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Sample records for acid oxidase activator

  1. 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.

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

    PubMed

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

    2014-08-06

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

  3. 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

  4. 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.

  5. 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

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

    PubMed

    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.

  7. 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.

  8. Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation).

    PubMed

    Vanlerberghe, G. C.; Day, D. A.; Wiskich, J. T.; Vanlerberghe, A. E.; McIntosh, L.

    1995-10-01

    Transgenic Nicotiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due to the introduction of a sense transgene(s) of Aox1, the nuclear gene encoding AOX, were used to investigate mechanisms regulating AOX activity. After purification of leaf mitochondria, a large proportion of the AOX protein was present as the oxidized (covalently associated and less active) dimer. High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and more active dimer) and its subsequent activation by certain [alpha]-keto acids, particularly pyruvate. Reduction of AOX to its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate. Our evidence suggests that NADPH may be specifically required for AOX reduction. All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well. Transgenic leaves lacking AOX due to the introduction of an Aox1 antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOX-regulatory mechanisms. Under conditions in which respiratory carbon metabolism is restricted by the capacity of mitochondrial electron transport, feed-forward activation of AOX by mitochondrial reducing power and pyruvate may act to prevent redirection of carbon metabolism, such as to fermentative pathways.

  9. Activation of polyphenol oxidase of chloroplasts.

    PubMed

    Tolbert, N E

    1973-02-01

    Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or -18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles x mg(-1) chlorophyll x hr(-1). Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes.Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30

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

    PubMed

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

    2016-11-29

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

  11. 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

    2011-11-09

    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.

  12. 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.

  13. THE PREPARATION AND PROPERTIES OF HIGHLY PURIFIED ASCORBIC ACID OXIDASE

    PubMed Central

    Powers, Wendell H.; Lewis, Stanley; Dawson, Charles R.

    1944-01-01

    1. A method is described for the preparation of a highly purified ascorbic acid oxidase containing 0.24 per cent copper. 2. Using comparable activity measurements, this oxidase is about one and a half times as active on a dry weight basis as the hitherto most highly purified preparation described by Lovett-Janison and Nelson. The latter contained 0.15 per cent copper. 3. The oxidase activity is proportional to the copper content and the proportionality factor is the same as that reported by Lovett-Janison and Nelson. 4. When dialyzed free of salt, the blue concentrated oxidase solutions precipitate a dark green-blue protein which carries the activity. This may be prevented by keeping the concentrated solutions about 0.1 M in Na2HPO4. 5. When highly diluted for activity measurements the oxidase rapidly loses activity (irreversibly) previous to the measurement, unless the dilution is made with a dilute inert protein (gelatin) solution. Therefore activity values obtained using such gelatin-stabilized dilute solutions of the oxidase run considerably higher than values obtained by the Lovett-Janison and Nelson technique. 6. The effect of pH and substrate concentration on the activity of the purified oxidase in the presence and absence of inert protein was studied. PMID:19873382

  14. Changes in D-aspartic acid and D-glutamic acid levels in the tissues and physiological fluids of mice with various D-aspartate oxidase activities.

    PubMed

    Han, Hai; Miyoshi, Yurika; Koga, Reiko; Mita, Masashi; Konno, Ryuichi; Hamase, Kenji

    2015-12-10

    D-Aspartic acid (D-Asp) and D-glutamic acid (D-Glu) are currently paid attention as modulators of neuronal transmission and hormonal secretion. These two D-amino acids are metabolized only by D-aspartate oxidase (DDO) in mammals. Therefore, in order to design and develop new drugs controlling the D-Asp and D-Glu amounts via regulation of the DDO activities, changes in these acidic D-amino acid amounts in various tissues are expected to be clarified in model animals having various DDO activities. In the present study, the amounts of Asp and Glu enantiomers in 6 brain tissues, 11 peripheral tissues and 2 physiological fluids of DDO(+/+), DDO(+/-) and DDO(-/-) mice were determined using a sensitive and selective two-dimensional HPLC system. As a result, the amounts of D-Asp were drastically increased with the decrease in the DDO activity in all the tested tissues and physiological fluids. On the other hand, the amounts of D-Glu were almost the same among the 3 strains of mice. The present results are useful for designing new drug candidates, such as DDO inhibitors, and further studies are expected.

  15. Involvement of NADPH oxidase in high-dose phenolic acid-induced pro-oxidant activity on rat mesenteric venules.

    PubMed

    Du, Wen-Yuan; Xiao, Ying; Yao, Jian-Jing; Hao, Zhe; Zhao, Yu-Bin

    2017-01-01

    In the present study, we investigated the potential role of phenolic acids in initiating oxidative damage to microvascular endothelial cells and the underlying mechanism mediating the pro-oxidant action. Male Wistar rats received high doses of phenolic acid [caffeic acid (CA), salvianolic acid B (SAB), chlorogenic acid (ChA) or ferulic acid (FA)]. The creation of reactive oxygen species in mesenteric microcirculation endothelial cells and adherent leukocytes along with venules were assessed using intravital microscopy. The expression levels of NADPH oxidase subunits (Nox4 and p22(phox)) in terminal ileum tissues were determined by western blot analysis. Intravenous injection of high-dose ChA or CA (7 mg/kg) markedly increased the peroxide production in the venular walls and upregulated the protein expression levels of Nox4 and p22(phox) in the ileum tissues, while the same dose of CA and SAB made no difference within the observation period. No changes were observed in the number of leukocytes adhering to the venular walls. High-dose ChA and FA led to an imbalance between the oxidant and antioxidant mechanism by boosting the expression levels of NADPH oxidase. Thus, we clarified the rationale behind the adverse effects of a herbal injection containing high levels of phenolic acid compounds.

  16. Retinoic acid modulation of thyroid dual oxidase activity in rats and its impact on thyroid iodine organification.

    PubMed

    Mühlbauer, Mônica; da Silva, Alba Cenélia Matos; Marassi, Michelle Porto; Lourenço, Alexandre Lopes; Ferreira, Andrea Claudia Freitas; de Carvalho, Denise Pires

    2010-06-01

    The sodium-iodide symporter (NIS) mediates iodide uptake into the thyrocytes, which is important for the diagnosis and therapy of thyroid disorders. Decreased ability to uptake iodide in thyroid carcinomas reduces the efficacy of radioiodine therapy, and retinoic acid (RA) treatment reinduces iodide uptake. The effectiveness of treatment depends not only on iodide uptake but also on the ability of thyrocytes to organify iodine, which is catalyzed by thyroperoxidase (TPO) in the presence of H(2)O(2). Our goal was to determine the influence of RA on thyroid iodide uptake, iodine organification, and TPO and dual oxidase (DuOx) activities. Normal rats were treated with all-trans-RA or 13-cis-RA (100 or 1500 microg/100 g body weight (b.w.), s.c.) for 14 and 28 days. The 2 h thyroid radioiodine content significantly decreased in rats treated with all-trans-RA (100 microg/100 g b.w.) for 14 days. In this group, NIS function and TPO activity were unchanged, whereas DuOx activity was significantly decreased, which might have contributed to the decrease in iodine organification. Both doses of 13-cis-RA for 28 days increased the 15 min thyroid radioiodine uptake, while the 2 h radioiodide uptake increased only in rats treated with the highest dose of 13-cis-RA. While TPO activity did not change, H(2)O(2) generation was increased in this group, and serum thyroxine levels were normal. Since radioiodine half-life in the thyroid gland is important for treatment efficacy, our results highlight the importance of correctly choosing the RA isomer, the time and the dose of treatment, in order to improve the efficacy of radioiodine therapy.

  17. 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

  18. Modulating NMDA Receptor Function with D-Amino Acid Oxidase Inhibitors: Understanding Functional Activity in PCP-Treated Mouse Model

    PubMed Central

    Sershen, Henry; Hashim, Audrey; Dunlop, David S.; Suckow, Raymond F.; Cooper, Tom B.; Javitt, Daniel C.

    2016-01-01

    Deficits in N-methyl-D-aspartate receptor (NMDAR) function are increasingly linked to persistent negative symptoms and cognitive deficits in schizophrenia. Accordingly, clinical studies have been targeting the modulatory site of the NMDA receptor, based on the decreased function of NMDA receptor, to see whether increasing NMDA function can potentially help treat the negative and cognitive deficits seen in the disease. Glycine and D-serine are endogenous ligands to the NMDA modulatory site, but since high doses are needed to affect brain levels, related compounds are being developed, for example glycine transport (GlyT) inhibitors to potentially elevate brain glycine or targeting enzymes, such as D-amino acid oxidase (DAAO) to slow the breakdown and increase the brain level of D-serine. In the present study we further evaluated the effect of DAAO inhibitors 5-chloro-benzo[d]isoxazol-3-ol (CBIO) and sodium benzoate (NaB) in a phencyclidine (PCP) rodent mouse model to see if the inhibitors affect PCP-induced locomotor activity, alter brain D-serine level, and thereby potentially enhance D-serine responses. D-Serine dose-dependently reduced the PCP-induced locomotor activity at doses above 1000 mg/kg. Acute CBIO (30 mg/kg) did not affect PCP-induced locomotor activity, but appeared to reduce locomotor activity when given with D-serine (600 mg/kg); a dose that by itself did not have an effect. However, the effect was also present when the vehicle (Trappsol®) was tested with D-serine, suggesting that the reduction in locomotor activity was not related to DAAO inhibition, but possibly reflected enhanced bioavailability of D-serine across the blood brain barrier related to the vehicle. With this acute dose of CBIO, D-serine level in brain and plasma were not increased. Another weaker DAAO inhibitor sodium benzoate (NaB) (400 mg/kg), and NaB plus D-serine also significantly reduced PCP-induced locomotor activity, but without affecting plasma or brain D-serine level

  19. Covalent immobilization of ascorbate oxidase onto polycarbonate strip for L-ascorbic acid detection.

    PubMed

    Kannoujia, Dileep Kumar; Kumar, Saroj; Nahar, Pradip

    2012-10-01

    Herein, a simple and rapid method is described for detection of L-ascorbic acid by ascorbate oxidase immobilized onto polycarbonate strip pre-activated by 1-fluoro-2-nitro-4-azidobenzene in photochemical reaction. Covalent attachment of ascorbate oxidase was confirmed by XPS studies. The immobilized-ascorbate oxidase shows higher pH, thermal and storage stability in comparison to free enzyme.

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

    PubMed

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

    2017-03-01

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

  1. Individual variation in hepatic aldehyde oxidase activity.

    PubMed

    Al-Salmy, H S

    2001-04-01

    Aldehyde oxidase (AO) is a molybdo-flavo enzyme expressed predominantly in the liver, lung, and kidney. AO plays a major role in oxidation of aldehydes, as well as oxidation of various N-heterocyclic compounds of pharmacological and toxicological importance including antiviral (famciclovir), antimalarial (quinine), antitumour (methotrexate), and nicotine. The aim of this study was to investigate cytosolic aldehyde oxidase activity in human liver. Cytosolic AO was characterised using both the metabolism of N-[(2-dimethylamino)ethyl] acridine-4-carboxamide (DACA) and benzaldehyde to form DACA-9(10H)-acridone (quantified by HPLC with fluorescence detection) and benzoic acid (quantified spectrophotometrically). Thirteen livers (10 female, 3 male) were examined. The intrinsic clearance (Vmax/Km) of DACA varied 18-fold (0.03-0.50 m/min/mg). Vmax ranged from 0.20-3.10 nmol/ min/mg, and Km ranged from 3.5-14.2 microM. In the same specimens, the intrinsic clearance for benzaldehyde varied 5-fold (0.40-1.8 ml/min/mg). Vmax ranged from 3.60-12.6 nmol/min/mg and Km ranged from 3.6-14.6 microM. Furthermore, there were no differences in AO activity between male and female human livers, nor was there any relationship to age of donor (range 29-73 years), smoking status, or disease status. In conclusion, our results showed that there are variations in AO activity in human liver. These variations in aldehyde oxidase activity might reflect individual variations or they might be due to AO stability during processing and storage.

  2. 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.

  3. Genistein effect on xanthine oxidase activity.

    PubMed

    Sumbayev, V V

    2001-01-01

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

  4. Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.) 1

    PubMed Central

    Lin, Liang-Shiou; Varner, Joseph E.

    1991-01-01

    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.” ImagesFigure 3Figure 4Figure 5 PMID:16668145

  5. Chlorpromazine oligomer is a potentially active substance that inhibits human D-amino acid oxidase, product of a susceptibility gene for schizophrenia.

    PubMed

    Iwana, Sanae; Kawazoe, Tomoya; Park, Hwan Ki; Tsuchiya, Koichiro; Ono, Koji; Yorita, Kazuko; Sakai, Takashi; Kusumi, Takenori; Fukui, Kiyoshi

    2008-12-01

    D-amino acid oxidase (DAO), a potential risk factor for schizophrenia, has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia. Here we show the inhibitory effect of an antipsychotic drug, chlorpromazine, on human DAO, which is consistent with previous reports using porcine DAO, although human DAO was inhibited to a lesser degree (K(i) = 0.7 mM) than porcine DAO. Since chlorpromazine is known to induce phototoxic or photoallergic reactions and also to be transformed into various metabolites, we examined the effects of white light-irradiated chlorpromazine on the enzymatic activity. Analytical methods including high-resolution mass spectrometry revealed that irradiation triggered the oligomerization of chlorpromazine molecules. The oligomerized chlorpromazine showed a mixed type inhibition with inhibition constants of low micromolar range, indicative of enhanced inhibition. Taken together, these results suggest that oligomerized chlorpromazine could act as an active substance that might contribute to the therapeutic effects of this drug.

  6. Effect of naphthalene on cytochrome oxidase activity

    SciTech Connect

    Harmon, H.J.

    1988-01-01

    Previous reports have demonstrated that naphthalene inhibits oxygen consumption in Daphnia magna tissue culture cells, and intact mitochondria and submitochondrial particles. These studies were extended to algal mitochondrial respiration as well as photosynthetic activity. The authors were able to demonstrate the specific site of apparent respiratory inhibition to be coenzyme Q (ubiquinone, UQ) and later to demonstrate the molecular basis of this inhibition at ubiquinone. The authors previously could not demonstrate an effect of naphthalene on cytochrome oxidase activity. However, the observation that naphthalene can stimulate respiration in algae prompted the reinvestigation of the effect of naphthalene on the kinetics of cytochrome oxidase. Cytochrome oxidase is a multi-subunit membranous protein responsible for the oxidation of cytochrome c and the reduction of molecular oxygen to water. Because of the complicated nature and mechanism of this enzyme, the potential exists for multiple and possibly opposite effects of naphthalene on its function.

  7. Involvement of phospholipase D and NADPH-oxidase in salicylic acid signaling cascade.

    PubMed

    Kalachova, Tetiana; Iakovenko, Oksana; Kretinin, Sergii; Kravets, Volodymyr

    2013-05-01

    Salicylic acid is associated with the primary defense responses to biotic stress and formation of systemic acquired resistance. However, molecular mechanisms of early cell reactions to phytohormone application are currently undisclosed. The present study investigates the participation of phospholipase D and NADPH-oxidase in salicylic acid signal transduction cascade. The activation of lipid signaling enzymes within 15 min of salicylic acid application was shown in Arabidopsis thaliana plants by measuring the phosphatidic acid accumulation. Adding of primary alcohol (1-butanol) to the incubation medium led to phosphatidylbutanol accumulation as a result of phospholipase D (PLD) action in wild-type and NADPH-oxidase RbohD deficient plants. Salicylic acid induced rapid increase in NADPH-oxidase activity in histochemical assay with nitroblue tetrazolium but the reaction was not observed in presence of 1-butanol and NADPH-oxidase inhibitor diphenylene iodide (DPI). The further physiological effect of salicylic acid and inhibitory analysis of the signaling cascade were made in the guard cell model. Stomatal closure induced by salicylic acid was inhibited by 1-butanol and DPI treatment. rbohD transgenic plants showed impaired stomatal reaction upon phytohormone effect, while the reaction to H2O2 did not differ from that of wild-type plants. Thus a key role of NADPH-oxidase D-isoform in the process of stomatal closure in response to salicylic acid has been postulated. It has enabled to predict a cascade implication of PLD and NADPH oxidase to salicylic acid signaling pathway.

  8. Multivalent Interactions of Human Primary Amine Oxidase with the V and C22 Domains of Sialic Acid-Binding Immunoglobulin-Like Lectin-9 Regulate Its Binding and Amine Oxidase Activity

    PubMed Central

    Fair-Mäkelä, Ruth; Salo-Ahen, Outi M. H.; Guédez, Gabriela; Bligt-Lindén, Eva; Grönholm, Janne; Jalkanen, Sirpa; Salminen, Tiina A.

    2016-01-01

    Sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) on leukocyte surface is a counter-receptor for endothelial cell surface adhesin, human primary amine oxidase (hAOC3), a target protein for anti-inflammatory agents. This interaction can be used to detect inflammation and cancer in vivo, since the labeled peptides derived from the second C2 domain (C22) of Siglec-9 specifically bind to the inflammation-inducible hAOC3. As limited knowledge on the interaction between Siglec-9 and hAOC3 has hampered both hAOC3-targeted drug design and in vivo imaging applications, we have now produced and purified the extracellular region of Siglec-9 (Siglec-9-EC) consisting of the V, C21 and C22 domains, modeled its 3D structure and characterized the hAOC3–Siglec-9 interactions using biophysical methods and activity/inhibition assays. Our results assign individual, previously unknown roles for the V and C22 domains. The V domain is responsible for the unusually tight Siglec-9–hAOC3 interactions whereas the intact C22 domain of Siglec-9 is required for modulating the enzymatic activity of hAOC3, crucial for the hAOC3-mediated leukocyte trafficking. By characterizing the Siglec-9-EC mutants, we could conclude that R120 in the V domain likely interacts with the terminal sialic acids of hAOC3 attached glycans whereas residues R284 and R290 in C22 are involved in the interactions with the active site channel of hAOC3. Furthermore, the C22 domain binding enhances the enzymatic activity of hAOC3 although the sialic acid-binding capacity of the V domain of Siglec-9 is abolished by the R120S mutation. To conclude, our results prove that the V and C22 domains of Siglec-9-EC interact with hAOC3 in a multifaceted and unique way, forming both glycan-mediated and direct protein-protein interactions, respectively. The reported results on the mechanism of the Siglec-9–hAOC3 interaction are valuable for the development of hAOC3-targeted therapeutics and diagnostic tools. PMID:27893774

  9. Polyphenol oxidase activity in annual forage clovers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO)-mediated phenol reactions in red clover (Trifolium pratense L.) bind forage protein and reduce proteolysis, producing beneficial effects on forage protein degradability, silage fermentation, and soil-N cycling. We evaluated PPO activity in seven previously untested annual c...

  10. Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus.

    PubMed

    Kasai, Kosuke; Ishikawa, Takashi; Komata, Takafumi; Fukuchi, Kaori; Chiba, Mitsuru; Nozaka, Hiroyuki; Nakamura, Toshiya; Sato, Tatsusuke; Miura, Tomisato

    2010-01-01

    Fish produce mucus substances as a defensive outer barrier against environmental xenobiotics and predators. Recently, we found a bioactive protein in the mucus layer of the flounder Platichthys stellatus, which showed antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and methicillin-resistant S. aureus. In this study, we isolated and identified the antibacterial protein from the mucus components of P. stellatus using a series of column chromatography steps. We then performed gel electrophoresis and cDNA cloning to characterize the protein. The antibacterial protein in the mucus had a molecular mass of approximately 52 kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it corresponded completely with the peptide sequence of antibacterial protein from the gill. A BLAST search suggested that the cDNA encoded an antibacterial protein sharing identity with a number of L-amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins. RT-PCR using a specific primer, and immunohistochemical analysis with anti-LAAO IgG, demonstrated tissue-specific expression and localization in the gill. Moreover, the anti-LAAO IgG was able to neutralize the antibacterial activity of the protein against methicillin-resistant S. aureus. Thus, we demonstrated that this antibacterial protein, identified from P. stellatus-derived epidermal mucus, is a novel LAAO-like protein with antibacterial activity, similar to snake LAAOs.

  11. Inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of small GTP-binding proteins. Lack of amino acid sequence specificity and importance of polybasic motif.

    PubMed

    Joseph, G; Gorzalczany, Y; Koshkin, V; Pick, E

    1994-11-18

    The small GTP-binding protein (G protein) Rac1 is an obligatory participant in the assembly of the superoxide (O2-.)-generating NADPH oxidase complex of macrophages. We investigated the effect of synthetic peptides, mapping within the near carboxyl-terminal domains of Rac1 and of related G proteins, on the activity of NADPH oxidase in a cell-free system consisting of solubilized guinea pig macrophage membrane, a cytosolic fraction enriched in p47phox and p67phox (or total cytosol), highly purified Rac1-GDP dissociation inhibitor for Rho (Rho GDI) complex, and the activating amphiphile, lithium dodecyl sulfate. Peptides Rac1-(178-188) and Rac1-(178-191), but not Rac2-(178-188), inhibited NADPH oxidase activity in a Rac1-dependent system when added prior to or simultaneously with the initiation of activation. However, undecapeptides corresponding to the near carboxyl-terminal domains of RhoA and RhoC and, most notably, a peptide containing the same amino acids as Rac1-(178-188), but in reversed orientation, were also inhibitory. Surprisingly, O2-. production in a Rac2-dependent cell-free system was inhibited by Rac1-(178-188) but not by Rac2-(178-188). Finally, basic polyamino acids containing lysine, histidine, or arginine, also inhibited NADPH oxidase activation. We conclude that inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of certain small G proteins is not amino acid sequence-specific but related to the presence of a polybasic motif. It has been proposed that such a motif serves as a plasma membrane targeting signal for a number of small G proteins (Hancock, J.F., Paterson, H., and Marshall, C.J. (1990) Cell 63, 133-139).

  12. 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.

  13. Effect of endogenous ascorbic acid oxidase activity and stability on vitamin C in carrots (Daucus carota subsp. sativus) during thermal treatment.

    PubMed

    Leong, Sze Ying; Oey, Indrawati

    2012-10-15

    The purpose of this research was to study the effect of endogenous ascorbic acid oxidase (AAO) on vitamin C in carrots (Daucus carota subsp. sativus), namely Nantes, Egmont Gold and baby carrots during thermal treatment. Enzyme-substrate reaction kinetics of AAO were described using Michaelis-Menten equation. The estimated K(m) and V(max) values of AAO ranged from 50.34 to 63.54 μM and 23.70 to 26.82 μmol/min, respectively. Nantes carrots had the lowest AAO activity. On the other hand, Egmont Gold had the highest V(max). AAO activity in all carrot cultivars was stable up to 50 °C and inactivated above 50 °C. Irreversible thermal inactivation of AAO followed first order kinetics (55-70 °C) and the estimated activation energy of the three carrot cultivars situated between 114.33 and 191.45 kJ/mol. Regarding vitamin C stability, thermal treatment at 60-70 °C has resulted in total conversion of l-AA to DHAA due to residual AAO activity; a complete AAO inactivation was found in 80 °C-treated carrots with high vitamin C retention predominantly in l-AA form, up to 90%. On average, the carrots had a total vitamin C content amounting from 368.24 to 379.87 μg/g dry matter and the Nantes carrots had the highest vitamin C content. The effectiveness of rapid inactivation of endogenous AAO via heating (>80 °C, 10 min) prior to matrix disruption gave protection to l-AA towards enzymatic oxidation, thus resulted in a higher vitamin C content and stability in carrots.

  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. Induction of the d-Amino Acid Oxidase from Trigonopsis variabilis

    PubMed Central

    Horner, R.; Wagner, F.; Fischer, L.

    1996-01-01

    Induction of the d-amino acid oxidase (EC. 1.4.3.3) from the yeast Trigonopsis variabilis was investigated by using a minimal medium containing glucose as the carbon and energy source, (NH(inf4))(inf2)SO(inf4) as the nitrogen source, and various d- and dl-amino acid derivatives as inducers. The best new inducers found were N-carbamoyl-d-alanine, N-acetyl-d-tryptophan, and N-chloroacetyl-d-(alpha)-aminobutyric acid; when the induction effects of these compounds were compared with the effects of d-alanine as the nitrogen source and inducer, the resulting activities of d-amino acid oxidase per gram of dried yeast were 4.2, 2.1, and 1.5 times higher, respectively. The optimum concentration of the best inducer, N-carbamoyl-d-alanine, was 5 mM. This inducer could also be used in its racemic form. The induction was pH dependent. After cultivation of the yeast in a 50-liter bioreactor, d-amino acid oxidase activity of about 3,850 (mu)kat (231,000 U) was obtained. In addition, production of the d-amino acid oxidase was found to be significantly dependent on the metal salt composition of the medium. Addition of zinc ions was required to obtain high d-amino acid oxidase levels in the cells. The optimum concentration of ZnSO(inf4) was about 140 (mu)M. PMID:16535339

  16. Early Effects of Boron Deficiency on Indoleacetic Acid Oxidase Levels of Squash Root Tips

    PubMed Central

    Bohnsack, Charles W.; Albert, Luke S.

    1977-01-01

    The indoleacetic acid (IAA) oxidase activity of root tips of boron-sufficient, -deficient, recovering, and IAA-treated boron-sufficient squash plants (Cucurbita pepo L.) was determined. Apical and subapical root sections displayed an increase in IAA oxidase activity between 6 and 9 hours after boron was withheld, and after 24 hours the activity of the apical sections showed a 20-fold increase over +B controls. Root elongation of -B plants was inhibited before an increase in oxidase activity could be detected. Roots of plants subjected to 12 hours of -B treatment and then transferred to +B treatment for recovery regained normal elongation rates and oxidase activity within 18 to 20 hours. IAA treatment of +B plants increased IAA oxidase activity of apical and subapical root sections and also inhibited root elongation and caused symptoms similar to -B treatments. These results have demonstrated the earliest enzymic change for intact boron-deficient plants. The results are in agreement with the theory that boron deficiency symptoms may be the result of supraoptimal endogenous levels of IAA. These high levels of IAA may inhibit cell division and lead to an induction of the IAA oxidase enzyme. PMID:16659990

  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. Snake venom L-amino acid oxidases: trends in pharmacology and biochemistry.

    PubMed

    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.

  19. 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

  20. 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

  1. Inhibition of apple polyphenol oxidase activity by procyanidins and polyphenol oxidation products.

    PubMed

    Le Bourvellec, Carine; Le Quéré, Jean-Michel; Sanoner, Philippe; Drilleau, Jean-François; Guyot, Sylvain

    2004-01-14

    The rate of consumption of dissolved oxygen by apple polyphenol oxidase in cider apple juices did not correlate with polyphenol oxidase activity in the fruits and decreased faster than could be explained by the decrease of its polyphenolic substrates. The kinetics parameters of a crude polyphenol oxidase extract, prepared from apple (Braeburn cultivar), were determined using caffeoylquinic acid as a substrate. Three apple procyanidin fractions of n 80, 10.5, and 4 were purified from the parenchyma of cider apples of various cultivars. Procyanidins, caffeoylquinic acid, (-)-epicatechin, and a mixture of caffeoylquinic acid and (-)-epicatechin were oxidized by reaction with caffeoylquinic acid o-quinone in order to form oxidation products. All the fractions were evaluated for their inhibitory effect on PPO activity. Native procyanidins inhibited polyphenol oxidase activity, the inhibition intensity increasing with n. The polyphenol oxidase activity decreased by 50% for 0.026 g/L of the fraction of n 80, 0.17 g/L of the fraction of n 10.5, and 1 g/L of the fraction of n 4. The inhibitory effect of oxidized procyanidins was twice that of native procyanidins. Oxidation products of caffeoylquinic acid and (-)-epicatechin also inhibited polyphenol oxidase.

  2. Lysyl oxidase activity in human normal skins and postburn scars.

    PubMed

    Hayakawa, T; Hino, N; Fuyamada, H; Nagatsu, T; Aoyama, H

    1976-09-06

    Lysyl oxidase activity of human normal skins derived from the frontal thighs of 33 subjects showed large variations and the mean value was 11 455 +/- 7 172 (S.D.) cpm/g of wet weight tissue. The age of lesion affected the lysyl oxidase activity in postburn scars. Granulation tissues showed a fairly low activity; however, the activity increased sharply within 2--3 months, and reached a significantly higher value than that of normal skin. The high level of activity continued for up to 2--3 years, then gradually decreased to normal range after 5 years or so. Lysyl oxidase activity was detected only after 4 M urea treatment of tissues. Benzylamine oxidase activity also showed large variations in both normal skins and postburn scars, with mean values of: 0.128 +/- 0.077 (S.D.) and 0.145 +/- 0.090 (S.D.) mmol/g of wet weight/h, respectively. No correlation was observed between lysyl oxidase and benzylamine oxidase activities. The granulation tissues showed significantly high values of benzylamine oxidase activity in contrast to the low values of lysyl oxidase activity.

  3. D-Amino acid oxidase and presence of D-proline in Xenopus laevis.

    PubMed

    Soma, Hiroki; Furuya, Ryuji; Kaneko, Ryo; Tsukamoto, Ayaka; Shirasu, Kazumitsu; Tanigawa, Minoru; Nagata, Yoko

    2013-10-01

    We purified D-amino acid oxidase (EC 1.4.3.3, DAO) from Xenopus laevis tadpoles. The optimal temperature and pH for enzyme activity were 35-40 °C and 8.3-9.0, respectively, depending on the substrate amino acids available to the enzyme; the highest activity was observed with D-proline followed by D-phenylalanine. Activity was significantly inhibited by p-hydroxymercuribenzoate, but only moderately by p-chloromercuribenzoate or benzoate. Enzyme activity was increased until the final tadpole stage, but was reduced to one-third in the adult and was localized primarily in the kidney. The tadpoles contained high concentrations of D-proline close to the final developmental stage and nearly no D-amino acids were detected in the adult frog, indicating that D-amino acid oxidase functions in metamorphosis.

  4. L-Amino acid oxidases from microbial sources: types, properties, functions, and applications.

    PubMed

    Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Liu, Long; Chen, Jian

    2014-02-01

    L-Amino acid oxidases (LAAOs), which catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids and ammonia, are flavin adenine dinucleotide-containing homodimeric proteins. L-Amino acid oxidases are widely distributed in diverse organisms and have a range of properties. Because expressing LAAOs as recombinant proteins in heterologous hosts is difficult, their biotechnological applications have not been thoroughly advanced. LAAOs are thought to contribute to amino acid catabolism, enhance iron acquisition, display antimicrobial activity, and catalyze keto acid production, among other roles. Here, we review the types, properties, structures, biological functions, heterologous expression, and applications of LAAOs obtained from microbial sources. We expect this review to increase interest in LAAO studies.

  5. Nitro-oleic acid ameliorates oxygen and glucose deprivation/re-oxygenation triggered oxidative stress in renal tubular cells via activation of Nrf2 and suppression of NADPH oxidase.

    PubMed

    Nie, Huibin; Xue, Xia; Liu, Gang; Guan, Guangju; Liu, Haiying; Sun, Lina; Zhao, Long; Wang, Xueling; Chen, Zhixin

    2016-01-01

    Nitroalkene derivative of oleic acid (OA-NO2), due to its ability to mediate revisable Michael addition, has been demonstrated to have various biological properties and become a therapeutic agent in various diseases. Though its antioxidant properties have been reported in different models of acute kidney injury (AKI), the mechanism by which OA-NO2 attenuates intracellular oxidative stress is not well investigated. Here, we elucidated the anti-oxidative mechanism of OA-NO2 in an in vitro model of renal ischemia/reperfusion (I/R) injury. Human tubular epithelial cells were subjected to oxygen and glucose deprivation/re-oxygenation (OGD/R) injury. Pretreatment with OA-NO2 (1.25 μM, 45 min) attenuated OGD/R triggered reactive oxygen species (ROS) generation and subsequent mitochondrial membrane potential disruption. This action was mediated via up-regulating endogenous antioxidant defense components including superoxide dismutase (SOD1), heme oxygenase 1 (HO-1), and γ-glutamyl cysteine ligase modulatory subunits (GCLM). Moreover, subcellular fractionation analyses demonstrated that OA-NO2 promoted nuclear translocation of nuclear factor-E2- related factor-2 (Nrf2) and Nrf2 siRNA partially abrogated these protective effects. In addition, OA-NO2 inhibited NADPH oxidase activation and NADPH oxidase 4 (NOX4), NADPH oxidase 2 (NOX2) and p22(phox) up-regulation after OGD/R injury, which was not relevant to Nrf2. These results contribute to clarify that the mechanism of OA-NO2 reno-protection involves both inhibition of NADPH oxidase activity and induction of SOD1, Nrf2-dependent HO-1, and GCLM.

  6. Spinach Thylakoid Polyphenol Oxidase : ISOLATION, ACTIVATION, AND PROPERTIES OF THE NATIVE CHLOROPLAST ENZYME.

    PubMed

    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. The higher molecular weight enzyme is the predominant form in freshly isolated preparations but on aging or further purification, the amount of lower molecular weight enzyme increases at the expense of the higher.Sonication releases polyphenol oxidase from the membrane largely in the latent state. C(18) fatty acids, especially linolenic acid, are potent activators of the enzymic activity. 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. The K(m) values for 3,4-dihydroxyphenylalanine and O(2) are 6.5 and 0.065 millimolar, respectively. 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(m) A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  7. High-level expression of Rhodotorula gracilis D-amino acid oxidase in Pichia pastoris.

    PubMed

    Abad, Sandra; Nahalka, Jozef; Winkler, Margit; Bergler, Gabriele; Speight, Robert; Glieder, Anton; Nidetzky, Bernd

    2011-03-01

    By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.

  8. Apoptosis induction in human breast cancer (MCF-7) cells by a novel venom L-amino acid oxidase (Rusvinoxidase) is independent of its enzymatic activity and is accompanied by caspase-7 activation and reactive oxygen species production.

    PubMed

    Mukherjee, Ashis K; Saviola, Anthony J; Burns, Patrick D; Mackessy, Stephen P

    2015-10-01

    We report the elucidation of a mechanism of apoptosis induction in breast cancer (MCF-7) cells by an L-amino acid oxidase (LAAO), Rusvinoxidase, purified from the venom of Daboia russelii russelii. Peptide mass fingerprinting analysis of Rusvinoxidase, an acidic monomeric glycoprotein with a mass of ~57 kDa, confirmed its identity as snake venom LAAO. The enzymatic activity of Rusvinoxidase was completely abolished after two cycles of freezing and thawing; however, its cytotoxicity toward MCF-7 cells remained unaffected. Dose- and time-dependent induction of apoptosis by Rusvinoxidase on MCF-7 cells was evident from changes in cell morphology, cell membrane integrity, shrinkage of cells and apoptotic body formation accompanied by DNA fragmentation. Rusvinoxidase induced apoptosis in MCF-7 cells by both the extrinsic (death-receptor) and intrinsic (mitochondrial) signaling pathways. The former pathway of apoptosis operated through activation of caspase-8 that subsequently activated caspase-7 but not caspase-3. Rusvinoxidase-induced intrinsic pathway of apoptosis was accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species, followed by a decrease in cellular glutathione content and catalase activity, and down-regulation of expression of anti-apoptotic proteins Bcl-XL and heat-shock proteins (HSP-90 and HSP-70). Rusvinoxidase treatment resulted in increase of the pro-apoptotic protein Bax, subsequently leading to the release of cytochrome c from mitochondria to the cytosol and activating caspase-9, which in turn stimulated effector caspase-7. Rusvinoxidase at a dose of 4 mg/kg was non-toxic in mice, indicating that it may be useful as a model for the development of peptide-based anticancer drugs.

  9. 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.

  10. 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

  11. Excess boron reduces polyphenol oxidase activities in embryo and endosperm of maize seed during germination.

    PubMed

    Olçer, Hillya; Kocaçaliskan, Ismail

    2007-01-01

    The effects of increasing concentrations of boron (0, 0.1, 1, 10 and 20 mM) as boric acid on the rate of germination and polyphenol oxidase activities in embryo and endosperm tissues of maize seeds (Zea mays L. cv. Arifiye) were studied. The germination percentage of maize seeds was not affected by boron concentrations up to 10 mM, and decreased by 20 mM. Distilled water and lower boron concentrations (0.1 and 1 mM) increased polyphenol oxidase activities at the beginning of germination up to 12 h whereas its excess levels (10 and 20 mM) decreased polyphenol oxidase activities in embryos and endosperm during germination. Polyphenol oxidase activities with o-diphenolic substrates (caffeic acid, catechol and dopa) were found to be higher than with a monophenolic substrat (tyrosine) in both embryos and endosperms. Further, caffeic acid oxidizing polyphenol oxidase was found to show more activity in embryos of the seeds germinating in distilled water when compared to other substrates.

  12. 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.

  13. 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.

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

    PubMed

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

    2002-12-04

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

  15. 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.

  16. Evidence for the involvement of GPR40 and NADPH oxidase in palmitic acid-induced superoxide production and insulin secretion.

    PubMed

    Graciano, Maria Fernanda; Valle, Maíra Mello; Curi, Rui; Carpinelli, Angelo Rafael

    2013-01-01

    G protein coupled receptor 40 (GPR40) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex have been shown to be involved in the fatty acid amplification of glucose-stimulated insulin secretion (GSIS). The effect of palmitic acid on superoxide production and insulin secretion by INS-1E cells and the possible involvement of GPR40 and NADPH oxidase in these processes were examined in this study. Cells were incubated during 1 h with palmitic acid in low and high glucose concentrations, a GPR40 agonist (GW9508) and inhibitors of NADPH oxidase (diphenyleneiodonium, DPI) and PKC (calphostin C). GW9508 induced superoxide production at 2.8 and 5.6 mM glucose concentrations and stimulated insulin secretion at 16.7 mM glucose concentration involving both PKC and NADPH oxidase activation. Palmitic acid induced superoxide production through NADPH oxidase and GPR40-dependent pathways and the stimulation of insulin secretion in the presence of a high glucose concentration was reduced by knockdown of GPR40 using siRNA. Our results suggest that palmitic acid induces superoxide production and potentiates GSIS through NADPH oxidase and GPR40 pathways in pancreatic ? cells.

  17. Rapid deactivation of NADPH oxidase in neutrophils: continuous replacement by newly activated enzyme sustains the respiratory burst.

    PubMed

    Akard, L P; English, D; Gabig, T G

    1988-07-01

    The cell-free system for activation of the neutrophil NADPH oxidase allowed us to examine activation of the oxidase in the absence of its NADPH-dependent turnover. The covalent sulfhydryl-modifying reagent N-ethylmaleimide completely inhibited the activation step (Ki = 40 mumol/L) in the cell-free system but had no effect on turnover of the preactivated particulate NADPH oxidase (up to 1 mmol/L). When N-ethylmaleimide was added to intact neutrophils during the period of maximal O2 generation in response to stimuli that activate the respiratory burst (phorbol myristate acetate, f-Met-Leu-Phe, opsonized zymosan, arachidonic acid), O2- generation ceased within seconds. Study of components of the cell-free activation system indicated that the cytosolic cofactor was irreversibly inhibited by N-ethylmaleimide whereas the N-ethylmaleimide-treated, membrane-associated oxidase could be activated by arachidonate and control cytosolic cofactor. Likewise, the cell-free system prepared from intact neutrophils that had been briefly exposed to N-ethylmaleimide and then washed reflected the effects of N-ethylmaleimide on the isolated cell-free components: cytosolic cofactor activity was absent, but the membrane oxidase remained fully activatable. Thus inhibition of oxidase activation by N-ethylamaleimide unmasked a rapid deactivation step that was operative in intact neutrophils but not in isolated particulate NADPH oxidase preparations. The demonstrated specificity of N-ethylmaleimide for oxidase activation and lack of effect on turnover of the NADPH oxidase suggested that sustained O2- generation by intact neutrophils was a result of continued replenishment of a small pool of active oxidase. The existence of an inactive pool of NADPH oxidase molecules in particulate preparations from stimulated neutrophils was supported more directly by activating these preparations again in the cell-free system.

  18. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity.

    PubMed

    Peng, Zeyu; Dittmer, Neal T; Lang, Minglin; Brummett, Lisa M; Braun, Caroline L; Davis, Lawrence C; Kanost, Michael R; Gorman, Maureen J

    2015-04-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism.

  19. 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

  20. 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

  1. CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity.

    PubMed

    Su, Jianmei; Bao, Peng; Bai, Tenglong; Deng, Lin; Wu, Hui; Liu, Fan; He, Jin

    2013-01-01

    Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10(-6)±0.21 M·min(-1) and 0.32±0.02 s(-1), respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a

  2. Isolation of oxalic acid tolerating fungi and decipherization of its potential to control Sclerotinia sclerotiorum through oxalate oxidase like protein.

    PubMed

    Yadav, Shivani; Srivastava, Alok K; Singh, Dhanajay P; Arora, Dilip K

    2012-11-01

    Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10 mM of oxalic acid whereas only 15 isolates were grow at 50 mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23 %) after 60 min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents.

  3. 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...

  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. 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.

  6. 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...

  7. [L-lysine-alpha-oxidase activity of some Trichoderma species].

    PubMed

    Smirnova, I P; Khaduev, S Kh

    1984-01-01

    Trichoderma cultures were tested for their ability to produce L-lysine-alpha-oxidase. The highest enzyme activity was manifested by T. harzianum (MGU), T. longibrachiatum Rifai VKM F-2025 and T. aureoviride Rifai VKM F-2026. The biosynthesis of the enzyme did not depend on the growth of the cultures and did not vary among the species.

  8. Dopa oxidase activity and ceruloplasmin in the sera of hamsters with melanoma.

    PubMed

    Vachtenheim, J; Pavel, S; Duchon, J

    1981-01-01

    Two simple spectrophotometric assays have been employed for the measurement of dopa oxidase activity and ceruloplasmin polyphenol oxidase activity in the sera from normal hamsters and hamsters bearing melanotic melanoma. Both activities were found to be augmented in tumor animals, the dopa oxidase activity much more prominently. The levels of the enzymes tested increased proportionally to the tumor mass.

  9. Synthesis, crystal structures, molecular docking, in vitro monoamine oxidase-B inhibitory activity of transition metal complexes with 2-{4-[bis (4-fluorophenyl)methyl]piperazin-1-yl} acetic acid

    NASA Astrophysics Data System (ADS)

    Yang, Dan-dan; Wang, Riu; Zhu, Jin-long; Cao, Qi-yue; Qin, Jie; Zhu, Hai-liang; Qian, Shao-song

    2017-01-01

    Three novel complexes, [Cu(L)2(H2O)](1), [Zn(L)2(H2O)2]·CH3OH·1.5H2O(2), and [Ni(L)2(H2O)1.8]·CH3OH·1.2H2O (3) (HL = 2-{4-[bis(4-fluorophenyl)methyl]pipera-zin-1-yl} acetic acid), were synthesized and structurally determined by single-crystal X-ray diffraction. Molecular docking study preliminarily revealed that complex 1 had potential Monoamine oxidase B inhibitory activity. All acquired compounds were tested against rat brain MAO-B in vitro. In accordance with the result of calculation, it showed complex 1 (IC50 = 1.85 ± 0.31 μM) have good inhibitory activity against MAO-B at the same micromolar concentrations with positive control Iproniazid Phosphate (IP, IC50 = 7.59 ± 1.17 μM). These results indicated that complex 1 was a potent MAO-B inhibitor.

  10. 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.

  11. Deletion of glucose oxidase changes the pattern of organic acid production in Aspergillus carbonarius

    PubMed Central

    2014-01-01

    Aspergillus carbonarius has potential as a cell factory for the production of different organic acids. At pH 5.5, A.carbonarius accumulates high amounts of gluconic acid when it grows on glucose based medium whereas at low pH, it produces citric acid. The conversion of glucose to gluconic acid is carried out by secretion of the enzyme, glucose oxidase. In this work, the gene encoding glucose oxidase was identified and deleted from A. carbonarius with the aim of changing the carbon flux towards other organic acids. The effect of genetic engineering was examined by testing glucose oxidase deficient (Δgox) mutants for the production of different organic acids in a defined production medium. The results obtained showed that the gluconic acid accumulation was completely inhibited and increased amounts of citric acid, oxalic acid and malic acid were observed in the Δgox mutants. PMID:25401063

  12. 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

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

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-08

    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.

  14. Amine oxidase activity regulates the development of pulmonary fibrosis.

    PubMed

    Marttila-Ichihara, Fumiko; Elima, Kati; Auvinen, Kaisa; Veres, Tibor Z; Rantakari, Pia; Weston, Christopher; Miyasaka, Masayuki; Adams, David; Jalkanen, Sirpa; Salmi, Marko

    2017-03-01

    In pulmonary fibrosis, an inflammatory reaction and differentiation of myofibroblasts culminate in pathologic deposition of collagen. Amine oxidase copper containing-3 (AOC3) is a cell-surface expressed oxidase that regulates leukocyte extravasation. Here we analyzed the potential role of AOC3 using gene-modified and inhibitor-treated mice in a bleomycin-induced pulmonary fibrosis model. Inflammation and fibrosis of lungs were assessed by histologic, flow cytometric, and quantitative PCR analysis. AOC3-deficient mice showed a 30-50% reduction in fibrosis, collagen synthesis, numbers of myofibroblasts, and accumulation of CD4(+) lymphocytes, NK T cells, macrophages, and type 2 innate lymphoid cells compared with wild-type control mice. AOC3 knock-in mice, which express a catalytically inactive form of AOC3, were also protected from lung fibrosis. In wild-type mice, a small-molecule AOC3 inhibitor treatment reduced leukocyte infiltration, myofibroblast differentiation, and fibrotic injury both in prophylactic and early therapeutic settings by about 50% but was unable to reverse the established fibrosis. AOC3 was also induced in myofibroblasts in human idiopathic pulmonary fibrosis. Thus, the oxidase activity of AOC3 contributes to the development of lung fibrosis mainly by regulating the accumulation of pathogenic leukocyte subtypes, which drive the fibrotic response.-Marttila-Ichihara, F., Elima, K., Auvinen, K., Veres, T. Z., Rantakari, P., Weston, C., Miyasaka, M., Adams, D., Jalkanen, S., Salmi, M. Amine oxidase activity regulates the development of pulmonary fibrosis.

  15. 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.

  16. 4-Hydroxyanisole: the most suitable monophenolic substrate for determining spectrophotometrically the monophenolase activity of polyphenol oxidase from fruits and vegetables.

    PubMed

    Espín, J C; Tudela, J; García-Cánovas, F

    1998-05-15

    A continuous spectrophotometric method for determining the monophenolase activity of polyphenol oxidase from several plant sources is described. This assay method is based on the coupling reaction between 3-methyl-2-benzothiazolinone hydrazone and the quinone product of the oxidation of 4-hydroxyanisole in the presence of polyphenol oxidase. 4-Hydroxyanisole proved to be the best monophenol assayed to measure the monophenolase activity of polyphenol oxidase from apple, artichoke, avocado, medlar, pear, and strawberry. Kinetic constants of 4-hydroxyanisole were compared to those of p-hydroxyphenyl propionic acid, a very sensitive monophenol previously reported to assay the monophenolase activity of polyphenol oxidase from apple, pear, and mushroom. The high values of the maximum steady state rate obtained for 4-hydroxyanisole suggest the existence of high catalytic constant toward this monophenol. These kinetic values were supported by nuclear magnetic resonance assays which predicted the highest reactivity of 4-hydroxyanisole. Therefore nuclear magnetic resonance assays proved to be a valuable and useful tool to predict the best monophenolic substrate for plant polyphenol oxidases. The 3-methyl-2-benzothiazlolinone-adduct for 4-hydroxyanisole was stable, with high molar absorptivity at the optimum pHs of the polyphenol oxidases assayed. All this together makes the use of 4-hydroxyanisol as monophenolic substrate and 3-methyl-2-benzothiazolinone as coupling reagent the most sensitive and precise assay method up to date reported in the literature to determine the monophenolas activity of polyphenol oxidase from fruits and vegetables.

  17. Nanoparticle strategies for cancer therapeutics: Nucleic acids, polyamines, bovine serum amine oxidase and iron oxide nanoparticles (Review).

    PubMed

    Agostinelli, Enzo; Vianello, Fabio; Magliulo, Giuseppe; Thomas, Thresia; Thomas, T J

    2015-01-01

    Nanotechnology for cancer gene therapy is an emerging field. Nucleic acids, polyamine analogues and cytotoxic products of polyamine oxidation, generated in situ by an enzyme-catalyzed reaction, can be developed for nanotechnology-based cancer therapeutics with reduced systemic toxicity and improved therapeutic efficacy. Nucleic acid-based gene therapy approaches depend on the compaction of DNA/RNA to nanoparticles and polyamine analogues are excellent agents for the condensation of nucleic acids to nanoparticles. Polyamines and amine oxidases are found in higher levels in tumours compared to that of normal tissues. Therefore, the metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine, can be targets for antineoplastic therapy since these naturally occurring alkylamines are essential for normal mammalian cell growth. Intracellular polyamine concentrations are maintained at a cell type-specific set point through the coordinated and highly regulated interplay between biosynthesis, transport, and catabolism. In particular, polyamine catabolism involves copper-containing amine oxidases. Several studies showed an important role of these enzymes in developmental and disease-related processes in animals through the control of polyamine homeostasis in response to normal cellular signals, drug treatment, and environmental and/or cellular stress. The production of toxic aldehydes and reactive oxygen species (ROS), H2O2 in particular, by these oxidases suggests a mechanism by which amine oxidases can be exploited as antineoplastic drug targets. The combination of bovine serum amine oxidase (BSAO) and polyamines prevents tumour growth, particularly well if the enzyme has been conjugated with a biocompatible hydrogel polymer. The findings described herein suggest that enzymatically formed cytotoxic agents activate stress signal transduction pathways, leading to apoptotic cell death. Consequently, superparamagnetic nanoparticles or other

  18. In Situ Click Chemistry for the Identification of a Potent D-Amino Acid Oxidase Inhibitor.

    PubMed

    Toguchi, Shohei; Hirose, Tomoyasu; Yorita, Kazuko; Fukui, Kiyoshi; Sharpless, K Barry; Ōmura, Satoshi; Sunazuka, Toshiaki

    2016-07-01

    In situ click chemistry is a target-guided synthesis approach for discovering novel lead compounds by assembling organic azides and alkynes into triazoles inside the affinity site of target biogenic molecules such as proteins. We report in situ click chemistry screening with human D-amino acid oxidase (hDAO), which led to the identification of a more potent hDAO inhibitor. The hDAO inhibitors have chemotherapeutic potential as antipsychotic agents. The new inhibitor displayed competitive inhibition of hDAO and showed significantly increased inhibitory activity against hDAO compared with that of an anchor molecule of in situ click chemistry.

  19. A continuous spectrophotometric method for determining the monophenolase and diphenolase activities of apple polyphenol oxidase.

    PubMed

    Espín, J C; Morales, M; Varón, R; Tudela, J; García-Cánovas, F

    1995-10-10

    A continuous spectrophotometric method for the determination of the monophenolase and diphenolase activities of apple polyphenol oxidase is described. The method is based on the coupling reaction between 3-methyl-2-benzothiazolinone hydrazone (MBTH) and the quinone product of the oxidation of p-hydroxyphenyl propionic acid and 3,4-dihydroxyphenyl propionic acid in the presence of polyphenol oxidase. The lambda(max) and molar absorptivity (epsilon) for the MBTH-quinone adduct have been calculated. The presence of MBTH in the reaction medium decreases the lag period during the expression of monophenolase activity. The high value of V(mas) suggests the existence of a high catalytic constant. This, together with the value of epsilon for the MBTH-quinone adduct, makes this method more sensitive than other continuous methods.

  20. Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

    NASA Astrophysics Data System (ADS)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei

    2016-03-01

    Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na2SeO3 as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant ( K m) values and maximal reaction velocity ( V max) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

  1. 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).

  2. Formate simultaneously reduces oxidase activity and enhances respiration in Campylobacter jejuni

    PubMed Central

    Kassem, Issmat I.; Candelero-Rueda, Rosario A.; Esseili, Kawthar A.; Rajashekara, Gireesh

    2017-01-01

    The foodborne microaerophilic pathogen, Campylobacter jejuni, possesses a periplasmic formate dehydrogenase and two terminal oxidases, which serve to metabolize formate and facilitate the use of oxygen as a terminal electron acceptor, respectively. Formate, a primary energy source for C. jejuni, inhibits oxidase activity in other bacteria. Here, we hypothesized that formate might affect both energy metabolism and microaerobic survival in C. jejuni. Subsequently, we showed that C. jejuni 81–176 (wildtype) exhibited enhanced chemoattraction to and respiration of formate in comparison to other organic acids. Formate also significantly increased C. jejuni’s growth, motility, and biofilm formation under microaerobic (5% O2) conditions. However, formate reduced oxidase activity under microaerobic conditions as well as aerotolerance and biofilm formation under ambient oxygen conditions. The expression of genes encoding the ribonucleotide reductase (RNR) and proteins that facilitate the use of alternative electron acceptors generally increased in the presence of formate. Taken together, formate might play a role in optimizing C. jejuni’s adaptation to the oxygen-limited gastrointestinal tract of the host. By affecting oxidase activity, formate possibly facilitates shuttling electrons to alternative acceptors, while likely conserving limited oxygen concentrations for other essential functions such as DNA synthesis via RNR which is required for C. jejuni’s growth. PMID:28091524

  3. Spectroscopic studies of the active site of galactose oxidase

    SciTech Connect

    Knowles, P.F.; Brown, R.D. III; Koenig, S.H.

    1995-07-19

    X-ray absorption and EPR spectroscopy have been used to probe the copper site structure in galactose oxidase at pH 4.5 and 7.0. the results suggest that there are no major differences in the structure of the tetragonal Cu(II) site at these pH values. Analysis of the extended X-ray absorption fine structure (EXAFS) indicates that four N,O scatterers are present at approximately 2 {Angstrom}; these are presumably the equatorial ligands. In addition, the EXAFS data establish that oxidative activation to produce the active-site tyrosine radical does not cause major changes in the copper coordination environment. Therefore results obtained on the one-electron reduced enzyme, containing Cu(II) but not the tyrosine radical, probably also apply to the catalytically active Cu(II)/tyrosine radical state. Solvent water exchange, inhibitor binding, and substrate binding have been probed via nuclear magnetic relaxation dispersion (NMRD) measurements. The NMRD profile of galactose oxidase is quantitatively consistent with the rapid exchange of a single, equatorial water ligand with a Cu(II)-O separation of about 2.4 {Angstrom}. Azide and cyanide displace this coordinated water. The binding of azide and the substrate dihydroxyacetone produce very similar effects on the NMRD profile of galactose oxidase, indicating that substrates also bind to the active site Cu(II) in an equatorial position.

  4. Effects of salicylic acid on alternative pathway respiration and alternative oxidase expression in tobacco calli.

    PubMed

    Lei, Tao; Yan, Ying-Cai; Xi, De-Hui; Feng, Hong; Sun, Xin; Zhang, Fan; Xu, Wei-Lin; Liang, Hou-Guo; Lin, Hong-Hui

    2008-01-01

    The alternative pathway (AP) respiration of plants is a cyanide-resistant and non-phosphorylating electron transport pathway in mitochondria. Alternative oxidase (AOX) is the terminal oxidase of the AP and exists in plant mitochondria as two states: the reduced, noncovalently linked state or the oxidized, covalently cross-linked state. In the present study, the effects of 20 microM exogenous salicylic acid (SA) on both AP activity and AOX expression in mitochondria of tobacco (Nicotiana rustica L. cv. yellow flower) calli were investigated. The results showed that SA treatment enhanced the AP activity. During the process of SA treatment, the AP activity increased dramatically and achieved the peak value after 8 h of treatment. Then it declined until 16 h, and maintained a steady level between 16 and 24 h. Changes in both the total AOX protein level and the reduced state were in accordance with the AP activity, but the oxidized state changed differently. The aox1 gene transcript level also showed a similar change as the AP activity and AOX protein level. The induction of AOX expression by low concentrations of SA was inferred through a reactive oxygen species (ROS)-independent pathway. These results indicate that the enhancement of AP activity in response to SA is correlated to the expression of AOX, and the reduced, non-covalently linked state of AOX plays an important role during this process.

  5. A novel D-amino acid oxidase from a contaminated agricultural soil metagenome and its characterization.

    PubMed

    Ou, Qian; Liu, Yao; Deng, Jie; Chen, Gao; Yang, Ying; Shen, Peihong; Wu, Bo; Jiang, Chengjian

    2015-06-01

    A novel D-amino acid oxidase (DAAO) gene designated as daoE was cloned by the sequence-based screening of a plasmid metagenomic library of uncultured microorganisms from contaminated agricultural soil. The deduced amino acid sequence comparison and phylogenetic analysis indicated that daoE and other putative DAAOs are closely related. The putative DAAO gene was subcloned into a pETBlue-2 vector and overexpressed in Escherichia coli Tunner(DE3)pLacI. The recombinant protein was purified to homogeneity. The maximum activity of DaoE protein occurred at pH 8.0 and 37 °C. DaoE recombinant protein had an apparent K m of 2.96 mM, V max of 0.018 mM/min, k cat of 10.9/min, and k cat/K m of 1.16 × 10(4)/mol/min. The identification of this novel DAAO gene demonstrated the importance of metagenomic libraries in exploring new D-amino acid oxidases from environmental microorganisms to optimize their applications.

  6. D-amino acid oxidase: its potential in the production of 7-aminocephalosporanic acid.

    PubMed

    Mujawar, S K

    1999-01-01

    D-Amino acid oxidase (DAAO) used in the preparation of alpha-keto acids, in the determination of D-amino acids and in the resolution of racemic mixture of amino acids is produced by a wide range of microorganisms. In the recent past this enzyme is being recognized for its potential in the commercial production of 7-aminocephalosporanic acid (7-ACA), a starting material for various semisynthetic cephalosporins. Though this enzyme is widespread among microorganisms, very few microbial species have been explored for the production of 7-ACA; this is because cephalosporin C is quantitatively deaminated by limited microbial DAAOs. Comparison of physico-chemical properties of enzyme preparations indicate wide variations, however in general DAAOs are specific for D-configuration of amino acids. Both immobilized enzyme and cell preparations are developed for its various applications. The advantages of DAAO in the production of 7-ACA are discussed.

  7. Genipin Cross-Linked Glucose Oxidase and Catalase Multi-enzyme for Gluconic Acid Synthesis.

    PubMed

    Cui, Caixia; Chen, Haibin; Chen, Biqiang; Tan, Tianwei

    2017-02-01

    In this work, glucose oxidase (GOD) and catalase (CAT) were used simultaneously to produce gluconic acid from glucose. In order to reduce the distance between the two enzymes, and therefore improve efficiency, GOD and CAT were cross-linked together using genipin. Improvements in gluconic acid production were due to quick removal of harmful intermediate hydrogen peroxide by CAT. GOD activity was significantly affected by the proportion of CAT in the system, with GOD activity in the cross-linked multi-enzyme (CLME) being 10 times higher than that in an un-cross-linked GOD/CAT mixture. The glucose conversion rate after 15 h using 15 % glucose was also 10 % higher using the CLME than was measured using a GOD/CAT mixture.

  8. 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.

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

    PubMed

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

    2014-05-01

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

  10. 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.

  11. 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.

  12. 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.

  13. Studies on polyphenol content, activities and isozymes of polyphenol oxidase and peroxidase during air-curing in three tobacco types.

    PubMed

    Sheen, S J; Calvert, J

    1969-02-01

    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.

  14. [Enzymatic production of α-ketoglutaric acid by L-glutamate oxidase from L-glutamic acid].

    PubMed

    Niu, Panqing; Zhang, Zhenyu; Liu, Liming

    2014-08-01

    We produced α-ketoglutaric acid (α-KG) from L-glutamic acid, using enzymatic transformation approach with L-glutamate oxidase (LGOX). First, wild strain Streptomyces sp. FMME066 was mutated with NTG, a genetically stable mutant Streptomyces sp. FMME067 was obtained. Under the optimal nutrition conditions with fructose 10 g/L, peptone 7.5 g/L, KH2PO4 1 g/L and CaCl2 0.05 g/L, the maximum LGOX activity reached 0.14 U/mL. The LGOX was stable to pH and temperature, and Mn2+ had a stimulating effect. Finally, after 24 h enzymatic conversion under the optimal conditions, the maximum titer of α-KG reached 38.1 g/L from 47 g/L L-glutamic acid. Enzymatic transformation by LGOX is a potential approach for α-KG production.

  15. Discovery of pyrazole carboxylic acids as potent inhibitors of rat long chain L-2-hydroxy acid oxidase.

    PubMed

    Barawkar, Dinesh A; Bandyopadhyay, Anish; Deshpande, Anil; Koul, Summon; Kandalkar, Sachin; Patil, Pradeep; Khose, Goraksha; Vyas, Samir; Mone, Mahesh; Bhosale, Shubhangi; Singh, Umesh; De, Siddhartha; Meru, Ashwin; Gundu, Jayasagar; Chugh, Anita; Palle, Venkata P; Mookhtiar, Kasim A; Vacca, Joseph P; Chakravarty, Prasun K; Nargund, Ravi P; Wright, Samuel D; Roy, Sophie; Graziano, Michael P; Cully, Doris; Cai, Tian-Quan; Singh, Sheo B

    2012-07-01

    Long chain L-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2.

  16. Purification and characterization of L-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation.

    PubMed

    Li, Z Y; Yu, T F; Lian, E C

    1994-11-01

    Venoms of several snake species contain large amounts of L-amino acid oxidase but its effects on human plasma coagulation and platelet aggregation have not been explored. We have purified L-amino acid oxidase from king cobra venom through CM-Sephadex C-25, Sephadex G-100 and DEAE Sephadex A-50 chromatographies. The purified enzyme has a mol. wt of 135,000 as determined by gel filtration and 65,000 by SDS-PAGE under non-reducing and reducing conditions. Incubation of plasma with L-amino acid oxidase at 200 micrograms/ml did not affect prothrombin time, activated partial thromboplastin time, or thrombin time. Upon addition of L-amino acid oxidase, platelets in platelet-rich plasma were aggregated. The enzyme-induced aggregation was abolished by catalase. The aggregation was also inhibited by indomethacin, aspirin, ethylenediaminetetraacetate, sodium nitroprusside, prostaglandin E1, mepacrine and verapamil, but not by heparin, hirudin, creatine phosphate/creatine phosphokinase or antimycin/2-deoxy-D-glucose. These results suggest that L-amino acid oxidase induces human platelet aggregation through the formation of H2O2, and subsequent thromboxane A2 synthesis requiring Ca2+ but independent of ADP release. The platelet aggregation caused by L-amino acid oxidase is likely to contribute to toxicity inflicted by cobra venom.

  17. Myeloperoxidase amplified high glucose-induced endothelial dysfunction in vasculature: Role of NADPH oxidase and hypochlorous acid.

    PubMed

    Tian, Rong; Ding, Yun; Peng, Yi-Yuan; Lu, Naihao

    2017-03-11

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H2O2), have emerged as important molecules in the pathogenesis of diabetic endothelial dysfunction. Additionally, neutrophils-derived myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) play important roles in the vascular injury. However, it is unknown whether MPO can use vascular-derived ROS to induce diabetic endothelial dysfunction. In the present study, we demonstrated that NADPH oxidase was the main source of ROS formation in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and played a critical role in high glucose-induced endothelial dysfunction such as cell apoptosis, loss of cell viability and reduction of nitric oxide (NO). However, the addition of MPO could amplify the high glucose-induced endothelial dysfunction which was inhibited by the presence of apocynin (NADPH oxidase inhibitor), catalase (H2O2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H2O2-MPO-HOCl pathway in the MPO/high glucose-induced vascular injury. In high glucose-incubated rat aortas, MPO also exacerbated the NADPH oxidase-induced impairment of endothelium-dependent relaxation. Consistent with these in vitro data, in diabetic rat aortas, both MPO expresion and NADPH oxidase activity were increased while the endothelial function was simultaneously impaired. The results suggested that vascular-bound MPO could amplify high glucose-induced vascular injury in diabetes. MPO-NADPH oxidase-HOCl may represent an important pathogenic pathway in diabetic vascular diseases.

  18. 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.

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

    PubMed

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

    2014-10-06

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

  20. Decavanadate inhibits the cell-free activation of neutrophil NADPH oxidase without affecting tyrosine phosphorylation.

    PubMed

    Okamura, N; Sakai, T; Nishimura, Y; Sakai, M; Araki, S; Yamaguchi, M; Ishibashi, S

    1999-08-01

    NADPH oxidase was activated by arachidonate in a cell-free system consisting of membrane and cytosol fractions prepared from guinea pig neutrophils. Vanadate apparently inhibited the NADPH oxidase activity in the cell-free system (IC50=2 microM) without phosphotyrosine accumulation. The pH dependency and stability of the inhibitory effect observed for vanadate solution indicated that decavanadate, an isopolyanion of vanadate, was responsible for the inhibition. Pervanadate (vanadyl hydroperoxide) also inhibited the oxidase activity but at a higher concentration (IC50=0.2 mM). Decavanadate lowered the Vmax but did not affect the Km value of NADPH oxidase for NADPH. Decavanadate inhibited the activation process of NADPH oxidase but not the oxidase activity itself. Decavanadate-pretreatment of membrane and cytosol fractions irreversibly decreased the abilities of both fractions to activate NADPH oxidase in the cell-free system. Translocation of p47-phox, one of the cytosolic activation factors of NADPH oxidase, from cytosol to membrane, was little affected by decavanadate. These results suggest that decavanadate inhibits the activation of NADPH oxidase in the cell-free system without affecting the phosphotyrosine phosphatase, and that decavanadate can bind to both the membrane and cytosolic activation factors when they are in a dormant state, but not to the active oxidase complex.

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

    PubMed

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

    2016-03-01

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

  2. 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.

  3. Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein.

    PubMed

    Cacas, Jean-Luc; Gerbeau-Pissot, Patricia; Fromentin, Jérôme; Cantrel, Catherine; Thomas, Dominique; Jeannette, Emmanuelle; Kalachova, Tetiana; Mongrand, Sébastien; Simon-Plas, Françoise; Ruelland, Eric

    2017-04-01

    Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [(33) P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.

  4. Recombinant expression and characterization of a L-amino acid oxidase from the fungus Rhizoctonia solani.

    PubMed

    Hahn, Katharina; Neumeister, Katrin; Mix, Andreas; Kottke, Tilman; Gröger, Harald; Fischer von Mollard, Gabriele

    2017-04-01

    L-Amino acid oxidases (L-AAOs) catalyze the oxidative deamination of L-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. L-AAOs are homodimeric enzymes with FAD as a non-covalently bound cofactor. They are of potential interest for biotechnological applications. However, heterologous expression has not succeeded in producing large quantities of active recombinant L-AAOs with a broad substrate spectrum so far. Here, we report the heterologous expression of an active L-AAO from the fungus Rhizoctonia solani in Escherichia coli as a fusion protein with maltose-binding protein (MBP) as a solubility tag. After purification, it was possible to remove the MBP-tag proteolytically without influencing the enzyme activity. MBP-rsLAAO1 and 9His-rsLAAO1 converted basic and large hydrophobic L-amino acids as well as methyl esters of these L-amino acids. The progress of the conversion of L-phenylalanine and L-leucine into the corresponding α-keto acids was determined by HPLC and (1)H-NMR analysis of reaction mixtures, respectively. Enzymatic activity was stimulated 50-100-fold by SDS treatment. K m values ranging from 0.9-10 mM and v max values from 3 to 10 U mg(-1) were determined after SDS activation of 9His-rsLAAO1 for the best substrates. The enzyme displayed a broad pH optimum between pH 7.0 and 9.5. In summary, a successful overexpression of recombinant L-AAO in E. coli was established that results in a promising enzymatic activity and a broad substrate spectrum for biotechnological application.

  5. 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

  6. 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.

  7. Contributions of spinal D-amino acid oxidase to chronic morphine-induced hyperalgesia.

    PubMed

    Ma, Shuai; Li, Xin-Yan; Gong, Nian; Wang, Yong-Xiang

    2015-12-10

    Spinal D-amino acid oxidase (DAAO) is an FAD-dependent peroxisomal flavoenzyme which mediates the conversion of neutral and polar D-amino acids (including D-serine) to the corresponding α-keto acids, and simultaneously produces hydrogen peroxide and ammonia. This study has aimed to explore the potential contributions of spinal DAAO and its mediated hydrogen peroxide/D-serine metabolism to the development of morphine-induced hyperalgesia. Bi-daily subcutaneous injections of morphine to mice over 7 days induced thermal hyperalgesia as measured by both the hot-plate and tail-immersion tests, and spinal astroglial activation with increased spinal gene expression of DAAO, glial fibrillary acidic protein (GFAP) and pro-inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)). Subcutaneous injections of the potent DAAO inhibitor CBIO (5-chloro-benzo[D]isoxazol-3-ol) prevented and reversed the chronic morphine-induced hyperalgesia. CBIO also inhibited both astrocyte activation and the expression of pro-inflammatory cytokines. Intrathecal injection of the hydrogen peroxide scavenger PBN (phenyl-N-tert-butylnitrone) and of catalase completely reversed established morphine hyperalgesia, whereas subcutaneous injections of exogenous D-serine failed to alter chronic morphine-induced hyperalgesia. These results provided evidence that spinal DAAO and its subsequent production of hydrogen peroxide rather than the D-serine metabolism contributed to the development of morphine-induced hyperalgesia.

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

    PubMed

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

    2015-12-17

    The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression.

  9. 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...

  10. Enzymatic production of α-ketoglutaric acid from l-glutamic acid via l-glutamate oxidase.

    PubMed

    Niu, Panqing; Dong, Xiaoxiang; Wang, Yuancai; Liu, Liming

    2014-06-10

    In this study, a novel strategy for α-ketoglutaric acid (α-KG) production from l-glutamic acid using recombinant l-glutamate oxidase (LGOX) was developed. First, by analyzing the molecular structure characteristics of l-glutamic acid and α-KG, LGOX was found to be the best catalyst for oxidizing the amino group of l-glutamic acid to a ketonic group without the need for exogenous cofactor. Then the LGOX gene was expressed in Escherichia coli BL21 (DE3) in a soluble and active form, and the recombinant LGOX activity reached to a maximum value of 0.59U/mL at pH 6.5, 30°C. Finally, the maximum α-KG concentration reached 104.7g/L from 110g/L l-glutamic acid in 24h, under the following optimum conditions: 1.5U/mL LGOX, 250U/mL catalase, 3mM MnCl2, 30°C, and pH 6.5.

