; Mauracher, Stephan Gerhard

    2016-01-01

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfat