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Sample records for acid oxidase activator

  1. Screening of Bothrops snake venoms for L-amino acid oxidase activity.

    PubMed

    Pessatti, M; Fontana, J D; Furtado, M F; Guimãraes, M F; Zanette, L R; Costa, W T; Baron, M

    1995-01-01

    Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use of biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom. PMID:7668847

  2. Screening of Bothrops snake venoms for L-amino acid oxidase activity

    SciTech Connect

    Pessati, M.L.; Fontana, J.D.; Guimaraes, M.F.

    1995-12-31

    Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom.

  3. 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

    2014-08-01

    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. PMID:25029106

  4. Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

    PubMed Central

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  5. Oleic, linoleic and linolenic acids increase ros production by fibroblasts via NADPH oxidase activation.

    PubMed

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47 (phox) phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47 (phox) mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  6. Monoamine Oxidase Inhibitory Activity of Ferulic Acid Amides: Curcumin-Based Design and Synthesis.

    PubMed

    Badavath, Vishnu N; Baysal, İpek; Uçar, Gülberk; Mondal, Susanta K; Sinha, Barij N; Jayaprakash, Venkatesan

    2016-01-01

    Ferulic acid has structural similarity with curcumin which is being reported for its monoamine oxidase (MAO) inhibitory activity. Based on this similarity, we designed a series of ferulic acid amides 6a-m and tested for their inhibitory activity on human MAO (hMAO) isoforms. All the compounds were found to inhibit the hMAO isoforms either selectively or non-selectively. Nine compounds (6a, 6b, 6g-m) were found to inhibit hMAO-B selectively, whereas the other four (6c-f) were found to be non-selective. There is a gradual shift from hMAO-B selectivity (6a,b) to non-selectivity (6c-f) as there is an increase in chain length at the amino terminus. In case of compounds having an aromatic nucleus at the amino terminus, increasing the carbon number between N and the aromatic ring increases the potency as well as selectivity toward hMAO-B. Compounds 6f, 6j, and 6k were subjected to membrane permeability and metabolic stability studies by in vitro assay methods. They were found to have a better pharmacokinetic profile than curcumin, ferulic acid, and selegiline. In order to understand the structural features responsible for the potency and selectivity of 6k, we carried out a molecular docking simulation study. PMID:26592858

  7. Activity of D-amino acid oxidase is widespread in the human central nervous system

    PubMed Central

    Sasabe, Jumpei; Suzuki, Masataka; Imanishi, Nobuaki; Aiso, Sadakazu

    2014-01-01

    It has been proposed that D-amino acid oxidase (DAO) plays an essential role in degrading D-serine, an endogenous coagonist of N-methyl-D-aspartate (NMDA) glutamate receptors. DAO shows genetic association with amyotrophic lateral sclerosis (ALS) and schizophrenia, in whose pathophysiology aberrant metabolism of D-serine is implicated. Although the pathology of both essentially involves the forebrain, in rodents, enzymatic activity of DAO is hindbrain-shifted and absent in the region. Here, we show activity-based distribution of DAO in the central nervous system (CNS) of humans compared with that of mice. DAO activity in humans was generally higher than that in mice. In the human forebrain, DAO activity was distributed in the subcortical white matter and the posterior limb of internal capsule, while it was almost undetectable in those areas in mice. In the lower brain centers, DAO activity was detected in the gray and white matters in a coordinated fashion in both humans and mice. In humans, DAO activity was prominent along the corticospinal tract, rubrospinal tract, nigrostriatal system, ponto-/olivo-cerebellar fibers, and in the anterolateral system. In contrast, in mice, the reticulospinal tract and ponto-/olivo-cerebellar fibers were the major pathways showing strong DAO activity. In the human corticospinal tract, activity-based staining of DAO did not merge with a motoneuronal marker, but colocalized mostly with excitatory amino acid transporter 2 and in part with GFAP, suggesting that DAO activity-positive cells are astrocytes seen mainly in the motor pathway. These findings establish the distribution of DAO activity in cerebral white matter and the motor system in humans, providing evidence to support the involvement of DAO in schizophrenia and ALS. Our results raise further questions about the regulation of D-serine in DAO-rich regions as well as the physiological/pathological roles of DAO in white matter astrocytes. PMID:24959138

  8. Cardiolipin linoleic acid content and mitochondrial cytochrome c oxidase activity are associated in rat skeletal muscle.

    PubMed

    Fajardo, Val Andrew; McMeekin, Lauren; Saint, Caitlin; LeBlanc, Paul J

    2015-04-01

    Cardiolipin (CL) is an inner-mitochondrial membrane phospholipid that is important for optimal mitochondrial function. Specifically, CL and CL linoleic (18:2ω6) content are known to be positively associated with cytochrome c oxidase (COX) activity. However, this association has not been examined in skeletal muscle. In this study, rats were fed high-fat diets with a naturally occurring gradient in linoleic acid (coconut oil [CO], 5.8%; flaxseed oil [FO], 13.2%; safflower oil [SO], 75.1%) in an attempt to alter both mitochondrial CL fatty acyl composition and COX activity in rat mixed hind-limb muscle. In general, mitochondrial membrane lipid composition was fairly resistant to dietary treatments as only modest changes in fatty acyl composition were detected in CL and other major mitochondrial phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). As a result of this resistance, CL 18:2ω6 content was not different between the dietary groups. Consistent with the lack of changes in CL 18:2ω6 content, mitochondrial COX activity was also not different between the dietary groups. However, correlational analysis using data obtained from rats across the dietary groups showed a significant relationship (p = 0.009, R(2) = 0.21). Specifically, our results suggest that CL 18:2ω6 content may positively influence mitochondrial COX activity thereby making this lipid molecule a potential factor related to mitochondrial health and function in skeletal muscle. PMID:25727371

  9. Activation of Membrane NADPH Oxidase Associated with Lysosome-Targeted Acid Sphingomyelinase in Coronary Endothelial Cells

    PubMed Central

    Bao, Jun-Xiang; Jin, Si; Zhang, Fan; Wang, Zheng-Chao; Li, Ningjun

    2010-01-01

    Abstract This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O2·− production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs. Antioxid. Redox Signal. 12, 703–712. PMID:19761405

  10. Characterization and cDNA sequence of Bothriechis schlegeliil-amino acid oxidase with antibacterial activity.

    PubMed

    Vargas Muñoz, Leidy Johana; Estrada-Gomez, Sebastian; Núñez, Vitelbina; Sanz, Libia; Calvete, Juan J

    2014-08-01

    Snake venoms are complex mixtures of proteins including l-amino acid oxidase (lAAO). A lAAO (named BslAAO) with a mass of 56kDa and a theoretical Ip of 5.79, was purified from Bothriechis schlegelii venom through size-exclusion, ion exchange and affinity chromatography. The entire protein sequence of 498 amino acids, was determined from cDNA using reverse-transcribed mRNA isolated from venom gland. The enzyme showed dose-dependent inhibition of bacterial growth. BslAAO showed inhibitory effect against S. aureus with a MIC of 4μg/mL and a MBC of 8μg/mL. Against Acinetobacter baumannii, showed a MIC of 2μg/mL and MBC of 4μg/mL, No effect was observed in Escherichia coli. This antibacterial activity was inhibited by catalase, indicating that antimicrobial activity was due to H2O2 production. BslAAO did not show any cytotoxic activity toward mouse myoblast cell line C2C12 or peripheral blood mononuclear cells. The enzyme oxidated l-Leu, with a Km of 16.37μM and a Vmax of 0.39μM/min. Snake venoms lAAOs, are potential frames of different therapeutics molecules since these enzymes exhibit low MICs and MBCs and show to be harmless to human cells due to microorganisms being generally several fold more sensitive to reactive oxygen species than human tissues. PMID:24875315

  11. Sensitive determination of D-amino acids in mammals and the effect of D-amino-acid oxidase activity on their amounts.

    PubMed

    Hamase, Kenji; Konno, Ryuichi; Morikawa, Akiko; Zaitsu, Kiyoshi

    2005-09-01

    The determination of small amounts of D-amino acids in mammalian tissues is still a challenging theme in the separation sciences. In this review, various gas-chromatographic and high-performance liquid chromatographic methods are discussed including highly selective and sensitive column-switching procedures. Based on these methods, the distributions of D-aspartic acid, D-serine, D-alanine, D-leucine and D-proline have been clarified in the mouse brain. As the regulation mechanisms of D-amino acid amounts in mammals, we focused on the D-amino-acid oxidase, which catalyzes the degradation of D-amino acids. Using the mutant mouse strain lacking D-amino-acid oxidase activity, the effects of the enzymatic activity on the amounts and distributions of various D-amino acids have been investigated. PMID:16141519

  12. Antiproliferative activity of king cobra (Ophiophagus hannah) venom L-amino acid oxidase.

    PubMed

    Li Lee, Mui; Chung, Ivy; Yee Fung, Shin; Kanthimathi, M S; Hong Tan, Nget

    2014-04-01

    King cobra (Ophiophagus hannah) venom L-amino acid oxidase (LAAO), a heat-stable enzyme, is an extremely potent antiproliferative agent against cancer cells when compared with LAAO isolated from other snake venoms. King cobra venom LAAO was shown to exhibit very strong antiproliferative activities against MCF-7 (human breast adenocarcinoma) and A549 (human lung adenocarcinoma) cells, with an IC50 value of 0.04±0.00 and 0.05±0.00 μg/mL, respectively, after 72-hr treatment. In comparison, its cytotoxicity was about 3-4 times lower when tested against human non-tumourigenic breast (184B5) and lung (NL 20) cells, suggesting selective antitumour activity. Furthermore, its potency in MCF-7 and A549 cell lines was greater than the effects of doxorubicin, a clinically established cancer chemotherapeutic agent, which showed an IC50 value of 0.18±0.03 and 0.63±0.21 μg/mL, respectively, against the two cell lines. The selective cytotoxic action of the LAAO was confirmed by phycoerythrin (PE) annexin V/7-amino-actinomycin (AAD) apoptotic assay, in which a significant increase in apoptotic cells was observed in LAAO-treated tumour cells than in their non-tumourigenic counterparts. The ability of LAAO to induce apoptosis in tumour cells was further demonstrated using caspase-3/7 and DNA fragmentation assays. We also determined that this enzyme may target oxidative stress in its killing of tumour cells, as its cytotoxicity was significantly reduced in the presence of catalase (a H2O2 scavenger). In view of its heat stability and selective and potent cytotoxic action on cancer cells, king cobra venom LAAO can be potentially developed for treating solid tumours. PMID:24118879

  13. Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation

    PubMed Central

    Terry-Lorenzo, Ryan T.; Chun, Lawrence E.; Brown, Scott P.; Heffernan, Michele L. R.; Fang, Q. Kevin; Orsini, Michael A.; Pollegioni, Loredano; Hardy, Larry W.; Spear, Kerry L.; Large, Thomas H.

    2014-01-01

    The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, ‘compound 2’ [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki=7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors. PMID:25001371

  14. Body pool and synthesis of ascorbic acid in adult sea lamprey (Petromyzon marinus): An agnathan fish with gulonolactone oxidase activity

    PubMed Central

    Moreau, Régis; Dabrowski, Konrad

    1998-01-01

    Although many vertebrates can synthesize ascorbic acid (vitamin C), it is still unclear from the evolutionary perspective when the ability to synthesize the vitamin first appeared in the animal kingdom and how frequently the trait has been lost. We report here ascorbic acid biosynthesis ability in sea lamprey (Petromyzon marinus) which represent the most ancient vertebrate lineage examined thus far for presence of gulonolactone oxidase, the enzyme catalyzing the terminal step in biosynthesis of vitamin C. This finding supports the view that the ancestors of living vertebrates were not scurvy prone and that the loss of gulonolactone oxidase activity subsequently occurred several times in vertebrate phylogeny. Adult sea lamprey allocate significant amounts of ascorbic acid to the gonads to guaranty high-quality gametes. Tissue stores of ascorbate were maintained by de novo synthesis (1.2–1.3 mg of ascorbic acid/300-g sea lamprey per day at 15°C) while sea lamprey fast during spawning migration. We estimate that the in vivo daily renewal rate of ascorbate is 4–5% of the whole-body ascorbate pool based on measurement of its biosynthesis and concentration in the whole animal. PMID:9707638

  15. Activation of Polyphenol Oxidase of Chloroplasts 1

    PubMed Central

    Tolbert, N. E.

    1973-01-01

    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 × mg−1 chlorophyll × 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

  16. Molecular cloning and expression in Escherichia coli of an active fused Zea mays L. D-amino acid oxidase.

    PubMed

    Gholizadeh, A; Kohnehrouz, B B

    2009-02-01

    D-Amino acid oxidase (DAAO) is an FAD-dependent enzyme that metabolizes D-amino acids in microbes and animals. However, such ability has not been identified in plants so far. We predicted a complete DAAO coding sequence consisting of 1158 bp and encoding a protein of 386 amino acids. We cloned this sequence from the leaf cDNA population of maize plants that could utilize D-alanine as a nitrogen source and grow normally on media containing D-Ala at the concentrations of 100 and 1000 ppm. For more understanding of DAAO ability in maize plant, we produced a recombinant plasmid by the insertion of isolated cDNA into the pMALc2X Escherichia coli expression vector, downstream of the maltose-binding protein coding sequence. The pMALc2X-DAAO vector was used to transform the TB1 strain of E. coli cells. Under normal growth conditions, fused DAAO (with molecular weight of about 78 kDa) was expressed up to 5 mg/liter of bacterial cells. The expressed product was purified by affinity chromatography and subjected to in vitro DAAO activity assay in the presence of five different D-amino acids. Fused DAAO could oxidize D-alanine and D-aspartate, but not D-leucine, D-isoleucine, and D-serine. The cDNA sequence reported in this paper has been submitted to EMBL databases under accession number AM407717. PMID:19267668

  17. Factors influencing diamine oxidase activity and γ-aminobutyric acid content of fava bean (Vicia faba L.) during germination.

    PubMed

    Yang, Runqiang; Chen, Hui; Gu, Zhenxin

    2011-11-01

    Factors (germination time, spectra, temperature, pH, and chemical inhibitors) influencing diamine oxidase (DAO, EC 1.4.3.6) activity and γ-aminobutyric acid (GABA) content of fava bean (Vicia faba L.) during germination were investigated in this study. DAO activity significantly increased in germinating seeds but varied with different organs. The enzyme activity was higher in shoot than that in cotyledon, hypocotyl, and radicle. When seeds were germinated in the dark, DAO activity was 2.35-, 2.00-, 2.36-, 4.40-, and 1.67-fold of that under white, red, blue, green, and yellow spectra, respectively. The optimum germination temperature and pH value for increasing DAO activity were 30 °C and 3.0, respectively. The DAO activity was inhibited significantly by aminoguanidine and sodium ethylenediamine tetracetate, while it was activated by CuCl(2) and CaCl(2). Germinating at an appropriate temperature and pH, 30% of GABA formation was supplied by DAO. Calcium was related to the regulation of DAO activity and GABA accumulation. PMID:21942768

  18. The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation.

    PubMed

    Kanade, Santosh R; Paul, Beena; Rao, A G Appu; Gowda, Lalitha R

    2006-05-01

    Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase)--a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen--and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1+/-2 to 75.9+/-0.6 A (1 A=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack. PMID:16393141

  19. Hypohalous acid-modified human serum albumin induces neutrophil NADPH oxidase activation, degranulation, and shape change.

    PubMed

    Gorudko, Irina V; Grigorieva, Daria V; Shamova, Ekaterina V; Kostevich, Valeria A; Sokolov, Alexey V; Mikhalchik, Elena V; Cherenkevich, Sergey N; Arnhold, Jürgen; Panasenko, Oleg M

    2014-03-01

    Halogenated lipids, proteins, and lipoproteins formed in reactions with myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) and hypobromous acid (HOBr) can contribute to the regulation of functional activity of cells and serve as mediators of inflammation. Human serum albumin (HSA) is the major plasma protein target of hypohalous acids. This study was performed to assess the potency of HSA modified by HOCl (HSA-Cl) and HOBr (HSA-Br) to elicit selected neutrophil responses. HSA-Cl/Br were found to induce neutrophil degranulation, generation of reactive oxygen intermediates, shape change, and actin cytoskeleton reorganization. Thus HSA-Cl/Br can initially act as a switch and then as a feeder of the "inflammatory loop" under oxidative stress. In HSA-Cl/Br-treated neutrophils, monoclonal antibodies against CD18, the β subunit of β2 integrins, reduced the production of superoxide anion radicals and hydrogen peroxide as well as MPO exocytosis, suggesting that CD18 contributed to neutrophil activation. HSA-Cl/Br-induced neutrophil responses were also inhibited by genistein, a broad-specificity tyrosine kinase inhibitor, and wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, supporting the notion that activation of both tyrosine kinase and PI3K may play a role in neutrophil activation by HSA modified in MPO-dependent reactions. These results confirm the hypothesis that halogenated molecules formed in vivo via MPO-dependent reactions can be considered as a new class of biologically active substances potentially able to contribute to activation of myeloid cells in sites of inflammation and serve as inflammatory response modulators. PMID:24384524

  20. Ascorbic acid and L-gulonolactone oxidase in lagomorphs.

    PubMed

    Jenness, R; Birney, E C; Ayaz, K L

    1978-01-01

    1. The activity of L-gulonolactone oxidase (EC 1.1.3.8) in the liver of eastern cottontail rabbits (Sylvilagus floridanus) is about 10-fold greater in winter than in summer. 2. L-gulonolactone oxidase activity is low and tissue ascorbate high during all seasons in snowshoe hares (Lepus americanus). 3. Liver contents of ascorbate fall to low levels in L. americanus fed on rabbit chow in the laboratory. 4. The activity of L-gulonolactone oxidase in liver of Sylvilagus and Oryctolagus is depressed by feeding high levels of L-ascorbic acid. 5. The New Zealand White breed of domestic rabbit (Oryctolagus cuniculus) has considerably higher levels of L-gulonolactone oxidase and liver ascorbate than does the Dutch breed. 6. In a wild population of Oryctolagus sampled in Australia L-gulonolactone oxidase levels were intermediate between those of the two domestic breeds and more variable than either. PMID:318384

  1. The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation

    PubMed Central

    Kanade, Santosh R.; Paul, Beena; Rao, A. G. Appu; Gowda, Lalitha R.

    2006-01-01

    Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase) – a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen – and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1±2 to 75.9±0.6 Å (1 Å=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack. PMID:16393141

  2. Uric Acid Promotes Apoptosis in Human Proximal Tubule Cells by Oxidative Stress and the Activation of NADPH Oxidase NOX 4

    PubMed Central

    Verzola, Daniela; Ratto, Elena; Villaggio, Barbara; Parodi, Emanuele Luigi; Pontremoli, Roberto; Garibotto, Giacomo; Viazzi, Francesca

    2014-01-01

    Mild hyperuricemia has been linked to the development and progression of tubulointerstitial renal damage. However the mechanisms by which uric acid may cause these effects are poorly explored. We investigated the effect of uric acid on apoptosis and the underlying mechanisms in a human proximal tubule cell line (HK-2). Increased uric acid concentration decreased tubule cell viability and increased apoptotic cells in a dose dependent manner (up to a 7-fold increase, p<0.0001). Uric acid up-regulated Bax (+60% with respect to Ctrl; p<0.05) and down regulated X-linked inhibitor of apoptosis protein. Apoptosis was blunted by Caspase-9 but not Caspase-8 inhibition. Uric acid induced changes in the mitochondrial membrane, elevations in reactive oxygen species and a pronounced up-regulation of NOX 4 mRNA and protein (p<0.05). In addition, both reactive oxygen species production and apoptosis was prevented by the NADPH oxidase inhibitor DPI as well as by Nox 4 knockdown. URAT 1 transport inhibition by probenecid and losartan and its knock down by specific siRNA, blunted apoptosis, suggesting a URAT 1 dependent cell death. In summary, our data show that uric acid increases the permissiveness of proximal tubule kidney cells to apoptosis by triggering a pathway involving NADPH oxidase signalling and URAT 1 transport. These results might explain the chronic tubulointerstitial damage observed in hyperuricaemic states and suggest that uric acid transport in tubular cells is necessary for urate-induced effects. PMID:25514209

  3. Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase

    PubMed Central

    Marín-Navarro, Julia; Roupain, Nicole; Talens-Perales, David; Polaina, Julio

    2015-01-01

    Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described. PMID:26642312

  4. Activation of human neutrophil nicotinamide adenine dinucleotide phosphate, reduced (triphosphopyridine nucleotide, reduced) oxidase by arachidonic acid in a cell-free system.

    PubMed Central

    Curnutte, J T

    1985-01-01

    Sonicates from unstimulated human neutrophils produce no measurable superoxide since the superoxide-generating enzyme, NADPH oxidase, is inactive in these preparations. Previous attempts to activate the oxidase in disrupted cells with conventional neutrophil stimuli have been unsuccessful. This report describes a cell-free system in which arachidonic acid (82 microM) was able to activate superoxide generation that was dependent upon the presence of NADPH and the sonicate. For activation to occur, both the particulate and supernatant fractions of the sonicate must be present. Calcium ions, which are required for activation of intact neutrophils by arachidonate, were not necessary in the cell-free system. In quantitative terms, the superoxide-generating activity in the cell-free system could account for at least 20-50% of the superoxide rate observed in intact neutrophils stimulated with arachidonate. Sonicates from patients with chronic granulomatous disease (CGD) could not be activated by arachidonic acid in the cell-free system. In three patients representing both genetic forms of CGD, the defect appeared to reside in the particulate fraction. The soluble cofactor was normal in all three patients and could be used to activate normal neutrophil pellets in the presence of arachidonic acid. Thus, at least a portion of the activation mechanism in the neutrophil, that residing in the soluble phase, appeared to be normal in patients with CGD. PMID:2987311

  5. Identification of L-amino acid oxidase (Mb-LAAO) with antibacterial activity in the venom of Montivipera bornmuelleri, a viper from Lebanon.

    PubMed

    Rima, Mohamad; Accary, Claudine; Haddad, Katia; Sadek, Riyad; Hraoui-Bloquet, Souad; Desfontis, Jean C; Fajloun, Ziad

    2013-10-01

    The L-amino acid oxidase (LAAO) is a multifunctional enzyme, able to partake in different activities including antibacterial activity. In this study, a novel LAAO (Mb-LAAO) was isolated from the venom of M. bornmuelleri snake using size exclusion chromatography followed by RP-HPLC and partially characterized. However, the molecular weight of the Mb-LAAO determined by ESI-MS and SDS-PAGE was 59 960.4 Da. Once the enzymatic activity test confirming the enzyme's identity (transformation of L-leucine) was done, the Mb-LAAO was evaluated for its antibacterial activity against Gram-negative bacteria. It showed a remarkable effect against M. morganii and K. pneumoniae. Moreover, no cytotoxic activity was observed for Mb-LAAO against human erythrocytes arguing for an exploration of its pharmaceutical interest. PMID:24712674

  6. Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.).

    PubMed

    Lin, L S; Varner, J E

    1991-05-01

    The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity and mRNA level were highest in the epidermis, and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, we have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall "loosening." PMID:16668145

  7. Bile acid reflux contributes to development of esophageal adenocarcinoma via activation of phosphatidylinositol-specific phospholipase Cgamma2 and NADPH oxidase NOX5-S.

    PubMed

    Hong, Jie; Behar, Jose; Wands, Jack; Resnick, Murray; Wang, Li Juan; Delellis, Ronald A; Lambeth, David; Cao, Weibiao

    2010-02-01

    Gastroesophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study, we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid-induced increase in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, hydrogen peroxide (H(2)O(2)) production, and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibitor of PI-PLC. PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, and PI-PLCgamma2, but not PI-PLCbeta2 and PI-PLCdelta1, were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLCgamma2 or extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein (MAP) kinase with small interfering RNAs (siRNA) significantly decreased TDCA-induced NOX5-S expression, H(2)O(2) production, and cell proliferation. In contrast, knockdown of PI-PLCbeta1, PI-PLCbeta3, PI-PLCbeta4, PI-PLCgamma1, or ERK1 MAP kinase had no significant effect. TDCA significantly increased ERK2 phosphorylation, an increase that was reduced by U73122 or PI-PLCgamma2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLCgamma2 and ERK2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with BE may increase reactive oxygen species production and cell proliferation via activation of PI-PLCgamma2, ERK2 MAP kinase, and NADPH oxidase NOX5-S, thereby contributing to the development of EA. PMID:20086178

  8. Expression of ascorbic acid oxidase in zucchini squash (Cucurbita pepo L. )

    SciTech Connect

    Lin, Liangshiou; Varner, J.E. )

    1991-05-01

    The expression of ascorbic acid oxidase was studied in zuchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, the authors have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall loosening.

  9. The relationship between hypertension and plasma allantoin, uric acid, xanthine oxidase activity and nitrite, and their predictive capacity in severe preeclampsia.

    PubMed

    Elmas, Oguz; Elmas, Onur; Aliciguzel, Yakup; Simsek, Tayyup

    2016-01-01

    It is controversial that uric acid (UA) levels are related to the severity of hypertension in preeclampsia (PE). Our aim in this study was to determine whether UA, xanthine oxidase activity (XOA), allantoin and nitrite levels are related to arterial blood pressure (BP) in PE. We formed a control group (n = 20) and a PE group (n = 20) for the study. Their BPs and plasma UA, XOA, allantoin and nitrite levels were measured. The values from the control and PE pregnant women were assessed via a Wilcoxon matched-pairs test. A Pearson correlation test was also performed. In addition, the diagnostic value of these tests was evaluated via receiver operating characteristic (ROC) analysis. The BP, UA, XOA and allantoin levels in the PE patients were found to be higher when compared with those of the pregnant controls. The UA, XOA and allantoin levels showed high correlations with BP in cases of PE. However, there was no superiority among the correlations. No differences were observed between the groups in terms of nitrite levels and the relationship between nitrite and BP. UA, XOA and allantoin levels may be high due to placental cell death because of abnormal trophoblastic activity observed in PE. Moreover, the reactive oxygen products that are created during the genetic material degradation may explain how UA, XOA and allantoin levels are related to BP. According to ROC analysis, UA, XOA and allantoin assays are reliable predictors for the determination of PE. PMID:26366935

  10. A high-performance liquid chromatography assay with a triazole-bonded column for evaluation of d-amino acid oxidase activity.

    PubMed

    Iwasaki, Megumi; Kashiwaguma, Yoshiyuki; Nagashima, Chihiro; Izumi, Mao; Uekusa, Ayano; Iwasa, Sumiko; Onozato, Mayu; Ichiba, Hideaki; Fukushima, Takeshi

    2016-03-01

    Elution profiles of kynurenic acid (KYNA) and 7-chlorokynurenic acid (Cl-KYNA) were examined by high-performance liquid chromatography (HPLC) using a triazole-bonded stationary phase column (Cosmosil® HILIC) under isocratic elution of a mobile phase consisting of CH3 CN-aqueous 10 mm ammonium formate between pH 3.0 and 6.0. The capacity factors of KYNA and Cl-KYNA varied with both the CH3 CN content and the pH of the mobile phase. The elution order of KYNA and Cl-KYNA was reversed between the CH3 CN- and H2 O-rich mobile phases, suggesting that hydrophilic interactions and anion-exchange interactions caused retention of KYNA and Cl-KYNA in the CH3 CN- and H2 O-rich mobile phases, respectively. The present HPLC method using a triazole-bonded column and fluorescence detection (excitation 250 nm, emission 398 nm) was applied to monitor in vitro production of KYNA from d-kynurenine (d-KYN) by d-amino acid oxidase (DAO) using Cl-KYNA as an internal standard. A single KYNA peak was clearly observed after enzymatic reaction of d-KYN with DAO. Production of KYNA from d-KYN was suppressed by the addition of commercial DAO inhibitors. The present HPLC method can be used to evaluate DAO activity and DAO inhibitory effects in candidate drugs for the treatment of schizophrenia. PMID:26174062

  11. Phosphoproteins and the activation of the neutrophil respiratory burst oxidase

    SciTech Connect

    Okamura, N.; Curnutte, J.T.; Babior, B.M.

    1987-05-01

    The respiratory burst oxidase is a neutrophil enzyme that converts oxygen to O/sub 2//sup -/. It is dormant in resting cells but is activated when the cells are exposed to phorbol myristate acetate (PMA). PMA also induces the incorporation of /sup 32/P into certain neutrophil proteins. To determine whether phosphorylation of these proteins is related to oxidase activation, protein phosphorylation was studied in patients with chronic granulomatous disease (GCD), a group of inherited conditions in which oxidase activity is missing. In normals, neutrophil activation by PMA is associated with the phosphorylation inter alia of 48K proteins at pI 7.3 and 7.8. There is also inconstant phosphorylation of a 48K protein at pI 6.8. In 4 patients with X-linked chronic granulomatous disease (CGD), phosphorylation of pp48/6.8 and pp48/7.3 was absent, while in autosomal recessive CGD, phosphorylation of all 3 of these proteins was absent in 3 patients and significantly diminished in a fourth. These results suggest that the phosphorylation of these proteins is related to the activation of the respiratory burst oxidase. By peptide mapping, these 3 proteins appear to consist of a single peptide species whose pI variability may be due to post-translational modification. The only phosphoamino acid found in pp48/7.3 was phosphoserine.

  12. Activation of microglia with zymosan promotes excitatory amino acid release via volume-regulated anion channels: the role of NADPH oxidases

    PubMed Central

    Harrigan, Timothy J.; Abdullaev, Iskandar F.; Jourd'heuil, David; Mongin, Alexander A.

    2008-01-01

    Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study we used an electrophysiological approach and a D-[3H]aspartate (glutamate) release assay to explore the ROS-dependent regulation of glutamate-permeable volume-regulated anion channels (VRACs). Exposure of rat microglia to hypoosmotic media stimulated Cl− currents and D-[3H]aspartate release, both of which were inhibited by the selective VRAC blocker DCPIB. Exogenously applied H2O2 potently increased swelling-activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger MnTMPyP, and by two inhibitors of NADPH oxidase (NOX) diphenyliodonium and thioridazine. However, zymosan-stimulated glutamate release was insensitive to other NOX blockers, apocynin and AEBSF. This pharmacological profile pointed to the potential involvement of apocynin-insensitive NOX4. Using RT-PCR we confirmed that NOX4 is expressed in rat microglial cells, along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2 we stimulated this isoform using protein kinase C (PKC) activator PMA, or inhibited it with the broad spectrum PKC blocker Gö6983. Both agents potently modulated endogenous ROS production by NOX2, but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release, and that its activity is regulated by endogenous ROS originating from NOX4. PMID:18624925

  13. Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus.

    PubMed

    Sakamoto, J; Koga, E; Mizuta, T; Sato, C; Noguchi, S; Sone, N

    1999-04-21

    Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. We previously identified and purified an alternative oxidase, cytochrome bd-type quinol oxidase, from a mutant of Bacillus stearothermophilus defective in the caa3-type oxidase activity (J. Sakamoto et al., FEMS Microbiol. Lett. 143 (1996) 151-158). Compared with proteobacterial counterparts, B. stearothermophilus cytochrome bd showed lower molecular weights of the two subunits, shorter wavelength of alpha-band absorption maximum due to heme D, and lower quinol oxidase activity. Preincubation with menaquinone-2 enhanced the enzyme activity up to 40 times, suggesting that, besides the catalytic site, there is another quinone-binding site which largely affects the enzyme activity. In order to clarify the molecular basis of the differences of cytochromes bd between B. stearothermophilus and proteobacteria, the genes encoding for the B. stearothermophilus bd was cloned based on its partial peptide sequences. The gene for subunit I (cbdA) encodes 448 amino acid residues with a molecular weight of 50195 Da, which is 14 and 17% shorter than those of Escherichia coli and Azotobacter vinelandii, respectively, and CbdA lacks the C-terminal half of the long hydrophilic loop between the putative transmembrane segments V and VI (Q loop), which has been suggested to include the substrate quinone-binding site for the E. coli enzyme. The gene for subunit II (cbdB) encodes 342 residues with a molecular weight of 38992 Da. Homology search indicated that the B. stearothermophilus cbdAB has the highest sequence similarity to ythAB in B. subtilis genome rather than to cydAB, the first set of cytochrome bd genes identified in the genome. Sequence comparison of cytochromes bd and their homologs from various organisms demonstrates that the proteins can be classified into two subfamilies, a proteobacterial type including E. coli bd and a

  14. Antimutagenic activity of oxidase enzymes

    SciTech Connect

    Agabeili, R.A.

    1986-11-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity.

  15. D-amino acid oxidase from the yeast Trigonopsis variabilis.

    PubMed

    Kubicek-Pranz, E M; Röhr, M

    1985-04-01

    D-Amino acid oxidase (EC 1.4.3.3) has been purified from the yeast Trigonopsis variabilis by the application of ion-exchange chromatography on DEAE-cellulose, salt precipitation, gel filtration, and hydroxyapatite adsorption. Alternatively the last two steps can be substituted by a single fast protein liquid chromatographic ion-exchange step (Mono Q). The enzyme appeared homogeneous in PAGE, but small amounts of impurities (not exceeding 5% of total protein) appeared in sodium dodecyl sulfate (SDS)-PAGE. Its Mr in SDS-PAGE is 39,000; it exhibits an isoelectric point of 4.8 and contains 7% (w/v) covalently bound carbohydrate. Its absorption spectrum is similar to hog kidney D-amino acid oxidase, indicating the presence of bound FAD, which, however, could not be separated from the enzyme under non-denaturing conditions. The enzyme is inhibited by SH-oxidizing agents, but not by metal-chelate formers and not by benzoate or toluene. It uses O2 exclusively as the only H acceptor. Km and Vmax values were determined for 15 D-amino acids, which, among 23 tested, were substrates of the enzyme. The enzyme has highest affinity for D-phenylalanine and D-leucine, but maximal activity is obtained with D-citrulline and D-isoleucine. The specific activity of the purified preparation is even higher than that of the commercially available hog kidney enzyme (21.7 vs 16 U/mg). The yeast enzyme may be a useful analytical and preparative tool in view of the difference between its substrate specificity and that of the hog enzyme. PMID:2865242

  16. Apoxin I, a novel apoptosis-inducing factor with L-amino acid oxidase activity purified from Western diamondback rattlesnake venom.

    PubMed

    Torii, S; Naito, M; Tsuruo, T

    1997-04-01

    Venom of the western diamondback rattlesnake (Crotalus atrox) induces apoptosis in human umbilical vein endothelial cells, which could result in hemorrhage in tissues bitten by the snake. To identify the hemorrhagic factor, we purified a novel protein, apoxin I, from rattlesnake venom. Apoxin I induced apoptosis in human umbilical vein endothelial, human promyelocytic leukemia HL-60, human ovarian carcinoma A2780, and mouse endothelial KN-3 cells. Amino acid sequence analysis of the apoxin I showed close similarity to L-amino acid oxidase from the Malayan pit viper (Calloselasma rhodostoma). The purified apoxin I oxidized L-leucine but not D-leucine to produce H2O2. The apoxin I-induced apoptosis was inhibited by catalase, a H2O2 scavenger. These results indicate that the H2O2 produced by L-amino acid oxidation by apoxin I is involved in the apoxin I-induced apoptosis and in hemorrhage caused by rattlesnake venom. PMID:9083096

  17. Snake Venom L-Amino Acid Oxidases: Trends in Pharmacology and Biochemistry

    PubMed Central

    Izidoro, Luiz Fernando M.; Sobrinho, Juliana C.; Mendes, Mirian M.; Costa, Tássia R.; Grabner, Amy N.; Rodrigues, Veridiana M.; da Silva, Saulo L.; Zanchi, Fernando B.; Zuliani, Juliana P.; Fernandes, Carla F. C.; Calderon, Leonardo A.; Stábeli, Rodrigo G.; Soares, Andreimar M.

    2014-01-01

    L-amino acid oxidases are enzymes found in several organisms, including venoms of snakes, where they contribute to the toxicity of ophidian envenomation. Their toxicity is primarily due to enzymatic activity, but other mechanisms have been proposed recently which require further investigation. L-amino acid oxidases exert biological and pharmacological effects, including actions on platelet aggregation and the induction of apoptosis, hemorrhage, and cytotoxicity. These proteins present a high biotechnological potential for the development of antimicrobial, antitumor, and antiprotozoan agents. This review provides an overview of the biochemical properties and pharmacological effects of snake venom L-amino acid oxidases, their structure/activity relationship, and supposed mechanisms of action described so far. PMID:24738050

  18. Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of formylphenylacetic acid ethyl ester.

    PubMed Central

    Kalyanaraman, V S; Mahadevan, S; Kumar, S A

    1975-01-01

    An enzyme system from Datura innoxia roots oxidizing formylphenylacetic acid ethyl ester was purified 38-fold by conventional methods such as (NH4)2SO4 fractionation, negative adsorption on alumina Cy gel and chromatography on DEAE-cellulose. The purified enzyme was shown to catalyse the stoicheiometric oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid, utilizing molecular O2. Substrate analogues such as phenylacetaldehyde and phenylpyruvate were oxidized at a very low rate, and formylphenylacetonitrile was an inhilating agents, cyanide, thiol compounds and ascorbic acid. This enzyme was identical with an oxidase-peroxidase isoenzyme. Another oxidase-peroxidase isoenzyme which separated on DEAE-chromatography also showed formylphenylacetic acid ethyl ester oxidase activity, albeit to a lesser extent. The properties of the two isoenzymes of the oxidase were compared and shown to differ in their oxidation and peroxidation properties. The oxidation of formylphenylacetic acid ethyl ester was also catalysed by horseradish peroxidase. The Datura isoenzymes exhibited typical haemoprotein spectra. The oxidation of formylphenylacetic acid ethyl ester was different from other peroxidase-catalysed reactions in not being activated by either Mn2+ or monophenols. The oxidation was inhibited by several mono- and poly-phenols and by catalase. A reaction mechanism for the oxidation is proposed. PMID:997

  19. Determination of free D-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity.

    PubMed

    Hamase, K; Inoue, T; Morikawa, A; Konno, R; Zaitsu, K

    2001-11-15

    A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This D-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for D-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free D-proline in eight brain regions and sera of mutant ddY/DAO- mice, lacking D-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free D-leucine was also investigated. The amount and distribution of D-proline in the brains of ddY/DAO+ mice and ddY/DAO- mice are almost the same, and relatively high amounts of D-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of D-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of D-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO- mice, D-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of L-proline and L-leucine are not significantly different between the two strains. In the serum, the amounts of both free D-proline and d-leucine are significantly higher in the ddY/DAO- mice than those obtained in ddY/DAO+ mice. PMID:11700980

  20. Phenol oxidase activity in secondary transformed peat-moorsh soils

    NASA Astrophysics Data System (ADS)

    Styła, K.; Szajdak, L.

    2009-04-01

    The chemical composition of peat depends on the geobotanical conditions of its formation and on the depth of sampling. The evolution of hydrogenic peat soils is closely related to the genesis of peat and to the changes in water conditions. Due to a number of factors including oscillation of ground water level, different redox potential, changes of aerobic conditions, different plant communities, and root exudes, and products of the degradation of plant remains, peat-moorsh soils may undergo a process of secondary transformation conditions (Sokolowska et al. 2005; Szajdak et al. 2007). Phenol oxidase is one of the few enzymes able to degrade recalcitrant phenolic materials as lignin (Freeman et al. 2004). Phenol oxidase enzymes catalyze polyphenol oxidation in the presence of oxygen (O2) by removing phenolic hydrogen or hydrogenes to from radicals or quinines. These products undergo nucleophilic addition reactions in the presence or absence of free - NH2 group with the eventual production of humic acid-like polymers. The presence of phenol oxidase in soil environments is important in the formation of humic substances a desirable process because the carbon is stored in a stable form (Matocha et al. 2004). The investigations were carried out on the transect of peatland 4.5 km long, located in the Agroecological Landscape Park host D. Chlapowski in Turew (40 km South-West of Poznań, West Polish Lowland). The sites of investigation were located along Wyskoć ditch. The following material was taken from four chosen sites marked as Zbechy, Bridge, Shelterbelt and Hirudo in two layers: cartel (0-50cm) and cattle (50-100cm). The object of this study was to characterize the biochemical properties by the determination of the phenol oxidize activity in two layers of the four different peat-moors soils used as meadow. The phenol oxidase activity was determined spectrophotometrically by measuring quinone formation at λmax=525 nm with catechol as substrate by method of Perucci

  1. Activation of antibacterial autophagy by NADPH oxidases

    PubMed Central

    Huang, Ju; Canadien, Veronica; Lam, Grace Y.; Steinberg, Benjamin E.; Dinauer, Mary C.; Magalhaes, Marco A. O.; Glogauer, Michael; Grinstein, Sergio; Brumell, John H.

    2009-01-01

    Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes, promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR ligands. Engagement of either TLRs or the Fcγ receptors (FcγRs) during phagocytosis induced recruitment of the autophagy protein LC3 to phagosomes with similar kinetics. Both receptors are known to activate the NOX2 NADPH oxidase, which plays a central role in microbial killing by phagocytes through the generation of reactive oxygen species (ROS). We found that NOX2-generated ROS are necessary for LC3 recruitment to phagosomes. Antibacterial autophagy in human epithelial cells, which do not express NOX2, was also dependent on ROS generation. These data reveal a coupling of oxidative and nonoxidative killing activities of the NOX2 NADPH oxidase in phagocytes through autophagy. Furthermore, our results suggest a general role for members of the NOX family in regulating autophagy. PMID:19339495

  2. 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

  3. Effect of contraceptive steroids on monoamine oxidase activity

    PubMed Central

    Southgate, Jennifer; Collins, G. G. S.; Pryse-Davies, J.; Sandler, M.

    1969-01-01

    Cyclical variations in monoamine oxidase activity during the human menstrual cycle, specific to the endometrium and modified in women undergoing contraceptive steroid treatment, may reflect changes in hormonal environment. Treatment of rats with individual constituents of the contraceptive pill causes analogous changes: oestrogens inhibit and progestogens potentiate uterine monoamine oxidase activity. ImagesFig. 2Fig. 3

  4. Catalase-peroxidases (KatG) exhibit NADH oxidase activity.

    PubMed

    Singh, Rahul; Wiseman, Ben; Deemagarn, Taweewat; Donald, Lynda J; Duckworth, Harry W; Carpena, Xavi; Fita, Ignacio; Loewen, Peter C

    2004-10-01

    Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG. PMID:15280362

  5. Renalase Prevents AKI Independent of Amine Oxidase Activity

    PubMed Central

    Wang, Ling; Velazquez, Heino; Moeckel, Gilbert; Chang, John; Ham, Ahrom; Lee, H. Thomas; Safirstein, Robert

    2014-01-01

    AKI is characterized by increased catecholamine levels and hypertension. Renalase, a secretory flavoprotein that oxidizes catecholamines, attenuates ischemic injury and the associated increase in catecholamine levels in mice. However, whether the amine oxidase activity of renalase is involved in preventing ischemic injury is debated. In this study, recombinant renalase protected human proximal tubular (HK-2) cells against cisplatin- and hydrogen peroxide–induced necrosis. Similarly, genetic depletion of renalase in mice (renalase knockout) exacerbated kidney injury in animals subjected to cisplatin-induced AKI. Interestingly, compared with the intact renalase protein, a 20–amino acid peptide (RP-220), which is conserved in all known renalase isoforms, but lacks detectable oxidase activity, was equally effective at protecting HK-2 cells against toxic injury and preventing ischemic injury in wild-type mice. Furthermore, in vitro treatment with RP-220 or recombinant renalase rapidly activated Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinases and downregulated c-Jun N-terminal kinase. In summary, renalase promotes cell survival and protects against renal injury in mice through the activation of intracellular signaling cascades, independent of its ability to metabolize catecholamines, and we have identified the region of renalase required for these effects. Renalase and related peptides show potential as therapeutic agents for the prevention and treatment of AKI. PMID:24511138

  6. Immobilization of Pichia pastoris cells containing alcohol oxidase activity

    PubMed Central

    Maleknia, S; Ahmadi, H; Norouzian, D

    2011-01-01

    Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

  7. Hyper-responsive Toll-like receptor 7 and 9 activation in NADPH oxidase-deficient B lymphoblasts.

    PubMed

    McLetchie, Shawna; Volpp, Bryan D; Dinauer, Mary C; Blum, Janice S

    2015-12-01

    Chronic granulomatous disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-subunit leucocyte NADPH oxidase. Myeloid-derived phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of reactive oxygen species to clear engulfed pathogens. In this study we show that oxidase also influences B-cell functions, including responses to single-stranded RNA or unmethylated DNA by endosomal Toll-like receptors (TLRs) 7 and 9. In response to TLR7/9 ligands, B-cell lines derived from patients with CGD with mutations in either the NADPH oxidase p40(phox) or p47(phox) subunits produced only low levels of reactive oxygen species. Remarkably, cytokine secretion and p38 mitogen-activated protein kinase activation by these oxidase-deficient B cells was significantly increased upon TLR7/9 activation when compared with oxidase-sufficient B cells. Increased TLR responsiveness was also detected in B cells from oxidase-deficient mice. NADPH oxidase-deficient patient-derived B cells also expressed enhanced levels of TLR7 and TLR9 mRNA and protein compared with the same cells reconstituted to restore oxidase activity. These data demonstrate that the loss of oxidase function associated with CGD can significantly impact B-cell TLR signalling in response to nucleic acids with potential repercussions for auto-reactivity in patients. PMID:26340429

  8. Xanthine oxidase inhibitory activity of Vietnamese medicinal plants.

    PubMed

    Nguyen, Mai Thanh Thi; Awale, Suresh; Tezuka, Yasuhiro; Tran, Quan Le; Watanabe, Hiroshi; Kadota, Shigetoshi

    2004-09-01

    Among 288 extracts, prepared from 96 medicinal plants used in Vietnamese traditional medicine to treat gout and related symptoms, 188 demonstrated xanthine oxidase (XO) inhibitory activity at 100 microg/ml, with 46 having greater than 50% inhibition. At 50 microg/ml, 168 of the extracts were active, with 21 possessing more than 50% inhibition. At 25 microg/ml, 146 extracts exhibited inhibitory activity, with 8 showing over 50% inhibition, while 126 extracts presented activity at 10 microg/ml, with 2 having greater than 50% inhibition. The MeOH extracts of Artemisia vulgaris, Caesalpinia sappan (collected at the Seven-Mountain area), Blumea balsamifera (collected in Lam Dong province), Chrysanthemum sinense and MeOH-H(2)O extract of Tetracera scandens (Khanh Hoa province) exhibited strong XO inhibitory activity with IC(50) values less than 20 microg/ml. The most active extract was the MeOH extract of the flower of C. sinense with an IC(50) value of 5.1 microg/ml. Activity-guided fractionation of the MeOH extract led to the isolation of caffeic acid (1), luteolin (2), eriodictyol (3), and 1,5-di-O-caffeoylquinic acid (4). All these compounds showed significant XO inhibitory activity in a concentration-dependent manner, and the activity of 2 was more potent (IC(50) 1.3 microM) than the clinically used drug, allopurinol (IC(50) 2.5 microM). PMID:15340229

  9. Diphenol activation of the monophenolase and diphenolase activities of field bean (Dolichos lablab) polyphenol oxidase.

    PubMed

    Gowda, Lalitha R; Paul, Beena

    2002-03-13

    This paper reports a study on the hydroxylation of ferulic acid and tyrosine by field bean (Dolichos lablab) polyphenol oxidase, a reaction that does not take place without the addition of catechol. A lag period similar to the characteristic lag of tyrosinase activity was observed, the length of which decreased with increasing catechol concentration and increased with increasing ferulic acid concentration. The activation constant K(a) of catechol for ferulic acid hydroxylation reaction was 5 mM. The kinetic parameters of field bean polyphenol oxidase toward ferulic acid and tyrosine were evaluated in the presence of catechol. 4-Methyl catechol, L-dihydroxyphenylalanine, pyrogallol, and 2,3,4-trihydroxybenzoic acid, substrates with high binding affinity to field bean polyphenol oxidase, could stimulate this hydroxylation reaction. In contrast, diphenols such as protocatechuic acid, gallic acid, chlorogenic acid, and caffeic acid, which were not substrates for the oxidation reaction, were unable to bring about this activation. It is most likely that only o-diphenols that are substrates for the diphenolase serve as cosubstrates by donating electrons at the active site for the monophenolase activity. The reaction mechanism for this activation is consistent with that proposed for tyrosinase (Sanchez-Ferrer, A.; Rodriguez-Lopez, J. N.; Garcia-Canovas, F.; Garcia-Carmona, F. Biochim. Biophys. Acta 1995, 1247, 1-11). The presence of o-diphenols, viz. catechol, L-dihydroxyphenylalanine, and 4-methyl catechol, is also necessary for the oxidation of the diphenols, caffeic acid, and catechin to their quinones by the field bean polyphenol oxidase. This oxidation reaction occurs immediately with no lag period and does not occur without the addition of diphenol. The kinetic parameters for caffeic acid (K(m) = 0.08 mM, V(max) = 32440 u/mg) in the presence of catechol and the activation constant K(a) of catechol (4.6 mM) for this reaction were enumerated. The absence of a lag

  10. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    PubMed Central

    Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji

    2012-01-01

    Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor. PMID:23203056

  11. Selective monoamine oxidase B inhibition by an Aphanizomenon flos-aquae extract and by its constitutive active principles phycocyanin and mycosporine-like amino acids.

    PubMed

    Scoglio, Stefano; Benedetti, Yanina; Benvenuti, Francesca; Battistelli, Serafina; Canestrari, Franco; Benedetti, Serena

    2014-06-15

    Aphanizomenon flos-aquae (AFA) is a fresh water unicellular blue-green alga that has been traditionally used for over 25 years for its health-enhancing properties. Recent studies have shown the ability of a proprietary AFA extract (Klamin(®)) to improve mood, counteract anxiety, and enhance attention and learning. Aim of this study was to test the monoamine oxidase (MAO) inhibition activity of the same AFA extract and of its constituents phycocyanin (AFA-PC) and mycosporine-like aminoacids (AFA-MAAs). All compounds showed a dose-dependent selective inhibition of MAO-B activity as compared to MAO-A. The IC50 values of the AFA extract (concentration 10 mg/ml), AFA-PC and AFA-MAAs were 6.4 μl/ml, 1.33 μM and 1.98 μM, respectively, evidencing a mixed-type of inhibition for the AFA extract (Ki 0.99 μl/ml), a non-competitive inhibition for AFA-PC (Ki 1.06 μM) and a competitive inhibition for AFA-MAAs (Ki 0.585 μM). These results are important to explain the neuromodulating properties of the AFA extract Klamin(®), which is rich in phenylethylamine, a general neuromodulator, that would nevertheless rapidly destroyed by MAO-B enzymes without the inhibitory activity of the synergic active principles AFA-PC and AFA-MAAs. The present investigation thus proposes the extract as potentially relevant in clinical areas such as mood disorders and neurodegenerative diseases. PMID:24690316

  12. L-amino acid oxidases with specificity for basic L-amino acids in cyanobacteria.

    PubMed

    Gau, Achim E; Heindl, Achim; Nodop, Anke; Kahmann, Uwe; Pistorius, Elfriede K

    2007-01-01

    The two closely related fresh water cyanobacteria Synechococcus elongatus PCC 6301 and Synechococcus elongatus PCC 7942 have previously been shown to constitutively express a FAD-containing L-amino acid oxidase with high specificity for basic L-amino acids (L-arginine being the best substrate). In this paper we show that such an enzyme is also present in the fresh water cyanobacterium Synechococcus cedrorum PCC 6908. In addition, an improved evaluation of the nucleotide/amino acid sequence of the L-amino acid oxidase of Synechococcus elongatus PCC 6301 (encoded by the aoxA gene) with respect to the FAD-binding site and a translocation pathway signal sequence will be given. Moreover, the genome sequences of 24 cyanobacteria will be evaluated for the occurrence of an aoxA-similar gene. In the evaluated cyanobacteria 15 genes encoding an L-amino acid oxidase-similar protein will be found. PMID:17542496

  13. Spinach thylakoid polyphenol oxidase isolation, activation, and properties of the native chloroplast enzyme

    SciTech Connect

    Golbeck, J.H.; Cammarata, K.V.

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14,18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. Sonication releases polyphenol oxidase from the membrane largely in the latent state. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time. Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K/sub m/. A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  14. Characterization of polyphenol oxidase activity in Ataulfo mango.

    PubMed

    Cheema, Summervir; Sommerhalter, Monika

    2015-03-15

    Crude extracts of Ataulfo exhibited polyphenol oxidase (PPO) activity with pyrogallol, 3-methylcatechol, catechol, gallic acid, and protocatechuic acid. The substrate dependent pH optima ranged from pH 5.4 to 6.4 with Michaelis-Menten constants between 0.84 ± 0.09 and 4.6 ± 0.7 mM measured in MES or phosphate buffers. The use of acetate buffers resulted in larger Michaelis-Menten constants, up to 14.62 ± 2.03 mM. Sodium ascorbate, glutathione, and kojic acid are promising inhibitors to prevent enzymatic browning in Ataulfo. PPO activity increased with ripeness and was always higher in the skin compared to the pulp. Sodium dodecyl sulphate (SDS) enhanced PPO activity, with pulp showing a stronger increase than skin. SDS-PAGE gels stained for catecholase activity showed multiple bands, with the most prominent bands at apparent molecular weights of 53, 112, and 144 kDa. PMID:25308684

  15. Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis.

    PubMed

    Wiel, C; Augert, A; Vincent, D F; Gitenay, D; Vindrieux, D; Le Calvé, B; Arfi, V; Lallet-Daher, H; Reynaud, C; Treilleux, I; Bartholin, L; Lelievre, E; Bernard, D

    2013-01-01

    Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability. PMID:24113189

  16. Monoamine oxidase inhibitory activities of heterocyclic chalcones.

    PubMed

    Minders, Corné; Petzer, Jacobus P; Petzer, Anél; Lourens, Anna C U

    2015-11-15

    Studies have shown that natural and synthetic chalcones (1,3-diphenyl-2-propen-1-ones) possess monoamine oxidase (MAO) inhibition activities. Of particular importance to the present study is a report that a series of furanochalcones acts as MAO-B selective inhibitors. Since the effect of heterocyclic substitution, other than furan (and more recently thiophene, piperidine and quinoline) on the MAO inhibitory properties of the chalcone scaffold remains unexplored, the aim of this study was to synthesise and evaluate further heterocyclic chalcone analogues as inhibitors of the human MAOs. For this purpose, heterocyclic chalcone analogues that incorporate pyrrole, 5-methylthiophene, 5-chlorothiophene and 6-methoxypyridine substitution were examined. Seven of the nine synthesised compounds exhibited IC50 values <1 μM for the inhibition of MAO-B, with all compounds exhibiting higher affinities for MAO-B compared to the MAO-A isoform. The most potent MAO-B inhibitor (4h) displays an IC50 value of 0.067 μM while the most potent MAO-A inhibitor (4e) exhibits an IC50 value of 3.81 μM. It was further established that selected heterocyclic chalcones are reversible and competitive MAO inhibitors. 4h, however, may exhibit tight-binding to MAO-B, a property linked to its thiophene moiety. We conclude that high potency chalcones such as 4h represent suitable leads for the development of MAO-B inhibitors for the treatment of Parkinson's disease and possibly other neurodegenerative disorders. PMID:26432037

  17. Comparative characterization of three D-aspartate oxidases and one D-amino acid oxidase from Caenorhabditis elegans.

    PubMed

    Katane, Masumi; Saitoh, Yasuaki; Seida, Yousuke; Sekine, Masae; Furuchi, Takemitsu; Homma, Hiroshi

    2010-06-01

    Previously, we cloned cDNAs for four Caenorhabditis elegans genes (F20 Hp, C47Ap, F18Ep, and Y69Ap genes) that were annotated in the database as encoding D-amino acid oxidase (DAO) or D-aspartate oxidase (DDO) proteins. These genes were expressed in Escherichia coli, and the recombinant C47Ap and F18Ep were shown to have functional DDO activities, while Y69Ap had functional DAO activity. In this study, we improved the E. coli culture conditions for the production of recombinant F20 Hp and, following purification of the protein, revealed that it has functional DDO activity. The kinetic properties of recombinant C47Ap (DDO-1), F18Ep (DDO-2), F20 Hp (DDO-3), and Y69Ap (DAO) were also determined and compared with recombinant human DDO and DAO. In contrast to the low catalytic efficiency of human DDO for D-Glu, all three C. elegans DDOs showed higher catalytic efficiencies for D-Glu than D-Asp or N-methyl-D-Asp. The catalytic efficiency of C. elegans DAO for D-Ser was substantially lower than that of human DAO, while the C. elegans DAO was more efficient at deamination of basic D-amino acids (D-Arg and D-His) than human DAO. Collectively, our results indicate that C. elegans contains at least three genes that encode functional DDOs, and one gene encoding a functional DAO, and that these enzymes have different and distinctive properties. PMID:20564561

  18. Identification in Marinomonas mediterranea of a novel quinoprotein with glycine oxidase activity

    PubMed Central

    Campillo-Brocal, Jonatan Cristian; Lucas-Elio, Patricia; Sanchez-Amat, Antonio

    2013-01-01

    Abstract A novel enzyme with lysine-epsilon oxidase activity was previously described in the marine bacterium Marinomonas mediterranea. This enzyme differs from other l-amino acid oxidases in not being a flavoprotein but containing a quinone cofactor. It is encoded by an operon with two genes lodA and lodB. The first one codes for the oxidase, while the second one encodes a protein required for the expression of the former. Genome sequencing of M. mediterranea has revealed that it contains two additional operons encoding proteins with sequence similarity to LodA. In this study, it is shown that the product of one of such genes, Marme_1655, encodes a protein with glycine oxidase activity. This activity shows important differences in terms of substrate range and sensitivity to inhibitors to other glycine oxidases previously described which are flavoproteins synthesized by Bacillus. The results presented in this study indicate that the products of the genes with different degrees of similarity to lodA detected in bacterial genomes could constitute a reservoir of different oxidases. PMID:23873697

  19. Isolation, characterization and screening of the in vitro cytotoxic activity of a novel L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom on human cancer cell lines.

    PubMed

    Teixeira, Tuila Leveghim; Oliveira Silva, Viviane Aline; da Cunha, Daniel Batista; Polettini, Flávia Lino; Thomaz, Camila Daniele; Pianca, Ariana Aparecida; Zambom, Fabiana Letícia; da Silva Leitão Mazzi, Denise Pimenta; Reis, Rui Manuel; Mazzi, Maurício Ventura

    2016-09-01

    An L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom was purified to homogeneity in a two-step procedure using molecular exclusion on Sephadex G-75, followed by Phenyl Sepharose FF chromatography. The molecular mass of the purified enzyme was 113 kDa, as determined by SDS-PAGE under reducing conditions. LAAOcdt showed amino acid homology to other L-amino acid oxidases isolated from different snake venoms. The comparative analysis of the internal peptide sequences of the NNPGILEYPVKPSEEGK fragments by LC-MS/MS spectrometry revealed 100% identity with C. durissus cumanensis LAAO. The purified protein catalyzed the oxidative deamination of L-amino acids, and the most specific substrates were L-Tyr and L-Phe. The enzyme presented optimum activity at pH 7.4 and at 44 °C. LAAOcdt also showed hemolytic activity (0.6-20 μg/μL) and induced both the formation plasma clots (5-100 μg/μL) and platelet aggregation (2.5 × 10(-3), 5.0 × 10(-3) and 10 × 10(-3) μg/mL), as well as bactericidal activity (2.5-10 μg/μL) against Staphylococcus aureus. Moreover, LAAOcdt exhibited cytotoxicity in distinct cancer cell lines, which presented a heterogeneous response profile. The mean IC50 value was 10.5 μg/mL. Glioma and pancreatic carcinoma cells were the most sensitive cell lines; they showed mean IC50 values of 7.2 μg/mL and 7.4 μg/mL, respectively. The exposure of the drug-sensitive cells to LAAOcdt for 24 h upregulated activated p-H2AX and efficiently decreased P42/P44 (ERK) activation in glioma cells (HCB151), which suggested an anti-proliferative effect. In addition, increased p21 expression was observed in SiHa cells, which showed a resistant phenotype. On the other hand, the flow cytometry and immunoblotting analyses showed that the enzyme did not induce cancer cell apoptosis. These results suggest that another cell death mechanism might contribute to the LAAOcdt-induced cytotoxicity. Taken together, this work may help to elucidate

  20. Cholesterol: A modulator of the phagocyte NADPH oxidase activity - A cell-free study

    PubMed Central

    Masoud, Rawand; Bizouarn, Tania; Houée-Levin, Chantal

    2014-01-01

    The NADPH oxidase Nox2, a multi-subunit enzyme complex comprising membrane and cytosolic proteins, catalyzes a very intense production of superoxide ions O2•−, which are transformed into other reactive oxygen species (ROS). In vitro, it has to be activated by addition of amphiphiles like arachidonic acid (AA). It has been shown that the membrane part of phagocyte NADPH oxidase is present in lipid rafts rich in cholesterol. Cholesterol plays a significant role in the development of cardio-vascular diseases that are always accompanied by oxidative stress. Our aim was to investigate the influence of cholesterol on the activation process of NADPH oxidase. Our results clearly show that, in a cell-free system, cholesterol is not an efficient activator of NADPH oxidase like arachidonic acid (AA), however it triggers a basal low superoxide production at concentrations similar to what found in neutrophile. A higher concentration, if present during the assembly process of the enzyme, has an inhibitory role on the production of O2•−. Added cholesterol acts on both cytosolic and membrane components, leading to imperfect assembly and decreasing the affinity of cytosolic subunits to the membrane ones. Added to the cytosolic proteins, it retains their conformations but still allows some conformational change induced by AA addition, indispensable to activation of NADPH oxidase. PMID:25462061

  1. 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

  2. Kinetics of Inhibition of Monoamine Oxidase Using Curcumin and Ellagic Acid

    PubMed Central

    Khatri, Dharmendra Kumar; Juvekar, Archana Ramesh

    2016-01-01

    Background: Curcumin and ellagic are the natural polyphenols having a wide range of pharmacological actions. They have been reported to have their use in various neurological disorders. Objective: This study was aimed to evaluate the effect of curcumin and ellagic acid on the activity of monoamine oxidase (MAO), the enzyme responsible for metabolism of monoamine neurotransmitters which are pivotal for neuronal development and function. Materials and Methods: The in vitro effects of these selected polyphenols on MAO activities in mitochondria isolated from rat brains were examined. Brain mitochondria were assayed for MAO type-B (MAO-B) using benzylamine as substrates. Rat brain mitochondrial MAO preparation was used to study the kinetics of enzyme inhibition using double reciprocal Lineweaver–Burk plot. Results: MAO activity was inhibited by curcumin and ellagic acid; however, higher half maximal inhibitory concentrations of curcumin (500.46 nM) and ellagic acid (412.24 nM) were required compared to the known MAO-B inhibitor selegiline. It is observed that the curcumin and ellagic acid inhibit the MAO activity with both the competitive and noncompetitive type of inhibitions. Conclusions: Curcumin and ellagic acid can be considered a possible source of MAO inhibitor used in the treatment of Parkinson's and other neurological disorders. SUMMARY Monoamine oxidase (MAO) is involved in a variety of neurological disorders including Parkinson's disease (PD)Curcumin and ellagic acid inhibit the monoamine oxidase activityEllagic acid revealed more potent MAO type-B (MAO-B) inhibitory activity than curcuminKinetic studies of MAO inhibition using different concentrations of curcumin and ellagic acid were plotted as double reciprocal Lineweaver–Burk plotThe mode of inhibition of both compounds toward MAO-B is mixed (competitive and uncompetitive) type of inhibition with both the competitive and noncompetitive type of inhibitions. Abbreviations used: MAO: Monoamine oxidase

  3. A Highly Stable D-Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus.

    PubMed

    Takahashi, Shouji; Furukawara, Makoto; Omae, Keishi; Tadokoro, Namiho; Saito, Yayoi; Abe, Katsumasa; Kera, Yoshio

    2014-12-01

    d-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic d-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested d-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain d-amino acids, with the highest specific activity toward d-valine and catalytic efficiency (kcat/Km) toward d-leucine. Substrate inhibition was observed in the case of d-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain d-amino acids, and as a scaffold to generate a novel DAO via protein engineering. PMID:25217016

  4. A Highly Stable d-Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

    PubMed Central

    Furukawara, Makoto; Omae, Keishi; Tadokoro, Namiho; Saito, Yayoi; Abe, Katsumasa; Kera, Yoshio

    2014-01-01

    d-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic d-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested d-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain d-amino acids, with the highest specific activity toward d-valine and catalytic efficiency (kcat/Km) toward d-leucine. Substrate inhibition was observed in the case of d-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain d-amino acids, and as a scaffold to generate a novel DAO via protein engineering. PMID:25217016

  5. Effects of anthocyanins from purple sweet potato (Ipomoea batatas L. cultivar Eshu No. 8) on the serum uric acid level and xanthine oxidase activity in hyperuricemic mice.

    PubMed

    Zhang, Zi-Cheng; Su, Guan-Hua; Luo, Chun-Li; Pang, Ya-Lu; Wang, Lin; Li, Xing; Wen, Jia-Hao; Zhang, Jiu-Liang

    2015-09-01

    This study was aimed at evaluating the hypouricemic effect of the anthocyanin-rich purple sweet potato extract (APSPE). In vitro, APSPE has been proved to significantly inhibit XO activity in a dose-dependent manner. In vivo, APSPE could not only inhibit the XO activity in mouse liver, but also reduce the serum uric acid level in hyperuricemic mice and affect the expression of mRNA levels of related renal transporters, such as mURAT1, mGLUT9, mOAT1 and mOCTN2. Moreover, APSPE could effectively regulate BUN and Cr levels to normal and decrease the inflammatory cellular influx in the tubule of the hyperuricemic mice. This study indicates the potential clinical utility of APSPE as a safe and effective anti-hyperuricemia bioactive agent or functional food. PMID:26201407

  6. CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity

    PubMed Central

    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

  7. 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...

  8. Lysyl oxidase binds transforming growth factor-beta and regulates its signaling via amine oxidase activity.

    PubMed

    Atsawasuwan, Phimon; Mochida, Yoshiyuki; Katafuchi, Michitsuna; Kaku, Masaru; Fong, Keith S K; Csiszar, Katalin; Yamauchi, Mitsuo

    2008-12-01

    Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-beta1 (TGF-beta1), a potent growth factor abundant in bone, the effect of LOX on TGF-beta1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-beta1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-beta1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-beta1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling. PMID:18835815

  9. Drugs related to monoamine oxidase activity.

    PubMed

    Fišar, Zdeněk

    2016-08-01

    Progress in understanding the role of monoamine neurotransmission in pathophysiology of neuropsychiatric disorders was made after the discovery of the mechanisms of action of psychoactive drugs, including monoamine oxidase (MAO) inhibitors. The increase in monoamine neurotransmitter availability, decrease in hydrogen peroxide production, and neuroprotective effects evoked by MAO inhibitors represent an important approach in the development of new drugs for the treatment of mental disorders and neurodegenerative diseases. New drugs are synthesized by acting as multitarget-directed ligands, with MAO, acetylcholinesterase, and iron chelation as targets. Basic information is summarized in this paper about the drug-induced regulation of monoaminergic systems in the brain, with a focus on MAO inhibition. Desirable effects of MAO inhibition include increased availability of monoamine neurotransmitters, decreased oxidative stress, decreased formation of neurotoxins, induction of pro-survival genes and antiapoptotic factors, and improved mitochondrial functions. PMID:26944656

  10. Stabilization of D-amino acid oxidase from Rhodosporidium toruloides by immobilization onto magnetic nanoparticles.

    PubMed

    Hsieh, Hao-Chieh; Kuan, I-Ching; Lee, Shiow-Ling; Tien, Gee-Yeng; Wang, Yi-Jen; Yu, Chi-Yang

    2009-04-01

    D-amino acid oxidase from Rhodosporidium toruloides was immobilized onto glutaraldehyde-activated magnetic nanoparticles. Approximately four enzyme molecules were attached to one magnetic nanoparticle when the weight ratio of the enzyme to the support was 0.12. After immobilization, the T(m) was increased from 45 degrees C of the free form to 55 degrees C. In the presence of 20 mM H2O2, the immobilized form retained 93% of its activity after 5 h while the free form was completely inactivated after 3.5 h. PMID:19066733

  11. Chemical Evidence for Potent Xanthine Oxidase Inhibitory Activity of Ethyl Acetate Extract of Citrus aurantium L. Dried Immature Fruits.

    PubMed

    Liu, Kun; Wang, Wei; Guo, Bing-Hua; Gao, Hua; Liu, Yang; Liu, Xiao-Hong; Yao, Hui-Li; Cheng, Kun

    2016-01-01

    Xanthine oxidase is a key enzyme which can catalyze hypoxanthine and xanthine to uric acid causing hyperuricemia in humans. Xanthine oxidase inhibitory activities of 24 organic extracts of four species belonging to Citrus genus of the family Rutaceae were assayed in vitro. Since the ethyl acetate extract of C. aurantium dried immature fruits showed the highest xanthine oxidase inhibitory activity, chemical evidence for the potent inhibitory activity was clarified on the basis of structure identification of the active constituents. Five flavanones and two polymethoxyflavones were isolated and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, hesperetin showed more potent inhibitory activity with an IC50 value of 16.48 μM. For the first time, this study provides a rational basis for the use of C. aurantium dried immature fruits against hyperuricemia. PMID:26950105

  12. 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. PMID:27125069

  13. Activity of glucose oxidase functionalized onto magnetic nanoparticles

    PubMed Central

    Kouassi, Gilles K; Irudayaraj, Joseph; McCarty, Gregory

    2005-01-01

    Background Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties. Methods Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy. Results The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94–100% while thiophene acetylation was 66–72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticles Conclusion Binding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological applications PMID:15762994

  14. In vitro oxidation of indoleacetic acid by soluble auxin-oxidases and peroxidases from maize roots. [Zea mays L

    SciTech Connect

    Beffa, R.; Martin, H.V.; Pilet, P.E. )

    1990-10-01

    Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl{sub 2} and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of ({sup 3}H)indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol.

  15. 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...

  16. Oxalic acid degradation by a novel fungal oxalate oxidase from Abortiporus biennis.

    PubMed

    Grąz, Marcin; Rachwał, Kamila; Zan, Radosław; Jarosz-Wilkołazka, Anna

    2016-01-01

    Oxalate oxidase was identified in mycelial extracts of a basidiomycete Abortiporus biennis strain. Intracellular enzyme activity was detected only after prior lowering of the pH value of the fungal cultures by using oxalic or hydrochloric acids. This enzyme was purified using size exclusion chromatography (Sephadex G-25) and ion-exchange chromatography (DEAE-Sepharose). This enzyme exhibited optimum activity at pH 2 when incubated at 40°C, and the optimum temperature was established at 60°C. Among the tested organic acids, this enzyme exhibited specificity only towards oxalic acid. Molecular mass was calculated as 58 kDa. The values of Km for oxalate and Vmax for the enzyme reaction were 0.015 M and 30 mmol min(-1), respectively. PMID:27337220

  17. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  18. Replacement of a terminal cytochrome c oxidase by ubiquinol oxidase during the evolution of acetic acid bacteria.

    PubMed

    Matsutani, Minenosuke; Fukushima, Kota; Kayama, Chiho; Arimitsu, Misato; Hirakawa, Hideki; Toyama, Hirohide; Adachi, Osao; Yakushi, Toshiharu; Matsushita, Kazunobu

    2014-10-01

    The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus, an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA, in addition to the UOX operon cyaBACD. We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA, which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost. PMID:24862920

  19. An alcohol oxidase of Phanerochaete chrysosporium with a distinct glycerol oxidase activity.

    PubMed

    Linke, Diana; Lehnert, Nicole; Nimtz, Manfred; Berger, Ralf G

    2014-01-01

    An intracellular alcohol oxidase (AOX) was isolated from the white-rot basidiomycete Phanerochaete chrysosporium (Pch), grown on l-lactate induction medium, and purified to electrophoretic homogeneity. The dimeric protein consisted of two identical 75kDa subunits. The open reading frame of 1,956bp resulted in a monomer consisting of 651 amino acids. The enzyme showed a pI at 5.4, a pH optimum of 9, a temperature optimum at 50°C, possessed putative conserved domains of the GMC superfamily, a FAD binding domain, and showed up to 86% homology to alcohol oxidase sequences of Gloeophyllum trabeum and Coprinopsis cinerea. As was shown for the first time for an AOX from a basidiomycete, not only methanol, but also lower primary alcohols and glycerol were accepted as substrates. An assay based on aldehyde dehydrogenase confirmed d-glyceraldehyde as the product of the reaction. A bioprocess based on this enzyme could alleviate the problems associated with the huge side-stream of glycerol occurring during the manufacture of biodiesel, yielding the green oxidant hydrogen peroxide. PMID:24910330

  20. Abscisic acid and aldehyde oxidase activity in maize ear leaf and grain relative to post-flowering photosynthetic capacity and grain-filling rate under different water/nitrogen treatments.

    PubMed

    Qin, Shujun; Zhang, Zongzheng; Ning, Tangyuan; Ren, Shizhong; Su, Licheng; Li, Zengjia

    2013-09-01

    This study investigated changes in leaf abscisic acid (ABA) concentrations and grain ABA concentrations in two maize cultivars and analyzed the following relationships under different water/nitrogen treatments: leaf ABA concentrations and photosynthetic parameters; leaf ABA concentrations and grain ABA concentrations; leaf/grain ABA concentrations and grain-filling parameters; and aldehyde oxidase (AO, EC 1.2.3.1) activities and ABA concentrations. The ear leaf average AO activities and ABA concentrations were lower in the controlled release urea treatments compared with the conventional urea treatments. The average AO activities in the grains were higher in the controlled release urea treatments, and the ABA concentrations were significantly increased at 11-30 DAF. The Pn and ABA concentrations in ear leaves were negatively correlated. And the Gmean were positively correlated with the grain ABA concentrations at 11-30 DAF and negatively correlated with the leaf ABA concentrations at 20 and 40-50 DAF. The grain ABA concentrations and leaf ABA concentrations were positively correlated. Thus, the Gmean were closely related to the AO activities and to the ear leaf and grain ABA concentrations. As compared to other treatments, the subsoiling and controlled release urea treatment promoted the uptake of water and nitrogen by maize, increased the photosynthetic capacity of the ear leaves, increased the grain-filling rate, and improved the movement of photosynthetic assimilates toward the developing grains. In the cultivar Z958, higher ABA concentrations in grains at 11-30 DAF and lower ABA concentrations in ear leaves during the late grain-filling stage, resulted in higher grain-filling rate and increased accumulation of photosynthetic products (relative to the cultivar D3). PMID:23770596

  1. Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

    PubMed Central

    Berthier, Sylvie; Hograindleur, Marc-André; Paclet, Marie-Hélène; Polack, Benoît; Morel, Françoise

    2012-01-01

    S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b558; and (iii) to determine the S100A8 consensus site involved in cytochrome b558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase activation

  2. Spatiotemporal Localization of d-Amino Acid Oxidase and d-Aspartate Oxidases during Development in Caenorhabditis elegans

    PubMed Central

    Saitoh, Yasuaki; Katane, Masumi; Kawata, Tomonori; Maeda, Kazuhiro; Sekine, Masae; Furuchi, Takemitsu; Kobuna, Hiroyuki; Sakamoto, Taro; Inoue, Takao; Arai, Hiroyuki; Nakagawa, Yasuhito

    2012-01-01

    Recent investigations have shown that a variety of d-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. d-Amino acid oxidase (DAO) and d-aspartate oxidase (DDO) are degradative enzymes stereospecific for d-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of d-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several d- and l-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and d-amino acids in multicellular organisms. PMID:22393259

  3. Spatiotemporal localization of D-amino acid oxidase and D-aspartate oxidases during development in Caenorhabditis elegans.

    PubMed

    Saitoh, Yasuaki; Katane, Masumi; Kawata, Tomonori; Maeda, Kazuhiro; Sekine, Masae; Furuchi, Takemitsu; Kobuna, Hiroyuki; Sakamoto, Taro; Inoue, Takao; Arai, Hiroyuki; Nakagawa, Yasuhito; Homma, Hiroshi

    2012-05-01

    Recent investigations have shown that a variety of D-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. D-Amino acid oxidase (DAO) and D-aspartate oxidase (DDO) are degradative enzymes stereospecific for D-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of D-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several D- and L-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and D-amino acids in multicellular organisms. PMID:22393259

  4. Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

    PubMed

    Gerdemann, C; Eicken, C; Magrini, A; Meyer, H E; Rompel, A; Spener, F; Krebs, B

    2001-07-01

    The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed. PMID:11451442

  5. Use of Glucose Oxidase in a Membrane Reactor for Gluconic Acid Production

    NASA Astrophysics Data System (ADS)

    Das Neves, Luiz Carlos Martins; Vitolo, Michele

    This article aims at the evaluation of the catalytic performance of glucose oxidase (GO) (EC.1.1.3.4) for the glucose/gluconic acid conversion in the ultrafiltration cell type membrane reactor (MB-CSTR). The reactor was coupled with a Millipore ultrafiltration-membrane (cutoff of 100 kDa) and operated for 24 h under agitation of 100 rpm, pH 5.5, and 30°C. The experimental conditions varied were the glucose concentration (2.5, 5.0, 10.0, 20.0, and 40.0 mM), the feeding rate (0.5, 1.0, 3.0, and 6.0/h), dissolved oxygen (8.0 and 16.0 mg/L), GO concentration (2.5, 5.0, 10.0, and 20.0 UGO/mL), and the glucose oxidase/catalase activity ratio (UGO/UCAT)(1∶0, 1∶10, 1∶20, and 1∶30). A conversion yield of 80% and specific reaction rate of 40×10-4 mmol/h·UGO were attained when the process was carried out under the following conditions: D=3.0/h, dissolved oxygen=16.0 mg/L, [G]=40 mM, and (UGO/UCAT)=1∶20. A simplified model for explaining the inhibition of GO activity by hydrogen peroxide, formed during the glucose/gluconic acid conversion, was presented.

  6. Ascorbyl palmitate-loaded chitosan nanoparticles: characteristic and polyphenol oxidase inhibitory activity.

    PubMed

    Kim, Mi Kyung; Lee, Ji-Soo; Kim, Kwang Yup; Lee, Hyeon Gyu

    2013-03-01

    The aim of this study was to produce ascorbyl palmitate (AP)-loaded nanoparticles in order to inhibit polyphenol oxidase (PPO) in bananas. AP-loaded chitosan nanoparticles were prepared using acetic acid and citric acid (denoted as CS/AA and CS/CA nanoparticles, respectively). As the initial AP concentration increases, the particle size significantly decreases, and the zeta potential, entrapment and loading efficiency significantly increases. The PPO inhibitory activity of AP was effectively improved when AP was nano-encapsulated by chitosan compared to no encapsulation. These results suggest that chitosan nano-encapsulation can be used to enhance the PPO inhibitory activity of AP. PMID:23247266

  7. Auxin-activated NADH oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

    NASA Technical Reports Server (NTRS)

    Morre, D. James; Morre, Dorothy M.; Ternes, Philipp

    2003-01-01

    The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.

  8. Activity of carbohydrate oxidases as influenced by wheat flour dough components.

    PubMed

    Degrand, L; Rakotozafy, L; Nicolas, J

    2015-08-15

    The carbohydrate oxidase (COXMn) from Microdochium nivale may well have desired functionalities as a dough and bread improver, similarly to Aspergillus niger glucose oxidase (GOX). COXMn catalyses the oxidation of various monosaccharides as well as maltooligosaccharides for which the best activity is obtained towards the maltooligosaccharides of polymerisation degrees 3 and 4. For the same activity towards glucose under air saturation, we show that COXMn exhibits a similar efficiency towards maltose as GOX towards glucose whatever the oxygen supply. Assays with COXMn show that no competition exists between carbohydrates naturally present in the wheat flour. We show that reaction products (d-glucono-δ-lactone and hydrogen peroxide) and the wheat flour dough component, ferulic acid, have no noticeable specific effect on the COXMn activity. The demonstrated differences in kinetics between COXMn and GOX allow predicting of differences in the functional behaviours of those enzymes during wheat flour dough formation. PMID:25794758

  9. Plant and animal glycolate oxidases have a common eukaryotic ancestor and convergently duplicated to evolve long-chain 2-hydroxy acid oxidases.

    PubMed

    Esser, Christian; Kuhn, Anke; Groth, Georg; Lercher, Martin J; Maurino, Veronica G

    2014-05-01

    Glycolate oxidase (GOX) is a crucial enzyme of plant photorespiration. The encoding gene is thought to have originated from endosymbiotic gene transfer between the eukaryotic host and the cyanobacterial endosymbiont at the base of plantae. However, animals also possess GOX activities. Plant and animal GOX belong to the gene family of (L)-2-hydroxyacid-oxidases ((L)-2-HAOX). We find that all (L)-2-HAOX proteins in animals and archaeplastida go back to one ancestral eukaryotic sequence; the sole exceptions are green algae of the chlorophyta lineage. Chlorophyta replaced the ancestral eukaryotic (L)-2-HAOX with a bacterial ortholog, a lactate oxidase that may have been obtained through the primary endosymbiosis at the base of plantae; independent losses of this gene may explain its absence in other algal lineages (glaucophyta, rhodophyta, and charophyta). We also show that in addition to GOX, plants possess (L)-2-HAOX proteins with different specificities for medium- and long-chain hydroxyacids (lHAOX), likely involved in fatty acid and protein catabolism. Vertebrates possess lHAOX proteins acting on similar substrates as plant lHAOX; however, the existence of GOX and lHAOX subfamilies in both plants and animals is not due to shared ancestry but is the result of convergent evolution in the two most complex eukaryotic lineages. On the basis of targeting sequences and predicted substrate specificities, we conclude that the biological role of plantae (L)-2-HAOX in photorespiration evolved by co-opting an existing peroxisomal protein. PMID:24408912

  10. Potential xanthine oxidase inhibitory activity of endophytic Lasiodiplodia pseudotheobromae.

    PubMed

    Kapoor, Neha; Saxena, Sanjai

    2014-07-01

    Xanthine oxidase is considered as a potential target for treatment of hyperuricemia. Hyperuricemia is predisposing factor for gout, chronic heart failure, atherosclerosis, tissue injury, and ischemia. To date, only two inhibitors of xanthine oxidase viz. allopurinol and febuxostat have been clinically approved for used as drugs. In the process of searching for new xanthine oxidase inhibitors, we screened culture filtrates of 42 endophytic fungi using in vitro qualitative and quantitative XO inhibitory assays. The qualitative assay exhibited potential XO inhibition by culture filtrates of four isolates viz. #1048 AMSTITYEL, #2CCSTITD, #6AMLWLS, and #96 CMSTITNEY. The XO inhibitory activity was present only in the chloroform extract of the culture filtrates. Chloroform extract of culture filtrate #1048 AMSTITYEL exhibited the highest inhibition of XO with an IC50 value of 0.61 μg ml(-1) which was better than allopurinol exhibiting an IC50 of 0.937 μg ml(-1) while febuxostat exhibited a much lower IC50 of 0.076 μg ml(-1). Further, molecular phylogenetic tools and morphological studies were used to identify #1048 AMSTITYEL as Lasiodiplodia pseudotheobromae. This is the first report of an endophytic Lasiodiplodia pseudotheobromae from Aegle marmelos exhibiting potential XO Inhibitory activity. PMID:24801403

  11. Substrate specificity of duckling hepatic and renal D-amino acid oxidase.

    PubMed

    Elkin, R G; Lyons, M L

    1988-05-01

    The substrate specificity of duckling hepatic and renal D-amino acid oxidase (DAAO; D-amino acid: O2 oxidoreductase [deaminating], E.C. 1.4.3.3) was determined using a method based on the combination of coupled enzyme reactions and a colorimetric procedure. When activities were averaged across tissues, D-proline was the most reactive substrate, followed by (in order) D-phenylalanine, D-alanine, D-methionine, D-leucine, D-isoleucine, D-valine, D-tryptophan, D-arginine, and D-lysine. Compared with D-alanine, duckling DAAO had minimal or no reactivity with D-asparagine, D-glutamine, D-histidine, D-threonine, D-cysteine, glycine, or D-serine. These results were in general agreement with data from other vertebrate species. PMID:2900508

  12. 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

    2014-10-01

    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. PMID:25113879

  13. A simple purification procedure of D-amino-acid oxidase from Candida guilliermondii H(see symbol)-4.

    PubMed

    Gevorgyan, G K; Davtyan, M A; Hambardzumyan, A A

    2012-01-01

    D-amino-acid oxidase (EC 1.4.3.3) was purified about 1480-fold from the yeast Candida guilliermondii H(see symbol)-4 using chromatofocusing method. The purification procedure gave an enzyme preparation which is greater than 90% homogenous on SDS-polyacrylamide gels with a specific activity of 11.54 U/mg at 30 degrees C with D-proline as substrate with the yield of total activity 9.3%. The molecular weights of subunit and native enzyme were determined to be 38.4 and 78.6 kDa by SDS-polyacrylamide gel electrophoresis and gel-filtration, respectively, suggesting that the native enzyme exists as a homodimer. A single molecular form with an isoelectric point of 6.85 was detected in analytical isoelectrofocusing. The optimum pH and temperature were 8.0 and 33 degrees C. An enzyme shows stability in the pH range from 7.4 to 9.0 and at the temperature no higher than 38 degrees C. Activation energy for D-amino-acid oxidase reaction was calculated to be 60 kJ/mol at 30 degrees C. The strict D-isomer specificity of the enzyme is confirmed, since no reaction could be detected with L-amino acids, and a large number of D-amino acids could be substrates for this enzyme. K(m) and V(max) values were determined for D-proline and D-alanine, which, among 22 tested, were the best substrates of the enzyme. D-amino-acid oxidase from the yeast C. guilliermondii is a flavoprotein oxidase in which the prosthetic group is tightly, but not covalently, bound FAD. The enzyme is completely inhibited by sodium benzoate, SH-oxidizing agents, but not by sodium azide, toluene or chloroform. PMID:23156699

  14. 4-Coumaroyl coenzyme A 3-hydroxylase activity from cell cultures of Lithospermum erythrorhizon and its relationship to polyphenol oxidase.

    PubMed

    Wang, Z X; Li, S M; Löscher, R; Heide, L

    1997-11-15

    A 4-coumaroyl-CoA 3-hydroxylase activity was purified 4600-fold from cell cultures of Lithospermum erythrorhizon. The enzyme showed a molecular mass of 42,400 +/- 1700 Da in gel chromatography and required ascorbate, NADH, or NADPH as cofactors. 4-Coumaroyl-CoA, 4-coumarate, p-cresol, and several other phenolic substances, but not tyrosine, were accepted as substrates for the hydroxylation. Besides hydroxylase activity, the enzyme showed diphenol oxidase activity. Both activities were inhibited by diethyldithiocarbamate or beta-mercaptoethanol, although at different concentrations. The enzyme showed striking similarity to a 4-coumaroyl-glucose 3-hydroxylase from sweet potato (Ipomoe batatas) roots, which has reportedly been purified to homogeneity and identified as a specific enzyme of chlorogenic acid biosynthesis. Close examination and comparison to a commercially available polyphenol oxidase, however, suggest that the enzyme activities purified from both Lithospermum and sweet potato are polyphenol oxidases rather than specific enzymes of secondary metabolism. PMID:9367532

  15. Structure and characterization of the glycan moiety of L-amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma.

    PubMed

    Geyer, A; Fitzpatrick, T B; Pawelek, P D; Kitzing, K; Vrielink, A; Ghisla, S; Macheroux, P

    2001-07-01

    Ophidian L-amino-acid oxidase (L-amino-acid oxygen:oxidoreductase, deaminating, EC 1.4.3.2) is found in the venom of many poisonous snakes (crotalids, elapids and viperids). This FAD-dependent glycoprotein has been studied from several snake species (e.g. Crotalus adamanteus, Crotalus atrox and Calloselasma rhodostoma) in detail with regard to the biochemical and enzymatic properties. The nature of glycosylation, however, as well as the chemical structure(s) of the attached oligosaccharide(s) are unknown. In view of the putative involvement of the glycan moiety in the biological effects of ophidian L-amino-acid oxidase, notably the apoptotic activity of the enzyme, structural knowledge is needed to evaluate its exact function. In this study we report on the glycosylation of L-amino-acid oxidase from the venom of the Malayan pit viper (Calloselasma rhodostoma). Its glycosylation is remarkably homogeneous with the major oligosaccharide accounting for approximately 90% of the total sugar content. Based on detailed analysis of the isolated oligosaccharide by 2D NMR spectroscopies and MALDI-TOF mass spectrometry the glycan is identified as a bis-sialylated, biantennary, core-fucosylated dodecasaccharide. The biological significance of this finding is discussed in light of the biological activities of the enzyme. PMID:11453999

  16. 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

  17. 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.

  18. Role of renal D-amino-acid oxidase in pharmacokinetics of D-leucine.

    PubMed

    Hasegawa, Hiroshi; Matsukawa, Takehisa; Shinohara, Yoshihiko; Konno, Ryuichi; Hashimoto, Takao

    2004-07-01

    d-Amino acids are now recognized to be widely present in mammals. Renal d-amino-acid oxidase (DAO) is associated with conversion of d-amino acids to the corresponding alpha-keto acids, but its contribution in vivo is poorly understood because the alpha-keto acids and/or l-amino acids formed are indistinguishable from endogenous compounds. First, we examined whether DAO is indispensable for conversion of d-amino acids to their alpha-keto acids by using the stable isotope tracer technique. After a bolus intravenous administration of d-[(2)H(7)]leucine to mutant mice lacking DAO activity (ddY/DAO(-)) and normal mice (ddY/DAO(+)), elimination of d-[(2)H(7)]leucine and formation of alpha-[(2)H(7)]ketoisocaproic acid ([(2)H(7)]KIC) and l-[(2)H(7)]leucine in plasma were determined. The ddY/DAO(-) mice, in contrast to ddY/DAO(+) mice, failed to convert d-[(2)H(7)]leucine to [(2)H(7)]KIC and l-[(2)H(7)]leucine. This result clearly revealed that DAO was indispensable for the process of chiral inversion of d-leucine. We further investigated the effect of renal mass reduction by partial nephrectomy on elimination of d-[(2)H(7)]leucine and formation of [(2)H(7)]KIC and l-[(2)H(7)]leucine. Renal mass reduction slowed down the elimination of d-[(2)H(7)]leucine. The fraction of conversion of d-[(2)H(7)]leucine to [(2)H(7)]KIC in sham-operated rats was 0.77, whereas that in five-sixths-nephrectomized rats was 0.25. The elimination behavior of d-[(2)H(7)]leucine observed in rats suggested that kidney was the principal organ responsible for converting d-leucine to KIC. PMID:15026304

  19. Salmonella Evades d-Amino Acid Oxidase To Promote Infection in Neutrophils

    PubMed Central

    Tuinema, Brian R.; Reid-Yu, Sarah A.

    2014-01-01

    ABSTRACT Neutrophils engulf and kill bacteria using oxidative and nonoxidative mechanisms. Despite robust antimicrobial activity, neutrophils are impaired in directing Salmonella clearance and harbor viable intracellular bacteria during early stages of infection that can subsequently escape to more-permissive cell types. The mechanisms accounting for this immune impairment are not understood. We report that Salmonella limits exposure to oxidative damage elicited by d-amino acid oxidase (DAO) in neutrophils by expressing an ABC importer specific for d-alanine, a DAO substrate found in peptidoglycan stem peptides. A Salmonella dalS mutant defective for d-alanine import was more susceptible to killing by DAO through exposure to greater oxidative stress during infection. This fitness defect was reversed by selective depletion of neutrophils or by inhibition of DAO in vivo with a small-molecule inhibitor. DalS-mediated subversion of neutrophil DAO is a novel host-pathogen interaction that enhances Salmonella survival during systemic infection. PMID:25425233

  20. Peroxidase and peroxidase-oxidase activities of isolated human myeloperoxidases.

    PubMed Central

    Svensson, B E; Domeij, K; Lindvall, S; Rydell, G

    1987-01-01

    Isolated neutrophils from healthy donors were used for the isolation of four highly purified forms of myeloperoxidase as determined by spectral (A430/A280 ratio 0.80-0.87) and enzyme-activity measurements. Although the myeloperoxidases exhibited different elution profiles on cation-exchange chromatography, gel filtration indicated similar relative molecular masses. When these forms were assayed for peroxidase and peroxidase-oxidase activities with several substrates, they all exhibited virtually the same specific activities. These results suggest that possible functional differences between the enzymes may be related to differences in their sites of action rather than to differences in enzyme activity. Myeloperoxidase from a patient with chronic myeloid leukaemia also revealed a similar heterogeneity on cation-exchange chromatography. However, this myeloperoxidase contained in addition one form with a lower and one form with a higher relative molecular mass, as indicated by gel-filtration chromatography. PMID:3036098

  1. Cell-free NADPH oxidase activation assays: "in vitro veritas".

    PubMed

    Pick, Edgar

    2014-01-01

    The superoxide (O2 (∙-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (∙-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (∙-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (∙-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (∙-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (∙-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount

  2. Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies

    PubMed Central

    Andjelković, Ana; Oliveira, Marcos T.; Cannino, Giuseppe; Yalgin, Cagri; Dhandapani, Praveen K.; Dufour, Eric; Rustin, Pierre; Szibor, Marten; Jacobs, Howard T.

    2015-01-01

    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. PMID:26672986

  3. 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...

  4. D-amino acid oxidase generates agonists of the aryl hydrocarbon receptor from D-tryptophan.

    PubMed

    Nguyen, Linh P; Hsu, Erin L; Chowdhury, Goutam; Dostalek, Miroslav; Guengerich, F Peter; Bradfield, Christopher A

    2009-12-01

    The aryl hydrocarbon receptor (AHR) is well-known for its role in mediating the toxic and adaptive responses to xenobiotic compounds. Recent studies also indicate that AHR ligands are endogenously produced and may be essential for normal development. Previously, we showed that the endogenous enzyme, aspartate aminotransferase (AST), generates the AHR proagonist, indole-3-pyruvic acid (I3P), by deamination of its substrate L-tryptophan. We hypothesized that other enzymatic pathways capable of producing I3P may generate AHR agonists in vivo. We now demonstrate that the enzyme d-amino acid oxidase (DAAO) catalyzes the production of AHR agonists through the enzymatic conversion of D-tryptophan to I3P. Moreover, we provide evidence that the nonenzymatic oxidation and condensation of I3P is a critical step in the generation of receptor agonists by DAAO and AST. Products of this process include two novel agonists, 1,3-di(1H-indol-3-yl)propan-2-one and 1-(1H-indol-3-yl)-3-(3H-indol-3-ylidene) propan-2-one [characterized in the accompanying paper, Chowdhury et al. ( 2009 ) Chem. Res. Toxicol. , DOI: 10.1021/tx9000418 ], both of which can potently activate the AHR at concentrations in the nanomolar range. These results show that endogenous AHR activity can be modulated by I3P production from amino acid precursors through multiple enzymatic pathways, including those catalyzed by DAAO and AST. PMID:19860415

  5. 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. PMID:26976571

  6. 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 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. PMID:26976571

  7. Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase: kinetics and operational stability.

    PubMed

    Nordkvist, Mikkel; Nielsen, Per Munk; Villadsen, John

    2007-07-01

    Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase was studied. The K(m)-value for lactose, obtained by a traditional enzymatic assay, was 0.066 mM at pH 6.4 and 38 degrees C. The effect of oxygen on the enzymatic rate of reaction as well as the operational stability of the enzyme was studied by performing reactions at constant pH and temperature in a stirred tank reactor. Catalase was included in all reactions to avoid inhibition and deactivation of the oxidase by hydrogen peroxide. At pH 6.4 and 38 degrees C, K(m) for oxygen was 0.97 mM, while the catalytical rate constant, k(cat), was 94 s(-1). Furthermore, we found that the operational stability of the oxidase was dependent on the type of base used for neutralization of the acid produced. Thus, when 2 M NaOH was used for neutralization of a reaction medium containing 50 mM phosphate buffer, significant deactivation of the oxidase was observed. Also, we found that the oxidase was protected against deactivation by base at high lactose concentrations. A simple model is proposed to explain the obtained results. PMID:17154316

  8. Effect of high pressure on peanut allergens in the presence of polyphenol oxidase and caffeic acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High pressure (HP) enhances enzymatic reactions. Because polyphenol oxidase (PPO) is an enzyme, and reduces IgE binding of peanut allergens in presence of caffeic acid (CA), we postulated that a further reduction in IgE binding can be achieved, using HP together with PPO and CA. Peanut extracts cont...

  9. Molecular characterization of L-amino acid oxidase from king cobra venom.

    PubMed

    Jin, Yang; Lee, Wen-Hui; Zeng, Lin; Zhang, Yun

    2007-09-15

    An L-amino acid oxidase from Ophiophagus hannah snake venom (Oh-LAAO) was purified by successive gel filtration, ion-exchange and heparin chromatography. Oh-LAAO did not induce platelet aggregation; however, it had potent inhibitory activity on platelet aggregation induced by ADP and U46619, but showed no effect on platelet aggregation induced by thrombin, mucetin, ristocetin and stejnulxin. By RT-PCR and 5'-RACE methods, the complete Oh-LAAO cDNA was cloned from the venom gland total RNA preparations. The cDNA sequence contains an open-reading frame (ORF) of 1476-bp, which encodes a protein of 491 amino acids comprising a signal peptide of 25 amino acids and 466-residue mature protein. The predicted protein sequence of Oh-LAAO was confirmed by N-terminal and trypsin-digested internal peptides sequencing together with peptide mass fingerprinting. cDNAs encoding for ORF of LAAOs from Bungarus fasciatus and B. multicinctus were cloned and reported in this study. In addition, partial cDNA encoding for Naja atra LAAO was also reported. Oh-LAAO shared approximately 50% protein sequence identity with other known snake venom LAAOs. Phylogenetic analysis indicated that Oh-LAAO is evolutionary distant to other snake venom LAAOs. PMID:17543361

  10. Defining the role of tyrosine and rational tuning of oxidase activity by genetic incorporation of unnatural tyrosine analogs.

    PubMed

    Yu, Yang; Lv, Xiaoxuan; Li, Jiasong; Zhou, Qing; Cui, Chang; Hosseinzadeh, Parisa; Mukherjee, Arnab; Nilges, Mark J; Wang, Jiangyun; Lu, Yi

    2015-04-15

    While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr. PMID:25672571

  11. Enhanced fatty acid accumulation in Isochrysis galbana by inhibition of the mitochondrial alternative oxidase pathway under nitrogen deprivation.

    PubMed

    Zhang, Litao; Liu, Jianguo

    2016-07-01

    The purpose of this study was to clarify the interrelation between the mitochondrial alternative oxidase (AOX) pathway and fatty acid accumulation in marine microalga Isochrysis galbana. Under normal conditions, the activity of the AOX pathway was maintained at a low level in I. galbana. Compared with the normal condition, nitrogen deprivation significantly increased the AOX pathway activity and fatty acid accumulation. Under nitrogen deprivation, the inhibition of the AOX pathway by salicylhydroxamic acid caused the accumulation of reducing equivalents and the over-reduction of chloroplasts in I. galbana cells, leading to a decrease in the photosynthetic O2 evolution rate. The over-production of reducing equivalents due to the inhibition of the AOX pathway under nitrogen deprivation further enhanced the accumulation of fatty acids in I. galbana cells. PMID:27068057

  12. Ataxia telangiectasia mutated influences cytochrome c oxidase activity.

    PubMed

    Patel, Akshar Y; McDonald, Todd M; Spears, Larry D; Ching, James Kain; Fisher, Jonathan S

    2011-02-25

    Cells lacking ataxia telangiectasia mutated (ATM) have impaired mitochondrial function. Furthermore, mammalian cells lacking ATM have increased levels of reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) deletions in the region encoding for cytochrome c oxidase (COX). We hypothesized that ATM specifically influences COX activity in skeletal muscle. COX activity was ∼40% lower in tibialis anterior from ATM-deficient mice than for wild-type mice (P < 0.01, n = 9/group). However, there were no ATM-related differences in activity of succinate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, or complex III. Incubation of wild-type extensor digitorum longus muscles for 1h with the ATM inhibitor KU55933 caused a ∼50% reduction (P<0.05, n = 5/group) in COX activity compared to muscles incubated with vehicle alone. Among the control muscles and muscles treated with the ATM inhibitor, COX activity was correlated (r = 0.61, P<0.05) with activity of glucose 6-phosphate dehydrogenase, a key determinant of antioxidant defense through production of NADPH. Overall, the findings suggest that ATM has a protective role for COX activity. PMID:21266166

  13. Quantitative structure-activity studies on monoamine oxidase inhibitors.

    PubMed

    Johnson, C L

    1976-05-01

    Quantitative structure-activity studies were carried out on a series of N-isopropylaryl hydrazides which inhibits monoamine oxidase (MAO). The inhibitory potencies of these compounds of MAO were found to correlate with the electron-withdrawing capacity of the aryl ring substituents as estimated by both empirical Hammet sigma constants and electronic indices from molecular orbital calculations. Based on these correlations and previously published data on other classes of MAO inhibitors, a general model for the inhibitor pharmacophore is proposed: potent MAO of an aromatic ring; electron-withdrawing groups on the aromatic ring or replacing the phenyl ring with certain types of heterocyclic rings will tend to increase the potency. PMID:1271400

  14. Distribution in Different Organisms of Amino Acid Oxidases with FAD or a Quinone As Cofactor and Their Role as Antimicrobial Proteins in Marine Bacteria

    PubMed Central

    Campillo-Brocal, Jonatan C.; Lucas-Elío, Patricia; Sanchez-Amat, Antonio

    2015-01-01

    Amino acid oxidases (AAOs) catalyze the oxidative deamination of amino acids releasing ammonium and hydrogen peroxide. Several kinds of these enzymes have been reported. Depending on the amino acid isomer used as a substrate, it is possible to differentiate between l-amino acid oxidases and d-amino acid oxidases. Both use FAD as cofactor and oxidize the amino acid in the alpha position releasing the corresponding keto acid. Recently, a novel class of AAOs has been described that does not contain FAD as cofactor, but a quinone generated by post-translational modification of residues in the same protein. These proteins are named as LodA-like proteins, after the first member of this group described, LodA, a lysine epsilon oxidase synthesized by the marine bacterium Marinomonas mediterranea. In this review, a phylogenetic analysis of all the enzymes described with AAO activity has been performed. It is shown that it is possible to recognize different groups of these enzymes and those containing the quinone cofactor are clearly differentiated. In marine bacteria, particularly in the genus Pseudoalteromonas, most of the proteins described as antimicrobial because of their capacity to generate hydrogen peroxide belong to the group of LodA-like proteins. PMID:26694422

  15. Following Glucose Oxidase Activity by Chemiluminescence and Chemiluminescence Resonance Energy Transfer (CRET) Processes Involving Enzyme-DNAzyme Conjugates

    PubMed Central

    Niazov, Angelica; Freeman, Ronit; Girsh, Julia; Willner, Itamar

    2011-01-01

    A hybrid consisting of glucose oxidase-functionalized with hemin/G-quadruplex units is used for the chemiluminescence detection of glucose. The glucose oxidase-mediated oxidation of glucose yields gluconic acid and H2O2. The latter in the presence of luminol acts as substrate for the hemin/G-quadruplex-catalyzed generation of chemiluminescence. The glucose oxidase/hemin G-quadruplex hybrid was immobilized on CdSe/ZnS quantum dots (QDs). The light generated by the hybrid, in the presence of glucose, activated a chemiluminescence resonance energy transfer process to the QDs, resulting in the luminescence of the QDs. The intensities of the luminescence of the QDs at different concentrations of glucose provided an optical means to detect glucose. PMID:22346648

  16. Following glucose oxidase activity by chemiluminescence and chemiluminescence resonance energy transfer (CRET) processes involving enzyme-DNAzyme conjugates.

    PubMed

    Niazov, Angelica; Freeman, Ronit; Girsh, Julia; Willner, Itamar

    2011-01-01

    A hybrid consisting of glucose oxidase-functionalized with hemin/G-quadruplex units is used for the chemiluminescence detection of glucose. The glucose oxidase-mediated oxidation of glucose yields gluconic acid and H(2)O(2). The latter in the presence of luminol acts as substrate for the hemin/G-quadruplex-catalyzed generation of chemiluminescence. The glucose oxidase/hemin G-quadruplex hybrid was immobilized on CdSe/ZnS quantum dots (QDs). The light generated by the hybrid, in the presence of glucose, activated a chemiluminescence resonance energy transfer process to the QDs, resulting in the luminescence of the QDs. The intensities of the luminescence of the QDs at different concentrations of glucose provided an optical means to detect glucose. PMID:22346648

  17. Thiolactomycin inhibits D-aspartate oxidase: a novel approach to probing the active site environment.

    PubMed

    Katane, Masumi; Saitoh, Yasuaki; Hanai, Toshihiko; Sekine, Masae; Furuchi, Takemitsu; Koyama, Nobuhiro; Nakagome, Izumi; Tomoda, Hiroshi; Hirono, Shuichi; Homma, Hiroshi

    2010-10-01

    D-Aspartate oxidase (DDO) and D-amino acid oxidase (DAO) are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of D-amino acids. While several functionally and structurally important amino acid residues have been identified in the DAO protein, little is known about the structure-function relationships of DDO. In the search for a potent DDO inhibitor as a novel tool for investigating its structure-function relationships, a large number of biologically active compounds of microbial origin were screened for their ability to inhibit the enzymatic activity of mouse DDO. We discovered several compounds that inhibited the activity of mouse DDO, and one of the compounds identified, thiolactomycin (TLM), was then characterized and evaluated as a novel DDO inhibitor. TLM reversibly inhibited the activity of mouse DDO with a mixed type of inhibition more efficiently than meso-tartrate and malonate, known competitive inhibitors of mammalian DDOs. The selectivity of TLM was investigated using various DDOs and DAOs, and it was found that TLM inhibits not only DDO, but also DAO. Further experiments with apoenzymes of DDO and DAO revealed that TLM is most likely to inhibit the activities of DDO and DAO by competition with both the substrate and the coenzyme, FAD. Structural models of mouse DDO/TLM complexes supported this finding. The binding mode of TLM to DDO was validated further by site-directed mutagenesis of an active site residue, Arg-237. Collectively, our findings show that TLM is a novel, active site-directed DDO inhibitor that will be useful for elucidating the molecular details of the active site environment of DDO. PMID:20603179

  18. [Sulfite oxidase activity deficiency caused by cofactor molybdenum deficiency: A case of early severe encephalopathy].

    PubMed

    Durousset, C; Gay, C; Magnin, S; Acquaviva, C; Patural, H

    2016-03-01

    Neonatal seizure incidence is approximately 3.5/1000 live births. Inborn metabolic diseases account for approximately 1-4% of neonatal seizure cases. Among them, the catabolism anomaly of sulfite to sulfate caused by sulfite oxidase or cofactor molybdenum deficiency (MoCD) is a rare metabolic disorder in which neurological damage is similar to that found in neonatal asphyxia. We report the case of a newborn child with a MoCD. Born of related parents, this child had intrauterine growth retardation predominating on size diagnosed in the third trimester of pregnancy. After an uneventful birth, he presented convulsions at the 12th hour of life, confirmed by an electroencephalogram. Anticonvulsants and adjuvant treatments were ineffective; the child then required intubation at day 5 of life. The initial biological assessment found an elevated blood lactate level and the chromatography of amino acids showed a significant decrease of cystine and the abnormal presence of sulfocysteine, suggestive of a lack of sulfite oxidase activity. The uric acid level measured secondarily was low, suggesting a MoCD. Brain MRI was performed at day 5 for diffuse ischemic injury of different ages. After limiting acute care, the child died at day 14 of life. The genetic study of the child found a homozygous mutation c.564+1G>A in the MOCS2 gene, confirming the diagnosis of MoCD, present in the heterozygous state in both parents. Investigations in a logical sequence quickly suggested the MoCD diagnosis in presence of a low plasma concentration of cysteine, the abnormal presence of sulfocysteine, and low uric acid levels. The diagnosis of sulfite oxidase deficiency was made. Until now, no treatment has proven effective but a new treatment appears to be effective in cases with a MOCS1 mutation. PMID:26775885

  19. Development of 2-(Substituted Benzylamino)-4-Methyl-1, 3-Thiazole-5-Carboxylic Acid Derivatives as Xanthine Oxidase Inhibitors and Free Radical Scavengers.

    PubMed

    Ali, Md Rahmat; Kumar, Suresh; Afzal, Obaid; Shalmali, Nishtha; Sharma, Manju; Bawa, Sandhya

    2016-04-01

    A series of 2-(substituted benzylamino)-4-methylthiazole-5-carboxylic acid was designed and synthesized as structural analogue of febuxostat. A methylene amine spacer was incorporated between the phenyl ring and thiazole ring in contrast to febuxostat in which the phenyl ring was directly linked with the thiazole moiety. The purpose of incorporating methylene amine was to provide a heteroatom which is expected to favour hydrogen bonding within the active site residues of the enzyme xanthine oxidase. The structure of all the compounds was established by the combined use of FT-IR, NMR and MS spectral data. All the compounds were screened in vitro for their ability to inhibit the enzyme xanthine oxidase as per the reported procedure along with DPPH free radical scavenging assay. Compounds 5j, 5k and 5l demonstrated satisfactory potent xanthine oxidase inhibitory activities with IC50 values, 3.6, 8.1 and 9.9 μm, respectively, whereas compounds 5k, 5n and 5p demonstrated moderate antioxidant activities having IC50 15.3, 17.6 and 19.6 μm, respectively, along with xanthine oxidase inhibitory activity. Compound 5k showed moderate xanthine oxidase inhibitory activity as compared with febuxostat along with antioxidant activity. All the compounds were also studied for their binding affinity in active site of enzyme (PDB ID-1N5X). PMID:26575582

  20. Changes in lysyl oxidase (LOX) distribution and its decreased activity in keratoconus corneas.

    PubMed

    Dudakova, Lubica; Liskova, Petra; Trojek, Tomas; Palos, Michalis; Kalasova, Sarka; Jirsova, Katerina

    2012-11-01

    Inadequate cross-linking between collagen lamellae is a characteristic feature of keratoconus corneas. The formation of covalent bonds between collagen and elastin fibrils, which maintain the biomechanical properties of the cornea, is mediated by the cuproenzyme lysyl oxidase and four lysyl oxidase-like enzymes. The aim of this study was to determine the distribution of lysyl oxidase and the total lysyl oxidase activity (lysyl oxidase and the four lysyl oxidase-like enzymes) in control and keratoconic corneas. Seven control and eight keratoconic corneas were used for the imunohistochemical detection of lysyl oxidase in corneal cryosections using two different antibodies. The total lysyl oxidase activity in the culture medium of corneal fibroblasts from six explanted keratoconic and four control corneas was measured using a fluorometric assay in the presence and absence of the lysyl oxidase inhibitor beta-aminopropionitrile and determined as the production of H(2)O(2) in nM per μg of total protein. In the control tissue, the most intense signal for lysyl oxidase was present in the corneal epithelium, in which perinuclear dots brightly projecting from more or less homogenous cytoplasmic staining may represent the lysyl oxidase propeptide. Less intense staining was present in keratocytes, the extracellular matrix and in the corneal endothelium. The epithelium of the limbus and the perilimbal conjunctiva showed intense to very intense staining. The distribution of lysyl oxidase was clearly decreased in at least five of the eight keratoconic specimens. The most marked signal reduction was observed in the stromal matrix and in keratocytes. Moreover, the signal in pathological specimens revealed a more irregular pattern, including the presence of intra- and extracellular clumps in the epithelium. Interestingly, endothelial cells showed no or very weak staining in areas just beneath negative stromal tissue. The mean activity of total lysyl oxidase in the keratoconic

  1. The stabilizing effects of immobilization in D-amino acid oxidase from Trigonopsis variabilis

    PubMed Central

    Dib, Iskandar; Nidetzky, Bernd

    2008-01-01

    Background Immobilization of Trigonopsis variabilis D-amino acid oxidase (TvDAO) on solid support is the key to a reasonably stable performance of this enzyme in the industrial process for the conversion of cephalosporin C as well as in other biocatalytic applications. Results To provide a mechanistic basis for the stabilization of the carrier-bound oxidase we analyzed the stabilizing effects of immobilization in TvDAO exposed to the stress of elevated temperature and operational conditions. Two different strategies of immobilization were used: multi-point covalent binding to epoxy-activated Sepabeads EC-EP; and non-covalent oriented immobilization of the enzyme through affinity of its N-terminal Strep-tag to Strep-Tactin coated on insoluble particles. At 50°C, the oriented immobilizate was not stabilized as compared to the free enzyme. The structure of TvDAO was stabilized via covalent attachment to Sepabeads EC-EP but concomitantly, binding of the FAD cofactor was weakened. FAD release from the enzyme into solution markedly reduced the positive effect of immobilization on the overall stability of TvDAO. Under conditions of substrate conversion in a bubble-aerated stirred tank reactor, both immobilization techniques as well as the addition of the surfactant Pluronic F-68 stabilized TvDAO by protecting the enzyme from the deleterious effect of gas-liquid interfaces. Immobilization of TvDAO on Sepabeads EC-EP however stabilized the enzyme beyond this effect and led to a biocatalyst that could be re-used in multiple cycles of substrate conversion. Conclusion Multi-point covalent attachment of TvDAO on an isoluble porous carrier provides stabilization against the denaturing effects of high temperature and exposure to a gas-liquid interface. Improvement of binding of the FAD cofactor, probably by using methods of protein engineering, would further enhance the stability of the immobilized enzyme. PMID:18798979

  2. Inhibition of monoamine oxidase-A activity in rat brain by synthetic hydrazines: structure-activity relationship (SAR).

    PubMed

    Dar, Ahsana; Khan, Khalid M; Ateeq, Humayun S; Khan, Shagufta; Rahat, Shagufta; Perveen, Shahnaz; Supuran, Claudiu T

    2005-06-01

    A series of hydrazine derivatives was synthesized in order to evaluate their monoamine oxidase A (MAO-A) inhibitory effects. MAO-A inhibitory activity of 4-tosyl benzoic acid carbohydrazide was quite potent, similarly to that of the corresponding 4-benzyloxy-benzoic acid carbohydrazide and its N-cyanoethylated derivative. Structural variations of these compounds, such as the replacement of the 4-substitutent, of the aromatic ring on which the carbohydrazide moiety is grafted, as well as cyclization of the hydrazide moiety in five- or six-membered rings caused either significant decline or complete loss of MAO inhibitory properties. The most active compound (4-tosyl benzoic acid carbohydrazide) was also subjected to the forced swim test, an animal model of depression, eliciting a marked reduction in immobility time in rats, without affecting the locomotor activity, implying that it possesses anti-depressant properties due to inhibition of MAO type-A. PMID:16119198

  3. The xanthine oxidase activity in different of secondary transformed peat-moorsh soils

    NASA Astrophysics Data System (ADS)

    Styła, Katarzyna; Wojciech Szajdak, Lech

    2010-05-01

    The investigations were carried out on the transect of peatland 4.5 km long, located in the Agroecological Landscape Park host D. Chlapowski in Turew (40 km South-West of Poznań, West Polish Lowland). The sites investigation were located along Wyskoć ditch. The following material was taken from four chosen sites marked as Zbęchy, Bridge, Shelterbelt and Hirudo in two layers: acrotelm (0-50 cm) and catotelm (50-100 cm). The object of this study was to characterize the biochemical properties by the determination of the xanthine oxidase activity in two layers (acrotelm and catotelm) of the four different peat-moorsh soils used as meadow. The xanthine oxidase activity was determined spectrophotometrically by measuring uric acid formation at λmax=290 nm with xanthine as substrate. In peat-moorsh soil the highest activities of xanthine oxidasewas observed in the Shelterbelt and whereas the lowest - in Zbęchy, Bridge and Hirudo. Activities of this enzyme in peat-moorsh soil ranged from 5.96 to 19.51 μmol h-1g d.m soil. Increased activities of xanthine oxidase have been recorded on the depth 50-100 cm - catotelm (from 11.71 to 19.51 μmol h-1g d.m soil) in comparison with the depth 0-50 cm - acrotelm (from 5.96 to 14.64 μmol h-1g d.m soil). This work was supported by a grant No. N N305 3204 36 founded by Polish Ministry of Education.

  4. Inhibition of apple polyphenol oxidase activity by sodium chlorite.

    PubMed

    Lu, Shengmin; Luo, Yaguang; Feng, Hao

    2006-05-17

    Sodium chlorite (SC) was shown to have strong efficacy both as a sanitizer to reduce microbial growth on produce and as a browning inhibitor on fresh-cut apples in previous experiments. This study was undertaken to investigate the inhibitory effect of SC on polyphenol oxidase (PPO) and the associated mechanisms. The experiment showed that SC had a strong inhibition of apple PPO. The extent of inhibition was influenced by SC concentration and pH. Inhibition was most prominent at pH 4.5, at which approximately 30% of enzyme activity was lost in the presence of 10 mM SC, followed closely by that at pH 4.0 with a 26% reduction in PPO activity. The inhibition mode was determined using Dixon and Lineweaver-Burk plots, which established SC to be a mixed inhibitor of apple PPO for the oxidation of catechol. Preincubation of PPO with 8 mM SC for 8 min caused a maximum of 46% activity reduction compared to noninhibited control. However, preincubation of SC with catechol for 8 min resulted in no additional loss of PPO activity. These findings provide further evidence that the inhibition of PPO activity by SC is due to the inhibition of the enzyme itself rather than removal of the substrate. PMID:19127746

  5. Wheat Germ Agglutinin Induces NADPH-Oxidase Activity in Human Neutrophils by Interaction with Mobilizable Receptors

    PubMed Central

    Karlsson, Anna

    1999-01-01

    Wheat germ agglutinin (WGA), a lectin with specificity for N-acetylglucosamine and sialic acid, was investigated with respect to its ability to activate the NADPH-oxidase of in vivo-exudated neutrophils (obtained from a skin chamber), and the activity was compared to that of peripheral blood neutrophils. The exudate cells responded to WGA, by both releasing reactive oxygen species into the extracellular milieu and producing oxygen metabolites intracellularly. The peripheral blood cells were unresponsive. To mimic the in vivo-exuded neutrophils with regards to receptor exposure, peripheral blood neutrophils were induced to mobilize their granules and vesicles to varying degrees (in vitro priming), prior to challenge with WGA. The oxidative response to WGA increased with increasing levels of granule mobilization, and the receptor(s) could be shown to reside in the secretory vesicles and/or the gelatinase granules in resting neutrophils. Several WGA-binding glycoproteins were detected in subcellular fractions containing these organelles. The extra- and intracellular NADPH-oxidase responses showed differences in sialic acid dependency, indicating that these two responses are mediated by different receptor structures. PMID:10377127

  6. Fermentation and alternative oxidase contribute to the action of amino acid biosynthesis-inhibiting herbicides.

    PubMed

    Zulet, Amaia; Gil-Monreal, Miriam; Zabalza, Ana; van Dongen, Joost T; Royuela, Mercedes

    2015-03-01

    Acetolactate synthase inhibitors (ALS-inhibitors) and glyphosate (GLP) are two classes of herbicide that act by the specific inhibition of an enzyme in the biosynthetic pathway of branched-chain or aromatic amino acids, respectively. The physiological effects that are detected after application of these two classes of herbicides are not fully understood in relation to the primary biochemical target inhibition, although they have been well documented. Interestingly, the two herbicides' toxicity includes some common physiological effects suggesting that they kill the treated plants by a similar pattern despite targeting different enzymes. The induction of aerobic ethanol fermentation and alternative oxidase (AOX) are two examples of these common effects. The objective of this work was to gain further insight into the role of fermentation and AOX induction in the toxic consequences of ALS-inhibitors and GLP. For this, Arabidopsis T-DNA knockout mutants of alcohol dehydrogenase (ADH) 1 and AOX1a were used. The results found in wild-type indicate that both GLP and ALS-inhibitors reduce ATP production by inducing fermentation and alternative respiration. The main physiological effects in the process of herbicide activity upon treated plants were accumulation of carbohydrates and total free amino acids. The effects of the herbicides on these parameters were less pronounced in mutants compared to wild-type plants. The role of fermentation and AOX regarding pyruvate availability is also discussed. PMID:25544587

  7. Low expression of the antibacterial factor L-amino acid oxidase in bovine mammary gland.

    PubMed

    Nagaoka, Kentaro; Zhang, Haolin; Arakuni, Masahiro; Taya, Kazuyoshi; Watanabe, Gen

    2014-12-01

    In the mouse, L-amino acid oxidase (LAO) produces hydrogen peroxide by utilizing free amino acids and is a proven antibacterial factor in mammary glands. Mastitis, a bacterial infection of the mammary gland, is the most frequent disease in dairy cattle. Here, we investigate whether LAO is expressed in the mammary gland of dairy cattle and is antibacterial. In dairy cattle, the expression level of LAO mRNA in the mammary gland was considerably lower than that in mice, and LAO activity was not observed in cattle milk that produced hydrogen peroxide. The expression of LAO mRNA was also low in Japanese Black cattle, the same as in Holstein cattle. A higher LAO mRNA expression was observed in the mastitis glands than in the lactating glands. Furthermore, spleen and lymph nodes expressed high levels of LAO mRNA in dairy cattle. We conclude that mammary glands in dairy cattle have lower ability to express the LAO gene compared to that in mice, which may result in a high incidence of mastitis. PMID:24961772

  8. Cost of a naturally occurring two-amino acid deletion in cytochrome c oxidase subunit 7A in Drosophila simulans.

    PubMed

    Ballard, J William O; Melvin, Richard G; Lazarou, Michael; Clissold, Fiona J; Simpson, Stephen J

    2010-10-01

    This study aimed to determine whether a naturally occurring (DeltaTrp85, DeltaVal86) deletion from a protein subunit of cytochrome c oxidase (complex IV) influenced cytochrome c oxidase activity, mRNA expression levels of electron transport chain genes, and aspects of adult female fitness in the fly Drosophila simulans. We modeled the tertiary structure of D. simulans cox7A containing the deletion by homology to the bovine cox7A structure and predicted that it would decrease the function of complex IV. This prediction led to the hypothesis that flies with the deletion would have lower cytochrome c oxidase activity and higher levels of mRNA expression from cox7A. This result was observed, but unexpectedly, elevated levels of mRNA expression were also observed in genes encoding subunits of complexes I, III, and IV. Together these data suggest that the deletion causes a high bioenergetic cost to the organism. To investigate the predicted cost at a physiological level, we assayed aspects of adult female fitness. Starvation sensitivity but not feeding rate was significantly influenced by the two-amino acid deletion. Further, we observed that carbohydrate and protein levels but not lipid levels were higher in the mutant flies. Together, these data show that quaternary structure modeling and biochemistry can be used to link the genotype with the organismal phenotype. PMID:20698788

  9. NAD(+)-independent aldehyde oxidase catalyzes cofactor balanced 3-hydroxypropionic acid production in Klebsiella pneumoniae.

    PubMed

    Li, Ying; Liu, Luo; Tian, Pingfang

    2014-11-01

    The limiting step for biosynthesis of 3-hydroxypropionic acid (3-HP) in Klebsiella pneumoniae is the conversion of 3-hydroxypropionaldehyde (3-HPA) to 3-HP. This reaction is catalyzed by aldehyde dehydrogenase (ALDH) with NAD(+) as a cofactor. Although NAD(+)-dependent ALDH overexpression facilitates 3-HP biosynthesis, ALDH activity decreases and 3-HP stops accumulation when NAD(+) is exhausted. Here, we show that an NAD(+)-independent aldehyde oxidase (AOX) from Pseudomonas sp. AIU 362 holds promise for cofactor-balanced 3-HP production in K. pneumoniae. The AOX coding gene, alod, was heterologously expressed in E. coli and K. pneumoniae, and their respective crude cell extracts showed 38.1 U/mg and 16.6 U/mg activities toward propionaldehyde. The recombinant K. pneumoniae expressing alod showed 13.7 U/mg activity toward 3-HPA; K m and V max were 6.7 mM and 42 μM/min/mg, respectively. In shake-flask cultures, the recombinant K. pneumoniae strain produced 0.89 g 3-HP/l, twice that of the control. Moreover, it produced 3 g 3-HP/l during 24 h fed-batch cultivation in a 5 l bioreactor. The results indicate that AOX can efficiently convert 3-HPA into 3-HP. PMID:24980850

  10. Suppression of NADPH Oxidase Activity May Slow the Expansion of Osteolytic Bone Metastases.

    PubMed

    McCarty, Mark F; DiNicolantonio, James

    2016-01-01

    Lysophosphatidic acid (LPA), generated in the microenvironment of cancer cells, can drive the proliferation, invasion, and migration of cancer cells by activating G protein-coupled LPA receptors. Moreover, in cancer cells that have metastasized to bone, LPA signaling can promote osteolysis by inducing cancer cell production of cytokines, such as IL-6 and IL-8, which can stimulate osteoblasts to secrete RANKL, a key promoter of osteoclastogenesis. Indeed, in cancers prone to metastasize to bone, LPA appears to be a major driver of the expansion of osteolytic bone metastases. Activation of NADPH oxidase has been shown to play a mediating role in the signaling pathways by which LPA, as well as RANKL, promote osteolysis. In addition, there is reason to suspect that Nox4 activation is a mediator of the feed-forward mechanism whereby release of TGF-beta from bone matrix by osteolysis promotes expression of PTHrP in cancer cells, and thereby induces further osteolysis. Hence, measures which can down-regulate NADPH oxidase activity may have potential for slowing the expansion of osteolytic bone metastases in cancer patients. Phycocyanin and high-dose statins may have utility in this regard, and could be contemplated as complements to bisphosphonates or denosumab for the prevention and control of osteolytic lesions. Ingestion of omega-3-rich flaxseed or fish oil may also have potential for controlling osteolysis in cancer patients. PMID:27571113

  11. The effects of ingested aluminium on brain cytochrome oxidase activity.

    PubMed

    Mohan, N; Alleyne, T; Adogwa, A

    2009-11-01

    Aluminium has a unique combination of physical and chemical properties which has enabled man to put this metal to very wide and varied use. However prolonged exposure to aluminium ions may lead to adverse health effects. In this study, we evaluated the effects of dietary aluminium on the protein composition and the intrinsic activity of cytochrome oxidase (COX) for brain mitochondria. New Zealand white rabbits were maintained on a diet of commercial rabbit pellets and distilled water for a period of 12 weeks. For the experimental group, AlCl3, 330 mg/kg/L was added to the drinking water. When compared to the control, mitochondria isolated from the brains of the AICl3 fed rabbits showed no change in Km but an approximate 35% decrease in both the low and high affinity Vmax values. Also, whereas the protein composition of the mitochondria from both sources appeared to be normal, isolation of highly purified COX proved to be difficult and for the AlCl3 fed rabbits, a number of the enzyme's low molecular weight subunits were absent. These results appear to confirm a relationship between long term aluminium consumption and low brain COX activity; they further suggest that an altered COX structure may be the cause of the low enzymic activity. PMID:20441059

  12. Electrochemical L-lactic acid sensor based on immobilized ZnO nanorods with lactate oxidase.

    PubMed

    Ibupoto, Zafar Hussain; Shah, Syed Muhammad Usman Ali; Khun, Kimleang; Willander, Magnus

    2012-01-01

    In this work, fabrication of gold coated glass substrate, growth of ZnO nanorods and potentiometric response of lactic acid are explained. The biosensor was developed by immobilizing the lactate oxidase on the ZnO nanorods in combination with glutaraldehyde as a cross linker for lactate oxidase enzyme. The potentiometric technique was applied for the measuring the output (EMF) response of l-lactic acid biosensor. We noticed that the present biosensor has wide linear detection range of concentration from 1 × 10(-4)-1 × 10(0) mM with acceptable sensitivity about 41.33 ± 1.58 mV/decade. In addition, the proposed biosensor showed fast response time less than 10 s, a good selectivity towards l-lactic acid in presence of common interfering substances such as ascorbic acid, urea, glucose, galactose, magnesium ions and calcium ions. The present biosensor based on immobilized ZnO nanorods with lactate oxidase sustained its stability for more than three weeks. PMID:22736960

  13. Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation.

    PubMed

    Arai, Yasuyuki; Nishinaka, Yoko; Arai, Toshiyuki; Morita, Makiko; Mizugishi, Kiyomi; Adachi, Souichi; Takaori-Kondo, Akifumi; Watanabe, Tomohiro; Yamashita, Kouhei

    2014-01-10

    Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-κB activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia. PMID:24326071

  14. Inhibition of acetylcholinesterase and cytochrome oxidase activity in Fasciola gigantica cercaria by phytoconstituents.

    PubMed

    Sunita, Kumari; Habib, Maria; Kumar, P; Singh, Vinay Kumar; Husain, Syed Akhtar; Singh, D K

    2016-02-01

    Fasciolosis is an important cattle and human disease caused by Fasciola hepatica and Fasciola gigantica. One of the possible methods to control this problem is to interrupt the life cycle of Fasciola by killing its larva (redia and cercaria) in host snail. Molecular identification of cercaria larva of F. gigantica was done by comparing the nucleotide sequencing with adult F. gigantica. It was noted that nucleotide sequencing of cercaria larva and adult F. gigantica were 99% same. Every month during the year 2011-2012, in vivo treatment with 60% of 4 h LC50 of phyto cercaricides citral, ferulic acid, umbelliferone, azadirachtin and allicin caused significant inhibition of acetylcholinesterase (AChE) and cytochrome oxidase activity in the treated cercaria larva of F. gigantica. Whereas, activity of both enzymes were not significantly altered in the nervous tissues of vector snail Lymnaea acuminata exposed to same treatments. Maximum reduction in AChE (1.35% of control in month of June) and cytochrome oxidase (3.71% of control in the month of July) activity were noted in the cercaria exposed to 60% of 4 h LC50 of azadirachtin and allicin, respectively. PMID:26536397

  15. Brain monoamine oxidase A activity predicts trait aggression.

    PubMed

    Alia-Klein, Nelly; Goldstein, Rita Z; Kriplani, Aarti; Logan, Jean; Tomasi, Dardo; Williams, Benjamin; Telang, Frank; Shumay, Elena; Biegon, Anat; Craig, Ian W; Henn, Fritz; Wang, Gene-Jack; Volkow, Nora D; Fowler, Joanna S

    2008-05-01

    The genetic deletion of monoamine oxidase A (MAO A), an enzyme that breaks down the monoamine neurotransmitters norepinephrine, serotonin, and dopamine, produces aggressive phenotypes across species. Therefore, a common polymorphism in the MAO A gene (MAOA, Mendelian Inheritance in Men database number 309850, referred to as high or low based on transcription in non-neuronal cells) has been investigated in a number of externalizing behavioral and clinical phenotypes. These studies provide evidence linking the low MAOA genotype and violent behavior but only through interaction with severe environmental stressors during childhood. Here, we hypothesized that in healthy adult males the gene product of MAO A in the brain, rather than the gene per se, would be associated with regulating the concentration of brain amines involved in trait aggression. Brain MAO A activity was measured in vivo in healthy nonsmoking men with positron emission tomography using a radioligand specific for MAO A (clorgyline labeled with carbon 11). Trait aggression was measured with the multidimensional personality questionnaire (MPQ). Here we report for the first time that brain MAO A correlates inversely with the MPQ trait measure of aggression (but not with other personality traits) such that the lower the MAO A activity in cortical and subcortical brain regions, the higher the self-reported aggression (in both MAOA genotype groups) contributing to more than one-third of the variability. Because trait aggression is a measure used to predict antisocial behavior, these results underscore the relevance of MAO A as a neurochemical substrate of aberrant aggression. PMID:18463263

  16. Brain Monoamine Oxidase-A Activity Predicts Trait Aggression

    PubMed Central

    Alia-Klein, Nelly; Goldstein, Rita Z.; Kriplani, Aarti; Logan, Jean; Tomasi, Dardo; Williams, Benjamin; Telang, Frank; Shumay, Elena; Biegon, Anat; Craig, Ian W.; Henn, Fritz; Wang, Gene-Jack; Volkow, Nora D.; Fowler, Joanna S.

    2008-01-01

    The genetic deletion of monoamine oxidase A (MAO A, an enzyme which breaks down the monoamine neurotransmitters norepinephrine, serotonin and dopamine) produces aggressive phenotypes across species. Therefore, a common polymorphism in the MAO A gene (MAOA, MIM 309850, referred to as high or low based on transcription in non-neuronal cells) has been investigated in a number of externalizing behavioral and clinical phenotypes. These studies provide evidence linking the low MAOA genotype and violent behavior but only through interaction with severe environmental stressors during childhood. Here, we hypothesized that in healthy adult males the gene product of MAO A in the brain, rather than the gene per se, would be associated with regulating the concentration of brain amines involved in trait aggression. Brain MAO A activity was measured in-vivo in healthy non-smoking men with positron emission tomography using a radioligand specific for MAO A (clorgyline labeled with carbon 11). Trait aggression was measured with the Multidimensional Personality Questionnaire (MPQ). Here we report for the first time that brain MAO A correlates inversely with the MPQ trait measure of aggression (but not with other personality traits) such that the lower the MAO A activity in cortical and subcortical brain regions the higher the self-reported aggression (in both MAOA genotype groups) contributing to more than a third of the variability. Since trait aggression is a measure used to predict antisocial behavior, these results underscore the relevance of MAO A as a neurochemical substrate of aberrant aggression. PMID:18463263

  17. Unfolding and refolding of active apple polyphenol oxidase.

    PubMed

    Mari, S; Marquès, L; Breton, F; Karamanos, Y; Macheix, J J

    1998-11-01

    For the first time, unfolding (6 M guanidine) and refolding of partially proteolysed purified polyphenol oxidase (PPOr) was achieved, with 88% of activity recovered. Optimal refolding conditions consisted in stepwise dialysis of guanidine treated extracts, the dialysis buffers containing 1 M (NH4)2SO4 and 100 microM CuSO4. However, CuSO4 had limited effect on the recovering of PPOr activity, whereas (NH4)2SO4 was essential. Concerning the PPO tertiary structure, denaturing conditions (combinations of boiling and reducing agent) used on SDS-PAGE have shown (i) a compact tertiary structure and (ii) the presence of disulfide bonds in PPOr, accounting for the shift between 27 and 41 kDa, and 41 and 42 kDa, respectively. Resistance to proteolytic cleavage was used to study the conformational changes induced by the denaturing treatments. Folded PPOr was resistant to further proteolysis whereas unfolded PPO was totally digested, indicating the role of tertiary structure of PPOr in the resistance to proteases. PMID:9842726

  18. A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential.

    PubMed

    Murata, M; Nishimura, M; Murai, N; Haruta, M; Homma, S; Itoh, Y

    2001-02-01

    Polyphenol oxidase (PPO) is responsible for enzymatic browning of apples. Apples lacking PPO activity might be useful not only for the food industry but also for studies of the metabolism of polyphenols and the function of PPO. Transgenic apple calli were prepared by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistant gene and antisense PPO gene. Four KM-resistant callus lines were obtained from 356 leaf explants. Among these transgenic calli, three calli grew on the medium containing KM at the same rate as non-transgenic callus on the medium without KM. One callus line had an antisense PPO gene, in which the amount and activity of PPO were reduced to half the amount and activity in non-transgenic callus. The browning potential of this line, which was estimated by adding chlorogenic acid, was also half the browning potential of non-transgenic callus. PMID:11302173

  19. Fabricating an Amperometric Cholesterol Biosensor by a Covalent Linkage between Poly(3-thiopheneacetic acid) and Cholesterol Oxidase

    PubMed Central

    Nien, Po-Chin; Chen, Po-Yen; Ho, Kuo-Chuan

    2009-01-01

    In this study, use of the covalent enzyme immobilization method was proposed to attach cholesterol oxidase (ChO) on a conducting polymer, poly(3-thiopheneacetic acid), [poly(3-TPAA)]. Three red-orange poly(3-TPAA) films, named electrodes A, B and C, were electropolymerized on a platinum electrode by applying a constant current of 1.5 mA, for 5, 20 and 100 s, respectively. Further, 1-ethyl-3-(3-dimethylamiopropyl)carbodiimide hydrochloride (EDC · HCl) and N-hydroxysuccinimide (NHS) were used to activate the free carboxylic groups of the conducting polymer. Afterwards, the amino groups of the cholesterol oxidase were linked on the activated groups to form peptide bonds. The best sensitivity obtained for electrode B is 4.49 mA M−1 cm−2, with a linear concentration ranging from 0 to 8 mM, which is suitable for the analysis of cholesterol in humans. The response time (t95) is between 70 and 90 s and the limit of detection is 0.42 mM, based on the signal to noise ratio equal to 3. The interference of species such as ascorbic acid and uric acid increased to 5.2 and 10.3% of the original current response, respectively, based on the current response of cholesterol (100%). With respect to the long-term stability, the sensing response retains 88% of the original current after 13 days. PMID:22573987

  20. Evaluation of Xanthine Oxidase Inhibitory Potential and In vivo Hypouricemic Activity of Dimocarpus longan Lour. Extracts

    PubMed Central

    Sheu, Shi-Yuan; Fu, Yuan-Tsung; Huang, Wen-Dar; Chen, Yung-Ann; Lei, Yi-Chih; Yao, Chun-Hsu; Hsu, Feng-Lin; Kuo, Tzong-Fu

    2016-01-01

    Background: Longan is a fruit tree known to contain many phenolic components, which are capable of protecting people from oxidative damage through an anti-inflammatory mechanism. It may be also worthwhile to study the effect on lowering uric acid activity. Materials and Methods: This study investigates the lowering of uric acid using longan extracts, including flowers, pericarps, seeds, leaves, and twigs, on potassium-oxonate-induced hyperuricemia mice and its inhibitory actions against xanthine oxidase (XO) activities. Results: The findings revealed that ethyl acetate fraction of longan extracts exhibited strong XO-inhibitory activity, and the flower extracts (IC50 = 115.8 μg/mL) revealed more potent XO-inhibitory activity to those of pericarps (118.9 μg/mL), twigs (125.3 μg/mL), seeds (262.5 μg/mL), and leaves (331.1 μg/mL) in vitro. In addition, different dosages of longan extract (50–100 mg/kg) were administered to hyperuricemic mice. The lowering effect of longan extracts on uric acid at 75 mg/kg markedly reduced plasma uric acid levels in decreasing order: Flowers (80%) > seeds (72%) > pericarps (64%) > twigs (59%) > leaves (41%), compared with allopurinol (89%). Finally, 10 isolated phytochemicals from longan flowers were then examined in vitro. The results indicated that proanthocyanidin A2 and acetonylgeraniin A significantly inhibited XO activity in vitro. This is the first report providing new insights into the urate-reducing effect of phenolic dimer and hydrolyzable tannin, which can be developed to potential hypouricemic agents. SUMMARY Longan flower extracts possess more potent XO-inhibitory activity than pericarps, twigs, seeds, and leaves in vitroThe lowering effect of longan flowers and seeds extracts markedly reduced plasma uric acid levels as compared to allopurinol in vivoThe extract proanthocyanidin A2 and acetonylgeraniin A were demonstrated potent XO inhibitory activity in vitro Abbreviations used: PO: Potassium-oxonate, XO: xanthine

  1. Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography.

    PubMed

    Yamamoto, T; Moriwaki, Y; Takahashi, S; Tsutsumi, Z; Yamakita, J; Nasako, Y; Hiroishi, K; Higashino, K

    1996-06-01

    An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 microliters of plasma and 240 microliters of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 microM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierke's disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects. PMID:8811453

  2. Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

    PubMed

    Strick, Christine A; Li, Cheryl; Scott, Liam; Harvey, Brian; Hajós, Mihály; Steyn, Stefanus J; Piotrowski, Mary A; James, Larry C; Downs, James T; Rago, Brian; Becker, Stacey L; El-Kattan, Ayman; Xu, Youfen; Ganong, Alan H; Tingley, F David; Ramirez, Andres D; Seymour, Patricia A; Guanowsky, Victor; Majchrzak, Mark J; Fox, Carol B; Schmidt, Christopher J; Duplantier, Allen J

    2011-01-01

    Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents. PMID:21763704

  3. Synthesis and evaluation of 1-hydroxy/methoxy-4-methyl-2-phenyl-1H-imidazole-5-carboxylic acid derivatives as non-purine xanthine oxidase inhibitors.

    PubMed

    Chen, Shaolei; Zhang, Tingjian; Wang, Jian; Wang, Fangyang; Niu, Handong; Wu, Chunfu; Wang, Shaojie

    2015-10-20

    Xanthine oxidase is a key enzyme that catalyses hypoxanthine and xanthine to uric acid, whose overproduction leads to the gout-causing hyperuricemia. In this study, a series of 1-hydroxy/methoxy-4-methyl-2-phenyl-1H-imidazole-5-carboxylic acid derivatives (4a-4k and 6a-6k) was synthesized and evaluated for their inhibitory potency against xanthine oxidase. The 1-hydroxyl substituted derivatives 4a-4k showed excellent inhibitory potency with IC50 values ranging from 0.003 μM to 1.2 μM, with compounds 4d (IC₅₀ = 0.003 μM), 4e (IC₅₀ = 0.003 μM), and 4f (IC₅₀ = 0.006 μM) manifesting the most potent xanthine oxidase inhibitory potency that were comparable with that of Febuxostat (IC₅₀ = 0.01 μM). Lineweaver-Burk plot analysis revealed that representative compound 4f acted as a mixed-type inhibitor for xanthine oxidase. The basis of significant inhibition of xanthine oxidase by 4f was rationalized by its molecular docking into the active site of xanthine dehydrogenase. PMID:26363870

  4. The Antimicrobial Activity of Marinocine, Synthesized by Marinomonas mediterranea, Is Due to Hydrogen Peroxide Generated by Its Lysine Oxidase Activity

    PubMed Central

    Lucas-Elío, Patricia; Gómez, Daniel; Solano, Francisco; Sanchez-Amat, Antonio

    2006-01-01

    Marinocine is a broad-spectrum antibacterial protein synthesized by the melanogenic marine bacterium Marinomonas mediterranea. This work describes the basis for the antibacterial activity of marinocine and the identification of the gene coding for this protein. The antibacterial activity is inhibited under anaerobic conditions and by the presence of catalase under aerobic conditions. Marinocine is active only in culture media containing l-lysine. In the presence of this amino acid, marinocine generates hydrogen peroxide, which causes cell death as confirmed by the increased sensitivity to marinocine of Escherichia coli strains mutated in catalase activity. The gene coding for this novel enzyme was cloned using degenerate PCR with primers designed based on conserved regions in the antimicrobial protein AlpP, synthesized by Pseudoalteromonas tunicata, and some hypothetical proteins. The gene coding for marinocine has been named lodA, standing for lysine oxidase, and it seems to form part of an operon with a second gene, lodB, that codes for a putative dehydrogenase flavoprotein. The identity of marinocine as LodA has been demonstrated by N-terminal sequencing of purified marinocine and generation of lodA mutants that lose their antimicrobial activity. This is the first report on a bacterial lysine oxidase activity and the first time that a gene encoding this activity has been cloned. PMID:16547036

  5. Mutation spectrum of homogentisic acid oxidase (HGD) in alkaptonuria

    PubMed Central

    Vilboux, Thierry; Kayser, Michael; Introne, Wendy; Suwannarat, Pim; Bernardini, Isa; Fischer, Roxanne; O’Brien, Kevin; Kleta, Robert; Huizing, Marjan; Gahl, William A.

    2009-01-01

    Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder, characterized by accumulation of homogentisic acid, leading to darkened urine, pigmentation of connective tissue (ochronosis), joint and spine arthritis, and destruction of cardiac valves. AKU is due to mutations in the homogentisate dioxygenase gene, HGD, that converts homogentisic acid to maleylacetoacetic acid in the tyrosine catabolic pathway. Here we report a comprehensive mutation analysis of 93 patients enrolled in our study, as well as an extensive update of all previously published HGD mutations associated with AKU. Within our patient cohort, we identified 52 HGD variants, of which 22 were novel. This yields a total of 91 identified HGD variations associated with AKU to date, including 62 missense, 13 splice site, 10 frameshift, 5 nonsense and 1 no-stop mutation. Most HGD variants reside in exons 3, 6, 8 and 13. We assessed the potential effect of all missense variations on protein function, using 5 bioinformatic tools specifically designed for interpretation of missense variants (SIFT, POLYPHEN, PANTHER, PMUT and SNAP). We also analyzed the potential effect of splice site variants using two different tools (BDGP and NetGene2). This study provides valuable resources for molecular analysis of alkaptonuria and expands our knowledge of the molecular basis of this disease. PMID:19862842

  6. Characterization of active site residues of nitroalkane oxidase.

    PubMed

    Valley, Michael P; Fenny, Nana S; Ali, Shah R; Fitzpatrick, Paul F

    2010-06-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  7. Sustained activation of proton channels and NADPH oxidase in human eosinophils and murine granulocytes requires PKC but not cPLA2α activity

    PubMed Central

    Morgan, Deri; Cherny, Vladimir V; Finnegan, Alison; Bollinger, James; Gelb, Michael H; DeCoursey, Thomas E

    2007-01-01

    The prevailing hypothesis that a signalling pathway involving cPLA2α is required to enhance the gating of the voltage-gated proton channel associated with NADPH oxidase was tested in human eosinophils and murine granulocytes. This hypothesis invokes arachidonic acid (AA) liberated by cPLA2α as a final activator of proton channels. In human eosinophils studied in the perforated-patch configuration, phorbol myristate acetate (PMA) stimulation elicited NADPH oxidase-generated electron current (Ie) and enhanced proton channel gating identically in the presence or absence of three specific cPLA2α inhibitors, Wyeth-1, pyrrolidine-2 and AACOCF3 (arachidonyl trifluoromethyl ketone). In contrast, PKC inhibitors GFX (GF109203X) or staurosporine prevented the activation of either proton channels or NADPH oxidase. PKC inhibition during the respiratory burst reversed the activation of both molecules, suggesting that ongoing phosphorylation is required. This effect of GFX was inhibited by okadaic acid, implicating phosphatases in proton channel deactivation. Proton channel activation by AA was partially reversed by GFX or staurosporine, indicating that AA effects are due in part to activation of PKC. In granulocytes from mice with the cPLA2α gene disrupted (knockout mice), PMA or fMetLeuPhe activated NADPH oxidase and proton channels in a manner indistinguishable from the responses of control cells. Thus, cPLA2α is not essential to activate the proton conductance or for a normal respiratory burst. Instead, phosphorylation of the proton channel or an activating molecule converts the channel to its activated gating mode. The existing paradigm for regulation of the concerted activity of proton channels and NADPH oxidase must be revised. PMID:17185330

  8. Lysyl oxidase-like 4 involvement in retinoic acid epithelial wound healing.

    PubMed

    Comptour, Aurélie; Rouzaire, Marion; Belville, Corinne; Bonnin, Nicolas; Daniel, Estelle; Chiambaretta, Frédéric; Blanchon, Loïc; Sapin, Vincent

    2016-01-01

    Vitamin A and its active forms (retinoic acids/RAs) are known to have pro-healing properties, but their mechanisms of action are still poorly understood. This work aimed to identify the cellular and molecular processes by which atRA (all-trans RA) improves wound healing, using an in vivo model of mouse corneal alkali burns and an in vitro cellular human corneal epithelial injury model. Regulation by atRA has been studied on most of the cellular events that occur in wound healing. We investigated the direct influence of atRA on a specific target gene known to be involved in the extracellular matrix (ECM) dynamics, one of the pathways contributing to epithelial repair. Our results demonstrate that atRA promotes corneal epithelial wound healing by acting preferentially on migration. The induction of lysyl oxidase-like 4 (LOXL4) expression by atRA in the corneal epithelium environment was established as essential in the mechanism of atRA-dependent wound healing. Our study describes for the first time a direct link between a retinoic-induced gene and protein, LOXL4, and its general clinical pro-healing properties in ECM dynamics. PMID:27597564

  9. Manageable cytotoxicity of nanocapsules immobilizing D-amino acid oxidase via exogenous administration of nontoxic prodrug

    NASA Astrophysics Data System (ADS)

    Zhao, Yang; Zhu, Yingchun; Fu, Jingke

    2014-02-01

    D-Amino acid oxidase (DAO), which could catalyze generation of hydrogen peroxide with strong oxidbility and cytotoxicity, has become of interest as a biocatalyst for therapeutic treatments. Herein we report that amino-functional hollow mesoporous silica with large pore size (10.27 nm) and positively charged surface effectively immobilize DAO with negative charge. The adsorption, activity and stability of DAO are demonstrated to depend mainly on the amino-functionalization of surface. Significant cancer cell killing effect is observed when the cells are treated by the nanocapsules entrapping DAO together with D-alanine, showing distinct dose-dependency on concentration of the nanocapsules entrapping DAO or D-alanine. Nevertheless, the toxicity is completely neutralized by the addition of catalase, and anti-tumor effect is not observed when either the nanocapsules entrapping DAO or D-alanine is applied alone. The results indicate that cytotoxicity of the nanocapsules entrapping DAO could be managed by exogenous administration of nontoxic prodrug to tumor tissue, due to the stereoselectivity of DAO and the scarcity of its substrates in mammalian organisms. Thus, the method might be exploited as a potential treatment for cancer therapy.

  10. Lysyl oxidase-like 4 involvement in retinoic acid epithelial wound healing

    PubMed Central

    Comptour, Aurélie; Rouzaire, Marion; Belville, Corinne; Bonnin, Nicolas; Daniel, Estelle; Chiambaretta, Frédéric; Blanchon, Loïc; Sapin, Vincent

    2016-01-01

    Vitamin A and its active forms (retinoic acids/RAs) are known to have pro-healing properties, but their mechanisms of action are still poorly understood. This work aimed to identify the cellular and molecular processes by which atRA (all-trans RA) improves wound healing, using an in vivo model of mouse corneal alkali burns and an in vitro cellular human corneal epithelial injury model. Regulation by atRA has been studied on most of the cellular events that occur in wound healing. We investigated the direct influence of atRA on a specific target gene known to be involved in the extracellular matrix (ECM) dynamics, one of the pathways contributing to epithelial repair. Our results demonstrate that atRA promotes corneal epithelial wound healing by acting preferentially on migration. The induction of lysyl oxidase-like 4 (LOXL4) expression by atRA in the corneal epithelium environment was established as essential in the mechanism of atRA-dependent wound healing. Our study describes for the first time a direct link between a retinoic-induced gene and protein, LOXL4, and its general clinical pro-healing properties in ECM dynamics. PMID:27597564

  11. Uric acid and xanthine oxidase in heart failure - Emerging data and therapeutic implications.

    PubMed

    Doehner, Wolfram; Jankowska, Ewa A; Springer, Jochen; Lainscak, Mitja; Anker, Stefan D

    2016-06-15

    The role of hyperuricaemia as cardiovascular risk factor has exhaustingly been debated for decades. While the association of elevated uric acid (UA) levels with increased mortality risk as convincingly been shown, the question whether UA is independently predictive of just a related effect within a more complex risk factor profile (including metabolic, inflammatory and haemodynamic risk factors) is still a matter of dispute. In heart failure the independent prognostic and functional impact of elevated UA has not only been shown but also the pathophysiologic mechanism(s) and the potential of targeted therapeutic interventions have been investigated in some detail. The emerging picture suggests the increased activity of the enzyme xanthine oxidase (XO) with corresponding increased production of free oxygen radical (ROS) as a main underlying principle with the resulting increase in UA levels being mostly a marker of this up-regulated pathway. While this concept will not diminish the value of UA as a prognostic marker, it provides the basis for a novel metabolic treatment option and the means to identify those patients most eligible for this tailored therapy. This review will summarize the recent evidence on XO as a novel and promising therapeutic target in heart failure. PMID:26318388

  12. Inhibition of polyphenol oxidases activity by various dipeptides.

    PubMed

    Girelli, Anna M; Mattei, Enrico; Messina, Antonella; Tarola, Anna M

    2004-05-19

    In an effort to develop natural and nontoxic inhibitors on the activity of mushroom polyphenol oxidase (PPO) the effect of various glycyl-dipeptides (GlyAsp, GlyGly, GlyHis, GlyLeu, GlyLys, GlyPhe, GlyPro, GlyTyr) was investigated. The inhibition study with dihydroxyphenylalanine (DOPA) as substrate is based on separation of the enzymatic reaction components by reversed phase HPLC and the UV detection of the dopachrome formed. The results have evidenced that several of tested dipeptides inhibited PPO activity in the range of 20-40% while GlyPro and GlyLeu had no effect. The study has also permitted the characterization of the following kinetic pattern: a linear-mixed-type mechanism for GlyAsp, GlyGly, GlyLys, and GlyPhe and a hyperbolic-mixed-type for GlyTyr. It was not possible to identify the inhibition mechanism for GlyHis, although it affects PPO activity. In addition the effects of GlyAsp, GlyLys and GlyHis were evaluated for lessening the browning of fresh Golden Delicious apple and Irish White Skinned potato. The effectiveness of such inhibitors was determined by the difference between the colors observed in the dipeptide-treated sample and the controls using the color space CIE-Lab system. The % browning inhibition on potato (20-50%) was greater than of apple (20-30%) by the all tested dipeptides. Only GlyLys presented the significant value of 50%. PMID:15137808

  13. Predicting Monoamine Oxidase Inhibitory Activity through Ligand-Based Models

    PubMed Central

    Vilar, Santiago; Ferino, Giulio; Quezada, Elias; Santana, Lourdes; Friedman, Carol

    2013-01-01

    The evolution of bio- and cheminformatics associated with the development of specialized software and increasing computer power has produced a great interest in theoretical in silico methods applied in drug rational design. These techniques apply the concept that “similar molecules have similar biological properties” that has been exploited in Medicinal Chemistry for years to design new molecules with desirable pharmacological profiles. Ligand-based methods are not dependent on receptor structural data and take into account two and three-dimensional molecular properties to assess similarity of new compounds in regards to the set of molecules with the biological property under study. Depending on the complexity of the calculation, there are different types of ligand-based methods, such as QSAR (Quantitative Structure-Activity Relationship) with 2D and 3D descriptors, CoMFA (Comparative Molecular Field Analysis) or pharmacophoric approaches. This work provides a description of a series of ligand-based models applied in the prediction of the inhibitory activity of monoamine oxidase (MAO) enzymes. The controlled regulation of the enzymes’ function through the use of MAO inhibitors is used as a treatment in many psychiatric and neurological disorders, such as depression, anxiety, Alzheimer’s and Parkinson’s disease. For this reason, multiple scaffolds, such as substituted coumarins, indolylmethylamine or pyridazine derivatives were synthesized and assayed toward MAO-A and MAO-B inhibition. Our intention is to focus on the description of ligand-based models to provide new insights in the relationship between the MAO inhibitory activity and the molecular structure of the different inhibitors, and further study enzyme selectivity and possible mechanisms of action. PMID:23231398

  14. Molecular Basis of Reduced Pyridoxine 5′-Phosphate Oxidase Catalytic Activity in Neonatal Epileptic Encephalopathy Disorder*

    PubMed Central

    Musayev, Faik N.; Di Salvo, Martino L.; Saavedra, Mario A.; Contestabile, Roberto; Ghatge, Mohini S.; Haynes, Alexina; Schirch, Verne; Safo, Martin K.

    2009-01-01

    Mutations in pyridoxine 5′-phosphate oxidase are known to cause neonatal epileptic encephalopathy. This disorder has no cure or effective treatment and is often fatal. Pyridoxine 5′-phosphate oxidase catalyzes the oxidation of pyridoxine 5′-phosphate to pyridoxal 5′-phosphate, the active cofactor form of vitamin B6 required by more than 140 different catalytic activities, including enzymes involved in amino acid metabolism and biosynthesis of neurotransmitters. Our aim is to elucidate the mechanism by which a homozygous missense mutation (R229W) in the oxidase, linked to neonatal epileptic encephalopathy, leads to reduced oxidase activity. The R229W variant is ∼850-fold less efficient than the wild-type enzyme due to an ∼192-fold decrease in pyridoxine 5′-phosphate affinity and an ∼4.5-fold decrease in catalytic activity. There is also an ∼50-fold reduction in the affinity of the R229W variant for the FMN cofactor. A 2.5 Å crystal structure of the R229W variant shows that the substitution of Arg-229 at the FMN binding site has led to a loss of hydrogen-bond and/or salt-bridge interactions between FMN and Arg-229 and Ser-175. Additionally, the mutation has led to an alteration of the configuration of a β-strand-loop-β-strand structure at the active site, resulting in loss of two critical hydrogen-bond interactions involving residues His-227 and Arg-225, which are important for substrate binding and orientation for catalysis. These results provide a molecular basis for the phenotype associated with the R229W mutation, as well as providing a foundation for understanding the pathophysiological consequences of pyridoxine 5′-phosphate oxidase mutations. PMID:19759001

  15. Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity1[C][W

    PubMed Central

    Kim, Dong Wook; Watanabe, Kanako; Murayama, Chihiro; Izawa, Sho; Niitsu, Masaru; Michael, Anthony J.; Berberich, Thomas; Kusano, Tomonobu

    2014-01-01

    The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development. PMID:24906355

  16. Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern 1

    PubMed Central

    Chatfield, J. Mark; Armstrong, Donald J.

    1986-01-01

    The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N6-(Δ2-isopentenyl)adenine-8-14C (i6 Ade-8-14C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i6 Ade-8-14C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded. PMID:16664650

  17. Inhibition of guinea pig aldehyde oxidase activity by different flavonoid compounds: An in vitro study.

    PubMed

    Siah, Maryam; Farzaei, Mohammad Hosein; Ashrafi-Kooshk, Mohammad Reza; Adibi, Hadi; Arab, Seyed Shahriar; Rashidi, Mohammad Reza; Khodarahmi, Reza

    2016-02-01

    Aldehyde oxidase (AO), a cytosolic molybdenum-containing hydroxylase, is predominantly active in liver and other tissues of mammalian species and involved in the metabolism of extensive range of aldehydes and nitrogen-containing compounds. A wide range of natural components including polyphenols are able to interfere with AO-catalyzed reactions. Polyphenols and flavonoids are one of the extensive secondary plant metabolites ubiquitously present in plants considered an important part of the human diet. The aim of the present study was to investigate inhibitory effect of selected phenolic compounds from three subclasses of aurone, flavanone and phenolic lactone compounds on the activity of AO, spectrophotometrically. AO enzyme was partially purified from liver of guinea pig. Then, inhibitory effects of 10 flavonoid compounds including 8 derivatives of 2-benzylidenebenzofuran-3(2H)-ones, as well as naringenin and ellagic acid on the activity of aldehyde oxidase were assessed compared with the specific inhibitor of AO, menadione. Among the phenolic compounds with inhibitory effects on the enzyme, ellagic acid (IC50=14.47 μM) was the most potent agent with higher inhibitory action than menadione (IC50=31.84 μM). The mechanisms by which flavonoid compounds inhibit AO activity have been also determined. The inhibitory process of the assessed compounds occurs via either a non-competitive or mixed mode. Although flavonoid compounds extensively present in the nature, mainly in dietary regimen, aurones with promising biological properties are not widely distributed in nature, so synthesis of aurone derivatives is of great importance. Additionally, aurones seem to provide a promising scaffold in medicinal chemistry for the skeleton of new developing drugs, so the results of the current study can be valuable in order to better understanding drug-food as well as drug-drug interaction and also appears to be worthwhile in drug development strategies. PMID:26722818

  18. Antioxidant, α-glucosidase and xanthine oxidase inhibitory activity of bioactive compounds from maize (Zea mays L.).

    PubMed

    Nile, Shivraj H; Park, Se W

    2014-01-01

    Chemical investigations into maize (Zea mays L.) kernels yielded phenolic compounds, which were structurally established using chromatographic and spectroscopic methods. The isolated phenolic compounds from maize kernel were examined in vitro for their antioxidant abilities by DPPH (2,2-diphenyl-1-picryl hydrazine) radical, OH radical scavenging activity, and reducing ability, along with α-glucosidase and xanthine oxidase (XO) inhibition. The isolated maize phenolics revealed significant xanthine oxidase and α-glucosidase inhibitory activity to that of allopurinol and acarbose in vitro and in vivo, respectively. The kinetics study with xanthine oxidase revealed competitive type of inhibition by isolated maize vanillic acid (M2), ferulic acid (M5), 3'-methoxyhirsutrin (M7), and peonidin-3-glucoside (M10) as compared to control allopurinol. Overall, with few exceptions, all the phenolic compounds from maize kernel revealed significant biological activities with all parameters examined. Also, the phenolic compounds from maize were found to be more reactive toward DPPH radical and had considerable reducing ability and OH radical scavenging activity. These findings suggest that maize kernel phenolic compounds can be considered as potential antioxidant, α-glucosidase, and XO inhibitory agents those might be further explored for the design of lead antioxidant, antidiabetic and antigout drug candidates using in vivo trials. PMID:23957301

  19. Studies on Polyphenol Content, Activities and Isozymes of Polyphenol Oxidase and Peroxidase During Air-Curing in Three Tobacco Types 1

    PubMed Central

    Sheen, S. J.; Calvert, J.

    1969-01-01

    The change in polyphenol content in the primed leaves of burley, flue-cured, and Turkish tobaccos during air-curing was related to the activities and isozymes of polyphenol oxidase and peroxidase. The quantity of chlorogenic acid was rapidly reduced during the first week of curing. The decrease in rutin content during curing was less significant, especially when the concentration of chlorogenic acid was high in leaf tissues. This result was further confirmed by in vitro assays with partially purified tobacco polyphenol oxidase. The polyphenol oxidase activity did not differ at any stage of curing in the 3 tobaccos. When the activity was measured by the oxidation of 3,4-dihydroxyphenylalanine it rose rapidly during the first day of curing and then decreased sharply so that in the fully cured leaf only 15% activity remained. The increase in activity was not observed when chlorogenic acid was used as the substrate. A similar level of peroxidase activity was found in the 3 tobaccos before curing. Peroxidase activities increased rapidly during the first 24 hr of curing, declined thereafter, and remained highest in the flue-cured tobacco, less in the Turkish line, and least in the burley at the end of curing process. By polyacrylamide gel block electrophoresis, 10 peroxidase isozyme bands, 2 cationic and 8 anionic, appeared identical in all 3 tobaccos. When catechol replaced benzidine-2 HCl as the electron donor, 1 cationic and 2 anionic peroxidase isozymes did not form. Of interest is that the same 10 peroxidase isozyme bands also exhibited polyphenol oxidase activities when treated with 3,4-dihydroxyphenylalanine or chlorogenic acid. Results suggest that in the crude tobacco leaf extract the peroxidase and polyphenol oxidase may associate as protein complexes, and peroxidase isozymes may differ in electron-donor requirements. Isozyme patterns for both oxidases at various curing intervals differed only quantitatively. Images PMID:16657046

  20. Thermal stability of ascorbic acid and ascorbic acid oxidase in african cowpea leaves ( Vigna unguiculata ) of different maturities.

    PubMed

    Wawire, Michael; Oey, Indrawati; Mathooko, Francis; Njoroge, Charles; Shitanda, Douglas; Hendrickx, Marc

    2011-03-01

    Cowpea, an African leafy vegetable ( Vigna unguiculata ), contains a high level of vitamin C. The leaves harvested at 4-9 weeks are highly prone to vitamin C losses during handling and processing. Therefore, the purpose of this research was to study the effect of thermal treatment on the stability of ascorbic acid oxidase (AAO), total vitamin C content (l-ascorbic acid, l-AA), and dehydroascorbic acid (DHAA) and l-AA/DHAA ratio in cowpea leaves harvested at different maturities (4, 6, and 8 weeks old). The results showed that AAO activity, total vitamin C content, and l-AA/DHAA ratio in cowpea leaves increased with increasing maturity (up to 8 weeks). Eight-week-old leaves were the best source of total vitamin C and showed a high ratio of l-AA/DHAA (4:1). Thermal inactivation of AAO followed first-order reaction kinetics. Heating at temperatures above 90 °C for short times resulted in a complete AAO inactivation, resulting in a protective effect of l-AA toward enzyme-catalyzed oxidation. Total vitamin C in young leaves (harvested at 4 and 6 weeks) was predominantly in the form of DHAA, and therefore temperature treatment at 30-90 °C for 10 min decreased the total vitamin C content, whereas total vitamin C in 8-week-old cowpea leaves was more than 80% in the form of l-AA, so that a high retention of the total vitamin C can be obtained even after heating and/or reheating (30-90 °C for 10 min) before consumption. The results indicated that the stability of total vitamin C in situ was strongly dependent on the plant maturity stage and the processing conditions applied. PMID:21309563

  1. Synthesis and theoretical calculations of carbazole substituted chalcone urea derivatives and studies their polyphenol oxidase enzyme activity.

    PubMed

    Nixha, Arleta Rifati; Arslan, Mustafa; Atalay, Yusuf; Gençer, Nahit; Ergün, Adem; Arslan, Oktay

    2013-08-01

    Synthesis of carbazole substituted chalcone urea derivatives and their polyphenol oxidase enzyme activity effects on the diphenolase activity of banana tyrosinase were evaluated. Tyrosinase has been purified from banana on an affinity gel comprised of Sepharose 4B-L-tyrosine-p-aminobenzoic acid. The results showed that most of the compounds (3,4,5a,5d-h) inhibited and some of them (5c,5i-l) activated the tyrosinase enzyme activity. The molecular calculations were performed using Gaussian software for the synthesized compounds to explain the experimental results. PMID:22803668

  2. Mouse aldehyde-oxidase-4 controls diurnal rhythms, fat deposition and locomotor activity

    PubMed Central

    Terao, Mineko; Barzago, Maria Monica; Kurosaki, Mami; Fratelli, Maddalena; Bolis, Marco; Borsotti, Andrea; Bigini, Paolo; Micotti, Edoardo; Carli, Mirjana; Invernizzi, Roberto William; Bagnati, Renzo; Passoni, Alice; Pastorelli, Roberta; Brunelli, Laura; Toschi, Ivan; Cesari, Valentina; Sanoh, Seigo; Garattini, Enrico

    2016-01-01

    Aldehyde-oxidase-4 (AOX4) is one of the mouse aldehyde oxidase isoenzymes and its physiological function is unknown. The major source of AOX4 is the Harderian-gland, where the enzyme is characterized by daily rhythmic fluctuations. Deletion of the Aox4 gene causes perturbations in the expression of the circadian-rhythms gene pathway, as indicated by transcriptomic analysis. AOX4 inactivation alters the diurnal oscillations in the expression of master clock-genes. Similar effects are observed in other organs devoid of AOX4, such as white adipose tissue, liver and hypothalamus indicating a systemic action. While perturbations of clock-genes is sex-independent in the Harderian-gland and hypothalamus, sex influences this trait in liver and white-adipose-tissue which are characterized by the presence of AOX isoforms other than AOX4. In knock-out animals, perturbations in clock-gene expression are accompanied by reduced locomotor activity, resistance to diet induced obesity and to hepatic steatosis. All these effects are observed in female and male animals. Resistance to obesity is due to diminished fat accumulation resulting from increased energy dissipation, as white-adipocytes undergo trans-differentiation towards brown-adipocytes. Metabolomics and enzymatic data indicate that 5-hydroxyindolacetic acid and tryptophan are novel endogenous AOX4 substrates, potentially involved in AOX4 systemic actions. PMID:27456060

  3. Mouse aldehyde-oxidase-4 controls diurnal rhythms, fat deposition and locomotor activity.

    PubMed

    Terao, Mineko; Barzago, Maria Monica; Kurosaki, Mami; Fratelli, Maddalena; Bolis, Marco; Borsotti, Andrea; Bigini, Paolo; Micotti, Edoardo; Carli, Mirjana; Invernizzi, Roberto William; Bagnati, Renzo; Passoni, Alice; Pastorelli, Roberta; Brunelli, Laura; Toschi, Ivan; Cesari, Valentina; Sanoh, Seigo; Garattini, Enrico

    2016-01-01

    Aldehyde-oxidase-4 (AOX4) is one of the mouse aldehyde oxidase isoenzymes and its physiological function is unknown. The major source of AOX4 is the Harderian-gland, where the enzyme is characterized by daily rhythmic fluctuations. Deletion of the Aox4 gene causes perturbations in the expression of the circadian-rhythms gene pathway, as indicated by transcriptomic analysis. AOX4 inactivation alters the diurnal oscillations in the expression of master clock-genes. Similar effects are observed in other organs devoid of AOX4, such as white adipose tissue, liver and hypothalamus indicating a systemic action. While perturbations of clock-genes is sex-independent in the Harderian-gland and hypothalamus, sex influences this trait in liver and white-adipose-tissue which are characterized by the presence of AOX isoforms other than AOX4. In knock-out animals, perturbations in clock-gene expression are accompanied by reduced locomotor activity, resistance to diet induced obesity and to hepatic steatosis. All these effects are observed in female and male animals. Resistance to obesity is due to diminished fat accumulation resulting from increased energy dissipation, as white-adipocytes undergo trans-differentiation towards brown-adipocytes. Metabolomics and enzymatic data indicate that 5-hydroxyindolacetic acid and tryptophan are novel endogenous AOX4 substrates, potentially involved in AOX4 systemic actions. PMID:27456060

  4. Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme d-amino acid oxidase. The catalytic and structural roles of Gly313 and Thr317.

    PubMed

    Setoyama, Chiaki; Nishina, Yasuzo; Tamaoki, Haruhiko; Mizutani, Hisashi; Miyahara, Ikuko; Hirotsu, Ken; Shiga, Kiyoshi; Miura, Retsu

    2002-01-01

    According to the three-dimensional structure of a porcine kidney D-amino acid oxidase-substrate (D-leucine) complex model, the G313 backbone carbonyl recognizes the substrate amino group by hydrogen bonding and the side-chain hydroxyl of T317 forms a hydrogen bond with C(2)=O of the flavin moiety of FAD [Miura et al. (1997) J. Biochem. 122, 825-833]. We have designed and expressed the G313A and T317A mutants and compared their enzymatic and spectroscopic properties with those of the wild type. The G313A mutant shows decreased activities to various D-amino acids, but the pattern of substrate specificity is different from that of the wild type. The results imply that the hydrogen bond between the G313 backbone carbonyl and the substrate amino group plays important roles in substrate recognition and in defining the substrate specificity of D-amino acid oxidase. The T317A mutant shows a decreased affinity for FAD. The steady-state kinetic measurements indicate diminished activities of T317A to substrate D-amino acids. The transient kinetic parameters measured by stopped-flow spectroscopy revealed that T317 plays key roles in stabilizing the purple intermediate, a requisite intermediate in the oxidative half-reaction, and in enhancing the release of the product from the active site, thereby optimizing the overall catalytic process of D-amino acid oxidase. PMID:11754736

  5. Manganese(IV) Oxide Production by Acremonium sp. Strain KR21-2 and Extracellular Mn(II) Oxidase Activity

    PubMed Central

    Miyata, Naoyuki; Tani, Yukinori; Maruo, Kanako; Tsuno, Hiroshi; Sakata, Masahiro; Iwahori, Keisuke

    2006-01-01

    Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; Km, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes. PMID:17021194

  6. Regional brain cytochrome oxidase activity in beta-amyloid precursor protein transgenic mice with the Swedish mutation.

    PubMed

    Strazielle, C; Sturchler-Pierrat, C; Staufenbiel, M; Lalonde, R

    2003-01-01

    Cytochrome oxidase activity was examined in a transgenic mouse model of Alzheimer's disease with overexpression of the 751 amino acid isoform of beta-amyloid precursor protein with the Swedish mutation under control of the murine thy-1 promoter. The neuritic plaques, abundantly localized in the hippocampus and anterior neocortical areas, showed a core devoid of enzymatic activity surrounded by higher cytochrome oxidase activity at the sites of the dystrophic neurites and activated glial cells. Quantitative measures, taken only in the healthy-appearing regional areas without neuritic plaques, were higher in numerous limbic and non-limbic regions of transgenic mice in comparison with controls. Enzymatic activity was higher in the dentate gyrus and CA2-CA3 region of the hippocampus, the anterior cingulate and primary visual cortex, two olfactory structures, the ventral part of the neostriatum, the parafascicularis nucleus of the thalamus, and the subthalamic nucleus. Brainstem regions anatomically related with altered forebrain regions were more heavily labeled as well, including the substantia nigra, the periaqueductal gray, the superior colliculus, the medial raphe, the locus coeruleus and the adjacent parabrachial nucleus, as well as the pontine nuclei, red nucleus, and trigeminal motor nucleus. Functional brain organization is discussed in the context of Alzheimer's disease. Although hypometabolism is generally observed in this pathology, the increased cytochrome oxidase activity obtained in these transgenic mice can be the result of a functional compensation on the surviving neurons, or of an early mitochondrial alteration related to increased oxidative damage. PMID:12732258

  7. Cholesterol oxidase with high catalytic activity from Pseudomonas aeruginosa: Screening, molecular genetic analysis, expression and characterization.

    PubMed

    Doukyu, Noriyuki; Nihei, Shyou

    2015-07-01

    An extracellular cholesterol oxidase producer, Pseudomonas aeruginosa strain PA157, was isolated by a screening method to detect 6β-hydroperoxycholest-4-en-3-one-forming cholesterol oxidase. On the basis of a putative cholesterol oxidase gene sequence in the genome sequence data of P. aeruginosa strain PAO1, the cholesterol oxidase gene from strain PA157 was cloned. The mature form of the enzyme was overexpressed in Escherichia coli cells. The overexpressed enzyme formed inclusion bodies in recombinant E. coli cells grown at 20 °C and 30 °C. A soluble and active PA157 enzyme was obtained when the recombinant cells were grown at 10 °C. The purified enzyme was stable at pH 5.5 to 10 and was most active at pH 7.5-8.0, showing optimal activity at pH 7.0 and 70 °C. The enzyme retained about 90% of its activity after incubation for 30 min at 70 °C. The enzyme oxidized 3β-hydroxysteroids such as cholesterol, β-cholestanol, and β-sitosterol at high rates. The Km value and Vmax value for the cholesterol were 92.6 μM and 15.9 μmol/min/mg of protein, respectively. The Vmax value of the enzyme was higher than those of commercially available cholesterol oxidases. This is the first report to characterize a cholesterol oxidase from P. aeruginosa. PMID:25573142

  8. Bacterial d-amino acid oxidases: Recent findings and future perspectives

    PubMed Central

    Takahashi, Shouji; Abe, Katsumasa; Kera, Yoshio

    2015-01-01

    D-Amino acid oxidase (DAO) is a flavin enzyme that catalyzes the oxidative deamination of d-amino acids. This enzyme has been studied extensively both biochemically and structurally as a model for the oxidase-dehydrogenase class of flavoproteins. This enzyme also has various applications, such as the determination of d-amino acids and production of building blocks for a number of pharmaceuticals. DAO has been found mainly in eukaryotic organisms and has been suggested to play a significant role in various cellular processes, one of which includes neurotransmission in the human brain. In contrast, this enzyme has not been identified in prokaryotic organisms. Some studies have recently identified and characterized DAO enzyme in some actinobacteria. In addition, a genome database search reveals a wide distribution of DAO homologous genes in this bacterial group. The bacterial DAOs characterized so far have certain distinct properties in comparison to eukaryotic DAOs. These enzymes also exhibit some important applicable properties, suggesting that bacteria could be used as a source for obtaining novel and useful DAOs. The physiological function of bacterial DAO have been proposed to include the degradation of non-canonical d-amino acids released from cell wall, but is still largely unknown and need to be studied in depth. PMID:25996186

  9. Expression, purification, and immobilization of His-tagged D-amino acid oxidase of Trigonopsis variabilis in Pichia pastoris.

    PubMed

    Zheng, Huabao; Wang, Xiaolan; Chen, Jun; Zhu, Ke; Zhao, Yuhua; Yang, Yunliu; Yang, Sheng; Jiang, Weihong

    2006-05-01

    High-level expression of D: -amino acid oxidase (DAO) has been reported in Pichia pastoris by integrating the DAO gene under the control of the alcohol oxidase promoter (PAOX1). However, the time taken to reach peak product concentration is usually long (approximately 43 h), and cultivation requires tight regulation of methanol feeding. In this paper, we describe the expression of His-tagged DAO (HDAO) in P. pastoris using the glyceraldehydes-3-phosphate dehydrogenase promoter (PGAP). The maximal level of HDAO expression using the PGAP integrant is attained in 13 h and is equal to that obtained using the PAOX1 integrant in 43 h. We also explored the possibility of secreting HDAO in P. pastoris. In-frame fusion of Saccharomyces cerevisiae alpha-factor secretion signal under a PGAP or PAOX1 resulted in low-level secretion of active HDAO, which was not of practical use. The intracellularly expressed HDAO under PGAP was purified by agar-based affinity support and then immobilized on Amberzyme oxirane resin. The immobilized HDAO, with specific activity of 75 U g-1 (wet weight), could be recycled more than 14 times without significant loss of activity. The data suggest that intracellular production of HDAO under PGAP, followed by affinity purification and immobilization on oxirane resin, may serve as an effective process for the manufacture of immobilized DAO for industrial application. PMID:16217653

  10. Methionine sulfoxide reductase: chemistry, substrate binding, recycling process and oxidase activity.

    PubMed

    Boschi-Muller, Sandrine; Branlant, Guy

    2014-12-01

    Three classes of methionine sulfoxide reductases are known: MsrA and MsrB which are implicated stereo-selectively in the repair of protein oxidized on their methionine residues; and fRMsr, discovered more recently, which binds and reduces selectively free L-Met-R-O. It is now well established that the chemical mechanism of the reductase step passes through formation of a sulfenic acid intermediate. The oxidized catalytic cysteine can then be recycled by either Trx when a recycling cysteine is operative or a reductant like glutathione in the absence of recycling cysteine which is the case for 30% of the MsrBs. Recently, it was shown that a subclass of MsrAs with two recycling cysteines displays an oxidase activity. This reverse activity needs the accumulation of the sulfenic acid intermediate. The present review focuses on recent insights into the catalytic mechanism of action of the Msrs based on kinetic studies, theoretical chemistry investigations and new structural data. Major attention is placed on how the sulfenic acid intermediate can be formed and the oxidized catalytic cysteine returns back to its reduced form. PMID:25108804

  11. Inhibition of chrysin on xanthine oxidase activity and its inhibition mechanism.

    PubMed

    Lin, Suyun; Zhang, Guowen; Liao, Yijing; Pan, Junhui

    2015-11-01

    Chrysin, a bioactive flavonoid, was investigated for its potential to inhibit the activity of xanthine oxidase (XO), a key enzyme catalyzing xanthine to uric acid and finally causing gout. The kinetic analysis showed that chrysin possessed a strong inhibition on XO ability in a reversible competitive manner with IC50 value of (1.26±0.04)×10(-6)molL(-1). The results of fluorescence titrations indicated that chrysin bound to XO with high affinity, and the interaction was predominately driven by hydrogen bonds and van der Waals forces. Analysis of circular dichroism demonstrated that chrysin induced the conformational change of XO with increases in α-helix and β-sheet and reductions in β-turn and random coil structures. Molecular simulation revealed that chrysin interacted with the amino acid residues Leu648, Phe649, Glu802, Leu873, Ser876, Glu879, Arg880, Phe1009, Thr1010, Val1011 and Phe1013 located within the active cavity of XO. The mechanism of chrysin on XO activity may be the insertion of chrysin into the active site occupying the catalytic center of XO to avoid the entrance of xanthine and causing conformational changes in XO. Furthermore, the interaction assays indicated that chrysin and its structural analog apigenin exhibited an additive effect on inhibition of XO. PMID:26275460

  12. Hemin/G-quadruplexes as DNAzymes for the fluorescent detection of DNA, aptamer-thrombin complexes, and probing the activity of glucose oxidase.

    PubMed

    Golub, Eyal; Freeman, Ronit; Niazov, Angelica; Willner, Itamar

    2011-11-01

    Hemin/G-quadruplex catalyzes the H(2)O(2)-mediated oxidation of Amplex Red to the fluorescent product resorufin. This process is implemented to develop hairpin nucleic acid structures for the detection of DNA, to probe the catalytic activity of glucose oxidase, to use the thrombin-aptamer complex as a catalytic readout structure, and to quantitatively analyze telomere chain composition. PMID:21881641

  13. Role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in Barrett's cells and Barrett's esophageal adenocarcinoma cells

    PubMed Central

    Li, Dan

    2014-01-01

    Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca2+ in FLO cells, an increase that is blocked by Ca2+-free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca2+ increase or knockdown of NOX5-S with NOX5 small-interfering RNA (siRNA) in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in BE acid reflux increases intracellular calcium, activates NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. PMID:24699332

  14. [The Xanthine Oxidase Inhibitory Activity and Hypouricemic Effects of Crude Drugs Obtained from the Silkworm in Mice].

    PubMed

    Tanaka, Ryuichirou; Miyata, Yuuma; Minakuchi, Naoki; Murakami, Ayako; Sakazaki, Fumitoshi

    2015-01-01

    This study evaluated the effects of crude drugs obtained from the silkworm in mice with oxonic acid-induced hyperuricemia using xanthine oxidase inhibitory activity and plasma uric acid levels. The plasma uric acid level was analyzed using an improved HPLC with UV detection (HPLC-UV) method, which enabled high-sensitivity analysis of a microliter of plasma. Using this method, we evaluated natural products administered orally to the hypouricemic mice. The plasma uric acid level of mice administered a water-soluble extract from silkworm larvae with botrytis (used in traditional Chinese medicine to reduce wind, lower blood pressure, and change platelet coagulation) was significantly lower than in the control group 1, 2, and 3 h after treatment. In addition, water soluble extracts from a fungus (NBRC 31161) metabolite and silkworm pupae and larvae reduced the plasma uric acid levels in mice compared with the control group. PMID:26423873

  15. Microwave assisted synthesis of naphthopyrans catalysed by silica supported fluoroboric acid as a new class of non purine xanthine oxidase inhibitors.

    PubMed

    Sharma, Sahil; Sharma, Kirti; Ojha, Ritu; Kumar, Dinesh; Singh, Gagandip; Nepali, Kunal; Bedi, P M S

    2014-01-15

    A series of naphthopyrans was synthesized employing silica supported fluoroboric acid under solvent free conditions in a microwave reactor. The catalytic influence of HBF4-SiO2 was investigated in detail to optimize the reaction conditions. The synthesised compounds were evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Structure-activity relationship analyses have also been presented. Among the synthesised compounds, NP-17, NP-19, NP-20, NP-23, NP-24, NP-25 and NP-26 were the active inhibitors with an IC50 ranging from 4 to 17 μM. Compound NP-19 with a thiophenyl ring at position 1 emerged as the most potent xanthine oxidase inhibitor (IC50=4 μM) in comparison to allopurinol (IC50=11.10 μM) and febuxostat (IC50=0.025 μM). The basis of significant inhibition of xanthine oxidase by NP-19 was rationalized by its molecular docking at MTE binding site of xanthine oxidase. PMID:24388807

  16. A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

    PubMed

    Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

    2015-11-10

    In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. PMID:26257292

  17. Involvement of NADPH oxidases in suppression of cyclooxygenase-2 promoter-dependent transcriptional activities by sesamol

    PubMed Central

    Shimizu, Satomi; Ishigamori, Rikako; Fujii, Gen; Takahashi, Mami; Onuma, Wakana; Terasaki, Masaru; Yano, Tomohiro; Mutoh, Michihiro

    2015-01-01

    Cyclooxygenase-2 (COX-2) has been shown to play an important role in colon carcinogenesis. Moreover, one of the components of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NADPH oxidase 1 (NOX1), dominantly expressed in the colon, is implicated in the pathogenesis of colon cancer. We have reported that sesamol, one of the lignans in sesame seeds, suppressed COX-2 gene transcriptional activity in human colon cancer cells, and also suppressed intestinal polyp formation in Apc-mutant mice. In the present study, we investigated the involvement of NADPH oxidase in the inhibition of COX-2 transcriptional activity by sesamol. We found that several NADPH oxidase inhibitors, such as apocynin, showed suppressive effects on COX-2 transcriptional activity. Moreover, sesamol significantly suppressed NOX1 mRNA levels in a dose-dependent manner. In addition, we demonstrated that knockdown of NOX1 successfully suppressed COX-2 transcriptional activity. These results suggest that inhibition of NADPH oxidase, especially NOX1, may be involved in the mechanism of the suppression of COX-2 transcriptional activity by sesamol. PMID:25759517

  18. Antibacterial efficacy of recombinant Siganus oramin L-amino acid oxidase expressed in Pichia pastoris.

    PubMed

    Li, Ruijun; Li, Anxing

    2014-12-01

    Siganus oraminl-amino acid oxidase is a novel natural protein (named SR-LAAO) isolated from serum of the rabbitfish (S. oramin), which showed antibacterial activity against both Gram-positive and Gram-negative bacteria and had a lethal effect on the parasites Cryptocaryon irritans, Trypanosoma brucei brucei and Ichthyophthirius multifiliis. In order to test whether recombinant SR-LAAO (rSR-LAAO) produced by the eukaryotic expression system also has antimicrobial activity, the yeast Pichia pastoris was used as the expression host to obtain rSR-LAAO in vitro. Crude rSR-LAAO produced by P. pastoris integrated with the SR-LAAO gene had antibacterial activity against both Gram-positive and Gram-negative bacteria as shown by inhibition zone assay of the antibacterial spectrum on agar plates. The average diameter of the inhibition zone of crude rSR-LAAO against the Gram-positive bacteria Staphylococcus aureus and Streptococcus agalactiae was 1.040 ± 0.045 cm and 1.209 ± 0.085 cm, respectively. For the Gram-negative bacteria Aeromonas sobria, Escherichia coli, Vibrio alginolyticus, Vibrio cholera and Photobacterium damselae subsp. piscicida, the average diameter of inhibition zone was 1.291 ± 0.089 cm, 0.943 ± 0.061 cm, 0.756 ± 0.057 cm, 0.834 ± 0.023 cm and 1.211 ± 0.026 cm, respectively. These results were obtained at the logarithmic growth phase of S. agalactiae and A. sobria cell suspensions after incubation with 0.5 mg/mL crude rSR-LAAO for 24 h. The final bacterial growth rate was decreased significantly. The relative inhibition rate can reach 50% compared to crude products from P. pastoris integrated with an empty vector at the same concentration of protein. The antimicrobial activity of crude rSR-LAAO was likely associated with H2O2 formation, because its inhibition zones were disturbed significantly by catalase. Scanning electron microscopy results showed crude rSR-LAAO-treated bacterial surfaces became rough and particles were attached, cell walls were

  19. Potato and mushroom polyphenol oxidase activities are differently modulated by natural plant extracts.

    PubMed

    Kuijpers, Tomas F M; van Herk, Teunie; Vincken, Jean-Paul; Janssen, Renske H; Narh, Deborah L; van Berkel, Willem J H; Gruppen, Harry

    2014-01-01

    Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the outcome of such screening, this study compared the effect of 60 plant extracts on the activity of PPO from mushroom ( Agaricus bisporus , AbPPO) and PPO from potato ( Solanum tuberosum , StPPO). Some plant extracts had different effects on the two PPOs: an extract that inhibited one PPO could be an activator for the other. As an example of this, the mate ( Ilex paraguariensis ) extract was investigated in more detail. In the presence of mate extract, oxygen consumption by AbPPO was found to be reduced >5-fold compared to a control reaction, whereas that of StPPO was increased >9-fold. RP-UHPLC-MS analysis showed that the mate extract contained a mixture of phenolic compounds and saponins. Upon incubation of mate extract with StPPO, phenolic compounds disappeared completely and saponins remained. Flash chromatography was used to separate saponins and phenolic compounds. It was found that the phenolic fraction was mainly responsible for inhibition of AbPPO and activation of StPPO. Activation of StPPO was probably caused by activation of latent StPPO by chlorogenic acid quinones. PMID:24344979

  20. Developmental onset of mixed-function oxidase activity in preimplantation mouse embryos

    SciTech Connect

    Filler, R.; Lew, K.J.

    1981-11-01

    Two-cell embryos, obtained from the C57BL/6N and DBA/2N strains, were cultured in media that supported in vitro differentiation and that contained (/sup 3/H)benzo(a)pyrene. High-pressure liquid chromatography of the activated intermediates formed during in vitro early embryonic development indicated that the onset of polynuclear aromatic hydrocarbon activation coincided with blastocyst formation. Comparison of individual oxygenated intermediates metabolically formed from embryos genetically ''responsive'' or ''nonresponsive'' to aromatic hydrocarbons revealed significant quantitative differences in the production of dihydrodiol, quinone, and phenolic derivatives. In addition to exhibiting basal mixed-function oxidase activity, blastocysts were also responsive to enzymatic induction when exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. The presence of operative metabolite-detoxifying pathways was also assayed. Enzymatic treatment of water-soluble metabolites with ..beta..-glucuronidase or arylsulfatase revealed that neither glucuronic acid conjugates nor sulfate ester derivatives were present. These data, therefore, provide direct evidence that late preimplanation mouse embryos (day 3 1/2 of gestation) are similar to later developmental stages in having the enzymatic capability for xenobiotic activation and enzyme induction but are dissimilar with respect to their detoxification mechanisms(s). Moreover, the ability of preimplantation embryos to activate directly polynuclear aromatic hydrocarbon to bioreactive intermediates may be of importance in assessing the ontological susceptibility of the developing embryo to carcinogenic or teratogenic chemicals.

  1. 1-Aminocyclopropane-1-Carboxylate Oxidase Activity Limits Ethylene Biosynthesis in Rumex palustris during Submergence

    PubMed Central

    Vriezen, Wim H.; Hulzink, Raymond; Mariani, Celestina; Voesenek, Laurentius A.C.J.

    1999-01-01

    Submergence strongly stimulates petiole elongation in Rumex palustris, and ethylene accumulation initiates and maintains this response in submerged tissues. cDNAs from R. palustris corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene (RP-ACO1) were isolated from elongating petioles and used to study the expression of the corresponding gene. An increase in RP-ACO1 messenger was observed in the petioles and lamina of elongating leaves 2 h after the start of submergence. ACC oxidase enzyme activity was measured in homogenates of R. palustris shoots, and a relevant increase was observed within 12 h under water with a maximum after 24 h. We have shown previously that the ethylene production rate of submerged shoots does not increase significantly during the first 24 h of submergence (L.A.C.J. Voesenek, M. Banga, R.H. Thier, C.M. Mudde, F.M. Harren, G.W.M. Barendse, C.W.P.M. Blom [1993] Plant Physiol 103: 783–791), suggesting that under these conditions ACC oxidase activity is inhibited in vivo. We found evidence that this inhibition is caused by a reduction of oxygen levels. We hypothesize that an increased ACC oxidase enzyme concentration counterbalances the reduced enzyme activity caused by low oxygen concentration during submergence, thus sustaining ethylene production under these conditions. Therefore, ethylene biosynthesis seems to be limited at the level of ACC oxidase activity rather than by ACC synthase in R. palustris during submergence. PMID:10482674

  2. Simulated ischaemia-reperfusion conditions increase xanthine dehydrogenase and oxidase activities in rat brain slices.

    PubMed

    Battelli, M G; Buonamici, L; Virgili, M; Abbondanza, A; Contestabile, A

    1998-01-01

    Xanthine dehydrogenase and oxidase activities increased by 87% in rat brain slices after 30 min in vitro ischaemia. A further 41% increase was induced by 30 min simulated reperfusion of ischaemic slices. No conversion from the dehydrogenase to the oxidase activity was observed. The increment of enzyme activity was not due to neosynthesis of the enzyme, since it was not affected by the addition of cycloheximide during the ischaemic incubation. The increased oxygen-dependent form of the enzyme could aggravate the ischaemic brain injury by free radicals production, in particular after reperfusion. PMID:9460697

  3. A description of an HPLC assay of coproporphyrinogen III oxidase activity in mononuclear cells.

    PubMed

    Gross, U; Gerlach, R; Kühnel, A; Seifert, V; Doss, M O

    2003-01-01

    Coproporphyrinogen III oxidase is deficient in hereditary coproporphyria. An activity assay for this enzyme in mononuclear cells, besides the preparation of the substrate, are presented. The separation conditions for the product of the test protoporphyrin IX by gradient, reversed-phase high-performance liquid chromatography are given. The normal value from mononuclear cells of healthy volunteers was 138 +/- 21 pkat/g total soluble protein (mean +/- SD). The enzyme activity of a family with hereditary coproporphyria was measured. The gene carriers exhibit a specific coproporphyrinogen III oxidase activity of 61-90 pkat/g total soluble protein. PMID:14605502

  4. Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction

    SciTech Connect

    Qiu, James A.; Wilson, Heather L.; Rajagopalan, K.V.

    2012-04-18

    Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conserved in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 {angstrom} resolution, respectively.

  5. Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane.

    PubMed

    El-Benna, Jamel; Dang, Pham My-Chan; Gougerot-Pocidalo, Marie-Anne

    2008-07-01

    Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation, neutrophils produce superoxide anion (O*2), which generates other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH*) and hypochlorous acid (HOCl), together with microbicidal peptides and proteases. The enzyme responsible for O2* production is called the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two trans-membrane proteins (p22phox and gp91phox/NOX2, which form the cytochrome b558), three cytosolic proteins (p47phox, p67phox, p40phox) and a GTPase (Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate factors. Three major events accompany NAPDH oxidase activation: (1) protein phosphorylation, (2) GTPase activation, and (3) translocation of cytosolic components to the plasma membrane to form the active enzyme. Actually, the neutrophil NADPH oxidase exists in different states: resting, primed, activated, or inactivated. The resting state is found in circulating blood neutrophils. The primed state can be induced by neutrophil adhesion, pro-inflammatory cytokines, lipopolysaccharide, and other agents and has been characterized as a "ready to go" state, which results in a faster and higher response upon exposure to a second stimulus. The active state is found at the inflammatory or infection site. Activation is induced by the pathogen itself or by pathogen-derived formylated peptides and other agents. Finally, inactivation of NADPH oxidase is induced by anti-inflammatory agents to limit inflammation. Priming is a "double-edged sword" process as it contributes to a rapid and efficient elimination of the pathogens but can also induce the generation of large quantities of toxic ROS by hyperactivation of

  6. Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing.

    PubMed

    Zhou, Xuechou; Tan, Bingcan; Zheng, Xinyu; Kong, Dexian; Li, Qinglu

    2015-11-15

    The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5-5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose. PMID:26278169

  7. 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.

  8. Inheritance of grain polyphenol oxidase (PPO) activity in multiple wheat (Triticum aestivum L.) genetic backgrounds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grain polyphenol oxidase (PPO) activity can cause discoloration of wheat (Triticum aestivum L.) food products. Five crosses (PI 117635/Antelope; Fielder/NW03681; Fielder/Antelope; NW07OR1070/Antelope; NW07OR1066/OR2050272H) were selected to study the genetic inheritance of PPO activity. STS marker...

  9. NADPH oxidase activity is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation

    PubMed Central

    MacFarlane, P M; Satriotomo, I; Windelborn, J A; Mitchell, G S

    2009-01-01

    Phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is a form of spinal, serotonin-dependent synaptic plasticity that requires reactive oxygen species (ROS) formation. We tested the hypothesis that spinal NADPH oxidase activity is a necessary source of ROS for pLTF. Sixty minutes post-AIH (three 5-min episodes of 11% O2, 5 min intervals), integrated phrenic and hypoglossal (XII) nerve burst amplitudes were increased from baseline, indicative of phrenic and XII LTF. Intrathecal injections (∼C4) of apocynin or diphenyleneiodonium chloride (DPI), two structurally and functionally distinct inhibitors of the NADPH oxidase complex, attenuated phrenic, but not XII, LTF. Immunoblots from soluble (cytosolic) and particulate (membrane) fractions of ventral C4 spinal segments revealed predominantly membrane localization of the NADPH oxidase catalytic subunit, gp91phox, whereas membrane and cytosolic expression were both observed for the regulatory subunits, p47phox and RAC1. Immunohistochemical analysis of fixed tissues revealed these same subunits in presumptive phrenic motoneurons of the C4 ventral horn, but not in neighbouring astrocytes or microglia. Collectively, these data demonstrate that NADPH oxidase subunits localized within presumptive phrenic motoneurons are a major source of ROS necessary for AIH-induced pLTF. Thus, NADPH oxidase activity is a key regulator of spinal synaptic plasticity, and may be a useful pharmaceutical target in developing therapeutic strategies for respiratory insufficiency in patients with, for example, cervical spinal injury. PMID:19237427

  10. Sulfite Oxidase Activity Is Essential for Normal Sulfur, Nitrogen and Carbon Metabolism in Tomato Leaves

    PubMed Central

    Brychkova, Galina; Yarmolinsky, Dmitry; Batushansky, Albert; Grishkevich, Vladislav; Khozin-Goldberg, Inna; Fait, Aaron; Amir, Rachel; Fluhr, Robert; Sagi, Moshe

    2015-01-01

    Plant sulfite oxidase [SO; E.C.1.8.3.1] has been shown to be a key player in protecting plants against exogenous toxic sulfite. Recently we showed that SO activity is essential to cope with rising dark-induced endogenous sulfite levels in tomato plants (Lycopersicon esculentum/Solanum lycopersicum Mill. cv. Rheinlands Ruhm). Here we uncover the ramifications of SO impairment on carbon, nitrogen and sulfur (S) metabolites. Current analysis of the wild-type and SO-impaired plants revealed that under controlled conditions, the imbalanced sulfite level resulting from SO impairment conferred a metabolic shift towards elevated reduced S-compounds, namely sulfide, S-amino acids (S-AA), Co-A and acetyl-CoA, followed by non-S-AA, nitrogen and carbon metabolite enhancement, including polar lipids. Exposing plants to dark-induced carbon starvation resulted in a higher degradation of S-compounds, total AA, carbohydrates, polar lipids and total RNA in the mutant plants. Significantly, a failure to balance the carbon backbones was evident in the mutants, indicated by an increase in tricarboxylic acid cycle (TCA) cycle intermediates, whereas a decrease was shown in stressed wild-type plants. These results indicate that the role of SO is not limited to a rescue reaction under elevated sulfite, but SO is a key player in maintaining optimal carbon, nitrogen and sulfur metabolism in tomato plants. PMID:27135342

  11. Cytochrome C oxidase activity in germinating Phaseolus vulgaris l. seeds: Effects of carbon monoxide

    SciTech Connect

    Caughey, W.S. ); Sowa, S.; Roos, E.E.

    1989-04-01

    Cytochrome c oxidase is a key bioenergetic enzyme required for seed germination. The enzyme was isolated from 2-day germinating beans and biochemically compared to its bovine heart counterpart. Carbon monoxide, which binds to the heme a{sub 3} site of cytochrome c oxidase, we used to probe O{sub 2} utilization activity in isolated enzyme, mitochondrial particles, and whole seeds. Bean seeds under 80% CO/20% O{sub 2} exhibited 46% growth inhibition as determined by root length. Reversible, dose-dependent partial inhibition of bean seed mitochondrial respiration was observed in the presence of CO; heart mitochondria had a more sensitive, less reversible response. Effects of CO on bean and bovine heart enzyme were similar. The close correlation of CO effects observed on seedling growth, mitochondrial respiration and cytochrome oxidase activity indicate an important role for this enzyme during the early stages of seed germination.

  12. Regional brain effects of sodium azide treatment on cytochrome oxidase activity: a quantitative histochemical study.

    PubMed

    Cada, A; Gonzalez-Lima, F; Rose, G M; Bennett, M C

    1995-12-01

    The objective of the present study was to determine if regional variation in brain cytochrome oxidase activity was observed following systemic administration of sodium azide. An image analysis system calibrated with internal standards of known cytochrome oxidase activity was used to quantify cytochrome oxidase in histochemically stained brain sections. Rats receiving chronic infusion of sodium azide (400 micrograms/hr), which were sacrificed after two weeks, showed a substantial decrease in brain cytochrome oxidase activity over those infused with saline. All of the 22 regions sampled from telencephalic, diencephalic, and mesencephalic levels, showed a significant activity reduction which ranged between 26% and 37%. The regions that appeared significantly more vulnerable to the sodium azide effects were the mesencephalic reticular formation and the central amygdala, which displayed the largest decrease in activity. In addition, interregional correlations of activity showed a deeply modified pattern of correlative metabolic activity between hippocampal, amygdaloid and cortical areas after azide treatment. The regional effects found were consistent with azide-induced learning and memory dysfunctions. PMID:8847994

  13. Exploring Regulation Genes Involved in the Expression of L-Amino Acid Oxidase in Pseudoalteromonas sp. Rf-1

    PubMed Central

    Wang, Ju; Lin, Jianxun; Zhao, Minyan

    2015-01-01

    Bacterial L-amino acid oxidase (LAAO) is believed to play important biological and ecological roles in marine niches, thus attracting increasing attention to understand the regulation mechanisms underlying its production. In this study, we investigated genes involved in LAAO production in marine bacterium Pseudoalteromonas sp. Rf-1 using transposon mutagenesis. Of more than 4,000 mutants screened, 15 mutants showed significant changes in LAAO activity. Desired transposon insertion was confirmed in 12 mutants, in which disrupted genes and corresponding functionswere identified. Analysis of LAAO activity and lao gene expression revealed that GntR family transcriptional regulator, methylase, non-ribosomal peptide synthetase, TonB-dependent heme-receptor family, Na+/H+ antiporter and related arsenite permease, N-acetyltransferase GCN5, Ketol-acid reductoisomerase and SAM-dependent methytransferase, and their coding genes may be involved in either upregulation or downregulation pathway at transcriptional, posttranscriptional, translational and/or posttranslational level. The nhaD and sdmT genes were separately complemented into the corresponding mutants with abolished LAAO-activity. The complementation of either gene can restore LAAO activity and lao gene expression, demonstrating their regulatory role in LAAO biosynthesis. This study provides, for the first time, insights into the molecular mechanisms regulating LAAO production in Pseudoalteromonas sp. Rf-1, which is important to better understand biological and ecological roles of LAAO. PMID:25815733

  14. Thioredoxin-1/peroxiredoxin-1 as sensors of oxidative stress mediated by NADPH oxidase activity in atherosclerosis.

    PubMed

    Madrigal-Matute, Julio; Fernandez-Garcia, Carlos-Ernesto; Blanco-Colio, Luis Miguel; Burillo, Elena; Fortuño, Ana; Martinez-Pinna, Roxana; Llamas-Granda, Patricia; Beloqui, Oscar; Egido, Jesus; Zalba, Guillermo; Martin-Ventura, José Luis

    2015-09-01

    To assess the potential association between TRX-1/PRX-1 and NADPH oxidase (Nox) activity in vivo and in vitro, TRX-1/PRX-1 levels were assessed by ELISA in 84 asymptomatic subjects with known phagocytic NADPH oxidase activity and carotid intima-media thickness (IMT). We found a positive correlation between TRX-1/PRX-1 and NADPH oxidase-dependent superoxide production (r=0.48 and 0.47; p<0.001 for both) and IMT (r=0.31 and 0.36; p<0.01 for both) adjusted by age and sex. Moreover, asymptomatic subjects with plaques have higher PRX-1 and TRX plasma levels (p<0.01 for both). These data were confirmed in a second study in which patients with carotid atherosclerosis showed higher PRX-1 and TRX plasma levels than healthy subjects (p<0.001 for both). In human atherosclerotic plaques, the NADPH oxidase subunit p22phox colocalized with TRX-1/PRX-1 in macrophages (immunohistochemistry). In monocytes and macrophages, phorbol 12-myristate 13-acetate (PMA) induced NADPH activation and TRX-1/PRX-1 release to the extracellular medium, with a concomitant decrease in their intracellular levels, which was reversed by the NADPH inhibitor apocynin (Western blot). In loss-of-function experiments, genetic silencing of the NADPH oxidase subunit Nox2 blocked PMA-induced intracellular TRX-1/PRX-1 downregulation in macrophages. Furthermore, the PMA-induced release of TRX-1/PRX-1 involves the modulation of their redox status and exosome-like vesicles. TRX-1/PRX-1 levels are associated with NADPH oxidase-activity in vivo and in vitro. These data could suggest a coordinated antioxidant response to oxidative stress in atherothrombosis. PMID:26117319

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

    PubMed

    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

  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. Virtual Screening Analysis and In-vitro Xanthine Oxidase Inhibitory Activity of Some Commercially Available Flavonoids.

    PubMed

    Umamaheswari, Muthuswamy; Madeswaran, Arumugam; Asokkumar, Kuppusamy

    2013-01-01

    Allopurinol, the xanthine oxidase inhibitor, is the only drug available for the treatment of gout. We examined the xanthine oxidase inhibitory activity of some commercially available flavonoids such asepigallocatechin, acacatechin, myricetin, naringenin, daidzein and glycitein by virtual screening and in-vitro studies. The interacting residues within the complex model and their contact types were identified. The virtual screening analysis were carried out using AutoDock 4.2 and in-vitro xanthine oxidase inhibitory activity was carried out using xanthine as the substrate. In addition, enzyme kinetics was performed using LineweaverBurkplot analysis. Allopurinol, a known xanthine oxidase inhibitor was used as the standard. The docking energy ofglycitein was found to be -8.49 kcal/mol which was less than that of the standard (-4.47 kcal/ mol). All the selected flavonoids were found to exhibit lower binding energy (-8.08 to -6.03 kcal/ mol) than allopurinol. The docking results confirm that flavonoids showed greater inhibition of xanthine oxidase due to their active binding sites and lesser binding energies compared to allopurinol. This may be attributed to the presence of benzopyran ring in the flavonoids. In the xanthine oxidase assay, IC50 value of glycitein was found to be 12±0.86 μg/mL, whereas that of allopurinol was 24±0.28 μg/mL. All the remaining compounds exhibited IC50 values ranging between 22±0.64 to 62±1.18 μg/mL. In the enzyme kinetic studies, flavonoids showed competitive type of enzyme inhibition. It can be concluded that flavonoids could be a promising remedy for the treatment of gout and related inflammatory disorders. Further in-vivo studies are required to develop potential compounds with lesser side effects. PMID:24250638

  18. Virtual Screening Analysis and In-vitro Xanthine Oxidase Inhibitory Activity of Some Commercially Available Flavonoids

    PubMed Central

    Umamaheswari, Muthuswamy; Madeswaran, Arumugam; Asokkumar, Kuppusamy

    2013-01-01

    Allopurinol, the xanthine oxidase inhibitor, is the only drug available for the treatment of gout. We examined the xanthine oxidase inhibitory activity of some commercially available flavonoids such asepigallocatechin, acacatechin, myricetin, naringenin, daidzein and glycitein by virtual screening and in-vitro studies. The interacting residues within the complex model and their contact types were identified. The virtual screening analysis were carried out using AutoDock 4.2 and in-vitro xanthine oxidase inhibitory activity was carried out using xanthine as the substrate. In addition, enzyme kinetics was performed using LineweaverBurkplot analysis. Allopurinol, a known xanthine oxidase inhibitor was used as the standard. The docking energy ofglycitein was found to be -8.49 kcal/mol which was less than that of the standard (-4.47 kcal/ mol). All the selected flavonoids were found to exhibit lower binding energy (-8.08 to -6.03 kcal/ mol) than allopurinol. The docking results confirm that flavonoids showed greater inhibition of xanthine oxidase due to their active binding sites and lesser binding energies compared to allopurinol. This may be attributed to the presence of benzopyran ring in the flavonoids. In the xanthine oxidase assay, IC50 value of glycitein was found to be 12±0.86 μg/mL, whereas that of allopurinol was 24±0.28 μg/mL. All the remaining compounds exhibited IC50 values ranging between 22±0.64 to 62±1.18 μg/mL. In the enzyme kinetic studies, flavonoids showed competitive type of enzyme inhibition. It can be concluded that flavonoids could be a promising remedy for the treatment of gout and related inflammatory disorders. Further in-vivo studies are required to develop potential compounds with lesser side effects. PMID:24250638

  19. Polyacrylic acid-coated cerium oxide nanoparticles: An oxidase mimic applied for colorimetric assay to organophosphorus pesticides.

    PubMed

    Zhang, Shi-Xiang; Xue, Shi-Fan; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

    2016-11-15

    It is important and urgent to develop reliable and highly sensitive methods that can provide on-site and rapid detection of extensively used organophosphorus pesticides (OPs) for their neurotoxicity. In this study, we developed a novel colorimetric assay for the detection of OPs based on polyacrylic acid-coated cerium oxide nanoparticles (PAA-CeO2) as an oxidase mimic and OPs as inhibitors to suppress the activity of acetylcholinesterase (AChE). Firstly, highly dispersed PAA-CeO2 was prepared in aqueous solution, which could catalyze the oxidation of TMB to produce a color reaction from colorless to blue. And the enzyme of AChE was used to catalyze the substrate of acetylthiocholine (ATCh) to produce thiocholine (TCh). As a thiol-containing compound with reducibility, TCh can decrease the oxidation of TMB catalyzed by PAA-CeO2. Upon incubated with OPs, the enzymatic activity of AChE was inhibited to produce less TCh, resulting in more TMB catalytically oxidized by PAA-CeO2 to show an increasing blue color. The two representative OPs, dichlorvos and methyl-paraoxon, were tested using our proposed assay. The novel assay showed notable color change in a concentration-dependent manner, and as low as 8.62 ppb dichlorvos and 26.73 ppb methyl-paraoxon can be readily detected. Therefore, taking advantage of such oxidase-like activity of PAA-CeO2, our proposed colorimetric assay can potentially be a screening tool for the precise and rapid evaluation of the neurotoxicity of a wealth of OPs. PMID:27208478

  20. Inhibitory effect of rice bran extracts and its phenolic compounds on polyphenol oxidase activity and browning in potato and apple puree.

    PubMed

    Sukhonthara, Sukhontha; Kaewka, Kunwadee; Theerakulkait, Chockchai

    2016-01-01

    Full-fatted and commercially defatted rice bran extracts (RBE and CDRBE) were evaluated for their ability to inhibit enzymatic browning in potato and apple. RBE showed more effective inhibition of polyphenol oxidase (PPO) activity and browning in potato and apple as compared to CDRBE. Five phenolic compounds in RBE and CDRBE (protocatechuic acid, vanillic acid, p-coumaric acid, ferulic acid and sinapic acid) were identified by HPLC. They were then evaluated for their important role in the inhibition using a model system which found that ferulic acid in RBE and p-coumaric acid in CDRBE were active in enzymatic browning inhibition of potato and apple. p-Coumaric acid exhibited the highest inhibitory effect on potato and apple PPO (p ⩽ 0.05). Almost all phenolic compounds showed higher inhibitory effect on potato and apple PPO than 100 ppm citric acid. PMID:26213057

  1. Exploring the arachidonic acid-induced structural changes in phagocyte NADPH oxidase p47(phox) and p67(phox) via thiol accessibility and SRCD spectroscopy.

    PubMed

    Bizouarn, Tania; Karimi, Gilda; Masoud, Rawand; Souabni, Hager; Machillot, Paul; Serfaty, Xavier; Wien, Frank; Réfrégiers, Matthieu; Houée-Levin, Chantal; Baciou, Laura

    2016-08-01

    The NADPH oxidase is the sole enzymatic complex that produces, in a controlled way, superoxide anions. In phagocytes, it is constituted by the assembly of four cytosolic (p67(phox) , p47(phox) , p40(phox) and Rac) and two membrane (p22(phox) and Nox2) proteins. In response to pro-inflammatory mediators, the NADPH oxidase is activated. In cells, arachidonic acid (cis-AA), released by activated phospholipase A2, also plays a role in activation of the NADPH oxidase complex, but the mechanism of action of cis-AA is still a matter for debate. In cell-free systems, cis-AA is commonly used for activation. We have shown previously that trans-AA isomers were unable to activate the NADPH oxidase complex. Here, we aim to evaluate the structural changes in p47(phox) and p67(phox) induced by AA. The structural impact of both AA isomers on both cytosolic proteins was investigated by the accessibility of the thiol group and by circular dichroism in the far-UV for global folds. cis-AA induces secondary structure changes of p47(phox) and p67(phox) , while the trans isomer does not, suggesting that the changes observed are of importance for the activation process of these proteins. While five of the nine thiol groups in p67(phox) and all of them in p47(phox) have low access to the solvent when proteins are alone in solution, all of them become fully accessible when proteins are together. In conclusion, the secondary structures of p47(phox) and p67(phox) are both dependent on the presence of the partner protein in solution and on the presence of the activator molecule cis-AA. PMID:27284000

  2. A facile and effective immobilization of glucose oxidase on tannic acid modified CoFe2O4 magnetic nanoparticles.

    PubMed

    Altun, Seher; Çakıroğlu, Bekir; Özacar, Münteha; Özacar, Mahmut

    2015-12-01

    This article presents a study of glucose oxidase (GOx) immobilization by employing tannic acid (TA) modified-CoFe2O4 (CFO) magnetic nanoparticles which demonstrates novel aspect for enzyme immobilization. By using the strong protein and tannic acid binding, GOx immobilization was carried out via physical adsorption in a simpler way compared with the other immobilization methods which require various chemicals and complicated procedures which is difficult, expensive, time-consuming, and destructive to the enzyme structure. CFO was synthesized by hydrothermal synthesis and modified with TA to immobilize GOx. The immobilized GOx demonstrated maximum catalytic activity at pH 6.5 and 45 °C. The samples were characterized by vibrating sample magnetometer (VSM), thermogravimetric analysis (TGA), zeta potential, and fourier transform infrared spectroscopy (FTIR), all of which confirm the surface modification of CFO and GOx immobilization. Also, field emission scanning electron microscope (FESEM) and X-ray diffraction (XRD) were performed to demonstrate the surface morphology and chemical structure of samples. According to the Lineweaver-Burk plot, GOx possessed lower affinity to glucose after immobilization, and the Michelis-Menten constant (KM) of immobilized and free GOx were found to be 50.05 mM and 28.00 mM, respectively. The immobilized GOx showed excellent reusability, and even after 8 consecutive activity assay runs, the immobilized GOx maintained ca. 60% of its initial activity. PMID:26562188

  3. Involvement of Peroxidase and Indole-3-acetic Acid Oxidase Isozymes from Pear, Tomato, and Blueberry Fruit in Ripening

    PubMed Central

    Frenkel, Chaim

    1972-01-01

    Protein extracts were obtained from climacteric fruits (pear, tomato) and nonclimacteric fruits (blueberry) during various stages of ripening. The use of a gel electrophoresis technique revealed a consistent reinforcement in indoleacetic acid oxidase but not in peroxidase isozymes during ripening. The significance of the results is discussed in relation to the resistance of fruits to ripening and ethylene action. Images PMID:16658043

  4. Listeriolysin O suppresses Phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection

    PubMed Central

    Lam, Grace Y.; Fattouh, Ramzi; Muise, Aleixo M.; Grinstein, Sergio; Higgins, Darren E.; Brumell, John H.

    2012-01-01

    Summary The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L. monocytogenes infection, a response that would adversely affect pathogen survival. However, secretion of LLO inhibits the NADPH oxidase by preventing its localization to phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O, a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit the NADPH oxidase. Our studies demonstrate that while the PLCs induce antimicrobial NADPH oxidase activity, this effect is alleviated by the pore-forming activity of LLO. Therefore, the combined activities of PLCs and LLO on membrane lysis and the inhibitory effects of LLO on NADPH oxidase activity allows L. monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst. PMID:22177565

  5. INVESTIGATIONS OF AMITRAZ NEUROTOXICITY IN RATS. III. EFFECTS ON MOTOR ACTIVITY AND INHIBITION OF MONOAMINE OXIDASE

    EPA Science Inventory

    The formamidine pesticide amitraz (AMZ) produces many behavioral and physiological changes in rats. o explore possible neurochemical mechanisms for the behavioral effects of AMZ, we examined the dose effect and time course of AMZ on motor activity, monoamine oxidase (MAO) and ace...

  6. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    SciTech Connect

    Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir; Orr, William C.; Sohal, Rajindar S.

    2010-10-08

    Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

  7. Gluconic acid production in bioreactor with immobilized glucose oxidase plus catalase on polymer membrane adjacent to anion-exchange membrane.

    PubMed

    Godjevargova, Tzonka; Dayal, Rajeshwar; Turmanova, Sevdalina

    2004-10-20

    Gluconic acid was obtained in the permeate side of the bioreactor with glucose oxidase (GOD) immobilized onto anion-exchange membrane (AEM) of low-density polyethylene grafted with 4-vinylpiridine. The electric resistance of the anion-exchange membranes was increased after the enzyme immobilization on the membrane. The gluconic acid productions were relatively low with the GOD immobilized by any method on the AEM. To increase the enzyme reaction efficiency, GOD was immobilized on membrane of AN copolymer (PAN) adjacent to an anion-exchange membrane in bioreactor. Uses of anion-exchange membrane led to selective removal of the gluconic acid from the glucose solution and reduce the gluconic acid inhibition. The amount of gluconic acid obtained in the permeate side of the bioreactor with the GOD immobilized on the PAN membrane adjacent to the AEM under electrodialysis was about 30 times higher than that obtained with enzyme directly bound to the AEM. The optimal substrate concentration in the feed side was found to be about 1 g/l. Further experiments were carried out with the co-immobilized GOD plus Catalase (CAT) on the PAN membrane adjacent to the AEM to improve the efficiency of the immobilize system. The yield of this process was at least 95%. The storage stability of the co-immobilized GOD and CAT was studied (lost 20% of initial activity for 90 d). The results obtained clearly showed the higher potential of the dual membrane bioreactor with GOD plus CAT bound to ultrafiltration polymer membrane adjacent to the AEM. Storage stability of GOD activity in GOD plus CAT immobilized on PAN//AEM membranes and on AEM. PMID:15497133

  8. Evolution of histamine oxidase activity for biotechnological applications.

    PubMed

    Rosini, Elena; Tonin, Fabio; Vasylieva, Natalia; Marinesco, Stephane; Pollegioni, Loredano

    2014-01-01

    Histamine is present to various degrees in many foods, and concentrations in fish samples are considered a good indicator of freshness and hygienic food quality. Seeking for innovative methods to quantify histamine in foods, we used a synthetic gene designed on the sequence of histamine oxidase from Arthrobacter crystallopoietes (HOD) as the starting point in this study to develop a biosensor. HOD was expressed in Escherichia coli cells with a yield of ∼7 mg protein/L of fermentation broth. Recombinant wild-type HOD oxidized histamine and tyramine whereas it was inactive toward putrescine and cadaverine (two amines present in fish samples). The putative residues involved in substrate binding were identified by an in silico docking procedure based on a model of the structure of HOD: site-saturation mutagenesis was performed on 8 positions. The most significant changes in kinetic properties were observed for the P143M HOD: this variant showed higher histamine affinity and lower substrate inhibition by tyramine than wild-type enzyme. Biosensor prototypes were produced using both the wild-type and the P143M variant HOD. These biosensors showed a good sensitivity and selectivity with respect to biogenic amines present in food specimens. Accordingly, the HOD-based biosensor was successfully used to assess histamine in fish samples, yielding values in good agreement with those obtained by HPLC analyses but in a few seconds and at a significantly lower cost per analysis. PMID:23995223

  9. A mitochondrial DNA variant, identified in Leber hereditary optic neuropathy patients, which extends the amino acid sequence of cytochrome c oxidase subunit I.

    PubMed Central

    Brown, M D; Yang, C C; Trounce, I; Torroni, A; Lott, M T; Wallace, D C

    1992-01-01

    A G-to-A transition at nucleotide pair (np) 7444 in the mtDNA was found to correlate with Leber hereditary optic neuropathy (LHON). The mutation eliminates the termination codon of the cytochrome c oxidase subunit I (COI) gene, extending the COI polypeptide by three amino acids. The mutation was discovered as an XbaI restriction-endonuclease-site loss present in 2 (9.1%) of 22 LHON patients who lacked the np 11778 LHON mutation and in 6 (1.1%) of 545 unaffected controls. The mutant polypeptide has an altered mobility on SDS-PAGE, suggesting a structural alteration, and the cytochrome c oxidase enzyme activity of patient lymphocytes is reduced approximately 40% relative to that in controls. These data suggest that the np 7444 mutation results in partial respiratory deficiency and thus contributes to the onset of LHON. Images Figure 1 Figure 3 PMID:1322638

  10. Active Site and Loop 4 Movements with Human Glycolate Oxidase: Implications for Substrate Specificity and Drug Design

    SciTech Connect

    Murray,M.; Holmes, R.; Lowther, W.

    2008-01-01

    Human glycolate oxidase (GO) catalyzes the FMN-dependent oxidation of glycolate to glyoxylate and glyoxylate to oxalate, a key metabolite in kidney stone formation. We report herein the structures of recombinant GO complexed with sulfate, glyoxylate, and an inhibitor, 4-carboxy-5-dodecylsulfanyl-1, 2,3-triazole (CDST), determined by X-ray crystallography. In contrast to most {alpha}-hydroxy acid oxidases including spinach glycolate oxidase, a loop region, known as loop 4, is completely visible when the GO active site contains a small ligand. The lack of electron density for this loop in the GO-CDST complex, which mimics a large substrate, suggests that a disordered to ordered transition may occur with the binding of substrates. The conformational flexibility of Trp110 appears to be responsible for enabling GO to react with a-hydroxy acids of various chain lengths. Moreover, the movement of Trp110 disrupts a hydrogen-bonding network between Trp110, Leu191, Tyr134, and Tyr208. This loss of interactions is the first indication that active site movements are directly linked to changes in the conformation of loop 4. The kinetic parameters for the oxidation of glycolate, glyoxylate, and 2-hydroxy octanoate indicate that the oxidation of glycolate to glyoxylate is the primary reaction catalyzed by GO, while the oxidation of glyoxylate to oxalate is most likely not relevant under normal conditions. However, drugs that exploit the unique structural features of GO may ultimately prove to be useful for decreasing glycolate and glyoxylate levels in primary hyperoxaluria type 1 patients who have the inability to convert peroxisomal glyoxylate to glycine.

  11. Semicarbazide-sensitive amine oxidase activation promotes adipose conversion of 3T3-L1 cells.

    PubMed Central

    Mercier, N; Moldes, M; El Hadri, K; Fève, B

    2001-01-01

    Semicarbazide-sensitive amine oxidase (SSAO) is an amine oxidase related to the copper-containing amine oxidase family. The tissular form of SSAO is located at the plasma membrane, and is mainly expressed in vascular smooth muscle cells and adipocytes. Recent studies have suggested that SSAO could activate glucose transport in fat cells. In the present work, we investigated the potential role of a chronic SSAO activation on adipocyte maturation of the 3T3-L1 pre-adipose cell line. Exposure of post-confluent 3T3-L1 pre-adipocytes to methylamine, a physiological substrate of SSAO, promoted adipocyte differentiation in a time- and dose-dependent manner. This effect could be related to SSAO activation, since it was antagonized in the presence of the SSAO inhibitor semicarbazide, but not in the presence of the monoamine oxidase inhibitor pargyline. In addition, methylamine-induced adipocyte maturation was mimicked by 3T3-L1 cell treatment with other SSAO substrates. Finally, the large reversion of methylamine action by catalase indicated that hydrogen peroxide generated by SSAO was involved, at least in part, in the modulation of adipocyte maturation. Taken together, our results suggest that SSAO may contribute to the control of adipose tissue development. PMID:11513731

  12. Irreversible inactivation of snake venom l-amino acid oxidase by covalent modification during catalysis of l-propargylglycine.

    PubMed

    Mitra, Jyotirmoy; Bhattacharyya, Debasish

    2013-01-01

    Snake venom l-amino acid oxidase (SV-LAAO, a flavor-enzyme) has attracted considerable attention due to its multifunctional nature, which is manifest in diverse clinical and biological effects such as inhibition of platelet aggregation, induction of cell apoptosis and cytotoxicity against various cells. The majority of these effects are mediated by H2O2 generated during the catalytic conversion of l-amino acids. The substrate analog l-propargylglycine (LPG) irreversibly inhibited the enzyme from Crotalus adamanteus and Crotalus atrox in a dose- and time-dependent manner. Inactivation was irreversible which was significantly protected by the substrate l-phenylalanine. A Kitz-Wilson replot of the inhibition kinetics suggested formation of reversible enzyme-LPG complex, which occurred prior to modification and inactivation of the enzyme. UV-visible and fluorescence spectra of the enzyme and the cofactor strongly suggested formation of covalent adduct between LPG and an active site residue of the enzyme. A molecular modeling study revealed that the FAD-binding, substrate-binding and the helical domains are conserved in SV-LAAOs and both His223 and Arg322 are the important active site residues that are likely to get modified by LPG. Chymotrypsin digest of the LPG inactivated enzyme followed by RP-HPLC and MALDI mass analysis identified His223 as the site of modification. The findings reported here contribute towards complete inactivation of SV-LAAO as a part of snake envenomation management. PMID:23772385

  13. Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase from Escherichia coli

    SciTech Connect

    Weidner, Annett; Neumann, Piotr; Wille, Georg; Stubbs, Milton T.; Tittmann, Kai

    2008-03-01

    The peripheral membrane flavoprotein pyruvate oxidase from E. coli has been crystallized in the full-length form and as a proteolytically activated truncation variant lacking the last 23 amino acids at the C-terminus. The thiamine diphosphate- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from Escherichia coli (EcPOX) has been crystallized in the full-length form and as a proteolytically activated C-terminal truncation variant which lacks the last 23 amino acids (Δ23 EcPOX). Crystals were grown by the hanging-drop vapour-diffusion method using either protamine sulfate (full-length EcPOX) or 2-methyl-2,4-pentanediol (Δ23 EcPOX) as precipitants. Native data sets were collected at a X-ray home source to a resolution of 2.9 Å. The two forms of EcPOX crystallize in different space groups. Whereas full-length EcPOX crystallizes in the tetragonal space group P4{sub 3}2{sub 1}2 with two monomers per asymmetric unit, the crystals of Δ23 EcPOX belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and contain 12 monomers per asymmetric unit.

  14. FcγR-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

    PubMed Central

    Tian, Wei; Li, Xing Jun; Stull, Natalie D.; Ming, Wenyu; Suh, Chang-Il; Bissonnette, Sarah A.; Yaffe, Michael B.; Grinstein, Sergio; Atkinson, Simon J.

    2008-01-01

    The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after phagosome internalization. We conclude that p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization. PMID:18711001

  15. 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.

  16. Colorimetric Glucose Assay Based on Magnetic Particles Having Pseudo-peroxidase Activity and Immobilized Glucose Oxidase.

    PubMed

    Martinkova, Pavla; Opatrilova, Radka; Kruzliak, Peter; Styriak, Igor; Pohanka, Miroslav

    2016-05-01

    Magnetic particles (MPs) are currently used as a suitable alternative for peroxidase in the construction of novel biosensors, analytic and diagnostic methods. Their better chemical and thermal stabilities predestine them as appropriate pseudo-enzymatic catalysts. In this point of view, our research was focused on preparation of simply and fast method for immobilization of glucose oxidase onto surface of MPs with peroxidase-like activity. Spectrophotometric method (wavelength 450 nm) optimized for glucose determination using modified MPs has been successfully developed. Concentration curve for optimization of method was assayed, and Michaelis-Menten constant (K m) calculated, maximum reaction rate (V max), limit of detection, and correlation coefficient were determined to be 0.13 mmol/l (2.34 mg/dl), 1.79 pkat, 3.74 µmol/l (0.067 mg/dl), and 0.996, respectively. Interferences of other sugars such as sucrose, sorbitol, deoxyribose, maltose, and fructose were determined as well as effect of substances presenting in plasma (ascorbic acid, reduced glutathione, trolox, and urea). Results in comparison with positive and negative controls showed no interferences of the other sugars and no influence of plasma substances to measuring of glucose. The constructed method showed corresponding results with linear dependence and a correlation coefficient of 0.997. Possibility of repeated use of modified MPs was successfully proved. PMID:27041274

  17. Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice

    PubMed Central

    Nagasu, Hajime; Satoh, Minoru; Kiyokage, Emi; Kidokoro, Kengo; Toida, Kazunori; Channon, Keith M; Kanwar, Yashpal S; Sasaki, Tamaki; Kashihara, Naoki

    2016-01-01

    Increased generation of reactive oxygen species (ROS) is a common denominative pathogenic mechanism underlying vascular and renal complications in diabetes mellitus. Endothelial NAD(P)H oxidase is a major source of vascular ROS, and it has an important role in endothelial dysfunction. We hypothesized that activation of endothelial NAD(P)H oxidase initiates and worsens the progression of diabetic nephropathy, particularly in the development of albuminuria. We used transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NAD(P)H oxidase, Nox2 (NOX2TG). NOX2TG mice were crossed with Akita insulin-dependent diabetic (Akita) mice that develop progressive hyperglycemia. We compared the progression of diabetic nephropathy in Akita versus NOX2TG-Akita mice. NOX2TG-Akita mice and Akita mice developed significant albuminuria above the baseline at 6 and 10 weeks of age, respectively. Compared with Akita mice, NOX2TG-Akita mice exhibited higher levels of NAD(P)H oxidase activity in glomeruli, developed glomerular endothelial perturbations, and attenuated expression of glomerular glycocalyx. Moreover, in contrast to Akita mice, the NOX2TG-Akita mice had numerous endothelial microparticles (blebs), as detected by scanning electron microscopy, and increased glomerular permeability. Furthermore, NOX2TG-Akita mice exhibited distinct phenotypic changes in glomerular mesangial cells expressing α-smooth muscle actin, and in podocytes expressing increased levels of desmin, whereas the glomeruli generated increased levels of ROS. In conclusion, activation of endothelial NAD(P)H oxidase in the presence of hyperglycemia initiated and exacerbated diabetic nephropathy characterized by the development of albuminuria. Moreover, ROS generated in the endothelium compounded glomerular dysfunctions by altering the phenotypes of mesangial cells and compromising the integrity of the podocytes. PMID:26552047

  18. Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility

    PubMed Central

    Smith, Madison A.; Gonzalez, Jesica; Hussain, Anjum; Oldfield, Rachel N.; Johnston, Kathryn A.; Lopez, Karlo M.

    2016-01-01

    Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM−1 cm−1. No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture. PMID:26942005

  19. Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility.

    PubMed

    Smith, Madison A; Gonzalez, Jesica; Hussain, Anjum; Oldfield, Rachel N; Johnston, Kathryn A; Lopez, Karlo M

    2016-01-01

    Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM(-1 )cm(-1). No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture. PMID:26942005

  20. Semicarbazide-sensitive amine oxidase activity of guinea pig dorsal skin.

    PubMed

    Buffoni, F; Cambi, S; Banchelli, G; Ignesti, G; Pirisino, R; Raimondi, L

    1994-01-01

    A semicarbazide-sensitive amine oxidase activity with a high affinity for benzylamine (Bz.SSAO) (E.C. 1.4.3.6) is present in guinea pig dorsal skin. This enzymic activity oxidized benzylamine, histamine, 1,4-methylhistamine and acetylputrescine and was inhibited by semicarbazide and by B24 (3,5-diethoxy-4-aminomethylpyridine), a selective inhibitor of Bz.SSAO enzymes. It cross reacted with the antibodies raised against pure pig plasma benzylamine oxidase. Immunohistochemistry showed that it was localized in fibroblasts. Bz.SSAO activity of guinea pig dorsal skin increased during the process of skin healing. A treatment of the wounds with 3 micrograms of b-FGF significantly accelerated the process of skin healing and the increase of Bz.SSAO activity. PMID:7931260

  1. Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells

    PubMed Central

    Kim, Da Jung; Kim, Yong Sik

    2015-01-01

    Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells. PMID:26221064

  2. An artificial di-iron oxo-protein with phenol oxidase activity

    PubMed Central

    Faiella, Marina; Andreozzi, Concetta; de Rosales, Rafael Torres Martin; Pavone, Vincenzo; Maglio, Ornella; Nastri, Flavia; DeGrado, William F; Lombardi, Angela

    2013-01-01

    Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site. PMID:19915535

  3. Differential expression of two 1-aminocyclopropane-1-carboxylic acid oxidase genes in broccoli after harvest.

    PubMed Central

    Pogson, B J; Downs, C G; Davies, K M

    1995-01-01

    Broccoli (Brassica oleracea L.) floral tissues rapidly differentiate and grow before harvest and then senesce rapidly after harvest. Associated with this postharvest deterioration is an increase in ethylene production by florets. Two cDNA clones having high nucleotide identity to sequences encoding 1-amino-cyclopropane-1-carboxylic acid (ACC) oxidase were isolated from senescing florets. The cDNAs, ACC Ox1 and ACC Ox2, apparently encode mRNAs from different genes. ACC Ox1 transcripts were found at low levels in whole florets at the time of harvest and increased markedly in abundance after harvest. ACC Ox1 transcript abundance also increased in sepals after harvest and in excised yellowing leaves. Transcripts corresponding to ACC Ox2 were found exclusively within the reproductive structures. These ACC Ox2 transcripts were absent at harvest but started to increase in abundance within 2 h of harvest and then accumulated to high levels. Hormone treatment did not alter the abundance of ACC Ox1 transcripts, whereas ACC Ox2 transcripts increased in abundance after treatment with abscisic acid and propylene. Wounding did not affect the levels of ACC Ox1 or Ox2 transcripts after harvest. At harvest, individual broccoli florets were closed and remained unpollinated. We propose a model whereby the rapid increase in ACC Ox1 and Ox2 transcript abundance after harvest contributes to increased ethylene production by florets. This ethylene may regulate aspects of postharvest senescence, in particular chlorophyll loss. PMID:7610162

  4. Pharmacokinetics of Oral d-Serine in d-Amino Acid Oxidase Knockout Mice

    PubMed Central

    Rais, Rana; Thomas, Ajit G.; Wozniak, Krystyna; Wu, Ying; Jaaro-Peled, Hanna; Sawa, Akira; Strick, Christine A.; Engle, Sandra J.; Brandon, Nicholas J.; Rojas, Camilo; Slusher, Barbara S.

    2012-01-01

    d-Amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-amino acids including d-serine, a full agonist at the glycine modulatory site of the N-methyl-d-aspartate (NMDA) receptor. To evaluate the significance of DAAO-mediated metabolism in the pharmacokinetics of oral d-serine, plasma d-serine levels were measured in both wild-type mice and transgenic mice lacking DAAO. Although d-serine levels were rapidly diminished in wild-type mice (t½ = 1.2 h), sustained drug levels over the course of 4 h (t½ > 10 h) were observed in mice lacking DAAO. Coadministration of d-serine with 6-chlorobenzo[d]isoxazol-3-ol (CBIO), a small-molecule DAAO inhibitor, in wild-type mice resulted in the enhancement of plasma d-serine levels, although CBIO seems to have only temporary effects on the plasma d-serine levels due to glucuronidation of the key hydroxyl group. These findings highlight the predominant role of DAAO in the clearance of d-serine from the systemic circulation. Thus, a potent DAAO inhibitor with a longer half-life should be capable of maintaining high plasma d-serine levels over a sustained period of time and might have therapeutic implications for the treatment of schizophrenia. PMID:22837388

  5. D-amino acid oxidase is expressed in the ventral tegmental area and modulates cortical dopamine.

    PubMed

    Betts, Jill F; Schweimer, Judith V; Burnham, Katherine E; Burnet, Philip W J; Sharp, Trevor; Harrison, Paul J

    2014-01-01

    D-amino acid oxidase (DAO, DAAO) degrades the NMDA receptor co-agonist D-serine, modulating D-serine levels and thence NMDA receptor function. DAO inhibitors are under development as a therapy for schizophrenia, a disorder involving both NMDA receptor and dopaminergic dysfunction. However, a direct role for DAO in dopamine regulation has not been demonstrated. Here, we address this question in two ways. First, using in situ hybridization and immunohistochemistry, we show that DAO mRNA and immunoreactivity are present in the ventral tegmental area (VTA) of the rat, in tyrosine hydroxylase (TH)-positive and -negative neurons, and in glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes. Second, we show that injection into the VTA of sodium benzoate, a DAO inhibitor, increases frontal cortex extracellular dopamine, as measured by in vivo microdialysis and high performance liquid chromatography. Combining sodium benzoate and D-serine did not enhance this effect, and injection of D-serine alone affected dopamine metabolites but not dopamine. These data show that DAO is expressed in the VTA, and suggest that it impacts on the mesocortical dopamine system. The mechanism by which the observed effects occur, and the implications of these findings for schizophrenia therapy, require further study. PMID:24822045

  6. Structural characterization of CalO2: A putative orsellinic acid P450 oxidase in the calicheamicin biosynthetic

    SciTech Connect

    McCoy, Jason G.; Johnson, Heather D.; Singh, Shanteri; Bingman, Craig A.; Lei, In-Kyoung; Thorson, Jon S.; Phillips, Jr., George N.

    2009-08-13

    Although bacterial iterative Type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products remains poorly understood. Toward this goal, we report the X-ray structure determination at 2.5 A resolution and preliminary characterization of the putative orsellenic acid P450 oxidase (CalO2) involved in calicheamicin biosynthesis. These studies represent the first crystal structure for a P450 involved in modifying a bacterial iterative Type I polyketide product and suggest the CalO2-catalyzed step may occur after CalO3-catalyzed iodination and may also require a coenzyme A- (CoA) or acyl carrier protein- (ACP) bound substrate. Docking studies also reveal a putative docking site within CalO2 for the CLM orsellinic acid synthase (CalO5) ACP domain which involves a well-ordered helix along the CalO2 active site cavity that is unique compared with other P450 structures.

  7. Modulation of NADPH oxidase activation in cerebral ischemia/reperfusion injury in rats.

    PubMed

    Genovese, Tiziana; Mazzon, Emanuela; Paterniti, Irene; Esposito, Emanuela; Bramanti, Placido; Cuzzocrea, Salvatore

    2011-02-01

    NADPH oxidase is a major complex that produces reactive oxygen species (ROSs) during the ischemic period and aggravates brain damage and cell death after ischemic injury. Although many approaches have been tested for preventing production of ROSs by NADPH oxidase in ischemic brain injury, the regulatory mechanisms of NADPH oxidase activity after cerebral ischemia are still unclear. The aim of this study is identifying apocynin as a critical modulator of NADPH oxidase and elucidating its role as a neuroprotectant in an experimental model of brain ischemia in rat. Treatment of apocynin 5min before of reperfusion attenuated cerebral ischemia in rats. Administration of apocynin showed marked reduction in infarct size compared with that of control rats. Medial carotid artery occlusion (MCAo)-induced cerebral ischemia was also associated with an increase in, nitrotyrosine formation, as well as IL-1β expression, IκB degradation and ICAM expression in ischemic regions. These expressions were markedly inhibited by the treatment of apocynin. We also demonstrated that apocynin reduces levels of apoptosis (TUNEL, Bax and Bcl-2 expression) resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. This new understanding of apocynin induced adaptation to ischemic stress and inflammation could suggest novel avenues for clinical intervention during ischemic and inflammatory diseases. PMID:21138737

  8. Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

    PubMed Central

    Moomaw, Ellen W.; Hoffer, Eric; Moussatche, Patricia; Salerno, John C.; Grant, Morgan; Immelman, Bridget; Uberto, Richard; Ozarowski, Andrew; Angerhofer, Alexander

    2013-01-01

    Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site. PMID:23469254

  9. 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.

  10. Improving Glyphosate Oxidation Activity of Glycine Oxidase from Bacillus cereus by Directed Evolution

    PubMed Central

    Zhan, Tao; Zhang, Kai; Chen, Yangyan; Lin, Yongjun; Wu, Gaobing; Zhang, Lili; Yao, Pei; Shao, Zongze; Liu, Ziduo

    2013-01-01

    Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg51 mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops. PMID:24223901

  11. Human spermatozoa possess an IL4I1 l-amino acid oxidase with a potential role in sperm function.

    PubMed

    Houston, B; Curry, B; Aitken, R J

    2015-06-01

    Reactive oxygen species (ROS) are known to play an important role in the regulation of human sperm function. In this study, we demonstrate for the first time that human spermatozoa possess interleukin-induced gene 1 (IL4I1), an l-amino acid oxidase (LAAO) which is capable of generating ROS on exposure to aromatic amino acids in the presence of oxygen. The preferred substrates were found to be phenylalanine and tryptophan while the enzyme was located in the acrosomal region and midpiece of these cells. In contrast to equine and bovine spermatozoa, enzyme activity was lost as soon as the spermatozoa became non-viable. On a cell-to-cell basis human spermatozoa were also shown to generate lower levels of hydrogen peroxide than their equine counterparts on exposure to phenylalanine. Stimulation of LAAO activity resulted in the induction of several hallmarks of capacitation including tyrosine phosphorylation of the sperm flagellum and concomitant activation of phospho-SRC expression. In addition, stimulation of LAAO resulted in an increase in the levels of acrosomal exocytosis in both the presence and absence of progesterone stimulation, via mechanisms that could be significantly reversed by the presence of catalase. As is often the case with free radical-mediated phenomena, prolonged exposure of human spermatozoa to phenylalanine resulted in the stimulation of apoptosis as indicated by significant increases in mitochondrial superoxide generation and the activation of intracellular caspases. These results confirm the existence of an LAAO in human spermatozoa with a potential role in driving the redox regulation of sperm capacitation and acrosomal exocytosis. PMID:25767141

  12. The xanthine oxidase inhibitor Febuxostat reduces tissue uric acid content and inhibits injury-induced inflammation in the liver and lung.

    PubMed

    Kataoka, Hiroshi; Yang, Ke; Rock, Kenneth L

    2015-01-01

    Necrotic cell death in vivo induces a robust neutrophilic inflammatory response and the resulting inflammation can cause further tissue damage and disease. Dying cells induce this inflammation by releasing pro-inflammatory intracellular components, one of which is uric acid. Cells contain high levels of intracellular uric acid, which is produced when purines are oxidized by the enzyme xanthine oxidase. Here we test whether a non-nucleoside xanthine oxidase inhibitor, Febuxostat (FBX), can reduce intracellular uric acid levels and inhibit cell death-induced inflammation in two different murine tissue injury models; acid-induced acute lung injury and acetaminophen liver injury. Infiltration of inflammatory cells induced by acid injection into lungs or peritoneal administration of acetaminophen was evaluated by quantification with flow cytometry and tissue myeloperoxidase activity in the presence or absence of FBX treatment. Uric acid levels in serum and tissue were measured before giving the stimuli and during inflammation. The impact of FBX treatment on the peritoneal inflammation caused by the microbial stimulus, zymosan, was also analyzed to see whether FBX had a broad anti-inflammatory effect. We found that FBX reduced uric acid levels in acid-injured lung tissue and inhibited acute pulmonary inflammation triggered by lung injury. Similarly, FBX reduced uric acid levels in the liver and inhibited inflammation in response to acetaminophen-induced hepatic injury. In contrast, FBX did not reduce inflammation to zymosan, and therefore is not acting as a general anti-inflammatory agent. These results point to the potential of using agents like FBX to treat cell death-induced inflammation. PMID:25449036

  13. Lactogenic hormone stimulation and epigenetic control of L-amino acid oxidase expression in lactating mammary glands.

    PubMed

    Fujii, Kazuki; Zhang, Haolin; Usuda, Kento; Watanabe, Gen; Nagaoka, Kentaro

    2015-11-01

    L-amino acid oxidase (LAO), a classic flavoprotein, shows antibacterial activity by producing hydrogen peroxide. LAO exists in many tissues such as salivary gland, thymus, spleen, small intestine and testis. In particular, LAO was highly expressed in mice milk and plays an important factor in innate immunity of mammary glands. However, the mechanism which LAO expression is regulated spatially and temporally in lactating mammary glands has been unclear. In this study, we showed the contribution of lactogenic hormone and epigenetic control on LAO gene expression. In monolayer of mammary epithelial cells, treatment of lactogenic hormone mixture, dexamethasone, insulin and prolactin, did not induce LAO mRNA expression and its promoter activity, even though one of milk protein β-casein expression was stimulated. However, increase of LAO expression was observed when the cells were treated with lactogenic hormones in a 3-dimensional culture. The results of chromatin immunoprecipitation analysis revealed that histone H3K18 acetylation increased and histone H3K27 tri-methylation decreased with lactation, which is associated with a period of high LAO expression. Moreover, the treatment of histone methylation inhibitor (DZNep) as well as histone deacetylation inhibitor (Trichostatine A) induced LAO expression in monolayer of mammary cells. Taken together, this is the first demonstration showing that LAO expression is induced in cell culture, and stimulation of lactogenic hormone and change of histone modification are promising signals to show highly expression of LAO in lactating mammary glands. PMID:25820447

  14. Catalytically active bovine serum amine oxidase bound to fluorescent and magnetically drivable nanoparticles

    PubMed Central

    Sinigaglia, Giulietta; Magro, Massimiliano; Miotto, Giovanni; Cardillo, Sara; Agostinelli, Enzo; Zboril, Radek; Bidollari, Eris; Vianello, Fabio

    2012-01-01

    Novel superparamagnetic surface-active maghemite nanoparticles (SAMNs) characterized by a diameter of 10 ± 2 nm were modified with bovine serum amine oxidase, which used rhodamine B isothiocyanate (RITC) adduct as a fluorescent spacer-arm. A fluorescent and magnetically drivable adduct comprised of bovine serum copper-containing amine oxidase (SAMN–RITC–BSAO) that immobilized on the surface of specifically functionalized magnetic nanoparticles was developed. The multifunctional nanomaterial was characterized using transmission electron microscopy, infrared spectroscopy, mass spectrometry, and activity measurements. The results of this study demonstrated that bare magnetic nanoparticles form stable colloidal suspensions in aqueous solutions. The maximum binding capacity of bovine serum amine oxidase was approximately 6.4 mg g−1 nanoparticles. The immobilization procedure reduced the catalytic activity of the native enzyme to 30% ± 10% and the Michaelis constant was increased by a factor of 2. We suggest that the SAMN–RITC–BSAO complex, characterized by a specific activity of 0.81 IU g−1, could be used in the presence of polyamines to create a fluorescent magnetically drivable H2O2 and aldehydes-producing system. Selective tumor cell destruction is suggested as a potential future application of this system. PMID:22619559

  15. Kinetic evidence that methionine sulfoxide reductase A can reveal its oxidase activity in the presence of thioredoxin.

    PubMed

    Kriznik, Alexandre; Boschi-Muller, Sandrine; Branlant, Guy

    2014-04-15

    The mouse methionine sulfoxide reductase A (MsrA) belongs to the subclass of MsrAs with one catalytic and two recycling Cys corresponding to Cys51, Cys198 and Cys206 in Escherichia coli MsrA, respectively. It was previously shown that in the absence of thioredoxin, the mouse and the E. coli MsrAs, which reduce two mol of methionine-O substrate per mol of enzyme, displays an in vitro S-stereospecific methionine oxidase activity. In the present study carried out with E. coli MsrA, kinetic evidence are presented which show that formation of the second mol of Ac-L-Met-NHMe is rate-limiting in the absence of thioredoxin. In the presence of thioredoxin, the overall rate-limiting step is associated with the thioredoxin-recycling process. Kinetic arguments are presented which support the accumulation of the E. coli MsrA under Cys51 sulfenic acid state in the presence of Trx. Thus, the methionine oxidase activity could be operative in vivo without the action of a regulatory protein in order to block the action of Trx as previously proposed. PMID:24632144

  16. Reduced cytochrome oxidase activity in the retrosplenial cortex after lesions to the anterior thalamic nuclei.

    PubMed

    Mendez-Lopez, Magdalena; Arias, Jorge L; Bontempi, Bruno; Wolff, Mathieu

    2013-08-01

    The anterior thalamic nuclei (ATN) make a critical contribution to hippocampal system functions. Growing experimental work shows that the effects of ATN lesions often resemble those of hippocampal lesions and both markedly reduce the expression of immediate-early gene markers in the retrosplenial cortex, which still appears normal by standard histological means. This study shows that moderate ATN damage was sufficient to produce severe spatial memory impairment as measured in a radial-arm maze. Furthermore, ATN rats exhibited reduced cytochrome oxidase activity in the most superficial cortical layers of the granular retrosplenial cortex, and, to a lesser extent, in the anterior cingulate cortex. By contrast, no change in cytochrome oxidase activity was observed in other limbic cortical regions or in the hippocampal formation. Altogether our results indicate that endogenous long-term brain metabolic capacity within the granular retrosplenial cortex is compromised by even limited ATN damage. PMID:23660649

  17. Effect of smoking on serum xanthine oxidase, malondialdehyde, ascorbic acid and α-tocopherol levels in healthy male subjects

    PubMed Central

    Shah, Ali Akbar; Khand, Fatehuddin; Khand, Tayyab Uddin

    2015-01-01

    Objective: To examine the effect of smoking on serum xanthine oxidase, malondialdehyde, α- tocopherol and ascorbic acid levels in healthy adult male subjects. Methods: This cross-sectional comparative study was carried out at Isra University Hyderabad from July 2012 to December 2012. One hundred and twenty apparently healthy adult male subjects (60 smokers and 60 non-smokers) included in present study, were recruited from Jaindal kot, a small village located midway between Hyderabad and Matiari. Serum samples from smokers and non-smokers were analyzed for xanthine oxidase and malondialdehyde levels by standard kit methods, while for ascorbic acid and alpha- tocopherol by spectrophotometric methods. Results: The mean xanthine oxidase and malondialdehyde levels measured in healthy smokers were 0.30±0.05 mg/dl and 37.50±4.05 µmoles/l respectively as against 0.25±0.04 mg/dl and 19.86±2.21µmoles/l in non-smokers. Both xanthine oxidase and malondialdehyde levels were significantly (p<0.001) raised in healthy smokers than in non-smokers. Likewise, mean vitamin E and vitamin C levels were respectively 0.69±0.37 mg/dl and 0.80±0.16 mg/dl in healthy smokers compared to 1.14±0.43 mg/dl and 1.22±0.29 mg/dl in non-smokers. The concentrations of both these vitamins were significantly (p<0.001) lower in smokers than in non-smokers. Conclusion: The results of present study demonstrate that smoking had significantly increased xanthine oxidase and malondialdehyde levels and decreased vitamins C and E (antioxidants) levels. These findings suggest that smokers have to take additional amounts of vitamins C and E in order to avoid deleterious effects of smoking on their health. PMID:25878632

  18. Coumarins with monoamine oxidase inhibitory activity and antioxidative coumarino-lignans from Hibiscus syriacus.

    PubMed

    Yun, B S; Lee, I K; Ryoo, I J; Yoo, I D

    2001-09-01

    A previously undescribed coumarin and a new coumarino-lignan, together with the known compounds scopoletin and cleomiscosins A, C, and D, have been isolated from the root bark of Hibiscus syriacus, and their structures were assigned on the basis of various spectral studies. The coumarin analogue and scopoletin inhibited monoamine oxidase with moderate IC(50) values. The new coumarino-lignan and cleomiscosin C showed lipid peroxidation inhibitory activity comparable to vitamin E. PMID:11575966

  19. Assessing gibberellins oxidase activity by anion exchange/hydrophobic polymer monolithic capillary liquid chromatography-mass spectrometry.

    PubMed

    Chen, Ming-Luan; Su, Xin; Xiong, Wei; Liu, Jiu-Feng; Wu, Yan; Feng, Yu-Qi; Yuan, Bi-Feng

    2013-01-01

    Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography--mass spectrometry (cLC-MS) method for the sensitive assay of in-vitro recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA1, GA4, GA9, GA20). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-co-divinylbenzene-co-ethylene glycol dimethacrylate)(META-co-DVB-co-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62-0.90 fmol. We determined the kinetic parameters (K m) of recombinant GA3-oxidase in Escherichia coli (E. coli) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA9 to GA4 in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology. PMID:23922762

  20. Assessing Gibberellins Oxidase Activity by Anion Exchange/Hydrophobic Polymer Monolithic Capillary Liquid Chromatography-Mass Spectrometry

    PubMed Central

    Liu, Jiu-Feng; Wu, Yan; Feng, Yu-Qi; Yuan, Bi-Feng

    2013-01-01

    Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography – mass spectrometry (cLC-MS) method for the sensitive assay of in-vitro recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA1, GA4, GA9, GA20). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-co-divinylbenzene-co-ethylene glycol dimethacrylate)(META-co-DVB-co-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62–0.90 fmol. We determined the kinetic parameters (Km) of recombinant GA3-oxidase in Escherichia coli (E. coli) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA9 to GA4 in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology. PMID:23922762

  1. Bile acid receptor TGR5, NADPH Oxidase NOX5-S and CREB Mediate Bile Acid-Induced DNA Damage In Barrett’s Esophageal Adenocarcinoma Cells

    PubMed Central

    Li, Dan; Cao, Weibiao

    2016-01-01

    The mechanisms whereby bile acid reflux may accelerate the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett’s patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA. PMID:27511066

  2. Bile acid receptor TGR5, NADPH Oxidase NOX5-S and CREB Mediate Bile Acid-Induced DNA Damage In Barrett's Esophageal Adenocarcinoma Cells.

    PubMed

    Li, Dan; Cao, Weibiao

    2016-01-01

    The mechanisms whereby bile acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett's patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA. PMID:27511066

  3. Propofol Attenuates Small Intestinal Ischemia Reperfusion Injury through Inhibiting NADPH Oxidase Mediated Mast Cell Activation

    PubMed Central

    Gan, Xiaoliang; Xing, Dandan; Su, Guangjie; Li, Shun; Luo, Chenfang; Irwin, Michael G.; Xia, Zhengyuan; Li, Haobo; Hei, Ziqing

    2015-01-01

    Both oxidative stress and mast cell (MC) degranulation participate in the process of small intestinal ischemia reperfusion (IIR) injury, and oxidative stress induces MC degranulation. Propofol, an anesthetic with antioxidant property, can attenuate IIR injury. We postulated that propofol can protect against IIR injury by inhibiting oxidative stress subsequent from NADPH oxidase mediated MC activation. Cultured RBL-2H3 cells were pretreated with antioxidant N-acetylcysteine (NAC) or propofol and subjected to hydrogen peroxide (H2O2) stimulation without or with MC degranulator compound 48/80 (CP). H2O2 significantly increased cells degranulation, which was abolished by NAC or propofol. MC degranulation by CP further aggravated H2O2 induced cell degranulation of small intestinal epithelial cell, IEC-6 cells, stimulated by tryptase. Rats subjected to IIR showed significant increases in cellular injury and elevations of NADPH oxidase subunits p47phox and gp91phox protein expression, increases of the specific lipid peroxidation product 15-F2t-Isoprostane and interleukin-6, and reductions in superoxide dismutase activity with concomitant enhancements in tryptase and β-hexosaminidase. MC degranulation by CP further aggravated IIR injury. And all these changes were attenuated by NAC or propofol pretreatment, which also abrogated CP-mediated exacerbation of IIR injury. It is concluded that pretreatment of propofol confers protection against IIR injury by suppressing NADPH oxidase mediated MC activation. PMID:26246867

  4. Determination of diamine oxidase in lentil seedlings by enzymic activity and immunoreactivity.

    PubMed

    Federico, R; Angelini, R; Cesta, A; Pini, C

    1985-09-01

    A competitive radioimmunoassay for the quantitation of diamine oxidase (EC 1.4.3.6) from Lens culinaris is reported. Specific antibodies raised in rabbits immunized with a homogeneous preparation of the enzyme were incubated with purified (125)I-enzyme and with either unlabeled diamine oxidase or plant material. Antigen-antibody complexes were isolated from the mixture by incubation with Staphylococcus protein A. The sensitivity of the test was about 5 nanograms in terms of enzyme protein. This assay was applied to the determination of the enzyme in extracts from lentil shoots grown either in the dark or in the light. Diamine oxidase activity and enzyme protein (as determined by radioimmunoassay) were measured during 7 days after germination. Both enzymic activity and enzyme protein declined slowly in the dark and rapidly in the light. These results indicate that fluctuation of the enzymic activity in this organ, both in the light and in the dark, are mediated via changes in the amount of the enzyme protein and not via the action of an inhibitor. PMID:16664402

  5. Effects of dietary carbohydrate on iron metabolism and cytochrome oxidase activity in copper-deficient rats

    SciTech Connect

    Johnson, M.A.; Henderson, J.

    1986-03-01

    The effects of dietary carbohydrate on the metabolism of iron and the activity of cytochrome oxidase were examined in Cu-deficient and Cu-adequate rats. Male rats (n = 36) were fed one of six diets which varied in copper level (Cu-: < 0.6 ppm or Cu+: 8.2 ppm) and carbohydrate type (cornstarch, sucrose or fructose). After 31 days, Cu- rats had 50% more iron in the liver and 38, 30 and 18% less iron in the tibia, spleen and kidneys, respectively, than Cu+ rats. The activity of cytochrome oxidase in the bone marrow, heart, and liver were 59%, 51%, and 43%, respectively, of the levels in Cu/sup +/ rats. The type of dietary carbohydrate significantly affected the development of anemia during copper deficiency. Cu-rats fed cornstarch, sucrose or fructose had hematocrit levels which were 92, 83 or 73%, respectively, of Cu+ rats. Similarly, the levels of iron in the tibias of Cu- rats fed cornstarch, sucrose or fructose were 69, 66 or 54%, respectively, of Cu+ rats. The hematocrit levels of Cu- rats were positively correlated to both tibia iron levels (r = 0.64, p < 0.005) and liver cytochrome oxidase activities (r = 0.50, p < 0.05). Thus, it appears that changes in the metabolism of iron may be involved with the development of anemia in Cu- rats fed fructose or sucrose.

  6. An antifungal gamma-pyrone and xanthones with monoamine oxidase inhibitory activity from Hypericum brasiliense.

    PubMed

    Rocha, L; Marston, A; Kaplan, M A; Stoeckli-Evans, H; Thull, U; Testa, B; Hostettmann, K

    1994-08-01

    A new gamma-pyrone (hyperbrasilone), three known xanthones (1,5-dihydroxyxanthone, 5-hydroxy-1-methoxyxanthone and 6-deoxyjacareubin) and betulinic acid have been isolated from a dichloromethane extract of stems and roots of Hypericum brasiliense. Their structures were established by spectroscopic methods (UV, EI-MS, 1H and 13C NMR) and that of the gamma-pyrone was confirmed by X-ray crystallography. Hyperbrasilone and the xanthones were all antifungal against Cladosporium cucumerinum, while the three xanthones showed differing degrees of inhibition of monoamine oxidase A and B. PMID:7765428

  7. Autophagy Protein Rubicon Mediates Phagocytic NADPH Oxidase Activation in Response to Microbial Infection or TLR Stimulation

    PubMed Central

    Yang, Chul-Su; Lee, Jong-Soo; Rodgers, Mary; Min, Chan-Ki; Lee, June-Yong; Kim, Hee Jin; Lee, Kwang-Hoon; Kim, Chul-Joong; Oh, Byungha; Zandi, Ebrahim; Yue, Zhenyu; Kramnik, Igor; Liang, Chengyu; Jung, Jae U.

    2013-01-01

    Summary Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicon's actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection. PMID:22423966

  8. 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. PMID:24530799

  9. SO-LAAO, a novel L-amino acid oxidase that enables Streptococcus oligofermentans to outcompete Streptococcus mutans by generating H2O2 from peptone.

    PubMed

    Tong, Huichun; Chen, Wei; Shi, Wenyuan; Qi, Fengxia; Dong, Xiuzhu

    2008-07-01

    We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H(2)O(2)) through lactate oxidase activity. In this study, we found that the lox mutant of S. oligofermentans regained the inhibition while growing on peptone-rich plates. Further studies demonstrated that the H(2)O(2) produced on peptone by S. oligofermentans was mainly derived from seven L-amino acids, i.e., L-aspartic acid, L-tryptophan, L-lysine, L-isoleucine, L-arginine, L-asparagine, and L-glutamine, indicating the possible existence of L-amino acid oxidase (LAAO) that can produce H(2)O(2) from L-amino acids. Through searching the S. oligofermentans genome for open reading frames with a conserved flavin adenine dinucleotide binding motif that exists in the known LAAOs, including those of snake venom, fungi, and bacteria, a putative LAAO gene, assigned as aao(So), was cloned and overexpressed in Escherichia coli. The purified protein, SO-LAAO, showed a molecular mass of 43 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and catalyzed H(2)O(2) formation from the seven L-amino acids determined above, thus confirming its LAAO activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known LAAOs in both substrate profile and sequence, suggesting that it could represent a novel LAAO. An aao(So) mutant of S. oligofermentans did lose H(2)O(2) formation from the seven L-amino acids, further verifying its function as an LAAO. Furthermore, the inhibition by S. oligofermentans of S. mutans in a peptone-rich mixed-species biofilm was greatly reduced for the aao(So) mutant, indicating the gene's importance in interspecies competition. PMID:18469105

  10. Enhanced hydrolysis of soluble cellulosic substrates by a metallocellulase with veratryl alcohol-oxidase activity

    SciTech Connect

    Evans, B.R.; Margalt, R.; Woodward, J.

    1995-12-31

    A cellulose enzyme fraction was separated from Trichoderma reesei Pulpzyme HA{trademark}, and its characteristics suggested that it was mainly composed of cellobiohydrolase II (CBH II). The covalent attachment of pentaammineruthenium (III) to this enzyme resulted in threefold and fourfold enhancements of its hydrolytic activity on carboxymethyl cellulose (CMC) and barley {beta}-glucan, respectively, as well as endowing it with veratryl alcohol-oxidase activity. Enhancement of hydrolysis was not affected by addition of tartrate or hydrogen peroxide to the reaction mixture. Both native and pentaammineruthenium modified enzymes had negligible activity on cellobiose and p-nitrophenyl {beta}-cellobioside (PNPC).

  11. Amperometric glucose biosensor with remarkable acid stability based on glucose oxidase entrapped in colloidal gold-modified carbon ionic liquid electrode.

    PubMed

    Liu, Xiaoying; Zeng, Xiandong; Mai, Nannan; Liu, Yong; Kong, Bo; Li, Yonghong; Wei, Wanzhi; Luo, Shenglian

    2010-08-15

    A colloidal gold-modified carbon ionic liquid electrode was constructed by mixing colloidal gold-modified graphite powder with a solid room temperature ionic liquid n-octyl-pyridinium hexafluorophosphate (OPPF(6)). Glucose oxidase (GOD) was entrapped in this composite matrix and maintained its bioactivity well and displayed excellent stability. The effect conditions of pH, applied potential and GOD loading were examined. Especially, the glucose oxidase entrapped in this carbon ionic liquid electrode fully retained its activity upon stressing in strongly acidic conditions (pH 2.0) for over one hour. The proposed biosensor responds to glucose linearly over concentration range of 5.0x10(-6) to 1.2x10(-3) and 2.6x10(-3) to 1.3x10(-2) M, and the detection limit is 3.5x10(-6) M. The response time of the biosensor is fast (within 10s), and the life time is over two months. The effects of electroactive interferents, such as ascorbic acid, uric acid, can be significantly reduced by a Nafion film casting on the surface of resulting biosensor. PMID:20510599

  12. NADH Oxidase Activity of Indoleamine 2,3-Dioxygenase*

    PubMed Central

    Rosell, Federico I.; Kuo, Hsin H.; Mauk, A. Grant

    2011-01-01

    The heme enzyme indoleamine 2,3-dioxygenase (IDO) was found to oxidize NADH under aerobic conditions in the absence of other enzymes or reactants. This reaction led to the formation of the dioxygen adduct of IDO and supported the oxidation of Trp to N-formylkynurenine. Formation of the dioxygen adduct and oxidation of Trp were accelerated by the addition of small amounts of hydrogen peroxide, and both processes were inhibited in the presence of either superoxide dismutase or catalase. Anaerobic reaction of IDO with NADH proceeded only in the presence of a mediator (e.g. methylene blue) and resulted in formation of the ferrous form of the enzyme. We propose that trace amounts of peroxide previously proposed to occur in NADH solutions as well as solid NADH activate IDO and lead to aerobic formation of superoxide and the reactive dioxygen adduct of the enzyme. PMID:21690092

  13. Regulation of Ascorbate Oxidase Expression in Pumpkin by Auxin and Copper 1

    PubMed Central

    Esaka, Muneharu; Fujisawa, Kouichi; Goto, Miwa; Kisu, Yasutomo

    1992-01-01

    Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:16652952

  14. Regulation of ascorbate oxidase expression in pumpkin by auxin and copper.

    PubMed

    Esaka, M; Fujisawa, K; Goto, M; Kisu, Y

    1992-09-01

    Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation. PMID:16652952

  15. Involvement of protein kinase D in Fc gamma-receptor activation of the NADPH oxidase in neutrophils.

    PubMed Central

    Davidson-Moncada, Jan K; Lopez-Lluch, Guillermo; Segal, Anthony W; Dekker, Lodewijk V

    2002-01-01

    Protein kinases involved in the activation of the NADPH oxidase by Fc gamma receptors in neutrophils were studied. Of three different protein kinase C (PKC) inhibitors, Gö 6976 inhibited the NADPH oxidase completely, whereas bisindolylmaleimide I and Ro 31-8220 caused a 70-80% inhibition. Thus a Gö 6976-sensitive, bisindolylmaleimide I/Ro 31-8220-insensitive component contributes to NADPH oxidase activation induced by Fc gamma receptors. Down-regulation of PKC isotypes resulted in inhibition of Fc gamma-receptor-activated NADPH oxidase, but a down-regulation-insensitive component was still present. This component was sensitive to Gö 6976, but insensitive to Ro 31-8220. It has been shown previously that protein kinase D/PKC-mu (PKD) shows this same pharmacology in vitro. We show that PKD is present in neutrophils and that, in contrast with PKC isotypes, PKD is not down-regulated. Therefore PKD may participate in NADPH oxidase activation. To obtain direct evidence for this we adopted an antisense approach. Antisense PKD inhibited NADPH oxidase induced by Fc gamma-receptor stimulation by 50% and the Ro 31-8220-insensitive component in the activation was inhibited by antisense PKD. In vitro kinase assays showed that PKD is activated by presenting IgG-opsonized particles to neutrophils. Furthermore, PKD localizes to the area of particle intake in the cell and phosphorylates two of the three cytosolic components of the NADPH oxidase, p40(phox) and p47(phox). Taken together, these data indicate that Fc gamma receptors engage PKD in the regulation of the NADPH oxidase. PMID:11903052

  16. Molecular mechanism of cell death induced by king cobra (Ophiophagus hannah) venom l-amino acid oxidase.

    PubMed

    Fung, Shin Yee; Lee, Mui Li; Tan, Nget Hong

    2015-03-01

    Snake venom LAAOs have been reported to exhibit a wide range of pharmacological activities, including cytotoxic, edema-inducing, platelet aggregation-inducing/platelet aggregation-inhibiting, bactericidal and antiviral activities. A heat-stable form of l-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom (OH-LAAO) has been shown to exhibit very potent cytotoxicity against human tumorigenic cells but not in their non-tumorigenic counterparts, and the cytotoxicity was due to the apoptosis-inducing effect of the enzyme. In this work, the molecular mechanism of cell death induced by OH-LAAO was investigated. The enzyme exerts its apoptosis-inducing effect presumably via both intrinsic and extrinsic pathways as suggested by the increase in caspase-8 and -9 activities. Oligonucleotide microarray analysis showed that the expression of a total of 178 genes was significantly altered as a result of oxidative stress induced by the hydrogen peroxide generated by the enzyme. Of the 178 genes, at least 27 genes are involved in apoptosis and cell death. These alterations of gene expression was presumably caused by the direct cytotoxic effect of H2O2 generated during the enzymatic reaction, as well as the non-specific oxidative modifications of signaling molecules that eventually lead to apoptosis and cell death. The very substantial up-regulation of cytochrome P450 genes may also contribute to the potent cytotoxic action of OH-LAAO by producing excessive reactive oxygen species (ROS). In conclusion, the potent apoptosis inducing activity of OH-LAAO was likely due to the direct cytotoxic effect of H2O2 generated during the enzymatic reaction, as well as the non-specific oxidation of signalling molecules. PMID:25615711

  17. Cytotoxic L-amino-acid oxidases from Amanita phalloides and Clitocybe geotropa induce caspase-dependent apoptosis

    PubMed Central

    Pišlar, A; Sabotič, J; Šlenc, J; Brzin, J; Kos, J

    2016-01-01

    L-amino-acid oxidases (LAO) purified from fungi induce cell death in various mammalian cells including human tumor cell lines. The mechanism, however, remains poorly understood. In this study, we aimed to define a precise mechanism of cell death induced in Jurkat and MCF7 cancer cell lines by ApLAO and CgLAO, LAOs isolated from Amanita phalloides and Clitocybe geotropa, respectively. Cell death induced by both LAOs is shown to be concentration- and time-dependent, with higher toxic effects in Jurkat cells. LAO activity is required for the cytotoxicity. Detailed study on Jurkat cells further demonstrated that ApLAO and CgLAO both induce the intrinsic mitochondrial pathway of apoptosis, accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species. Treatment with the LAOs resulted in an increased ratio of the expression of proapoptotic Bax to that of antiapoptotic Bcl-2, subsequently leading to the activation of caspase-9 and -3. However, the pancaspase inhibitor, Z-VAD-FMK, did not completely abolish the cell death induced by either ApLAO or CgLAO, suggesting an alternative pathway for LAO-induced apoptosis. Indeed, caspase-8 activity in ApLAO- and CgLAO-treated cells was increased. Further, Fas/FasL (Fas ligand) antagonist caused a slight reduction in toxin-induced cell death, supporting the involvement of ApLAO and CgLAO in death-receptor-mediated apoptosis. These results thus provide new evidence that ApLAO and CgLAO induce apoptosis in Jurkat cells via both the intrinsic and extrinsic pathways, although the significantly higher increase of caspase-9 over caspase-8 activity suggests that it is the intrinsic pathway that is the predominant mode of ApLAO- and CgLAO-induced apoptosis. PMID:27551514

  18. Cytotoxic L-amino-acid oxidases from Amanita phalloides and Clitocybe geotropa induce caspase-dependent apoptosis.

    PubMed

    Pišlar, A; Sabotič, J; Šlenc, J; Brzin, J; Kos, J

    2016-01-01

    L-amino-acid oxidases (LAO) purified from fungi induce cell death in various mammalian cells including human tumor cell lines. The mechanism, however, remains poorly understood. In this study, we aimed to define a precise mechanism of cell death induced in Jurkat and MCF7 cancer cell lines by ApLAO and CgLAO, LAOs isolated from Amanita phalloides and Clitocybe geotropa, respectively. Cell death induced by both LAOs is shown to be concentration- and time-dependent, with higher toxic effects in Jurkat cells. LAO activity is required for the cytotoxicity. Detailed study on Jurkat cells further demonstrated that ApLAO and CgLAO both induce the intrinsic mitochondrial pathway of apoptosis, accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species. Treatment with the LAOs resulted in an increased ratio of the expression of proapoptotic Bax to that of antiapoptotic Bcl-2, subsequently leading to the activation of caspase-9 and -3. However, the pancaspase inhibitor, Z-VAD-FMK, did not completely abolish the cell death induced by either ApLAO or CgLAO, suggesting an alternative pathway for LAO-induced apoptosis. Indeed, caspase-8 activity in ApLAO- and CgLAO-treated cells was increased. Further, Fas/FasL (Fas ligand) antagonist caused a slight reduction in toxin-induced cell death, supporting the involvement of ApLAO and CgLAO in death-receptor-mediated apoptosis. These results thus provide new evidence that ApLAO and CgLAO induce apoptosis in Jurkat cells via both the intrinsic and extrinsic pathways, although the significantly higher increase of caspase-9 over caspase-8 activity suggests that it is the intrinsic pathway that is the predominant mode of ApLAO- and CgLAO-induced apoptosis. PMID:27551514

  19. Coupling in cytochrome c oxidase

    PubMed Central

    Kessler, R. J.; Blondin, G. A.; Zande, H. Vande; Haworth, R. A.; Green, D. E.

    1977-01-01

    Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) can be resolved into an electron transfer complex (ETC) and an ionophore transfer complex (ITC). Coupling requires an interaction between the moving electron in the ETC and a moving, positively charged ionophore-cation adduct in the ITC. The duplex character of cytochrome oxidase facilitates this interaction. The ITC mediates cyclical cation transport. It can be replaced as the coupling partner by the combination of valinomycin and nigericin in the presence of K+ when cytochrome oxidase is incorporated into liposomes containing acidic phospholipids or by the combination of lipid cytochrome c and bile acids in an ITC-resolved preparation of the ETC. Respiratory control can be induced by incorporating cytochrome oxidase into vesicles of unfractionated whole mitochondrial lipid. The activity of the ITC is suppressed by such incorporation and this suppression leads to the emergence of respiratory control. The ionophoroproteins of the ITC can be extracted into organic solvents; some 50% of the total protein of cytochrome oxidase is extractable. The release of free ionophore is achieved by tryptic digestion of the ionophoroprotein. Preliminary to this release the ionophoroprotein is degraded to an ionophoropeptide. Electrogenic ionophores, as well as uncoupler, are liberated by such proteolysis. The ITC contains a set of ionophoroproteins imbedded in a matrix of phospholipid. Images PMID:198794

  20. Bioactive Compounds, Antioxidant, Xanthine Oxidase Inhibitory, Tyrosinase Inhibitory and Anti-Inflammatory Activities of Selected Agro-Industrial By-products

    PubMed Central

    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 IC50 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. PMID:22272095

  1. Effect of architecture on the activity of glucose oxidase/horseradish peroxidase/carbon nanoparticle conjugates.

    PubMed

    Ciaurriz, Paula; Bravo, Ernesto; Hamad-Schifferli, Kimberly

    2014-01-15

    We investigate the activity of glucose oxidase (GOx) together with horseradish peroxidase (HRP) on carbon nanoparticles (CNPs). Because GOx activity relies on HRP, we probe how the arrangement of the enzymes on the CNPs affects enzymatic behavior. Colorimetric assays to probe activity found that the coupling strategy affects activity of the bienzyme-nanoparticle complex. GOx is more prone than HRP to denaturation on the CNP surface, where its activity is compromised, while HRP activity is enhanced when interfaced to the CNP. Thus, arrangements where HRP is directly on the surface of the CNP and GOx is not are more favorable for overall activity. Coverage also influenced activity of the bienzyme complex, but performing the conjugation in the presence of glucose did not improve GOx activity. These results show that the architecture of the assembly is an important factor in optimization of nanoparticle-protein interfaces. PMID:24231087

  2. Regulation of cytochrome c oxidase activity by c-Src in osteoclasts

    PubMed Central

    Miyazaki, Tsuyoshi; Neff, Lynn; Tanaka, Sakae; Horne, William C.; Baron, Roland

    2003-01-01

    The function of the nonreceptor tyrosine kinase c-Src as a plasma membrane–associated molecular effector of a variety of extracellular stimuli is well known. Here, we show that c-Src is also present within mitochondria, where it phosphorylates cytochrome c oxidase (Cox). Deleting the c-src gene reduces Cox activity, and this inhibitory effect is restored by expressing exogenous c-Src. Furthermore, reducing endogenous Src kinase activity down-regulates Cox activity, whereas activating Src has the opposite effect. Src-induced Cox activity is required for normal function of cells that require high levels of ATP, such as mitochondria-rich osteoclasts. The peptide hormone calcitonin, which inhibits osteoclast function, also down-regulates Cox activity. Increasing Src kinase activity prevented the inhibitory effect of calcitonin on Cox activity and osteoclast function. These results suggest that c-Src plays a previously unrecognized role in maintaining cellular energy stores by activating Cox in mitochondria. PMID:12615910

  3. Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization

    PubMed Central

    Wang, Haibo; Jiang, Yanchao; Shi, Dallas; Quilliam, Lawrence A.; Chrzanowska-Wodnicka, Magdalena; Wittchen, Erika S.; Li, Dean Y.; Hartnett, M. Elizabeth

    2014-01-01

    Activation of Rap1 GTPase can improve the integrity of the barrier of the retina pigment epithelium (RPE) and reduce choroidal neovascularization (CNV). Inhibition of NADPH oxidase activation also reduces CNV. We hypothesize that Rap1 inhibits NADPH oxidase-generated ROS and thereby reduces CNV formation. Using a murine model of laser-induced CNV, we determined that reduced Rap1 activity in RPE/choroid occurred with CNV formation and that activation of Rap1 by 2′-O-Me-cAMP (8CPT)-reduced laser-induced CNV via inhibiting NADPH oxidase-generated ROS. In RPE, inhibition of Rap1 by Rap1 GTPase-activating protein (Rap1GAP) increased ROS generation, whereas activation of Rap1 by 8CPT reduced ROS by interfering with the assembly of NADPH oxidase membrane subunit p22phox with NOX4 or cytoplasmic subunit p47phox. Activation of NADPH oxidase with Rap1GAP reduced RPE barrier integrity via cadherin phosphorylation and facilitated choroidal EC migration across the RPE monolayer. Rap1GAP-induced ROS generation was inhibited by active Rap1a, but not Rap1b, and activation of Rap1a by 8CPT in Rap1b−/− mice reduced laser-induced CNV, in correlation with decreased ROS generation in RPE/choroid. These findings provide evidence that active Rap1 reduces CNV by interfering with the assembly of NADPH oxidase subunits and increasing the integrity of the RPE barrier.—Wang, H., Jiang, Y., Shi, D., Quilliam, L. A., Chrzanowska-Wodnicka, M., Wittchen, E. S., Li, D. Y., Hartnett, M. E. Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization. PMID:24043260

  4. Revisitation of the βCl-Elimination Reaction of d-Amino Acid Oxidase

    PubMed Central

    Ghisla, Sandro; Pollegioni, Loredano; Molla, Gianluca

    2011-01-01

    d-Amino acid oxidase (DAAO) from pig has been reported to catalyze the β-elimination of Cl− from βCl-d-alanine via abstraction of the substrate α-H as H+ (“carbanion mechanism”) (Walsh, C. T., Schonbrunn, A., and Abeles, R. H. (1971) J. Biol. Chem. 246, 6855–6866). In view of the fundamental mechanistic importance of this reaction and of the recent reinterpretation of the DAAO dehydrogenation step as occurring via a hydride mechanism, we reinvestigated the elimination reaction using yeast DAAO. That enzyme catalyzes the same reactions as the pig enzyme but with a much higher efficiency and a substantially different kinetic behavior. The reaction is initiated by a very rapid and fully reversible dehydrogenation step. This leads to an equilibrium (kon ≈ kreverse) between the complexes of oxidized enzyme-βCl-d-alanine and reduced enzyme-βCl-iminopyruvate. In the presence of O2 the latter complex can partition between an oxidative half-reaction and elimination of Cl−, which proceeds at a rate of ≈50 s−1. This step forms a complex between oxidized enzyme and enamine that is characterized by a charge transfer absorption (which describes its rates of formation and decay). A minimal scheme that lists relevant steps of the reductive and oxidative half-reactions and elimination pathways along with the estimate of the corresponding rate constants is presented. β-Elimination of Cl− is proposed to originate at the locus of the enzyme-βCl-iminopyruvate complex. A chemical mechanism that can account for elimination is discussed in detail. PMID:21949129

  5. Effect of Soy Sauce on Serum Uric Acid Levels in Hyperuricemic Rats and Identification of Flazin as a Potent Xanthine Oxidase Inhibitor.

    PubMed

    Li, Huipin; Zhao, Mouming; Su, Guowan; Lin, Lianzhu; Wang, Yong

    2016-06-15

    This is the first report on the ability of soy sauce to effectively reduce the serum uric acid levels and xanthine oxidase (XOD) activities of hyperuricemic rats. Soy sauce was partitioned sequentially into ethyl acetate and water fractions. The ethyl acetate fraction with strong XOD inhibition effect was purified further. On the basis of xanthine oxidase inhibitory (XOI) activity-guided purification, nine compounds including 3,4-dihydroxy ethyl cinnamate, diisobutyl terephthalate, harman, daidzein, flazin, catechol, thymine, genistein, and uracil were obtained. It was the first time that 3,4-dihydroxy ethyl cinnamate and diisobutyl terephthalate had been identified from soy sauce. Flazin with hydroxymethyl furan ketone group at C-1 and carboxyl at C-3 exhibited the strongest XOI activity (IC50 = 0.51 ± 0.05 mM). According to fluorescence quenching and molecular docking experiments, flazin could enter into the catalytic center of XOD to interact with Lys1045, Gln1194, and Arg912 mainly by hydrophobic forces and hydrogen bonds. Flazin, catechol, and genistein not only were potent XOD inhibitors but also held certain antioxidant activities. According to ADME (absorption, distribution, metabolism, and excretion) simulation in silico, flazin had good oral bioavailability in vivo. PMID:27181598

  6. Regulation of cytochrome c- and quinol oxidases, and piezotolerance of their activities in the deep-sea piezophile Shewanella violacea DSS12 in response to growth conditions.

    PubMed

    Ohke, Yoshie; Sakoda, Ayaka; Kato, Chiaki; Sambongi, Yoshihiro; Kawamoto, Jun; Kurihara, Tatsuo; Tamegai, Hideyuki

    2013-01-01

    The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O2 concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O2 concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O2 concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments. PMID:23832349

  7. 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.

  8. Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

    SciTech Connect

    Mo Yiqun; Wan Rong; Chien Sufan; Tollerud, David J.; Zhang Qunwei

    2009-04-15

    Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O{sub 2}{sup {center_dot}}{sup -} generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67{sup phox} siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91{sup phox} knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47{sup phox}, p67{sup phox} and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67{sup phox} siRNA. Exposure of MPMVEC obtained from gp91{sup phox} knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly

  9. Treatment with polyamine oxidase inhibitor reduces microglial activation and limits vascular injury in ischemic retinopathy.

    PubMed

    Patel, C; Xu, Z; Shosha, E; Xing, J; Lucas, R; Caldwell, R W; Caldwell, R B; Narayanan, S P

    2016-09-01

    Retinal vascular injury is a major cause of vision impairment in ischemic retinopathies. Insults such as hyperoxia, oxidative stress and inflammation contribute to this pathology. Previously, we showed that hyperoxia-induced retinal neurodegeneration is associated with increased polyamine oxidation. Here, we are studying the involvement of polyamine oxidases in hyperoxia-induced injury and death of retinal vascular endothelial cells. New-born C57BL6/J mice were exposed to hyperoxia (70% O2) from postnatal day (P) 7 to 12 and were treated with the polyamine oxidase inhibitor MDL 72527 or vehicle starting at P6. Mice were sacrificed after different durations of hyperoxia and their retinas were analyzed to determine the effects on vascular injury, microglial cell activation, and inflammatory cytokine profiling. The results of this analysis showed that MDL 72527 treatment significantly reduced hyperoxia-induced retinal vascular injury and enhanced vascular sprouting as compared with the vehicle controls. These protective effects were correlated with significant decreases in microglial activation as well as levels of inflammatory cytokines and chemokines. In order to model the effects of polyamine oxidation in causing microglial activation in vitro, studies were performed using rat brain microvascular endothelial cells treated with conditioned-medium from rat retinal microglia stimulated with hydrogen peroxide. Conditioned-medium from activated microglial cultures induced cell stress signals and cell death in microvascular endothelial cells. These studies demonstrate the involvement of polyamine oxidases in hyperoxia-induced retinal vascular injury and retinal inflammation in ischemic retinopathy, through mechanisms involving cross-talk between endothelial cells and resident retinal microglia. PMID:27239699

  10. Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental diabetes.

    PubMed

    Di Noia, Maria Antonietta; Van Driesche, Sarah; Palmieri, Ferdinando; Yang, Li-Ming; Quan, Shuo; Goodman, Alvin I; Abraham, Nader G

    2006-06-01

    Up-regulation of heme oxygenase (HO-1) by either cobalt protoporphyrin (CoPP) or human gene transfer improves vascular and renal function by several mechanisms, including increases in antioxidant levels and decreases in reactive oxygen species (ROS) in vascular and renal tissue. The purpose of the present study was to determine the effect of HO-1 overexpression on mitochondrial transporters, cytochrome c oxidase, and anti-apoptotic proteins in diabetic rats (streptozotocin, (STZ)-induced type 1 diabetes). Renal mitochondrial carnitine, deoxynucleotide, and ADP/ATP carriers were significantly reduced in diabetic compared with nondiabetic rats (p < 0.05). The citrate carrier was not significantly decreased in diabetic tissue. CoPP administration produced a robust increase in carnitine, citrate, deoxynucleotide, dicarboxylate, and ADP/ATP carriers and no significant change in oxoglutarate and aspartate/glutamate carriers. The increase in mitochondrial carriers (MCs) was associated with a significant increase in cytochrome c oxidase activity. The administration of tin mesoporphyrin (SnMP), an inhibitor of HO-1 activity, prevented the restoration of MCs in diabetic rats. Human HO-1 cDNA transfer into diabetic rats increased both HO-1 protein and activity, and restored mitochondrial ADP/ATP and deoxynucleotide carriers. The increase in HO-1 by CoPP administration was associated with a significant increase in the phosphorylation of AKT and levels of BcL-XL proteins. These observations in experimental diabetes suggest that the cytoprotective mechanism of HO-1 against oxidative stress involves an increase in the levels of MCs and anti-apoptotic proteins as well as in cytochrome c oxidase activity. PMID:16595661

  11. Cr(VI) reduction by gluconolactone and hydrogen peroxide, the reaction products of fungal glucose oxidase: Cooperative interaction with organic acids in the biotransformation of Cr(VI).

    PubMed

    Romo-Rodríguez, Pamela; Acevedo-Aguilar, Francisco Javier; Lopez-Torres, Adolfo; Wrobel, Kazimierz; Wrobel, Katarzyna; Gutiérrez-Corona, J Félix

    2015-09-01

    The Cr(VI) reducing capability of growing cells of the environmental A. tubingensis Ed8 strain is remarkably efficient compared to reference strains A. niger FGSC322 and A. tubingensis NRRL593. Extracellular glucose oxidase (GOX) activity levels were clearly higher in colonies developed in solid medium and in concentrated extracts of the spent medium of liquid cultures of the Ed8 strain in comparison with the reference strains. In addition, concentrated extracts of the spent medium of A. tubingensis Ed8, but not those of the reference strains, exhibited the ability to reduce Cr(VI). In line with this observation, it was found that A. niger purified GOX is capable of mediating the conversion of Cr(VI) to Cr(III) in a reaction dependent on the presence of glucose that is stimulated by organic acids. Furthermore, it was found that a decrease in Cr(VI) may occur in the absence of the GOX enzyme, as long as the reaction products gluconolactone and hydrogen peroxide are present; this conversion of Cr(VI) is stimulated by organic acids in a reaction that generates hydroxyl radicals, which may involve the formation of an intermediate peroxichromate(V) complex. These findings indicated that fungal glucose oxidase acts an indirect chromate reductase through the formation of Cr(VI) reducing molecules, which interact cooperatively with other fungal metabolites in the biotransformation of Cr(VI). PMID:25577697

  12. Spatio-Temporal Detection of the Thiomonas Population and the Thiomonas Arsenite Oxidase Involved in Natural Arsenite Attenuation Processes in the Carnoulès Acid Mine Drainage.

    PubMed

    Hovasse, Agnès; Bruneel, Odile; Casiot, Corinne; Desoeuvre, Angélique; Farasin, Julien; Hery, Marina; Van Dorsselaer, Alain; Carapito, Christine; Arsène-Ploetze, Florence

    2016-01-01

    The acid mine drainage (AMD) impacted creek of the Carnoulès mine (Southern France) is characterized by acid waters with a high heavy metal content. The microbial community inhabiting this AMD was extensively studied using isolation, metagenomic and metaproteomic methods, and the results showed that a natural arsenic (and iron) attenuation process involving the arsenite oxidase activity of several Thiomonas strains occurs at this site. A sensitive quantitative Selected Reaction Monitoring (SRM)-based proteomic approach was developed for detecting and quantifying the two subunits of the arsenite oxidase and RpoA of two different Thiomonas groups. Using this approach combined with FISH and pyrosequencing-based 16S rRNA gene sequence analysis, it was established here for the first time that these Thiomonas strains are ubiquitously present in minor proportions in this AMD and that they express the key enzymes involved in natural remediation processes at various locations and time points. In addition to these findings, this study also confirms that targeted proteomics applied at the community level can be used to detect weakly abundant proteins in situ. PMID:26870729

  13. Spatio-Temporal Detection of the Thiomonas Population and the Thiomonas Arsenite Oxidase Involved in Natural Arsenite Attenuation Processes in the Carnoulès Acid Mine Drainage

    PubMed Central

    Hovasse, Agnès; Bruneel, Odile; Casiot, Corinne; Desoeuvre, Angélique; Farasin, Julien; Hery, Marina; Van Dorsselaer, Alain; Carapito, Christine; Arsène-Ploetze, Florence

    2016-01-01

    The acid mine drainage (AMD) impacted creek of the Carnoulès mine (Southern France) is characterized by acid waters with a high heavy metal content. The microbial community inhabiting this AMD was extensively studied using isolation, metagenomic and metaproteomic methods, and the results showed that a natural arsenic (and iron) attenuation process involving the arsenite oxidase activity of several Thiomonas strains occurs at this site. A sensitive quantitative Selected Reaction Monitoring (SRM)-based proteomic approach was developed for detecting and quantifying the two subunits of the arsenite oxidase and RpoA of two different Thiomonas groups. Using this approach combined with FISH and pyrosequencing-based 16S rRNA gene sequence analysis, it was established here for the first time that these Thiomonas strains are ubiquitously present in minor proportions in this AMD and that they express the key enzymes involved in natural remediation processes at various locations and time points. In addition to these findings, this study also confirms that targeted proteomics applied at the community level can be used to detect weakly abundant proteins in situ. PMID:26870729

  14. A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit.

    PubMed

    Garchery, Cécile; Gest, Noé; Do, Phuc T; Alhagdow, Moftah; Baldet, Pierre; Menard, Guillaume; Rothan, Christophe; Massot, Capucine; Gautier, Hélène; Aarrouf, Jawad; Fernie, Alisdair R; Stevens, Rebecca

    2013-01-01

    The regulation of carbon allocation between photosynthetic source leaves and sink tissues in response to stress is an important factor controlling plant yield. Ascorbate oxidase is an apoplastic enzyme, which controls the redox state of the apoplastic ascorbate pool. RNA interference was used to decrease ascorbate oxidase activity in tomato (Solanum lycopersicum L.). Fruit yield was increased in these lines under three conditions where assimilate became limiting for wild-type plants: when fruit trusses were left unpruned, when leaves were removed or when water supply was limited. Several alterations in the transgenic lines could contribute to the improved yield and favour transport of assimilate from leaves to fruits in the ascorbate oxidase lines. Ascorbate oxidase plants showed increases in stomatal conductance and leaf and fruit sugar content, as well as an altered apoplastic hexose:sucrose ratio. Modifications in gene expression, enzyme activity and the fruit metabolome were coherent with the notion of the ascorbate oxidase RNAi lines showing altered sink strength. Ascorbate oxidase may therefore be a target for strategies aimed at improving water productivity in crop species. PMID:22725103

  15. Cocaine reduces cytochrome oxidase activity in the prefrontal cortex and modifies its functional connectivity with brainstem nuclei

    PubMed Central

    Vélez-Hernández, M.E.; Padilla, E.; Gonzalez-Lima, F.; Jiménez-Rivera, C.A.

    2014-01-01

    Cocaine-induced psychomotor stimulation may be mediated by metabolic hypofrontality and modification of brain functional connectivity. Functional connectivity refers to the pattern of relationships among brain regions, and one way to evaluate this pattern is using interactivity correlations of the metabolic marker cytochrome oxidase among different regions. This is the first study of how repeated cocaine modifies: (1) mean cytochrome oxidase activity in neural areas using quantitative enzyme histochemistry, and (2) functional connectivity among brain regions using inter-correlations of cytochrome oxidase activity. Rats were injected with 15 mg/kg i.p. cocaine or saline for 5 days, which lead to cocaine-enhanced total locomotion. Mean cytochrome oxidase activity was significantly decreased in cocaine-treated animals in the superficial dorsal and lateral frontal cortical association areas Fr2 and Fr3 when compared to saline-treated animals. Functional connectivity showed that the cytochrome oxidase activity of the noradrenergic locus coeruleus and the infralimbic cortex were positively inter-correlated in cocaine but not in control rats. Positive cytochrome oxidase activity inter-correlations were also observed between the dopaminergic substantia nigra compacta and Fr2 and Fr3 areas and the lateral orbital cortex in cocaine-treated animals. In contrast, cytochrome oxidase activity in the interpeduncular nucleus was negatively correlated with that of Fr2, anterior insular cortex, and lateral orbital cortex in saline but not in cocaine groups. After repeated cocaine specific prefrontal areas became hypometabolic and their functional connectivity changed in networks involving noradrenergic and dopaminergic brainstem nuclei. We suggest that this pattern of hypofrontality and altered functional connectivity may contribute to cocaine-induced psychomotor stimulation. PMID:24505625

  16. beta-aminobutyric acid primes an NADPH oxidase-dependent reactive oxygen species production during grapevine-triggered immunity.

    PubMed

    Dubreuil-Maurizi, Carole; Trouvelot, Sophie; Frettinger, Patrick; Pugin, Alain; Wendehenne, David; Poinssot, Benoît

    2010-08-01

    The molecular mechanisms underlying the process of priming are poorly understood. In the present study, we investigated the early signaling events triggered by beta-aminobutyric acid (BABA), a well-known priming-mediated plant resistance inducer. Our results indicate that, in contrast to oligogalacturonides (OG), BABA does not elicit typical defense-related early signaling events nor defense-gene expression in grapevine. However, in OG-elicited cells pretreated with BABA, production of reactive oxygen species (ROS) and expression of the respiratory-burst oxidase homolog RbohD gene were primed. In response to the causal agent of downy mildew Plasmopara viticola, a stronger ROS production was specifically observed in BABA-treated leaves. This process was correlated with an increased resistance. The NADPH oxidase inhibitor diphenylene iodonium (DPI) abolished this primed ROS production and reduced the BABA-induced resistance (BABA-IR). These results suggest that priming of an NADPH oxidase-dependent ROS production contributes to BABA-IR in the Vitis-Plasmopara pathosystem. PMID:20615112

  17. Diamine oxidase plasma activities after treatment with heparin and jejunal morphometry in untreated coeliac disease.

    PubMed Central

    Corazza, G R; Ginaldi, L; Falasca, A; Strocchi, A; Rossi, C A; Quaglino, D; Gasbarrini, G

    1989-01-01

    Diamine oxidase plasma concentrations after treatment with heparin were measured and compared with the surface to volume ratio of jejunal biopsy samples assessed by a morphometric technique in patients with untreated and treated coeliac disease and in biopsied controls. As expected, enzyme activity was significantly lower in patients with untreated coeliac disease than in patients on a gluten-free diet and in biopsied controls. No difference was found between treated patients and biopsied controls. There was a significant overall correlation between plasma enzyme activity and surface to volume ratio of jejunal mucosa, although two untreated patients without an overt malabsorption syndrome but with a very low surface to volume ratio had normal enzyme activity. This study shows that in coeliac disease plasma diamine oxidase activity after treatment with heparin does not always mirror the extent of the jejunal lesions, particularly in those patients with minimal or unrelated symptoms who would benefit most from a valid screening test to identify their condition. PMID:2511229

  18. Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

    SciTech Connect

    Doonan, C.J.; Wilson, H.L.; Rajagopalan, K.V.; Garrett, R.M.; Bennett, B.; Prince, R.C.; George, G.N.

    2009-06-02

    The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme sulfite oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.

  19. Preliminary pharmacological studies on Eugenia uniflora leaves: xanthine oxidase inhibitory activity.

    PubMed

    Schmeda-Hirschmann, G; Theoduloz, C; Franco, L; Ferro, E; de Arias, A R

    1987-11-01

    Eugenia uniflora is widely used in Paraguayan folk medicine. A hydroalcoholic extract of the leaves showed some central nervous system activity in hippocratic screening when given intraperitoneally, but little to no acute or subacute toxicity in doses up to 4200 mg/kg orally in BALB c mice. The LD50 of the extract was 220 mg/kg i.p. in mice. A decoction or infusion of the leaves is recommended for treating gout by native herbalists. The known flavonoids quercitrin, quercetin, myricitrin and myricetin were found to be responsible for the xanthine oxidase inhibitory action of the plant extract. PMID:3437769

  20. Direct spectrophotometric assay of monooxygenase and oxidase activities of mushroom tyrosinase in the presence of synthetic and natural substrates.

    PubMed

    Haghbeen, Kamahldin; Wue Tan, Eng

    2003-01-01

    Alternative substrates were synthesized to allow direct and continuous spectrophotometric assay of both monooxygenase (cresolase) and oxidase (catecholase) activities of mushroom tyrosinase (MT). Using diazo derivatives of phenol, 4-[(4-methoxybenzo)azo]-phenol, 4-[(4-methylphenyl)azo]-phenol, 4-(phenylazo)-phenol, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide, and diazo derivatives of catechol 4-[(4-methylbenzo)azo]-1,2-benzenediol, 4-(phenylazo)-1,2-benzenediol, and 4-[(4-sulfonamido)azo]-1,2 benzenediol (SACat), as substrates allows measurement of the rates of the corresponding enzymatic reactions through recording of the depletion rates of substrates at their lambda(max)(s) with the least interference of the intermediates' or products' absorption. Parallel attempts using natural compounds, p-coumaric acid and caffeic acid, as substrates for assaying both activities of MT were comparable approaches. Based on the ensuing data, the electronic effect of the substituent on the substrate activity and the affinity of the enzyme for the substrate are reviewed. Kinetic parameters extracted from the corresponding Lineweaver-Burk plots and advantages of these substrates over the previously used substrates in similar assays of tyrosinases are also presented. PMID:12479831

  1. Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity

    PubMed Central

    Liu, Jian; Zhou, Jun; Xing, Da

    2012-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination. PMID:22448275

  2. NADPH oxidase activation is required for pentylenetetrazole kindling-induced hippocampal autophagy.

    PubMed

    Zhu, Xinjian; Shen, Kai; Bai, Ying; Zhang, Aifeng; Xia, Zhengrong; Chao, Jie; Yao, Honghong

    2016-05-01

    Growing evidence indicates that alterations in autophagy are present in a variety of neurological disorders, ranging from neurodegenerative diseases to acute neurological insults. Only recently has the role of autophagy in epilepsy started to be recognized. In this study, we used pentylenetetrazole (PTZ) kindling, which provides a model of chronic epilepsy, to investigate the involvement of autophagy in the hippocampus and the possible mechanisms involved. Our western blot results showed that autophagy-related proteins were significantly increased after the mice were fully kindled. In addition, immunofluorescence studies revealed a significant increase in the punctate accumulation of LC3 in the hippocampal CA1 region of fully PTZ-kindled mice. Consistent with the upregulation of ATG proteins and punctate accumulation of LC3 in the hippocampal CA1 region, autophagosomal vacuole formation was observed by an ultrastructural analysis, verifying the presence of a hippocampal autophagic response in PTZ-kindled mice. Increased oxidative stress has been postulated to play an important role in the pathogenesis of a number of neurological diseases, including epilepsy. In this study, we demonstrate that PTZ kindling induced reactive oxygen species (ROS) production and lipid peroxidation, which were accompanied by mitochondrial ultrastructural damage due to the activation of NADPH oxidase. Pharmacological inhibition of NADPH oxidase by apocynin significantly suppressed the oxidative stress and ameliorated the hippocampal autophagy in PTZ-kindled mice. Interestingly, pharmacological induction of autophagy suppressed PTZ-kindling progress and reduced PTZ-kindling-induced oxidative stress while inhibition of autophagy accelerated PTZ kindling progress and increased PTZ-kindling-induced oxidative stress. These results suggest that the oxidative stress induced by NADPH oxidase activation may play a pivotal role in PTZ-kindling process as well as in PTZ kindling-induced hippocampal

  3. Dietary Phenolic Compounds Interfere with the Fate of Hydrogen Peroxide in Human Adipose Tissue but Do Not Directly Inhibit Primary Amine Oxidase Activity

    PubMed Central

    Carpéné, Christian; Hasnaoui, Mounia; Balogh, Balázs; Matyus, Peter; Fernández-Quintela, Alfredo; Rodríguez, Víctor; Mercader, Josep; Portillo, Maria P.

    2016-01-01

    Resveratrol has been reported to inhibit monoamine oxidases (MAO). Many substrates or inhibitors of neuronal MAO interact also with other amine oxidases (AO) in peripheral organs, such as semicarbazide-sensitive AO (SSAO), known as primary amine oxidase, absent in neurones, but abundant in adipocytes. We asked whether phenolic compounds (resveratrol, pterostilbene, quercetin, and caffeic acid) behave as MAO and SSAO inhibitors. AO activity was determined in human adipose tissue. Computational docking and glucose uptake assays were performed in 3D models of human AO proteins and in adipocytes, respectively. Phenolic compounds fully inhibited the fluorescent detection of H2O2 generated during MAO and SSAO activation by tyramine and benzylamine. They also quenched H2O2-induced fluorescence in absence of biological material and were unable to abolish the oxidation of radiolabelled tyramine and benzylamine. Thus, phenolic compounds hampered H2O2 detection but did not block AO activity. Only resveratrol and quercetin partially impaired MAO-dependent [14C]-tyramine oxidation and behaved as MAO inhibitors. Phenolic compounds counteracted the H2O2-dependent benzylamine-stimulated glucose transport. This indicates that various phenolic compounds block downstream effects of H2O2 produced by biogenic or exogenous amine oxidation without directly inhibiting AO. Phenolic compounds remain of interest regarding their capacity to limit oxidative stress rather than inhibiting AO. PMID:26881018

  4. Monoamine oxidase B and free radical scavenging activities of natural flavonoids in Melastoma candidum D. Don.

    PubMed

    Lee, M H; Lin, R D; Shen, L Y; Yang, L L; Yen, K Y; Hou, W C

    2001-11-01

    Monoamine oxidase type B (MAO-B) activity and free radicals are elevated in certain neurological diseases. Four natural flavonoids, quercitrin, isoquercitrin, rutin, and quercetin, were isolated for the first time from the leaves of Melastoma candidum D. Don. They exhibited an inhibitory effect on MAO-B. These potent flavonoids were purified using bioassay-guided fractionation and were separated by Diaion, Sephadex LH-20, and MCI CHP20P columns. The IC(50) values of the four potent flavonoids, quercitrin, isoquercitrin, rutin, and quercetin on monoamine oxidase were 19.06, 11.64, 3.89, and 10.89 microM and enzyme kinetics analysis revealed apparent inhibition constants (K(i)) of 21.01, 2.72, 1.83, and 7.95 microM, respectively, on the substrate, benzylamine. The four potent compounds also exhibited hydroxyl radical scavenging activity as determined using a spin trapping electron spin resonance method. This suggests that the four flavonoids from M. candidum possess both MAO-B inhibitory and free radical scavenging activities. These important properties may be used for preventing some neurodegenerative diseases in the future. PMID:11714358

  5. Activity-stability relationships revisited in blue oxidases catalyzing electron transfer at extreme temperatures.

    PubMed

    Roulling, Frédéric; Godin, Amandine; Cipolla, Alexandre; Collins, Tony; Miyazaki, Kentaro; Feller, Georges

    2016-09-01

    Cuproxidases are a subset of the blue multicopper oxidases that catalyze the oxidation of toxic Cu(I) ions into less harmful Cu(II) in the bacterial periplasm. Cuproxidases from psychrophilic, mesophilic, and thermophilic bacteria display the canonical features of temperature adaptation, such as increases in structural stability and apparent optimal temperature for activity with environmental temperature as well as increases in the binding affinity for catalytic and substrate copper ions. In contrast, the oxidative activities at 25 °C for both the psychrophilic and thermophilic enzymes are similar, suggesting that the nearly temperature-independent electron transfer rate does not require peculiar adjustments. Furthermore, the structural flexibilities of both the psychrophilic and thermophilic enzymes are also similar, indicating that the firm and precise bindings of the four catalytic copper ions are essential for the oxidase function. These results show that the requirements for enzymatic electron transfer, in the absence of the selective pressure of temperature on electron transfer rates, produce a specific adaptive pattern, which is distinct from that observed in enzymes possessing a well-defined active site and relying on conformational changes such as for the induced fit mechanism. PMID:27315165

  6. Estrogen Attenuates Ischemic Oxidative Damage via an ERα-Mediated Inhibition of NADPH Oxidase Activation

    PubMed Central

    Zhang, Quan-Guang; Raz, Limor; Wang, Ruimin; Han, Dong; De Sevilla, Liesl; Yang, Fang; Vadlamudi, Ratna K.; Brann, Darrell W.

    2009-01-01

    The goal of this study was to elucidate the mechanisms of 17β-estradiol (E2) antioxidant and neuroprotective actions in stroke. The results reveal a novel extranuclear receptor-mediated antioxidant mechanism for E2 during stroke, as well as a hypersensitivity of the CA3/CA4 region to ischemic injury after prolonged hypoestrogenicity. E2 neuroprotection was shown to involve a profound attenuation of NADPH oxidase activation and superoxide production in hippocampal CA1 pyramidal neurons after stroke, an effect mediated by extranuclear ERα-mediated nongenomic signaling, involving Akt activation and subsequent phosphorylation/inactivation of Rac1, a factor critical for activation of NOX2 NADPH oxidase. Intriguingly, E2 nongenomic signaling, antioxidant action and neuroprotection in the CA1 region were lost after long-term E2 deprivation; and this loss was tissue-specific, as the uterus remained responsive to E2. Correspondingly, a remarkable loss of ERα, but not ERβ, was observed in the CA1 following long-term E2 deprivation, with no change observed in the uterus. As a whole, the study reveals a novel, membrane-mediated antioxidant mechanism in neurons by E2, provides support and mechanistic insights for a “critical period” of E2 replacement in the hippocampus, and demonstrates a heretofore unknown hypersensitivity of the CA3/CA4 to ischemic injury after prolonged hypoestrogenicity. PMID:19889994

  7. Histamine content, diamine oxidase activity and histamine methyltransferase activity in human tissues: fact or fictions?

    PubMed

    Hesterberg, R; Sattler, J; Lorenz, W; Stahlknecht, C D; Barth, H; Crombach, M; Weber, D

    1984-04-01

    To understand the role of histamine in the aetiology and pathogenesis of human diseases reliable data are urgently needed for the histamine content and for the activities of histamine-forming and -inactivating enzymes in human tissues. In order to make a substantial progress toward this aim a tissue-sampling programme during surgical interventions was carefully conceived and conducted. From March 1982 until January 1983 106 tissue specimens were taken from 56 patients who underwent surgery. Only healthy tissues, not injured or oedematous, and without adherent structures were taken by only one surgeon who was interested in this research and experienced in tissue preparation procedures in biochemistry. The times of 'warm' ischaemia during the operative procedures were visually estimated, the times between resection of the organs or specimens and deep-freezing of the tissues were precisely recorded. Compared to previous work in the literature and especially to our own work using the same assays for determination higher histamine contents were found in this study in most of the tissues, in particular in the gastrointestinal tract. Also the diamine oxidase activities were considerably higher in many organs, e.g. 3-4 times higher in the gastrointestinal tract when compared with those in publications of our group who used always the same analytical test. However, the histamine methyltransferase activities in this study were not at variance to those determined in previous investigations. Many of them were reported in this communication for the first time. Since the methods for histamine determination and those for measuring enzymic activities were not different in this study and in previous communications of our group we are convinced that the optimized tissue-sampling and -preparation techniques were responsible for the higher values in this communication. But the problem of the 'warm' ischaemia period could not be solved by sample-taking procedures of this type during

  8. Enhanced Expression and Activation of the Alternative Oxidase during Infection of Arabidopsis with Pseudomonas syringae pv tomato1

    PubMed Central

    Simons, Bert H.; Millenaar, Frank F.; Mulder, Lonneke; Van Loon, Leendert C.; Lambers, Hans

    1999-01-01

    Cyanide-resistant (“alternative”) respiration was studied in Arabidopsis during incompatible and compatible infection with Pseudomonas syringae pv tomato DC3000. Total leaf respiration increased as the leaves became necrotic, as did the cyanide-resistant component that was sensitive to salicylhydroxamic acid. Infiltration of leaves with an avirulent strain rapidly induced alternative oxidase (AOX) mRNA, whereas the increase was delayed in the compatible combination. The increase in mRNA correlated with the increase in AOX protein. Increased expression was confined to the infected leaves, in contrast to the pathogenesis-related protein-1, which was induced systemically. Virtually all of the AOX protein was in the reduced (high-activity) form. Using transgenic NahG and mutant npr1-1 and etr1-1 plants, we established that the rapid induction of the AOX was associated with necrosis and that ethylene, but not salicylic acid, was required for its induction. Increased pyruvate levels in the infected leaves suggested that increased substrate levels were respired through the alternative pathway; however, in the control leaves and the infected leaves, respiration was not inhibited by salicylhydroxamic acid alone. Increased respiration appeared to be associated primarily with symptom expression rather than resistance reactions. PMID:10364404

  9. Listeriolysin O suppresses phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection.

    PubMed

    Lam, Grace Y; Fattouh, Ramzi; Muise, Aleixo M; Grinstein, Sergio; Higgins, Darren E; Brumell, John H

    2011-12-15

    The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L. monocytogenes infection, a response that would adversely affect pathogen survival. However, secretion of LLO inhibits the NADPH oxidase by preventing its localization to phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O, a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit the NADPH oxidase. Our studies demonstrate that while the PLCs induce antimicrobial NADPH oxidase activity, this effect is alleviated by the pore-forming activity of LLO. Therefore, the combined activities of PLCs and LLO on membrane lysis and the inhibitory effects of LLO on NADPH oxidase activity allow L. monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst. PMID:22177565

  10. THREE POLYPHENOL OXIDASES FROM RED CLOVER (TRIFOLIUM PRATENSE) DIFFER IN ENZYMATIC ACTIVITIES AND ACTIVATION PROPERTIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidases (PPOs) oxidize o-diphenols to o-quinones, which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense L.). Production of o-quinones in red clover inhibits postharvest proteolysis during the ensiling proces...

  11. A cerium method for the ultracytochemical localization of monoamine oxidase activity.

    PubMed

    Fujimoto, T; Inomata, K; Ogawa, K

    1982-01-01

    A cytochemical method based on the complex formation between cerous ions and hydrogen peroxide is described for the ultrastructural localization of monoamine oxidase (MAO). First, the residual MAO activity after fixation was measured by a radiochemical assay technique and was found to be sufficiently retained for cytochemical detection. Although the Tris buffer used in the present method was found to be inhibitory to MAO, considerable activity was still retained after fixation and incubation in Tris. MAO activity, detected as precipitates of cerium perhydroxide, was observed in the mitochondrial outer compartment, mitochondrial cristae and perinuclear space of myocardial cells and endothelial cells of rat heart. MAO activity was also found along the plasma membrane of capillary endothelia. Omission of substrate from the incubation medium or pre-incubation with pargyline, a specific MAO inhibitor, drastically reduced the amount of deposits. The present cerium method seems promising because of its reproducibility and the high electron density of the reaction products. PMID:6174485

  12. Boosting the oxidase mimicking activity of nanoceria by fluoride capping: rivaling protein enzymes and ultrasensitive F(-) detection.

    PubMed

    Liu, Biwu; Huang, Zhicheng; Liu, Juewen

    2016-07-14

    Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ∼15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F(-) capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F(-) in water and in toothpastes, while no other tested anions can achieve the activity enhancement. PMID:27378306

  13. Cigarette smoke-induced kinin B1 receptor promotes NADPH oxidase activity in cultured human alveolar epithelial cells.

    PubMed

    Talbot, Sébastien; Lin, James Chi-Jen; Lahjouji, Karim; Roy, Jean-Philippe; Sénécal, Jacques; Morin, André; Couture, Réjean

    2011-07-01

    Pulmonary inflammation is an important pathological feature of tobacco smoke-related lung diseases. Kinin B1 receptor (B1R) is up-regulated in the rat trachea chronically exposed to cigarette-smoke. This study aimed at determining (1) whether exposure to total particulate matter of the cigarette smoke (TPM) can induce B1R in human alveolar epithelial A549 cells, (2) the mechanism of B1R induction, (3) the functionality of de novo synthesized B1R, and (4) the role of B1R in TPM-induced increase of superoxide anion (O₂(●⁻)) level. Results show that A549 cells exposed to 10 μg/ml TPM increased O₂(●⁻) level along with B1R (protein and mRNA) and IL-1β mRNA. In contrast, B2R and TNF-α mRNA were not affected by TPM. The increasing effect of TPM on O₂(●⁻) level was not significantly affected by the B1R antagonist SSR240612. TPM-increased B1R mRNA was prevented by co-treatments with N-acetyl-l-cysteine (potent antioxidant), diphenyleneiodonium (NADPH oxidase inhibitor), IL-1Ra (interleukin-1R antagonist) and SN-50 (specific inhibitor of NF-kB activation) but not by pentoxifylline (TNF-α release inhibitor), indomethacin and niflumic acid (COX-1 and -2 inhibitors). Stimulation of B1R with a selective agonist (des-Arg⁹-BK, 10 μM; 30 min) increased O₂(●⁻)production which was prevented by apocynin and diphenyleneiodonium (NADPH oxidase inhibitors). Data suggest that the increased expression of B1R by TPM in A549 cells is mediated by oxidative stress, IL-1β and NF-kB but not by cyclooxygenases or TNF-α. The amplification of O₂(●⁻) levels via the activation of B1R-NADPH oxidase may exacerbate pulmonary inflammation and contribute to the chronicity of tobacco smoke-related lung diseases. PMID:21600945

  14. Temperature dependence of the activity of polyphenol peroxidases and polyphenol oxidases in modern and buried soils

    NASA Astrophysics Data System (ADS)

    Yakushev, A. V.; Kuznetsova, I. N.; Blagodatskaya, E. V.; Blagodatsky, S. A.

    2014-05-01

    Under conditions of the global climate warming, the changes in the reserves of soil humus depend on the temperature sensitivities of polyphenol peroxidases (PPPOs) and polyphenol oxidases (PPOs). They play an important role in lignin decomposition, mineralization, and humus formation. The temperature dependence of the potential enzyme activity in modern and buried soils has been studied during incubation at 10 or 20°C. The experimental results indicate that it depends on the availability of the substrate and the presence of oxygen. The activity of PPOs during incubation in the absence of oxygen for two months decreases by 2-2.5 times, which is balanced by an increase in the activity of PPPOs by 2-3 times. The increase in the incubation temperature to 20°C and the addition of glucose accelerates this transition due to the more abrupt decrease in the activity of PPOs. The preincubation of the soil with glucose doubles the activity of PPPOs but has no significant effect on the activity of PPOs. The different effects of temperature on two groups of the studied oxidases and the possibility of substituting enzymes by those of another type under changing aeration conditions should be taken into consideration in predicting the effect of the climate warming on the mineralization of the soil organic matter. The absence of statistically significant differences in the enzymatic activity between the buried and modern soil horizons indicates the retention by the buried soil of some of its properties (soil memory) and the rapid restoration of high enzymatic activity during the preincubation.

  15. Boosting the oxidase mimicking activity of nanoceria by fluoride capping: rivaling protein enzymes and ultrasensitive F- detection

    NASA Astrophysics Data System (ADS)

    Liu, Biwu; Huang, Zhicheng; Liu, Juewen

    2016-07-01

    Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement.Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement. Electronic supplementary information (ESI) available: Methods, TMB oxidation kinetics and control experiments. See DOI: 10.1039/c6nr02730j

  16. Dithiocarbamates are teratogenic to developing zebrafish through inhibition of lysyl oxidase activity

    SciTech Connect

    Boxtel, Antonius L. van; Kamstra, Jorke H.; Fluitsma, Donna M.; Legler, Juliette

    2010-04-15

    Dithiocarbamates (DTCs) are a class of compounds that are extensively used in agriculture as pesticides. As such, humans and wildlife are undoubtedly exposed to these chemicals. Although DTCs are thought to be relatively safe due to their short half lives, it is well established that they are teratogenic to vertebrates, especially to fish. In zebrafish, these teratogenic effects are characterized by distorted notochord development and shortened anterior to posterior axis. DTCs are known copper (Cu) chelators but this does not fully explain the observed teratogenic effects. We show here that DTCs cause malformations in zebrafish that highly resemble teratogenic effects observed by direct inhibition of a group of cuproenzymes termed lysyl oxidases (LOX). Additionally, we demonstrate that partial knockdown of three LOX genes, lox, loxl1 and loxl5b, sensitizes the developing embryo to DTC exposure. Finally, we show that DTCs directly inhibit zebrafish LOX activity in an ex vivo amine oxidase assay. Taken together, these results provide the first evidence that DTC induced teratogenic effects are, at least in part, caused by direct inhibition of LOX activity.

  17. Exogenous methyl jasmonate regulates cytokinin content by modulating cytokinin oxidase activity in wheat seedlings under salinity.

    PubMed

    Avalbaev, Azamat; Yuldashev, Ruslan; Fedorova, Kristina; Somov, Kirill; Vysotskaya, Lidiya; Allagulova, Chulpan; Shakirova, Farida

    2016-02-01

    The treatment of 4-days-old wheat seedlings with methyl jasmonate (MeJA) in concentration optimal for their growth (0.1 μM) resulted in a rapid transient almost two-fold increase in the level of cytokinins (CKs). MeJA-induced accumulation of CKs was due to inhibition of both cytokinin oxidase (CKX) (cytokinin oxidase/dehydrogenase, EC 1.5.99.12) gene expression and activity of this enzyme. Pretreatment of wheat seedlings with MeJA decreased the growth-retarding effect of sodium chloride salinity and accelerated growth recovery after withdrawal of NaCl from the incubation medium. We speculate that this protective effect of the hormone might be due to MeJA's ability to prevent the salinity-induced decline in CK concentration that was caused by inhibition of gene expression and activity of CKX in wheat seedlings. The data might indicate an important role for endogenous cytokinins in the implementation of growth-promoting and protective effects of exogenous MeJA application on wheat plants. PMID:26748373

  18. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    PubMed Central

    Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir; Orr, William C.; Sohal, Rajindar S.

    2010-01-01

    The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H2O2 generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle- and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc and VIII, indicated that the abundance these polypeptides decreased, ranging from 11 to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology. PMID:20833144

  19. Induction of hepatoma carcinoma cell apoptosis through activation of the JNK-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-ROS self-driven death signal circuit.

    PubMed

    Zeng, Ke-Wu; Song, Fang-Jiao; Wang, Ying-Hong; Li, Ning; Yu, Qian; Liao, Li-Xi; Jiang, Yong; Tu, Peng-Fei

    2014-10-28

    As an efficient method for inducing tumor cell apoptosis, ROS can be constantly formed and accumulated in NADPH oxidase overactivated-cells, resulting in further mitochondrial membrane damage and mitochondria-dependent apoptosis. In addition, JNK mitogen-activated protein kinase (JNK MAPK) signal also acts as a vital candidate pathway for inducing tumor cell apoptosis by targeting mitochondrial death pathway. However, the relationship between NADPH oxidase-ROS and JNK MAPK signal still remains unclear. Here, we discovered a novel self-driven signal circuit between NADPH oxidase-ROS and JNK MAPK, which was induced by a cytotoxic steroidal saponin (ASC) in hepatoma carcinoma cells. NADPH oxidase-dependent ROS production was markedly activated by ASC and directly led to JNK MAPK activation. Moreover, antioxidant, NADPH oxidase inhibitor and specific knock-out for p47 subunit of NADPH oxidase could effectively block NADPH oxidase-ROS-dependent JNK activation, suggesting that NADPH oxidase is an upstream regulator of JNK MAPK. Conversely, a specific JNK inhibitor could inhibit ASC-induced NADPH oxidase activation and down-regulate ROS levels as well, indicating that JNK might also regulate NADPH oxidase activity to some extent. These observations indicate that NADPH oxidase and JNK MAPK activate each other as a signal circuit. Furthermore, drug pretreatment experiments with ASC showed this signal circuit operated continuously via a self-driven mode and finally induced apoptosis in hepatoma carcinoma cells. Taken together, we provide a proof for inducing hepatoma carcinoma cell apoptosis by activating the JNK-NADPH oxidase-ROS-dependent self-driven signal circuit pathway. PMID:25064608

  20. (/sup 11/C)clorgyline and (/sup 11/C)-L-deprenyl and their use in measuring functional monoamine oxidase activity in the brain using positron emission tomography

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1986-04-17

    This invention involves a new strategy for imaging the activity of the enzyme monoamine oxidase in the living body by using /sup 11/C-labeled enzyme inhibitors which bind irreversibly to an enzyme as a result of catalysis. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  1. Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia

    PubMed Central

    2010-01-01

    Background Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton. Results Here, we in addition to toxins use conditional expression of the major actin regulatory protein LIM kinase-1 (LIMK1), and shRNA knock-down of cofilin to modulate the cellular F/G-actin ratio in the Ra2 microglia cell line, and we use Fluorescence Recovery after Photobleaching (FRAP) in β-actin-YFP-transduced cells to obtain a dynamic measure of actin recovery rates (actin turn-over rates) in different F/G-actin states of the actin cytoskeleton. Our data demonstrate that stimulated NADPH oxidase function was severely impaired only at extreme actin recovery rates and F/G-actin ratios, and surprisingly, that any moderate changes of these parameters of the actin cytoskeleton invariably resulted in an increased NADPH oxidase activity. Conclusion moderate actin polymerization and depolymerization both increase the FMLP and PMA-stimulated NADPH oxidase activity of microglia, which is directly correlated with neither actin recovery rate nor F/G- actin ratio. Our results indicate that NADPH oxidase functions in an enhanced state of activity in stimulated phagocytes despite widely different states of the actin cytoskeleton. PMID:20825680

  2. Oxidase, superoxide dismutase, and hydrogen peroxide reductase activities of methanobactin from types I and II methanotrophs.

    PubMed

    Choi, Dong W; Semrau, Jeremy D; Antholine, William E; Hartsel, Scott C; Anderson, Ryan C; Carey, Jeffrey N; Dreis, Ashley M; Kenseth, Erik M; Renstrom, Joel M; Scardino, Lori L; Van Gorden, Garrett S; Volkert, Anna A; Wingad, Aaron D; Yanzer, Paul J; McEllistrem, Marcus T; de la Mora, Arlene M; DiSpirito, Alan A

    2008-08-01

    Methanobactin (mb) is a copper-binding chromopeptide that appears to be involved in oxidation of methane by the membrane-associated or particulate methane monooxygenase (pMMO). To examine this potential physiological role, the redox and catalytic properties of mb from three different methanotrophs were examined in the absence and presence of O(2). Metal free mb from the type II methanotroph Methylosinus trichosporium OB3b, but not from the type I methanotrophs Methylococcus capsulatus Bath or Methylomicrobium album BG8, were reduced by a variety of reductants, including NADH and duroquinol, and catalyzed the reduction of O(2) to O(2)(-). Copper-containing mb (Cu-mb) from all three methanotrophs showed several interesting properties, including reductase dependent oxidase activity, dismutation of O(2)(-) to H(2)O(2), and the reductant dependent reduction of H(2)O(2) to H(2)O. The superoxide dismutase-like and hydrogen peroxide reductase activities of Cu-mb were 4 and 1 order(s) of magnitude higher, respectively, than the observed oxidase activity. The results demonstrate that Cu-mb from all three methanotrophs are redox-active molecules and oxygen radical scavengers, with the capacity to detoxify both superoxide and hydrogen peroxide without the formation of the hydroxyl radicals associated with Fenton reactions. As previously observed with Cu-mb from Ms. trichosporium OB3b, Cu-mb from both type I methanotrophs stimulated pMMO activity. However, in contrast to previous studies using mb from Ms. trichosporium OB3b, pMMO activity was not inhibited by mb from the two type I methanotrophs at low copper to mb ratios. PMID:18372044

  3. Essential oil from leaves of Cinnamomum osmophloeum acts as a xanthine oxidase inhibitor and reduces the serum uric acid levels in oxonate-induced mice.