  11. Identification of the Atlantic cod L-amino acid oxidase and its alterations following bacterial exposure.

    PubMed

    Kitani, Yoichiro; Fernandes, Jorge M O; Kiron, Viswanath

    2015-06-01

    Antibacterial factors that are present in epidermal mucus of fish have a potential role in the first line of host defence to bacterial pathogens. This study reports the identification of L-amino acid oxidase (LAO) in Atlantic cod (GmLao) and the changes in the molecule following bacterial exposure. The gmlao transcripts and LAO activity were present on both the body surface and in the internal organs of the fish. Relative mRNA level of gmlao increased significantly in the gills, the spleen and the head kidney (up to 8-fold) of fish that were challenged with the pathogen Vibrio anguillarum. The gmlao expression in skin was 4-fold higher in challenged fish. Our data indicate that LAO may be an important effector of antibacterial defence in Atlantic cod.

  12. 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.

  13. 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

  14. Synthesis and evaluation of quinazoline amino acid derivatives as mono amine oxidase (MAO) inhibitors.

    PubMed

    Khattab, Sherine Nabil; Haiba, Nesreen Saied; Asal, Ahmed Mosaad; Bekhit, Adnan A; Amer, Adel; Abdel-Rahman, Hamdy M; El-Faham, Ayman

    2015-07-01

    A series of quinazolinone amino acid ester and quinazolinone amino acid hydrazides were prepared under microwave irradiation as well as conventional condition. The microwave irradiation afforded the product in less reaction time, higher yield and purity. The structures of the synthesized compounds were confirmed by IR, NMR, and elemental analysis. The new synthesized compounds were studied for their monoamine oxidase inhibitory activity. They showed more selective inhibitory activity toward MAO-A than MAO-B. Compounds 7, 10, and 15 showed MAO-A inhibition activity (IC50=3.6×10(-9), 2.8×10(-9), 2.1×10(-9) M, respectively) comparable to that of the standard clorgyline (IC50=2.9×10(-9)M). 2-(2-(Benzo[d][1,3]dioxol-5-yl)-4-oxo-1,2-dihydroquinazolin-3(4H)-yl)acetohydrazide 15 showed selective MAO-A inhibition activity (SI=39524) superior to that of the standard clorgyline (SI=33793). The acute toxicity of the synthesized compounds was determined. In addition, computer-assisted simulated docking experiments were performed to rationalize the biological activity.

  15. The insert region of the Rac GTPases is dispensable for activation of superoxide-producing NADPH oxidases.

    PubMed

    Miyano, Kei; Koga, Hirofumi; Minakami, Reiko; Sumimoto, Hideki

    2009-08-13

    Rac1 and Rac2, which belong to the Rho subfamily of Ras-related GTPases, play an essential role in activation of gp91phox/Nox2 (cytochrome b-245, beta polypeptide; also known as Cybb), the catalytic core of the superoxide-producing NADPH oxidase in phagocytes. Rac1 also contributes to activation of the non-phagocytic oxidases Nox1 (NADPH oxidase 1) and Nox3 (NADPH oxidase 3), each related closely to gp91phox/Nox2. It has remained controversial whether the insert region of Rac (amino acids 123-135), unique to the Rho subfamily proteins, is involved in gp91phox/Nox2 activation. In the present study we show that removal of the insert region from Rac1 neither affects activation of gp91phox/Nox2, which is reconstituted under cell-free and whole-cell conditions, nor blocks its localization to phagosomes during ingestion of IgG-coated beads by macrophage-like RAW264.7 cells. The insert region of Rac2 is also dispensable for gp91phox/Nox2 activation at the cellular level. Although Rac2, as well as Rac1, is capable of enhancing superoxide production by Nox1 and Nox3, the enhancements by the two GTPases are both independent of the insert region. We also demonstrate that Rac3, a third member of the Rac family in mammals, has an ability to activate the three oxidases and that the activation does not require the insert region. Thus the insert region of the Rac GTPases does not participate in regulation of the Nox family NADPH oxidases.

  16. A colorimetric method for lysyl oxidase activity in copper deficient rats fed a high fructose diet

    SciTech Connect

    Werman, M.J.; Bhathena, S.J. )

    1991-03-11

    Lysyl oxidase is involved in initiating cross link formation in collagen and elastin. The activity of lysyl oxidase is traditionally assessed by the tritium released assay. The authors describe a simplified and modified method for measuring lysyl oxidase activity in rats, based on measuring ammonia release according to the Bertholet colorimetric reaction. Lysyl oxidase activity was measured in copper deficient rats using this method. Sixteen weanling Sprague Dawley male rats were fed for four weeks either copper adequate or copper deficient diets containing 62% fructose. Copper deficiency was confirmed by significant low copper levels in heart, brain, liver and skin, and by nondetectable levels of ceruloplasmin. Lysyl oxidase activity was significantly lower in heart and skin of rats fed a copper deficient diet compared to those fed a copper adequate diet. No significant difference in activity was observed in brain tissue. A correlation was not observed between decreased tissue copper levels and decreased lysyl oxidase activity. Thus, the determination of ammonia liberated during lysyl oxidase activity may serve as an effective tool in assessing lysyl oxidase activity.

  17. 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...

  18. 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.

  19. Polyphenol oxidase activity and antioxidant properties of Yomra apple (Malus communis L.) from Turkey.

    PubMed

    Can, Zehra; Dincer, Barbaros; Sahin, Huseyin; Baltas, Nimet; Yildiz, Oktay; Kolayli, Sevgi

    2014-12-01

    In this study, firstly, antioxidant and polyphenol oxidase (PPO) properties of Yomra apple were investigated. Seventeen phenolic constituents were measured by reverse phase-high-performance liquid chromatography (RP-HPLC). Total phenolic compounds (TPCs), ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activities were performed to measure antioxidant capacity. Some kinetic parameters (Km, Vmax), and inhibition behaviors against five different substrates were measured in the crude extract. Catechin and chlorogenic acid were found as the major components in the methanolic extract, while ferulic acid, caffeic acid, p-hydroxybenzoic acid, quercetin and p-coumaric acid were small quantities. Km values ranged from 0.70 to 10.10 mM in the substrates, and also 3-(4-hydroxyphenyl) propanoic acid (HPPA) and L-DOPA showed the highest affinity. The inhibition constant of Ki were ranged from 0.05 to 14.90 mM against sodium metabisulphite, ascorbic acid, sodium azide and benzoic acid, while ascorbic acid and sodium metabisulphite were the best inhibitors.

  20. The oxidase activity of vascular adhesion protein-1 (VAP-1) is essential for function.

    PubMed

    Noonan, Thomas; Lukas, Susan; Peet, Gregory W; Pelletier, Josephine; Panzenbeck, Mark; Hanidu, Adedayo; Mazurek, Suzanne; Wasti, Ruby; Rybina, Irina; Roma, Teresa; Kronkaitis, Anthony; Shoultz, Alycia; Souza, Donald; Jiang, Huiping; Nabozny, Gerald; Modis, Louise Kelly

    2013-01-01

    Vascular adhesion protein-1 (VAP-1) has been implicated in the pathogenesis of inflammatory diseases and is suggested to play a role in immune cell trafficking. It is not clear whether this effect is mediated by the oxidase activity or by other features of the protein such as direct adhesion. In order to study the role of VAP-1 oxidase activity in vivo, we have generated mice carrying an oxidase activity-null VAP-1 protein. We demonstrate that the VAP-1 oxidase null mutant mice have a phenotype similar to the VAP-1 null mice in animal models of sterile peritonitis and antibody induced arthritis suggesting that the oxidase activity is responsible for the inflammatory function of VAP-1.

  1. The oxidase activity of vascular adhesion protein-1 (VAP-1) is essential for function

    PubMed Central

    Noonan, Thomas; Lukas, Susan; Peet, Gregory W; Pelletier, Josephine; Panzenbeck, Mark; Hanidu, Adedayo; Mazurek, Suzanne; Wasti, Ruby; Rybina, Irina; Roma, Teresa; Kronkaitis, Anthony; Shoultz, Alycia; Souza, Donald; Jiang, Huiping; Nabozny, Gerald; Modis, Louise Kelly

    2013-01-01

    Vascular adhesion protein-1 (VAP-1) has been implicated in the pathogenesis of inflammatory diseases and is suggested to play a role in immune cell trafficking. It is not clear whether this effect is mediated by the oxidase activity or by other features of the protein such as direct adhesion. In order to study the role of VAP-1 oxidase activity in vivo, we have generated mice carrying an oxidase activity-null VAP-1 protein. We demonstrate that the VAP-1 oxidase null mutant mice have a phenotype similar to the VAP-1 null mice in animal models of sterile peritonitis and antibody induced arthritis suggesting that the oxidase activity is responsible for the inflammatory function of VAP-1. PMID:23885334

  2. Inhibition and oxygen activation in copper amine oxidases.

    PubMed

    Shepard, Eric M; Dooley, David M

    2015-05-19

    Copper-containing amine oxidases (CuAOs) use both copper and 2,4,5-trihydroxyphenylalanine quinone (TPQ) to catalyze the oxidative deamination of primary amines. The CuAO active site is highly conserved and comprised of TPQ and a mononuclear type II copper center that exhibits five-coordinate, distorted square pyramidal coordination geometry with histidine ligands and equatorially and axially bound water in the oxidized, resting state. The active site is buried within the protein, and CuAOs from various sources display remarkable diversity with respect to the composition of the active site channel and cofactor accessibility. Structural and mechanistic factors that influence substrate preference and inhibitor sensitivity and selectivity have been defined. This Account summarizes the strategies used to design selective CuAO inhibitors based on active site channel characteristics, leading to either enhanced steric fits or the trapping of reactive electrophilic products. These findings provide a framework to support the future development of candidate molecules aimed at minimizing the negative side effects associated with drugs containing amine functionalities. This is vital given the existence of human diamine oxidase and vascular adhesion protein-1, which have distinct amine substrate preferences and are associated with different metabolic processes. Inhibition of these enzymes by antifungal or antiprotozoal agents, as well as classic monoamine oxidase (MAO) inhibitors, may contribute to the adverse side effects associated with drug treatment. These observations provide a rationale for the limited clinical value associated with certain amine-containing pharmaceuticals and emphasize the need for more selective AO inhibitors. This Account also discusses the novel roles of copper and TPQ in the chemistry of O2 activation and substrate oxidation. Reduced CuAOs exist in a redox equilibrium between the Cu(II)-TPQAMQ (aminoquinol) and Cu(I)-TPQSQ (semiquinone). Elucidating

  3. The antiviral drug acyclovir is a slow-binding inhibitor of (D)-amino acid oxidase.

    PubMed

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

    2013-08-20

    d-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for d-amino acids, including d-serine and d-alanine, which are believed to be coagonists of the N-methyl-d-aspartate (NMDA) receptor. To identify a new class of DAO inhibitor(s) that can be used to elucidate the molecular details of the active site environment of DAO, manifold biologically active compounds of microbial origin and pre-existing drugs were screened for their ability to inhibit DAO activity, and several compounds were identified as candidates. One of these compounds, acyclovir (ACV), a well-known antiviral drug used for the treatment of herpesvirus infections, was characterized and evaluated as a novel DAO inhibitor in vitro. Analysis showed that ACV acts on DAO as a reversible slow-binding inhibitor, and interestingly, the time required to achieve equilibrium between DAO, ACV, and the DAO/ACV complex was highly dependent on temperature. The binding mechanism of ACV to DAO was investigated in detail by several approaches, including kinetic analysis, structural modeling of DAO complexed with ACV, and site-specific mutagenesis of an active site residue postulated to be involved in the binding of ACV. The results confirm that ACV is a novel, active site-directed inhibitor of DAO that can be a valuable tool for investigating the structure-function relationships of DAO, including the molecular details of the active site environment of DAO. In particular, it appears that ACV can serve as an active site probe to study the structural basis of temperature-induced conformational changes of DAO.

  4. 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

  5. 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

  6. Hypoxia inhibits semicarbazide-sensitive amine oxidase activity in adipocytes.

    PubMed

    Repessé, Xavier; Moldes, Marthe; Muscat, Adeline; Vatier, Camille; Chetrite, Gérard; Gille, Thomas; Planes, Carole; Filip, Anna; Mercier, Nathalie; Duranteau, Jacques; Fève, Bruno

    2015-08-15

    Semicarbazide-sensitive amine oxidase (SSAO), an enzyme highly expressed on adipocyte plasma membranes, converts primary amines into aldehydes, ammonium and hydrogen peroxide, and is likely involved in endothelial damage during the course of diabetes and obesity. We investigated whether in vitro, adipocyte SSAO was modulated under hypoxic conditions that is present in adipose tissue from obese or intensive care unit. Physical or pharmacological hypoxia decreased SSAO activity in murine adipocytes and human adipose tissue explants, while enzyme expression was preserved. This effect was time-, dose-dependent and reversible. This down-regulation was confirmed in vivo in subcutaneous adipose tissue from a rat model of hypoxia. Hypoxia-induced suppression in SSAO activity was independent of the HIF-1-α pathway or of oxidative stress, but was partially antagonized by medium acidification. Hypoxia-induced down-regulation of SSAO activity could represent an adaptive mechanism to lower toxic molecules production, and may thus protect from tissue injury during these harmful conditions.

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

    PubMed Central

    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

  8. 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.

  9. 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.

  10. Gene expression and distribution of antibacterial L-amino acid oxidase in the rockfish Sebastes schlegeli.

    PubMed

    Kitani, Yoichiro; Mori, Tsukasa; Nagai, Hiroshi; Toyooka, Keiko; Ishizaki, Shoichiro; Shimakura, Kuniyoshi; Shiomi, Kazuo; Nagashima, Yuji

    2007-12-01

    Antibacterial factors in the epidermal mucus of fish have a potential importance in the first line of the host defense response to bacterial pathogens. We previously isolated a novel antibacterial protein termed SSAP (Sebastes schlegeli antibacterial protein) from the skin mucus of the rockfish S. schlegeli and identified it as a new member of the L-amino acid oxidase (LAO) family. In the present study, the localization of SSAP in S. schlegeli was investigated by reverse transcription (RT)-PCR, quantitative real time RT-PCR, Western blotting and measurements of LAO and antibacterial activities. SSAP mRNA was expressed dominantly in skin and gill and weakly in ovary or kidney as shown by RT-PCR and real time RT-PCR. The quantity of SSAP mRNA in skin varied among the individuals, ranging from 1.1 to 13.9 ng microg(-1) total RNA, although no relationship was found between the size of fish and gene expression. SSAP was exclusively detected in skin and gill by Western blotting using a specific anti-SSAP antiserum. In addition, the extracts of both tissues apparently showed LAO activity and antibacterial activity against Photobacterium damselae subsp. piscicida. This study demonstrates that SSAP is predominantly synthesized in skin and gill and probably functions as an antibacterial LAO in both tissues.

  11. The effect of an NADH oxidase inhibitor (hydrocortisone) on polymorphonuclear leukocyte bactericidal activity

    PubMed Central

    Mandell, Gerald L.; Rubin, Walter; Hook, Edward W.

    1970-01-01

    Polymorphonuclear neutrophils (PMN) from patients with chronic granulomatous disease of childhood have impaired bactericidal activity and are deficient in diphosphopyridine nucleotide, reduced form of, (NADH) oxidase. Since hydrocortisone had been shown to inhibit NADH oxidation, experiments were undertaken to determine the effect of hydrocortisone on several parameters of human PMN function. The phagocytic and bactericidal capacity of PMN with or without hydrocortisone (2.1 mM) was determined by quantitation of cell-free, cell-associated, and total bacteria. Phagocytosis of Staphylococcus aureus and several gram-negative rods was unimpaired by the presence of hydrocortisone in the media. In contrast, killing of bacteria was markedly impaired by hydrocortisone. After 30 min of incubation, there were 20-400 times as many bacteria surviving in hydrocortisone-treated PMN as in simultaneously run controls without hydrocortisone. The defect of intracellular killing noted in the presence of hydrocortisone was not related to impaired degranulation. Quantitative kinetic studies of degranulation revealed no difference in the release of granule associated acid phosphatase in hydrocortisone-treated and control PMN after phagocytosis. Electron microscopy of PMN also indicated that the presence of hydrocortisone had no effect on the extent of degranulation after phagocytosis. These observations were confirmed by studies using histochemical techniques to detect lysosomal enzymes. After phagocytosis, hydrocortisone-treated PMN demonstrated less NADH oxidase activity, oxygen consumption, and hydrogen peroxide production than postphagocytic control PMN. In addition, Nitro blue tetrazolium dye reduction was diminished in hydrocortisone-treated PMN. Thus, impairment of NADH oxidase activity in normal human PMN by hydrocortisone results in reduced intracellular killing of bacteria, diminished postphagocytic oxygen consumption, decreased ability to reduce Nitro blue tetrazolium, and

  12. Engineering the properties of D-amino acid oxidases by a rational and a directed evolution approach.

    PubMed

    Pollegioni, Loredano; Sacchi, Silvia; Caldinelli, Laura; Boselli, Angelo; Pilone, Mirella S; Piubelli, Luciano; Molla, Gianluca

    2007-12-01

    D-amino acid oxidase (DAAO) is a FAD-containing flavoprotein that dehydrogenates the D-isomer of amino acids to the corresponding imino acids, coupled with the reduction of FAD. The cofactor then reoxidizes on molecular oxygen and the imino acid hydrolyzes spontaneously to the alpha-keto acid and ammonia. In vitro DAAO displays broad substrate specificity, acting on several neutral and basic D-amino acids: the most efficient substrates are amino acids with hydrophobic side chains. D-aspartic acid and D-glutamic acid are not substrates for DAAO. Through the years, it has been the subject of a number of structural, functional and kinetic investigations. The most recent advances are represented by site-directed mutagenesis studies and resolution of the 3D-structure of the enzymes from pig, human and yeast. The two approaches have given us a deeper understanding of the structure-function relationships and promoted a number of investigations aimed at the modulating the protein properties. By a rational and/or a directed evolution approach, DAAO variants with altered substrate specificity (e.g., active on acidic or on all D-amino acids), increased stability (e.g., stable up to 60 degrees C), modified interaction with the flavin cofactor, and altered oligomeric state were produced. The aim of this paper is to provide an overview of the most recent research on the engineering of DAAOs to illustrate their new intriguing properties, which also have enabled us to pursue new biotechnological applications.

  13. 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.

  14. Induction of apoptosis in yeast by L-amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma.

    PubMed

    Ande, Sudharsana Rao; Fussi, Heike; Knauer, Heide; Murkovic, Michael; Ghisla, Sandro; Fröhlich, Kai-Uwe; Macheroux, Peter

    2008-05-01

    Here we report for the first time that L-amino acid oxidase (LAAO), a major component of snake venom, induces apoptosis in yeast. The causative agent for induction of apoptosis has been shown to be hydrogen peroxide, produced by the enzymatic activity of LAAO. However, the addition of catalase, a specific hydrogen peroxide scavenger, does not prevent cell demise completely. Intriguingly, depletion of leucine from the medium by LAAO and the interaction of LAAO with yeast cells are shown to be the major factors responsible for cell demise in the presence of catalase.

  15. 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

  16. 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%.

  17. Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells.

    PubMed

    Gesell, Andreas; Chávez, Maria Luisa Díaz; Kramell, Robert; Piotrowski, Markus; Macheroux, Peter; Kutchan, Toni M

    2011-06-01

    Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.

  18. 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.

  19. Investigating antimicrobial activity in Rheinheimera sp. due to hydrogen peroxide generated by l-lysine oxidase activity.

    PubMed

    Chen, Wen Ming; Lin, Chang Yi; Sheu, Shih Yi

    2010-05-05

    A greenish yellow pigmented bacterial strain, designated GR5, was recently isolated from a freshwater culture pond for a soft-shell turtle. Phylogenetic analyses based on 16S rRNA gene sequences indicate that strain GR5 belongs to the genus Rheinheimera and its only closest neighbor is the type strain of Rheinheimera texasensis (98.2%). Based on the antibiogram assay, strain GR5 possesses a broad spectrum of antimicrobial activity including Gram-positive and Gram-negative bacteria, yeast, algae, and strain GR5 itself. Strain GR5 can synthesize a macromolecule with antimicrobial activity due to the generation of hydrogen peroxide and this antimicrobial effect can be inhibited by catalase. This antimicrobial activity is active only in complex culture media or chemically defined culture media containing l-lysine. This antimicrobial macromolecule in strain GR5 is shown to be a monomeric protein with a molecular mass of 71kDa and isoelectric point of approximately 3.68. Liquid chromatography-tandem mass spectrometry analyses reveal close similarity of a 19-amino acid fragment derived from this protein to the antibacterial protein, AlpP from the marine bacterium Pseudoalteromonas tunicata D2, and to the antibacterial protein, marinocine, from the marine bacterium Marinomonas mediterranea. This study explores the nature of antimicrobial macromolecule such as l-lysine oxidase. This is the first report on a freshwater bacterium producing antimicrobial activity by generating hydrogen peroxide through its enzymatic activity of l-lysine oxidase.

  20. Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes.

    PubMed

    Hutzler, J Matthew; Yang, Young-Sun; Albaugh, Daniel; Fullenwider, Cody L; Schmenk, Jennifer; Fisher, Michael B

    2012-02-01

    Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O⁶-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min⁻¹ · kg⁻¹, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H₂¹⁸O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (∼5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O⁶-benzylguanine was within ∼80% of the observed total clearance in humans after intravenous administration (15 ml · min⁻¹ · kg⁻¹), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.

  1. 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

  2. Endoplasmic Reticulum Thiol Oxidase Deficiency Leads to Ascorbic Acid Depletion and Noncanonical Scurvy in Mice

    PubMed Central

    Zito, Ester; Hansen, Henning Gram; Yeo, Giles S.H.; Fujii, Junichi; Ron, David

    2012-01-01

    Summary Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1β, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H2O2-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy. PMID:22981861

  3. Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice.

    PubMed

    Zito, Ester; Hansen, Henning Gram; Yeo, Giles S H; Fujii, Junichi; Ron, David

    2012-10-12

    Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1β, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H(2)O(2)-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.

  4. 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

  5. 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.

  6. THE METABOLISM OF THE ISCHEMIC KIDNEY : I. THE RESPIRATION AND THE OXIDASE ACTIVITY OF THE ISCHEMIC KIDNEY.

    PubMed

    Raska, S B

    1943-07-01

    The consumption of oxygen by slices of kidney tissue of dogs made hypertensive by the Goldblatt technique was studied manometrically. The respiration of the ischemic kidney tissue was found to be much less than that of the normal kidney. Further, a marked reduction in oxidizing ability, as measured by the oxygen uptake and ammonia formation in the presence of the added amines and amino acids, tyramine, isoamylamine, dl-alanine, and l-aspartic acid, was observed. Extracts of the kidneys were made and tested for amine oxidase, amino acid oxidase, and polyphenol oxidase activity by measuring the increased oxygen consumption and ammonia formation in the presence of the substrates listed above with the addition of l-epinephrine, histamine, and dl- and l-dihydroxyphenylalanine. The preparations from ischemic kidneys of dogs and rabbits showed much lower activity. Animals with varying degrees of constriction of the renal arteries and therefore varying degrees of renal ischemia were prepared and studied. The results with these animals suggested a direct relationship between the degree of renal ischemia and the decrease in oxidizing power of the tissue. The product of the enzymic oxidation of tyramine was identified as p-hydroxyphenylacetaldehyde by isolation as the dinitrophenylhydrazone of p-hydroxyphenylacetaldehyde.

  7. 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

  8. Effects of Seselin and Coumarin on Growth, Indoleacetic Acid Oxidase, and Peroxidase, with Special Reference to Cucumber (Cucumis sativa L.) Radicles

    PubMed Central

    Goren, Raphael; Tomer, Eliahu

    1971-01-01

    Seselin, a natural coumarin derivative isolated from citrus roots, inhibited radicle growth in seedlings of cucumber (Cucumis sativa), lettuce (Lactuca sativum), radish (Raphanus sativus), and wheat (Triticum aestivum) grown in the dark. Coumarin similarly inhibited radicle growth of cucumber seedlings. Growth retardation of the cucumber radicles was accompanied by an increased activity of peroxidase and indole-3-acetic acid oxidase. Both compounds antagonized indole-3-acetic acid-induced growth of wheat coleoptiles, whereas coumarin was much less effective than seselin in antagonizing gibberellic acid-induced release of reducing sugars from barley endosperm. It is suggested that seselin plays an important role in the regulation of root growth, and that it is the indole-3-acetic acid oxidase cofactor previously detected in citrus roots. Images PMID:16657614

  9. Effect of afforestation on urate oxidase activity in two kinds of soils

    NASA Astrophysics Data System (ADS)

    Meysner, Teresa; Wojciech Szajdak, Lech

    2010-05-01

    Researches were carried out in soils under a 125-m-long the afforestation located in the Kościan Plain in Turew, which is a part of West Poland Lowland. Soil samples were taken from four chosen sites marked as Nos. 1, 2, 3 and 4 near wells. One part of this afforestation was allocated on mineral, whereas the second part was on mineral-organic soil. Times of sampling were from March to November in 2009 from the layer at 0-20 cm depth after removing leaf litter. Urate oxidase activity in soils was determined colorimetrically by measuring the absorbance at λ=293 nm. Urate oxidase is a homotetrameric enzyme containing four identical active sites situated at the interfaces between its four subunits. This enzyme catalyzes the oxidation of uric acid, a final product of purine catabolism to 5-hydroxyisourate, which is non-enzymatically transformed into allantoin, carbon dioxide and hydrogen peroxide. Uricase is also an essential enzyme in the ureide pathway, where nitrogen fixation occurs in the root nodules of legumes. Nitrogen heterocyclic compounds such as allantoin may serve as nitrogen sources or nitrogen transport compounds in plants that are not able to fix nitrogen. It has been estimated that heterocyclic nitrogen compounds represent about 30% of the reduced nitrogen in soils. These studies indicated that the flow of ground water was accompanied by an increase of uricase activity from 16 to 71% (from point 1 to point 2) in all periods of sampling in mineral soils. Similar trend was shown in mineral-organic soils. There was an increase of uricase activity from the point 3 to 4 and ranged from 13 to 37% similar to the direction of the flow of ground water. However, no significant differences of urate oxidase activity between two kinds of soils were observed. This study showed that the uricase activity ranged from 1.99 to 7.16 μmol×h-1×g-1 in the mineral soils and from 1.79 to 8.36 μmol×h-1×g-1. The study indicated an impact of the afforestation located on

  10. Modulation of p47PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

    PubMed Central

    Hoyal, Carolyn R.; Gutierrez, Abel; Young, Brandon M.; Catz, Sergio D.; Lin, Jun-Hsiang; Tsichlis, Philip N.; Babior, Bernard M.

    2003-01-01

    The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document} at the expense of NADPH. Extensive phosphorylation of the oxidase subunit p47PHOX occurs during the activation of the enzyme in intact cells. p47PHOX carrying certain serine-to-alanine mutations fails to support NADPH oxidase activity in intact cells, suggesting that the phosphorylation of specific serines on p47PHOX is required for the activation of the oxidase. Earlier studies with both intact cells and a kinase-dependent, cell-free system have suggested that protein kinase C can phosphorylate those serines of p47PHOX whose phosphorylation is necessary for its activity. Work with inhibitors suggested that a phosphatidylinositol 3-kinase-dependent pathway also can activate the oxidase. Phosphorylation of p47PHOX by Akt (protein kinase B), whose activation depends on phosphatidylinositol 3-kinase, could be the final step in such a pathway. We now find that Akt activates the oxidase in vitro by phosphorylating serines S304 and S328 of p47PHOX. These results suggest that Akt could participate in the activation of the leukocyte NADPH oxidase. PMID:12704229

  11. 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

    2011-03-09

    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.

  12. 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.

  13. Catalase activity of cytochrome C oxidase assayed with hydrogen peroxide-sensitive electrode microsensor.

    PubMed

    Bolshakov, I A; Vygodina, T V; Gennis, R; Karyakin, A A; Konstantinov, A A

    2010-11-01

    An iron-hexacyanide-covered microelectrode sensor has been used to continuously monitor the kinetics of hydrogen peroxide decomposition catalyzed by oxidized cytochrome oxidase. At cytochrome oxidase concentration ~1 µM, the catalase activity behaves as a first order process with respect to peroxide at concentrations up to ~300-400 µM and is fully blocked by heat inactivation of the enzyme. The catalase (or, rather, pseudocatalase) activity of bovine cytochrome oxidase is characterized by a second order rate constant of ~2·10(2) M(-1)·sec(-1) at pH 7.0 and room temperature, which, when divided by the number of H2O2 molecules disappearing in one catalytic turnover (between 2 and 3), agrees reasonably well with the second order rate constant for H2O2-dependent conversion of the oxidase intermediate F(I)-607 to F(II)-580. Accordingly, the catalase activity of bovine oxidase may be explained by H2O2 procession in the oxygen-reducing center of the enzyme yielding superoxide radicals. Much higher specific rates of H2O2 decomposition are observed with preparations of the bacterial cytochrome c oxidase from Rhodobacter sphaeroides. The observed second order rate constants (up to ~3000 M(-1)·sec(-1)) exceed the rate constant of peroxide binding with the oxygen-reducing center of the oxidized enzyme (~500 M(-1)·sec(-1)) several-fold and therefore cannot be explained by catalytic reaction in the a(3)/Cu(B) site of the enzyme. It is proposed that in the bacterial oxidase, H2O2 can be decomposed by reacting with the adventitious transition metal ions bound by the polyhistidine-tag present in the enzyme, or by virtue of reaction with the tightly-bound Mn2+, which in the bacterial enzyme substitutes for Mg2+ present in the mitochondrial oxidase.

  14. An automated sequential injection spectrophotometric method for evaluation of tyramine oxidase inhibitory activity of some flavonoids.

    PubMed

    Moonrungsee, Nuntaporn; Shimamura, Tomoko; Kashiwagi, Takehiro; Jakmunee, Jaroon; Higuchi, Keiro; Ukeda, Hiroyuki

    2014-05-01

    An automated sequential injection (SI) spectrophotometric system has been developed for evaluation of tyramine oxidase (TOD) inhibitory activity. The method is based on the inhibition of TOD that catalyzes the oxidation of tyramine substrate to produce aldehyde and hydrogen peroxide (H₂O₂). The produced H₂O₂ reacts with vanillic acid and 4-aminoantipyrine (4-AA) in the presence of peroxidase (POD) to form a quinoneimine dye, the absorbance of which is measured of absorbance at wavelength of 490 nm. The decrease of the quinoneimine dye is related to an increase of TOD inhibitory activity. Under the optimum conditions: 1.0 mM tyramine, 8 U mL(-1) TOD, 1.0 mM vanillic acid, 1.0 mM 4-AA and delay time of 10 s, some flavonoid compounds were examined for the TOD inhibitory activity expressed as IC₅₀ value. It was found that flavonols (quercetin and myricetin) and flavans (epicatechin gallate (ECG) and epigallocatechin (EGC)) showed higher TOD inhibitory activity than flavones and flavanones. The results of IC₅₀ values obtained from the proposed method and a batch-wise method were not significantly different from each other. Moreover, the SI system enabled automation of the analysis, leading to more convenient, more sensitive and faster analysis than the batch-wise method. A precise timing of the system also improves precision and accuracy of the assay, especially when the measurement of absorbance at non-steady state condition is involved.

  15. Salicylic acid inhibits enzymatic browning of fresh-cut Chinese chestnut (Castanea mollissima) by competitively inhibiting polyphenol oxidase.

    PubMed

    Zhou, Dan; Li, Lin; Wu, Yanwen; Fan, Junfeng; Ouyang, Jie

    2015-03-15

    The inhibitory effect and associated mechanisms of salicylic acid (SA) on the browning of fresh-cut Chinese chestnut were investigated. Shelled and sliced chestnuts were immersed in different concentrations of an SA solution, and the browning of the chestnut surface and interior were inhibited. The activities of polyphenol oxidase (PPO) and peroxidase (POD) extracted from chestnuts were measured in the presence and absence of SA. SA at concentrations higher than 0.3g/L delayed chestnut browning by significantly inhibiting the PPO activity (P<0.01), and the POD activity was not significantly affected (P>0.05). The binding and inhibition modes of SA with PPO and POD, determined by AUTODOCK 4.2 and Lineweaver-Burk plots, respectively, established SA as a competitive inhibitor of PPO.

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

    PubMed

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

    2013-12-01

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

  17. Mapping of functional domains in p47(phox) involved in the activation of NADPH oxidase by "peptide walking".

    PubMed

    Morozov, I; Lotan, O; Joseph, G; Gorzalczany, Y; Pick, E

    1998-06-19

    The superoxide generating NADPH oxidase of phagocytes consists, in resting cells, of a membrane-associated electron transporting flavocytochrome (cytochrome b559) and four cytosolic proteins as follows: p47(phox), p67(phox), p40(phox), and the small GTPase, Rac(1 or 2). Activation of the oxidase is consequent to the assembly of a membrane-localized multimolecular complex consisting of cytochrome b559 and the cytosolic components. We used "peptide walking" (Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082) for mapping domains in the amino acid sequence of p47(phox) participating in the molecular events leading to the activation of NADPH oxidase. Ninety-five overlapping pentadecapeptides, with a four-residue offset between neighboring peptides, spanning the complete p47(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in a cell-free system. This consisted of solubilized macrophage membranes, recombinant p47(phox), p67(phox), and Rac1, and lithium dodecyl sulfate, as the activator. Eight functional domains were identified and labeled a-h. These were (N- and C-terminal residue numbers are given for each domain) as follows: a (21-35); b (105-119); c (149-159); d (193-207); e (253-267); f (305-319); g (325-339), and h (373-387). Four of these domains (c, d, e, and g) correspond to or form parts of regions shown before to participate in NADPH oxidase assembly. Thus, domain c corresponds to a region on the N-terminal boundary of the first src homology 3 (SH3) domain, whereas domains d and e represent more precisely defined sites within the full-length first and second SH3 domains, respectively. Domain g overlaps an extensively investigated arginine-rich region. Domains a and b, in the N-terminal half of p47(phox), and domains f and h, in the C-terminal half, represent newly identified entities, for which there is no earlier experimental evidence of involvement in NADPH oxidase activation. "Peptide walking" was also applied to

  18. 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.

  19. Ascorbate Oxidase-Based Amperometric Biosensor for l-Ascorbic Acid Determination in Beverages.

    PubMed

    Csiffáry, Gábor; Fűtő, Péter; Adányi, Nóra; Kiss, Attila

    2016-03-01

    A novel biosensor for l-ascorbic acid determination in different beverages was elaborated. The ascorbate oxidase enzyme (AAO) from Cucurbita sp., EC 1.10.3.3, was immobilized on a screen-printed carbon electrode with poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) as a crosslinking agent. The standards and samples were measured first with a blank electrode. An inert protein, bovine serum albumin (BSA), was immobilized on the surface of this electrode with PEGDGE. The BSA mass was equivalent to the mass of 10 U of AAO enzyme immobilized on the electrodes (0.021 mg). The linear measuring range for l-ascorbic acid was between 5 and 150 µmol/L. As l-ascorbic acid is a vital vitamin and a common antioxidant used in food industry, fruit juices and vitamin C effervescent tablets were examined. The results were compared to HPLC measurements.

  20. Crystal structure and molecular dynamics studies of L-amino acid oxidase from Bothrops atrox.

    PubMed

    Feliciano, Patricia R; Rustiguel, Joane K; Soares, Ricardo O S; Sampaio, Suely V; Cristina Nonato, M

    2017-03-15

    L-amino acid oxidases (LAAOs) are dimeric flavoproteins that catalyze the deamination of L-amino acid to α-keto acid, producing ammonia and hydrogen peroxide. In this study, we report the crystal structure and molecular dynamics simulations of LAAO from the venom of Bothrops atrox (BatroxLAAO). BatroxLAAO presents several biological and pharmacological properties with promising biomedical applications. BatroxLAAO structure contains the highly conserved structural pattern of LAAOs comprising a FAD-binding domain, substrate-binding domain and helical domain, and a dimeric arrangement that can be stabilized by zinc. Also, molecular dynamics results show an asymmetric behavior, and a direct communication between FAD- and substrate-binding domains of counterpart subunits. These findings shed light on the structural role of dimerization to catalytic mechanism of SV-LAAOs.

  1. Ascorbate Oxidase-Based Amperometric Biosensor for l-Ascorbic Acid Determination in Beverages

    PubMed Central

    Csiffáry, Gábor; Fűtő, Péter; Adányi, Nóra; Kiss, Attila

    2016-01-01

    Summary A novel biosensor for l-ascorbic acid determination in different beverages was elaborated. The ascorbate oxidase enzyme (AAO) from Cucurbita sp., EC 1.10.3.3, was immobilized on a screen-printed carbon electrode with poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) as a crosslinking agent. The standards and samples were measured first with a blank electrode. An inert protein, bovine serum albumin (BSA), was immobilized on the surface of this electrode with PEGDGE. The BSA mass was equivalent to the mass of 10 U of AAO enzyme immobilized on the electrodes (0.021 mg). The linear measuring range for l-ascorbic acid was between 5 and 150 µmol/L. As l-ascorbic acid is a vital vitamin and a common antioxidant used in food industry, fruit juices and vitamin C effervescent tablets were examined. The results were compared to HPLC measurements. PMID:27904390

  2. NADPH oxidase plays a crucial role in the activation of pancreatic stellate cells.

    PubMed

    Masamune, Atsushi; Watanabe, Takashi; Kikuta, Kazuhiro; Satoh, Kennichi; Shimosegawa, Tooru

    2008-01-01

    Activated pancreatic stellate cells (PSCs) play an important role in pancreatic fibrosis and inflammation, where oxidative stress is implicated in the pathogenesis. NADPH oxidase might be a source of reactive oxygen species (ROS) in the injured pancreas. This study aimed to clarify the expression and regulation of cell functions by NADPH oxidase in PSCs. PSCs were isolated from rat and human pancreas tissues. Expression of NADPH oxidase was assessed by reverse transcription-PCR and immunostaining. Intracellular ROS production was assessed using 2',7'-dichlorofluorescin diacetate. The effects of diphenylene iodonium (DPI) and apocynin, inhibitors of NADPH oxidase, on key parameters of PSC activation were evaluated in vitro. In vivo, DPI (at 1 mg.kg body wt(-1).day(-1)) was administered in drinking water to 10-wk-old male Wistar Bonn/Kobori rats for 10 wk and to rats with chronic pancreatitis induced by dibutyltin dichloride (DBTC). PSCs expressed key components of NADPH oxidase (p22(phox), p47(phox), NOX1, gp91(phox)/NOX2, NOX4, and NOX activator 1). PDGF-BB, IL-1beta, and angiotensin II induced ROS production, which was abolished by DPI and apocynin. DPI inhibited PDGF-induced proliferation, IL-1beta-induced chemokine production, and expression of alpha-smooth muscle actin and collagen. DPI inhibited transformation of freshly isolated cells to a myofibroblast-like phenotype. In addition, DPI inhibited the development of pancreatic fibrosis in Wistar Bonn/Kobori rats and in rats with DBTC-induced chronic pancreatitis. In conclusion, PSCs express NADPH oxidase to generate ROS, which mediates key cell functions and activation of PSCs. NADPH oxidase might be a potential target for the treatment of pancreatic fibrosis.

  3. 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

    2014-01-08

    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.

  4. 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.

  5. Platelet monoamine oxidase activity predicts alcohol sensitivity and voluntary alcohol intake in rhesus monkeys.

    PubMed

    Wargelius, Hanna-Linn; Fahlke, Claudia; Suomi, Stephen J; Oreland, Lars; Higley, James Dee

    2010-02-01

    Platelet monoamine oxidase B (MAO-B) has been proposed to be a biological marker for the properties of monoamine systems, with low activity being associated with vulnerability for high scores on personality traits such as sensation seeking, monotony avoidance, and impulsiveness, as well as for vulnerability for alcoholism. In the present study, platelet MAO-B activity was analysed in 78 rhesus macaques, and its relation to voluntary alcohol intake and behaviours after intravenous alcohol administration was observed. Monkeys with low platelet MAO-B activity had low levels of 5-hydroxyindole acetic acid in cerebrospinal fluid and showed excessive aggression after alcohol administration. A novel finding was that animals with low platelet MAO-B activity showed less intoxication following alcohol administration. As we have shown previously, they also voluntarily consumed more alcohol. We here replicate results from studies on both humans and non-human primates, showing the utility of platelet MAO as a marker for risk behaviours and alcohol abuse. Furthermore, we link platelet MAO activity to alcohol sensitivity.

  6. 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

  7. Diphosphatidylglycerol is required for optimal activity of beef heart cytochrome c oxidase.

    PubMed Central

    Vik, S B; Georgevich, G; Capaldi, R A

    1981-01-01

    Isolated beef heart cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) contains four or five molecules of tightly bound diphosphatidylglycerol per monomer (2-heme complex). This lipid could be removed in part, or wholly, by mixing the enzyme with high concentrations of Triton X-100 and then centrifuging the mixture through a glycerol gradient equilibrated in the same detergent. Cytochrome c oxidase retaining three or more diphosphatidylglycerol molecules per monomer was fully active when assayed in 1-oleoyl lysophosphatidylcholine. Upon removal of one or more of these diphosphatidylglycerols, enzymic activity was lost. Full activation could be obtained by adding diphosphatidylglycerol to the assay mixture along with lysophosphatidylcholine but not by adding phosphatidylcholine or phosphatidylethanolamine. Direct binding experiments, kinetic studies, and previous work using arylazidocytochrome c derivatives [Bisson, R., Jacobs, B. & Capaldi, R. A. (1980) Biochemistry 10, 4173-4178], indicate that diphosphatidylglycerol is involved in binding of substrate cytochrome c to cytochrome c oxidase. PMID:6262802

  8. [Hydrazine derivative modification of the activity of brain mitochondrial monoamine oxidases].

    PubMed

    Kalninia, I E; Baumanas, E A; Kaĭrane, Ch B; Gorkin, V Z

    1981-01-01

    Treatment of bovine brain stem mitochondria with hydrazine derivatives, which inhibited the monoamine oxidase activity (substrate: 5-hydroxytryptamine), was accompanied by appearance of the properties to deaminate histamine and cadaverine at a high rate. The same phenomenon was observed in vivo after treatment of mice with the hydrazine derivatives. The dramatic increase in histamine deaminating activity in brain was accompanied by a decrease in the tissue concentration of histamine. The hydrazine derivatives are considered as prooxidants stimulating via a free-radical mechanism lipid peroxidation in methyloleate solutions and in biomembranes (ref. 5) and causing qualitative alteration (transformation) in catalytic properties of monoamine oxidases of the tyre A, which acquire the histamine deaminating activity. A certain correlation was noted between the prooxidant effect of the hydrazine derivatives and the modification of catalytic properties of the membrane bound monoamine oxidases of brain mitochondria in vitro and in vivo.

  9. 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.

  10. 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...

  11. Kinetic and thermodynamic investigation on ascorbate oxidase activity and stability of a Cucurbita maxima extract.

    PubMed

    Porto, Tatiana S; Porto, Camila S; Cavalcanti, Maria T H; Filho, José L Lima; Perego, Patrizia; Porto, Ana L F; Converti, Attilio; Pessoa, Adalberto

    2006-01-01

    The kinetic and thermodynamic properties of ascorbate oxidase (AO) activity and stability of a Cucurbita maxima extract were investigated. Activity tests performed at 25 degrees C using initial ascorbic acid concentration in the range 50-750 M allowed estimating the Michaelis constant for this substrate (Km = 126 microM) and the maximum initial rate of ascorbic acid oxidation (A0,max = 1.57 mM min-1). The main thermodynamic parameters of the enzyme reaction (DeltaH* = 10.3 kJ mol-1; DeltaG* = 87.2 kJ mol-1; DeltaS* = -258 J mol-1 K-1) were estimated through activity tests performed at 25-48 C. Within such a temperature range, no decrease in the initial reaction rate was detected. The long-term thermostability of the raw extract was then investigated by means of residual activity tests carried out at 10-70 degrees C, which allowed estimating the thermodynamic parameters of the irreversible enzyme inactivation as well (DeltaH*D = 51.7 kJ mol-1; DeltaG*D = 103 kJ mol-1; S*D = -160 J mol-1 K-1). Taking into account the specific rate of AO inactivation determined at different temperatures, we also estimated the enzyme half-life (1047 min at 10 degrees C and 21.2 min at 70 degrees C) and predicted the integral activity of a continuous system using this enzyme preparation. This work should be considered as a preliminary attempt to characterize the AO activity of a C. maxima extract before its concentration by liquid-liquid extraction techniques.

  12. 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.

  13. Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation.

    PubMed

    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

    2012-03-15

    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.

  14. 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.

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

    PubMed

    Nile, Shivraj H; Khobragade, Chandrahasy N

    2011-10-01

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

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

    PubMed

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

    2009-03-16

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

  17. 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

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

    PubMed

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

    2012-03-01

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

  19. 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.

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

    PubMed

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

    2016-09-01

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

  1. Oxalate decarboxylase and oxalate oxidase activities can be interchanged with a specificity switch of up to 282,000 by mutating an active site lid.

    PubMed

    Burrell, Matthew R; Just, Victoria J; Bowater, Laura; Fairhurst, Shirley A; Requena, Laura; Lawson, David M; Bornemann, Stephen

    2007-10-30

    Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features: a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share common catalytic steps that diverge when a carboxylate radical intermediate becomes protonated. The Bacillus subtilis decarboxylase has two manganese binding sites, and we proposed that Glu162 on a flexible lid is the site 1 general acid. We now demonstrate that a decarboxylase can be converted into an oxidase by mutating amino acids of the lid that include Glu162 with specificity switches of 282,000 (SEN161-3DAS), 275,000 (SENS161-4DSSN), and 225,000 (SENS161-4DASN). The structure of the SENS161-4DSSN mutant showed that site 2 was not affected. The requirement for substitutions other than of Glu162 was, at least in part, due to the need to decrease the Km for dioxygen for the oxidase reaction. Reversion of decarboxylase activity could be achieved by reintroducing Glu162 to the SENS161-4DASN mutant to give a relative specificity switch of 25,600. This provides compelling evidence for the crucial role of Glu162 in the decarboxylase reaction consistent with it being the general acid, for the role of the lid in controlling the Km for dioxygen, and for site 1 being the sole catalytically active site. We also report the trapping of carboxylate radicals produced during turnover of the mutant with the highest oxidase activity. Such radicals were also observed with the wild-type decarboxylase.

  2. 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.

  3. 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

  4. 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

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

    PubMed Central

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

    2016-01-01

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

  6. Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils.

    PubMed Central

    Dekker, L V; Leitges, M; Altschuler, G; Mistry, N; McDermott, A; Roes, J; Segal, A W

    2000-01-01

    We have analysed the involvement of the beta isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-beta is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcgamma-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-beta specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-beta, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-beta also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-beta isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcgamma receptors in neutrophils. PMID:10727429

  7. 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.

  8. The active site of cytochrome P-450 nifedipine oxidase: a model-building study.

    PubMed

    Ferenczy, G G; Morris, G M

    1989-12-01

    A model of the active site of cytochrome P-450 nifedipine oxidase is built on the basis of sequence homology with cytochrome P-450CAM. Substrates are docked into the binding pocket, and molecular mechanical energy minimization is performed to analyze the forces between the substrates and the enzyme.

  9. Season-controlled changes in biochemical constituents and oxidase enzyme activities in tomato (Lycopersicon esculentum Mill.).

    PubMed

    Sen, Supatra; Mukherji, S

    2009-07-01

    Season-controlled changes in biochemical constituents viz. carotenoids (carotene and xanthophyll) and pectic substances along with IAA-oxidase and polyphenol oxidase (PPO) enzyme activities were estimated/assayed in leaves of Lycopersicon esculentum Mill. (tomato) in two developmental stages--pre-flowering (35 days after sowing) and post-flowering (75 days after sowing) in three different seasons--summer rainy and winter Carotenoid content along with pectic substances were highest in winter and declined significantly in summer followed by rainy i.e. winter > summer > rainy. Carotenoid content was significantly higher in the pre-flowering as compared to post-flowering in all three seasons while pectic substances increased in the post-flowering as compared to pre-flowering throughout the annual cycle. IAA oxidase and PPO enzyme activities were enhanced in rainy and decreased sharply in summer and winter i.e. rainy > summer > winter. Both the enzymes exhibited higher activity in the post-flowering stage as compared to pre-flowering in all three seasons. These results indicate winter to be the most favourable season for tomato plants while rainy season environmental conditions prove to be unfavourable (stressful) with diminished content of carotenoid and pectic substances and low activities of IAA oxidase and PPO, ultimately leading to poor growth and productivity.

  10. 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.

  11. 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.

  12. Antioxidant effect of imperatorin from Angelica dahurica in hypertension via inhibiting NADPH oxidase activation and MAPK pathway.

    PubMed

    Cao, Yanjun; Zhang, Yanmin; Wang, Nan; He, Langchong

    2014-08-01

    Imperatorin (IMP) is an active furocoumarin in the traditional Chinese medicine Angelica dahurica and has been demonstrated to have vasodilatory activity. In the present study, we investigated the effect of IMP on blood pressure (BP) and antioxidant effects in spontaneously hypertensive rats (SHR) and human embryonic kidney 293 cells. SHR were administered IMP (6.25, 12.5, and 25 mg/kg/d) or tempol (18 mg/kg/d) daily by gavage for 12 weeks. Thiobarbituric acid-reactive substances, proteinuria levels, and superoxide dismutase activity were evaluated with commercial kits. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits of the renal cortical tissues were determined by reverse transcriptase polymerase chain reaction and Western blot. Twenty-four hour urinary 8-Iso-prostaglandin F2α was measured by enzyme linked immunosorbent assay. Systolic BP and diastolic BP were significantly reduced by treatment with IMP (6.25, 12.5, and 25 mg/kg/d) in SHR. Meanwhile, we found that renal cortical superoxide dismutase activities were significantly increased in IMP-treated groups. Renal cortical and urinary thiobarbituric acid-reactive substances' levels, the 24-hour urinary excretion of 8-Iso-prostaglandin F2α, and proteinuria in the IMP-treated group, were lower than SHR group. After that, we found the messenger RNA expressions and protein levels of NADPH oxidase subunits were markedly reduced after IMP treated in SHR. IMP also reduced the phosphorylation of protein kinase B, extracellular signal-regulated kinase1/2, p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase in renal cortical in SHR. In addition, H2O2-induced ROS production in human embryonic kidney 293 cells was markedly attenuated by IMP. H2O2-induced activation of MAPK, protein kinase B, and expression of NADPH oxidase were also attenuated by pretreatment of IMP. In summary, IMP showed antihypertensive effect via prevention of renal injury not only by reducing NADPH

  13. Effect of L-amino acid oxidase from Calloselasma rhodosthoma snake venom on human neutrophils.

    PubMed

    Pontes, Adriana S; da S Setúbal, Sulamita; Xavier, Caroline V; Lacouth-Silva, Fabianne; Kayano, Anderson M; Pires, Weverson L; Nery, Neriane Monteiro; Boeri de Castro, Onassis; da Silva, Silvana D; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Zuliani, Juliana P

    2014-03-01

    The in vitro effects of LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function were investigated. LAAO showed no toxicity on neutrophils. At non-cytotoxic concentrations, LAAO induced the superoxide anion production by isolated human neutrophil. This toxin, in its native form, is also able to stimulate the production of hydrogen peroxide in neutrophils, suggesting that its primary structure is essential for stimulation the cell. Moreover, the incubation of LAAO and phenol red medium did not induce the production of hydrogen peroxide. Furthermore, LAAO was able to stimulate neutrophils to release proinflammatory mediators such as IL-8 and TNF-α as well as NETs liberation. Together, the data showed that the LAAO triggers relevant proinflammatory events. Particular regions of the molecule distinct from the LAAO catalytic site may be involved in the onset of inflammatory events.

  14. Identification, cloning, and expression of L-amino acid oxidase from marine Pseudoalteromonas sp. B3.

    PubMed

    Yu, Zhiliang; Zhou, Ning; Qiao, Hua; Qiu, Juanping

    2014-01-01

    L-amino acid oxidase (LAAO) is attracting more attentions due to its broad and important biological functions. Recently, an LAAO-producing marine microorganism (strain B3) was isolated from the intertidal zone of Dinghai sea area, China. Physiological, biochemical, and molecular identifications together with phylogenetic analysis congruously suggested that it belonged to the genus Pseudoalteromonas. Therefore, it was designated as Pseudoalteromonas sp. B3. Its capability of LAAO production was crossly confirmed by measuring the products of H2O2, a-keto acids, and NH4+ in oxidization reaction. Two rounds of PCR were performed to gain the entire B3-LAAO gene sequence of 1608 bps in length encoding for 535 amino acid residues. This deduced amino acid sequence showed 60 kDa of the calculated molecular mass, supporting the SDS-PAGE result. Like most of flavoproteins, B3-LAAO also contained two conserved typical motifs, GG-motif and βαβ-dinucleotide-binding domain motif. On the other hand, its unique substrate spectra and sequence information suggested that B3-LAAO was a novel LAAO. Our results revealed that it could be functionally expressed in E. coli BL21(DE3) using vectors, pET28b(+) and pET20b(+). However, compared with the native LAAO, the expression level of the recombinant one was relatively low, most probably due to the formation of inclusion bodies. Several solutions are currently being conducted in our lab to increase its expression level.

  15. Implications of terminal oxidase function in regulation of salicylic acid on soybean seedling photosynthetic performance under water stress.

    PubMed

    Tang, Yanping; Sun, Xin; Wen, Tao; Liu, Mingjie; Yang, Mingyan; Chen, Xuefei

    2017-03-01

    The aim of this study is to investigate whether exogenous application of salicylic acid (SA) could modulate the photosynthetic capacity of soybean seedlings in water stress tolerance, and to clarify the potential functions of terminal oxidase (plastid terminal oxidase (PTOX) and alternative oxidase (AOX)) in SA' s regulation on photosynthesis. The effects of SA and water stress on gas exchange, pigment contents, chlorophyll fluorescence, enzymes (guaiacol peroxidase (POD; EC 1.11.1.7), superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11) and NADP-malate dehydrogenase (NADP-MDH; EC1.1.1.82)) activity and transcript levels of PTOX, AOX1, AOX2a, AOX2b were examined in a hydroponic cultivation system. Results indicate that water stress significantly decreased the photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (E), pigment contents (Chla + b, Chla/b, Car), maximum quantum yield of PSⅡphotochemistry (Fv/Fm), efficiency of excitation capture of open PSⅡcenter (Fv'/Fm'), quantum efficiency of PSⅡphotochemistry (ΦPSⅡ), photochemical quenching (qP), and increased malondialdehyde (MDA) content and the activity of all the enzymes. SA pretreatment led to significant decreases in Ci and MDA content, and increases in Pn, Gs, E, pigment contents, Fv/Fm, Fv'/Fm', ΦPSⅡ, qP, and the activity of all the enzymes. SA treatment and water stress alone significantly up-regulated the expression of PTOX, AOX1 and AOX2b. SA pretreatment further increased the transcript levels of PTOX and AOX2b of soybean seedling under water stress. These results indicate that SA application alleviates the water stress-induced decrease in photosynthesis may mainly through maintaining a lower reactive oxygen species (ROS) level, a greater PSⅡefficiency, and an enhanced alternative respiration and chlororespiration. PTOX and AOX may play important roles in SA-mediated resistance to water stress.

  16. 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.

  17. Artificial Warming and Rain Addition Increase Phenol Oxidase Activity in Arctic Soils

    NASA Astrophysics Data System (ADS)

    Kang, H.; Seo, J.; Jang, I.; Lee, Y. K.

    2014-12-01

    Artic tundra is one of the largest carbon stocks, of which amount is estimated up to 1,600 Pg. Global climate change models predict surface temperature rise and higher precipitation during summer in Arctic regions, raising concerns about faster decomposition of organic carbon and consequent releases of CO2, CH4 and DOC. Microorganisms are directly involved in decomposition process by releasing various extracellular enzymes. In particular, phenol oxidase was noted to play a key role because it is related to dynamics of highly recalcitrant carbon, which often represents a rate-limiting step of overall decomposition. In this study, we monitored phenol oxidase activity, hydrolases (β-glucosidase, cellobiohydrolase, N-acetylglucosaminidase and aminopeptidase), microbial abundance (qPCR) and chemical properties (δ13C and δ15N signatures) of tundra soils exposed to artificial warming and rain addition, by employing a passive chamber method in Cambridge Bay, Canada. Warming and rain addition combinedly increased phenol oxidase activity while no such changes were discernible for other hydrolases. Stable isotope signature indicates that warming induced water stress to the ecosystem and that nitrogen availability may be enhanced, which is partially responsible for the changes in enzyme activities. A short-term warming (2 years) may not accelerate mineralization of easily decomposable carbon, but may affect phenol oxidase which has the longer-term influence on recalcitrant carbon.

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

    PubMed

    Frenkel, C

    1972-05-01

    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.

  19. Fabrication of enzyme reactor utilizing magnetic porous polymer membrane for screening D-Amino acid oxidase inhibitors.

    PubMed

    Jiang, Jun Fang; Qiao, Juan; Mu, Xiao Yu; Moon, Myeong Hee; Qi, Li

    2017-04-01

    In this work, a unique D-amino acid oxidase reactor for enhanced enzymolysis efficiency is presented. A kind of magnetic polymer matrices, composed of iron oxide nanoparticles and porous polymer membrane (poly styrene-co-maleic anhydride), was prepared. With covalent bonding D-Amino acid oxidase on the surface of the matrices and characterization of scanning electron microscope and vibrating sample magnetometer, it demonstrated that the membrane enzyme reactor was successfully constructed. The enzymolysis efficiency of the enzyme reactor was evaluated and the apparent Michaelis-Menten constants of D-Amino acid oxidase were determined (Km was 1.10mM, Vmax was 23.8mMmin(-1)) by a chiral ligand exchange capillary electrophoresis protocol with methionine as the substrate. The results indicated that the enzyme reactor could exhibit good stability and excellent reusability. Importantly, because the enzyme and the substrate could be confined into the pores of the matrices, the enzyme reactor displayed the improved enzymolysis efficiency due to the confinement effect. Further, the prepared enzyme reactor was applied for D-Amino acid oxidase inhibitors screening. It has displayed that the proposed protocol could pave a new way for fabrication of novel porous polymer membrane based enzyme reactors to screen enzyme inhibitors.

  20. THE RESPIRATION AND CYTOCHROME OXIDASE ACTIVITY OF RAT AORTA IN EXPERIMENTAL HYPERTENSION

    PubMed Central

    Daly, Marie M.; Gurpide, E. Gambetta

    1959-01-01

    Oxygen consumption and cytochrome oxidase activity of aortas of rats with experimental hypertension were found to be higher than the corresponding values for aortas of normotensive animals. The higher metabolic activity of aortas of hypertensive animals appeared to be due both to an increase in the proportion of muscle cells to connective tissue fibers and to a higher activity of the intracellular portion of the tissue. PMID:13620848

  1. 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.

  2. 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.

  3. 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.

  4. Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein.

    PubMed

    Eklund, E A; Marshall, M; Gibbs, J B; Crean, C D; Gabig, T G

    1991-07-25

    Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric GTP-binding protein that is sensitive to pertussis toxin. Activation of this NADPH oxidase by arachidonate in a cell-free system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T. G., English, D., Akard, L. P., and Schell, M. J. (1987) (J. Biol. Chem. 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig, T. G., Eklund, E. A., Potter, G. B., and Dykes, J. R. (1990) J. Immunol. 145, 945-951). In the present study, immunodepletion of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH oxidase in the membrane fraction. The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction from neutrophil cytosol that contained a 21-kDa GTP-binding protein. Purified human recombinant Krev-1 p21 also completely reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive activity. Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43) completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide. An inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation. Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot. Thus a G protein in the cytosol of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for NADPH oxidase activation. This system represents a unique model to study molecular interactions of a ras-like G

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

    PubMed

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

    2010-11-15

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

  6. 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

  7. [Activity of nonspecific hepatic oxidases and the biological effects of the antineoplastic antibiotic adriamycin].

    PubMed

    Bogush, T A; Syrkin, A B; Donenko, F V

    1981-10-01

    It was shown in male CBA mice that toxic doses (15 and 20 mg/kg) of adriamycin (AD) inhibited the activity of nonspecific liver oxidases and noticeably increased the duration of the animals' sleep after injection of hexenal which is a substrate of this enzymatic system. The inhibitory effect of AD remained unchanged in the course of 9 days of the experiment. The nontoxic dose of AD (5 mg/kg) inhibited the activity of the enzymatic system on the 2nd--3rd days after the injection of the drug. Meanwhile the activity of the enzymatic system returned to the level seen in intact animals by days 5--6. The toxic action of AD declined on activation of nonspecific liver oxidases with phenobarbital and rose as a result of administering the inhibitor SKF 525-A. The authors discuss whether it is possible to use the data obtained for clinical application of AD.

  8. 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

  9. 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

  10. NADPH oxidase activity in allergenic pollen grains of different plant species.

    PubMed

    Wang, Xiao-Ling; Takai, Toshiro; Kamijo, Seiji; Gunawan, Hendra; Ogawa, Hideoki; Okumura, Ko

    2009-09-25

    Pollen is an important trigger of allergic diseases. Recent studies have shown that ragweed pollen NAD(P)H oxidase generates reactive oxygen species (ROS) and plays a prominent role in the pathogenesis of allergies in mouse models. Here, we demonstrated that allergenic pollen grains showed NAD(P)H oxidase activity that differed in intensity and localization according to the plant families. The activity occurred at the surface or in the cytoplasm in pollen of grasses, birch, and ragweed; in subpollen particles released from ragweed pollen; and at the inner surface or in the cytoplasm but not on the outer wall, which was sloughed off after the rupture, of pollen of Japanese cedar and Japanese cypress. The activity was mostly concentrated within insoluble fractions, suggesting that it facilitates the exposure of tissues to ROS generated by this enzyme. The extent of exposure to pollen-generated ROS could differ among the plant families.

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

    PubMed

    Metz, Sebastian; Thiel, Walter

    2010-01-28

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

  12. 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.

  13. Bio-inspired amino acid oxidation by a non-heme iron catalyst modeling the action of 1-aminocyclopropane-1-carboxylic acid oxidase.

    PubMed

    Baráth, Gábor; Kaizer, József; Pap, József Sándor; Speier, Gábor; El Bakkali-Taheri, Nadia; Simaan, A Jalila

    2010-10-21

    In this communication we describe the first example of a biomimetic mononuclear iron complex, [Fe(III)(Salen)Cl] (Salen = N,N'-bis(salicylidene)-ethylenediaminato), that highly selectively and efficiently catalyzes the oxidation of 1-aminocyclopropane-1-carboxylic acid (ACCH), α-aminoisobutyric acid (AIBH), and alanine (ALAH) to ethylene or the corresponding carbonyl compounds, mimicking the action of the non-heme iron enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO).

  14. 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.

  15. Mechanism of the cytotoxic effect of l-amino acid oxidase isolated from Bothrops alternatus snake venom.

    PubMed

    Ribeiro, Patrícia H; Zuliani, Juliana P; Fernandes, Carla F C; Calderon, Leonardo A; Stábeli, Rodrigo G; Nomizo, Auro; Soares, Andreimar M

    2016-11-01

    BaltLAAO-I, an L-amino acid oxidase isolated from Bothrops alternatus, is a glycoprotein enzyme with a pI-5.3, 15% sugar and a related molecular mass of 66,000Da in its monomeric form, and 123,000Da in its dimeric form. The objective of this study is to describe the cytotoxicity activity induced by BaltLAAO-I isolated from Bothrops alternatus venom and its possible mechanism of action on tumor cells. Our results clearly depict that BaltLAAO-I has a strong selective cytotoxic activity on tumor cell lines (JURKAT, SK-BR-3 and B16F10). On the other hand, the results show low cytotoxicity on human peripheral blood mononuclear cells. Furthermore, our findings demonstrate that BaltLAAO-I induces the apoptosis of tumor cell lines through a cytotoxic activity exerted by a generation of reactive oxygen intermediates. All in all, the data indicate that LAAOs exert a selective cytotoxic role on tumor cells, demonstrating a great potential for future use in clinical therapy.

  16. RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes.

    PubMed

    Filina, Julia V; Gabdoulkhakova, Aida G; Safronova, Valentina G

    2014-10-01

    Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1μM fMLF and 1μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells.

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

    PubMed Central

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

    1988-01-01

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

  18. 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.

  19. Elucidation of the factors affecting the oxidative activity of Acremonium sp. HI-25 ascorbate oxidase by an electrochemical approach.

    PubMed

    Murata, Kenichi; Nakamura, Nobuhumi; Ohno, Hiroyuki

    2008-03-07

    Steady-state kinetics of Acremonium sp. HI-25 ascorbate oxidase toward p-hydroquinone derivatives have been examined by using an electrochemical analysis based on the theory of steady-state bioelectrocatalysis. The electrochemical technique has enabled one to examine the influence of electronic and chemical properties of substrates on the activity. It was proven that the oxidative activity of ascorbate oxidase was dominated by the highly selective substrate-binding affinity based on electrostatic interaction beyond the one-electron redox potential difference between ascorbate oxidase's type 1 copper site and substrate.

  20. Elucidation of the factors affecting the oxidative activity of Acremonium sp. HI-25 ascorbate oxidase by an electrochemical approach

    SciTech Connect

    Murata, Kenichi; Nakamura, Nobuhumi Ohno, Hiroyuki

    2008-03-07

    Steady-state kinetics of Acremonium sp. HI-25 ascorbate oxidase toward p-hydroquinone derivatives have been examined by using an electrochemical analysis based on the theory of steady-state bioelectrocatalysis. The electrochemical technique has enabled one to examine the influence of electronic and chemical properties of substrates on the activity. It was proven that the oxidative activity of ascorbate oxidase was dominated by the highly selective substrate-binding affinity based on electrostatic interaction beyond the one-electron redox potential difference between ascorbate oxidase's type 1 copper site and substrate.

  1. 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

  2. Why copper is preferred over iron for oxygen activation and reduction in haem-copper oxidases

    NASA Astrophysics Data System (ADS)

    Bhagi-Damodaran, Ambika; Michael, Matthew A.; Zhu, Qianhong; Reed, Julian; Sandoval, Braddock A.; Mirts, Evan N.; Chakraborty, Saumen; Moënne-Loccoz, Pierre; Zhang, Yong; Lu, Yi

    2016-11-01

    Haem-copper oxidase (HCO) catalyses the natural reduction of oxygen to water using a haem-copper centre. Despite decades of research on HCOs, the role of non-haem metal and the reason for nature's choice of copper over other metals such as iron remains unclear. Here, we use a biosynthetic model of HCO in myoglobin that selectively binds different non-haem metals to demonstrate 30-fold and 11-fold enhancements in the oxidase activity of Cu- and Fe-bound HCO mimics, respectively, as compared with Zn-bound mimics. Detailed electrochemical, kinetic and vibrational spectroscopic studies, in tandem with theoretical density functional theory calculations, demonstrate that the non-haem metal not only donates electrons to oxygen but also activates it for efficient O-O bond cleavage. Furthermore, the higher redox potential of copper and the enhanced weakening of the O-O bond from the higher electron density in the d orbital of copper are central to its higher oxidase activity over iron. This work resolves a long-standing question in bioenergetics, and renders a chemical-biological basis for the design of future oxygen-reduction catalysts.

  3. Aryl hydrocarbon receptor modulates NADPH oxidase activity via direct transcriptional regulation of p40phox expression.

    PubMed

    Wada, Taira; Sunaga, Hiroshi; Ohkawara, Reiko; Shimba, Shigeki

    2013-05-01

    A member of the NADPH oxidase subunits, p40(phox) plays an important role in the regulation of NADPH oxidase activity and the subsequent production of reactive oxygen species (ROS). In this study, we show that mouse p40(phox) is a novel transcriptional target of the aryl hydrocarbon receptor (AhR), known as a dioxin receptor or xenobiotic receptor, in the liver. Treatment of mice with 3-methylcholanthrene (3MC) increased p40(phox) gene expression in the liver, but this induction of p40(phox) gene expression was diminished by the deletion of the AhR gene in the liver. Consistent with the in vivo results, the expression of the p40(phox) gene was increased in 3MC-treated Hepa1c1c7 cells in an AhR-dependent manner. In addition, promoter analysis established p40(phox) as a transcriptional target of AhR. Studies using the RNA-interference technique revealed that p40(phox) is involved in the increase of NADPH oxidase activity and the subsequent ROS production in AhR-activated Hepa1c1c7 cells. Consequently, the results obtained here may provide a novel molecular mechanism for ROS production after exposure to dioxins.

  4. Why copper is preferred over iron for oxygen activation and reduction in haem-copper oxidases.

    PubMed

    Bhagi-Damodaran, Ambika; Michael, Matthew A; Zhu, Qianhong; Reed, Julian; Sandoval, Braddock A; Mirts, Evan N; Chakraborty, Saumen; Moënne-Loccoz, Pierre; Zhang, Yong; Lu, Yi

    2017-03-01

    Haem-copper oxidase (HCO) catalyses the natural reduction of oxygen to water using a haem-copper centre. Despite decades of research on HCOs, the role of non-haem metal and the reason for nature's choice of copper over other metals such as iron remains unclear. Here, we use a biosynthetic model of HCO in myoglobin that selectively binds different non-haem metals to demonstrate 30-fold and 11-fold enhancements in the oxidase activity of Cu- and Fe-bound HCO mimics, respectively, as compared with Zn-bound mimics. Detailed electrochemical, kinetic and vibrational spectroscopic studies, in tandem with theoretical density functional theory calculations, demonstrate that the non-haem metal not only donates electrons to oxygen but also activates it for efficient O-O bond cleavage. Furthermore, the higher redox potential of copper and the enhanced weakening of the O-O bond from the higher electron density in the d orbital of copper are central to its higher oxidase activity over iron. This work resolves a long-standing question in bioenergetics, and renders a chemical-biological basis for the design of future oxygen-reduction catalysts.