    PubMed

    Wang, S Y; Yang, C W; Liao, J W; Zhen, W W; Chu, F H; Chang, S T

    2008-11-01

    The xanthine oxidase (XOD) inhibitory activity and anti-hyperuricemia effect in mice of Cinnamomum osmophloeum, which is an endemic tree in Taiwan, were evaluated in this study. The results demonstrated that the essential oil of C. osmophloeum leaves presented the strongest XOD inhibition activity (IC(50)=16.3 μg/ml); however, no significant XOD inhibition activities were found in ethanolic and hot water extracts. Furthermore, among the main compounds of essential oil, the cinnamaldehyde exhibited the potent XOD inhibition activity with an IC(50)=8.4 μg/ml. Besides, the reducing serum uric acid levels in oxonate-induced mice by cinnamaldehyde were further investigated. The hyperuricemic mice were oral administrated cinnamaldehyde at a dosage of 150 mg/kg, the uric acid value in serum was reduced from 5.25±0.63 to 2.10±0.04 mg/dl, the levels of serum uric acid in mice was lowered down by 84.48% as compared to the hyperuricemic control group. Based on the results obtained in this study, cinnamaldehyde may be a potential lead compound for developing the pharmaceutic for anti-hyperuricemia agent. PMID:18693097

  4. Correlation of active site metal content in human diamine oxidase with trihydroxyphenylalanine quinone cofactor biogenesis .

    PubMed

    McGrath, Aaron P; Caradoc-Davies, Tom; Collyer, Charles A; Guss, J Mitchell

    2010-09-28

    Copper-containing amine oxidases (CAOs) require a protein-derived topaquinone cofactor (TPQ) for activity. TPQ biogenesis is a self-processing reaction requiring the presence of copper and molecular oxygen. Recombinant human diamine oxidase (hDAO) was heterologously expressed in Drosophila S2 cells, and analysis indicates that the purified hDAO contains substoichiometric amounts of copper and TPQ. The crystal structure of a complex of an inhibitor, aminoguanidine, and hDAO at 2.05 Å resolution shows that the aminoguanidine forms a covalent adduct with the TPQ and that the site is ∼75% occupied. Aminoguanidine is a potent inhibitor of hDAO with an IC(50) of 153 ± 9 nM. The structure indicates that the catalytic metal site, normally occupied by copper, is fully occupied. X-ray diffraction data recorded below the copper edge, between the copper and zinc edges, and above the zinc edge have been used to show that the metal site is occupied approximately 75% by copper and 25% by zinc and the formation of the TPQ cofactor is correlated with copper occupancy. PMID:20722416

  5. Crystallization and preliminary analysis of active nitroalkane oxidase in three crystal forms.

    PubMed

    Nagpal, Akanksha; Valley, Michael P; Fitzpatrick, Paul F; Orville, Allen M

    2004-08-01

    Nitroalkane oxidase (NAO), a flavoprotein cloned and purified from Fusarium oxysporum, catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones, with the production of H2O2 and nitrite. In this paper, the crystallization and preliminary X-ray data analysis of three crystal forms of active nitroalkane oxidase are described. The first crystal form belongs to a trigonal space group (either P3(1)21 or P3(2)21, with unit-cell parameters a = b = 103.8, c = 487.0 A) and diffracts to at least 1.6 A resolution. Several data sets were collected using 2theta and kappa geometry in order to obtain a complete data set to 2.07 A resolution. Solvent-content and Matthews coefficient analysis suggests that crystal form 1 contains two homotetramers per asymmetric unit. Crystal form 2 (P2(1)2(1)2(1); a = 147.3, b = 153.5, c = 169.5 A) and crystal form 3 (P3(1) or P3(2); a = b = 108.9, c = 342.5 A) are obtained from slightly different conditions and also contain two homotetramers per asymmetric unit, but have different solvent contents. A three-wavelength MAD data set was collected from selenomethionine-enriched NAO (SeMet-NAO) in crystal form 3 and will be used for phasing. PMID:15272176

  6. A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity

    PubMed Central

    Larrondo, Luis F.; Salas, Loreto; Melo, Francisco; Vicuña, Rafael; Cullen, Daniel

    2003-01-01

    Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe2+, with a Km close to 2 μM. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity. PMID:14532088

  7. Genomic organisation, activity and distribution analysis of the microbial putrescine oxidase degradation pathway.

    PubMed

    Foster, Alexander; Barnes, Nicole; Speight, Robert; Keane, Mark A

    2013-10-01

    The catalytic action of putrescine specific amine oxidases acting in tandem with 4-aminobutyraldehyde dehydrogenase is explored as a degradative pathway in Rhodococcus opacus. By limiting the nitrogen source, increased catalytic activity was induced leading to a coordinated response in the oxidative deamination of putrescine to 4-aminobutyraldehyde and subsequent dehydrogenation to 4-aminobutyrate. Isolating the dehydrogenase by ion exchange chromatography and gel filtration revealed that the enzyme acts principally on linear aliphatic aldehydes possessing an amino moiety. Michaelis-Menten kinetic analysis delivered a Michaelis constant (K(M)=0.014 mM) and maximum rate (Vmax=11.2 μmol/min/mg) for the conversion of 4-aminobutyraldehyde to 4-aminobutyrate. The dehydrogenase identified by MALDI-TOF mass spectrometric analysis (E value=0.031, 23% coverage) belongs to a functionally related genomic cluster that includes the amine oxidase, suggesting their association in a directed cell response. Key regulatory, stress and transport encoding genes have been identified, along with candidate dehydrogenases and transaminases for the further conversion of 4-aminobutyrate to succinate. Genomic analysis has revealed highly similar metabolic gene clustering among members of Actinobacteria, providing insight into putrescine degradation notably among Micrococcaceae, Rhodococci and Corynebacterium by a pathway that was previously uncharacterised in bacteria. PMID:23906496

  8. A newly identified fatty alcohol oxidase gene is mainly responsible for the oxidation of long-chain ω-hydroxy fatty acids in Yarrowia lipolytica.

    PubMed

    Gatter, Michael; Förster, André; Bär, Kati; Winter, Miriam; Otto, Christina; Petzsch, Patrick; Ježková, Michaela; Bahr, Katrin; Pfeiffer, Melanie; Matthäus, Falk; Barth, Gerold

    2014-09-01

    Nine potential (fatty) alcohol dehydrogenase genes and one alcohol oxidase gene were identified in Yarrowia lipolytica by comparative sequence analysis. All relevant genes were deleted in Y. lipolytica H222ΔP which is lacking β-oxidation. Resulting transformants were tested for their ability to accumulate ω-hydroxy fatty acids and dicarboxylic acids in the culture medium. The deletion of eight alcohol dehydrogenase genes (FADH, ADH1-7), which may be involved in ω-oxidation, led only to a slightly increased accumulation of ω-hydroxy fatty acids, whereas the deletion of the fatty alcohol oxidase gene (FAO1), which has not been described yet in Y. lipolytica, exhibited a considerably higher effect. The combined deletion of the eight (fatty) alcohol dehydrogenase genes and the alcohol oxidase gene further reduced the formation of dicarboxylic acids. These results indicate that both (fatty) alcohol dehydrogenases and an alcohol oxidase are involved in ω-oxidation of long-chain fatty acids whereby latter plays the major role. This insight marks the first step toward the biotechnological production of long-chain ω-hydroxy fatty acids with the help of the nonconventional yeast Y. lipolytica. The overexpression of FAO1 can be further used to improve existing strains for the production of dicarboxylic acids. PMID:24931727

  9. Study of a reagent- and mediator-less biosensor for D-amino acids based on co-immobilized D-amino acid oxidase and peroxidase in carbon paste electrodes.

    PubMed

    Johansson, E; Marko-Varga, G; Gorton, L

    1993-10-01

    A biosensor for the analysis of D-amino acids is described. Carbon paste (graphite/paraffin oil) was chemically modified with immobilized D-amino acid oxidase and either horse-radish peroxidase or fungal peroxidase from Arthromyces ramosus. The two enzymes dissolved in buffer, together with an amine containing oligomer or polymer, were adsorbed on dry graphite. Prior to immobilization, the graphite was heat treated at 700 degrees C for 15 s to promote an efficient electron transfer between graphite and the peroxidase. The mixture was dried before addition of the pasting liquid. The sensor is based on the fact that the hydrogen peroxide produced by the action of D-amino acid oxidase is electrocatalytically reduced through the action of the peroxidase. The amine containing compound acted as a stabilizer and activator of the enzymes in the paste. The enzyme electrode was investigated as a sensor for D-phenylalanine and hydrogen peroxide in a flow through electrochemical cell connected to a single line flow injection system. The influences on the response by different additives to the paste and pH are reported. Linear calibration curves were obtained between 0.1 and 1.4 mM for D-phenylalanine and 5 and 1000 microM for hydrogen peroxide at an applied potential of -50 mV vs. Ag/AgCl. The sensor was also active for the following D-amino acids: D-alanine, D-valine, D-leucine, D-isoleucine, D-serine, D-aspartic acid, D-glutamic acid, D-lysine, D-histidine, D-arginine, D-tryptophan, D-methionine, and D-proline. PMID:7902430

  10. Abortiporus biennis tolerance to insoluble metal oxides: oxalate secretion, oxalate oxidase activity, and mycelial morphology.

    PubMed

    Graz, Marcin; Jarosz-Wilkołazka, Anna; Pawlikowska-Pawlega, Bozena

    2009-06-01

    The ability of Abortiporus biennis to tolerate and solubilize toxic metal oxides (Cu(2)O, Al(2)O(3), ZnO, CuFe(2)O(4)Zn, CdO, and MnO(2)) incorporated into agar media was investigated and the growth rate, oxalic acid secretion, and mycelial morphology were monitored. Among the tested metal oxides, formation of clear zones underneath the mycelium growing on Cu(2)O- and ZnO-amended plates was observed. ZnO, CdO and Cu(2)O caused the highest rate of fungal growth inhibition. An increased level of oxalic acid concentration was detected as a response of A. biennis to the presence of Cu(2)O, MnO(2), ZnO and CuFe(2)O(4)Zn in growth medium. The oxalate oxidase (OXO) was found to be responsible for oxalic acid degradation in A. biennis cultivated in metal-amended media. An increased level of OXO was observed in media amended with Cu(2)O, ZnO and MnO(2). Confocal microscopy used in this study revealed changes in mycelial morphology which appeared as increased hyphal branching, increased septation and increased spore number. PMID:18985279

  11. Evaluation of antioxidant and xanthine oxidase inhibitory activity of different solvent extracts of leaves of Citrullus colocynthis

    PubMed Central

    Nessa, Fazilatun; Khan, Saeed A.

    2014-01-01

    Background: Citrullus colocynthis is a folk medicinal plan of United Arab Emirates. Several studies on this plant reported and focused on the biological and toxicological profile of fruits pulp. The present study focused on the antioxidant potency of leaf extract of this plant. Aim: To evaluate the antioxidant and xanthine oxidase (XO) inhibitory activities of C. colocynthis by chemical method. Materials and Methods: Four different solvent extracts (methanol-CCM, methanol: water (1:1)-CCMW, chloroform-CCC and hexane-CCH) of leaves of C. colocynthis were investigated for their free radical scavenging activity using DPPH radical as a substrate, lipid peroxidation (LPO) inhibitory activity using a model system consisting of β-carotene-linoleic acid, superoxide radical scavenging activity (enzymatically/nonenzymatically) and XO inhibitory activity. A dose response curve was plotted for determining SC50 and IC50 values for expressing the results of free radical scavenging activity and XO inhibitory activities respectively. Results: The high polyphenolic content of CCM and CCMW extract showed highest antioxidant activity irrespective the method used for this investigation. The overall results decreased in the order of: CCM > CCMW > CCC > CCH. CCH extract was inactive towards chemically generated superoxide radical and poor DPPH radical scavengers. The results of LPO inhibitory activities of leaves extract (0.1, 0.5 and 1.0 mg/mL) also decreased in the order of: CCM > CCMW > CCC > CCH. Overall 1.0 mg/mL leaves extract showed highest antioxidant potency amongst the studied concentration. Conclusion: CCMW and CCM extract of C. colocynthis exhibited promising antioxidants and XO inhibitory activities. PMID:25002802

  12. Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells.

    PubMed

    Pitari, G; D'Andrea, G; Salucci, M L; Rossi, A; Avigliano, L

    1998-08-01

    Ascorbate oxidase activity and immunoreactivity were evaluated in crude tissue extracts obtained from callus cell cultures induced by green zucchini sarcocarp and grown in the presence of tunicamycin, a powerful N-glycosylation inhibitor. Tunicamycin at 2 or 4 microg ml(-1) blocked cell growth within a couple of weeks, although a sustained cell viability was observed in the same period. A significant inhibition of total protein synthesis was observed at 10 and 15 days of culture time, with a decrease of 30% and 43% respectively when cells were grown in the presence of 2 microg ml(-1) tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 microg ml(-1). After the same culture times ascorbate oxidase specific activity assayed in crude tissue extracts showed increases of about 1.9-fold and 3.5-fold (10 days) and 1.7-fold and 3.1-fold (15 days) at 2 and 4 microg ml(-1) tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the same growing times. Lectin-blot, based on the use of concanavalin A, indicated a marked decrease of glycosylated proteins in tunicamycin-treated cultures. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarcely recognized the enzyme expressed in tunicamycin-treated cells; on the contrary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures. PMID:9870353

  13. Copper supplementation restores cytochrome c oxidase activity in cultured cells from patients with SCO2 mutations.

    PubMed Central

    Salviati, Leonardo; Hernandez-Rosa, Evelyn; Walker, Winsome F; Sacconi, Sabrina; DiMauro, Salvatore; Schon, Eric A; Davidson, Mercy M

    2002-01-01

    Human SCO2 is a nuclear-encoded Cu-binding protein, presumed to be responsible for the insertion of Cu into the mitochondrial cytochrome c oxidase (COX) holoenzyme. Mutations in SCO2 are associated with cardioencephalomyopathy and COX deficiency. Studies in yeast and bacteria have shown that Cu supplementation can restore COX activity in cells harbouring mutations in genes involving Cu transport. Therefore we investigated whether Cu supplementation could restore COX activity in cultured cells from patients with SCO2 mutations. Our data demonstrate that the COX deficiency observed in fibroblasts, myoblasts and myotubes from patients with SCO2 mutations can be restored to almost normal levels by the addition of CuCl(2) to the growth medium. PMID:11931660

  14. Loss of NADH Oxidase Activity in Streptococcus mutans Leads to Rex-Mediated Overcompensation in NAD+ Regeneration by Lactate Dehydrogenase

    PubMed Central

    Baker, J. L.; Derr, A. M.; Faustoferri, R. C.

    2015-01-01

    ABSTRACT Previous studies of the oral pathogen Streptococcus mutans have determined that this Gram-positive facultative anaerobe mounts robust responses to both acid and oxidative stresses. The water-forming NADH oxidase (Nox; encoded by nox) is thought to be critical for the regeneration of NAD+, for use in glycolysis, and for the reduction of oxygen, thereby preventing the formation of damaging reactive oxygen species. In this study, the free NAD+/NADH ratio in a nox deletion strain (Δnox) was discovered to be remarkably higher than that in the parent strain, UA159, when the strains were grown in continuous culture. This unanticipated result was explained by significantly elevated lactate dehydrogenase (Ldh; encoded by ldh) activity and ldh transcription in the Δnox strain, which was mediated in part by the redox-sensing regulator Rex. cDNA microarray analysis of S. mutans cultures exposed to simultaneous acid stress (growth at a low pH) and oxidative stress (generated through the deletion of nox or the addition of exogenous oxygen) revealed a stress response synergistically heightened over that with either stress alone. In the Δnox strain, this elevated stress response included increased glucose phosphoenolpyruvate phosphotransferase system (PTS) activity, which appeared to be due to elevated manL transcription, mediated in part, like elevated ldh transcription, by Rex. While the Δnox strain does possess a membrane composition different from that of the parent strain, it did not appear to have defects in either membrane permeability or ATPase activity. However, the altered transcriptome and metabolome of the Δnox strain were sufficient to impair its ability to compete with commensal peroxigenic oral streptococci during growth under aerobic conditions. IMPORTANCE Streptococcus mutans is an oral pathogen whose ability to outcompete commensal oral streptococci is strongly linked to the formation of dental caries. Previous work has demonstrated that the S

  15. NADPH Oxidase Dependent NLRP3 Inflammasome Activation Plays an Important Role in Lung Fibrosis by Multi-Walled Carbon Nanotubes

    PubMed Central

    Sun, Bingbing; Wang, Xiang; Ji, Zhaoxia; Wang, Meiying; Liao, Yu-Pei; Chang, Chong Hyun; Li, Ruibin; Zhang, Haiyuan; Nel, André E.; Xia, Tian

    2015-01-01

    The purpose of this communication is to elucidate the key role of NADPH oxidase in NLRP3 inflammasome activation and generation of pulmonary fibrosis by multi-walled carbon nanotubes (MWCNTs). Although it is known that oxidative stress plays a role in pulmonary fibrosis by single-walled CNTs, the role of specific sources of reactive oxygen species (ROS), including NADPH oxidase, in inflammasome activation remains to be clarified. In this study, three long aspect ratio (LAR) materials (MWCNTs, SWCNTs, and silver nanowires) are used to compare with spherical carbon black and silver nanoparticles for their ability to trigger oxygen burst activity and NLRP3 assembly. All LAR materials but not spherical nanoparticles induce robust NADPH oxidase activation and respiratory burst activity in THP-1 cells, which are blunted in p22phox deficient cells. NADPH oxidase is directly involved in lysosome damage by LAR materials, as demonstrated by decreased cathepsin B release and IL-1β production in p22phox deficient cells. Reduced respiratory burst activity and inflammasome activation are also observed in bone marrow-derived macrophages from p47phox deficient mice. Moreover, p47phox deficient mice have reduced IL-1β production and lung collagen deposition in response to MWCNTs. Lung fibrosis is also suppressed by N-acetyl-cysteine (NAC) in wild type animals exposed to MWCNTs. PMID:25581126

  16. Probing Oxygen Activation Sites in Two Flavoprotein Oxidases Using Chloride as an Oxygen Surrogate

    SciTech Connect

    Kommoju, Phaneeswara-Rao; Chen, Zhi-wei; Bruckner, Robert C.; Mathews, F. Scott; Jorns, Marilyn Schuman

    2011-08-16

    A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX-chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX-chloride complex and a ternary MSOX-chloride-MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.

  17. Polyphenolic composition, antioxidant activity, and polyphenol oxidase (PPO) activity of quince (Cydonia oblonga Miller) varieties.

    PubMed

    Wojdyło, Aneta; Oszmiański, Jan; Bielicki, Paweł

    2013-03-20

    Phytochemical profiles (phenolic compounds, L-ascorbic acid, antioxidant and PPO activities) of 13 different quince varieties and 5 genotypes were studied. Polyphenols were identified by LC-PDA-QTof/MS and quantified by UPLC-PDA and UPLC-FL. A total of 26 polyphenolic compounds found in quince tissues were identified and presented: 9 flavan-3-ols ((-)-epicatechin, procyanidin B2, 3 procyanidin dimers and trimers, and 1 tetramer); 8 hydroxycinnamates, derivatives of caffeoylquinic and coumaroylquinic acid; and 9 kaempferol and quercetin derivatives. The content of total polyphenols was between 1709.43 (genotype 'S1') and 3436.56 mg/100 g dry weight ('Leskovač'). Flavan-3-ols, which are the major class of quince polyphenols, represented between 78 and 94% of the total polyphenolic compounds. The activity of PPO enzyme ranged from 709.85 to 1284.59 ΔU/min, and that of L-ascorbic acid ranged from 5.86 to 26.42 mg/100 g. Some quince varieties and their products characterized by a higher content of phenolic compounds may be selected to promote their positive effect on health. PMID:23461298

  18. Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom.

    PubMed

    Rodrigues, Renata S; da Silva, Juliana F; Boldrini França, Johara; Fonseca, Fernando P P; Otaviano, Antônio R; Henrique Silva, Flávio; Hamaguchi, Amélia; Magro, Angelo J; Braz, Antônio Sérgio K; dos Santos, Juliana I; Homsi-Brandeburgo, Maria Inês; Fontes, Marcos R M; Fuly, André L; Soares, Andreimar M; Rodrigues, Veridiana M

    2009-04-01

    An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL-4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAO-cDNA of 1548bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Bp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents. PMID:19135502

  19. Aldehyde oxidase 1 is highly abundant in hepatic steatosis and is downregulated by adiponectin and fenofibric acid in hepatocytes in vitro

    SciTech Connect

    Neumeier, Markus; Weigert, Johanna; Schaeffler, Andreas; Weiss, Thomas S.; Schmidl, Christian; Buettner, Roland; Bollheimer, Cornelius; Aslanidis, Charalampos; Schoelmerich, Juergen; Buechler, Christa . E-mail: christa.buechler@klinik.uni-regensburg.de

    2006-11-24

    Adiponectin protects the liver from steatosis caused by obesity or alcohol and therefore the influence of adiponectin on human hepatocytes was analyzed. GeneChip experiments indicated that recombinant adiponectin downregulates aldehyde oxidase 1 (AOX1) expression and this was confirmed by real-time RT-PCR and immunoblot. AOX1 is a xenobiotic metabolizing protein and produces reactive oxygen species (ROS), that promote cell damage and fibrogenesis. Adiponectin and fenofibric acid activate peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) and both suppress AOX1 protein and this is blocked by the PPAR-{alpha} antagonist RU486. Obesity is associated with low adiponectin, reduced hepatic PPAR-{alpha} activity and fatty liver, and AOX1 was found induced in the liver of rats on a high-fat diet when compared to controls. Free fatty acids and leptin, that are elevated in obesity, failed to upregulate AOX1 in vitro. The current data indicate that adiponectin reduces AOX1 by activating PPAR-{alpha} whereas fatty liver disease is associated with elevated hepatic AOX1. High AOX1 may be associated with higher ROS well described to induce fibrogenesis in liver tissue but may also influence drug metabolism and activity.

  20. Structural Insights into Sulfite Oxidase Deficiency

    SciTech Connect

    Karakas,E.; Wilson, H.; Graf, T.; Xiang, S.; Jaramillo-Busquets, S.; Rajagopalan, K.; Kisker, C.

    2005-01-01

    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.

  1. Early Events Induced by the Elicitor Cryptogein in Tobacco Cells: Involvement of a Plasma Membrane NADPH Oxidase and Activation of Glycolysis and the Pentose Phosphate Pathway.

    PubMed Central

    Pugin, A.; Frachisse, J. M.; Tavernier, E.; Bligny, R.; Gout, E.; Douce, R.; Guern, J.

    1997-01-01

    Application of the elicitor cryptogein to tobacco (cv Xanthi) is known to evoke external medium alkalinization, active oxygen species production, and phytoalexin synthesis. These are all dependent on an influx of calcium. We show here that cryptogein also induces calcium-dependent plasma membrane depolarization, chloride efflux, cytoplasm acidification, and NADPH oxidation without changes in NAD+ and ATP levels, indicating that the elicitor-activated redox system, responsible for active oxygen species production, uses NADPH in vivo. NADPH oxidation activates the functioning of the pentose phosphate pathway, leading to a decrease in glucose 6-phosphate and to the accumulation of glyceraldehyde 3-phosphate, 3- and 2-phosphoglyceric acid, and phosphoenolpyruvate. By inhibiting the pentose phosphate pathway, we demonstrate that the activation of the plasma membrane NADPH oxidase is responsible for active oxygen species production, external alkalinization, and acidification of the cytoplasm. A model is proposed for the organization of the cryptogein responses measured to date. PMID:12237354

  2. Synergistic effect of Aspergillus tubingensis CTM 507 glucose oxidase in presence of ascorbic acid and alpha amylase on dough properties, baking quality and shelf life of bread.

    PubMed

    Kriaa, Mouna; Ouhibi, Rabeb; Graba, Héla; Besbes, Souhail; Jardak, Mohamed; Kammoun, Radhouane

    2016-02-01

    The impact of Aspergillus tubingensis glucose oxidase (GOD) in combination with α-amylase and ascorbic acid on dough properties, qualities and shelf life of bread was investigated. Regression models of alveograph and texture parameters of dough and bread were adjusted. Indeed, the mixture of GOD (44 %) and ascorbic acid (56 %) on flour containing basal improver showed its potential as a corrective action to get better functional and rheological properties of dough and bread texture. Furthermore, wheat flour containing basal additives and enriched with GOD (63.8 %), ascorbic acid (32 %) and α- amylase (4.2 %) led to high technological bread making parameters, to decrease the crumb firmness and chewiness and to improve elasticity, adhesion, cohesion and specific volume of bread. In addition to that, the optimized formulation addition significantly reduced water activity and therefore decreased bread susceptibility to microbial spoilage. These findings demonstrated that GOD could partially substitute not only ascorbic acid but also α-amylase. The generated models allowed to predict the behavior of wheat flour containing additives in the range of values tested and to define the additives formula that led to desired rheological and baking qualities of dough. This fact provides new perspectives to compensate flour quality deficiencies at the moment of selecting raw materials and technological parameters reducing the production costs and facilitating gluten free products development. Graphical abstractᅟ. PMID:27162406

  3. NADPH Oxidase- and Mitochondria-derived Reactive Oxygen Species in Proinflammatory Microglial Activation: A Bipartisan Affair?

    PubMed Central

    Bordt, Evan A.; Polster, Brian M.

    2014-01-01

    Microglia are the resident immune cells of the brain and play major roles in central nervous system development, maintenance, and disease. Brain insults cause microglia to proliferate, migrate, and transform into one or more activated states. Classical M1 activation triggers the production of proinflammatory factors such as tumor necrosis factor- α (TNF-α), interleukin-1β (IL-1β), nitric oxide (NO), and reactive oxygen species which, in excess, can exacerbate brain injury. The mechanisms underlying microglial activation are not fully understood, yet reactive oxygen species are increasingly implicated as mediators of microglial activation. In this review, we highlight studies linking reactive oxygen species, in particular hydrogen peroxide derived from NADPH oxidase-generated superoxide, to the classical activation of microglia. In addition, we critically evaluate controversial evidence suggesting a specific role for mitochondrial reactive oxygen species in the activation of the NLRP3 inflammasome, a multiprotein complex that mediates the production of IL-1β and IL-18. Finally, the limitations of common techniques used to implicate mitochondrial ROS in microglial and inflammasome activation, such as the use of the mitochondrially-targeted ROS indicator MitoSOX and the mitochondrially-targeted antioxidant MitoTEMPO, are also discussed. PMID:25091898

  4. Recombinant human diamine oxidase activity is not inhibited by ethanol, acetaldehyde, disulfiram, diethyldithiocarbamate or cyanamide.

    PubMed

    Bartko, Johann; Gludovacz, Elisabeth; Petroczi, Karin; Borth, Nicole; Jilma, Bernd; Boehm, Thomas

    2016-08-01

    Human diamine oxidase (hDAO, EC 1.4.3.22) is the key enzyme in the degradation of extracellular histamine. Consumption of alcohol is a known trigger of mast cell degranulation in patients with mast cell activation syndrome. Ethanol may also interfere with enzymatic histamine degradation, but reports on the effects on DAO activity are controversial. There are also conflicting reports whether disulfiram, an FDA-approved agent in the treatment of alcohol dependence, inhibits DAO. We therefore investigated the inhibitory potential of ethanol and disulfiram and their metabolites on recombinant human DAO (rhDAO) in three different assay systems. Relevant concentrations of ethanol, acetaldehyde, and acetate did not inhibit rhDAO activity in an in vitro assay system using horseradish peroxidase (HRP) -mediated luminol oxidation. The aldehyde dehydrogenase (ALDH; EC 1.2.1.3) inhibitors cyanamide and its dimer dicyanamide also had no effect on DAO activity. In one assay system, the irreversible ALDH inhibitor disulfiram and its main metabolite diethyldithiocarbamate seemed to inhibit DAO activity. However, the decreased product formation was not due to a direct block of DAO activity but resulted from inhibition of peroxidase employed in the coupled system. Our in vitro data do not support a direct blocking effect of ethanol, disulfiram, and their metabolites on DAO activity in vivo. PMID:27401969

  5. Sphingomyelinase promotes oxidant production and skeletal muscle contractile dysfunction through activation of NADPH oxidase

    PubMed Central

    Loehr, James A.; Abo-Zahrah, Reem; Pal, Rituraj; Rodney, George G.

    2015-01-01

    Elevated concentrations of sphingomyelinase (SMase) have been detected in a variety of diseases. SMase has been shown to increase muscle derived oxidants and decrease skeletal muscle force; however, the sub-cellular site of oxidant production has not been elucidated. Using redox sensitive biosensors targeted to the mitochondria and NADPH oxidase (Nox2), we demonstrate that SMase increased Nox2-dependent ROS and had no effect on mitochondrial ROS in isolated FDB fibers. Pharmacological inhibition and genetic knockdown of Nox2 activity prevented SMase induced ROS production and provided protection against decreased force production in the diaphragm. In contrast, genetic overexpression of superoxide dismutase within the mitochondria did not prevent increased ROS production and offered no protection against decreased diaphragm function in response to SMase. Our study shows that SMase induced ROS production occurs in specific sub-cellular regions of skeletal muscle; however, the increased ROS does not completely account for the decrease in muscle function. PMID:25653619

  6. Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation.

    PubMed

    Zielonka, Jacek; Zielonka, Monika; VerPlank, Lynn; Cheng, Gang; Hardy, Micael; Ouari, Olivier; Ayhan, Mehmet Menaf; Podsiadły, Radosław; Sikora, Adam; Lambeth, J David; Kalyanaraman, Balaraman

    2016-03-25

    Using high throughput screening-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of the NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O2 (̇̄)), hydrogen peroxide (H2O2), and peroxynitrite (ONOO(-)), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO(-)formation in activated macrophages. A new diagnostic marker product for ONOO(-)is reported. We conclude that the newly developed high throughput screening/reactive oxygen species assays could also be used to identify potential inhibitors of ONOO(-)formed from Nox2-derived O2 (̇̄)and nitric oxide synthase-derived nitric oxide. PMID:26839313

  7. Dopamine-β-oxidase activity in man, using hydroxyamphetamine as substrate

    PubMed Central

    Sjoerdsma, A.; von Studnitz, W.

    1963-01-01

    Hydroxyamphetamine was administered orally to five human subjects in daily doses of 26 to 118 mg. Approximately half the dosage administered was recoverable in the urine as unchanged drug (free plus conjugated) and 3.7 to 9.1% was excreted as the β-hydroxylated metabolite, hydroxynorephedrine (free plus conjugated). Since conversion of hydroxyamphetamine to hydroxynorephedrine occurs in vitro by the action of dopamine-β-oxidase, a simple method is suggested for measuring the activity of this enzyme and the effect of its inhibitors in man. No impairment of β-hydroxylation was noted in an adrenalectomized subject. The β-hydroxylation of hydroxyamphetamine in vivo has not been described previously. PMID:13977820

  8. Activation of NADPH oxidase 1 increases intracellular calcium and migration of smooth muscle cells.

    PubMed

    Zimmerman, Matthew C; Takapoo, Maysam; Jagadeesha, Dammanahalli K; Stanic, Bojana; Banfi, Botond; Bhalla, Ramesh C; Miller, Francis J

    2011-09-01

    Redox-dependent migration and proliferation of vascular smooth muscle cells (SMCs) are central events in the development of vascular proliferative diseases; however, the underlying intracellular signaling mechanisms are not fully understood. We tested the hypothesis that activation of Nox1 NADPH oxidase modulates intracellular calcium ([Ca(2+)](i)) levels. Using cultured SMCs from wild-type and Nox1 null mice, we confirmed that thrombin-dependent generation of reactive oxygen species requires Nox1. Thrombin rapidly increased [Ca(2+)](i), as measured by fura-2 fluorescence ratio imaging, in wild-type but not Nox1 null SMCs. The increase in [Ca(2+)](i) in wild-type SMCs was inhibited by antisense to Nox1 and restored by expression of Nox1 in Nox1 null SMCs. Investigation into potential mechanisms by which Nox1 modulates [Ca(2+)](i) showed that thrombin-induced inositol triphosphate generation and thapsigargin-induced intracellular calcium mobilization were similar in wild-type and Nox1 null SMCs. To examine the effects of Nox1 on Ca(2+) entry, cells were either bathed in Ca(2+)-free medium or exposed to dihydropyridines to block L-type Ca(2+) channel activity. Treatment with nifedipine or removal of extracellular Ca(2+) reduced the thrombin-mediated increase of [Ca(2+)](i) in wild-type SMCs, whereas the response in Nox1 null SMCs was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases, restored the thrombin-induced increase of [Ca(2+)](i) in Nox1 null SMCs. Migration of SMCs was impaired with deficiency of Nox1 and restored with expression of Nox1 or the addition of sodium vanadate. In summary, we conclude that Nox1 NADPH oxidase modulates Ca(2+) mobilization in SMCs, in part through regulation of Ca(2+) influx, to thereby promote cell migration. PMID:21810651

  9. Cdc42-Dependent Activation of NADPH Oxidase Is Involved in Ethanol-Induced Neuronal Oxidative Stress

    PubMed Central

    Wang, Xin; Ke, Zunji; Chen, Gang; Xu, Mei; Bower, Kimberly A.; Frank, Jacqueline A.; Zhang, Zhuo; Shi, Xianglin; Luo, Jia

    2012-01-01

    It has been suggested that excessive reactive oxygen species (ROS) and oxidative stress play an important role in ethanol-induced damage to both the developing and mature central nervous system (CNS). The mechanisms underlying ethanol-induced neuronal ROS, however, remain unclear. In this study, we investigated the role of NADPH oxidase (NOX) in ethanol-induced ROS generation. We demonstrated that ethanol activated NOX and inhibition of NOX reduced ethanol-promoted ROS generation. Ethanol significantly increased the expression of p47phox and p67phox, the essential subunits for NOX activation in cultured neuronal cells and the cerebral cortex of infant mice. Ethanol caused serine phosphorylation and membrane translocation of p47phox and p67phox, which were prerequisites for NOX assembly and activation. Knocking down p47phox with the small interfering RNA was sufficient to attenuate ethanol-induced ROS production and ameliorate ethanol-mediated oxidative damage, which is indicated by a decrease in protein oxidation and lipid peroxidation. Ethanol activated cell division cycle 42 (Cdc42) and overexpression of a dominant negative (DN) Cdc42 abrogate ethanol-induced NOX activation and ROS generation. These results suggest that Cdc42-dependent NOX activation mediates ethanol-induced oxidative damages to neurons. PMID:22662267

  10. Measurement of polyphenol oxidase activity using optical waveguide lightmode spectroscopy-based immunosensor.

    PubMed

    Kim, Namsoo; Kim, Woo-Yeon

    2015-02-15

    Polyphenol oxidase (PPO) is an important quality index during food processing involving heat-treatment and sensitive determination of PPO activity has been a critical concern in the food industry. In this study, a new measurement of PPO activity exploiting an optical waveguide lightmode spectroscopy-based immunosensor is presented using a polyclonal anti-PPO antibody that was immobilized in situ to the surface of a 3-aminopropyltriethoxysilane-treated optical grating coupler activated with glutaraldehyde. When analysed with a purified PPO fraction from potato tubers, a linear relationship was found between PPO activities of 0.0005607-560.7U/mL and the sensor responses obtained. The sensor was applicable to measurement of PPO activity in real samples that were prepared from potato tubers, grapes and Kimchi cabbage, and the analytical results were compared with those obtained by a conventional colorimetric assay measuring PPO activity. When tested for long-term stability, the sensor was reusable up to 10th day after preparation. PMID:25236218

  11. Blueberry polyphenol oxidase: Characterization and the kinetics of thermal and high pressure activation and inactivation.

    PubMed

    Terefe, Netsanet Shiferaw; Delon, Antoine; Buckow, Roman; Versteeg, Cornelis

    2015-12-01

    Partially purified blueberry polyphenol oxidase (PPO) in Mcllvaine buffer (pH=3.6, typical pH of blueberry juice) was subjected to processing at isothermal-isobaric conditions at temperatures from 30 to 80 °C and pressure from 0.1 to 700 MPa. High pressure processing at 30-50 °C at all pressures studied caused irreversible PPO activity increase with a maximum of 6.1 fold increase at 500 MPa and 30 °C. Treatments at mild pressure-mild temperature conditions (0.1-400 MPa, 60 °C) also caused up to 3 fold PPO activity increase. Initial activity increase followed by a decrease occurred at relatively high pressure-mild temperature (400-600 MPa, 60 °C) and mild pressure-high temperature (0.1-400 MPa, 70-80 °C) combinations. At temperatures higher than 76 °C, monotonic decrease in PPO activity occurred at 0.1 MPa and pressures higher than 500 MPa. The activation/inactivation kinetics of the enzyme was successfully modelled assuming consecutive reactions in series with activation followed by inactivation. PMID:26041182

  12. Activation of Polyphenol Oxidase in Dormant Wild Oat Caryopses by a Seed-Decay Isolate of Fusarium avenaceum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Incubation of dormant wild oat (Avena fatua L., isoline M73) caryopses for 1 to 3 days with Fusarium avenaceum seed-decay isolate F.a.1 induced activity of the plant defense enzyme polyphenol oxidase (PPO). Both extracts and leachates obtained from F.a.1-treated caryopses had decreased abundance of ...

  13. Organochlorine insecticides induce NADPH oxidase-dependent reactive oxygen species in human monocytic cells via phospholipase A2/arachidonic acid.

    PubMed

    Mangum, Lee C; Borazjani, Abdolsamad; Stokes, John V; Matthews, Anberitha T; Lee, Jung Hwa; Chambers, Janice E; Ross, Matthew K

    2015-04-20

    Bioaccumulative organohalogen chemicals, such as organochlorine (OC) insecticides, have been increasingly associated with disease etiology; however, the mechanistic link between chemical exposure and diseases, such as atherosclerosis, cancer, and diabetes, is complex and poorly defined. Systemic oxidative stress stemming from OC exposure might play a vital role in the development of these pathologies. Monocytes are important surveillance cells of the innate immune system that respond to extracellular signals possessing danger-associated molecular patterns by synthesizing oxyradicals, such as superoxide, for the purpose of combating infectious pathogens. We hypothesized that OC chemicals can be toxic to monocytes because of an inappropriate elevation in superoxide-derived reactive oxygen species (ROS) capable of causing cellular oxidative damage. Reactive oxyradicals are generated in monocytes in large part by NADPH oxidase (Nox). The present study was conducted to examine the ability of two chlorinated cyclodiene compounds, trans-nonachlor and dieldrin, as well as p,p'-DDE, a chlorinated alicyclic metabolite of DDT, to stimulate Nox activity in a human monocytic cell line and to elucidate the mechanisms for this activation. Human THP-1 monocytes treated with either trans-nonachlor or dieldrin (0.1-10 μM in the culture medium) exhibited elevated levels of intracellular ROS, as evidenced by complementary methods, including flow cytometry analysis using the probe DCFH-DA and hydroethidine-based fluorometric and UPLC-MS assays. In addition, the induced reactive oxygen flux caused by trans-nonachlor was also observed in two other cell lines, murine J774 macrophages and human HL-60 cells. The central role of Nox in OC-mediated oxidative stress was demonstrated by the attenuated superoxide production in OC-exposed monocytes treated with the Nox inhibitors diphenyleneiodonium and VAS-2870. Moreover, monocytes challenged with OCs exhibited increased phospho-p47(phox

  14. Peroxygenase and Oxidase Activities of Dehaloperoxidase-Hemoglobin from Amphitrite ornata

    PubMed Central

    2015-01-01

    The marine globin dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of monohaloindoles, a previously unknown class of substrate for DHP. Using 5-Br-indole as a representative substrate, the major monooxygenated products were found to be 5-Br-2-oxindole and 5-Br-3-oxindolenine. Isotope labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H2O2, indicative of a previously unreported peroxygenase activity for DHP. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. It was found that 5-Br-3-oxindole, a precursor of the product 5-Br-3-oxindolenine, readily reduced the ferric enzyme to the oxyferrous state, demonstrating an unusual product-driven reduction of the enzyme. As such, DHP returns to the globin-active oxyferrous form after peroxygenase activity ceases. Reactivity with 5-Br-3-oxindole in the absence of H2O2 also yielded 5,5′-Br2-indigo above the expected reaction stoichiometry under aerobic conditions, and O2-concentration studies demonstrated dioxygen consumption. Nonenzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5′-Br2-indigo, and together suggest a newly identified oxidase activity for DHP. PMID:24791647

  15. Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production.

    PubMed

    Bal-Price, Anna; Matthias, Anita; Brown, Guy C

    2002-01-01

    Cultured rat microglia produced extracellular superoxide at a rate of 814 +/- 52 pmol/min/million cells when stimulated with phorbol 12-myristate 13-acetate (PMA) as measured by extracellular cytochrome c reduction. This superoxide production resulted in a rapid rate of superoxide dismutase-sensitive nitric oxide (NO) breakdown (155 +/- 30 pmol of NO/min/million cells) when NO was added to PMA stimulated microglia. Lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-activated microglia produce NO at the rate of 145 +/- 42 pmol/min/million cells and activated astrocytes at the rate of 51 +/- 9 pmol/min/million cells as estimated by NO electrode. Both types of cells maintained a steady-state level of 0.5-0.7 microm NO, only in the presence of L-arginine. Addition of PMA to activated microglia (but not activated astrocytes) caused the rapid and complete disappearance of all extracellular NO (but was restored in the presence of superoxide dismutase) followed by the production of peroxynitrite (as measured by urate-sensitive oxidation of dihydrorhodamine). Co-incubation of activated microglia with cerebellar granule neurones resulted in NO inhibition of neuronal respiration, but this was rapidly removed by PMA-induced breakdown of the NO. Thus, microglial NADPH oxidase can regulate the bioavailability of NO and the production of peroxynitrite. PMID:11796745

  16. 2-Arylquinazolin-4(3H)-ones: Inhibitory Activities Against Xanthine Oxidase.

    PubMed

    Zafar, Humaira; Saad, Syed M; Perveen, Shahnaz; Arshia; Malik, Rizwana; Khan, Ajmal; Khan, Khalid M; Choudhary, Muhammad I

    2016-01-01

    2-Arylquinazolin-4(3H)-ones (1-25) were synthesized, and evaluated for their xanthine oxidase inhibitory activity. Significant to moderate activities were exhibited by the compounds 1-3, 7, 9, 13-15, 19-21, and 23 with IC50 between 2.80 - 28.13 µM as compared to the standard allopurinol (IC50 (IC50 = 2.01 ± 0.01 µM). Compounds 4-6, 8, 11-12, 16-18, 22, and 24 demonstrated a weak activity with IC50 values 44.60 - 112.60 µM. Nonetheless, compounds 10 and 25 did not show any activity. Amongst all derivatives, compound 2, containing a C-4´ dimethylamino group, was the most potent inhibitor of the enzyme with an IC50 value comparable to the standard. Kinetics studies on the most active compounds (2, 7, 9, 14, 15, 19, and 20) were conducted in order to determine their modes of inhibition and dissociation constants Ki. Some of the compounds of 2-arylquinazolin-4(3H)-one series were thus identified as potential leads for further studies towards the treatment of hyperuricemia and gout. PMID:26256588

  17. Alteration of serum semicarbazide-sensitive amine oxidase activity in chronic renal failure.

    PubMed

    Nemcsik, J; Szökö, E; Soltész, Zs; Fodor, E; Toth, L; Egresits, J; Tábi, T; Magyar, K; Kiss, I

    2007-01-01

    Despite recent intensive investigations, physiological and pathological role of semicarbazide-sensitive amine oxidase (SSAO) is far from clear. In this study, serum SSAO activity was determined, radiochemically, in various groups of uremic patients: haemodialysed (HD), peritoneally dialysed (PD) and those receiving conservative treatment but still not dialysed (ND), as well as in controls. Reduced enzyme activity was found in HD uremic patients before and after dialysis treatment, compared to controls (5260 +/- 862 and 6011 +/- 958 pmol/h/ml vs. 8601 +/- 283 pmol/h/ml, p < 0.01 and p < 0.05, respectively). The activity was slightly lower in PD, and normal in ND patients. In HD patients SSAO activity was also determined by an assay based on the formation of hydrogen peroxide, and was found to be elevated compared to controls (2384 +/- 323 pmol/h/ml vs. 1437 +/- 72 pmol/h/ml, p < 0.05). The elevated serum SSAO activity measured through the detection of the enzyme-generated hydrogen peroxide in HD patients might indicate its contribution to the accelerated atherosclerotic disease observed in uremia. PMID:17431736

  18. Role of reactive oxygen species produced by NADPH oxidase in gibberellin biosynthesis during barley seed germination.

    PubMed

    Kai, Kyohei; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Watabe, Gaku; Yuasa, Takashi; Iwaya-Inoue, Mari; Ishibashi, Yushi

    2016-05-01

    NADPH oxidase catalyzes the production of the superoxide anion (O2(-)), a reactive oxygen species (ROS), and regulates the germination of barley (Hordeum vulgare L.). Diphenyleneiodonium (DPI) chloride, an NADPH oxidase inhibitor, delayed barley germination, and exogenous H2O2 (an ROS) partially rescued it. Six enzymes, ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA20-oxidase (GA20ox) and GA3-oxidase (GA3ox), catalyze the transformation of trans-geranylgeranyl diphosphate to active gibberellin, which promotes germination. Exogenous H2O2 promoted the expressions of HvKAO1 and HvGA3ox1 in barley embryos. These results suggest that ROS produced by NADPH oxidase are involved in gibberellin biosynthesis through the regulation of HvKAO1 and HvGA3ox1. PMID:27110861

  19. Mutation at a Strictly-Conserved, Active-Site Tyrosine in the Copper Amine Oxidase Leads to Uncontrolled Oxygenase Activity†

    PubMed Central

    Chen, Zhi-wei; Datta, Saumen; DuBois, Jennifer L.; Klinman, Judith P.; Mathews, F. Scott

    2010-01-01

    The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ), and the subsequent oxidative deamination of substrate amines. Because the same active-site pocket must facilitate both reactions, individual active-site residues may serve multiple roles. We have examined the roles of a strictly-conserved active-site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically, and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild-type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy-intermediates in cofactor biogenesis, but also indicates the critical control of oxidation chemistry that can be conferred by a single active-site residue. PMID:20684524

  20. Serotonin 2A and 2B receptor-induced phrenic motor facilitation: differential requirement for spinal NADPH oxidase activity

    PubMed Central

    MacFarlane, P.M.; Vinit, S.; Mitchell, G.S.

    2011-01-01

    Acute intermittent hypoxia (AIH) facilitates phrenic motor output by a mechanism that requires spinal serotonin (type 2) receptor activation, NADPH oxidase activity and formation of reactive oxygen species (ROS). Episodic spinal serotonin (5-HT) receptor activation alone, without changes in oxygenation, is sufficient to elicit NADPH oxidase-dependent phrenic motor facilitation (pMF). Here we investigated: 1) whether serotonin 2A and/or 2B (5-HT2a/b) receptors are expressed in identified phrenic motor neurons, and 2) which receptor subtype is capable of eliciting NADPH-oxidase-dependent pMF. In anesthetized, artificially ventilated adult rats, episodic C4 intrathecal injections (3 × 6µl injections, 5 min intervals) of a 5-HT2a (DOI) or 5-HT2b (BW723C86) receptor agonist elicited progressive and sustained increases in integrated phrenic nerve burst amplitude (i.e. pMF), an effect lasting at least 90 minutes post-injection for both receptor subtypes. 5-HT2a and 5-HT2b receptor agonist-induced pMF were both blocked by selective antagonists (ketanserin and SB206553, respectively), but not by antagonists to the other receptor subtype. Single injections of either agonist failed to elicit pMF, demonstrating a need for episodic receptor activation. Phrenic motor neurons retrogradely labeled with cholera toxin B fragment expressed both 5-HT2a and 5-HT2b receptors. Pre-treatment with NADPH oxidase inhibitors (apocynin and DPI) blocked 5-HT2b, but not 5-HT2a-induced pMF. Thus, multiple spinal type 2 serotonin receptors elicit pMF, but they act via distinct mechanisms that differ in their requirement for NADPH oxidase activity. PMID:21223996

  1. p38 MAPK is involved in human neutrophil chemotaxis induced by L-amino acid oxidase from Calloselasma rhodosthoma.

    PubMed

    Pontes, Adriana S; Setúbal, Sulamita da S; Nery, Neriane Monteiro; da Silva, Francisquinha Souza; da Silva, Silvana D; Fernandes, Carla F C; Stábeli, Rodrigo G; Soares, Andreimar M; Zuliani, Juliana P

    2016-09-01

    The action of LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function was investigated. Cr-LAAO showed no toxicity on neutrophils. Cr-LAAO in its native form induced the neutrophil chemotaxis, suggesting that its primary structure is essential for stimulation the cell. p38 MAPK and PI3K have a role as signaling pathways of CR-LAAO induced chemotaxis. This toxin also induced the production of hydrogen peroxide and stimulated phagocytosis in neutrophils. Furthermore, Cr-LAAO was able to stimulate neutrophils to release IL-6, IL-8, MPO, LTB4 and PGE2. Together, the data showed that the Cr-LAAO triggers relevant proinflammatory events. PMID:27242041

  2. The control of polyphenol oxidase activity in fruits and vegetables. A study of the interactions between the chemical compounds used and heat treatment.

    PubMed

    Almeida, M E; Nogueira, J N

    1995-04-01

    Objective of this research was to find alternative methods for the control of polyphenol oxidase (PPO) activity in fruits and vegetables with the purpose of reducing or eliminating the use of SO2 for this purpose. Interactions between the use of ascorbic acid, citric acid, EDTA, sodium metabisulphite and heat treatment (70 degrees C for 2 min) in the control of PPO activity were studied in avocado (var. Fortuna), banana (var. Nanica), apple (var. Ana, Fuji, Gala & Golden), pear (var. D'Agua), peach (var. Réal), potato (var. Bintje), eggplant (var. Super F100), mushroom (Agaricus bisporus) and hearts-of-palm (Euterpe edulis Mart). The results demonstrated that PPO of avocado and eggplant was most resistant to inhibition by the methods used. The least efficient method tested for the control of PPO was the addition of ascorbic acid and EDTA, while the most efficient methods investigated included the use of ascorbic acid, citric acid, sodium metabisulphite and heat treatment. The results indicated that, with the exception of PPO from avocado, the most adequate alternative method to substitute for the use of SO2 in the control of PPO was a combination of ascorbic acid, citric acid and heat treatment. PMID:7659702

  3. Methadone, monoamine oxidase, and depression: opioid distribution and acute effects on enzyme activity

    SciTech Connect

    Kaufmann, C.A.; Kreek, M.J.; Raghunath, J.; Arns, P.

    1983-09-01

    Narcotic withdrawal is often accompanied by an atypical depression which responds to resumption of narcotics. It was hypothesized that methadone might exert its antidepressant effects through monoamine oxidase (MAO) inhibition. The current study examined /sub 3/H-methadone distribution in rat brain and effects on regional MAO activity with acute doses (2.5 mg/kg) which approximate those found during chronic methadone maintenance in man. Limbic areas (amygdala, basomedial hypothalamus, caudate-putamen, hippocampus, preoptic nucleus), as well as pituitary and liver were assayed for MAO activity and methadone concentration. MAO activities did not differ significantly in acute methadone or saline-treated cage-mates at 1 or 24 hr. The concentrations of methadone at 1 hr ranged between 17 and 223 ng/100 mg wet wt tissue in the preoptic nucleus and pituitary, respectively. No significant correlation was found between change in MAO activity (MAO methadone/MAO saline) and methadone concentration in any region at 1 or 24 hr. This study does not support the hypothesis that methadone acts as an antidepressant through MAO inhibition, at least not following acute administration of this exogenous opioid.

  4. Xanthine oxidase inhibitory activities and crystal structures of methoxyflavones from Kaempferia parviflora rhizome.

    PubMed

    Nakao, Kikuyo; Murata, Kazuya; Deguchi, Takahiro; Itoh, Kimihisa; Fujita, Takanori; Higashino, Masayuki; Yoshioka, Yuri; Matsumura, Shin-Ichi; Tanaka, Rika; Shinada, Tetsuro; Ohfune, Yasufumi; Matsuda, Hideaki

    2011-01-01

    Kaempferia parviflora (KP), a Zingiberaceae plant, is used as a folk medicine in Thailand for the treatment of various symptoms, including general pains, colic gastrointestinal disorders, and male impotence. In this study, the inhibitory activities of KP against xanthine oxidase (XOD) were investigated. The extract of KP rhizomes showed more potent inhibitory activity (38% at 500 µg/ml) than those of the other Zingiberaceae plants tested. Ten methoxyflavones were isolated from the KP extract as the major chemical components and their chemical structures were elucidated by X-ray crystallography. The structurally confirmed methoxyflavones were subjected to the XOD inhibitory test. Among them, 3,5,7,4',5'-pentamethoxyflavone and 3',4',5,7-tetramethoxyflavone showed inhibitory activities (IC(50) of 0.9 and >4 mM, respectively) and their modes of inhibition are clarified as competitive/non-competitive mixed type. To the best of our knowledge, this is the first report to present the inhibitory activities of KP, 3,5,7,4',5'-pentamethoxyflavone and 3',4',5,7-tetramethoxyflavone against XOD. PMID:21720029

  5. A sensitive method to assay the xanthine oxidase activity in primary cultures of cerebellar granule cells.

    PubMed

    Atlante, A; Valenti, D; Gagliardi, S; Passarella, S

    2000-11-01

    Since xanthine oxidase (XO, Xanthine:oxidoreductase, E.C.1.2.3.22) is a key enzyme in reactive oxygen specie formation which plays a major role in cell oxidative stress, the availability of a sensitive and simple assay useful to detect its activity in monolayer cell cultures is worthwhile. In order to achieve this, we developed a method in which the conversion of pterine into isoxanthopterin is monitored fluorimetrically. Temperature assay was 50 degrees C. The activity of XO was detected in cerebellar granule cells exposed to glutamate. Since XO is formed from protease-dependent xanthine dehydrogenase processing, its activity appearance was found to be prevented by the protease inhibitor, leupeptin, as well as the glutamate NMDA-receptor inhibitor, MK-801, and the Ca(++) complexing agent, EGTA. The reported novel protocol, at variance with a conventional method, is shown to be a simple, fast, sensitive and relatively cheap method to assay XO activity. In addition, the reported assay can be applied to any cell type in culture. PMID:11086257

  6. NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A*S⃞

    PubMed Central

    Frasch, S. Courtney; Berry, Karin Zemski; Fernandez-Boyanapalli, Ruby; Jin, Hyun-Sun; Leslie, Christina; Henson, Peter M.; Murphy, Robert C.; Bratton, Donna L.

    2008-01-01

    Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation. PMID:18824544

  7. ASIC-like Currents in Freshly Isolated Cerebral Artery Smooth Muscle Cells are Inhibited by Endogenous Oxidase Activity

    PubMed Central

    Chung, Wen-Shuo; Farley, Jerry M.; Drummond, Heather A.

    2011-01-01

    Background/Aims: The aim of this study was to determine if VSMC ASIC-like currents are regulated by oxidative state. Methods: We used whole-cell patch clamp of isolated mouse cerebral VSMCs to determine if 1) reducing agents, such as DTT and GSH, and 2) inhibition of endogenous oxidase activity from NADPH and Xanthine oxidases potentiate active currents and activate electrically silent currents. Results: Pretreatment with 2 mM DTT or GSH, increased the mean peak amplitude of ASIC-like currents evoked by pH 6.0 from 0.4 ± 0.1 to 14.9 ± 3.6 pA/pF, and from 0.9 ± 0.3 to 11.3 ± 2.4 pA/pF, respectively. Pretreatment with apocynin, a NADPH oxidase inhibitor, mimics the effect of the reducing agents, with the mean peak current amplitude increased from 0.9 ± 0.5 to 7.0 ± 2.6 pA/pF and from 0.5 ± 0.2 to 26.4 ± 6.8 pA/pF by 50 and 200 μM apocynin, respectively. Pretreatment with allopurinol, a xanthine oxidase inhibitor, also potentiates the VSMC ASIC-like activity. Conclusion: These findings suggest that VSMC ASIC-like channels are regulated by oxidative state and may be inhibited by basal endogenous oxidative sources such as NADPH and xanthine oxidase. PMID:21325830

  8. Induction of mixed-function oxidase activity in mouse lymphoid tissues by polycyclic aromatic hydrocarbons

    SciTech Connect

    Griffin, G.D.; Egan, B.Z.; Lee, N.E.; Burtis, C.A.

    1986-01-01

    Polycyclic aromatic hydrocarbon (PAH) exposure can cause mixed-function oxidase (MFO) enzyme induction in certain tissues of various organisms. Measurements of such induction might serve as a useful bioindicator of human exposure to PAHs, provided readily obtainable human tissues can be utilized for such measurements. The authors have investigated the MFO activity in various lymphoid tissues of the C3H mouse as a model system and have studied the effect of systemic PAH treatment on such enzyme activity. An MFO enzyme assay was used to measure the activity of 7-ethoxyresorufin deethylase, an enzyme activity that may be specific for the cytochrome P-448 subset of MFO enzymes (those enzymes that are induced in cells or tissues following PAH administration). Intraperitoneal injection of mice with 180 mg/kg (4.6 mg) benzo(a)pyrene (BaP) or 160 mg/kg (4.0 mg) 3-methylcholanthrene (MC) produced a significant induction in MFO activity in mouse spleen S9 fractions 48 h after the injection. Induction ratios (induced activity/control activity) between 4 and 5 were seen with BaP; MC produced induction ratios of 2.5-3.0. Enzyme activity was not induced in the spleen within 16 h following BaP or MC administration. Other experiments indicated that MFO activity could be induced in thymus cells 48 h after either BaP or MC treatment. Treatment with BaP or MC did produce significant enzyme induction in the liver and lung tissues from the animals both 16 and 48 h after chemical treatment.

  9. Flagellin-induced NADPH oxidase 4 activation is involved in atherosclerosis

    PubMed Central

    Kim, Jinoh; Seo, Misun; Kim, Su Kyung; Bae, Yun Soo

    2016-01-01

    It is widely accepted that bacterial infection-mediated inflammation facilitates development of atherosclerosis by activating toll-like receptor (TLR) signaling system. We reasoned that NADPH oxidases (Nox), required for TLR-mediated inflammatory response, are involved in atherogenesis. Here, we show that the activation of Nox4 through TLR5 regulates the inflammation of the endothelium and in atherogenesis. Flagellin-induced interaction between the COOH region of Nox4 and the TIR domain of TLR5 led to H2O2 generation, which in turn promoted the secretion of pro-inflammatory cytokines including IL-8, as well as the expression of ICAM-1 in human aortic endothelial cells (HAECs). Knockdown of the Nox4 in HAECs resulted in attenuated expressions of IL-8 and ICAM-1 leading to a reduction in the adhesion and trans-endothelial migration of monocytes. Challenge of recombinant FliC (rFliC) to the ApoE KO mice with high-fat diet (HFD) resulted in significantly increased atherosclerotic plaque sizes compared to the saline-injected mice. However, an injection of rFliC into the Nox4ApoE DKO mice with HFDs failed to generate atherosclerotic plaque, suggesting that Nox4 deficiency resulted in significant protections against rFliC-mediated atherogenesis. We conclude that TLR5-dependent Nox4 activation and subsequent H2O2 generation play critical roles for the development of atherosclerosis. PMID:27146088

  10. Characterization of germin-like protein with polyphenol oxidase activity from Satsuma mandarine.

    PubMed

    Cheng, Xi; Huang, Xingjian; Liu, Siyu; Tang, Mi; Hu, Wanfeng; Pan, Siyi

    2014-07-01

    Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris-HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65°C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity. PMID:24845377

  11. Flagellin-induced NADPH oxidase 4 activation is involved in atherosclerosis.

    PubMed

    Kim, Jinoh; Seo, Misun; Kim, Su Kyung; Bae, Yun Soo

    2016-01-01

    It is widely accepted that bacterial infection-mediated inflammation facilitates development of atherosclerosis by activating toll-like receptor (TLR) signaling system. We reasoned that NADPH oxidases (Nox), required for TLR-mediated inflammatory response, are involved in atherogenesis. Here, we show that the activation of Nox4 through TLR5 regulates the inflammation of the endothelium and in atherogenesis. Flagellin-induced interaction between the COOH region of Nox4 and the TIR domain of TLR5 led to H2O2 generation, which in turn promoted the secretion of pro-inflammatory cytokines including IL-8, as well as the expression of ICAM-1 in human aortic endothelial cells (HAECs). Knockdown of the Nox4 in HAECs resulted in attenuated expressions of IL-8 and ICAM-1 leading to a reduction in the adhesion and trans-endothelial migration of monocytes. Challenge of recombinant FliC (rFliC) to the ApoE KO mice with high-fat diet (HFD) resulted in significantly increased atherosclerotic plaque sizes compared to the saline-injected mice. However, an injection of rFliC into the Nox4ApoE DKO mice with HFDs failed to generate atherosclerotic plaque, suggesting that Nox4 deficiency resulted in significant protections against rFliC-mediated atherogenesis. We conclude that TLR5-dependent Nox4 activation and subsequent H2O2 generation play critical roles for the development of atherosclerosis. PMID:27146088

  12. Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells.

    PubMed

    Isoai, Atsushi; Kimura, Hidetoshi; Reichert, Arno; Schörgendorfer, Kurt; Nikaido, Kiyokazu; Tohda, Hideki; Giga-Hama, Yuko; Mutoh, Norihiro; Kumagai, Hiromichi

    2002-10-01

    The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe. A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to alpha-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Biocatalysts were prepared using ASP327-10 and T. variabilis, and evaluated to demonstrate the feasibility of recombinant S. pombe for industrial application. The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) fixation and permeabilization by alkaline treatment. Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T. variabilis decreased to 20-30%. Heat treatment was required before cell fixation by GDA to inactivate the catalase in S. pombe. This improved the efficiency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S. pombe. To overcome this weakness, a catalase-deficient host strain was obtained by ethylmethansulfate mutagenesis. Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S. pombe even in the absence of G418. The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required. The obtained integrant and its biocatalyst were significantly improved in GL-7ACA conversion ability and mechanical strength. This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme. PMID:12209783

  13. Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor

    NASA Technical Reports Server (NTRS)

    Morre, D. James

    2002-01-01

    The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

  14. Nitric Oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation

    PubMed Central

    Qian, Jin; Chen, Feng; Kovalenkov, Yevgeniy; Pandey, Deepesh; Moseleley, M. Arthur; Foster, Matthew W.; Black, Stephen M.; Venema, Richard C.; Stepp, David W.; Fulton, David J.R.

    2012-01-01

    The NADPH oxidases (Nox) are a family of transmembrane oxidoreductases that produce superoxide and other reactive oxygen species (ROS). Nox5 was the last of the conventional Nox isoforms to be identified and is a calcium-dependent enzyme that does not depend on accessory subunits for activation. Recently, Nox5 was shown to be expressed in human blood vessels and therefore the goal of current study was to determine whether nitric oxide (NO) can modulate Nox5 activity. Endogenously produced NO potently inhibited basal and stimulated Nox5 activity and inhibition was reversible with chronic, but not acute exposure to L-NAME. Nox5 activity was reduced by NO donors, iNOS, eNOS and in endothelial cells and LPS-stimulated smooth muscle cells in a manner dependent on NO concentration. ROS production was diminished by NO in an isolated enzyme activity assay replete with surplus calcium and NADPH. There was no evidence for NO-dependent changes in tyrosine nitration, glutathiolation or phosphorylation of Nox5. In contrast, there was evidence for the increased nitrosylation of Nox5 as determined by the biotin-switch assay and mass spectrometry. Four S-nitrosylation sites were identified and of these, mutation of C694 dramatically lowered Nox5 activity, NO-sensitivity and biotin-labeling. Furthermore, co-expression of the denitrosylation enzymes thioredoxin (Trx1) and GSNO reductase (GSNOR) prevented NO-dependent inhibition of Nox5. The potency of NO against other Nox enzymes was Nox1≥Nox3>Nox5>Nox2 whereas Nox4 was refractory. Collectively, these results suggest that endogenously produced NO can directly S-nitrosylate and inhibit the activity of Nox5. PMID:22387196

  15. The Role of NADPH Oxidases (NOXs) in Liver Fibrosis and the Activation of Myofibroblasts

    PubMed Central

    Liang, Shuang; Kisseleva, Tatiana; Brenner, David A.