  5. Planarian D-amino acid oxidase is involved in ovarian development during sexual induction.

    PubMed

    Maezawa, Takanobu; Tanaka, Hiroyuki; Nakagawa, Haruka; Ono, Mizuki; Aoki, Manabu; Matsumoto, Midori; Ishida, Tetsuo; Horiike, Kihachiro; Kobayashi, Kazuya

    2014-05-01

    To elucidate the molecular mechanisms underlying switching from asexual to sexual reproduction, namely sexual induction, we developed an assay system for sexual induction in the hermaphroditic planarian species Dugesia ryukyuensis. Ovarian development is the initial and essential step in sexual induction, and it is followed by the formation of other reproductive organs, including the testes. Here, we report a function of a planarian D-amino acid oxidase, Dr-DAO, in the control of ovarian development in planarians. Asexual worms showed significantly more widespread expression of Dr-DAO in the parenchymal space than did sexual worms. Inhibition of Dr-DAO by RNAi caused the formation of immature ovaries. In addition, we found that feeding asexual worms 5 specific D-amino acids could induce the formation of immature ovaries that are similar to those observed in Dr-DAO knockdown worms, suggesting that Dr-DAO inhibits the formation of immature ovaries by degrading these D-amino acids. Following sexual induction, Dr-DAO expression was observed in the ovaries. The knockdown of Dr-DAO during sexual induction delayed the maturation of the other reproductive organs, as well as ovary. These findings suggest that Dr-DAO acts to promote ovarian maturation and that complete sexual induction depends on the production of mature ovaries. We propose that Dr-DAO produced in somatic cells prevents the onset of sexual induction in the asexual state, and then after sexual induction, the female germ cells specifically produce Dr-DAO to induce full maturation. Therefore, Dr-DAO produced in somatic and female germline cells may play different roles in sexual induction.

  6. Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas

    PubMed Central

    1987-01-01

    Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta- aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space. PMID:2888772

  7. Inhibition of NADPH oxidase activation in endothelial cells by ortho-methoxy-substituted catechols.

    PubMed

    Johnson, David K; Schillinger, Kurt J; Kwait, David M; Hughes, Chambers V; McNamara, Erin J; Ishmael, Fauod; O'Donnell, Robert W; Chang, Ming-Mei; Hogg, Michael G; Dordick, Jonathan S; Santhanam, Lakshmi; Ziegler, Linda M; Holland, James A

    2002-01-01

    NADPH oxidase is a major enzymatic source of oxygen free radicals in stimulated endothelial cells (ECs). The ortho-methoxy-substituted catechol, apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, inhibits the release of superoxide anion (O2*-) by this enzyme. The compound acts by blocking the assembly of a functional NADPH oxidase complex. The underlying chemistry of this inhibitory activity, and its physiological significance to EC proliferation, have been investigated. A critical event is the reaction of ortho-methoxy-substituted catechols with reactive oxygen species (ROS) and peroxidase. Analysis of this reaction reveals that apocynin is converted to a symmetrical dimer through the formation of a 5,5' carbon-carbon bond. Both reduced glutathione and L-cysteine inhibit this dimerization process. Catechols without the ortho-methoxy-substituted group do not undergo this chemical reaction. Superoxide production by an endothelial cell-free system incubated with apocynin was nearly completely inhibited after a lagtime for inhibition of ca. 2 min. Conversely, O2*- production was nearly completely inhibited, without a lagtime, by incubation with the dimeric form of apocynin. The apocynin dimer undergoes a two-electron transfer reaction with standard redox potentials of -0.75 and -1.34 V as determined by cyclic voltammetry. Inhibition of endothelial NADPH oxidase by apocynin caused a dose-dependent inhibition of cell proliferation. These findings identify a metabolite of an ortho-methoxy-substituted catechol, which may be the active compound formed within stimulated ECs that prevents NADPH oxidase complex assembly and activation.

  8. Cell-free activation of phagocyte NADPH-oxidase: tissue and differentiation-specific expression of cytosolic cofactor activity.

    PubMed

    Parkinson, J F; Akard, L P; Schell, M J; Gabig, T G

    1987-06-30

    We examined a variety of tissues for the presence of cytosolic cofactor activity that would support arachidonate-dependent cell-free activation of NADPH-oxidase in isolated human neutrophil membranes. Cofactor activity was not found in cytosol isolated from erythrocytes, lymphocytes, placenta, brain, liver, or the human promyelocytic leukemic cell line HL-60. Induction of differentiation in HL-60 cells led to expression of cytosolic cofactor activity. In dimethylsulphoxide-induced HL-60 cells the level of cytosolic cofactor activity was closely correlated with phorbol myristate acetate-stimulated whole cell superoxide production. These results strongly suggest that the cytosolic cofactor is a phagocyte-specific regulatory protein of physiologic importance in NADPH-oxidase activation.

  9. CR-LAAO, an L-amino acid oxidase from Calloselasma rhodostoma venom, as a potential tool for developing novel immunotherapeutic strategies against cancer

    PubMed Central

    Costa, Tássia R.; Menaldo, Danilo L.; Zoccal, Karina F.; Burin, Sandra M.; Aissa, Alexandre F.; Castro, Fabíola A. de; Faccioli, Lúcia H.; Greggi Antunes, Lusânia M.; Sampaio, Suely V.

    2017-01-01

    L-amino acid oxidases from snake venoms have been described to possess various biological functions. In this study, we investigated the inflammatory responses induced in vivo and in vitro by CR-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodostoma venom, and its antitumor potential. CR-LAAO induced acute inflammatory responses in vivo, with recruitment of neutrophils and release of IL-6, IL-1β, LTB4 and PGE2. In vitro, IL-6 and IL-1β production by peritoneal macrophages stimulated with CR-LAAO was dependent of the activation of the Toll-like receptors TLR2 and TLR4. In addition, CR-LAAO promoted apoptosis of HL-60 and HepG2 tumor cells mediated by the release of hydrogen peroxide and activation of immune cells, resulting in oxidative stress and production of IL-6 and IL-1β that triggered a series of events, such as activation of caspase 8, 9 and 3, and the expression of the pro-apoptotic gene BAX. We also observed that CR-LAAO modulated the cell cycle of these tumor cells, promoting delay in the G0/G1 and S phases. Taken together, our results suggest that CR-LAAO could serve as a potential tool for the development of novel immunotherapeutic strategies against cancer, since this toxin promoted apoptosis of tumor cells and also activated immune cells against them. PMID:28205610

  10. A rational protocol for the successful crystallization of l-amino-acid oxidase from Bothrops atrox

    PubMed Central

    Alves, Raquel Melo; Feliciano, Patricia Rosa; Sampaio, Suely Vilela; Nonato, Maria Cristina

    2011-01-01

    Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme l-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 73.64, b = 123.92, c = 105.08 Å, β = 96.03°. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient V M of 2.12 Å3 Da−1, corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques. PMID:21505245

  11. [Liver monoamine oxidase activity of the lamprey Lampetra fluviatilis. the substrate-inhibitory specificity].

    PubMed

    Iagodina, O V; Basova, I N

    2013-01-01

    Based on data of substrate-inhibitory analysis with use of specific inhibitors--deprenyl, chlorgi-lin--and specific substrates--serotonin, noradrenalin, benzylamine, beta-phenylethylamine, and N-methylhistamine--a suggestion is put forward about the possible existence of one molecular form of monoamine oxidase (MAO) in liver of mature individuals of the European lamprey Lampetra fluviatilis. There are determined kinetic parameters of monoamine oxidase deamination of eight substrates, which indicates the large spectrum of substrate specificity of the lamprey liver MAO. The studied enzyme does not deaminate histamine and putrescine and is not sensitive to 10(-2) M semicarbaside. Results of study of the substrate-inhibitor specificity allow us to suggest some resemblance of catalytic properties of the lamprey liver MAO and the mammalian form A MAO. The revealed low activity of the enzyme at deamination of all used substrates seems to be connected with low detoxational functional of the lamprey liver.

  12. Synthetic models of the active site of catechol oxidase: mechanistic studies.

    PubMed

    Koval, Iryna A; Gamez, Patrick; Belle, Catherine; Selmeczi, Katalin; Reedijk, Jan

    2006-09-01

    The ability of copper proteins to process dioxygen at ambient conditions has inspired numerous research groups to study their structural, spectroscopic and catalytic properties. Catechol oxidase is a type-3 copper enzyme usually encountered in plant tissues and in some insects and crustaceans. It catalyzes the conversion of a large number of catechols into the respective o-benzoquinones, which subsequently auto-polymerize, resulting in the formation of melanin, a dark pigment thought to protect a damaged tissue from pathogens. After the report of the X-ray crystal structure of catechol oxidase a few years earlier, a large number of publications devoted to the biomimetic modeling of its active site appeared in the literature. This critical review (citing 114 references) extensively discusses the synthetic models of this enzyme, with a particular emphasis on the different approaches used in the literature to study the mechanism of the catalytic oxidation of the substrate (catechol) by these compounds. These are the studies on the substrate binding to the model complexes, the structure-activity relationship, the kinetic studies of the catalytic oxidation of the substrate and finally the substrate interaction with (per)oxo-dicopper adducts. The general overview of the recognized types of copper proteins and the detailed description of the crystal structure of catechol oxidase, as well as the proposed mechanisms of the enzymatic cycle are also presented.

  13. Coproporphyrinogen oxidase activity and porphyrin concentrations in peripheral red blood cells in hereditary sideroblastic anaemia.

    PubMed

    Pasanen, A V; Eklöf, M; Tenhunen, R

    1985-03-01

    The activity of coproporphyrinogen oxidase and the concentrations of coproporphyrin and protoporphyrin (measured by HPLC) in peripheral red blood cells were established in 2 families with different types of hereditary sideroblastic anaemia. 2 males and 4 females were members of a family with an X-chromosome-linked and pyridoxine-responsive HSA, and 3 females were members of another family where the mode of inheritance is not clear and where pyridoxine did not produce a haematological response. Coproporphyrinogen oxidase activity was normal in 8 of 9 patients and slightly decreased only in 1 patient. All patients had normal red cell coproporphyrin concentrations, but red cell protoporphyrin concentration was decreased in 4 patients. These findings indicate that in vivo haem synthesis was not impaired at the step of coproporphyrinogen oxidase, hence enzymatic defects in earlier steps of haem synthesis are more evident. Earlier suggestions of impaired haem synthesis at this level, based on observed increased concentrations of coproporphyrin in peripheral red blood cells might be explained by the use of unspecific methods.

  14. 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).

  15. 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.

  16. NADPH oxidase activation played a critical role in the oxidative stress process in stable coronary artery disease

    PubMed Central

    Zhang, Jiefang; Wang, Meihui; Li, Zhengwei; Bi, Xukun; Song, Jiale; Weng, Shaoxiang; Fu, Guosheng

    2016-01-01

    Objectives: The study was designed to investigate the oxidative stress levels of endothelial progenitor cells (EPCs) in stable coronary artery disease (CAD) and to explore the underlying mechanisms of NADPH oxidase activation and subsequent EPCs dysfunction. Methods: EPCs were isolated from patients with stable CAD (n=50) and matched healthy volunteers (n=50). NADPH oxidase activation was detected by measuring the expression of each subunit using western blotting and qPCR analyses and the membrane translocation of p47phox using immunofluorescence. The in vivo angiogenesis capacity was evaluated using immunofluorescence by transplanting EPCs into a rat hind limb ischemia model. The PKC inhibitor GÖ-6983 was used to determine the role of PKC in NADPH oxidase activation. Results: Oxidative stress level was increased and the in vivo angiogenesis capacity was impaired in EPCs obtained from CAD subjects with the activation of NADPH oxidase. P47phox membrane translocation increased in CAD group vs controls. These effects were resolved by NADPH oxidase inhibition. Up-regulation of PKCα/β2 was found in EPCs from CAD subjects, PKC inhibition GÖ-6983 could reduce the expression and activity of NADPH oxidation. Conclusions: NADPH oxidase activation via p47phox membrane translocation played a critical role in the initiation and progression of CAD, and the PKCα/β2 signaling pathway might be involved. PMID:28077995

  17. Characterization and cytotoxicity of L-amino acid oxidase from the venom of king cobra (Ophiophagus hannah).

    PubMed

    Ahn, M Y; Lee, B M; Kim, Y S

    1997-06-01

    The aim of this project was to determine the cytotoxic components from the venom of king cobra, Ophiophagus hannah. Venom was purified by a combination of gel-filtration, ion-exchange and reversed-phase chromatographic steps. The biochemical properties of the cytotoxic component were consistent with those of L-amino acid oxidase. The molecular weight of the enzyme was estimated to be 150,000 by gel filtration and 70,000 under the denaturing conditions of SDS-PAGE, indicating a dimer. It has an isoelectric point of 4.5 and is a glycoprotein. The N-terminal sequence of L-amino acid oxidase from the king cobra venom was determined to be SVINLEESFQEPEYE. The cytotoxicity of L-amino acid oxidase was observed in stomach cancer, murine melanoma, fibrosarcoma, colorectal cancer and Chinese hamster ovary cell lines. Cytotoxicity resulted in the loss of ability in attachment and inhibition of cell proliferation. The cytotoxic protein decreased the level of cell proliferation by 74% according to [3H]thymidine uptake assay. The mechanism of enzyme action may be related to the inhibition of thymidine incorporation and an interaction with DNA.

  18. Cloning and characterization of the gene for L-amino acid oxidase in hybrid tilapia.

    PubMed

    Shen, Yubang; Fu, Gui Hong; Liu, Feng; Yue, Gen Hua

    2015-12-01

    Tilapia is the common name for a group of cichlid fishes. Identification of DNA markers significantly associated with important traits in candidate genes may speed up genetic improvement. L-Amino acid oxidase (LAO) plays a crucial role in the innate immune defences of animals. Previously, whether LAO variants were associated with economic traits had not been studied in fish. We characterized the cDNA sequence of the LAO gene of hybrid tilapia (Oreochromis spp.). Its ORF was 1536 bp, encoding a flavoenzyme of 511 amino acids. This gene consisted of seven exons and six introns. Its expression was detected in the intestine, blood, kidney, skin, liver. It was highly expressed in the intestine. After a challenge with a bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the liver, intestine and spleen (P < 0.05). We identified one SNP in the genomic sequence of the gene and found that this SNP was associated significantly with body length (P < 0.05), but not with resistance to S. agalactiae. The results of this study suggest that the LAO gene plays an important role in innate immune responses to the bacterial pathogen in tilapia. The investigation of relationship between polymorphism of LAO gene and disease resistance and growth in tilapia showed that one SNP was associated significantly with body length. Further experiments on whether SNPs in the LAO gene are associated with growth in tilapia and other populations could be useful in understanding more functions of the LAO gene.

  19. 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.

  20. Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing L-amino acid oxidase from ink of the sea hare Aplysia californica.

    PubMed

    Yang, Hsiuchin; Johnson, Paul Micah; Ko, Ko-Chun; Kamio, Michiya; Germann, Markus W; Derby, Charles D; Tai, Phang C

    2005-09-01

    A 60 kDa monomeric protein isolated from the defensive purple ink secretion of the sea hare Aplysia californica was cloned and sequenced, and is the first sea hare antimicrobial protein to be functionally expressed in E. coli. Sequence analysis suggested that this protein is a flavin-containing l-amino acid oxidase (LAAO), with one predicted potential glycosylation site, although the glycosylation could not be experimentally confirmed. This protein, which we call ;escapin', has high sequence similarity to several other gastropod proteins. Escapin was verified by NMR, mass spectroscopy and HPLC to have FAD as its flavin cofactor. Escapin's antimicrobial effects, bacteriostasis and bactericidal, were determined using a combination of two assays: (1) incubation of bacteria on solid media followed by assessment of inhibition by direct observation of zones of inhibition or by turbidity measurements; and (2) incubation of bacteria in liquid media followed by counting viable colonies after growing on agar plates. Native escapin inhibited the growth of Gram-positive and Gram-negative bacteria, including marine bacteria (Vibrio harveyii and Staphylococcus aureus) and pathogenic bacteria (Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa). Escapin also inhibited the growth of yeast and fungi, with different efficacies. Escapin's antimicrobial activity was concentration dependent and did not decrease when stored for more than 5 months at room temperature. Escapin was bacteriostatic and not bactericidal in minimal media (e.g. salt media) with glucose, yeast extract, and a mixture of 20 amino acids each at 50 micromol l(-1), but was bactericidal in media enriched with Tryptone Peptone. Escapin was also strongly bactericidal in media with l-lysine at concentrations as low as 3 mmol l(-1) and slightly bactericidal in 50 mmol l(-1) l-arginine, but not in most other amino acids even at 50 mmol l(-1). Escapin had high oxidase activity (producing hydrogen

  1. Size-selective QD@MOF core-shell nanocomposites for the highly sensitive monitoring of oxidase activities.

    PubMed

    Wang, Ke; Li, Nan; Zhang, Jing; Zhang, Zhiqi; Dang, Fuquan

    2017-01-15

    In this work, we proposed a novel and facile method to monitor oxidase activities based on size-selective fluorescent quantum dot (QD)@metal-organic framework (MOF) core-shell nanocomposites (CSNCPs). The CSNCPs were synthesized from ZIF-8 and CdTe QDs in aqueous solution in 40min at room temperature with stirring. The prepared CdTe@ZIF-8 CSNCPs , which have excellent water dispersibility and stability, displays distinct fluorescence responses to hole scavengers of different molecular sizes (e.g., H2O2, substrate, and oxidase) due to the aperture limitation of the ZIF-8 shell. H2O2 can efficiently quench the fluorescence of CdTe@ZIF-8 CSNCPs over a linearity range of 1-100nM with a detection limit of 0.29nM, whereas large molecules such as substrate and oxidase have very little effect on its fluorescence. Therefore, the highly sensitive detection of oxidase activities was achieved by monitoring the fluorescence quenching of CdTe@ZIF-8 CSNCPs by H2O2 produced in the presence of substrate and oxidase, which is proportional to the oxidase activities. The linearity ranges of the uricase and glucose oxidase activity are 0.1-50U/L and 1-100U/L, respectively, and their detection limits are 0.024U/L and 0.26U/L, respectively. Therefore, the current QD@MOF CSNCPs based sensing system is a promising, widely applicable means of monitoring oxidase activities in biochemical research.

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

    PubMed

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

    2011-01-01

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

  3. [Alternative oxidase in industrial fungi].

    PubMed

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

    2015-01-01

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

  4. Polyphenol oxidase activity and differential accumulation of polyphenolics in seed coats of pinto bean (Phaseolus vulgaris L.) characterize postharvest color changes.

    PubMed

    Marles, M A Susan; Vandenberg, Albert; Bett, Kirstin E

    2008-08-27

    Postharvest darkening of pinto bean (Phaseolus vulgaris L.) was evaluated in a population of recombinant inbred lines derived from a cross between CDC Pintium (a regular-darkening line) and 1533-15 (a slow-darkening line). Flavonoid metabolite concentrations, polyphenol oxidase activity, lignin concentration, and seed coat anatomy characteristics were assessed for cosegregation with the darkening phenotype. Significantly lower kaempferol concentrations (p = 0.00001) together with differences in polyphenol oxidase activity (p = 0.0045) were two of the key findings associated with these recombinant inbred lines. In addition, two different assays (thioglycolic acid and Klason lignin) to quantify lignin together with an assessment of extractable condensed tannin were used to estimate the contribution of these polymers to changes in the seed coat tissue. This is the first report of precise biochemical characterization of polyphenolics that associate with postharvest darkening in legumes.

  5. Functional biomimetic models for the active site in the respiratory enzyme cytochrome c oxidase.

    PubMed

    Collman, James P; Decréau, Richard A

    2008-11-07

    A functional analog of the active site in the respiratory enzyme, cytochrome c oxidase (CcO) reproduces every feature in CcO's active site: a myoglobin-like heme (heme a3), a distal tridentate imidazole copper complex (Cu(B)), a phenol (Tyr244), and a proximal imidazole. When covalently attached to a liquid-crystalline SAM film on an Au electrode, this functional model continuously catalyzes the selective four-electron reduction of dioxygen at physiological potential and pH, under rate-limiting electron flux (as occurs in CcO).

  6. Comparison of brain mitochondrial cytochrome c oxidase activity with cyanide LD(50) yields insight into the efficacy of prophylactics.

    PubMed

    Marziaz, Mandy L; Frazier, Kathryn; Guidry, Paul B; Ruiz, Robyn A; Petrikovics, Ilona; Haines, Donovan C

    2013-01-01

    Cyanide inhibits cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory pathway, therefore inhibiting the cell oxygen utilization and resulting in the condition of histotoxic anoxia. The enzyme rhodanese detoxifies cyanide by utilizing sulfur donors to convert cyanide to thiocyanate, and new and improved sulfur donors are actively sought as researchers seek to improve cyanide prophylactics. We have determined brain cytochrome c oxidase activity as a marker for cyanide exposure for mice pre-treated with various cyanide poisoning prophylactics, including sulfur donors thiosulfate (TS) and thiotaurine (TT3). Brain mitochondria were isolated by differential centrifugation, the outer mitochondrial membrane was disrupted by a maltoside detergent, and the decrease in absorbance at 550 nm as horse heart ferrocytochrome c (generated by the dithiothreitol reduction of ferricytochrome c) was oxidized was monitored. Overall, the TS control prophylactic treatment provided significant protection of the cytochrome c oxidase activity. The TT3-treated mice showed reduced cytochrome c oxidase activity even in the absence of cyanide. In both treatment series, addition of exogenous Rh did not significantly enhance the prevention of cytochrome c oxidase inhibition, but the addition of sodium nitrite did. These findings can lead to a better understanding of the protection mechanism by various cyanide antidotal systems.

  7. 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.

  8. 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

  9. Acute Ethanol Intake Induces NAD(P)H Oxidase Activation and Rhoa Translocation in Resistance Arteries

    PubMed Central

    Simplicio, Janaina A.; Hipólito, Ulisses Vilela; do Vale, Gabriel Tavares; Callera, Glaucia Elena; Pereira, Camila André; Touyz, Rhian M; Tostes, Rita de Cássia; Tirapelli, Carlos R.

    2016-01-01

    Background The mechanism underlying the vascular dysfunction induced by ethanol is not totally understood. Identification of biochemical/molecular mechanisms that could explain such effects is warranted. Objective To investigate whether acute ethanol intake activates the vascular RhoA/Rho kinase pathway in resistance arteries and the role of NAD(P)H oxidase-derived reactive oxygen species (ROS) on such response. We also evaluated the requirement of p47phox translocation for ethanol-induced NAD(P)H oxidase activation. Methods Male Wistar rats were orally treated with ethanol (1g/kg, p.o. gavage) or water (control). Some rats were treated with vitamin C (250 mg/kg, p.o. gavage, 5 days) before administration of water or ethanol. The mesenteric arterial bed (MAB) was collected 30 min after ethanol administration. Results Vitamin C prevented ethanol-induced increase in superoxide anion (O2-) generation and lipoperoxidation in the MAB. Catalase and superoxide dismutase activities and the reduced glutathione, nitrate and hydrogen peroxide (H2O2) levels were not affected by ethanol. Vitamin C and 4-methylpyrazole prevented the increase on O2- generation induced by ethanol in cultured MAB vascular smooth muscle cells. Ethanol had no effect on phosphorylation levels of protein kinase B (Akt) and eNOS (Ser1177 or Thr495 residues) or MAB vascular reactivity. Vitamin C prevented ethanol-induced increase in the membrane: cytosol fraction ratio of p47phox and RhoA expression in the rat MAB. Conclusion Acute ethanol intake induces activation of the RhoA/Rho kinase pathway by a mechanism that involves ROS generation. In resistance arteries, ethanol activates NAD(P)H oxidase by inducing p47phox translocation by a redox-sensitive mechanism. PMID:27812679

  10. Inhibition of NADPH oxidase activation by 4-(2-aminoethyl)-benzenesulfonyl fluoride and related compounds.

    PubMed

    Diatchuk, V; Lotan, O; Koshkin, V; Wikstroem, P; Pick, E

    1997-05-16

    The elicitation of an oxidative burst in phagocytes rests on the assembly of a multicomponental complex (NADPH oxidase) consisting of a membrane-associated flavocytochrome (cytochrome b559), representing the redox element responsible for the NADPH-dependent reduction of oxygen to superoxide (O-2), two cytosolic components (p47(phox), p67(phox)), and the small GTPase Rac (1 or 2). We found that 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), an irreversible serine protease inhibitor, prevented the elicitation of O-2 production in intact macrophages and the amphiphile-dependent activation of NADPH oxidase in a cell-free system, consisting of solubilized membrane or purified cytochrome b559 combined with total cytosol or a mixture of recombinant p47(phox), p67(phox), and Rac1. AEBSF acted at the activation step and did not interfere with the ensuing electron flow. It did not scavenge oxygen radicals and did not affect assay reagents. Five other serine protease inhibitors (three irreversible and two reversible) were found to lack an inhibitory effect on cell-free activation of NADPH oxidase. A structure-function study of AEBSF analogues demonstrated that the presence of a sulfonyl fluoride group was essential for inhibitory activity and that compounds containing an aminoalkylbenzene moiety were more active than amidinobenzene derivatives. Exposure of the membrane fraction or of purified cytochrome b559, but not of cytosol or recombinant cytosolic components, to AEBSF, in the presence of a critical concentration of the activating amphiphile lithium dodecyl sulfate, resulted in a marked impairment of their ability to support cell-free NADPH oxidase activation upon complementation with untreated cytosol or cytosolic components. Kinetic analysis of the effect of varying the concentration of each of the three cytosolic components on the inhibitory potency of AEBSF indicated that this was inversely related to the concentrations of p47(phox) and, to a lesser degree, p67

  11. Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production*

    PubMed Central

    Meijles, Daniel N.; Fan, Lampson M.; Howlin, Brendan J.; Li, Jian-Mei

    2014-01-01

    Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox−/− coronary microvascular cells. Compared with wild-type p47phox cDNA transfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2⨪ production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells. PMID:24970888

  12. Molecular insights of p47phox phosphorylation dynamics in the regulation of NADPH oxidase activation and superoxide production.

    PubMed

    Meijles, Daniel N; Fan, Lampson M; Howlin, Brendan J; Li, Jian-Mei

    2014-08-15

    Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47(phox) is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47(phox) phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47(phox) protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47(phox) is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22(phox) binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47(phox-/-) coronary microvascular cells. Compared with wild-type p47(phox) cDNA transfected cells, the single mutation of S379A completely blocked p47(phox) membrane translocation, binding to p22(phox) and endothelial O2(·-) production in response to acute stimulation of PKC. p47(phox) C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47(phox) conformational changes and NADPH oxidase-dependent superoxide production by cells.

  13. 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.

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

    PubMed

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

    2014-04-01

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

  15. 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

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

    PubMed Central

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

    2016-01-01

    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. Newborn 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

  17. Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity.

    PubMed

    Güllçin, Ilhami; Küfrevioğlu, O Irfan; Oktay, Münir

    2005-06-01

    Polyphenol oxidase (PPO) of nettle (Urtica dioica L.) was extracted and purified through (NH4)2SO4 precipitation, dialysis, and CM-Sephadex ion-exchange chromatography and was used for its characterization. The PPO showed activity to catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), L-tyrosine, p-cresol, pyrogallol, catechin and trans-cinnamic acid. For each of these eight substrates, optimum conditions such as pH and temperature were determined and L-tyrosine was found to be one of the most suitable substrates. Optimum pH and temperature were found at pH 4.5 and 30 degrees C respectively and Km and Vmax values were 7.90 x 10(-4) M, and 11290 EU/mL for with L-tyrosine as substrate. The inhibitory effect of several inhibitors, L-cysteine chloride, sodium azide, sodium cyanide, benzoic acid, salicylic acid, L-ascorbic acid, glutathione, thiourea, sodium diethyl dithiocarbamate, beta-mercaptoethanol and sodium metabisulfite were tested. The most effective was found to be sodium diethyl dithiocarbamate which acted as a competitive inhibitor with a Ki value of 1.79 x 10(-9)M. In addition one isoenzyme of PPO was detected by native polacrylamide slab gel electrophoresis.

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

    PubMed

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

    2015-09-01

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

  19. 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.

  20. 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

  1. Diagnosis of cyanide intoxication by measurement of cytochrome c oxidase activity.

    PubMed

    Ikegaya, H; Iwase, H; Hatanaka, K; Sakurada, K; Yoshida, K; Takatori, T

    2001-02-28

    Cytochrome c oxidase (CCO), a mitochondrial enzyme, is inactivated by cyanide or carbon monoxide (CO) intoxication. We measured CCO activity, in the major organs of the rat at various times after death caused by cyanide intoxication. Tissue samples were homogenized, and the CCO activity in the mitochondrial fraction was measured using ferrous cytochrome c as the substrate. The CCO activity inhibition was highest in the brain, although the cyanide concentration was lowest level. As a result of this and the clinical symptoms displayed, we consider the brain to be the primary organ of cyanide intoxication. As cyanide is highly toxic to humans, in small amounts and many patients and victims have already had some medical care, it is difficult to detect cyanide in criminal investigations. The CCO activities in various organs remained significantly low for 2 days after the cyanide intoxication, suggesting that the diagnosis may be possible by measuring not only the cyanide concentration but also the CCO activity.

  2. Characteristics of cytochrome oxidase activity in visual system neurons in kittens reared in conditions of flashing illumination.

    PubMed

    Merkul'eva, N S; Makarov, F N

    2005-10-01

    The studies reported here addressed the effects of flashing (15 Hz) lights on the metabolic activity of visual system neurons in animals reared in condition of crepuscular illumination. Activity of the respiratory enzyme cytochrome oxidase was detected in the cortex of visual areas 17 and 18 and in the lateral geniculate body in kittens. The results showed that kittens subjected to this stimulation, unlike intact kittens and kittens reared in conditions of crepuscular illumination, showed a change in the pattern of cytochrome oxidase distribution in cortical field 17 consisting of the appearance of alternating areas of increased and decreased enzyme activity in layers III and IV. In cortical field 18 and the lateral geniculate body, experimental kittens showed no changes in the cytochrome oxidase activity distribution pattern. It is suggested that flashing illumination leads to disturbance of the balance in activity in the Y and X conducting channels of the visual system.

  3. 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

  4. Phosphatidylinositol 3-kinase plays a vital role in regulation of rice seed vigor via altering NADPH oxidase activity.

    PubMed

    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.

  5. Retinal NMDA receptor function and expression are altered in a mouse lacking d-amino acid oxidase

    PubMed Central

    Morgans, Catherine W.; Tekmen, Merve; Sullivan, Steven J.; Esguerra, Manuel; Konno, Ryuichi; Miller, Robert F.

    2013-01-01

    d-serine is present in the vertebrate retina and serves as a coagonist for the N-methyl-d-aspartate (NMDA) receptors of ganglion cells. Although the enzyme d-amino acid oxidase (DAO) has been implicated as a pathway for d-serine degradation, its role in the retina has not been established. In this study, we investigated the role of DAO in regulating d-serine levels using a mutant mouse line deficient in DAO (ddY/DAO−) and compared these results with their wild-type counterparts (ddY/DAO+). Our results show that DAO is functionally present in the mouse retina and normally serves to reduce the background levels of d-serine. The enzymatic activity of DAO was restricted to the inner plexiform layer as determined by histochemical analysis. Using capillary electrophoresis, we showed that mutant mice had much higher levels of d-serine. Whole cell recordings from identified retinal ganglion cells demonstrated that DAO-deficient animals had light-evoked synaptic activity strongly biased toward a high NMDA-to-AMPA receptor ratio. In contrast, recordings from wild-type ganglion cells showed a more balanced ratio between the two receptor subclasses. Immunostaining for AMPA and NMDA receptors was carried out to compare the two receptor ratios by quantitative immunofluorescence. These studies revealed that the mutant mouse had a significantly higher representation of NMDA receptors compared with the wild-type controls. We conclude that 1) DAO is an important regulatory enzyme and normally functions to reduce d-serine levels in the retina, and 2) d-serine levels play a role in the expression of NMDA receptors and the NMDA-to-AMPA receptor ratio. PMID:24068757

  6. 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

  7. 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).

  8. Dinuclear copper complexes with imidazole derivative ligands: a theoretical study related to catechol oxidase activity.

    PubMed

    Martínez, Ana; Membrillo, Ingrid; Ugalde-Saldívar, Victor M; Gasque, Laura

    2012-07-19

    Catechol oxidase is a very important and interesting metalloprotein. In spite of the efforts to understand the reaction mechanism of this protein, there are important questions that remain unanswered concerning the catalytic mechanism of this enzyme. In this article, dinuclear copper compounds are used as biomimetic models of catechol oxidase to study plausible reaction paths. These dinuclear copper(II) complexes have distant metal centers (of 7.5 Å approximately) and superior catalytic activity to that of many dicopper complexes with shorter Cu-Cu distances. One mononuclear copper(II) complex is also analyzed in this investigation in order to see the influence of the two metal centers in the catalytic activity. Density functional theory calculations were performed to obtain optimized structures, vertical ionization energies, vertical electron affinities, the electrodonating power (ω(-)), the electroaccepting power (ω(+)) and the energy difference of several reaction paths. The K(M) experimental results that were previously reported compare well with the electroaccepting power (ω(+)) of the copper compounds that are included in this article, indicating that this index is useful for the interpretation of the electron transfer capacity and therefore the catalytic activity. The catechol moiety coordinates to only one Cu ion, but two metal atoms are needed in order to have a good electron acceptor capacity of the biomimetic models.

  9. Structure, recombinant expression and mutagenesis studies of the catalase with oxidase activity from Scytalidium thermophilum.

    PubMed

    Yuzugullu, Yonca; Trinh, Chi H; Smith, Mark A; Pearson, Arwen R; Phillips, Simon E V; Sutay Kocabas, Didem; Bakir, Ufuk; Ogel, Zumrut B; McPherson, Michael J

    2013-03-01

    Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon-optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9 Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis-hydroxychlorin γ-spirolactone, which is rotated 180° with respect to small-subunit catalases. The haem-binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d-type haem to a b-type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide.

  10. 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.

  11. 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.

  12. p47phox Molecular Activation for Assembly of the Neutrophil NADPH Oxidase Complex*

    PubMed Central

    Marcoux, Julien; Man, Petr; Petit-Haertlein, Isabelle; Vivès, Corinne; Forest, Eric; Fieschi, Franck

    2010-01-01

    The p47phox cytosolic factor from neutrophilic NADPH oxidase has always been resistant to crystallogenesis trials due to its modular organization leading to relative flexibility. Hydrogen/deuterium exchange coupled to mass spectrometry was used to obtain structural information on the conformational mechanism that underlies p47phox activation. We confirmed a relative opening of the protein with exposure of the SH3 Src loops that are known to bind p22phox upon activation. A new surface was shown to be unmasked after activation, representing a potential autoinhibitory surface that may block the interaction of the PX domain with the membrane in the resting state. Within this surface, we identified 2 residues involved in the interaction with the PX domain. The double mutant R162A/D166A showed a higher affinity for specific phospholipids but none for the C-terminal part of p22phox, reflecting an intermediate conformation between the autoinhibited and activated forms. PMID:20592030

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

    PubMed

    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 [(14)C]-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.

  14. 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

  15. [Peculiarities of cytochrome oxidase activity in the visual cortex neurons of kittens reared under the conditions of flickering illumination].

    PubMed

    Merkul'eva, N S; Makarov, F N

    2004-01-01

    The objective of this study was to analyze the influence of the light flickering with the frequency of 15 Hz, on the neuronal metabolic activity in kittens reared under the conditions of mesopic illumination. The method for demonstration of a respiratory enzyme cytochrome oxidase was used to study the visual cortex areas 17 and 18 and the lateral geniculate nucleus. It was shown that in kittens that were subjected to this stimulation as opposed to intact animals and to kittens reared under the conditions of mesopic illumination, specific changes in pattern of cytochrome oxidase distribution in area 17 took place. This change was manifested by the appearance of alternating zones of increased and decreased enzyme activity in layers III and IV. Within the cortical area 18 and lateral geniculate nucleus, no changes in the pattern of cytochrome oxidase activity distribution were detected in experimental kittens. It is suggested that flickering illumination results in disequilibrium of Y- and X-visual pathway activity.

  16. Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymatic activity.

    PubMed

    Müller, M; Schlapfer, B; Azzi, A

    1988-09-20

    Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.

  17. 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.

  18. 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.

  19. 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

  20. On the reaction of D-amino acid oxidase with dioxygen: O2 diffusion pathways and enhancement of reactivity.

    PubMed

    Rosini, Elena; Molla, Gianluca; Ghisla, Sandro; Pollegioni, Loredano

    2011-02-01

    Evidence is accumulating that oxygen access in proteins is guided and controlled. We also have recently described channels that might allow access of oxygen to pockets at the active site of the flavoprotein D-amino acid oxidase (DAAO) that have a high affinity for dioxygen and are in close proximity to the flavin. With the goal of enhancing the reactivity of DAAO with oxygen, we have performed site-saturation mutagenesis at three positions that flank the putative oxygen channels and high-affinity sites. The most interesting variants at positions 50, 201 and 225 were identified by a screening procedure at low oxygen concentration. The biochemical properties of these variants have been studied and compared with those of wild-type DAAO, with emphasis on the reactivity of the reduced enzyme species with dioxygen. The substitutions at positions 50 and 225 do not enhance this reaction, but mainly affect the protein conformation and stability. However, the T201L variant shows an up to a threefold increase in the rate constant for reaction of O(2) with reduced flavin, together with a fivefold decrease in the K(m) for dioxygen. This effect was not observed when a valine is located at position 201, and is thus attributed to a specific alteration in the micro-environment of one high-affinity site for dioxygen (site B) close to the flavin that plays an important role in the storage of oxygen. The increase in O(2) reactivity observed for T201L DAAO is of great interest for designing new flavoenzymes for biotechnological applications.

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

    PubMed

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

    2003-10-01

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

  2. Selected biochemical properties of polyphenol oxidase in butter lettuce leaves (Lactuca sativa L. var. capitata) elicited with dl-β-amino-n-butyric acid.

    PubMed

    Złotek, Urszula; Gawlik-Dziki, Urszula

    2015-02-01

    The study concentrated on changes in certain biochemical parameters of polyphenol oxidase (PPO) from lettuce leaves caused by dl-β-amino-n-butyric acid (BABA) elicitation. PPO from control plants demonstrated the highest affinity toward catechol, whereas PPO from BABA-elicited lettuce showed the highest affinity to 4-methylcatechol. The optimum temperature for enzymes from control plants was 35°C, whereas from plants elicited with 1mM BABA this was 25°C. PPO from plants elicited with BABA was also more sensitive to the tested inhibitors than PPO from control plants. l-Cysteine was the most effective inhibitor. Native gel stained for PPO activity in control samples showed two isoforms. However, in BABA-treated lettuce three bands visualising PPO activity were observed. The information obtained in this study will be valuable for the development of treatment technology and storage conditions to control undesirable browning reactions in elicited lettuce.

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

    PubMed

    Lavelli, Vera

    2008-08-27

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

  4. (/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.

  5. NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury.

    PubMed

    Fischer, Marie T; Sharma, Rakhi; Lim, Jamie L; Haider, Lukas; Frischer, Josa M; Drexhage, Joost; Mahad, Don; Bradl, Monika; van Horssen, Jack; Lassmann, Hans

    2012-03-01

    Multiple sclerosis is a chronic inflammatory disease of the central nervous system, associated with demyelination and neurodegeneration. The mechanisms of tissue injury are poorly understood, but recent data suggest that mitochondrial injury may play an important role in this process. Mitochondrial injury can be triggered by reactive oxygen and nitric oxide species, and we recently provided evidence for oxidative damage of oligodendrocytes and dystrophic axons in early stages of active multiple sclerosis lesions. In this study, we identified potential sources of reactive oxygen and nitrogen species through gene expression in carefully staged and dissected lesion areas and by immunohistochemical analysis of protein expression. Genome-wide microarrays confirmed mitochondrial injury in active multiple sclerosis lesions, which may serve as an important source of reactive oxygen species. In addition, we found differences in the gene expression levels of various nicotinamide adenine dinucleotide phosphate oxidase subunits between initial multiple sclerosis lesions and control white matter. These results were confirmed at the protein level by means of immunohistochemistry, showing upregulation of the subunits gp91phox, p22phox, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 in activated microglia in classical active as well as slowly expanding lesions. The subunits gp91phox and p22phox were constitutively expressed in microglia and were upregulated in the initial lesion. In contrast, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 expression were more restricted to the zone of initial damage or to lesions from patients with acute or early relapsing/remitting multiple sclerosis. Double labelling showed co-expression of the nicotinamide adenine dinucleotide phosphate oxidase subunits in activated microglia and

  6. NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury

    PubMed Central

    Fischer, Marie T.; Sharma, Rakhi; Lim, Jamie L.; Haider, Lukas; Frischer, Josa M.; Drexhage, Joost; Mahad, Don; Bradl, Monika; van Horssen, Jack

    2012-01-01

    Multiple sclerosis is a chronic inflammatory disease of the central nervous system, associated with demyelination and neurodegeneration. The mechanisms of tissue injury are poorly understood, but recent data suggest that mitochondrial injury may play an important role in this process. Mitochondrial injury can be triggered by reactive oxygen and nitric oxide species, and we recently provided evidence for oxidative damage of oligodendrocytes and dystrophic axons in early stages of active multiple sclerosis lesions. In this study, we identified potential sources of reactive oxygen and nitrogen species through gene expression in carefully staged and dissected lesion areas and by immunohistochemical analysis of protein expression. Genome-wide microarrays confirmed mitochondrial injury in active multiple sclerosis lesions, which may serve as an important source of reactive oxygen species. In addition, we found differences in the gene expression levels of various nicotinamide adenine dinucleotide phosphate oxidase subunits between initial multiple sclerosis lesions and control white matter. These results were confirmed at the protein level by means of immunohistochemistry, showing upregulation of the subunits gp91phox, p22phox, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 in activated microglia in classical active as well as slowly expanding lesions. The subunits gp91phox and p22phox were constitutively expressed in microglia and were upregulated in the initial lesion. In contrast, p47phox, nicotinamide adenine dinucleotide phosphate oxidase 1 and nicotinamide adenine dinucleotide phosphate oxidase organizer 1 expression were more restricted to the zone of initial damage or to lesions from patients with acute or early relapsing/remitting multiple sclerosis. Double labelling showed co-expression of the nicotinamide adenine dinucleotide phosphate oxidase subunits in activated microglia and

  7. Evaluation of the oxidase like activity of nanoceria and its application in colorimetric assays.

    PubMed

    Hayat, Akhtar; Cunningham, Jessica; Bulbul, Gonca; Andreescu, Silvana

    2015-07-23

    Nanomaterial-based enzyme mimics have attracted considerable interest in chemical analysis as alternative catalysts to natural enzymes. However, the conditions in which such particles can replace biological catalysts and their selectivity and reactivity profiles are not well defined. This work explored the oxidase like properties of nanoceria particles in the development of colorimetric assays for the detection of dopamine and catechol. Selectivity of the system with respect to several phenolic compounds, the effect of interferences and real sample analysis are discussed. The conditions of use such as buffer composition, selectivity, pH, reaction time and particle type are defined. Detection limits of 1.5 and 0.2μM were obtained with nanoceria for dopamine and catechol. The same assay could be used as a general sensing platform for the detection of other phenolics. However, the sensitivity of the method varies significantly with the particle type, buffer composition, pH and with the structure of the phenolic compound. The results demonstrate that nanoceria particles can be used for the development of cost effective and sensitive methods for the detection of these compounds. However, the selection of the particle system and experimental conditions is critical for achieving high sensitivity. Recommendations are provided on the selection of the particle system and reaction conditions to maximize the oxidase like activity of nanoceria.

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

    PubMed Central

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

    2006-01-01

    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 P3121 or P3221, with unit-cell parameters a = b = 103.8, c = 487.0 Å) and diffracts to at least 1.6 Å resolution. Several data sets were collected using 2θ and κ geometry in order to obtain a complete data set to 2.07 Å resolution. Solvent-content and Matthews coefficient analysis suggests that crystal form 1 contains two homotetramers per asymmetric unit. Crystal form 2 (P212121; a = 147.3, b = 153.5, c = 169.5 Å) and crystal form 3 (P31 or P32; a = b = 108.9, c = 342.5 Å) 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

  9. 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.

  10. Ovarian dual oxidase (Duox) activity is essential for insect eggshell hardening and waterproofing.

    PubMed

    Dias, Felipe A; Gandara, Ana Caroline P; Queiroz-Barros, Fernanda G; Oliveira, Raquel L L; Sorgine, Marcos H F; Braz, Glória R C; Oliveira, Pedro L

    2013-12-06

    In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H2O2 and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H2O2 that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H2O2 generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H2O2 injection into the female hemocoel. Taken together, our results show that Duox-generated H2O2 fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing.

  11. Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade

    NASA Astrophysics Data System (ADS)

    Zhang, Yifei; Tsitkov, Stanislav; Hess, Henry

    2016-12-01

    A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.

  12. NADPH oxidase inhibitor DPI is neuroprotective at femtomolar concentrations through inhibition of microglia over-activation.

    PubMed

    Qian, Li; Gao, Xi; Pei, Zhong; Wu, Xuefei; Block, Michelle; Wilson, Belinda; Hong, Jau-Shyong; Flood, Patrick M

    2007-01-01

    In this paper we report that diphenyliodonium (DPI), a NADPH oxidase inhibitor, shows potent anti-inflammatory and neuroprotective effects at femtomolar concentrations (10(-13) to 10(-14) M) in primary midbrain cultures. Mechanistic studies revealed that DPI-elicited effects were mediated by the inhibition of LPS-induced microglial ROS production and the subsequent release of pro-inflammatory cytokine TNFa, and the production of nitric oxide. Further studies showed that 10(-14) M DPI significantly reduced LPS-induced ERK phosphorylation. Taken together, our results demonstrate that femtomolar concentrations of DPI exert potent anti-inflammatory and neuroprotective effects by inhibiting microglial activation through the inhibition of ERK-regulated PHOX activity.

  13. 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.

  14. NADPH Oxidase-Dependent NLRP3 Inflammasome Activation and its Important Role in Lung Fibrosis by Multiwalled Carbon Nanotubes.

    PubMed

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

    2015-05-06

    The purpose of this paper 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, including NADPH oxidase, in inflammasome activation remains to be clarified. In this study, three long aspect ratio (LAR) materials (MWCNTs, single-walled carbon nanotubes, 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 p22(phox) -deficient cells. The NADPH oxidase is directly involved in lysosomal damage by LAR materials, as demonstrated by decreased cathepsin B release and IL-1β production in p22(phox) -deficient cells. Reduced respiratory burst activity and inflammasome activation are also observed in bone marrow-derived macrophages from p47(phox) -deficient mice. Moreover, p47(phox) -deficient mice have reduced IL-1β production and lung collagen deposition in response to MWCNTs. Lung fibrosis is also suppressed by N-acetyl-cysteine in wild-type animals exposed to MWCNTs.

  15. 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.

  16. A novel L-amino acid oxidase from Trichoderma harzianum ETS 323 associated with antagonism of Rhizoctonia solani.

    PubMed

    Yang, Chia-Ann; Cheng, Chi-Hua; Lo, Chaur-Tsuen; Liu, Shu-Ying; Lee, Jeng-Woei; Peng, Kou-Cheng

    2011-05-11

    Trichoderma spp. are used as biocontrol agents against phytopathogens such as Rhizoctonia solani, but their biocontrol mechanisms are poorly understood. A novel L-amino oxidase (Th-LAAO) was identified from the extracellular proteins of Trichoderma harzianum ETS 323. Here, we show a FAD-binding glycoprotein with the best substrate specificity constant for L-phenylalanine. Although the amino acid sequence of Th-LAAO revealed limited homology (16-24%) to other LAAO members, a highly conserved FAD-binding motif was identified in the N-terminus. Th-LAAO was shown to be a homodimeric protein, but the monomeric form was predominant when grown in the presence of deactivated Rhizoctonia solani. Furthermore, in vitro assays demonstrated that Th-LAAO had an antagonistic effect against Rhizoctonia solani and a stimulatory one on hyphal density and sporulation in T. harzianum ETS 323. These findings further our understanding of T. harzianum as a biocontrol agent and provide insight into the biological function of l-amino acid oxidase.

  17. Effect of maturity on chlorophyll, tannin, color, and polyphenol oxidase (PPO) activity of sugarcane juice (Saccharum officinarum Var. Yellow Cane).

    PubMed

    Qudsieh, Hanan Yassin M; Yusof, Salmah; Osman, Azizah; Rahman, Russly Abdul

    2002-03-13

    A study was conducted to determine the effect of sugarcane maturation on the contents of chlorophyll, tannin, and polyphenol oxidase (PPO) activity and on color change of sugarcane juice. The maturation period of the cane studied was between 3 and 10 months after planting. Different parts of the cane, namely, the top, middle, and bottom portions, were analyzed. Results obtained indicated that there were significant (P < 0.01) decreases in total chlorophyll a and b and tannin contents during maturity followed by slower rates of decrease of both parameters at the end of maturity stages. There were no significant differences (P > 0.05) in chlorophyll and tannin contents between the middle and bottom portions. On the other hand, the top portion of the stem had a significantly (P < 0.01) lower concentration of chlorophyll and a significantly (P < 0.01) higher content of tannin. PPO activity of sugarcane juice was determined using chlorogenic acid as a substrate. There was a highly significant difference (P < 0.01) in PPO activity of cane juice during maturity. PPO activity was high at the early development stage, decreased during maturation, and then remained relatively constant at the end of maturity. PPO activity was higher when chlorogenic acid was used as substrate. There were also significant differences (P < 0.01) in juice color (L*, a*, b* values) from different portions at different maturity stages. At the early stages, the color of extracted juice was dark, and then the juice turned to yellowish green during maturity. The decrease in green color or the increase in the yellow color could be associated with the decline in chlorophyll. The overall color change (DeltaE) at maturity indicated that the color of the middle and bottom portions was lower than that of the top portion.

  18. Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression.

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    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 lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.

  19. Surface modification of polyvinyl alcohol/malonic acid nanofibers by gaseous dielectric barrier discharge plasma for glucose oxidase immobilization

    NASA Astrophysics Data System (ADS)

    Afshari, Esmail; Mazinani, Saeedeh; Ranaei-Siadat, Seyed-Omid; Ghomi, Hamid

    2016-11-01

    Polymeric nanofiber prepares a suitable situation for enzyme immobilization for variety of applications. In this research, we have fabricated polyvinyl alcohol (PVA)/malonic acid nanofibers using electrospinning. After fabrication of nanofibers, the effect of air, nitrogen, CO2, and argon DBD (dielectric barrier discharge) plasmas on PVA/malonic acid nanofibers were analysed. Among them, air plasma had the most significant effect on glucose oxidase (GOx) immobilization. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrum analysis and X-ray photoelectron spectroscopy (XPS) results revealed that in case of air plasma modified nanofibers, the carboxyl groups on the surface are increased. The scanning electron microscopy (SEM) images showed that, after GOx immobilization, the modified nanofibers with plasma has retained its nanofiber structure. Finally, we analysed reusability and storage stability of GOx immobilized on plasma modified and unmodified nanofibers. The results were more satisfactory for modified nanofibers with respect to unmodified ones.

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

    PubMed

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

    2012-09-01

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

  1. Purification and partial characterization of an L-amino acid oxidase from bushmaster snake (Surucucu Pico de Jaca) Lachesis muta muta venom.

    PubMed

    Sánchez, E O; Magalhães, A

    1991-01-01

    1. L-amino acid oxidase (L-AO) from the venom of Lachesis muta muta was purified 72 times (38%) by gel filtration on Sephadex G-100, followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-300. 2. The protein was shown to be homogeneous by polyacrylamide gel electrophoresis at pH 8.5, immunoelectrophoresis, immunodiffusion and isoelectric focusing. Its specific activity was 44.4 units/mg protein, using 7.5 mM L-leucine as substrate and O-dianisidine as electron donor, at pH 7.6 and 25 degrees C. The increase in absorbance at 436 nm was recorded. 3. The enzyme was characterized as a glycoprotein with an S20,w = 6.72, MW = 138,000 and pI = 5.2. It presented maxima at 389 and 460 nm and contained 2 mol of FAD per mole protein.

  2. Structural insights into sulfite oxidase deficiency.

    PubMed

    Karakas, Erkan; Wilson, Heather L; Graf, Tyler N; Xiang, Song; Jaramillo-Busquets, Sandra; Rajagopalan, K V; Kisker, Caroline

    2005-09-30

    Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

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

    PubMed

    Nilthong, Somrudee; Graybosch, R A; Baenziger, P S

    2012-12-01

    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 markers, PPO18, PPO29 and STS01, were used to identify lines with putative alleles at the Ppo-A1 and Ppo-D1 loci conditioning low or high PPO activity. ANOVA showed significant genotypic effects on PPO activity (P < 0.0001) in all populations. The generations and generation × genotype effects were not significant in any population. A putative third (null) genotype at Ppo-A1 (no PCR fragments for PPO18) was discovered in NW07OR1066 and NW07OR1070 derived populations, and these had the lowest mean PPO activities. Results demonstrated that both Ppo-A1 and Ppo-D1 loci affect the kernel PPO activity, but the Ppo-A1 has the major effect. In three populations, contrary results were observed to those predicted from previous work with Ppo-D1 alleles, suggesting the markers for Ppo-D1 allele might give erroneous results in some genetic backgrounds or lineages. Results suggest that selection for low or null alleles only at Ppo-A1 might allow development of low PPO wheat cultivars.

  4. 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.

  5. 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.

  6. 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.

  7. Role of a strictly conserved active site tyrosine in cofactor genesis in the copper amine oxidase from Hansenula polymorpha.

    PubMed

    DuBois, Jennifer L; Klinman, Judith P

    2006-03-14

    The copper amine oxidases catalyze the O(2)-dependent, two-electron oxidation of amines to aldehydes at an active site that contains Cu(II) and topaquinone (TPQ) cofactor. TPQ arises from the autocatalytic, post-translational oxidation of a tyrosine side chain within the same active site. The contributions of individual active site amino acids to each of these chemical processes are being delineated. Previously, using the amine oxidase from the yeast Hansenula polymorpha (HPAO), mutations of a strictly conserved and structurally pivotal active site tyrosine (Y305) were studied and their effects on the catalytic cycle demonstrated [Hevel, J. M., Mills, S. A., and Klinman, J. P. (1999) Biochemistry 38, 3683-3693]. This study examines mutations at the same position for their effects on cofactor generation. While the Y305A mutation had moderate effects on the kinetics of catalysis (2.5- and 8-fold effects on k(cat) using ethylamine and benzylamine as substrates), the same mutation slows cofactor formation by approximately 45-fold relative to that of the wild-type (WT). Additionally, the Y305A mutant forms at least two species: primarily TPQ at lower pH and a species with a blue-shifted absorbance at high pH (lambda(max) = 400 nm). The 400 nm species does not react with phenylhydrazine or ethylamine and is stable toward pH buffer exchange, long-term storage (>3 weeks), incubation at high temperatures, or incubation with reductants and colorimetric peroxide quenching reagents. A similar species accumulates appreciably even at approximately neutral pH in the Y305F mutant, despite the fact that the rate of TPQ formation is reduced only 3-fold relative to that of WT HPAO. This small impact of Y305F on the rate of biogenesis contracts with a decrease in k(cat) (using ethylamine as the substrate) of 125-fold. The opposing effects of mutations at position 305 in biogenesis versus catalysis indicate that a single residue can be recruited for different roles during these

  8. A biochemical approach to study the role of the terminal oxidases in aerobic respiration in Shewanella oneidensis MR-1.

    PubMed

    Le Laz, Sébastien; Kpebe, Arlette; Bauzan, Marielle; Lignon, Sabrina; Rousset, Marc; Brugna, Myriam

    2014-01-01

    The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb 3-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb 3-type oxidase is the major terminal oxidase under aerobic conditions while both cbb 3-type and bd-type oxidases are involved in respiration at low-O2 tensions. On the contrary, the low O2-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa 3-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa 3-type oxidase in S. oneidensis MR-1 are discussed.

  9. 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 ...

  10. Conformational plasticity surrounding the active site of NADH oxidase from Thermus thermophilus

    PubMed Central

    Miletti, Teresa; Di Trani, Justin; Jr Levros, Louis-Charles; Mittermaier, Anthony

    2015-01-01

    Biotechnological applications of enzymes can involve the use of these molecules under nonphysiological conditions. Thus, it is of interest to understand how environmental variables affect protein structure and dynamics and how this ultimately modulates enzyme function. NADH oxidase (NOX) from Thermus thermophilus exemplifies how enzyme activity can be tuned by reaction conditions, such as temperature, cofactor substitution, and the addition of cosolutes. This enzyme catalyzes the oxidation of reduced NAD(P)H to NAD(P)+ with the concurrent reduction of O2 to H2O2, with relevance to biosensing applications. It is thermophilic, with an optimum temperature of approximately 65°C and sevenfold lower activity at 25°C. Moderate concentrations (≈1M) of urea and other chaotropes increase NOX activity by up to a factor of 2.5 at room temperature. Furthermore, it is a flavoprotein that accepts either FMN or the much larger FAD as cofactor. We have used nuclear magnetic resonance (NMR) titration and 15N spin relaxation experiments together with isothermal titration calorimetry to study how NOX structure and dynamics are affected by changes in temperature, the addition of urea and the substitution of the FMN cofactor with FAD. The majority of signals from NOX are quite insensitive to changes in temperature, cosolute addition, and cofactor substitution. However, a small cluster of residues surrounding the active site shows significant changes. These residues are implicated in coupling changes in the solution conditions of the enzyme to changes in catalytic activity. PMID:25970557

  11. Glycerate-oxidizing activity of glycolate oxidase from leaves of Spinacia oleracea.

    PubMed

    Huang, Jiu-Jiu; Wang, Wei-Jun; Ye, Jin-Quan; Peng, Xin-Xiang

    2006-04-01

    Glycolate oxidase (GO) was purified to homogeneity from leaves of spinach (Spinacia oleracea). Through detecting the consumption of oxygen and the formation of hydrogen peroxide in the assay solution, it was found that GO could also oxidize glycerate, another metabolite in the photorespiratory pathway, and use FMN and FAD, but not riboflavin and lumiflavin, as its cofactors. The optimum reaction pH, Km for glycerate, k(cat) and activation energy of this oxidizing reaction were determined to be 8.0, 7.14 mmol/L, 1.04 s(-1) and 17.29 kJ/mol, respectively. Oxalate and pyruvate at 5.0 mmol/L could inhibit the glycerate-oxidizing activity by 34% and 26%, and oxalate acted as a competitive inhibitor of the glycerate oxidation reaction with a K(i) of 0.75 mmol/L. By the competition plotting with mixed-substrates, it was indicated that glycolate-oxidizing activity and glycerate-oxidizing activity of GO shared the same active site.

  12. Polyphenol oxidase activity as a potential intrinsic index of adequate thermal pasteurization of apple cider.

    PubMed

    Chen, L; Ingham, B H; Ingham, S C

    2004-05-01

    In response to increasing concerns about microbial safety of apple cider, the U.S. Food and Drug Administration has mandated treatment of cider sufficient for a 5-log reduction of the target pathogen. Pasteurization has been suggested as the treatment most likely to achieve a 5-log reduction, with Escherichia coli O157:H7 as the target pathogen. Regulators and processors need a reliable method for verifying pasteurization, and apple cider polyphenol oxidase (PPO) activity was studied as a potential intrinsic index for thermal pasteurization. The effect of pasteurization conditions and apple cider properties on PPO activity and survival of three pathogens (E. coli O157:H7, Salmonella, and Listeria monocytogenes) was studied using a Box-Behnken response surface design. Factors considered in the design were pasteurization conditions, i.e., hold temperature (60, 68, and 76 degrees C), preheat time (10, 20, 30 s), and hold time (0, 15, 30 s), pH, and sugar content ((o)Brix) of apple cider. Response surface contour plots were constructed to illustrate the effect of these factors on PPO activity and pathogen survival. Reduction in PPO activity of at least 50% was equivalent to a 5-log reduction in E. coli O157:H7 or L. monocytogenes for cider at pH 3.7 and 12.5 (o)Brix. Further studies, however, are needed to verify the relationship between PPO activity and pathogen reduction in cider with various pH and (o)Brix values.

  13. Time-dependent activation of the semicarbazide-sensitive amine oxidase (SSAO) from ox lung microsomes.

    PubMed Central

    Lizcano, J M; Tipton, K F; Unzeta, M

    2000-01-01

    The activity of ox lung microsomal semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) towards benzylamine increased 20-fold during incubation at 37 degrees C. After an initial lag-period, activation was first-order with time and complete after approx. 20 h. No significant changes in activity towards methylamine, histamine or 2-phenylethylamine were observed, although mixed-substrate experiments were consistent with the same enzyme being involved in the oxidation of all these substrates, both before and after time-dependent activation. The enzyme-tryptophan fluorescence increased on incubation at 37 degrees C in parallel with the increase in activity towards benzylamine. Treatment of the activated-enzyme preparation with 6 M guanidinium chloride followed by dialysis, caused both the activity towards benzylamine and the fluorescence to fall to that occurring before activation. However, incubation of this preparation at 37 degrees C resulted in increases in fluorescence and activity similar to those seen with the unactivated enzyme. Benzylamine oxidation was inhibited, uncompetitively with respect to oxygen, by high substrate concentrations but no such inhibition was observed with the other amines. Activation resulted in an increase in V(max) for benzylamine oxidation, with no significant alterations in the K(m) or the K(si) for high-substrate inhibition. Kinetic studies were consistent with sequential mechanisms being followed for the oxidation of both benzylamine and methylamine but the dependence on oxygen concentration was complex. These results might indicate that benzylamine follows a different reaction pathway from the other substrates, with substrate-specific activation involving a reaction step that is rate-limiting for benzylamine oxidation but not for the others. PMID:11042135

  14. 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.

  15. Two pathways of activation of the superoxide-generating NADPH oxidase of phagocytes in vitro--distinctive effects of inhibitors.

    PubMed

    Sigal, Natalia; Gorzalczany, Yara; Pick, Edgar

    2003-06-01

    The NADPH oxidase complex of phagocytes comprises a membrane-associated flavocytochrome b559, and 4 cytosolic components: p47phox, p67phox, p40phox, and the small GTPase Rac. Activation of the oxidase in vivo is the result of assembly of the cytosolic components with cytochrome b559 and is mimicked in vitro by a cell-free system consisting of membranes, p47phox, p67phox, nonprenylated or prenylated Rac, and an anionic amphiphile as activator (defined as "p47phox and amphiphile-dependent" or canonical pathway). We reported that prenylated Rac1 is capable of activating the NADPH oxidase in vitro in the absence of p47phox and amphiphile (defined as "p47phox and amphiphile-independent" pathway). We now demonstrate that the 2 pathways exhibit distinctive susceptibilities to inhibitors: 1) The anionic amphiphile lithium dodecyl sulfate, an activator of the canonical pathway, has the opposite effect (inhibition) on oxidase activation by prenylated Rac and p67phox; 2) GDP and, paradoxically, GTP (but not GMP, ATP, ADP, and AMP) prevent oxidase activation by the p47phox and amphiphile-independent pathway but do not affect activation by the canonical pathway; 3) The Rac-binding domain of p21-activated kinase is a potent inhibitor of activation by the p47phox and amphiphile-independent pathway while exerting a milder inhibitory effect on the canonical pathway; 4) The C-terminal polybasic Rac1 peptide 177-191 and the cationic antibiotic neomycin sulfate inhibit activation by the canonical pathway but do not affect activation by the p47phox and amphiphile-independent pathway; 5) Binding of prenylated Rac1 to membrane-mimicking phospholipid vesicles is, nevertheless, enhanced when these contain negatively charged lipids. It is proposed that preferential inhibition of oxidase activation, via the p47phox and amphiphile-independent pathway, is a reflection of interference by the inhibitors with Rac-dependent recruitment of p67phox to the membrane.

  16. 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.

  17. Psychological traits and platelet monoamine oxidase activity in eating disorder patients: their relationship and stability.

    PubMed

    Podar, Iris; Jaanisk, Maiken; Allik, Jüri; Harro, Jaanus

    2007-01-30

    Self-reported behavior and attitudes towards eating [Eating Disorder Inventory-2; Garner DM (1991). Eating Disorder Inventory-2: Professional Manual. Odessa, Fl.: Psychological Assessment Resources; Estonian version Podar I, Hannus A, Allik J (1999). Personality and Affectivity Characteristics Associated With Eating Disorders: a Comparison of Eating Disordered, Weight-Preoccupied, and Normal Samples. J Pers Assess; 73(1), 133-147] and the activity of platelet monoamine oxidase (MAO) was studied in 11 patients with anorexia nervosa (AN), 43 patients with bulimia nervosa (BN) and a healthy control group (n=138). Nineteen patients filled in the EDI-2 questionnaire and donated blood samples three times with three month intervals in order to determine platelet MAO activity. Eating disordered (ED) patients scored higher on all EDI-2 subscales and had lower MAO activity compared to the control group. They also scored higher than the control group on the Neuroticism domain but lower on the Extraversion, Openness, and Conscientiousness domains of the NEO-PI-R questionnaire. The average stability of MAO on different occasions (r=.56) was slightly smaller than the stability of the EDI-2 scores (r=.70). The lack of correlations between personality dispositions and MAO activity indicates that they have independent influence on eating disorders. A possible relationship between neurochemical mechanisms and psychological symptoms of eating disordered behavior is discussed.

  18. Extra virgin olive oil rich in polyphenols modulates VEGF-induced angiogenic responses by preventing NADPH oxidase activity and expression.

    PubMed

    Calabriso, Nadia; Massaro, Marika; Scoditti, Egeria; D'Amore, Simona; Gnoni, Antonio; Pellegrino, Mariangela; Storelli, Carlo; De Caterina, Raffaele; Palasciano, Giuseppe; Carluccio, Maria Annunziata

    2016-02-01

    Previous studies have shown the antiinflammatory, antioxidant and antiangiogenic properties by pure olive oil polyphenols; however, the effects of olive oil phenolic fraction on the inflammatory angiogenesis are unknown. In this study, we investigated the effects of the phenolic fraction (olive oil polyphenolic extract, OOPE) from extra virgin olive oil and related circulating metabolites on the VEGF-induced angiogenic responses and NADPH oxidase activity and expression in human cultured endothelial cells. We found that OOPE (1-10 μg/ml), at concentrations achievable nutritionally, significantly reduced, in a concentration-dependent manner, the VEGF-induced cell migration, invasiveness and tube-like structure formation through the inhibition of MMP-2 and MMP-9. OOPE significantly (P<0.05) reduced VEGF-induced intracellular reactive oxygen species by modulating NADPH oxidase activity, p47phox membrane translocation and the expression of Nox2 and Nox4. Moreover, the treatment of endothelial cells with serum obtained 4 h after acute intake of extra virgin olive oil, with high polyphenol content, decreased VEGF-induced NADPH oxidase activity and Nox4 expression, as well as, MMP-9 expression, as compared with fasting control serum. Overall, native polyphenols and serum metabolites of extra virgin olive oil rich in polyphenols are able to lower the VEGF-induced angiogenic responses by preventing endothelial NADPH oxidase activity and decreasing the expression of selective NADPH oxidase subunits. Our results provide an alternative mechanism by which the consumption of olive oil rich in polyphenols may account for a reduction of oxidative stress inflammatory-related sequelae associated with chronic degenerative diseases.

  19. Probiotic yogurts manufactured with increased glucose oxidase levels: postacidification, proteolytic patterns, survival of probiotic microorganisms, production of organic acid and aroma compounds.

    PubMed

    Cruz, A G; Castro, W F; Faria, J A F; Lollo, P C B; Amaya-Farfán, J; Freitas, M Q; Rodrigues, D; Oliveira, C A F; Godoy, H T

    2012-05-01

    We investigated the effect of increased glucose oxidase concentration as a technological option to decrease oxidative stress during the processing of probiotic yogurts. Probiotic yogurts were produced with increased concentrations of glucose oxidase (0, 250, 500, 750, or 1,000 mg/kg) and submitted to physicochemical and microbiological analysis at 1, 15, and 30 d of refrigerated storage. Higher concentrations of glucose oxidase (750 and 1,000 mg/kg) and a longer storage time were found to have an influence on the characteristics of the probiotic yogurt, contributing to more extensive postacidification, an increase in the dissolved oxygen level, and higher proteolysis. In addition, increased production of aroma compounds (diacetyl and acetaldehyde) and organic acids (mainly lactic acid) and a decrease in the probiotic bacteria count were reported. The use of glucose oxidase was a feasible option to minimize oxidative stress in probiotic yogurts. However, supplementation with excessive amounts of the enzyme may be ineffective, because insufficient substrate (glucose) is present for its action. Consumer tests should be performed to evaluate changes in the sensory attributes of the probiotic yogurts with increased supplementation of glucose oxidase. In addition, packaging systems with different permeability to oxygen should be evaluated.

  20. Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.

    PubMed Central

    Lambowitz, A M; Sabourin, J R; Bertrand, H; Nickels, R; McIntosh, L

    1989-01-01

    Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species. Images PMID:2524649

  1. 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.

  2. Bioseparation of Four Proteins from Euphorbia characias Latex: Amine Oxidase, Peroxidase, Nucleotide Pyrophosphatase/Phosphodiesterase, and Purple Acid Phosphatase

    PubMed Central

    Medda, Rosaria; Pintus, Francesca; Spanò, Delia; Floris, Giovanni

    2011-01-01

    This paper deals with the purification of four proteins from Euphorbia characias latex, a copper amine oxidase, a nucleotide pyrophosphatase/phosphodiesterase, a peroxidase, and a purple acid phosphatase. These proteins, very different in molecular weight, in primary structure, and in the catalyzed reaction, are purified using identical preliminary steps of purification and by chromatographic methods. In particular, the DEAE-cellulose chromatography is used as a useful purification step for all the four enzymes. The purification methods here reported allow to obtain a high purification of all the four proteins with a good yield. This paper will give some thorough suggestions for researchers busy in separation of macromolecules from different sources. PMID:22013530

  3. 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.