    2016-01-01

    Chronic liver injury, resulted from different etiologies (e.g., virus infection, alcohol abuse, nonalcoholic steatohepatitis (NASH) and cholestasis) can lead to liver fibrosis characterized by the excess accumulation of extracellular matrix (ECM) proteins (e.g., type I collagen). Hepatic myofibroblasts that are activated upon liver injury are the key producers of ECM proteins, contributing to both the initiation and progression of liver fibrosis. Hepatic stellate cells (HSCs) and to a lesser extent, portal fibroblast, are believed to be the precursor cells that give rise to hepatic myofibroblasts in response to liver injury. Although, much progress has been made toward dissecting the lineage origin of myofibroblasts, how these cells are activated and become functional producers of ECM proteins remains incompletely understood. Activation of myofibroblasts is a complex process that involves the interactions between parenchymal and non-parenchymal cells, which drives the phenotypic change of HSCs from a quiescent stage to a myofibroblastic and active phenotype. Accumulating evidence has suggested a critical role of NADPH oxidase (NOX), a multi-component complex that catalyzes reactions from molecular oxygen to reactive oxygen species (ROS), in the activation process of hepatic myofibroblasts. NOX isoforms, including NOX1, NOX2 and NOX4, and NOX-derived ROS, have all been implicated to regulate HSC activation and hepatocyte apoptosis, both of which are essential steps for initiating liver fibrosis. This review highlights the importance of NOX isoforms in hepatic myofibroblast activation and the progression of liver fibrosis, and also discusses the therapeutic potential of targeting NOXs for liver fibrosis and associated hepatic diseases. PMID:26869935

  16. The Role of NADPH Oxidases (NOXs) in Liver Fibrosis and the Activation of Myofibroblasts.

    PubMed

    Liang, Shuang; Kisseleva, Tatiana; Brenner, David A

    2016-01-01

    Chronic liver injury, resulted from different etiologies (e.g., virus infection, alcohol abuse, nonalcoholic steatohepatitis (NASH) and cholestasis) can lead to liver fibrosis characterized by the excess accumulation of extracellular matrix (ECM) proteins (e.g., type I collagen). Hepatic myofibroblasts that are activated upon liver injury are the key producers of ECM proteins, contributing to both the initiation and progression of liver fibrosis. Hepatic stellate cells (HSCs) and to a lesser extent, portal fibroblast, are believed to be the precursor cells that give rise to hepatic myofibroblasts in response to liver injury. Although, much progress has been made toward dissecting the lineage origin of myofibroblasts, how these cells are activated and become functional producers of ECM proteins remains incompletely understood. Activation of myofibroblasts is a complex process that involves the interactions between parenchymal and non-parenchymal cells, which drives the phenotypic change of HSCs from a quiescent stage to a myofibroblastic and active phenotype. Accumulating evidence has suggested a critical role of NADPH oxidase (NOX), a multi-component complex that catalyzes reactions from molecular oxygen to reactive oxygen species (ROS), in the activation process of hepatic myofibroblasts. NOX isoforms, including NOX1, NOX2 and NOX4, and NOX-derived ROS, have all been implicated to regulate HSC activation and hepatocyte apoptosis, both of which are essential steps for initiating liver fibrosis. This review highlights the importance of NOX isoforms in hepatic myofibroblast activation and the progression of liver fibrosis, and also discusses the therapeutic potential of targeting NOXs for liver fibrosis and associated hepatic diseases. PMID:26869935

  17. Elevated NADPH oxidase activity contributes to oxidative stress and cell death in Huntington's disease

    PubMed Central

    Valencia, Antonio; Sapp, Ellen; Kimm, Jeffrey S.; McClory, Hollis; Reeves, Patrick B.; Alexander, Jonathan; Ansong, Kwadwo A.; Masso, Nicholas; Frosch, Matthew P.; Kegel, Kimberly B.; Li, Xueyi; DiFiglia, Marian

    2013-01-01

    A mutation in the huntingtin (Htt) gene produces mutant Htt and Huntington's disease (HD), a neurodegenerative disorder. HD patients have oxidative damage in the brain, but the causes are unclear. Compared with controls, we found brain levels of NADPH oxidase (NOX) activity, which produces reactive oxygen species (ROS), elevated in human HD postmortem cortex and striatum and highest in striatum of presymptomatic individuals. Synaptosome fractions from cortex and striatum of HD140Q/140Q mice had elevated NOX activity at 3 months of age and a further rise at 6 and 12 months compared with synaptosomes of age-matched wild-type (WT) mice. High NOX activity in primary cortical and striatal neurons of HD140Q/140Q mice correlated with more ROS and neurite swellings. These features and neuronal cell death were markedly reduced by treatment with NOX inhibitors such as diphenyleneiodonium (DPI), apocynin (APO) and VAS2870. The rise in ROS levels in mitochondria of HD140Q/140Q neurons followed the rise in NOX activity and inhibiting only mitochondrial ROS was not neuroprotective. Mutant Htt colocalized at plasma membrane lipid rafts with gp91-phox, a catalytic subunit for the NOX2 isoform. Assembly of NOX2 components at lipid rafts requires activation of Rac1 which was also elevated in HD140Q/140Q neurons. HD140Q/140Q mice bred to gp91-phox knock-out mice had lower NOX activity in the brain and in primary neurons, and neurons had normal ROS levels and significantly improved survival. These findings suggest that increased NOX2 activity at lipid rafts is an early and major source of oxidative stress and cell death in HD140Q/140Q neurons. PMID:23223017

  18. Evaluation of oxalate decarboxylase and oxalate oxidase for industrial applications.

    PubMed

    Cassland, Pierre; Sjöde, Anders; Winestrand, Sandra; Jönsson, Leif J; Nilvebrant, Nils-Olof

    2010-05-01

    Increased recirculation of process water has given rise to problems with formation of calcium oxalate incrusts (scaling) in the pulp and paper industry and in forest biorefineries. The potential in using oxalate decarboxylase from Aspergillus niger for oxalic acid removal in industrial bleaching plant filtrates containing oxalic acid was examined and compared with barley oxalate oxidase. Ten different filtrates from chemical pulping were selected for the evaluation. Oxalate decarboxylase degraded oxalic acid faster than oxalate oxidase in eight of the filtrates, while oxalate oxidase performed better in one filtrate. One of the filtrates inhibited both enzymes. The potential inhibitory effect of selected compounds on the enzymatic activity was tested. Oxalate decarboxylase was more sensitive than oxalate oxidase to hydrogen peroxide. Oxalate decarboxylase was not as sensitive to chlorate and chlorite as oxalate oxidase. Up to 4 mM chlorate ions, the highest concentration tested, had no inhibitory effect on oxalate decarboxylase. Analysis of the filtrates suggests that high concentrations of chlorate present in some of the filtrates were responsible for the higher sensitivity of oxalate oxidase in these filtrates. Oxalate decarboxylase was thus a better choice than oxalate oxidase for treatment of filtrates from chlorine dioxide bleaching. PMID:19763895

  19. Identification of two promoters for human D-amino acid oxidase gene: implication for the differential promoter regulation mediated by PAX5/PAX2.

    PubMed

    Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

    2015-05-01

    D-amino acid oxidase (DAO) is a flavoenzyme that metabolizes d-amino acids. Until now, the DAO expression mechanism is still unclear. Our assessment of human DAO (hDAO) promoter activity using luciferase reporter system indicated the proximal upstream region of exon1 (-237/+1) has promoter activity (P1). Interestingly, we identified an alternative promoter in the proximal upstream region of exon2 (+4,126/+4,929) (P2). This alternative promoter has stronger activity than that of P1. Our results also revealed a negative regulatory segment (+1,163/+1,940) in intron1; that would act in concert with P1 and P2. Bioinformatics analyses elucidated the conservation of transcription factor PAX5 family binding sites among species. These sites (-60/-31) and (+4,464/+4,493), locate in P1 and P2 of hDAO, respectively. Gel shift assays demonstrated P1 contains a site (-60/-31) for PAX5 binding while P2 has three sites for both paired box gene 2 (PAX2) and paired box gene 5 (PAX5) binding. The dual roles of PAX5 family in regulating hDAO transcription by modulating promoter activity of P1 and activating promoter activity of P2 were implicated based on the site-directed mutagenesis experiment. Altogether, our data suggested the differential regulation of hDAO expression by two promoters whose activities may be modulated by the binding of PAX2 and PAX5. PMID:25500505

  20. 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

  1. Coimmobilization of acetylcholinesterase and choline oxidase on gold nanoparticles: stoichiometry, activity, and reaction efficiency.

    PubMed

    Keighron, Jacqueline D; Åkesson, Sebastian; Cans, Ann-Sofie

    2014-09-30

    Hybrid structures constructed from biomolecules and nanomaterials have been used in catalysis and bioanalytical applications. In the design of many chemically selective biosensors, enzymes conjugated to nanoparticles or carbon nanotubes have been used in functionalization of the sensor surface for enhancement of the biosensor functionality and sensitivity. The conditions for the enzyme:nanomaterial conjugation should be optimized to retain maximal enzyme activity, and biosensor effectiveness. This is important as the tertiary structure of the enzyme is often altered when immobilized and can significantly alter the enzyme catalytic activity. Here we show that characterization of a two-enzyme:gold nanoparticle (AuNP) conjugate stoichiometry and activity can be used to gauge the effectiveness of acetylcholine detection by acetylcholine esterase (AChE) and choline oxidase (ChO). This was done by using an analytical approach to quantify the number of enzymes bound per AuNP and monitor the retained enzyme activity after the enzyme:AuNP synthesis. We found that the amount of immobilized enzymes differs from what would be expected from bulk solution chemistry. This analysis was further used to determine the optimal ratio of AChE:ChO added at synthesis to achieve optimum sequential enzyme activity for the enzyme:AuNP conjugates, and reaction efficiencies of greater than 70%. We here show that the knowledge of the conjugate stoichiometry and retained enzyme activity can lead to more efficient detection of acetylcholine by controlling the AChE:ChO ratio bound to the gold nanoparticle material. This approach of optimizing enzyme gold nanoparticle conjugates should be of great importance in the architecture of enzyme nanoparticle based biosensors to retain optimal sensor sensitivity. PMID:25167196

  2. Localization of hydrogen peroxide accumulation and diamine oxidase activity in pea root nodules under aluminum stress.

    PubMed

    Sujkowska-Rybkowska, Marzena; Borucki, Wojciech

    2014-02-01

    Aluminum (Al) is one of the environmental stressors that induces formation of reactive oxygen species (ROS) in plants. Hydrogen peroxide (H2O2) and H2O2-generated apoplast diamine oxidase (DAO) activity were detected cytochemically via transmission electron microscopy (TEM), in pea (Pisum sativum L.) root nodules exposed to high (50 μM AlCl3, for 2 and 24h) Al stress. The nodules were shown to respond to Al stress by disturbances in infection thread (IT) growth, bacteria endocytosis, premature degeneration of bacteroidal tissue and generation of H2O2 in nodule apoplast. Large amounts of peroxide were found at the same sites as high DAO activity under Al stress, suggesting that DAO is a major source of Al-induced peroxide accumulation in the nodules. Peroxide distribution and DAO activity in the nodules of both control plants and Al-treated ones were typically found in the plant cell walls, intercellular spaces and infection threads. However, 2 h Al treatment increased DAO activity and peroxide accumulation in the nodule apoplast and bacteria within threads. A prolonged Al treatment (24 h) increased the H2O2 content and DAO activity in the nodule apoplast, especially in the thread walls, matrix and bacteria within infection threads. In addition to ITs, prematurely degenerated bacteroids, which occurred in response to Al, were associated with intense staining for H2O2 and DAO activity. These results suggest the involvement of DAO in the production of a large amount of H2O2 in the nodule apoplast under Al stress. The role of reactive oxygen species in pea-Rhizobium symbiosis under Al stress is discussed. PMID:24246127

  3. The substrate tolerance of alcohol oxidases.

    PubMed

    Pickl, Mathias; Fuchs, Michael; Glueck, Silvia M; Faber, Kurt

    2015-08-01

    Alcohols are a rich source of compounds from renewable sources, but they have to be activated in order to allow the modification of their carbon backbone. The latter can be achieved via oxidation to the corresponding aldehydes or ketones. As an alternative to (thermodynamically disfavoured) nicotinamide-dependent alcohol dehydrogenases, alcohol oxidases make use of molecular oxygen but their application is under-represented in synthetic biotransformations. In this review, the mechanism of copper-containing and flavoprotein alcohol oxidases is discussed in view of their ability to accept electronically activated or non-activated alcohols and their propensity towards over-oxidation of aldehydes yielding carboxylic acids. In order to facilitate the selection of the optimal enzyme for a given biocatalytic application, the substrate tolerance of alcohol oxidases is compiled and discussed: Substrates are classified into groups (non-activated prim- and sec-alcohols; activated allylic, cinnamic and benzylic alcohols; hydroxy acids; sugar alcohols; nucleotide alcohols; sterols) together with suitable alcohol oxidases, their microbial source, relative activities and (stereo)selectivities. PMID:26153139

  4. Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase.

    PubMed

    Li, Jian; Lawton, Thomas J; Kostecki, Jan S; Nisthal, Alex; Fang, Jia; Mayo, Stephen L; Rosenzweig, Amy C; Jewett, Michael C

    2016-02-01

    Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an alternative to traditional recombinant protein expression, we show the ability to use cell-free protein synthesis (CFPS) to produce complex MCO proteins with high soluble titers. Specifically, we report the production of MCOs in an Escherichia coli-based cell-free transcription-translation system. Total yields as high as 1.2 mg mL(-1) were observed after a 20-h batch reaction. More than 95% of the protein was soluble and activity was obtained by simple post-CFPS addition of copper ions in the form of CuSO4 . Scale-up reactions were achieved from 15 to 100 µL without a decrease in productivity and solubility. CFPS titers were higher than in vivo expression titers and more soluble, avoiding the formation of inclusion bodies. Our work extends the utility of the cell-free platform to the production of active proteins containing copper cofactors and demonstrates a simple method for producing MCOs. PMID:26356243

  5. Adsorption and Catalytic Activity of Glucose Oxidase Accumulated on OTCE upon the Application of External Potential

    PubMed Central

    Benavidez, Tomás E.; Torrente, Daniel; Marucho, Marcelo; Garcia, Carlos D.

    2014-01-01

    This article describes the adsorption of glucose oxidase (GOx) onto optically transparent carbon electrodes (OTCE) under the effect of applied potential and the analysis of the enzymatic activity of the resulting GOx/OTCE substrates. In order to avoid electrochemical interferences with the enzyme redox center, control electrochemical experiments were performed using flavin adenine dinucleotide (FAD) and GOx/OTCE substrates. Then, the enzyme adsorption experiments were carried out as a function of the potential applied (ranged from the open circuit potential to +950 mV), the pH solution, the concentration of enzyme, and the ionic strength on the environment. The experimental results demonstrated that an increase in the adsorbed amount of GOx on the OTCE can be achieved when the potential was applied. Although the increase in the adsorbed amount was examined as a function of the potential, a maximum enzymatic activity was observed in the GOx/OTCE substrate achieved at +800 mV. These experiments suggest that although an increase in the amount of enzyme adsorbed can be obtained by the application of an external potential to the electrode, the magnitude of such potential can produce detrimental effects in the conformation of the adsorbed protein and should be carefully considered. As such, the article describes a simple and rational approach to increase the amount of enzyme adsorbed on a surface and can be applied to improve the sensitivity of a variety of biosensors. PMID:25261840

  6. Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice.

    PubMed

    Hosseini, Mohsen; Mahfouf, Walid; Serrano-Sanchez, Martin; Raad, Houssam; Harfouche, Ghida; Bonneu, Marc; Claverol, Stephane; Mazurier, Frederic; Rossignol, Rodrigue; Taieb, Alain; Rezvani, Hamid Reza

    2015-04-01

    Xeroderma pigmentosum type C (XP-C) is characterized mostly by a predisposition to skin cancers and accelerated photoaging, but little is known about premature skin aging in this disease. By comparing young and old mice, we found that the level of progerin and p16(INK4a) expression, β-galactosidase activity, and reactive oxygen species, which increase with age, were higher in young Xpc(-/-) mice than in young Xpc(+/+) ones. The expression level of mitochondrial complexes and mitochondrial functions in the skin of young Xpc(-/-) was as low as in control aged Xpc(+/+)animals. Furthermore, the metabolic profile in young Xpc(-/-) mice resembled that found in aged Xpc(+/+) mice. Furthermore, premature skin aging features in young Xpc(-/-) mice were mostly rescued by inhibition of nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) activity by using a NOX1 peptide inhibitor, suggesting that the continuous oxidative stress due to overactivation of NOX1 has a causative role in the underlying pathophysiology. PMID:25437426

  7. Is Xanthine oxidase activity in polycystic ovary syndrome associated with inflammatory and cardiovascular risk factors?

    PubMed

    Isık, Hatice; Aynıoglu, Oner; Tımur, Hakan; Sahbaz, Ahmet; Harma, Muge; Can, Murat; Guven, Berrak; Alptekin, Husnu; Kokturk, Furuzan

    2016-08-01

    The aim of this study is to examine women with polycystic ovary syndrome (PCOS) to determine the relationship between xanthine oxidase (XO) and oxidative stress, inflammatory status, and various clinical and biochemical parameters. In this cross-sectional study a total of 83 women including 45 PCOS patients and 38 healthy women were enrolled. We collected blood samples for XO and superoxide dismutase (SOD) activity, hormone levels, cholesterol values, and inflammatory markers. Body mass index (BMI) , waist-to-hip ratio (WHR), and blood pressure were assessed. Blood samples were taken for hormonal levels, cholesterol levels, fasting plasma glucose (FPG), fasting plasma insulin (FPI), homeostatic model assessment-insulin resistance (HOMA-IR) index, quantitative insulin sensitivity check index (QUICKI), C-reactive protein (CRP), white blood cell and neutrophil counts, XO and SOD activities. The basal hormone levels, triglyceride (TG) levels, TG/HDL-C (high density lipoprotein-cholesterol) ratios FPG, FPI and HOMA-IR levels were higher in PCOS patients compared to controls (p<0.05). Platelet and plateletcrit (PCT) values, CRP, and XO activity were significantly increased, however SOD activity was decreased in PCOS patients (p<0.001). XO activity was positively correlated with LH/FSH and TG/HDL ratios, CRP, PCT, FPG, FPI, and HOMA-IR, and negatively correlated with QUICKI levels. In conclusion, XO is a useful marker to assess oxidative stress in PCOS patients. Positive correlations between XO and inflammatory markers and cardiovascular disease risk factors suggest that XO plays an important role in the pathogenesis of PCOS and its metabolic complications. PMID:27295433

  8. NMDA Receptor-Mediated Activation of NADPH Oxidase and Glomerulosclerosis in Hyperhomocysteinemic Rats

    PubMed Central

    Zhang, Chun; Yi, Fan; Xia, Min; Boini, Krishna M.; Zhu, Qing; Laperle, Laura A.; Abais, Justine M.; Brimson, Christopher A.

    2010-01-01

    Abstract This study investigated the role of NMDA receptor in hyperhomocyteinemia (hHcys)-induced NADPH oxidase (Nox) activation and glomerulosclerosis. Sprague–Dawley rats were fed a folate-free (FF) diet to produce hHcys, and a NMDA receptor antagonist, MK-801, was administrated. Rats fed the FF diet exhibited significantly increased plasma homocysteine levels, upregulated NMDA receptor expression, enhanced Nox activity and Nox-dependent O2.− production in the glomeruli, which were accompanied by remarkable glomerulosclerosis. MK-801 treatment significantly inhibited Nox-dependent O2.− production induced by hHcys and reduced glomerular damage index as compared with vehicle-treated hHcys rats. Correspondingly, glomerular deposition of extracellular matrix components in hHcys rats was ameliorated by the administration of MK-801. Additionally, hHcys induced an increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) expression and a decrease in matrix metalloproteinase (MMP)-1 and MMP-9 activities, all of which were abolished by MK-801 treatment. In vitro studies showed that homocysteine increased Nox-dependent O2.− generation in rat mesangial cells, which was blocked by MK-801. Pretreatment with MK-801 also reversed homocysteine-induced decrease in MMP-1 activity and increase in TIMP-1 expression. These results support the view that the NMDA receptor may mediate Nox activation in the kidney during hHcys and thereby play a critical role in the development of hHcys-induced glomerulosclerosis. Antioxid. Redox Signal. 13, 975–986. PMID:20406136

  9. The Arabidopsis COX11 Homolog is Essential for Cytochrome c Oxidase Activity

    PubMed Central

    Radin, Ivan; Mansilla, Natanael; Rödel, Gerhard; Steinebrunner, Iris

    2015-01-01

    Members of the ubiquitous COX11 (cytochrome c oxidase 11) protein family are involved in copper delivery to the COX complex. In this work, we characterize the Arabidopsis thaliana COX11 homolog (encoded by locus At1g02410). Western blot analyses and confocal microscopy identified Arabidopsis COX11 as an integral mitochondrial protein. Despite sharing high sequence and structural similarities, the Arabidopsis COX11 is not able to functionally replace the Saccharomyces cerevisiae COX11 homolog. Nevertheless, further analysis confirmed the hypothesis that Arabidopsis COX11 is essential for COX activity. Disturbance of COX11 expression through knockdown (KD) or overexpression (OE) affected COX activity. In KD lines, the activity was reduced by ~50%, resulting in root growth inhibition, smaller rosettes and leaf curling. In OE lines, the reduction was less pronounced (~80% of the wild type), still resulting in root growth inhibition. Additionally, pollen germination was impaired in COX11 KD and OE plants. This effect on pollen germination can only partially be attributed to COX deficiency and may indicate a possible auxiliary role of COX11 in ROS metabolism. In agreement with its role in energy production, the COX11 promoter is highly active in cells and tissues with high-energy demand for example shoot and root meristems, or vascular tissues of source and sink organs. In COX11 KD lines, the expression of the plasma-membrane copper transporter COPT2 and of several copper chaperones was altered, indicative of a retrograde signaling pathway pertinent to copper homeostasis. Based on our data, we postulate that COX11 is a mitochondrial chaperone, which plays an important role for plant growth and pollen germination as an essential COX complex assembly factor. PMID:26734017

  10. Rotenone Activates Phagocyte NADPH Oxidase through Binding to Its Membrane Subunit gp91phox

    PubMed Central

    Zhou, Hui; Zhang, Feng; Chen, Shih-heng; Zhang, Dan; Wilson, Belinda; Hong, Jau-shyong; Gao, Hui-Ming

    2011-01-01

    Rotenone, a widely used pesticide, reproduces Parkinsonism in rodents and associates with increased risk for Parkinson’s disease. We previously reported rotenone increased superoxide production through stimulating microglial phagocyte NADPH oxidase (PHOX). The present study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91phox, the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91phox. Functional studies showed both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91phox/p22phox) and cytosolic subunits (p67phox and p47phox). Rotenone-elicited extracellular superoxide release in p47phox-deficient macrophages suggested rotenone enabled to activate PHOX through a p47phox-independent mechanism. Increased membrane translocation of p67phox, elevated binding of p67phox to rotenone-treated membrane fractions, and co-immunoprecipitation of p67phox and gp91phox in rotenone-treated wild-type and p47phox-deficient macrophages indicated p67phox played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91phox. Rac1, a Rho-like small GTPase, enhanced p67phox-gp91phox interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91phox; such an interaction triggered membrane translocation of p67phox, leading to PHOX activation and superoxide production. PMID:22094225

  11. The Arabidopsis COX11 Homolog is Essential for Cytochrome c Oxidase Activity.

    PubMed

    Radin, Ivan; Mansilla, Natanael; Rödel, Gerhard; Steinebrunner, Iris

    2015-01-01

    Members of the ubiquitous COX11 (cytochrome c oxidase 11) protein family are involved in copper delivery to the COX complex. In this work, we characterize the Arabidopsis thaliana COX11 homolog (encoded by locus At1g02410). Western blot analyses and confocal microscopy identified Arabidopsis COX11 as an integral mitochondrial protein. Despite sharing high sequence and structural similarities, the Arabidopsis COX11 is not able to functionally replace the Saccharomyces cerevisiae COX11 homolog. Nevertheless, further analysis confirmed the hypothesis that Arabidopsis COX11 is essential for COX activity. Disturbance of COX11 expression through knockdown (KD) or overexpression (OE) affected COX activity. In KD lines, the activity was reduced by ~50%, resulting in root growth inhibition, smaller rosettes and leaf curling. In OE lines, the reduction was less pronounced (~80% of the wild type), still resulting in root growth inhibition. Additionally, pollen germination was impaired in COX11 KD and OE plants. This effect on pollen germination can only partially be attributed to COX deficiency and may indicate a possible auxiliary role of COX11 in ROS metabolism. In agreement with its role in energy production, the COX11 promoter is highly active in cells and tissues with high-energy demand for example shoot and root meristems, or vascular tissues of source and sink organs. In COX11 KD lines, the expression of the plasma-membrane copper transporter COPT2 and of several copper chaperones was altered, indicative of a retrograde signaling pathway pertinent to copper homeostasis. Based on our data, we postulate that COX11 is a mitochondrial chaperone, which plays an important role for plant growth and pollen germination as an essential COX complex assembly factor. PMID:26734017

  12. D-Amino acid oxidase-induced oxidative stress, 3-bromopyruvate and citrate inhibit angiogenesis, exhibiting potent anticancer effects.

    PubMed

    El Sayed, S M; El-Magd, R M Abou; Shishido, Y; Yorita, K; Chung, S P; Tran, D H; Sakai, T; Watanabe, H; Kagami, S; Fukui, K

    2012-10-01

    Angiogenesis is critical for cancer growth and metastasis. Steps of angiogenesis are energy consuming, while vascular endothelial cells are highly glycolytic. Glioblastoma multiforme (GBM) is a highly vascular tumor and this enhances its aggressiveness. D-amino acid oxidase (DAO) is a promising therapeutic protein that induces oxidative stress upon acting on its substrates. Oxidative stress-energy depletion (OSED) therapy was recently reported (El Sayed et al., Cancer Gene Ther, 19, 1-18, 2012). OSED combines DAO-induced oxidative stress with energy depletion caused by glycolytic inhibitors such as 3-bromopyruvate (3BP), a hexokinase II inhibitor that depleted ATP in cancer cells and induced production of hydrogen peroxide. 3BP disturbs the Warburg effect and antagonizes effects of lactate and pyruvate (El Sayed et al., J Bioenerg Biomembr, 44, 61-79, 2012). Citrate is a natural organic acid capable of inhibiting glycolysis by targeting phosphofructokinase. Here, we report that DAO, 3BP and citrate significantly inhibited angiogenesis, decreased the number of vascular branching points and shortened the length of vascular tubules. OSED delayed the growth of C6/DAO glioma cells. 3BP combined with citrate delayed the growth of C6 glioma cells and decreased significantly the number and size of C6 glioma colonies in soft agar. Human GBM cells (U373MG) were resistant to chemotherapy e.g. cisplatin and cytosine arabinoside, while 3BP was effective in decreasing the viability and disturbing the morphology of U373MG cells. PMID:22802136

  13. Substance P Exacerbates Dopaminergic Neurodegeneration through Neurokinin-1 Receptor-Independent Activation of Microglial NADPH Oxidase

    PubMed Central

    Chu, Chun-Hsien; Qian, Li; Chen, Shih-Heng; Wilson, Belinda; Oyarzabal, Esteban; Jiang, Lulu; Ali, Syed; Robinson, Bonnie; Kim, Hyoung-Chun

    2014-01-01

    Although dysregulated substance P (SP) has been implicated in the pathophysiology of Parkinson's disease (PD), how SP affects the survival of dopaminergic neurons remains unclear. Here, we found that mice lacking endogenous SP (TAC1−/−), but not those deficient in the SP receptor (neurokinin-1 receptor, NK1R), were more resistant to lipopolysaccharide (LPS)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic neurodegeneration than wild-type controls, suggesting a NK1R-independent toxic action of SP. In vitro dose–response studies revealed that exogenous SP enhanced LPS- and 1-methyl-4-phenylpyridinium (MPP+)-induced dopaminergic neurodegeneration in a bimodal manner, peaking at submicromolar and subpicomolar concentrations, but was substantially less effective at intermediate concentrations. Mechanistically, the actions of submicromolar levels of SP were NK1R-dependent, whereas subpicomolar SP-elicited actions required microglial NADPH oxidase (NOX2), the key superoxide-producing enzyme, but not NK1R. Subpicomolar concentrations of SP activated NOX2 by binding to the catalytic subunit gp91phox and inducing membrane translocation of the cytosolic subunits p47phox and p67phox. The importance of NOX2 was further corroborated by showing that inhibition or disruption of NOX2 blocked subpicomolar SP-exacerbated neurotoxicity. Together, our findings revealed a critical role of microglial NOX2 in mediating the neuroinflammatory and dopaminergic neurodegenerative effects of SP, which may provide new insights into the pathogenesis of PD. PMID:25209287

  14. Improvement of the stability and activity of immobilized glucose oxidase on modified iron oxide magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Abbasi, Mahboube; Amiri, Razieh; Bordbar, Abdol-Kalegh; Ranjbakhsh, Elnaz; Khosropour, Ahmad-Reza

    2016-02-01

    Immobilized proteins and enzymes are widely investigated in the medical field as well as the food and environmental fields. In this study, glucose oxidase (GOX) was covalently immobilized on the surface of modified iron oxide magnetic nanoparticles (MIMNs) to produce a bioconjugate complex. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to the size, shape and structure characterization of the MIMNs. Binding of GOX to these MIMNs was confirmed by using FT-IR spectroscopy. The stability of the immobilized and free enzyme at different temperature and pH values was investigated by measuring the enzymatic activity. These studies reveal that the enzyme's stability is enhanced by immobilization. Further experiments showed that the storage stability of the enzyme is improved upon binding to the MIMNs. The results of kinetic measurements suggest that the effect of the immobilization process on substrate and product diffusion is small. Such bioconjugates can be considered as a catalytic nanodevice for accelerating the glucose oxidation reaction for biotechnological purposes.

  15. Magnetic colorimetric immunoassay for human interleukin-6 based on the oxidase activity of ceria spheres.

    PubMed

    Peng, Juan; Guan, Jufang; Yao, Huiqin; Jin, Xiaoyong

    2016-01-01

    A novel magnetic colorimetric immunoassay strategy was designed for sensitive detection of human interleukin-6 (IL-6) using ceria spheres as labels. Ceria spheres showed excellent oxidase activity, which can directly catalyze the oxidation of substrate o-phenylenediamine (OPD) to a stable yellow product, 2,3-diaminophenazine (oxOPD). The absorbance of oxOPD was recorded to reflect the level of IL-6. The relatively mild conditions made the immunoassay strategy more robust, reliable, and easy. A linear relationship between absorbance intensity and the logarithm of IL-6 concentrations was obtained in the range of 0.0001-10 ng mL(-1) with a detection limit of 0.04 pg mL(-1) (S/N = 3). The colorimetric immunoassay exhibited high sensitivity and specificity for the detection of IL-6. This immunoassay has been successfully applied in the detection of IL-6 in serum samples and can be readily extended toward the on-site monitoring of cancer biomarkers in serum samples. PMID:26416691

  16. Inhibition of polyamine oxidase activity affects tumor development during the maize-Ustilago maydis interaction.

    PubMed

    Jasso-Robles, Francisco Ignacio; Jiménez-Bremont, Juan Francisco; Becerra-Flora, Alicia; Juárez-Montiel, Margarita; Gonzalez, María Elisa; Pieckenstain, Fernando Luis; García de la Cruz, Ramón Fernando; Rodríguez-Kessler, Margarita

    2016-05-01

    Ustilago maydis is a biotrophic plant pathogenic fungus that leads to tumor development in the aerial tissues of its host, Zea mays. These tumors are the result of cell hypertrophy and hyperplasia, and are accompanied by the reprograming of primary and secondary metabolism of infected plants. Up to now, little is known regarding key plant actors and their role in tumor development during the interaction with U. maydis. Polyamines are small aliphatic amines that regulate plant growth, development and stress responses. In a previous study, we found substantial increases of polyamine levels in tumors. In the present work, we describe the maize polyamine oxidase (PAO) gene family, its contribution to hydrogen peroxide (H2O2) production and its possible role in tumor development induced by U. maydis. Histochemical analysis revealed that chlorotic lesions and maize tumors induced by U. maydis accumulate H2O2 to significant levels. Maize plants inoculated with U. maydis and treated with the PAO inhibitor 1,8-diaminooctane exhibit a notable reduction of H2O2 accumulation in infected tissues and a significant drop in PAO activity. This treatment also reduced disease symptoms in infected plants. Finally, among six maize PAO genes only the ZmPAO1, which encodes an extracellular enzyme, is up-regulated in tumors. Our data suggest that H2O2 produced through PA catabolism by ZmPAO1 plays an important role in tumor development during the maize-U. maydis interaction. PMID:26926794

  17. LRPPRC mutation suppresses cytochrome oxidase activity by altering mitochondrial RNA transcript stability in a mouse model.

    PubMed

    Xu, Fenghao; Addis, Jane B L; Cameron, Jessie M; Robinson, Brian H

    2012-01-01

    LRPPRC (leucine-rich pentatricopeptide repeat-containing) has been shown to be essential for the maturation of COX (cytochrome c oxidase), possibly by stabilizing RNA transcripts of COXI, COXII and COXIII genes encoded in mtDNA (mitochondrial DNA). We established a mouse 'gene-trap' model using ES cells (embryonic stem cells) in which the C-terminus of LRPPRC has been replaced with a β-geo construct. Mice homozygous for this modification were found to be subject to embryonic lethality, with death before 12.5 dpc (days post-coitum). Biochemical analysis of MEFs (mouse embryonic fibroblasts) isolated from homozygous mutants showed a major decrease in COX activity, with slight reductions in other respiratory chain complexes with mtDNA encoded components. Constructs of LRPPRC containing different numbers of PPRs (pentatricopeptide repeats) were expressed as recombinant proteins and tested for their ability to bind to the COXI mRNA transcript. Full binding required the first 19 PPR motifs. A specific segment of COXI mRNA was identified as the binding target for LRPPRC, encoded by mouse mtDNA nucleotides 5961-6020. These data strongly suggest that LRPPRC is involved in the maturation of COX, and is involved in stabilizing of mitochondrial mRNAs encoding COX transcripts. PMID:21880015

  18. Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities

    SciTech Connect

    Kajikawa, Takao; Kataoka, Kunishige; Sakurai, Takeshi

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Proton transfer pathway to dioxygen in CueO was identified. Black-Right-Pointing-Pointer Glu506 is the key amino acid to transport proton. Black-Right-Pointing-Pointer The Ala mutation at Glu506 formed a compensatory proton transfer pathway. Black-Right-Pointing-Pointer The Ile mutation at Glu506 shut down the hydrogen bond network. -- Abstract: CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase.

  19. Growth arrest of lung carcinoma cells (A549) by polyacrylate-anchored peroxovanadate by activating Rac1-NADPH oxidase signalling axis.

    PubMed

    Chatterjee, Nirupama; Anwar, Tarique; Islam, Nashreen S; Ramasarma, T; Ramakrishna, Gayatri

    2016-09-01

    Hydrogen peroxide is often required in sublethal, millimolar concentrations to show its oxidant effects on cells in culture as it is easily destroyed by cellular catalase. Previously, we had shown that diperoxovanadate, a physiologically stable peroxovanadium compound, can substitute H2O2 effectively in peroxidation reactions. We report here that peroxovanadate when anchored to polyacrylic acid (PAPV) becomes a highly potent inhibitor of growth of lung carcinoma cells (A549). The early events associated with PAPV treatment included cytoskeletal modifications, increase in GTPase activity of Rac1, accumulation of the reactive oxygen species, and also increase in phosphorylation of H2AX (γH2AX), a marker of DNA damage. These effects persisted even at 24 h after removal of the compound and culminated in increased levels of p53 and p21 together with growth arrest. The PAPV-mediated growth arrest was significantly abrogated in cells pre-treated with the N-acetylcysteine, Rac1 knocked down by siRNA and DPI an inhibitor of NADPH oxidase. In conclusion, our results show that polyacrylate derivative of peroxovanadate efficiently arrests growth of A549 cancerous cells by activating the axis of Rac1-NADPH oxidase leading to oxidative stress and DNA damage. PMID:27435854

  20. Comparison of the Inhibition of Monoamine Oxidase and Butyrylcholinesterase Activities by Infusions from Green Tea and Some Citrus Peels

    PubMed Central

    Ademosun, Ayokunle O.

    2014-01-01

    This study sought to investigate the effect of infusions from green tea (Camellia sinensis) and some citrus peels [shaddock (Citrus maxima), grapefruit (Citrus paradisi), and orange (Citrus sinensis)] on key enzymes relevant to the management of neurodegenerative conditions [monoamine oxidase (MAO) and butyrylcholinesterase (BChE)]. The total phenol contents and antioxidant activities as typified by their 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals scavenging abilities, ferric reducing antioxidant properties, and Fe2+ chelating abilities were also investigated. Green tea had the highest total phenol (43.3 mg/g) and total flavonoid (16.4 mg/g) contents, when compared to orange [total phenol (19.6 mg/g), total flavonoid (6.5 mg/g)], shaddock [total phenol (16.3 mg/g), total flavonoid (5.2 mg/g)], and grapefruit [total phenol (17.7 mg/g), total flavonoid (5.9 mg/g)]. Orange (EC50 = 1.78 mg/mL) had the highest MAO inhibitory ability, while green tea had the least MAO inhibitory ability (EC50 = 2.56 mg/mL). Similarly, green tea had the least BChE inhibitory ability (EC50 = 5.43 mg/mL) when compared to the citrus peels' infusions. However, green tea infusions had the strongest highest ABTS radical scavenging ability, reducing power, and Fe2+ chelating ability. The inhibition of MAO and BChE activities by the green tea and citrus peels infusions could make them good dietary means for the prevention/management of neurodegenerative conditions. PMID:25243093

  1. Inactivation of nitroalkane oxidase upon mutation of the active site base and rescue with a deprotonated substrate.

    PubMed

    Valley, Michael P; Fitzpatrick, Paul F

    2003-07-23

    Mutation of Asp402 in nitroalkane oxidase to Asn or Ala inactivates the enzyme with neutral nitroethane as substrate, but the activity can be rescued with the nitroethane anion. The V/K values of the D402N and D402A enzymes with the nitroethane anion are independent of pH, whereas the V/K values of the wild-type and D402E enzymes are pH dependent with both the protonated and the deprotonated forms of nitroethane. Moreover, although the V/K value of the D402E enzyme with neutral nitroethane is 20-fold less than that of the wild-type enzyme, there is only a 2-fold difference in the V/K values with the nitroethane anion. These results are fully consistent with a primary role for Asp402 as the active site base in nitroalkane oxidase which abstracts the substrate alpha-proton. PMID:12862464

  2. Purification, characterization, and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum.

    PubMed

    Sutay Kocabas, Didem; Bakir, Ufuk; Phillips, Simon E V; McPherson, Michael J; Ogel, Zumrut B

    2008-06-01

    A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 +/- 0.2 and 10.1 +/- 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases. PMID:18369615

  3. Contribution of NADPH Oxidase to Membrane CD38 Internalization and Activation in Coronary Arterial Myocytes

    PubMed Central

    Xu, Ming; Li, Xiao-Xue; Ritter, Joseph K.; Abais, Justine M.; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    The CD38-ADP-ribosylcyclase-mediated Ca2+ signaling pathway importantly contributes to the vasomotor response in different arteries. Although there is evidence indicating that the activation of CD38-ADP-ribosylcyclase is associated with CD38 internalization, the molecular mechanism mediating CD38 internalization and consequent activation in response to a variety of physiological and pathological stimuli remains poorly understood. Recent studies have shown that CD38 may sense redox signals and is thereby activated to produce cellular response and that the NADPH oxidase isoform, NOX1, is a major resource to produce superoxide (O2·−) in coronary arterial myocytes (CAMs) in response to muscarinic receptor agonist, which uses CD38-ADP-ribosylcyclase signaling pathway to exert its action in these CAMs. These findings led us hypothesize that NOX1-derived O2·− serves in an autocrine fashion to enhance CD38 internalization, leading to redox activation of CD38-ADP-ribosylcyclase activity in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2·− production and increased CD38-ADP-ribosylcyclase activity in CAMs, while activation of NOX1 by overexpression of Rac1 or Vav2 or administration of exogenous O2·− significantly increased CD38 internalization in CAMs. Lastly, ET-1 was found to markedly increase membrane raft clustering as shown by increased colocalization of cholera toxin-B with CD38 and NOX1. Taken together, these results provide direct evidence that Rac1-NOX1-dependent O2·− production mediates CD38 internalization in CAMs, which may represent an important mechanism linking receptor activation with CD38 activity in these cells. PMID:23940720

  4. Arsenic, cadmium, mercury and nickel stimulate cell growth via NADPH oxidase activation.

    PubMed

    Mohammadi-Bardbori, Afshin; Rannug, Agneta

    2014-11-10

    Exposure to metals and metalloids including arsenic, cadmium, mercury, and nickel has been a worldwide health problem for several decades. The aim of this study was to learn how metal-induced oxidative stress triggers cell proliferation, a process of great significance for cancer. NADPH oxidase (NOX) activity and cell proliferation were measured as endpoints in both NOX-deficient and NOX-proficient cells. The X chromosome linked CGD (X-CGD) human promyelocytic leukemia PLB-985 cells lacking gp91phox and the X-CGD cells re-transfected with gp91phox (X-CGD-gp91(phox)) were used together with immortalized human keratinocyte cells (HaCaT). The cells were exposed to different concentrations of the metals alone or together with the NOX inhibitor, diphenyleneiodonium (DPI). We found that the studied metals increased NOX activity. They stimulated cell proliferation in HaCaT and X-CGD-gp91(phox) cells at concentrations below 1μM but not in the X-CGD cells that lack functional NOX. Addition of DPI attenuated the metal-induced cell proliferation. At concentrations above 1μM these metals inhibited cell proliferation. Based on these findings, we propose that many environmental pollutants, including metals and also endogenous NOX-activators such as oxidants and growth factors, interfere with cell growth kinetics by increasing the levels of the diffusible molecule H2O2. Here, we provide evidence that NOXs is central to the mechanism of metal-mediated reactive oxygen species production and stimulation of cell proliferation. PMID:25446860

  5. The monoamine oxidase inhibitory activity of essential oils obtained from Eryngium species and their chemical composition.

    PubMed

    Klein-Júnior, Luiz Carlos; Dos Santos Passos, Carolina; Tasso de Souza, Tiago Juliano; Gobbi de Bitencourt, Fernanda; Salton, Juliana; de Loreto Bordignon, Sérgio Augusto; Henriques, Amélia Teresinha

    2016-06-01

    Context Monoamine oxidase (MAO) inhibitors are used in the treatment of depression, anxiety disorders, and the symptomatic treatment of Parkinson's disease. Eryngium, the most representative of the Apiaceae family, is well known for the presence of essential oils (EOs), which have already demonstrated MAO inhibitory potential. Objective The objective of this study is to evaluate the MAO inhibitory capacity of the EOs obtained from Eryngium floribundum Cham. & Schlecht. (EF), E. eriophorum Cham. & Schlecht. (EE), E. nudicaule Lam. (EN), E. horridum Malme (EH), and E. pandanifolium Cham. & Schlecht. (EP). Materials and methods EOs were obtained from fresh whole plants by hydrodistillation (3 h). Chemical analyses were performed by GC/MS using apolar and polar columns, with oven temperature from 60 to 300 °C at 3 °C/min. The MAO-A and -B activities were evaluated in vitro by an end-point method using kynuramine as the substrate and mitochondrial suspension or human recombinant enzymes as the enzymatic source. DMSO 2%, clorgyline 10(-7) M, and pargyline 10(-6) M were used as controls. Results and discussion EFEO, EEEO, ENEO, EHEO, and EPEO GC/MS analysis showed (E)-caryophyllene (4.9-10.8%), germacrene D (0.6-35.1%), bicyclogermacrene (10.4-17.2), spathulenol (0.4-36.0%), and globulol (1.4-18.6%) as main constituents. None of the EOs inhibited MAO-A activity (4 and 40 μg/mL). However, EHEO inhibited MAO-B activity with an IC50 value of 5.65 μg/mL (1-200 μg/mL). Pentadecane (10 μM), its major constituent (53.5%), did not display significant MAO-B inhibition. Conclusion The study demonstrates the promising application of Eryngium species as a source of potential central nervous system bioactive secondary metabolites, specially related to neurodegenerative disorders. PMID:26810928

  6. Epithelial-to-Mesenchymal Transition in Podocytes Mediated by Activation of NADPH Oxidase in Hyperhomocysteinemia

    PubMed Central

    Zhang, Chun; Xia, Min; Boini, Krishna M.; Li, Cai-Xia; Abais, Justine M.; Li, Xiao-Xue; Laperle, Laura A.; Li, Pin-Lan

    2012-01-01

    The present study tested the hypothesis that hyperhomocysteinemia (hHcys) induces podocytes to undergo epithelial-to-mesenchymal transition (EMT) through the activation of NADPH oxidase (Nox). It was found that increased homocysteine (Hcys) level suppressed the expression of slit diaphragm-associated proteins, P-cadherin and zonula occludens-1 (ZO-1) in conditionally immortalized mouse podocytes, indicating the loss of their epithelial features. Meanwhile, Hcys remarkably increased the abundance of mesenchymal markers, such as fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA). These phenotype changes in podocytes induced by Hcys were accompanied by enhanced superoxide (O2.−) production, which was substantially suppressed by inhibition of Nox activity. Functionally, Hcys significantly enhanced the permeability of the podocyte monolayer coupled with increased EMT, and this EMT-related increase in cell permeability could be restored by Nox inhibitors. In mice lacking gp91phox (gp91−/−), an essential Nox subunit gene, hHcys-enhanced podocyte EMT and consequent glomerular injury were examined. In wild-type (gp91+/+) mice, hHcys induced by a folate-free (FF) diet markedly enhanced expression of mesenchymal markers (FSP-1 and α-SMA) but decreased expression of epithelial markers of podocytes in glomeruli, which were not observed in gp91−/− mouse glomeruli. Podocyte injury, glomerular sclerotic pathology, and marked albuminuria observed in gp91+/+ mice with hHcys were all significantly attenuated in gp91−/− mice. These results suggest that hHcys induces EMT of podocytes through activation of Nox, which represents a novel mechanism of hHcys-associated podocyte injury. PMID:21647593

  7. 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....

  8. AMPK activation by glucagon-like peptide-1 prevents NADPH oxidase activation induced by hyperglycemia in adult cardiomyocytes.

    PubMed

    Balteau, Magali; Van Steenbergen, Anne; Timmermans, Aurélie D; Dessy, Chantal; Behets-Wydemans, Gaetane; Tajeddine, Nicolas; Castanares-Zapatero, Diego; Gilon, Patrick; Vanoverschelde, Jean-Louis; Horman, Sandrine; Hue, Louis; Bertrand, Luc; Beauloye, Christophe

    2014-10-15

    Exposure of cardiomyocytes to high glucose concentrations (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase (NOX2). NOX2 activation is triggered by enhanced glucose transport through a sodium-glucose cotransporter (SGLT) but not by a stimulation of glucose metabolism. The aim of this work was to identify potential therapeutic approaches to counteract this glucotoxicity. In cultured adult rat cardiomyocytes incubated with 21 mM glucose (HG), AMP-activated protein kinase (AMPK) activation by A769662 or phenformin nearly suppressed ROS production. Interestingly, glucagon-like peptide 1 (GLP-1), a new antidiabetic drug, concomitantly induced AMPK activation and prevented the HG-mediated ROS production (maximal effect at 100 nM). α2-AMPK, the major isoform expressed in cardiomyocytes (but not α1-AMPK), was activated in response to GLP-1. Anti-ROS properties of AMPK activators were not related to changes in glucose uptake or glycolysis. Using in situ proximity ligation assay, we demonstrated that AMPK activation prevented the HG-induced p47phox translocation to caveolae, whatever the AMPK activators used. NOX2 activation by either α-methyl-d-glucopyranoside, a glucose analog transported through SGLT, or angiotensin II was also counteracted by GLP-1. The crucial role of AMPK in limiting HG-mediated NOX2 activation was demonstrated by overexpressing a constitutively active form of α2-AMPK using adenoviral infection. This overexpression prevented NOX2 activation in response to HG, whereas GLP-1 lost its protective action in α2-AMPK-deficient mouse cardiomyocytes. Under HG, the GLP-1/AMPK pathway inhibited PKC-β2 phosphorylation, a key element mediating p47phox translocation. In conclusion, GLP-1 induces α2-AMPK activation and blocks HG-induced p47phox translocation to the plasma membrane, thereby preventing glucotoxicity. PMID:25128166

  9. NADPH Oxidase and Neurodegeneration

    PubMed Central

    Hernandes, Marina S; Britto, Luiz R G

    2012-01-01

    NADPH oxidase (Nox) is a unique, multi-protein, electron transport system that produces large amounts of superoxide via the reduction of molecular oxygen. Nox-derived reactive oxygen species (ROS) are known to be involved in a variety of physiological processes, including host defense and signal transduction. However, over the past decade, the involvement of (Nox)-dependent oxidative stress in the pathophysiology of several neurodegenerative diseases has been increasingly recognized. ROS produced by Nox proteins contribute to neurodegenerative diseases through distinct mechanisms, such as oxidation of DNA, proteins, lipids, amino acids and metals, in addition to activation of redox-sensitive signaling pathways. In this review, we discuss the recent literature on Nox involvement in neurodegeneration, focusing on Parkinson and Alzheimer diseases. PMID:23730256

  10. Exosomes from hypoxic endothelial cells have increased collagen crosslinking activity through up-regulation of lysyl oxidase-like 2.

    PubMed

    de Jong, Olivier G; van Balkom, Bas W M; Gremmels, Hendrik; Verhaar, Marianne C

    2016-02-01

    Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations, research on exosome-ECM interactions is limited. Here, we investigate whether the exosome-associated lysyl oxidase family member lysyl oxidase-like 2 (LOXL2) is involved in ECM remodelling. We found that LOXL2 is present on the exterior of endothelial cell (EC)-derived exosomes, placing it in direct vicinity of the ECM. It is up-regulated twofold in EC-derived exosomes cultured under hypoxic conditions. Intact exosomes from hypoxic EC and LOXL2 overexpressing EC show increased activity in a fluorometric lysyl oxidase enzymatic activity assay as well as in a collagen gel contraction assay. Concordantly, knockdown of LOXL2 in exosome-producing EC in both normal and hypoxic conditions reduces activity of exosomes in both assays. Our findings show for the first time that ECM crosslinking by EC-derived exosomes is mediated by LOXL2 under the regulation of hypoxia, and implicate a role for exosomes in hypoxia-regulated focal ECM remodelling, a key process in both fibrosis and wound healing. PMID:26612622

  11. Reductive half-reaction of nitroalkane oxidase: effect of mutation of the active site aspartate to glutamate.

    PubMed

    Valley, Michael P; Fitzpatrick, Paul F

    2003-05-20

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707]. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the DeltaG(++) for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase. PMID:12741843

  12. PHARMACOLOGICAL ACTIVITIES OF PROTOCATECHUIC ACID.

    PubMed

    Khan, Abida Kalsoom; Rashid, Rehana; Fatima, Nighat; Mahmood, Sadaf; Mir, Sadullah; Khan, Sara; Jabeen, Nyla; Murtaza, Ghulam

    2015-01-01

    Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a simple phenolic acid. It is found in a large variety of edible plants and possesses various pharmacological activities. This article aims to review the modern trends in phytochemical isolation and extraction of PCA from plants and other natural resources. Moreover, this article also encompasses pharmacological and biological activities of PCA. It is well known to have anti-inflammatory, antioxidant, anti-hyperglycemia, antibacterial, anticancer, anti-ageing, anti-athro- genic, anti-tumoral, anti-asthma, antiulcer, antispasmodic and neurological properties. PMID:26647619

  13. Analysis of the Role of the Active Site Residue Arg98 in the Flavoprotein Tryptophan 2-Monooxygenase, a Member of the l-Amino Oxidase Family†

    PubMed Central

    Sobrado, Pablo; Fitzpatrick, Paul F.

    2006-01-01

    The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide. We have previously identified tryptophan 2-monooxygenase as a homologue of l-amino acid oxidase [Sobrado, P., and Fitzpatrick, P. F. (2002) Arch. Biochem. Biophys. 402, 24–30]. On the basis of the sequence comparisons of the different LAAO family members, Arg98 of tryptophan 2-monooxygenase can be identified as an active site residue which interacts with the carboxylate of the amino acid substrate. The catalytic properties of R98K and R98A tryptophan 2-monooxygenase have been characterized to evaluate the role of this residue. Mutation of Arg98 to lysine decreases the first-order rate constant for flavin reduction by 180-fold and the second-order rate constant for flavin oxidation by 26-fold, has no significant effect on the Kd value for tryptophan or the Ki value for the competitive inhibitor indoleacetamide, and increases the Ki value for indolepyruvate less than 2-fold. Mutation of this residue to alanine decreases the rate constants for reduction and oxidation an additional 5- and 2-fold, respectively, and increases the Kd value for tryptophan and the Ki value for indolepyruvate by 31- and 17-fold, respectively, while having an only 2-fold effect on the Ki value for indoleacetamide. Both mutations increase the value of the primary deuterium isotope effect with tryptophan as a substrate, consistent with a later transition state. Both mutant enzymes catalyze a simple oxidase reaction, producing indolepyruvate and hydrogen peroxide. The pH dependences of the V/Ktrp values for the mutant enzymes show that the anionic form of the substrate is preferred but that the zwitterionic form is a substrate. The results are consistent with the interaction between Arg98 and the carboxylate of the amino acid substrate being critical for correct positioning of the substrate in the active site for efficient catalysis. PMID:14636049

  14. High glycolate oxidase activity is required for survival of maize in normal air.

    PubMed

    Zelitch, Israel; Schultes, Neil P; Peterson, Richard B; Brown, Patrick; Brutnell, Thomas P

    2009-01-01

    A mutant in the maize (Zea mays) Glycolate Oxidase1 (GO1) gene was characterized to investigate the role of photorespiration in C4 photosynthesis. An Activator-induced allele of GO1 conditioned a seedling lethal phenotype when homozygous and had 5% to 10% of wild-type GO activity. Growth of seedlings in high CO2 (1%-5%) was sufficient to rescue the mutant phenotype. Upon transfer to normal air, the go1 mutant became necrotic within 7 d and plants died within 15 d. Providing [1-14C]glycolate to leaf tissue of go1 mutants in darkness confirmed that the substrate is inefficiently converted to 14CO2, but both wild-type and GO-deficient mutant seedlings metabolized [1-14C]glycine similarly to produce [14C]serine and 14CO2 in a 1:1 ratio, suggesting that the photorespiratory pathway is otherwise normal in the mutant. The net CO2 assimilation rate in wild-type leaves was only slightly inhibited in 50% O2 in high light but decreased rapidly and linearly with time in leaves with low GO. When go1 mutants were shifted from high CO2 to air in light, they accumulated glycolate linearly for 6 h to levels 7-fold higher than wild type and 11-fold higher after 25 h. These studies show that C4 photosynthesis in maize is dependent on photorespiration throughout seedling development and support the view that the carbon oxidation pathway evolved to prevent accumulation of toxic glycolate. PMID:18805949

  15. Structural Characterization of Mutations at the Oxygen Activation Site in Monomeric Sarcosine Oxidase

    SciTech Connect

    Schuman Jorns, Marilyn; Chen, Zhi-wei; Mathews, F. Scott

    2010-04-30

    Oxygen reduction and sarcosine oxidation in monomeric sarcosine oxidase (MSOX) occur at separate sites above the si- and re-faces, respectively, of the flavin ring. Mutagenesis studies implicate Lys265 as the oxygen activation site. Substitution of Lys265 with a neutral (Met, Gln, or Ala) or basic (Arg) residue results in an {approx}10{sup 4}- or 250-fold decrease, respectively, in the reaction rate. The overall structure of MSOX and residue conformation in the sarcosine binding cavity are unaffected by replacement of Lys265 with Met or Arg. The side chain of Met265 exhibits the same configuration in each molecule of Lys265Met crystals and is nearly congruent with Lys265 in wild-type MSOX. The side chain of Arg265 is, however, dramatically shifted (4-5 {angstrom}) compared with Lys265, points in the opposite direction, and exhibits significant conformational variability between molecules of the same crystal. The major species in solutions of Lys265Arg is likely to contain a 'flipped-out' Arg265 and exhibit negligible oxygen activation, similar to Lys265Met. The 400-fold higher oxygen reactivity observed with Lys265Arg is attributed to a minor (<1%) 'flipped-in' Arg265 conformer whose oxygen reactivity is similar to that of wild-type MSOX. A structural water (WAT1), found above the si-face of the flavin ring in all previously determined MSOX structures, is part of an apparent proton relay system that extends from FAD N(5) to bulk solvent. WAT1 is strikingly absent in Lys265Met and Lys265Arg, a feature that may account for the apparent kinetic stabilization of a reductive half-reaction intermediate that is detectable with the mutants but not wild-type MSOX.

  16. NADPH Oxidase NOX4 Mediates Stellate Cell Activation and Hepatocyte Cell Death during Liver Fibrosis Development

    PubMed Central

    Sancho, Patricia; Mainez, Jèssica; Crosas-Molist, Eva; Roncero, César; Fernández-Rodriguez, Conrado M.; Pinedo, Fernando; Huber, Heidemarie; Eferl, Robert; Mikulits, Wolfgang; Fabregat, Isabel

    2012-01-01

    A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2−/−/p19ARF−/−, Stat3Δhc/Mdr2−/−) and a model of experimental induced fibrosis (CCl4) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes. PMID:23049784

  17. Synthesis and characterization of microparticles based on poly-methacrylic acid with glucose oxidase for biosensor applications.

    PubMed

    Hervás Pérez, J P; López-Ruiz, B; López-Cabarcos, E

    2016-03-01

    In the line of the applicability of biocompatible monomers pH and temperature dependent, we assayed poly-methacrylic acid (p-MAA) microparticles as immobilization system in the design of enzymatic biosensors. Glucose oxidase was used as enzyme model for the study of microparticles as immobilization matrices and as biological material in the performance of glucose biosensors. The enzyme immobilization method was optimized by investigating the influence of monomer concentration and cross-linker content (N',N'-methylenebisacrylamide), used in the preparation of the microparticles in the response of the biosensors. The kinetics of the polymerization and the effects of the temperature were studied, also the conversion of the polymerization was determinates by a weight method. The structure of the obtained p-MAA microparticles were studied through scanning electron microscopy (SEM) and differential scanning microscopy (DSC). The particle size measurements were performed with a Galai-Cis 1 particle analyzer system. Furthermore, the influence of the swelling behavior of hydrogel matrix as a function of pH and temperature were studied. Analytical properties such as sensitivity, linear range, response time and detection limit were studied for the glucose biosensors. The sensitivity for glucose detection obtained with poly-methacrylic acid (p-MAA) microparticles was 11.98mAM(-1)cm(-2) and 10μM of detection limit. A Nafion® layer was used to eliminate common interferents of the human serum such as uric and ascorbic acids. The biosensors were used to determine glucose in human serum samples with satisfactory results. When stored in a frozen phosphate buffer solution (pH 6.0) at -4°C, the useful lifetime of all biosensors was at least 550 days. PMID:26717846

  18. Microfluidic Devices Integrating Microcavity Surface-Plasmon-Resonance Sensors: Glucose Oxidase Binding-Activity Detection

    PubMed Central

    Amarie, Dragos; Alileche, Abdelkrim; Dragnea, Bogdan; Glazier, James A.

    2010-01-01

    We have developed miniature (≈1 μm diameter) microcavity surface-plasmon-resonance sensors (MSPRS), integrated them with microfluidics and tested their sensitivity to refractive-index changes. We tested their biosensing capability by distinguishing the interaction of glucose oxidase (Mr 160 kDa) with its natural substrate (β-D-glucose, Mr 180 Da) from its interactions with non-specific substrates (L-glucose, D-mannose and 2-deoxy-D-glucose). We ran the identical protocol we had used with the MSPRS on a Biacore 3000 instrument using their bare gold chip. Only the MSPRS was able to detect β-D-glucose binding to glucose oxidase. Each MSPRS can detect the binding to its surface of fewer than 35,000 glucose-oxidase molecules (representing 9.6 fg or 60 zmol of protein), about 106 times fewer than classical surface-plasmon-resonance biosensors. PMID:19968248

  19. Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr(215) in Aerococcus viridans lactate oxidase.

    PubMed

    Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K; Nidetzky, Bernd

    2016-01-01

    L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr(215) in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr(215), effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr(215) can thus lead to a kinetic bottleneck in product release. PMID:27302031

  20. Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr215 in Aerococcus viridans lactate oxidase

    PubMed Central

    Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K.; Nidetzky, Bernd

    2016-01-01

    L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr215 in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr215, effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr215 can thus lead to a kinetic bottleneck in product release. PMID:27302031

  1. Increasing the catalytic activity of Bilirubin oxidase from Bacillus pumilus: Importance of host strain and chaperones proteins.

    PubMed

    Gounel, Sébastien; Rouhana, Jad; Stines-Chaumeil, Claire; Cadet, Marine; Mano, Nicolas

    2016-07-20

    Aggregation of recombinant proteins into inclusion bodies (IBs) is the main problem of the expression of multicopper oxidase in Escherichia coli. It is usually attributed to inefficient folding of proteins due to the lack of copper and/or unavailability of chaperone proteins. The general strategies reported to overcome this issue have been focused on increasing the intracellular copper concentration. Here we report a complementary method to optimize the expression in E. coli of a promising Bilirubin oxidase (BOD) isolated from Bacillus pumilus. First, as this BOD has a disulfide bridge, we switched E.coli strain from BL21 (DE3) to Origami B (DE3), known to promote the formation of disulfide bridges in the bacterial cytoplasm. In a second step, we investigate the effect of co-expression of chaperone proteins on the protein production and specific activity. Our strategy allowed increasing the final amount of enzyme by 858% and its catalytic rate constant by 83%. PMID:27165502

  2. Cytotoxicity of, and DNA damage by, active oxygen species produced by xanthine oxidase.

    PubMed

    Chiricolo, M; Tazzari, P L; Abbondanza, A; Dinota, A; Battelli, M G

    1991-10-21

    Toxicity to Raji cells of the xanthine oxidase/hypoxanthine system is related to the formation of single-strand DNA breaks. DNA damage was proportional to the concentration of xanthine oxidase and to the time of exposure. It was prevented by the absence of hypoxanthine, or by the presence of allopurinol, or both superoxide dismutase and catalase. The release of 51Cr from damaged cells was detectable 12 h after the inhibition of cloning efficiency and the production of DNA breakage. These data suggest that DNA damage induced by the oxygen products precedes the severe lesion to the cellular membrane. PMID:1936259

  3. Type I Helicobacter pylori Lipopolysaccharide Stimulates Toll-Like Receptor 4 and Activates Mitogen Oxidase 1 in Gastric Pit Cells

    PubMed Central

    Kawahara, Tsukasa; Teshima, Shigetada; Oka, Ayuko; Sugiyama, Toshiro; Kishi, Kyoichi; Rokutan, Kazuhito

    2001-01-01

    Guinea pig gastric pit cells express an isozyme of gp91-phox, mitogen oxidase 1 (Mox1), and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phox). Helicobacter pylori lipopolysaccharide (LPS) and Escherichia coli LPS have been shown to function as potent activators for the Mox1 oxidase. These cells spontaneously secreted about 10 nmol of superoxide anion (O2−)/mg of protein/h under LPS-free conditions. They expressed the mRNA and protein of Toll-like receptor 4 (TLR4) but not those of TLR2. LPS from type I H. pylori at 2.1 endotoxin units/ml or higher stimulated TLR4-mediated phosphorylations of transforming growth factor β-activated kinase 1 and its binding protein 1 induced TLR4 and p67-phox and up-regulated O2− production 10-fold. In contrast, none of these events occurred with H. pylori LPS from complete or partial deletion mutants of the cag pathogenicity island. Lipid A was confirmed to be a bioactive component for the priming effects, while removal of bisphosphates from lipid A completely eliminated the effects, suggesting the importance of the phosphorylation pattern besides the acylation pattern for the bioactivity. H. pylori LPS is generally accepted as having low toxicity; however, our results suggest that type I H. pylori lipid A may be a potent stimulator for innate immune responses of gastric mucosa by stimulating the TLR4 cascade and Mox1 oxidase in pit cells. PMID:11401977

  4. Augmented EGF receptor tyrosine kinase activity impairs vascular function by NADPH oxidase-dependent mechanism in type 2 diabetic mouse.

    PubMed

    Kassan, Modar; Ait-Aissa, Karima; Ali, Maha; Trebak, Mohamed; Matrougui, Khalid

    2015-10-01

    We previously determined that augmented EGFR tyrosine kinase (EGFRtk) impairs vascular function in type 2 diabetic mouse (TD2). Here we determined that EGFRtk causes vascular dysfunction through NADPH oxidase activity in TD2. Mesenteric resistance arteries (MRA) from C57/BL6 and db-/db- mice were mounted in a wired myograph and pre-incubated for 1h with either EGFRtk inhibitor (AG1478) or exogenous EGF. The inhibition of EGFRtk did not affect the contractile response to phenylephrine-(PE) and thromboxane-(U46619) or endothelium-dependent relaxation (EDR) to acetylcholine in MRA from control group. However, in TD2 mice, AG1478 reduced the contractile response to U46619, improved vasodilatation and reduced p22phox-NADPH expression, but had no effect on the contractile response to PE. The incubation of MRA with exogenous EGF potentiated the contractile response to PE in MRA from control and diabetic mice. However, EGF impaired the EDR and potentiated the vasoconstriction to U46619 only in the control group. Interestingly, NADPH oxidase inhibition in the presence of EGF restored the normal contraction to PE and improved the EDR but had no effect on the potentiated contraction to U46619. Vascular function improvement was associated with the rescue of eNOS and Akt and reduction in phosphorylated Rho-kinase, NOX4 mRNA levels, and NADPH oxidase activity. MRA from p47phox-/- mice incubated with EGF potentiated the contraction to U46619 but had no effect to PE or ACh responses. The present study provides evidence that augmented EGFRtk impairs vascular function by NADPH oxidase-dependent mechanism. Therefore, EGFRtk and oxidative stress should be potential targets to treat vascular dysfunction in TD2. PMID:26036345

  5. D-amino acid oxidase gene therapy sensitizes glioma cells to the antiglycolytic effect of 3-bromopyruvate.

    PubMed

    El Sayed, S M; Abou El-Magd, R M; Shishido, Y; Chung, S P; Sakai, T; Watanabe, H; Kagami, S; Fukui, K

    2012-01-01

    Glioma tumors are refractory to conventional treatment. Glioblastoma multiforme is the most aggressive type of primary brain tumors in humans. In this study, we introduce oxidative stress-energy depletion (OSED) therapy as a new suggested treatment for glioblastoma. OSED utilizes D-amino acid oxidase (DAO), which is a promising therapeutic protein that induces oxidative stress and apoptosis through generating hydrogen peroxide (H2O2). OSED combines DAO with 3-bromopyruvate (3BP), a hexokinase II (HK II) inhibitor that interferes with Warburg effect, a metabolic alteration of most tumor cells that is characterized by enhanced aerobic glycolysis. Our data revealed that 3BP induced depletion of energetic capabilities of glioma cells. 3BP induced H2O2 production as a novel mechanism of its action. C6 glioma transfected with DAO and treated with D-serine together with 3BP-sensitized glioma cells to 3BP and decreased markedly proliferation, clonogenic power and viability in a three-dimensional tumor model with lesser effect on normal astrocytes. DAO gene therapy using atelocollagen as an in vivo transfection agent proved effective in a glioma tumor model in Sprague-Dawley (SD) rats, especially after combination with 3BP. OSED treatment was safe and tolerable in SD rats. OSED therapy may be a promising therapeutic modality for glioma. PMID:21921941

  6. The composition of milk xanthine oxidase

    PubMed Central

    Hart, L. I.; McGartoll, Mary A.; Chapman, Helen R.; Bray, R. C.

    1970-01-01

    The composition of milk xanthine oxidase has been reinvestigated. When the enzyme is prepared by methods that include a selective denaturation step in the presence of sodium salicylate the product is obtained very conveniently and in high yield, and is homogeneous in the ultracentrifuge and in recycling gel filtration. It has specific activity higher than previously reported preparations of the enzyme and its composition approximates closely to 2mol of FAD, 2g-atoms of Mo and 8g-atoms of Fe/mol of protein (molecular weight about 275000). In contrast, when purely conventional preparative methods are used the product is also homogeneous by the above criteria but has a lower specific activity and is generally comparable to the crystallized enzyme described previously. Such samples also contain 2mol of FAD/mol of protein but they have lower contents of Mo (e.g. 1.2g-atom/mol). Amino acid compositions for the two types of preparation are indistinguishable. These results confirm the previous conclusion that conventional methods give mixtures of xanthine oxidase with an inactive modification of the enzyme now termed `de-molybdo-xanthine oxidase', and show that salicylate can selectively denature the latter. The origin of de-molybdo-xanthine oxidase was investigated. FAD/Mo ratios show that it is present not only in enzyme purified by conventional methods but also in `milk microsomes' (Bailie & Morton, 1958) and in enzyme samples prepared without proteolytic digestion. We conclude that it is secreted by cows together with the active enzyme and we discuss its occurrence in the preparations of other workers. Studies on the milks of individual cows show that nutritional rather than genetic factors determine the relative amounts of xanthine oxidase and de-molybdo-xanthine oxidase. A second inactive modification of the enzyme, now termed `inactivated xanthine oxidase', causes variability in activity relative to E450 or to Mo content and formation of it decreases these ratios

  7. Development of new chiral ligand exchange capillary electrophoresis system with amino acid ionic liquids ligands and its application in studying the kinetics of L-amino acid oxidase.

    PubMed

    Sun, Bingbing; Mu, Xiaoyu; Qi, Li

    2014-04-22

    New kinds of amino acid ionic liquids (AAILs) with pyridinium as cations and L-lysine (L-Lys) as anion have been developed as the available chiral ligands coordinated with Zn(II) in chiral ligand-exchange capillary electrophoresis (CLE-CE). Four kinds of AAILs, including [1-ethylpyridinium][L-lysine], 1-butylpyridinium][L-lysine], [1-hexylpyridinium][L-lysine] and 1-[octylpyridinium][L-lysine], were successfully synthesized and characterized by nuclear magnetic resonance and mass spectrometry. Compared with other AAILs, the best chiral separation of Dns-D, L-amino acids could be achieved when [1-ethylpyridinium][L-lysine] was chosen as the chiral ligand. It has been found that after investigating the influence of key factors on the separation efficiency, such as pH of buffer solution, the ratio of Zn(II) to ligand and complex concentration, eight pairs of Dns-D, L-AAs enantiomers could be baseline separated and three pairs were partly separated under the optimum conditions. The proposed CLE-CE method also exhibited favorable quantitative analysis property of Dns-D, L-Met with good linearity (r(2)=0.998) and favorable repeatability (RSD≤1.5%). Furthermore, the CLE-CE system was applied in investigating the kinetic contents of L-amino acid oxidase, which implied that the proposed system has the potential in studying the enzymatic reaction mechanism. PMID:24703219

  8. Changes in Cytokinin Content and Cytokinin Oxidase Activity in Response to Derepression of ipt Gene Transcription in Transgenic Tobacco Calli and Plants.

    PubMed Central

    Motyka, V.; Faiss, M.; Strand, M.; Kaminek, M.; Schmulling, T.

    1996-01-01

    Metabolic control of cytokinin oxidase by its substrate was investigated in planta using wild-type (WT) and conditionally ipt gene-expressing transgenic (IPT) tobacco (Nicotiana tabacum L.) callus cultures and plants. The derepression of the tetracycline (Tc)-dependent ipt gene transcription was followed by a progressive, more than 100-fold increase in total cytokinin content in IPT calli. The activity of cytokinin oxidase extracted from these calli began to increase 16 to 20 h after gene derepression, and after 13 d it was 10-fold higher than from Tc-treated WT calli. An increase in cytokinin oxidase activity, as a consequence of elevated cytokinin levels, was found in detached leaves (8-fold after 4 d) and in roots of intact plants (4-fold after 3 d). The partially purified cytokinin oxidase from WT, repressed IPT, and Tc-derepressed IPT tobacco calli exhibited similar characteristics. It had the same broad pH optimum (pH 6.5-8.5), its activity in vitro was enhanced 4-fold in the presence of copper-imidazole, and the apparent Km(N6-[[delta]2iso-pentenyl]adenine) values were in the range of 3.1 to 4.9 [mu]M. The increase in cytokinin oxidase activity in cytokinin-overproducing tissue was associated with the accumulation of a glycosylated form of the enzyme. The present data indicate the substrate induction of cytokinin oxidase activity in different tobacco tissues, which may contribute to hormone homeostasis. PMID:12226431

  9. A chiral ligand exchange CE essay with zinc(II)-L-valine complex for determining enzyme kinetic constant of L-amino acid oxidase.

    PubMed

    Qi, Li; Yang, Gengliang; Zhang, Haizhi; Qiao, Juan

    2010-06-15

    A new strategy for the enantioseparation of D,L-amino acids employing the principle of ligand exchange capillary electrophoresis with Zn(II)-L-valine complex as a chiral selecting system in the presence of beta-cyclodextrin has been designed. Successful enantioseparation of label free and labeled amino acids have been achieved with a buffer of 100.0mM boric acid, 5.0mM ammonium acetate, 4.0mM beta-cyclodextrin, 4.0mM ZnSO(4) and 8.0mM L-valine at pH 8.1. This new method was shown to be applicable to the quantitative analysis of label free D- and L-aromatic amino acids. Furthermore, the expanding enzymatic use of L-amino acid oxidase to incubate with different L-amino acids has allowed understanding of the substrate's specificity. An on-column incubation assay has been developed to study the L-amino acid oxidase's catalytic efficiency. It was demonstrated that the enzyme kinetic constant could be determined by using this new method. PMID:20441938

  10. Cloning, characterization and mutagenesis of Russell's viper venom L-amino acid oxidase: Insights into its catalytic mechanism.

    PubMed

    Chen, Hong-Sen; Wang, Ying-Ming; Huang, Wan-Ting; Huang, Kai-Fa; Tsai, Inn-Ho

    2012-02-01

    To investigate the structure-function relationships and geographic variations of L-amino acid oxidase (LAAO) from Daboia venoms, a single LAAO (designated as DrLAO) was purified from eastern Indian Daboia russelii venom and characterized. The purified DrLAO showed subunit molecular mass of 60-64kDa; its N-terminal sequence (1-20) was identical to those of several true viper LAAOs. Its preferred substrates were hydrophobic l-amino acids and the kinetic specificities were ordered as follows: Phe, Tyr, Met, Leu, and Trp. Enzyme assay and Western blotting showed that the venom LAAO contents of D. russelii were higher than those of Daboia siamensis. DrLAO dose-dependently inhibited ADP- and collagen-induced platelet aggregation with IC(50) values of 0.27 and 0.82μM, respectively. Apparently, DrLAO may synergize with other venom components to prolong and enhance bleeding symptoms after Daboia envenoming. The full sequence of DrLAO was deduced from its cDNA sequence and then confirmed by peptide mass fingerprinting. Molecular phylogenetic analysis revealed that SV-LAAO family members could be differentiated not only by snake taxonomy but also by the variations at position 223, and they divided into H223, S223, N223, and D223 subclasses. We have further prepared recombinant DrLAO and mutants by the Pichia expression system. Mutagenic analyses of DrLAO His223 revealed that this residue bound substrates instead of serving as an essential base in the catalytic steps. Our results suggest a direct hydride transfer from substrate to FAD as the mechanism for SV-LAAOs. PMID:21802487

  11. Potato tuber cytokinin oxidase/dehydrogenase genes: Biochemical properties, activity, and expression during tuber dormancy progression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in meristems isolated from field-g...

  12. Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells

    PubMed Central

    Cao, Weibiao

    2016-01-01

    Mechanisms of the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA. PMID:26901778

  13. Intestinal NADPH oxidase 2 activity increases in a neonatal rat model of necrotizing enterocolitis.

    PubMed

    Welak, Scott R; Rentea, Rebecca M; Teng, Ru-Jeng; Heinzerling, Nathan; Biesterveld, Ben; Liedel, Jennifer L; Pritchard, Kirkwood A; Fredrich, Katherine M; Gourlay, David M

    2014-01-01

    Necrotizing enterocolitis (NEC) is a complication of prematurity. The etiology is unknown, but is related to enteral feeding, ischemia, infection, and inflammation. Reactive oxygen species production, most notably superoxide, increases in NEC. NADPH oxidase (NOX) generates superoxide, but its activity in NEC remains unknown. We hypothesize that NOX-derived superoxide production increases in NEC. Newborn Sprague-Dawley rats were divided into control, formula-fed, formula/LPS, formula/hypoxia, and NEC (formula, hypoxia, and LPS). Intestinal homogenates were analyzed for NADPH-dependent superoxide production. Changes in superoxide levels on days 0-4 were measured. Inhibitors for nitric oxide synthase (L-NAME) and NOX2 (GP91-ds-tat) were utilized. RT-PCR for eNOS, NOX1, GP91phox expression was performed. Immunofluorescence studies estimated the co-localization of p47phox and GP91phox in control and NEC animals on D1, D2, and D4. NEC pups generated more superoxide than controls on D4, while all other groups were unchanged. NADPH-dependent superoxide production was greater in NEC on days 0, 3, and 4. GP91-ds-tat decreased superoxide production in both groups, with greater inhibition in NEC. L-NAME did not alter superoxide production. Temporally, superoxide production varied minimally in controls. In NEC, superoxide generation was decreased on day 1, but increased on days 3-4. GP91phox expression was higher in NEC on days 2 and 4. NOX1 and eNOS expression were unchanged from controls. GP91phox and p47phox had minimal co-localization in all control samples and NEC samples on D1 and D2, but had increased co-localization on D4. In conclusion, this study proves that experimentally-induced NEC increases small intestinal NOX activity. All components of NEC model are necessary for increased NOX activity. NOX2 is the major source, especially as the disease progresses. PMID:25517730

  14. CHARACTERISTICS OF POLYPHENOL OXIDASES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO, EC 1.14.18.1 or EC 1.10.3.1) catalyzes the oxidation of o-diphenols to o-quinones. Highly reactive o-quinones couple with phenolics and specific amino acids on proteins to form the characteristic browning products in many wounded fruits, vegetables, and leaf tissues of plant...

  15. Xanthine oxidase, but not neutrophils, contributes to activation of cardiac sympathetic afferents during myocardial ischaemia in cats

    PubMed Central

    Tjen-A-Looi, Stephanie C; Fu, Liang-Wu; Longhurst, John C

    2002-01-01

    Activation of cardiac sympathetic afferents during myocardial ischaemia causes angina and induces important cardiovascular reflex responses. Reactive oxygen species (ROS) are important chemical stimuli of cardiac afferents during and after ischaemia. Iron-catalysed Fenton chemistry constitutes one mechanism of production of hydroxyl radicals. Another potential source of these species is xanthine oxidase-catalysed oxidation of purines. Polymorphonuclear leukocytes (PMNs) also contribute to the production of ROS in some conditions. The present study tested the hypothesis that both xanthine oxidase-catalysed oxidation of purines and neutrophils provide a source of ROS sufficient to activate cardiac afferents during ischaemia. We recorded single-unit activity of cardiac afferents innervating the ventricles recorded from the left thoracic sympathetic chain (T1-5) of anaesthetized cats to identify the afferents' responses to ischaemia. The role of xanthine oxidase in activation of these afferents was determined by infusion of oxypurinol (10 mg kg−1, i.v.), an inhibitor of xanthine oxidase. The importance of neutrophils as a potential source of ROS in the activation of cardiac afferents during ischaemia was assessed by the infusion of a polyclonal antibody (3 mg ml−1 kg−1, i.v.) raised in rabbits immunized with cat PMNs. This antibody decreased the number of circulating PMNs and, to a smaller extent, platelets. Since previous data suggest that platelets release serotonin (5-HT), which activates cardiac afferents through a serotonin receptor (subtype 3,5-HT3 receptor) mechanism, before treatment with the antibody in another group, we blocked 5-HT3 receptors on sensory nerve endings with tropisetron (300 μg kg−1, i.v.). We observed that oxypurinol significantly decreased the activity of cardiac afferents during myocardial ischaemia from 1.5 ± 0.4 to 0.8 ± 0.4 impulses s−1. Similarly, the polyclonal antibody significantly reduced the discharge frequency of

  16. Chcanges in Germinability and Activities of Polyphenol Oxidase and Peroxidase in Seeds of Pentaclethramacrophylla During Lowtemperature Treatment

    NASA Astrophysics Data System (ADS)

    Udosen, I. R.; Nkang, A. E.; Sam, S. M.

    2012-07-01

    Activities of peroxidase (POD) and polyphenol Oxidase (PPO) were investigated in seeds of Pentaclethramacrophylla during low temperature treatment. The seeds from the small-sized fruits (variety A) and those of the big-sized fruits (variety B) showed high germination, with maximum germination values ranging between 60 ñ 90%. Low temperature treatment did not significantly (P< 0.5) affect maximum germination values. Activities of POD and PPO increased initially (2-4 days) but declined with prolonged (6ñ8 days) low temperature treatment.

  17. NADPH Oxidase Activity in Cerebral Arterioles Is a Key Mediator of Cerebral Small Vessel Disease—Implications for Prevention

    PubMed Central

    McCarty, Mark F.

    2015-01-01

    Cerebral small vessel disease (SVD), a common feature of brain aging, is characterized by lacunar infarcts, microbleeds, leukoaraiosis, and a leaky blood-brain barrier. Functionally, it is associated with cognitive decline, dementia, depression, gait abnormalities, and increased risk for stroke. Cerebral arterioles in this syndrome tend to hypertrophy and lose their capacity for adaptive vasodilation. Rodent studies strongly suggest that activation of Nox2-dependent NADPH oxidase activity is a crucial driver of these structural and functional derangements of cerebral arterioles, in part owing to impairment of endothelial nitric oxide synthase (eNOS) activity. This oxidative stress may also contribute to the breakdown of the blood-brain barrier seen in SVD. Hypertension, aging, metabolic syndrome, smoking, hyperglycemia, and elevated homocysteine may promote activation of NADPH oxidase in cerebral arterioles. Inhibition of NADPH oxidase with phycocyanobilin from spirulina, as well as high-dose statin therapy, may have potential for prevention and control of SVD, and high-potassium diets merit study in this regard. Measures which support effective eNOS activity in other ways—exercise training, supplemental citrulline, certain dietary flavonoids (as in cocoa and green tea), and capsaicin, may also improve the function of cerebral arterioles. Asian epidemiology suggests that increased protein intakes may decrease risk for SVD; conceivably, arginine and/or cysteine—which boosts tissue glutathione synthesis, and can be administered as N-acetylcysteine—mediate this benefit. Ameliorating the risk factors for SVD—including hypertension, metabolic syndrome, hyperglycemia, smoking, and elevated homocysteine—also may help to prevent and control this syndrome, although few clinical trials have addressed this issue to date.

  18. Effects of IFN-γ on intracellular trafficking and activity of macrophage NADPH oxidase flavocytochrome b558

    PubMed Central

    Casbon, Amy-Jo; Long, Matthew E.; Dunn, Kenneth W.; Allen, Lee-Ann H.; Dinauer, Mary C.

    2012-01-01

    Flavocytochrome b558, the catalytic core of the phagocyte NADPH oxidase (NOX2), mediates electron transfer from NADPH to molecular oxygen to generate superoxide, the precursor of highly ROS for host defense. Flavocytochrome b558 is an integral membrane heterodimer consisting of a large glycosylated subunit, gp91phox, and a smaller subunit, p22phox. We recently showed in murine macrophages that flavocytochrome b558 localizes to the PM and Rab11-positive recycling endosomes, whereas in primary hMDMs, gp91phox and p22phox reside in the PM and the ER. The antimicrobial activity of macrophages, including ROS production, is greatly enhanced by IFN-γ, but how this is achieved is incompletely understood. To further define the mechanisms by which IFN-γ enhances macrophage NADPH oxidase activity, we evaluated changes in flavocytochrome b558 expression and localization, along with NADPH oxidase activity, in IFN-γ stimulated RAW 264.7 cells and primary murine BMDMs and hMDMs. We found that enhanced capacity for ROS production is, in part, a result of increased protein expression of gp91phox and p22phox but also demonstrate that IFN-γ induced a shift in the predominant localization of gp91phox and p22phox from intracellular membrane compartments to the PM. Our results are the first to show that a cytokine can change the distribution of macrophage flavocytochrome b558 and provide a potential, new mechanism by which IFN-γ modulates macrophage antimicrobial activity. Altogether, our data suggest that the mechanisms by which IFN-γ regulates antimicrobial activity of macrophages are more complex than previously appreciated. PMID:22822009

  19. Advanced glycation endproducts induce apoptosis of endothelial progenitor cells by activating receptor RAGE and NADPH oxidase/JNK signaling axis

    PubMed Central

    Chen, Jianfei; Jing, Jun; Yu, Shiyong; Song, Minbao; Tan, Hu; Cui, Bin; Huang, Lan

    2016-01-01

    Elevated levels of advanced glycation endproducts (AGEs) is an important risk factor for atherosclerosis. Dysfunction of endothelial progenitor cells (EPCs), which is essential for re-endothelialization and neovascularization, is a hallmark of atherosclerosis. However, it remains unclear whether and how AGEs acts on EPCs to promote pathogenesis of atherosclerosis. In this study, EPCs were exposed to different concentrations of AGEs. The expression of NADPH and Rac1 was measured to investigate the involvement of NADPH oxidase pathway. ROS was examined to indicate the level of oxidative stress in EPCs. Total JNK and p-JNK were determined by Western blotting. Cell apoptosis was evaluated by both TUNEL staining and flow cytometry. Cell proliferation was measured by 3H thymidine uptake. The results showed that treatment of EPCs with AGEs increased the levels of ROS in EPCs. Mechanistically, AGEs increased the activity of NADPH oxidase and the expression of Rac1, a major component of NADPH. Importantly, treatment of EPCs with AGEs activated the JNK signaling pathway, which was closely associated with cell apoptosis and inhibition of proliferation. Our results suggest that the RAGE activation by AGEs in EPCs upregulates intracellular ROS levels, which contributes to increased activity of NADPH oxidase and expression of Rac1, thus promoting cellular apoptosis and inhibiting proliferation. Mechanistically, AGEs binding to the receptor RAGE in EPCs is associated with hyperactivity of JNK signaling pathway, which is downstream of ROS. Our findings suggest that dysregulation of the AGEs/RAGE axis in EPCs may promote atherosclerosis and identify the NADPH/ROS/JNK signaling axis as a potential target for therapeutic intervention. PMID:27347324

  20. Genetic Inactivation of D-Amino Acid Oxidase Enhances Extinction and Reversal Learning in Mice

    ERIC Educational Resources Information Center

    Labrie, Viviane; Duffy, Steven; Wang, Wei; Barger, Steven W.; Baker, Glen B.; Roder, John C.

    2009-01-01

    Activation of the N-methyl-d-aspartate receptor (NMDAR) glycine site has been shown to accelerate adaptive forms of learning that may benefit psychopathologies involving cognitive and perseverative disturbances. In this study, the effects of increasing the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic…

  1. Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes.

    PubMed

    Kasi, Viswanath; Bodiga, Sreedhar; Kommuguri, Upendra Nadh; Sankuru, Suneetha; Bodiga, Vijaya Lakshmi

    2011-07-01

    Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47(phox) phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress. PMID:21651898

  2. Haplotypes of the D-Amino Acid Oxidase Gene Are Significantly Associated with Schizophrenia and Its Neurocognitive Deficits

    PubMed Central

    Hwu, Hai-Gwo; Fann, Cathy Shen-Jang; Yang, Ueng-Cheng; Yang, Wei-Chih; Hsu, Pei-Chun; Chang, Chien-Ching; Wen, Chun-Chiang; Tsai-Wu, Jyy-Jih; Hwang, Tzung-Jeng; Hsieh, Ming H.; Liu, Chen-Chung; Chien, Yi-Ling; Fang, Chiu-Ping; Faraone, Stephen V.; Tsuang, Ming T.; Chen, Wei J.; Liu, Chih-Min

    2016-01-01

    D-amino acid oxidase (DAO) has been reported to be associated with schizophrenia. This study aimed to search for genetic variants associated with this gene. The genomic regions of all exons, highly conserved regions of introns, and promoters of this gene were sequenced. Potentially meaningful single-nucleotide polymorphisms (SNPs) obtained from direct sequencing were selected for genotyping in 600 controls and 912 patients with schizophrenia and in a replicated sample consisting of 388 patients with schizophrenia. Genetic associations were examined using single-locus and haplotype association analyses. In single-locus analyses, the frequency of the C allele of a novel SNP rs55944529 located at intron 8 was found to be significantly higher in the original large patient sample (p = 0.016). This allele was associated with a higher level of DAO mRNA expression in the Epstein-Barr virus-transformed lymphocytes. The haplotype distribution of a haplotype block composed of rs11114083-rs2070586-rs2070587-rs55944529 across intron 1 and intron 8 was significantly different between the patients and controls and the haplotype frequencies of AAGC were significantly higher in patients, in both the original (corrected p < 0.0001) and replicated samples (corrected p = 0.0003). The CGTC haplotype was specifically associated with the subgroup with deficits in sustained attention and executive function and the AAGC haplotype was associated with the subgroup without such deficits. The DAO gene was a susceptibility gene for schizophrenia and the genomic region between intron 1 and intron 8 may harbor functional genetic variants, which may influence the mRNA expression of DAO and neurocognitive functions in schizophrenia. PMID:26986737

  3. d-Amino acid oxidase-mediated increase in spinal hydrogen peroxide is mainly responsible for formalin-induced tonic pain

    PubMed Central

    Lu, Jin-Miao; Gong, Nian; Wang, Yan-Chao; Wang, Yong-Xiang

    2012-01-01

    BACKGROUND AND PURPOSE Spinal reactive oxygen species (ROS) are critically involved in chronic pain. d-Amino acid oxidase (DAAO) oxidizes d-amino acids such as d-serine to form the byproduct hydrogen peroxide without producing other ROS. DAAO inhibitors are specifically analgesic in tonic pain, neuropathic pain and cancer pain. This study examined the role of spinal hydrogen peroxide in pain and the mechanism of the analgesic effects of DAAO inhibitors. EXPERIMENTAL APPROACH Formalin-induced pain behaviours and spinal hydrogen peroxide levels were measured in rodents. KEY RESULTS Formalin injected into the paw increased spinal hydrogen peroxide synchronously with enhanced tonic pain; both were effectively prevented by i.t. fluorocitrate, a selective astrocyte metabolic inhibitor. Given systemically, the potent DAAO inhibitor CBIO (5-chloro-benzo[d]isoxazol-3-ol) blocked spinal DAAO enzymatic activity and specifically prevented formalin-induced tonic pain in a dose-dependent manner. Although CBIO maximally inhibited tonic pain by 62%, it completely prevented the increase in spinal hydrogen peroxide. I.t. catalase, an enzyme specific for decomposition of hydrogen peroxide, completely depleted spinal hydrogen peroxide and prevented formalin-induced tonic pain by 65%. Given systemically, the ROS scavenger PBN (phenyl-N-tert-butylnitrone) also inhibited formalin-induced tonic pain and increase in spinal hydrogen peroxide. Formalin-induced tonic pain was potentiated by i.t. exogenous hydrogen peroxide. CBIO did not increase spinal d-serine level, and i.t. d-serine did not alter either formalin-induced tonic pain or CBIO's analgesic effect. CONCLUSIONS AND IMPLICATIONS Spinal hydrogen peroxide is specifically and largely responsible for formalin-induced pain, and DAAO inhibitors produce analgesia by blocking spinal hydrogen peroxide production rather than interacting with spinal d-serine. PMID:21950354

  4. Searching for cognitive enhancement in the Morris water maze: better and worse performance in D-amino acid oxidase knockout (Dao(-/-)) mice.

    PubMed

    Pritchett, David; Taylor, Amy M; Barkus, Christopher; Engle, Sandra J; Brandon, Nicholas J; Sharp, Trevor; Foster, Russell G; Harrison, Paul J; Peirson, Stuart N; Bannerman, David M

    2016-04-01

    A common strategy when searching for cognitive-enhancing drugs has been to target the N-methyl-d-aspartate receptor (NMDAR), given its putative role in synaptic plasticity and learning. Evidence in favour of this approach has come primarily from studies with rodents using behavioural assays like the Morris water maze. D-amino acid oxidase (DAO) degrades neutral D-amino acids such as D-serine, the primary endogenous co-agonist acting at the glycine site of the synaptic NMDAR. Inhibiting DAO could therefore provide an effective and viable means of enhancing cognition, particularly in disorders like schizophrenia, in which NMDAR hypofunction is implicated. Indirect support for this notion comes from the enhanced hippocampal long-term potentiation and facilitated water maze acquisition of ddY/Dao(-) mice, which lack DAO activity due to a point mutation in the gene. Here, in Dao knockout (Dao(-/-) ) mice, we report both better and worse water maze performance, depending on the radial distance of the hidden platform from the side wall of the pool. Dao(-/-) mice displayed an increased innate preference for swimming in the periphery of the maze (possibly due to heightened anxiety), which facilitated the discovery of a peripherally located platform, but delayed the discovery of a centrally located platform. By contrast, Dao(-/-) mice exhibited normal performance in two alternative assays of long-term spatial memory: the appetitive and aversive Y-maze reference memory tasks. Taken together, these results question the proposed relationship between DAO inactivation and enhanced long-term associative spatial memory. They also have generic implications for how Morris water maze studies are performed and interpreted. PMID:26833794

  5. NecroX-7 prevents oxidative stress-induced cardiomyopathy by inhibition of NADPH oxidase activity in rats

    SciTech Connect

    Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun; Kim, Hyoung Jin; Park, Ji-hoon; Koo, Sun Young; Kwak, Hyo-Shin; Park, Heui Sul; Kim, Dong Wook; Song, Myoungsub; Yim, Hyeon Joo; Seo, Dong Ook; Kim, Soon Ha

    2012-08-15

    Oxidative stress is one of the causes of cardiomyopathy. In the present study, NecroXs, novel class of mitochondrial ROS/RNS scavengers, were evaluated for cardioprotection in in vitro and in vivo model, and the putative mechanism of the cardioprotection of NecroX-7 was investigated by global gene expression profiling and subsequent biochemical analysis. NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced death of H9C2 rat cardiomyocytes at EC{sub 50} = 0.057 μM. In doxorubicin (DOX)-induced cardiomyopathy in rats, NecroX-7 significantly reduced the plasma levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) which were increased by DOX treatment (p < 0.05). Microarray analysis revealed that 21 genes differentially expressed in tBHP-treated H9C2 cells were involved in ‘Production of reactive oxygen species’ (p = 0.022), and they were resolved by concurrent NecroX-7 treatment. Gene-to-gene networking also identified that NecroX-7 relieved cell death through Ncf1/p47phox and Rac2 modulation. In subsequent biochemical analysis, NecroX-7 inhibited NADPH oxidase (NOX) activity by 53.3% (p < 0.001). These findings demonstrate that NecroX-7, in part, provides substantial protection of cardiomyopathy induced by tBHP or DOX via NOX-mediated cell death. -- Highlights: ► NecroX-7 prevented tert-butyl hydroperoxide-induced in vitro cardiac cell death. ► NecroX-7 ameliorated doxorubicin-induced in vivo cardiomyopathy. ► NecroX-7 prevented oxidative stress and necrosis-enriched transcriptional changes. ► NecroX-7 effectively inhibited NADPH oxidase activation. ► Cardioprotection of Necro-7 was brought on by modulation of NADPH oxidase activity.

  6. Synthesis, structure and catechol-oxidase activity of copper(II) complexes of 17-hydroxy-16-(N-3-oxo-prop-1-enyl)amino steroids.

    PubMed

    Wegner, Rainer; Dubs, Manuela; Görls, Helmar; Robl, Christian; Schönecker, Bruno; Jäger, Ernst-G

    2002-09-01

    Copper is next to iron the most important element in the biological transport, storage and in redox reactions of dioxygen. A bioanalogous activation of dioxygen with copper complexes is used for catalytical epoxidation, allylic hydroxylation and oxidative coupling of aromatic substrates, for example. With stereochemical information in form of chiral ligands, enantioselective reactions may be possible. Another aspect of interest on copper catalyzed reactions with dioxygen is that the exact mechanism and biological function of some enzymes (especially catechol oxidase) is yet not fully clear. For studies mimicking the copper-containing catechol oxidase appropriate chiral steroid ligands with defined stereochemistry and conformation have been synthesized. The four diastereomeric 16,17-aminoalcohols of the 3-methoxy-estra-1,3,5(10)-triene series have been condensed with salicylic aldehyde and different beta-ketoenols to the chiral ligand types 1-5. These compounds with different steric and electronic properties and different arrangements of the neighboring hydroxy and nitrogen functions were reacted with copper(II) acetate to copper complexes. The structure of these complexes will be discussed. The bioanalogous oxidation of 3,5-di-tbutyl-catechol (dtbc) to the corresponding quinone was catalyzed by most of the complexes, indicating their ability to activate dioxygen. The trans configurations c and d showed an activity one magnitude higher than the cis configurations a and b. Comparing compounds with the same diastereomeric configuration, the main influence was that of the peripheral R(1-3) substituents at the beta-ketoenaminic group which are useful for the fine-tuning of the properties of the copper atoms like redox potential and Lewis acidity. PMID:12231119

  7. Involvement of Pyruvate Oxidase Activity and Acetate Production in the Survival of Lactobacillus plantarum during the Stationary Phase of Aerobic Growth▿ †

    PubMed Central

    Goffin, Philippe; Muscariello, Lidia; Lorquet, Frederique; Stukkens, Aline; Prozzi, Deborah; Sacco, Margherita; Kleerebezem, Michiel; Hols, Pascal

    2006-01-01

    In addition to the previously characterized pyruvate oxidase PoxB, the Lactobacillus plantarum genome encodes four predicted pyruvate oxidases (PoxC, PoxD, PoxE, and PoxF). Each pyruvate oxidase gene was individually inactivated, and only the knockout of poxF resulted in a decrease in pyruvate oxidase activity under the tested conditions. We show here that L. plantarum has two major pyruvate oxidases: PoxB and PoxF. Both are involved in lactate-to-acetate conversion in the early stationary phase of aerobic growth and are regulated by carbon catabolite repression. A strain devoid of pyruvate oxidase activity was constructed by knocking out the poxB and poxF genes. In this mutant, acetate production was strongly affected, with lactate remaining the major end product of either glucose or maltose fermentation. Notably, survival during the stationary phase appeared to be dramatically improved in the poxB poxF double mutant. PMID:17012588

  8. Effect of ethanol, carbon tetrachloride, and methyl ethyl ketone on butanol oxidase activity in rat lung and liver

    SciTech Connect

    Carlson, G.P. )

    1989-01-01

    Tha ability of the rat liver to oxidize 2-butanol via a cytochrome P-450-mediated mixed-function oxidase reaction is well known. The purpose of this study was to examine this microsomal alcohol oxidizing system in rat lung and determine if it could be altered by treatments that inhibit or induce this activity. 2-Butanol was incubated with microsomal preparations from male rats, and methyl ethyl ketone production was measured by gas chromatography. The rate was six to eight times lower in lung than in liver. Administration of low doses of ethanol (0.5 ml/kg and 1.0 ml/kg) ip for 7 d did not alter activity in the liver but was inhibitory in the lung, as was a high dose of 3.0 ml/kg in the liver. Carbon tetrachloride (1.0 ml/kg, ip) decreased activity in both tissues, especially the lung. The effects of the two inhibitors were not additive. Methyl ethyl ketone induced 2-butanol oxidation in both tissues. The lung possesses butanol oxidase activity that is alterable by both inhibitors and inducers.

  9. NADPH oxidase activation contributes to native low-density lipoprotein-induced proliferation of human aortic smooth muscle cells

    PubMed Central

    Park, Il Hwan; Hwang, Hye Mi; Jeon, Byeong Hwa; Kwon, Hyung-Joo; Hoe, Kwang Lae; Kim, Young Myeong; Ryoo, Sungwoo

    2015-01-01

    Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-θ (PKCθ) and protein kinase C-β (PKCβ) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCθ and PKCβ stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox−/− mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated h

  10. Variation of the Phytochemical Constituents and Antioxidant Activities of Zingiber officinale var. rubrum Theilade Associated with Different Drying Methods and Polyphenol Oxidase Activity.

    PubMed

    Ghasemzadeh, Ali; Jaafar, Hawa Z E; Rahmat, Asmah

    2016-01-01

    The effects of different drying methods (freeze drying, vacuum oven drying, and shade drying) on the phytochemical constituents associated with the antioxidant activities of Z. officinale var. rubrum Theilade were evaluated to determine the optimal drying process for these rhizomes. Total flavonoid content (TFC), total phenolic content (TPC), and polyphenol oxidase (PPO) activity were measured using the spectrophotometric method. Individual phenolic acids and flavonoids, 6- and 8-gingerol and shogaol were identified by ultra-high performance liquid chromatography method. Ferric reducing antioxidant potential (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were used for the evaluation of antioxidant activities. The highest reduction in moisture content was observed after freeze drying (82.97%), followed by vacuum oven drying (80.43%) and shade drying (72.65%). The highest TPC, TFC, and 6- and 8-shogaol contents were observed in samples dried by the vacuum oven drying method compared to other drying methods. The highest content of 6- and 8-gingerol was observed after freeze drying, followed by vacuum oven drying and shade drying methods. Fresh samples had the highest PPO activity and lowest content of flavonoid and phenolic acid compounds compared to dried samples. Rhizomes dried by the vacuum oven drying method represent the highest DPPH (52.9%) and FRAP activities (566.5 μM of Fe (II)/g DM), followed by freeze drying (48.3% and 527.1 μM of Fe (II)/g DM, respectively) and shade drying methods (37.64% and 471.8 μM of Fe (II)/g DM, respectively) with IC50 values of 27.2, 29.1, and 34.8 μg/mL, respectively. Negative and significant correlations were observed between PPO and antioxidant activity of rhizomes. Vacuum oven dried rhizomes can be utilized as an ingredient for the development of value-added food products as they contain high contents of phytochemicals with valuable antioxidant potential. PMID:27322227

  11. NOX2β: A Novel Splice Variant of NOX2 That Regulates NADPH Oxidase Activity in Macrophages

    PubMed Central

    Guida, Elizabeth; King, Paul T.; Sobey, Christopher G.; Drummond, Grant R.

    2012-01-01

    Nox2 oxidase is one isoform in a family of seven NADPH oxidases that generate reactive oxygen species (ROS) and thereby contribute to physiological and pathological processes including host defense, redox signaling and oxidative tissue damage. While alternative mRNA splicing has been shown to influence the activity of several Nox-family proteins, functionally relevant splice variants of Nox2 have not previously been identified. We immunoscreened several mouse tissues and cells for the presence of truncated Nox2 proteins and identified a 30 kDa protein in lung, spleen and macrophages. RT-PCR analysis of mRNA from primary and immortalised (RAW264.7) mouse macrophages, and from human alveolar macrophages, identified a truncated Nox2 transcript which, upon sequence analysis, was found to be a product of the ‘exon skipping’ mode of alternative splicing, lacking exons 4–10 of the Nox2 gene. The predicted protein is comparable in size to that identified by immunoscreening and contains two transmembrane helices and an extended cytosolic C-terminus with binding sites for NADPH and the Nox organiser protein p47phox. Importantly, selective siRNA-mediated knockdown of the transcript reduced expression of the 30 kDa protein in macrophages, and suppressed phorbol ester-stimulated ROS production by 50%. We thus provide the first evidence that Nox2 undergoes alternative mRNA splicing to yield a 30 kDa protein – herein termed Nox2β – that regulates NADPH oxidase activity in macrophages from mice and humans. The discovery of Nox2β paves the way for future examination of its role in physiological and pathological processes. PMID:23118986

  12. Hexose Oxidase-Mediated Hydrogen Peroxide as a Mechanism for the Antibacterial Activity in the Red Seaweed Ptilophora subcostata.

    PubMed

    Ogasawara, Kimi; Yamada, Kenji; Hatsugai, Noriyuki; Imada, Chiaki; Nishimura, Mikio

    2016-01-01

    Marine algae have unique defense strategies against microbial infection. However, their mechanisms of immunity remain to be elucidated and little is known about the similarity of the immune systems of marine algae and terrestrial higher plants. Here, we suggest a possible mechanism underlying algal immunity, which involves hexose oxidase (HOX)-dependent production of hydrogen peroxide (H2O2). We examined crude extracts from five different red algal species for their ability to prevent bacterial growth. The extract from one of these algae, Ptilophora subcostata, was particularly active and prevented the growth of gram-positive and -negative bacteria, which was completely inhibited by treatment with catalase. The extract did not affect the growth of either a yeast or a filamentous fungus. We partially purified from P. subcostata an enzyme involved in its antibacterial activity, which shared 50% homology with the HOX of red seaweed Chondrus crispus. In-gel carbohydrate oxidase assays revealed that P. subcostata extract had the ability to produce H2O2 in a hexose-dependent manner and this activity was highest in the presence of galactose. In addition, Bacillus subtilis growth was strongly suppressed near P. subcostata algal fronds on GYP agar plates. These results suggest that HOX plays a role in P. subcostata resistance to bacterial attack by mediating H2O2 production in the marine environment. PMID:26867214

  13. Hexose Oxidase-Mediated Hydrogen Peroxide as a Mechanism for the Antibacterial Activity in the Red Seaweed Ptilophora subcostata

    PubMed Central

    Ogasawara, Kimi; Yamada, Kenji; Hatsugai, Noriyuki; Imada, Chiaki; Nishimura, Mikio

    2016-01-01

    Marine algae have unique defense strategies against microbial infection. However, their mechanisms of immunity remain to be elucidated and little is known about the similarity of the immune systems of marine algae and terrestrial higher plants. Here, we suggest a possible mechanism underlying algal immunity, which involves hexose oxidase (HOX)-dependent production of hydrogen peroxide (H2O2). We examined crude extracts from five different red algal species for their ability to prevent bacterial growth. The extract from one of these algae, Ptilophora subcostata, was particularly active and prevented the growth of gram-positive and -negative bacteria, which was completely inhibited by treatment with catalase. The extract did not affect the growth of either a yeast or a filamentous fungus. We partially purified from P. subcostata an enzyme involved in its antibacterial activity, which shared 50% homology with the HOX of red seaweed Chondrus crispus. In-gel carbohydrate oxidase assays revealed that P. subcostata extract had the ability to produce H2O2 in a hexose-dependent manner and this activity was highest in the presence of galactose. In addition, Bacillus subtilis growth was strongly suppressed near P. subcostata algal fronds on GYP agar plates. These results suggest that HOX plays a role in P. subcostata resistance to bacterial attack by mediating H2O2 production in the marine environment. PMID:26867214

  14. Catalytic activities of fungal oxidases in hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate-based microemulsion.

    PubMed

    Zhou, Gui-Ping; Zhang, Yun; Huang, Xi-Rong; Shi, Chuan-Hong; Liu, Wei-Feng; Li, Yue-Zhong; Qu, Yin-Bo; Gao, Pei-Ji

    2008-10-01

    For hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF(6)]), an H(2)O-in-[BMIM][PF(6)] microemulsion could be formed in the presence of nonionic surfactant Triton X-100 (TX-100). In such a medium, both lignin peroxidase (LiP) and laccase could express their catalytic activity with the optimum molar ratio of H(2)O to TX-100 at 8.0 for LiP and >20 for laccase, and the optimum pH values at 3.2 for LiP and 4.2 for laccase, respectively. As compared with pure or water saturated [BMIM][PF(6)], in which the two oxidases had negligible catalytic activity due to the strong inactivating effect of [BMIM][PF(6)] on both enzymes, the use of the [BMIM][PF(6)]-based microemulsion had some advantages. Not only the catalytic activities of both fungal oxidases greatly enhanced, but also the apparent viscosity of the medium decreased. PMID:18602799

  15. Tryptamine serves as a proligand of the AhR transcriptional pathway whose activation is dependent of monoamine oxidases.

    PubMed

    Vikström Bergander, Linda; Cai, Wen; Klocke, Bernward; Seifert, Martin; Pongratz, Ingemar

    2012-09-01

    The function of the aryl hydrocarbon receptor (AhR) in mediating the biological effect to environmental pollutants is well established. However, accumulated evidence indicates a wide range of physiological and pathological functions mediated by the AhR, suggesting the existence of endogenous AhR ligand(s). The nature of an AhR ligand remain elusive; however, it is known that the AhR is activated by several compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin or the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole. In this study, we show that physiological concentrations of tryptamine (TA) lead to induction of cytochrome P4501A1 transcription through an AhR-dependent mechanism. In addition, we show that activation of the AhR by TA requires a functional monoamino oxidase system, suggesting that TA acts as an AhR proligand possibly by converting to a high-affinity AhR ligand. Taken together, we show a possible mechanism, through which AhR signaling is activated by endogenous conversion of TA involving monoamine oxidases. PMID:22865928

  16. The effect of high polyphenol oxidase grass silage on metabolism of polyunsaturated fatty acids and nitrogen across the rumen of beef steers.

    PubMed

    Lee, M R F; Theobald, V J; Gordon, N; Leyland, M; Tweed, J K S; Fychan, R; Scollan, N D

    2014-11-01

    Polyphenol oxidase (PPO) activity in red clover (Trifolium pratense) has been reported to reduce both proteolysis and lipolysis, resulting in greater N use efficiency and protection of PUFA across the rumen. Although high levels of PPO have been reported in grasses such as cocksfoot (orchard grass; Dactylis glomerata), no in vivo research has determined whether grass PPO elicits the same response as red clover PPO. To test the hypothesis that silage ensiled from grass with high levels of PPO protects N and PUFA across the rumen, 6 steers with ruminal and duodenal cannulas were offered cocksfoot silage (CO; high-PPO grass), perennial ryegrass silage (PR; Lolium perenne; low-PPO grass), or red clover silage (RC; high-PPO control) at 16 g DM/kg BW daily with the experiment consisting of two 3 × 3 Latin squares with 21-d periods, consisting of 12 d of diet adaptation, 6 d of duodenal marker infusion, 2 d of duodenal sampling, and 1 d of ruminal sampling. All silages were well preserved, with DM of 34.4, 55.3, and 45.4% for CO, PR, and RC. Activity of PPO in silages was low due to deactivation but was greater in CO than either PR or RC (0.15 vs. 0.05 and 0.08 μkatal/g DM). Protein-bound phenol (mg/g DM) as a measure of the degree of oxidation and an indication of PPO protection was greatest for RC (15.9) but comparable for PR (10.1) and CO (12.2). Biohydrogenation of C18 PUFA was significantly lower on RC compared to the 2 grass silages with CO greater than PR. Despite lower levels of total fatty acid intake and subsequent duodenal flow, CO resulted in greater levels of phytanic acid and total branched and odd chain fatty acids in duodenal digesta than RC or PR. Ruminal ammonia concentration was greatest for RC, with no difference between the grasses. Duodenal flow of microbial N and efficiency of microbial protein synthesis were lowest for CO and comparable for RC and PR. The CO (high-grass PPO) did not result in elevated levels of C18 PUFA escaping the rumen or

  17. Kaempferol modulates pro-inflammatory NF-κB activation by suppressing advanced glycation endproducts-induced NADPH oxidase

    PubMed Central

    Kim, Ji Min; Lee, Eun Kyeong; Kim, Dae Hyun; Yu, Byung Pal

    2010-01-01

    Advanced glycation endproducts (AGE) are oxidative products formed from the reaction between carbohydrates and a free amino group of proteins that are provoked by reactive species (RS). It is also known that AGE enhance the generation of RS and that the binding of AGE to a specific AGE receptor (RAGE) induces the activation of the redox-sensitive, pro-inflammatory transcription factor, nuclear factor-kappa B (NF-ĸB). In this current study, we investigated the anti-oxidative effects of short-term kaempferol supplementation on the age-related formation of AGE and the binding activity of RAGE in aged rat kidney. We further investigated the suppressive action of kaempferol against AGE's ability to stimulate activation of pro-inflammatory NF-ĸB and its molecular mechanisms. For this study, we utilized young (6 months old), old (24 months old), and kaempferol-fed (2 and 4 mg/kg/day for 10 days) old rats. In addition, for the molecular work, the rat endothelial cell line, YPEN-1 was used. The results show that AGE and RAGE were increased during aging and that these increases were blunted by kaempferol. In addition, dietary kaempferol reduced age-related increases in NF-κB activity and NF-ĸB-dependant pro-inflammatory gene activity. The most significant new finding from this study is that kaempferol supplementation prevented age-related NF-κB activation by suppressing AGE-induced nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). Taken together, our results demonstrated that dietary kaempferol exerts its anti-oxidative and anti-inflammatory actions by modulating the age-related NF-κB signaling cascade and its pro-inflammatory genes by suppressing AGE-induced NADPH oxidase activation. Based on these data, dietary kaempferol is proposed as a possible anti-AGE agent that may have the potential for use in anti-inflammation therapies. PMID:20431987

  18. Extraction of rice bran extract and some factors affecting its inhibition of polyphenol oxidase activity and browning in potato.

    PubMed

    Boonsiripiphat, Kunnikar; Theerakulkait, Chockchai

    2009-01-01

    The extraction conditions of rice bran extract (RBE), including extraction ratio, extraction time, and extraction temperature, were studied in relation to enzymatic browning inhibition in potato. The inhibitory effect of RBE on potato polyphenol oxidase (PPO) activity and its total phenolic compound content were highest at an extraction ratio of 1:3 (rice bran:water, w/v), extraction time of 30 min, and extraction temperature of 40 degrees C. RBE showed the most inhibitory effect on PPO activity at pH 6.5. However, the inhibitory effect of RBE on potato PPO activity and its total phenolic compound content were decreased at the higher temperature and longer time. PMID:19291577

  19. Urate oxidase: primary structure and evolutionary implications.

    PubMed Central

    Wu, X W; Lee, C C; Muzny, D M; Caskey, C T

    1989-01-01

    Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyzes the oxidation of uric acid to allantoin in most mammals. In humans and certain other primates, however, the enzyme has been lost by some unknown mechanism. To identify the molecular basis for this loss, urate oxidase cDNA clones were isolated from pig, mouse, and baboon, and their DNA sequences were determined. The mouse urate oxidase open reading frame encodes a 303-amino acid polypeptide, while the pig and baboon urate oxidase cDNAs encode a 304-amino acid polypeptide due to a single codon deletion/insertion event. The authenticity of this single additional codon was confirmed by sequencing the mouse and pig genomic copies of the gene. The urate oxidase sequence contains a domain similar to the type 2 copper binding motif found in other copper binding proteins, suggesting that the copper ion in urate oxidase is coordinated as a type 2 structure. Based upon a comparison of the NH2-terminal peptide and deduced sequences, we propose that the maturation of pig urate oxidase involves the posttranslational cleavage of a six-amino acid peptide. Two nonsense mutations were found in the human urate oxidase gene, which confirms, at the molecular level, that the urate oxidase gene in humans is nonfunctional. The sequence comparisons favor the hypothesis that the loss of urate oxidase in humans is due to a sudden mutational event rather than a progressive mutational process. Images PMID:2594778

  20. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination

    PubMed Central

    Ishibashi, Yushi; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Kai, Kyohei; Yuasa, Takashi; Hanada, Atsushi; Yamaguchi, Shinjiro; Iwaya-Inoue, Mari

    2015-01-01

    Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells. PMID:26579718

  1. A Role for Reactive Oxygen Species Produced by NADPH Oxidases in the Embryo and Aleurone Cells in Barley Seed Germination.

    PubMed

    Ishibashi, Yushi; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Kai, Kyohei; Yuasa, Takashi; Hanada, Atsushi; Yamaguchi, Shinjiro; Iwaya-Inoue, Mari

    2015-01-01

    Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells. PMID:26579718

  2. Decreased glycolate oxidase activity leads to altered carbon allocation and leaf senescence after a transfer from high CO2 to ambient air in Arabidopsis thaliana.

    PubMed

    Dellero, Younès; Jossier, Mathieu; Glab, Nathalie; Oury, Céline; Tcherkez, Guillaume; Hodges, Michael

    2016-05-01

    Metabolic and physiological analyses of Arabidopsis thaliana glycolate oxidase (GOX) mutant leaves were performed to understand the development of the photorespiratory phenotype after transfer from high CO2 to air. We show that two Arabidopsis genes, GOX1 and GOX2, share a redundant photorespiratory role. Air-grown single gox1 and gox2 mutants grew normally and no significant differences in leaf metabolic levels and photosynthetic activities were found when compared with wild-type plants. To study the impact of a highly reduced GOX activity on plant metabolism, both GOX1 and GOX2 expression was knocked-down using an artificial miRNA strategy. Air-grown amiRgox1/2 plants with a residual 5% GOX activity exhibited a severe growth phenotype. When high-CO2-grown adult plants were transferred to air, the photosynthetic activity of amiRgox1/2 was rapidly reduced to 50% of control levels, and a high non-photochemical chlorophyll fluorescence quenching was maintained. (13)C-labeling revealed that daily assimilated carbon accumulated in glycolate, leading to reduced carbon allocation to sugars, organic acids, and amino acids. Such changes were not always mirrored in leaf total metabolite levels, since many soluble amino acids increased after transfer, while total soluble protein, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), and chlorophyll amounts decreased in amiRgox1/2 plants. The senescence marker, SAG12, was induced only in amiRgox1/2 rosettes after transfer to air. The expression of maize photorespiratory GOX in amiRgox1/2 abolished all observed phenotypes. The results indicate that the inhibition of the photorespiratory cycle negatively impacts photosynthesis, alters carbon allocation, and leads to early senescence in old rosette leaves. PMID:26896850

  3. Glutathione and cinnamic acid: natural dietary components used in preventing the process of browning by inhibition of Polyphenol Oxidase in apple juice.

    PubMed

    Gacche, R N; Warangkar, S C; Ghole, V S

    2004-04-01

    Consumer demands for 'freshness' in processed foods has been given increasing attention by food processing industries by searching for minimally processed products. Polyphenol Oxidase (PPO) mediated browning is a major cause of undesirable flavors and nutritional losses in fruit juices. Here the anti-browning efficiency of glutathione (GSH, reduced form) and cinnamic acid (CA) in apple juice is evaluated. It was observed that the rate of the browning reaction could be efficiently delayed using GSH and CA, which act as inhibitors of PPO. Kinetic studies confirm that GSH and CA are non-competitive and competitive inhibitors of PPO respectively. PMID:15449733

  4. A fifth member of the tomato 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene family harbours a leucine zipper and is anaerobically induced.

    PubMed

    Sell, Simone; Hehl, Reinhard

    2005-02-01

    Using the leucine zipper domain of a small anaerobically induced bZIP transcription factor in a yeast two hybrid screen, anaerobically induced genes were identified. One peptide corresponds to an anaerobically induced IDS4-like protein that maybe involved in G-protein signaling. Surprisingly, another interacting peptide corresponds to a novel anaerobically induced 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, designated ACO5. ACO5 harbours a leucine zipper and transcription is mainly induced in fruits and to a lesser extend in leaves. The role of ACO5 in the low oxygen response of tomato is discussed. PMID:16040352

  5. 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. PMID:25724690

  6. Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom.

    PubMed

    Lee, Mui Li; Tan, Nget Hong; Fung, Shin Yee; Sekaran, Shamala Devi

    2011-03-01

    The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme. PMID:21059402

  7. Binding geometry, stoichiometry, and thermodynamics of cyclomalto-oligosaccharide (cyclodextrin) inclusion complex formation with chlorogenic acid, the major substrate of apple polyphenol oxidase.

    PubMed

    Irwin, P L; Pfeffer, P E; Doner, L W; Sapers, G M; Brewster, J D; Nagahashi, G; Hicks, K B

    1994-03-18

    The inclusion complexes of cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD), cyclomaltooctaose (gamma-CD), and polymerized beta-CD (beta-CDn) with chlorogenic acid (CA), the major substrate of apple fruit polyphenol oxidase (PPO), were studied with regard to pH, ionic strength, and temperature in model buffer systems and apple juice. The thermodynamics of CD.CA inclusion complex formation, which were studied in solution using UV spectrophotometry, displayed enthalpy-entropy compensation typical of processes driven by solvation phenomena. We also found that the apparent association constants (K) of the CD.CA equilibrium were relatively insensitive to pH for beta-CD, compared to alpha- and gamma-CDs, but were subject to substantial enhancement at low ionic strengths. The beta-CD.CA inclusion complex was also characterized for binding geometry and stoichiometry at 9.4 T and 25 degrees C in 0.05 M Na phosphate buffer by 1H NMR spectroscopy. A 1:1 stoichiometric ratio for the complex was found using the method of continuous variations. 1H Spin-lattice relaxation and chemical-shift data indicate that the phenolic ring of CA docks within the cavity of beta-CD. The Ks for beta-, alpha-, and gamma-CD determined in apple juice, which contains a mixture of PPO substrates, were found to correlate with PPO activity-related data. Apple juice, treated with beta-CDn, did not brown until CA was added back. These latter findings strongly argue that the mechanism for inhibition of juice browning with cyclodextrins was mainly due to the binding of PPO substrates and not some other means such as enzyme inactivation via sequestration of Cu2+ by CDs. PMID:8194069

  8. Arsenite Oxidase Also Functions as an Antimonite Oxidase

    PubMed Central

    Wang, Qian; Warelow, Thomas P.; Kang, Yoon-Suk; Romano, Christine; Osborne, Thomas H.; Lehr, Corinne R.; Bothner, Brian; McDermott, Timothy R.

    2015-01-01

    Arsenic and antimony are toxic metalloids and are considered priority environmental pollutants by the U.S. Environmental Protection Agency. Significant advances have been made in understanding microbe-arsenic interactions and how they influence arsenic redox speciation in the environment. However, even the most basic features of how and why a microorganism detects and reacts to antimony remain poorly understood. Previous work with Agrobacterium tumefaciens strain 5A concluded that oxidation of antimonite [Sb(III)] and arsenite [As(III)] required different biochemical pathways. Here, we show with in vivo experiments that a mutation in aioA [encoding the large subunit of As(III) oxidase] reduces the ability to oxidize Sb(III) by approximately one-third relative to the ability of the wild type. Further, in vitro studies with the purified As(III) oxidase from Rhizobium sp. strain NT-26 (AioA shares 94% amino acid sequence identity with AioA of A. tumefaciens) provide direct evidence of Sb(III) oxidation but also show a significantly decreased Vmax compared to that of As(III) oxidation. The aioBA genes encoding As(III) oxidase are induced by As(III) but not by Sb(III), whereas arsR gene expression is induced by both As(III) and Sb(III), suggesting that detection and transcriptional responses for As(III) and Sb(III) differ. While Sb(III) and As(III) are similar with respect to cellular extrusion (ArsB or Acr3) and interaction with ArsR, they differ in the regulatory mechanisms that control the expression of genes encoding the different Ars or Aio activities. In summary, this study documents an enzymatic basis for microbial Sb(III) oxidation, although additional Sb(III) oxidation activity also is apparent in this bacterium. PMID:25576601

  9. The urinary MHPG/creatinine ratio and its relationship to platelet monoamine oxidase activity in abstinent alcoholics.

    PubMed

    Farren, C K; Tipton, K F

    1999-01-01

    This study was designed to assess the baseline noradrenergic turnover of subgroups of postwithdrawal abstinent alcoholics and healthy controls. The method chosen was an overnight fasting urine sample of the breakdown product of norepinephrine, MHPG, related to urinary creatinine. A comparison was made with platelet monoamine oxidase activity and also within subgroups of the study population. This study found no difference between alcoholics and controls, nor between subgroups of postwithdrawal alcoholics in their level of urinary MHPG corrected for creatinine, and no significant correlation with major subject characteristics or with platelet monoamine oxidase. There was a trend, however, towards a significant correlation with duration of abstinence from alcohol, and there was a correlation with a history of fighting when drinking alcohol, but not with sociopathic traits overall. Within the type 2 alcoholics there was a significant correlation with a history of fighting when drinking and a negative correlation with behavioral tolerance to alcohol. It is possible that only the subset of type 2 alcoholics with certain antisocial characteristics have noradrenergic abnormalities. Although no statistical difference was found between the different groups under study, the information is helpful in increasing understanding of the noradrenergic system in abstinent alcoholics. PMID:20575773

  10. [Effect of abiotic elicitation on the sanguinarine production and polyphenol oxidase activity in the suspension culture of Eschscholtzia californica CHAM].

    PubMed

    Bilka, František; Balážová, Andrea; Bilková, Andrea; Holková, Ivana

    2013-08-01

    Elicitation of plant in vitro cultures represents a biotechnological tool to improve the production of secondary metabolites. In this study, the effect of AgNO3 and CdCl2 on the sanguinarine production by the suspension culture of Eschscholtzia californica CHAM. was investigated. Elicitors were added to the cultures at the 14th day of subcultivation and their effect on the sanguinarine production was evaluated after a 48 h exposure. AgNO3 at the concentration of 0.075 mmol.l-1 and CdCl2 at the concentration of 4 mmol.l-1 induced a ca. 5.2- and 5.6-multiple increase in sanguinarine synthesis, respectively. This amount represents probably the maximal production, because a further increase in the elicitors concentrations did not increase sanguinarine production. Both abiotic elicitors induced a polyphenol oxidase specific activity increase. Polyphenol oxidase is probably involved in the biosynthesis of sanguinarine at the level of dopamine formation. Dopamine is a precursor of (S)-norcoclaurine, the first intermediate with the benzylisoquinoline structure. PMID:24047145

  11. Pigment epithelium-derived factor stimulates skeletal muscle glycolytic activity through NADPH oxidase-dependent reactive oxygen species production.

    PubMed

    Carnagarin, Revathy; Carlessi, Rodrigo; Newsholme, Philip; Dharmarajan, Arun M; Dass, Crispin R

    2016-09-01

    Pigment epithelium-derived factor is a multifunctional serpin implicated in insulin resistance in metabolic disorders. Recent evidence suggests that exposure of peripheral tissues such as skeletal muscle to PEDF has profound metabolic consequences with predisposition towards chronic conditions such as obesity, type 2 diabetes, metabolic syndrome and polycystic ovarian syndrome. Chronic inflammation shifts muscle metabolism towards increased glycolysis and decreased oxidative metabolism. In the present study, we demonstrate a novel effect of PEDF on cellular metabolism in mouse cell line (C2C12) and human primary skeletal muscle cells. PEDF addition to skeletal muscle cells induced enhanced phospholipase A2 activity. This was accompanied with increased production of reactive oxygen species in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner that triggered a shift towards a more glycolytic phenotype. Extracellular flux analysis and glucose consumption assays demonstrated that PEDF treatment resulted in enhanced glycolysis but did not change mitochondrial respiration. Our results demonstrate that skeletal muscle cells express a PEDF-inducible oxidant generating system that enhances glycolysis but is sensitive to antioxidants and NADPH oxidase inhibition. PMID:27343430

  12. Enzyme-mimetic effects of gold@platinum nanorods on the antioxidant activity of ascorbic acid

    NASA Astrophysics Data System (ADS)

    Zhou, Yu-Ting; He, Weiwei; Wamer, Wayne G.; Hu, Xiaona; Wu, Xiaochun; Lo, Y. Martin; Yin, Jun-Jie

    2013-01-01

    Au@Pt nanorods were prepared by growing platinum nanodots on gold nanorods. Using electron spin resonance (ESR), we determined that the mechanisms for oxidation of ascorbic acid (AA) by Au@Pt nanorods and ascorbic acid oxidase (AAO) were kinetically similar and yielded similar products. In addition we observed that Au@Pt nanorods were stable with respect to temperature and pH. Using UV-VIS spectroscopy, the apparent kinetics of enzyme-mimetic activity of Au@Pt nanorods were studied and compared with the activity of AAO. With the help of ESR, we found that Au@Pt nanorods did not scavenge hydroxyl radicals but inhibited the antioxidant ability of AA for scavenging hydroxyl radicals produced by photoirradiating solutions containing titanium dioxide and zinc oxide. Moreover, the Au@Pt nanorods reduced the ability of AA to scavenge DPPH radicals and superoxide radicals. These results demonstrate that Au@Pt nanorods can reduce the antioxidant activity of AA. Therefore, it is necessary to consider the effects of using Pt nanoparticles together with other reducing agents or antioxidants such as AA due to the oxidase-like property of Au@Pt nanorods.Au@Pt nanorods were prepared by growing platinum nanodots on gold nanorods. Using electron spin resonance (ESR), we determined that the mechanisms for oxidation of ascorbic acid (AA) by Au@Pt nanorods and ascorbic acid oxidase (AAO) were kinetically similar and yielded similar products. In addition we observed that Au@Pt nanorods were stable with respect to temperature and pH. Using UV-VIS spectroscopy, the apparent kinetics of enzyme-mimetic activity of Au@Pt nanorods were studied and compared with the activity of AAO. With the help of ESR, we found that Au@Pt nanorods did not scavenge hydroxyl radicals but inhibited the antioxidant ability of AA for scavenging hydroxyl radicals produced by photoirradiating solutions containing titanium dioxide and zinc oxide. Moreover, the Au@Pt nanorods reduced the ability of AA to scavenge

  13. Type IX Ehlers-Danlos syndrome and Menkes syndrome: the decrease in lysyl oxidase activity is associated with a corresponding deficiency in the enzyme protein.

    PubMed Central

    Kuivaniemi, H; Peltonen, L; Kivirikko, K I

    1985-01-01

    Type IX of the Ehlers-Danlos syndrome (E-D IX) and the Menkes syndrome are X-linked recessively inherited disorders characterized by abnormalities in copper metabolism. These abnormalities are associated with a severe reduction in the activity of lysyl oxidase, the extracellular copper enzyme that initiates crosslinking of collagens and elastin. No increase in this deficient enzyme activity was obtained when culture media from fibroblasts of patients with E-D IX or the Menkes syndrome were incubated with copper under various conditions in vitro. A distinct, although small, increase in lysyl oxidase activity was obtained, however, when copper-supplemented media were used during culturing of the fibroblasts, although even under these conditions, the enzyme activity in the media from the affected cells remained markedly below that of the controls. Immunoprecipitation, dot-blotting, and immunoperoxidase staining experiments with antisera to human lysyl oxidase indicated that fibroblasts from patients with E-D IX or the Menkes syndrome do not secrete into their medium, or contain inside the cell, any significant amounts of a copper-deficient, catalyt