  4. Impaired NADPH oxidase activity in peripheral blood lymphocytes of galactosemia patients.

    PubMed

    Al-Essa, Mazen; Dhaunsi, Gursev S; Al-Qabandi, Wafa'a; Khan, Islam

    2013-07-01

    Galactosemia is an autosomal recessive disorder with a wide range of clinical abnormalities. Cellular oxidative stress is considered as one of the pathogenic mechanisms of galactosemia. In this study, we examined the activity of NADPH oxidase (NOX), a major superoxide-generating enzyme system, in peripheral blood lymphocytes (PBL) from galactosemia patients. PBL were isolated from galactosemia patients and healthy control subjects and used for cell culture studies and biochemical assays. PBL were cultured in the presence or absence of galactose or galactose-1-phosphate (Gal-1-P), and enzyme activities and/or gene expression of NOX, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in the cell homogenates. PBL isolated from galactosemia patients showed significantly reduced (P < 0.01) activities of catalase and GPx; however SOD activity remained unaltered. Galactosemia patients were found to have significantly (P < 0.01) increased levels of malondialdehyde (MDA) in blood lymphocytes. Enzymatic activity of NOX was significantly (P < 0.001) reduced in galactosemia patients; however, Western blotting revealed that NOX-1 protein was not significantly altered. Interestingly, levels of NOX activity in lymphocytes isolated from galactosemia patients significantly increased but remained subnormal when cultured in galactose-deficient medium for two weeks, indicating a galactose-mediated inhibition of NOX. Lymphocytes isolated from control subjects were found to have significantly (P < 0.01) reduced NOX activity when cultured in the presence of galactose or Gal-1-P for two weeks. These results show that galactose-induced cellular oxidative stress is not NOX mediated. However, impairment of the NOX system might be responsible for some of the clinical complications in galactosemia patients.

  5. 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.

  6. Latent and active polyphenol oxidase (PPO) in red clover (Trifolium pratense) and use of a low PPO mutant to study the role of PPO in proteolysis reduction.

    PubMed

    Winters, Ana L; Minchin, Frank R; Michaelson-Yeates, Terry P T; Lee, Michael R F; Morris, Phillip

    2008-04-23

    Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzyme's own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.

  7. Spectroscopic and crystallographic characterization of "alternative resting" and "resting oxidized" enzyme forms of bilirubin oxidase: implications for activity and electrochemical behavior of multicopper oxidases.

    PubMed

    Kjaergaard, Christian H; Durand, Fabien; Tasca, Federico; Qayyum, Munzarin F; Kauffmann, Brice; Gounel, Sébastien; Suraniti, Emmanuel; Hodgson, Keith O; Hedman, Britt; Mano, Nicolas; Solomon, Edward I

    2012-03-28

    While there is broad agreement on the catalytic mechanism of multicopper oxidases (MCOs), the geometric and electronic structures of the resting trinuclear Cu cluster have been variable, and their relevance to catalysis has been debated. Here, we present a spectroscopic characterization, complemented by crystallographic data, of two resting forms occurring in the same enzyme and define their interconversion. The resting oxidized form shows similar features to the resting form in Rhus vernicifera and Trametes versicolor laccase, characterized by "normal" type 2 Cu electron paramagnetic resonance (EPR) features, 330 nm absorption shoulder, and a short type 3 (T3) Cu-Cu distance, while the alternative resting form shows unusually small A(||) and high g(||) EPR features, lack of 330 nm absorption intensity, and a long T3 Cu-Cu distance. These different forms are evaluated with respect to activation for catalysis, and it is shown that the alternative resting form can only be activated by low-potential reduction, in contrast to the resting oxidized form which is activated via type 1 Cu at high potential. This difference in activity is correlated to differences in redox states of the two forms and highlights the requirement for efficient sequential reduction of resting MCOs for their involvement in catalysis.

  8. Effect of disease and drug treatment on blood serotonin and monoamine oxidase B activity in Parkinson's disease.

    PubMed

    van Kempen, G M; Janjua, R; Roos, R A

    1995-05-01

    Serotonin (5-hydroxytryptamine, 5HT) content and monoamine oxidase (MAO) activity were determined in whole blood of patients with Parkinson's disease (PD) with and without drug treatment and compared with controls. From that comparison a significant reduction in platelet 5HT became apparent in PD. Selegiline, which was always used in combination with L-dopa, not only inhibited MAO activity, as expected, but it also appeared to induce an increase in 5HT content.

  9. A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction.

    PubMed

    Gabig, T G; Eklund, E A; Potter, G B; Dykes, J R

    1990-08-01

    The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.

  10. 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

  11. Methodology to assay CYP2E1 mixed function oxidase catalytic activity and its induction

    PubMed Central

    Cederbaum, Arthur I.

    2014-01-01

    The cytochrome P450 mixed function oxidase enzymes are the major catalysts involved in drug metabolism. There are many forms of P450. CYP2E1 metabolizes many toxicologically important compounds including ethanol and is active in generating reactive oxygen species. Since several of the contributions in the common theme series “Role of CYP2E1 and Oxidative/Nitrosative Stress in the Hepatotoxic Actions of Alcohol” discuss CYP2E1, this methodology review describes assays on how CYP2E1 catalytic activity and its induction by ethanol and other inducers can be measured using substrate probes such as the oxidation of para-nitrophenol to para-nitrocatechol and the oxidation of ethanol to acetaldehyde. Approaches to validate that a particular reaction e.g. oxidation of a drug or toxin is catalyzed by CYP2E1 or that induction of that reaction is due to induction of CYP2E1 are important and specific examples using inhibitors of CYP2E1, anti-CYP2E1 IgG or CYP2E1 knockout and knockin mice will be discussed. PMID:25454746

  12. Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6

    PubMed Central

    Schüll, S; Günther, S D; Brodesser, S; Seeger, J M; Tosetti, B; Wiegmann, K; Pongratz, C; Diaz, F; Witt, A; Andree, M; Brinkmann, K; Krönke, M; Wiesner, R J; Kashkar, H

    2015-01-01

    Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction. PMID:25766330

  13. Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6.

    PubMed

    Schüll, S; Günther, S D; Brodesser, S; Seeger, J M; Tosetti, B; Wiegmann, K; Pongratz, C; Diaz, F; Witt, A; Andree, M; Brinkmann, K; Krönke, M; Wiesner, R J; Kashkar, H

    2015-03-12

    Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction.

  14. Nebivolol prevents ethanol-induced reactive oxygen species generation and lipoperoxidation in the rat kidney by regulating NADPH oxidase activation and expression.

    PubMed

    do Vale, Gabriel T; Gonzaga, Natália A; Simplicio, Janaina A; Tirapelli, Carlos R

    2017-03-15

    We studied whether the β1-adrenergic antagonist nebivolol would prevent ethanol-induced reactive oxygen species generation and lipoperoxidation in the rat renal cortex. Male Wistar rats were treated with ethanol (20% v/v) for 2 weeks. Nebivolol (10mg/kg/day; p.o. gavage) prevented both the increase in superoxide anion (O2(-)) generation and thiobarbituric acid reactive substances (TBARS) concentration induced by ethanol in the renal cortex. Ethanol decreased nitrate/nitrite (NOx) concentration in the renal cortex, and nebivolol prevented this response. Nebivolol did not affect the reduction of hydrogen peroxide (H2O2) concentration induced by ethanol. Nebivolol prevented the ethanol-induced increase of catalase (CAT) activity. Both SOD activity and the levels of reduced glutathione (GSH) were not affected by treatment with nebivolol or ethanol. Neither ethanol nor nebivolol affected the expression of Nox1, Nox4, eNOS, nNOS, CAT, Nox organizer 1 (Noxo1), c-Src, p47(phox) or superoxide dismutase (SOD) isoforms in the renal cortex. On the other hand, treatment with ethanol increased Nox2 expression, and nebivolol prevented this response. Finally, nebivolol reduced the expression of protein kinase (PK) Cδ and Rac1. The major finding of our study is that nebivolol prevented ethanol-induced reactive oxygen species generation and lipoperoxidation in the kidney by a mechanism that involves reduction on the expression of Nox2, a catalytic subunit of NADPH oxidase. Additionally, we demonstrated that nebivolol reduces NADPH oxidase-derived reactive oxygen species by decreasing the expression of PKCδ and Rac1, which are important activators of NADPH oxidase.

  15. Parameters that enhance the bacterial expression of active plant polyphenol oxidases.

    PubMed

    Dirks-Hofmeister, Mareike E; Kolkenbrock, Stephan; Moerschbacher, Bruno M

    2013-01-01

    Polyphenol oxidases (PPOs, EC 1.10.3.1) are type-3 copper proteins that enzymatically convert diphenolic compounds into their corresponding quinones. Although there is significant interest in these enzymes because of their role in food deterioration, the lack of a suitable expression system for the production of soluble and active plant PPOs has prevented detailed investigations of their structure and activity. Recently we developed a bacterial expression system that was sufficient for the production of PPO isoenzymes from dandelion (Taraxacum officinale). The system comprised the Escherichia coli Rosetta 2 (DE3) [pLysSRARE2] strain combined with the pET-22b(+)-vector cultivated in auto-induction medium at a constant low temperature (26 °C). Here we describe important parameters that enhance the production of active PPOs using dandelion PPO-2 for proof of concept. Low-temperature cultivation was essential for optimal yields, and the provision of CuCl2 in the growth medium was necessary to produce an active enzyme. By increasing the copper concentration in the production medium to 0.2 mM, the yield in terms of PPO activity per mol purified protein was improved 2.7-fold achieving a v(max) of 0.48 ± 0.1 µkat per mg purified PPO-2 for 4-methylcatechol used as a substrate. This is likely to reflect the replacement of an inactive apo-form of the enzyme with a correctly-folded, copper-containing counterpart. We demonstrated the transferability of the method by successfully expressing a PPO from tomato (Solanum lycopersicum) showing that our optimized system is suitable for the analysis of further plant PPOs. Our new system therefore provides greater opportunities for the future of research into this economically-important class of enzymes.

  16. NADPH oxidase and aging drive microglial activation, oxidative stress, and dopaminergic neurodegeneration following systemic LPS administration.

    PubMed

    Qin, Liya; Liu, Yuxin; Hong, Jau-Shyong; Crews, Fulton T

    2013-06-01

    Parkinson's disease is characterized by a progressive degeneration of substantia nigra (SN) dopaminergic neurons with age. We previously found that a single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) injection caused a slow progressive loss of tyrosine hydroxylase immunoreactive (TH+IR) neurons in SN associated with increasing motor dysfunction. In this study, we investigated the role of NADPH oxidase (NOX) in inflammation-mediated SN neurotoxicity. A comparison of control (NOX2(+/+) ) mice with NOX subunit gp91(phox) -deficient (NOX2(-/-) ) mice 10 months after LPS administration (5 mg/kg, i.p.) resulted in a 39% (P < 0.01) loss of TH+IR neurons in NOX2(+/+) mice, whereas NOX2(-/-) mice did not show a significant decrease. Microglia (Iba1+IR) showed morphological activation in NOX2(+/+) mice, but not in NOX2(-/-) mice at 1 hr. Treatment of NOX2(+/+) mice with LPS resulted in a 12-fold increase in NOX2 mRNA in midbrain and 5.5-6.5-fold increases in NOX2 protein (+IR) in SN compared with the saline controls. Brain reactive oxygen species (ROS), determined using diphenyliodonium histochemistry, was increased by LPS in SN between 1 hr and 20 months. Diphenyliodonium (DPI), an NOX inhibitor, blocked LPS-induced activation of microglia and production of ROS, TNFα, IL-1β, and MCP-1. Although LPS increased microglial activation and ROS at all ages studied, saline control NOX2(+/+) mice showed age-related increases in microglial activation, NOX, and ROS levels at 12 and 22 months of age. Together, these results suggest that NOX contributes to persistent microglial activation, ROS production, and dopaminergic neurodegeneration that persist and continue to increase with age.

  17. NADPH oxidase and aging drive microglial activation, oxidative stress and dopaminergic neurodegeneration following systemic LPS administration

    PubMed Central

    Qin, Liya; Liu, Yuxin; Hong, Jau-Shyong; Crews, Fulton T.

    2013-01-01

    Parkinson’s disease is characterized by a progressive degeneration of substantia nigra (SN) dopaminergic neurons with age. We previously found that a single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) injection caused a slow progressive loss of tyrosine hydroxylase immunoreactive (TH+IR) neurons in SN associated with increasing motor dysfunction. In this study, we investigated the role of NADPH oxidase (NOX) in inflammation-mediated SN neurotoxicity. A comparison of control (NOX2+/+) mice with NOX subunit gp91phox-deficient (NOX2−/−) mice 10 months after LPS administration (5 mg/kg, i.p.) resulted in a 39% (p<0.01) loss of TH+IR neurons in NOX2+/+ mice, whereas, NOX2−/− mice did not show a significant decrease. Microglia (Iba1+IR) showed morphological activation in NOX2+/+ mice, but not in NOX2−/− mice at 1 hour. Treatment of NOX2+/+ mice with LPS resulted in a 12 fold increase in NOX2 mRNA in midbrain and 5.5–6.5 fold increases in NOX2 protein (+IR) in SN compared to the saline controls. Brain reactive oxygen species (ROS), determined by hydroethidine histochemistry, was increased by LPS in SN between 1 hour and 20 months. Diphenyliodonium (DPI), a NOX inhibitor, blocked LPS-induced activation of microglia and production of ROS, TNFα, IL-1β, and MCP-1. Although LPS increased microglial activation and ROS at all ages studied, saline control NOX2+/+ mice showed age-related increases in microglial activation, NOX and ROS levels at 12 and 22 months of age. Together, these results suggest that NOX contributes to persistent microglial activation, ROS production and dopaminergic neurodegeneration that persist and continue to increase with age. PMID:23536230

  18. Identification of potent bactericidal compounds produced by escapin, an L-amino acid oxidase in the ink of the sea hare Aplysia californica.

    PubMed

    Ko, Ko-Chun; Wang, Binghe; Tai, Phang C; Derby, Charles D

    2008-12-01

    The ink of sea hares (Aplysia californica) contains escapin, an L-amino acid oxidase that metabolizes L-lysine, thereby producing a mixture that kills microbes and deters attacking predators. This secretion contains H2O2,ammonia, and an equilibrium mixture of "escapin intermediate product" (EIP-K) that includes alpha-keto-epsilon-aminocaproic acid and several other molecules. Components of the equilibrium mixture react nonenzymatically with H2O2 to form "escapin end product" (EEP-K), which contains delta-aminovaleric acid and delta-valerolactam. The proportions of the molecules in this equilibrium mixture change with pH, and this is biologically important because the secretion is pH 5 when released but becomes pH 8 when fully diluted in seawater. The goal of the current study was to identify which molecules in this equilibrium mixture are bactericidal. We show that a mixture of H2O2 and EIP-K, but not EEP-K, at low mM concentrations is synergistically responsible for most of the bactericidal activity of the secretion against Escherichia coli, Vibrio harveyi, Staphylococcus aureus,and Pseudomonas aeruginosa. Low pH enhances the bactericidal effect, and this does not result from stress associated with low pH itself. Sequential exposure to low mM concentrations of EIP-K and H2O2, in either order, does not kill E. coli. Reaction products formed when L-arginine is substituted for L-lysine have almost no bactericidal activity. Our results favor the idea that the bactericidal activity is due to unstable intermediates of the reaction of alpha-keto-epsilon-aminocaproic acid with H2O2.

  19. 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

  20. 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.

  1. 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

  2. NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice

    PubMed Central

    Song, Hongman; Vijayasarathy, Camasamudram; Zeng, Yong; Marangoni, Dario; Bush, Ronald A.; Wu, Zhijian; Sieving, Paul A.

    2016-01-01

    Purpose The active form of small GTPase RAC1 is required for activation of NADPH oxidase (NOX), which in turn generates reactive oxygen species (ROS) in nonphagocytic cells. We explored whether NOX-induced oxidative stress contributes to rod degeneration in retinas expressing constitutively active (CA) RAC1. Methods Transgenic (Tg)–CA-RAC1 mice were given apocynin (10 mg/kg, intraperitoneal), a NOX inhibitor, or vehicle daily for up to 13 weeks. Superoxide production and oxidative damage were assessed by dihydroethidium staining and by protein carbonyls and malondialdehyde levels, respectively. Outer nuclear layer (ONL) cells were counted and electroretinogram (ERG) amplitudes measured in Tg-CA-RAC1 mice. Outer nuclear layer cells were counted in wild-type (WT) mice after transfer of CA-Rac1 gene by subretinal injection of AAV8-pOpsin-CA Rac1-GFP. Results Transgenic-CA-RAC1 retinas had significantly fewer photoreceptor cells and more apoptotic ONL cells than WT controls from postnatal week (Pw) 3 to Pw13. Superoxide accumulation and protein and lipid oxidation were increased in Tg-CA-RAC1 retinas and were reduced in mice treated with apocynin. Apocynin reduced the loss of photoreceptors and increased the rod ERG a- and b-wave amplitudes when compared with vehicle-injected transgenic controls. Photoreceptor loss was also observed in regions of adult WT retina transduced with AAV8-pOpsin-CA Rac1-GFP but not in neighboring regions that were not transduced or in AAV8-pOpsin-GFP–transduced retinas. Conclusions Constitutively active RAC1 promotes photoreceptor cell death by oxidative damage that occurs, at least partially, through NOX-induced ROS. Reactive oxygen species are likely involved in multiple forms of retinal degenerations, and our results support investigating RAC1 inhibition as a therapeutic approach that targets this disease pathway. PMID:27233035

  3. Successful Treatment of Intracranial Glioblastoma Xenografts With a Monoamine Oxidase B-Activated Pro-Drug

    PubMed Central

    Sharpe, Martyn A.; Livingston, Andrew D.; Gist, Taylor L.; Ghosh, Pardip; Han, Junyan; Baskin, David S.

    2015-01-01

    The last major advance in the treatment of glioblastoma multiforme (GBM) was the introduction of temozolomide in 1999. Treatment with temozolomide following surgical debulking extends survival rate compared to radiotherapy and debulking alone. However, virtually all glioblastoma patients experience disease progression within 7 to 10 months. Although many salvage treatments, including bevacizumab, rechallenge with temozolomide, and other alkylating agents, have been evaluated, none of these clearly improves survival. Monoamine oxidase B (MAOB) is highly expressed in glioblastoma cell mitochondria, and mitochondrial function is intimately tied to treatment-resistant glioblastoma progression. These glioblastoma properties provide a strong rationale for pursuing a MAOB-selective pro-drug treatment approach that, upon drug activation, targets glioblastoma mitochondria, especially mitochondrial DNA. MP-MUS is the lead compound in a family of pro-drugs designed to treat GBM that is converted into the mature, mitochondria-targeting drug, P+-MUS, by MAOB. We show that MP-MUS can successfully kill primary gliomas in vitro and in vivo mouse xenograft models. PMID:26501110

  4. 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.

  5. CO-dynamics in the active site of cytochrome c oxidase

    NASA Astrophysics Data System (ADS)

    Soloviov, Maksym; Meuwly, Markus

    2014-04-01

    The transfer of CO from heme a3 to the CuB site in Cytochrome c oxidase (CcO) after photolysis is studied using molecular dynamics simulations using an explicitly reactive, parametrized potential energy surface based on density functional theory calculations. After photodissociation from the heme-Fe, the CO ligand rebinds to the CuB site on the sub-picosecond time scale. Depending on the simulation protocol the characteristic time ranges from 260 fs to 380 fs which compares with an estimated 450 fs from experiment based on the analysis of the spectral changes as a function of time delay after the photodissociating pulse. Following photoexcitation ≈90% of the ligands are found to rebind to either the CuB (major component, 85%) or the heme-Fe (minor component, 2%) whereas about 10% remain in an unbound state. The infrared spectra of unbound CO in the active site is broad and featureless and no appreciable shift relative to gas-phase CO is found, which is in contrast to the situation in myoglobin. These observations explain why experimentally, unbound CO in the binuclear site of CcO has not been found as yet.

  6. High Osmolarity Environments Activate the Mitochondrial Alternative Oxidase in Debaryomyces Hansenii

    PubMed Central

    Garcia-Neto, Wilson; Cabrera-Orefice, Alfredo; Uribe-Carvajal, Salvador; Kowaltowski, Alicia J.; Alberto Luévano-Martínez, Luis

    2017-01-01

    The oleaginous yeast Debaryomyces hansenii is a good model to understand molecular mechanisms involved in halotolerance because of its impressive ability to survive under a wide range of salt concentrations. Several cellular adaptations are implicated in this response, including the presence of a cyanide-insensitive ubiquinol oxidase (Aox). This protein, which is present in several taxonomical orders, has been related to different stress responses. However, little is known about its role in mitochondria during transitions from low to high saline environments. In this report, we analyze the effects of Aox in shifts from low to high salt concentrations in the culture media. At early stages of a salt insult, we observed that this protein prevents the overflow of electrons on the mitochondrial respiratory chain, thus, decreasing the production of reactive oxygen species. Interestingly, in the presence of high osmolite concentrations, Aox activity is able to sustain a stable membrane potential when coupled to complex I, despite a compromised cytochrome pathway. Taken together, our results suggest that under high osmolarity conditions Aox plays a critical role regulating mitochondrial physiology. PMID:28060946

  7. Concomitant Prevalence of Low Serum Diamine Oxidase Activity and Carbohydrate Malabsorption

    PubMed Central

    Meinitzer, Andreas; Mangge, Harald; Kriegshäuser, Gernot; Halwachs-Baumann, Gabriele; Reininghaus, Eva Z.; Bengesser, Susanne A.; Schnedl, Wolfgang J.

    2016-01-01

    The aim of this retrospective study was to analyze the concomitant prevalence rates for lactose malabsorption (LM), fructose malabsorption (FM), and histamine intolerance (HI) in patients with so far unexplained gastrointestinal (GI) symptoms. A total of 439 outpatients, who presented unclear abdominal discomfort, underwent lactose (50 g) and fructose (25 g) hydrogen (H2) breath tests. Additionally, serum diamine oxidase (DAO) measurements were performed. Individuals with low serum DAO activity (<10 U/mL), GI symptoms, and response to histamine-free diet were diagnosed with HI. Of all 439 patients, 341 (77.7%) were found with 7 various GI conditions. In total, 94 (21.4%), 31 (7.1%), and 100 (22.8%) individuals presented LM, FM, or HI only, whereas 116 (26.4%) patients showed an overlap of GI entities investigated here. Interestingly, 89 out of 241 (36.9%) individuals with carbohydrate malabsorption were also diagnosed with HI (LM + HI: 52 [11.8%], FM + HI: 23 [5.2%], and LM + FM + HI 14 [3.2%] individuals). In conclusion different combinations of LM, FM, and HI are present in individuals with unclear abdominal discomfort/pain. In clinical practice we suggest testing for LM, FM, and additional HI in the diagnostic work-up of these patients. Depending on these various diagnoses possible, patients should get an individualized dietary advice. PMID:28042564

  8. 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

  9. 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.

  10. Successful Treatment of Intracranial Glioblastoma Xenografts With a Monoamine Oxidase B-Activated Pro-Drug.

    PubMed

    Sharpe, Martyn A; Livingston, Andrew D; Gist, Taylor L; Ghosh, Pardip; Han, Junyan; Baskin, David S

    2015-09-01

    The last major advance in the treatment of glioblastoma multiforme (GBM) was the introduction of temozolomide in 1999. Treatment with temozolomide following surgical debulking extends survival rate compared to radiotherapy and debulking alone. However, virtually all glioblastoma patients experience disease progression within 7 to 10 months. Although many salvage treatments, including bevacizumab, rechallenge with temozolomide, and other alkylating agents, have been evaluated, none of these clearly improves survival. Monoamine oxidase B (MAOB) is highly expressed in glioblastoma cell mitochondria, and mitochondrial function is intimately tied to treatment-resistant glioblastoma progression. These glioblastoma properties provide a strong rationale for pursuing a MAOB-selective pro-drug treatment approach that, upon drug activation, targets glioblastoma mitochondria, especially mitochondrial DNA. MP-MUS is the lead compound in a family of pro-drugs designed to treat GBM that is converted into the mature, mitochondria-targeting drug, P(+)-MUS, by MAOB. We show that MP-MUS can successfully kill primary gliomas in vitro and in vivo mouse xenograft models.

  11. 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.

  12. 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

    2016-06-17

    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.

  13. Sildenafil Promotes eNOS Activation and Inhibits NADPH Oxidase in the Transgenic Sickle Cell Mouse Penis

    PubMed Central

    Musicki, Biljana; Bivalacqua, Trinity J.; Champion, Hunter C.; Burnett, Arthur L.

    2014-01-01

    Introduction Sickle cell disease (SCD)-associated vasculopathy in the penis is characterized by aberrant nitric oxide and phosphodiesterase (PDE) 5 signaling, and by increased oxidative stress. Preliminary clinical trials show that continuous treatment with PDE5 inhibitor sildenafil unassociated with sexual activity decreases priapic activity in patients with SCD. However, the mechanism of its vasculoprotective effect in the penis remains unclear. Aims We evaluated whether continuous administration of PDE5 inhibitor sildenafil promotes eNOS function at posttranslational levels and decreases superoxide-producing enzyme NADPH oxidase activity in the sickle cell mouse penis. Methods SCD transgenic mice were used as an animal model of SCD. WT mice served as controls. Mice received treatment with the PDE5 inhibitor sildenafil (100 mg/kg/day) or vehicle for 3 weeks. eNOS phosphorylation on Ser-1177 (positive regulatory site), eNOS interactions with heat-shock protein 90 (HSP90) (positive regulator), phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177), an NADPH oxidase catalytic subunit gp91(phox), and a marker of oxidative stress (4-hydroxy-2-nonenal [HNE]) were measured by Western blot. Main Outcome Measures Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse. Results Continuous treatment with sildenafil reversed (P < 0.05) the abnormalities in protein expressions of P-eNOS (Ser-1177), eNOS/HSP90 interaction, P-AKT, protein expression of gp91(phox), and 4-HNE, in the sickle cell mouse penis. Sildenafil treatment of WT mice did not affect any of these parameters. Conclusion Our findings that sildenafil enhances eNOS activation and inhibits NADPH oxidase function in the sickle cell mouse penis offers a vasculoprotective molecular basis for the therapeutic effect of sildenafil in the penis in association with SCD. PMID:24251665

  14. Activation of TRPM2 and TRPV1 Channels in Dorsal Root Ganglion by NADPH Oxidase and Protein Kinase C Molecular Pathways: a Patch Clamp Study.

    PubMed

    Nazıroğlu, Mustafa

    2017-03-01

    Despite considerable research, the mechanisms of neuropathic pain induced by excessive oxidative stress production and overload calcium ion (Ca(2+)) entry in dorsal root ganglion (DRG) remain substantially unidentified. The transient receptor potential melastatin 2 (TRPM2) and vanilloid 1 (TRPV1) channels are activated with different stimuli including oxidative stress. TRPM2 and TRPV1 have been shown to be involved in induction of neuropathic pain. However, the activation mechanisms of TRPM2 and TRPV1 via NADPH oxidase and protein kinase C (PKC) pathways are poorly understood. In this study, I investigated the roles of NADPH oxidase and PKC on Ca(2+) entry through TRPM2 and TRPV1 channels in in vitro DRG neurons of rats. Rat DRG neurons were used in whole-cell patch clamp experiments. The H2O2-induced TRPM2 current densities were decreased by N-(p-amylcinnamoyl)anthranilic acid (ACA), and dose-dependent capsaicin (CAP) and H2O2-induced TRPV1 currents were inhibited by capsazepine (CPZ). The TRPV1 channel is activated in the DRG neurons by 0.01 mM capsaicin but not 0.001 mM or 0.05 mM capsaicin. TRPM2 and TRPV1 currents were increased by the PKC activator, phorbol myristate acetate (PMA), although the currents were decreased by ACA, CPZ, and the PKC inhibitor, bisindolylmaleimide I (BIM). Both channel currents were further increased by PMA + H2O2 as compared to H2O2 only. In the combined presence of PMA + BIM, no TRPM2 or TRPV1 currents were observed. The CAP and H2O2-induced TRPM2 current densities were also decreased by the NADPH oxidase inhibitors apocynin and N-Acetylcysteine. In conclusion, these results demonstrate a protective role for NADPH oxidase and PKC inhibitors on Ca(2+) entry through TRPM2 and TRPV1 channels in DRG neurons. Since excessive oxidative stress production and Ca(2+) entry are implicated in the pathophysiology of neuropathic pain, the findings may be relevant to the etiology and treatment of neuropathology in DRG neurons.

  15. 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

  16. Activity of glucose oxidase immobilized onto Fe3+ attached hydroxypropyl methylcellulose films.

    PubMed

    Sözügeçer, Sevgi; Bayramgil, Nursel Pekel

    2013-01-01

    Hydroxypropyl methylcellulose (HMPC) insoluble films were prepared by (60)Co-γ irradiation of 10% (w/w) aqueous solutions of hydroxypropyl methylcellulose. The adsorption of Fe(3+) onto HPMC films was studied in the range of pH 3.0-7.0. The effect of initial concentrations of Fe(3+) solutions on adsorption capacity was studied in the range of 100-1000 ppm. Maximum adsorption capacity was found as 250 mg Fe(3+)/g dry HPMC film at pH 5.0. The structure and the morphology of Fe(3+)-attached HPMC film were evaluated by using FTIR/ATR and SEM-EDX methods. Glucose oxidase (GOX) immobilization on both pristine HPMC and maximum Fe(3+)-attached HPMC film was investigated in aqueous solutions containing different amount of GOX and at different pHs. Maximum GOX adsorption capacity was found as 500 mg/g Fe(3+)-attached HPMC film. Residual activity of GOX on pristine HPMC and Fe(3+)-attached HPMC films was investigated with changing pH. While maximum residual GOX activity was observed at pH 6.0 for free enzyme, it was obtained by HPMC and Fe(3+)-attached HPMC at pH 7.0. GOX desorption studies were achieved by using pH 6.0 buffer (I=0.02 M) and 0.1 M EDTA solutions. The long-term stability and activity studies of GOX, which is immobilized onto Fe(3+)-attached HPMC films are still under our investigation.

  17. In Vitro Effects of Cognitives and Nootropics on Mitochondrial Respiration and Monoamine Oxidase Activity.

    PubMed

    Singh, Namrata; Hroudová, Jana; Fišar, Zdeněk

    2016-09-23

    Impairment of mitochondrial metabolism, particularly the electron transport chain (ETC), as well as increased oxidative stress might play a significant role in pathogenesis of Alzheimer's disease (AD). Some effects of drugs used for symptomatic AD treatment may be related to their direct action on mitochondrial function. In vitro effects of pharmacologically different cognitives (galantamine, donepezil, rivastigmine, 7-MEOTA, memantine) and nootropic drugs (latrepirdine, piracetam) were investigated on selected mitochondrial parameters: activities of ETC complexes I, II + III, and IV, citrate synthase, monoamine oxidase (MAO), oxygen consumption rate, and hydrogen peroxide production of pig brain mitochondria. Complex I activity was decreased by galantamine, donepezil, and memantine; complex II + III activity was increased by galantamine. None of the tested drugs caused significant changes in the rate of mitochondrial oxygen consumption, even at high concentrations. Except galantamine, all tested drugs were selective MAO-A inhibitors. Latrepirdine, donepezil, and 7-MEOTA were found to be the most potent MAO-A inhibitors. Succinate-induced mitochondrial hydrogen peroxide production was not significantly affected by the drugs tested. The direct effect of cognitives and nootropics used in the treatment of AD on mitochondrial respiration is relatively small. The safest drugs in terms of disturbing mitochondrial function appear to be piracetam and rivastigmine. The MAO-A inhibition by cognitives and nootropics may also participate in mitochondrial neuroprotection. The results support the future research aimed at measuring the effects of currently used drugs or newly synthesized drugs on mitochondrial functioning in order to understand their mechanism of action.

  18. The NADPH oxidase inhibitor diphenyleneiodonium activates the human TRPA1 nociceptor.

    PubMed

    Suzuki, Hiroka; Hatano, Noriyuki; Muraki, Yukiko; Itoh, Yuka; Kimura, Satoko; Hayashi, Hidetoshi; Onozaki, Kikuo; Ohi, Yoshiaki; Haji, Akira; Muraki, Katsuhiko

    2014-08-15

    Transient receptor potential ankyrin 1 (TRPA1) is a Ca(2+)-permeable nonselective cation channel expressed in neuronal and nonneuronal cells and plays an important role in acute and inflammatory pain. Here, we show that an NADPH oxidase (NOX) inhibitor, diphenyleneiodonium (DPI), functions as a TRPA1 activator in human embryonic kidney cells expressing human TRPA1 (HEK-TRPA1) and in human fibroblast-like synoviocytes. Application of DPI at 0.03-10 μM induced a Ca(2+) response in HEK-TRPA1 cells in a concentration-dependent manner. The Ca(2+) response was effectively blocked by a selective TRPA1 antagonist, HC-030031 (HC). In contrast, DPI had no effect on HEK cells expressing TRPV1-V4 or TRPM8. Four other NOX inhibitors, apocynin (APO), VAS2870 (VAS), plumbagin, and 2-acetylphenothiazine, also induced a Ca(2+) response in HEK-TRPA1 cells, which was inhibited by pretreatment with HC. In the presence of 5 mM glutathione, the Ca(2+) response to DPI was effectively reduced. Moreover, mutation of cysteine 621 in TRPA1 substantially inhibited the DPI-induced Ca(2+) response, while it did not inhibit the APO- and VAS-induced responses. The channel activity was induced by DPI in excised membrane patches with both outside-out and inside-out configurations. Internal application of neomycin significantly inhibited the DPI-induced inward currents. In inflammatory synoviocytes with TRPA1, DPI evoked a Ca(2+) response that was sensitive to HC. In mice, intraplantar injection of DPI caused a pain-related response which was inhibited by preadministration with HC. Taken together, our findings demonstrate that DPI and other NOX inhibitors activate human TRPA1 without mediating NOX.

  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.

  20. 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.

  1. 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.

  2. Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate† ,‡

    PubMed Central

    Valley, Michael P.; Fitzpatrick, Paul F.

    2006-01-01

    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 ΔG‡ 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

  3. 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.

  4. Polyphenols enhance platelet nitric oxide by inhibiting protein kinase C-dependent NADPH oxidase activation: effect on platelet recruitment.

    PubMed

    Pignatelli, P; Di Santo, S; Buchetti, B; Sanguigni, V; Brunelli, A; Violi, F

    2006-06-01

    Several studies demonstrated an inverse association between polyphenol intake and cardiovascular events. Platelet recruitment is an important phase of platelet activation at the site of vascular injury, but it has never been investigated whether polyphenols influence platelet recruitment. The aim of the study was to analyze in vitro whether two polyphenols, quercetin and catechin, were able to affect platelet recruitment. Platelet recruitment was reduced by NO donors and by NADPH oxidase inhibitors and was enhanced by L-NAME, an inhibitor of NO synthase. Quercetin and catechin, but not single polyphenol, significantly inhibited platelet recruitment in a concentration-dependent fashion. The formation of superoxide anion was significantly inhibited in platelets incubated with quercetin and catechin but was unaffected by a single polyphenol. Incubation of platelets with quercetin and catechin resulted in inhibition of PKC and NADPH oxidase activation. Treatment of platelets with quercetin and catechin resulted in an increase of NO and also down-regulated the expression of GpIIb/IIIa glycoprotein. This study shows that the polyphenols quercetin and catechin synergistically act in reducing platelet recruitment via inhibition of PKC-dependent NADPH oxidase activation. This effect, resulting in NO-mediated platelet glycoprotein GpIIb/IIIa down-regulation, could provide a novel mechanism through which polyphenols reduce cardiovascular disease.

  5. Protein kinase C epsilon activates lens mitochondrial cytochrome c oxidase subunit IV during hypoxia.

    PubMed

    Barnett, Michael; Lin, Dingbo; Akoyev, Vladimir; Willard, Lloyd; Takemoto, Dolores

    2008-02-01

    Protein kinase C (PKC) isoforms have been identified as major cellular signaling proteins that act directly in response to oxidation conditions. In retina and lens two isoforms of PKC respond to changes in oxidative stress, PKCgamma and PKCepsilon, while only PKCepsilon is found in heart. In heart the PKCepsilon acts on connexin 43 to protect from hypoxia. The presence of both isoforms in the lens led to this study to determine if lens PKCepsilon had unique targets. Both lens epithelial cells in culture and whole mouse lens were examined using PKC isoform-specific enzyme activity assays, co-immunoprecipitation, confocal microscopy, immunoblots, and light and electron microscopy. PKCepsilon was found in lens epithelium and cortex but not in the nucleus of mouse lens. The PKCepsilon isoform was activated in both epithelium and whole lens by 5% oxygen when compared to activity at 21% oxygen. In hypoxic conditions (5% oxygen) the PKCepsilon co-immunoprecipitated with the mitochondrial cytochrome c oxidase IV subunit (CytCOx). Concomitant with this the CytCOx enzyme activity was elevated and increased co-localization of CytCOx with PCKvarepsilon was observed using immunolabeling and confocal microscopy. In contrast, no hypoxia-induced activation of CytCOx was observed in lenses from the PKCepsilon knockout mice. Lens from 6-week-old PKCepsilon knockout mice had a disorganized bow region which was filled with vacuoles indicating a possible loss of mitochondria but the size of the lens was not altered. Electron microscopy demonstrated that the nuclei of the PCKepsilon knockout mice were abnormal in shape. Thus, PKCepsilon is found to be activated by hypoxia and this results in the activation of the mitochondrial protein CytCOx. This could protect the lens from mitochondrial damage under the naturally hypoxic conditions observed in this tissue. Lens oxygen levels must remain low. Elevation of oxygen which occurs during vitreal detachment or liquification is associated with

  6. 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.

  7. Mitotane alters mitochondrial respiratory chain activity by inducing cytochrome c oxidase defect in human adrenocortical cells.

    PubMed

    Hescot, Ségolène; Slama, Abdelhamid; Lombès, Anne; Paci, Angelo; Remy, Hervé; Leboulleux, Sophie; Chadarevian, Rita; Trabado, Séverine; Amazit, Larbi; Young, Jacques; Baudin, Eric; Lombès, Marc

    2013-06-01

    Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane is the most effective medical therapy for adrenocortical carcinoma, but its molecular mechanism of action remains poorly understood. Although mitotane is known to have mitochondrial (mt) effects, a direct link to mt dysfunction has never been established. We examined the functional consequences of mitotane exposure on proliferation, steroidogenesis, and mt respiratory chain, biogenesis and morphology, in two human adrenocortical cell lines, the steroid-secreting H295R line and the non-secreting SW13 line. Mitotane inhibited cell proliferation in a dose- and a time-dependent manner. At the concentration of 50 μM (14 mg/l), which corresponds to the threshold for therapeutic efficacy, mitotane drastically reduced cortisol and 17-hydroxyprogesterone secretions by 70%. This was accompanied by significant decreases in the expression of genes encoding mt proteins involved in steroidogenesis (STAR, CYP11B1, and CYP11B2). In both H295R and SW13 cells, 50 μM mitotane significantly inhibited (50%) the maximum velocity of the activity of the respiratory chain complex IV (cytochrome c oxidase (COX)). This effect was associated with a drastic reduction in steady-state levels of the whole COX complex as revealed by blue native PAGE and reduced mRNA expression of both mtDNA-encoded COX2 (MT-CO2) and nuclear DNA-encoded COX4 (COX4I1) subunits. In contrast, the activity and expression of respiratory chain complexes II and III were unaffected by mitotane treatment. Lastly, mitotane exposure enhanced mt biogenesis (increase in mtDNA content and PGC1α (PPARGC1A) expression) and triggered fragmentation of the mt network. Altogether, our results provide first evidence that mitotane induced a mt respiratory chain defect in human adrenocortical cells.

  8. 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

  9. 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....

  10. 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%.

  11. Phagocyte NADPH-oxidase. Studies with flavin analogues as active site probes in triton X-100-solubilized preparations.

    PubMed

    Parkinson, J F; Gabig, T G

    1988-06-25

    NADPH-oxidase of stimulated human neutrophil membranes was solubilized in Triton X-100 and activity reconstituted with FAD, 8-F-FAD, 8-phenyl-S-FAD, and 8-S-FAD. The enzyme had similar affinities for all the flavins with Km values in the 60-80 nM range. Vmax was found to increase 4-fold with increasing redox midpoint potential of the flavin. 8-F-FAD reconstituted with the enzyme was reactive toward thiophenol, suggesting exposure of the 8-position to solvent, a finding supported by unsuccessful attempts to label the enzyme with the photoaffinity probe 8-N3-[32P]FAD. Solubilized oxidase stabilized the red thiolate form of 8-S-FAD, a characteristic of flavoproteins of the dehydrogenase/electron transferase classes which stabilize the blue neutral form of the flavin semiquinone radical.

  12. Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning deficit and inhibiting cytochrome oxidase activity.

    PubMed Central

    Bennett, M C; Mlady, G W; Fleshner, M; Rose, G M

    1996-01-01

    Previously, we developed a rat model of persistent mitochondrial dysfunction based upon the chronic partial inhibition of the mitochondrial enzyme cytochrome oxidase (EC 1.9.3.1). Continuous systemic infusion of sodium azide at approximately 1 mg/kg per hr inhibited cytochrome oxidase activity and produced a spatial learning deficit. In other laboratories, glucocorticoids have been reported to exacerbate neuronal damage from various acute metabolic insults. Therefore, we tested the hypothesis that corticosterone, the primary glucocorticoid in the rat, would potentiate the sodium azide-induced learning deficit. To this end, we first identified nonimpairing doses of sodium azide (approximately 0.75 mg/kg per hr) and corticosterone (100-mg pellet, 3-week sustained-release). We now report that chronic co-administration of these individually nonimpairing treatments produced a severe learning deficit. Moreover, the low dose of corticosterone, which did not elevate serum corticosterone, acted synergistically with sodium azide to inhibit cytochrome oxidase activity. The latter result represents a previously unidentified effect of glucocorticoids that provides a candidate mechanism for glucocorticoid potentiation of neurotoxicity induced by metabolic insult. These results may have the clinical implication of expanding the definition of hypercortisolism in patient populations with compromised oxidative metabolism. Furthermore, they suggest that glucocorticoid treatment may contribute to pathology in disease or trauma conditions that involve metabolic insult. PMID:8577764

  13. 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

  14. 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.

  15. Increased Expression and Cellular Localization of Spermine Oxidase in Ulcerative Colitis and Relationship to Disease Activity

    PubMed Central

    Hong, Shih-Kuang S.; Chaturvedi, Rupesh; Blanca Piazuelo, M.; Coburn, Lori A.; Williams, Christopher S.; Delgado, Alberto G.; Casero, Robert A.; Schwartz, David A.; Wilson, Keith T.

    2010-01-01

    Background Polyamines are important in cell growth and wound repair, but have also been implicated in inflammation-induced carcinogenesis. Polyamine metabolism includes back-conversion of spermine to spermidine by the enzyme spermine oxidase (SMO), which produces hydrogen peroxide that causes oxidative stress. In ulcerative colitis (UC), levels of spermine are decreased compared to spermidine. Therefore, we sought to determine if SMO is involved in UC. Methods Colon biopsies and clinical information from subjects undergoing colonoscopy for evaluation of UC or colorectal cancer screening were utilized from 16 normal controls and 53 UC cases. Histopathologic disease severity was graded and the Mayo Disease Activity Index (DAI) and endoscopy subscore assessed. SMO mRNA expression was measured in frozen biopsies by Taq-Man-based real-time polymerase chain reaction (PCR). Formalin-fixed tissues were used for SMO immunohistochemistry. Results There was a 3.1-fold upregulation of SMO mRNA levels in UC patients compared to controls (P = 0.044), and a 3.7-fold increase in involved left colon versus paired uninvolved right colon (P < 0.001). With worsening histologic injury in UC there was a progressive increase in SMO staining of mononuclear inflammatory cells. There was a similar increase in SMO staining with worsening endoscopic disease severity and strong correlation with the DAI (r = 0.653, P < 0.001). Inflammatory cell SMO staining was increased in involved left colon versus uninvolved right colon. Conclusions SMO expression is upregulated in UC tissues, deriving from increased levels in mononuclear inflammatory cells. Dysregulated polyamine homeostasis may contribute to chronic UC by altering immune responses and increasing oxidative stress. PMID:20127992

  16. 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

    2016-01-01

    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.

  17. ATL9, a RING Zinc Finger Protein with E3 Ubiquitin Ligase Activity Implicated in Chitin- and NADPH Oxidase-Mediated Defense Responses

    PubMed Central

    Berrocal-Lobo, Marta; Stone, Sophia; Yang, Xin; Antico, Jay; Callis, Judy; Ramonell, Katrina M.; Somerville, Shauna

    2010-01-01

    Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 ( = ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst. PMID:21203445

  18. Differences in Monoamine Oxidase Activity in the Brain of Wistar and August Rats with High and Low Locomotor Activity: A Cytochemical Study.

    PubMed

    Sergutina, A V; Rakhmanova, V I

    2016-06-01

    Monoamine oxidase activity was quantitatively assessed by cytochemical method in brain structures (layers III and V of the sensorimotor cortex, caudate nucleus, nucleus accumbens, hippocampal CA3 field) of rats of August line and Wistar population with high and low locomotor activity in the open fi eld test. Monoamine oxidase activity (substrate tryptamine) predominated in the nucleus accumbens of Wistar rats with high motor activity in comparison with rats with low locomotor activity. In August rats, enzyme activity (substrates tryptamine and serotonin) predominated in the hippocampus of animals with high motor activity. Comparison of August rats with low locomotor activity and Wistar rats with high motor activity (i.e. animals demonstrating maximum differences in motor function) revealed significantly higher activity of the enzyme (substrates tryptamine and serotonin) in the hippocampus of Wistar rats. The study demonstrates clear-cut morphochemical specificity of monoaminergic metabolism based on the differences in the cytochemical parameter "monoamine oxidase activity", in the studied brain structures, responsible for the formation and realization of goal-directed behavior in Wistar and August rats.

  19. Use of a quantitative oxidase test for characterizing oxidative metabolism in bacteria.

    PubMed Central

    Jurtshuk, P; McQuitty, D N

    1976-01-01

    It was possible to quantitate the terminal oxidase(s) reaction using bacterial resting-cell suspensions and demonstrate the usefulness of this reaction for taxonomic purposes. Resting-cell suspensions of physiologically diverse bacteria were examined for their capabilities of oxidizing N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) using a manometric assay. For organisms having this capability, it was possible to calculate the conventional TMPD oxidase Q(O2) value (microliters of O2 consumed per hour per milligram [dry weight]). All cultures were grown heterotrophically at 30 C, under identical nutritional conditions, and were harvested at the late-logarithmic growth phase. The TMPD oxidase Q(O2) values showed perfect correlation with the Kovacs oxidase test and, in addition, it was possible to define quantitatively that point which separated oxidase-positive from oxidase-negative bacteria. Oxidase-negative bacteria exhibited a TMPD oxidase Q(O2) value (after correcting for the endogenous by substraction) of less than or equal 33 and had an uncorrected TMPD/endogenous ratio of less than or equal 5. The TMPD oxidase Q(O2) values were also correlated with the data obtained for the Hugh-Leifson Oxferm test. In general, bacteria that exhibited a respiratory mechanism had high TMPD oxidase values, whereas fermentative organsims had low TMPD oxidase activity. All exceptions to this are noted. This quantitative study also demonstrated that organisms that (i) lack a type c cytochrome, or (ii) lack a cytochrome-containing electron transport system, like the lactic acid bacteria, exhibited low or negligible TMPD oxidase Q(O2) values. From the 79 bacterial species (36 genera) examined, it appears that this quantitative oxidase test has taxonomic value that can differentiate the oxidative relationships between bacteria at the subspecies, species, and genera levels. PMID:1275489

  20. 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.

  1. Characterization of two amine oxidases from Aspergillus carbonarius AIU 205.

    PubMed

    Sugawara, Asami; Matsui, Daisuke; Yamada, Miwa; Asano, Yasuhisa; Isobe, Kimiyasu

    2015-06-01

    We have reported that Aspergillus carbonarius AIU 205, which was isolated by our group, produced three enzymes exhibiting oxidase activity for 4-aminobutanamide (4-ABAD) (J. Biosci. Bioeng., 117, 263-268, 2014). Among three enzymes, characteristics of enzyme I have been revealed, but those of the other two enzymes have not. In this study, we purified enzymes II and III, and compared their characteristics with those of enzyme I. Enzymes II and III also oxidized aliphatic monoamines, aromatic amines, and aliphatic aminoalcohols. In addition, the oxidase activity of both enzymes was strongly inhibited by carbonyl reagents and specific inhibitors for copper-containing amine oxidases. Thus, enzymes II and III were also classified into the copper-containing amine oxidase group (EC 1.4.3.6) along with enzyme I. However, these three enzymes differed from each other in their enzymatic, kinetic, and physicochemical properties. The N-terminal amino acid sequences also differed from each other; that of enzyme I was modified, that of enzyme II was similar to those of peroxisomal copper-containing amine oxidases, and that of enzyme III was similar to those of copper-containing amine oxidases from the genus Aspergillus. Therefore, we concluded that A. carbonarius AIU 205 produced three different types of amine oxidase in the mycelia.

  2. Involvement of activation of NADPH oxidase and extracellular signal-regulated kinase (ERK) in renal cell injury induced by zinc.

    PubMed

    Matsunaga, Yoshiko; Kawai, Yoshiko; Kohda, Yuka; Gemba, Munekazu

    2005-05-01

    Zinc is employed as a supplement; however, zinc-related nephropathy is not generally known. In this study, we investigated zinc-induced renal cell injury using a pig kidney-derived cultured renal epithelial cell line, LLC-PK(1), with proximal kidney tubule-like features, and examined the involvement of free radicals and extracellular signal-regulated kinase (ERK) in the cell injury. The LLC-PK(1) cells showed early uptake of zinc (30 microM), and the release of lactate dehydrogenase (LDH), an index of cell injury, was observed 24 hr after uptake. Three hours after zinc exposure, generation of reactive oxygen species (ROS) was increased. An antioxidant, N, N'-diphenyl-p-phenylenediamine (DPPD), inhibited a zinc-related increase in ROS generation and zinc-induced renal cell injury. An NADPH oxidase inhibitor, diphenyleneiodonium (DPI), inhibited a zinc-related increase in ROS generation and cell injury. We investigated translocation from the cytosol fraction of the p67(phox) subunit, which is involved in the activation of NADPH oxidase, to the membrane fraction, and translocation was induced 3 hr after zinc exposure. We examined the involvement of ERK1/2 in the deterioration of zinc-induced renal cell injury, and the association between ERK1/2 and an increase in ROS generation. Six hours after zinc exposure, the activation (phosphorylation) of ERK1/2 was observed. An antioxidant, DPPD, inhibited the zinc-related activation of ERK1/2. An MAPK/ERK kinase (MEK1/2) inhibitor, U0126, almost completely inhibited zinc-related cell injury (the release of LDH), but did not influence ROS generation. These results suggest that early intracellular uptake of zinc by LLC-PK(1) cells causes the activation of NADPH oxidase, and that ROS generation by the activation of the enzyme leads to the deterioration of renal cell injury via the activation of ERK1/2.

  3. Association-dissociation of the flavoprotein hog kidney D-amino acid oxidase. Determination of the monomer-dimer equilibrium constant and the energetics of subunit association.

    PubMed

    Horiike, K; Shiga, K; Nishina, Y; Isomoto, A; Yamano, T

    1977-11-01

    The enzyme concentration dependence of spectrophotometric titrations of hog kidney D-amino acid oxidase [EC 1.4.3.3] with p-aminobenzoate was studied. The monomer-dimer equilibrium constant of the oxidized holoenzyme at 25 degrees C was estimated to be 7 X 10(5)M-1 at pH 7.5 and 4X 10(6)M-1 at pH 8.3. The energetics of subunit association are discussed.

  4. 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.

  5. DPPH radical scavenging and semicarbazide-sensitive amine oxidase inhibitory and cytotoxic activities of Taiwanofungus camphoratus (Chang-chih).

    PubMed

    Wang, Guei-Jane; Lin, Shyr-Yi; Wu, Wen-Chun; Hou, Wen-Chi

    2007-08-01

    Wild, liquid state culture and solid state culture of Taiwanofungus camphoratus (Chang-chih) were sequentially extracted with cold water, methanol, and hot water to get cold water soluble, methanol soluble, and hot water soluble extracts respectively. The extracts from three Chang-chih were used to determine 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, semicarbazide sensitive amine oxidase inhibitory, and cytotoxic activities against B16-F10 and HT-1080 cell lines. It was found that extracted fractions from three Chang-chih exhibited the different levels of biological activities.

  6. Interplay between microbial d-amino acids and host d-amino acid oxidase modifies murine mucosal defence and gut microbiota.

    PubMed

    Sasabe, Jumpei; Miyoshi, Yurika; Rakoff-Nahoum, Seth; Zhang, Ting; Mita, Masashi; Davis, Brigid M; Hamase, Kenji; Waldor, Matthew K

    2016-07-25

    L-Amino acids are the building blocks for proteins synthesized in ribosomes in all kingdoms of life, but d-amino acids (d-aa) have important non-ribosome-based functions(1). Mammals synthesize d-Ser and d-Asp, primarily in the central nervous system, where d-Ser is critical for neurotransmission(2). Bacteria synthesize a largely distinct set of d-aa, which become integral components of the cell wall and are also released as free d-aa(3,4). However, the impact of free microbial d-aa on host physiology at the host-microbial interface has not been explored. Here, we show that the mouse intestine is rich in free d-aa that are derived from the microbiota. Furthermore, the microbiota induces production of d-amino acid oxidase (DAO) by intestinal epithelial cells, including goblet cells, which secrete the enzyme into the lumen. Oxidative deamination of intestinal d-aa by DAO, which yields the antimicrobial product H2O2, protects the mucosal surface in the small intestine from the cholera pathogen. DAO also modifies the composition of the microbiota and is associated with microbial induction of intestinal sIgA. Collectively, these results identify d-aa and DAO as previously unrecognized mediators of microbe-host interplay and homeostasis on the epithelial surface of the small intestine.

  7. Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers.

    PubMed Central

    Keller, H; Dreyer, C; Medin, J; Mahfoudi, A; Ozato, K; Wahli, W

    1993-01-01

    The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPAR alpha heterodimerizes with retinoid X receptor beta (RXR beta; RXR is the receptor for 9-cis-retinoic acid) and that the two receptors cooperate for the activation of the acyl-CoA oxidase gene promoter. The strongest stimulation of this promoter was obtained when both receptors were exposed simultaneously to their cognate activators. Furthermore, we show that natural fatty acids, and especially polyunsaturated fatty acids, activate PPARs as potently as does the hypolipidemic drug Wy 14,643, the most effective activator known so far. Moreover, we discovered that the synthetic arachidonic acid analogue 5,8,11,14-eicosatetraynoic acid is 100 times more effective than Wy 14,643 in the activation of PPAR alpha. In conclusion, our data demonstrate a convergence of the PPAR and RXR signaling pathways in the regulation of the peroxisomal beta-oxidation of fatty acids by fatty acids and retinoids. Images Fig. 1 Fig. 2 PMID:8384714

  8. Monomeric L-amino acid oxidase-induced mitochondrial dysfunction in Rhizoctonia solani Reveals a novel antagonistic mechanism of Trichoderma harzianum ETS 323.

    PubMed

    Yang, Chia-Ann; Cheng, Chi-Hua; Lee, Jeng-Woei; Lo, Chaur-Tsuen; Liu, Shu-Ying; Peng, Kou-Cheng

    2012-03-14

    The monomeric L-amino acid oxidase (mTh-LAAO) of Trichoderma harzianum ETS 323 has been suggested to antagonize Rhizoctonia solani by an unknown mechanism. Here, the mTh-LAAO-treated R. solani exhibited hyphal lysis and apoptotic characteristics such as DNA fragmentation, reactive oxygen species (ROS) accumulation, lipid peroxidation, and mitochondrial membrane potential depolarization. This hyphal lysis was suppressed by the mitochondria-dependent apoptosis inhibitor oligomycin while accompanied by reduction of ROS accumulation. This result suggested that mitochondria-mediated apoptosis in R. solani was involved in mTh-LAAO-induced growth inhibition, which was supported by the evidence of cytocheome c release and activation of caspases 9 and 3. Furthermore, the data indicated that the mTh-LAAO-induced fungal cell death was also closely interrelated with the interaction of mTh-LAAO with R. solani hyphal cell wall proteins. These results illuminate the biological function and mechanism underlying the antagonistic action of T. harzianum mTh-LAAO against fungal pathogens.

  9. NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL-1β secretion but not in inflammasome activation.

    PubMed

    Gabelloni, María Laura; Sabbione, Florencia; Jancic, Carolina; Fuxman Bass, Juan; Keitelman, Irene; Iula, Leonardo; Oleastro, Matías; Geffner, Jorge R; Trevani, Analía S

    2013-12-01

    Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin-1 beta (IL-1β) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL-1β processing is still controversial and has not been previously studied in neutrophils. We report here that IL-1β processing in human neutrophils is dependent on caspase-1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase-1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL-1β; whereas ROS negatively controlled caspase-1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL-1β out of the cell, a role never previously described. The complex ROS-mediated regulation of neutrophil IL-1β secretion might constitute a physiological mechanism to control IL-1β-dependent inflammatory processes where neutrophils play a crucial role.

  10. 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.

  11. Electrochemical activity of glucose oxidase on a poly(ionic liquid) - Au nanoparticle composite.

    SciTech Connect

    Lee, S.; Ringstrand, B. S.; Stone, D. A.; Firestone, M. A.

    2012-01-01

    Glucose oxidase (GOx) adsorbed on an ionic liquid-derived polymer containing internally organized columns of Au nanoparticles exhibits direct electron transfer and bioelectrocatalytic properties towards the oxidation of glucose. The cationic poly(ionic liquid) provides an ideal substrate for the electrostatic immobilization of GOx. The encapsulated Au nanoparticles serve to both promote the direct electron transfer with the recessed enzyme redox centers and impart electronic conduction to the composite, allowing it to function as an electrode for electrochemical detection.

  12. 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.

  13. 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…

  14. The oxidase activity of vascular adhesion protein-1 (VAP-1) induces endothelial E- and P-selectins and leukocyte binding.

    PubMed

    Jalkanen, Sirpa; Karikoski, Marika; Mercier, Nathalie; Koskinen, Kaisa; Henttinen, Tiina; Elima, Kati; Salmivirta, Katriina; Salmi, Marko

    2007-09-15

    Leukocyte migration from the blood into tissues is pivotal in immune homeostasis and in inflammation. During the multistep extravasation cascade, endothelial selectins (P- and E-selectin) and vascular adhesion protein-1 (VAP-1), a cell-surface-expressed oxidase, are important in tethering and rolling. Here, we studied the signaling functions of the catalytic activity of VAP-1. Using human endothelial cells transfected with wild-type VAP-1 and an enzymatically inactive VAP-1 point mutant, we show that transcription and translation of E- and P-selectins are induced through the enzymatic activity of VAP-1. Moreover, use of VAP-1-deficient animals and VAP-1-deficient animals carrying the human VAP-1 as a transgene show a VAP-enzyme activity-dependent induction of P-selectin in vivo. Up-regulation of P-selectin was found both in high endothelial venules in lymphoid tissues and in flat-walled vessels in noninflamed tissues. VAP-1 activity in vivo led to increased P-selectin-dependent binding of lymphocytes to endothelial cells. These data show that the oxidase reaction catalyzed by VAP-1 alters the expression of other molecules involved in the leukocyte extravasation cascade. Our findings indicate cross-talk between adhesion molecules involved in the tethering and rolling of leukocytes and show that VAP-1-dependent signaling can prime the vessels for an enhanced inflammatory response.

  15. STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defense.

    PubMed

    Zhang, Hong; Clemens, Regina A; Liu, Fengchun; Hu, Yongmei; Baba, Yoshihiro; Theodore, Pierre; Kurosaki, Tomohiro; Lowell, Clifford A

    2014-04-03

    The stromal-interacting molecule 1 (STIM1) is a potent sensor of intracellular calcium, which in turn regulates entry of external calcium through plasma membrane channels to affect immune cell activation. Although the contribution of STIM1 to calcium signaling in lymphocytes has been well studied, the role of this protein in neutrophil-mediated inflammation and host defense is unknown. We report that STIM1-deficient murine neutrophils show loss of store-operated calcium entry (SOCE) in response to both soluble ligands that activate G-proteins as well as Fcγ-receptor or integrin ligation that activates tyrosine kinase signaling. This results in modest defects in phagocytosis and degranulation responses but a profound block in superoxide production by the phagocyte oxidase. We trace the primary intracellular target of calcium to be protein kinase C isoforms α and β (PKCα and PKCβ), which in turn phosphorylate subunits of the oxidase leading to superoxide production. In vivo the loss of SOCE in stim1(-/-) chimeric mice results in marked susceptibility to bacterial infections but also protection from tissue injury in hepatic ischemia/reperfusion injury. These results demonstrate the critical role of STIM1-mediated SOCE and define major protein targets of calcium signaling in neutrophil activation during inflammatory disease.

  16. Cytokinin oxidase from Phaseolus vulgaris callus tissues. Enhanced in vitro activity of the enzyme in the presence of copper-imidazole complexes

    SciTech Connect

    Chatfield, J.M.; Armstrong, D.J.

    1987-07-01

    The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N/sup 6/-(..delta../sup 2/-isopentenyl)-adenine-2,8-/sup 3/H (i/sup 6/ Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N/sup 6/-side chain of i/sup 6/ Ade.

  17. Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues.

    PubMed

    Fitzpatrick, Paul F; Chadegani, Fatemeh; Zhang, Shengnan; Dougherty, Vi

    2017-02-14

    The flavoenzyme l-6-hydroxynicotine oxidase is a member of the monoamine oxidase family that catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during microbial catabolism of nicotine. While the enzyme has long been understood to catalyze oxidation of the carbon-carbon bond, it has recently been shown to catalyze oxidation of a carbon-nitrogen bond [Fitzpatrick, P. F., et al. (2016) Biochemistry 55, 697-703]. The effects of pH and mutagenesis of active site residues have now been utilized to study the mechanism and roles of active site residues. Asn166 and Tyr311 bind the substrate, while Lys287 forms a water-mediated hydrogen bond with flavin N5. The N166A and Y311F mutations result in ∼30- and ∼4-fold decreases in kcat/Km and kred for (S)-6-hydroxynicotine, respectively, with larger effects on the kcat/Km value for (S)-6-hydroxynornicotine. The K287M mutation results in ∼10-fold decreases in these parameters and a 6000-fold decrease in the kcat/Km value for oxygen. The shapes of the pH profiles are not altered by the N166A and Y311F mutations. There is no solvent isotope effect on the kcat/Km value for amines. The results are consistent with a model in which both the charged and neutral forms of the amine can bind, with the former rapidly losing a proton to a hydrogen bond network of water and amino acids in the active site prior to the transfer of hydride to the flavin.

  18. 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

  19. Evidence that d-cysteine protects mice from gastric damage via hydrogen sulfide produced by d-amino acid oxidase.

    PubMed

    Souza, Luan Kelves M; Araújo, Thiago S L; Sousa, Nayara A; Sousa, Francisca Beatriz M; Nogueira, Kerolayne M; Nicolau, Lucas A D; Medeiros, Jand Venes R

    2017-04-01

    Hydrogen sulfide (H2S) is a signaling molecule in the gastrointestinal tract. H2S production can derive from d-cysteine via various pathways, thus pointing to a new therapeutic approach: delivery of H2S to specific tissues. This study was designed to evaluate the concentration and effects of H2S (generated by d-amino acid oxidase [DAO] from d-cysteine) in the gastric mucosa and the protective effects against ethanol-induced lesions in mice. Mice were treated with l-cysteine or d-cysteine (100 mg/kg per os). Other groups received oral l-propargylglycine (cystathionine γ-lyase inhibitor, 100 mg/kg) or indole-2-carboxylate (DAO inhibitor), and 30 min later, received d- or l-cysteine. After 30 min, 50% ethanol (2.5 mL/kg, per os) was administered. After 1 h, the mice were euthanized and their stomachs excised and analyzed. Pretreatment with either l-cysteine or d-cysteine significantly reduced ethanol-induced lesions. Pretreatment of d-cysteine- or l-cysteine-treated groups with indole-2-carboxylate reversed the gastroprotective effects of d-cysteine but not l-cysteine. Histological analysis revealed that pretreatment with d-cysteine decreased hemorrhagic damage, edema, and the loss of the epithelium, whereas the administration of indole-2-carboxylate reversed these effects. d-Cysteine also reduced malondialdehyde levels but maintained the levels of reduced glutathione. Furthermore, pretreatment with d-cysteine increased the synthesis of H2S. Thus, an H2S-generating pathway (involving d-cysteine and DAO) is present in the gastric mucosa and protects this tissue from ethanol-induced damage by decreasing direct oxidative damage.

  20. Pear ACO genes encoding putative 1-aminocyclopropane-1-carboxylate oxidase homologs are functionally expressed during fruit ripening and involved in response to salicylic acid.

    PubMed

    Shi, Hai-Yan; Zhang, Yu-Xing

    2012-10-01

    1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final reaction of the ethylene biosynthetic pathway, converting ACC into ethylene. Past studies have shown a possible link between ACC oxidase and salicylic acid during fruit ripening in pear, but the relationship has received no more than modest study at the gene expression level. In this study, two cDNA clones encoding putative ACC oxidase, PpACO1 and PpACO2, were isolated from a cDNA library constructed by our own laboratory and produced using mRNA from mesocarp of pear (Pyrus pyrifolia Nakai. cv.Whangkeumbae). One cDNA clone, designated PpACO1 (GenBank accession No. JN807390), comprised an open reading frame of 945 bp encoding a protein of 314 amino acids. The other cDNA, designated PpACO2 (GenBank accession No. JN807392), encodes a protein with 322 amino acids that shares high similarity with the known plant ACOs. Using PCR amplification techniques, two genomic clones corresponding to PpACO1 and PpACO2 were isolated and shown to contain independently three introns with typical GT/AG boundaries defining the splice junctions. The PpACO1 gene product shared 99 % identity with an ACC oxidase from pear (Pyrus × bretschneideri Rehd.cv.Yali), and phylogenetic analyses clearly placed the gene product in the ACC oxidase cluster of the pear 2-oxoglutarate-dependent dioxygenase superfamily tree. Quantitative RT-PCR analysis indicated that the two PpACO genes are differentially expressed in pear tissues. PpACO1 and PpACO2 were predominantly expressed in fruit. The transcripts of PpACO1 were accumulated at relatively low levels in early fruit, but strongly high levels in fruit ripening and senescence stages, while the transcripts of PpACO2 were accumulated at higher levels in early fruit and much lower levels with further fruit cell development than the transcripts of PpACO1. In addition, PpACO1 gene was down-regulated in fruit by salicylic acid (SA). Nevertheless, PpACO2 gene was dramatically up-regulated in

  1. Influence of altered gravity on the cytochemical localization of cytochrome oxidase activity in central and peripheral gravisensory systems in developing cichlid fish

    NASA Astrophysics Data System (ADS)

    Paulus, U.; Nindl, G.; Körtje, K. H.; Slenzka, K.; Neubert, J.; Rahmann, H.

    Cichlid fish larvae were reared from hatching to active free swimming under different gravity conditions: natural environment, increased acceleration in a centrifuge, simulated weightlessness in a clinostat and near weightlessness during space flight. Cytochrome oxidase activity was analyzed semiquantitatively on the ultrastructural level as a marker of regional neuronal activity in a primary, vestibular brainstem nucleus and in gravity receptive epithelia in the inner ear. Our results show, that gravity seems to be positively correlated with cytochrome oxidase activity in the magnocellular nucleus of developing fish brain. In the inner ear the energy metabolism is decreased under microgravity concerning utricle but not saccule. Hypergravity has not effect on cytochrome oxidase activity in sensory inner ear epithelia.

  2. 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.

  3. 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.

  4. 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

  5. A Penicillium expansum glucose oxidase-encoding gene, GOX2, is essential for gluconic acid production and acidification during colonization of deciduous fruit.

    PubMed

    Barad, Shiri; Horowitz, Sigal Brown; Moscovitz, Oren; Lichter, Amnon; Sherman, Amir; Prusky, Dov

    2012-06-01

    Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest maceration of fruit through secretion of total, d-gluconic acid (GLA). Two P. expansum glucose oxidase (GOX)-encoding genes, GOX1 and GOX2, were analyzed. GOX activity and GLA accumulation were strongly related to GOX2 expression, which increased with pH to a maximum at pH 7.0, whereas GOX1 was expressed at pH 4.0, where no GOX activity or extracellular GLA were detected. This differential expression was also observed at the leading edge of the decaying tissue, where GOX2 expression was dominant. The roles of the GOX genes in pathogenicity were further studied through i) development of P. expansum goxRNAi mutants exhibiting differential downregulation of GOX2, ii) heterologous expression of the P. expansum GOX2 gene in the nondeciduous fruit-pathogen P. chrysogenum, and iii) modulation of GLA production by FeSO(4) chelation. Interestingly, in P. expansum, pH and GLA production elicited opposite effects on germination and biomass accumulation: 26% of spores germinated at pH 7.0 when GOX activity and GLA were highest whereas, in P. chrysogenum at the same pH, when GLA did not accumulate, 72% of spores germinated. Moreover, heterologous expression of P. expansum GOX2 in P. chrysogenum resulted in enhanced GLA production and reduced germination, suggesting negative regulation of spore germination and GLA production. These results demonstrate that pH modulation, mediated by GLA accumulation, is an important factor in generating the initial signal or signals for fungal development leading to host-tissue colonization by P. expansum.

  6. Glucocorticoid and androgen activation of monoamine oxidase A is regulated differently by R1 and Sp1.

    PubMed

    Ou, Xiao-Ming; Chen, Kevin; Shih, Jean C

    2006-07-28

    Monoamine oxidase (MAO) A is a key enzyme for the degradation of neurotransmitters serotonin, norepinephrine, and dopamine. There are three consensus glucocorticoid/androgen response elements and four Sp1-binding sites in the human monoamine oxidase A 2-kb promoter. A novel transcription factor R1 (RAM2/CDCA7L) interacts with Sp1-binding sites and represses MAO A gene expression. Luciferase assays show that glucocorticoid (dexamethasone) and androgen (R1881) increase MAO A promoter and catalytic activities in human neuroblastoma and glioblastoma cells. Gel-shift analysis demonstrates that glucocorticoid/androgen receptors interact directly with the third glucocorticoid/androgen response element. Glucocorticoid/androgen receptors also interact with Sp1-binding sites indirectly via transcription factor Sp1. In addition, dexamethasone induces R1 translocation from the cytosol to the nucleus in a time-dependent manner in both the neuroblastoma and wild-type UW228 cell lines but not in R1 knock-down UW228 cells. In summary, this study shows that glucocorticoid enhances monoamine oxidase A gene expression by 1) regulation of R1 translocation; 2) direct interaction of the glucocorticoid receptor with the third glucocorticoid/androgen response element; and 3) indirect interaction of glucocorticoid receptor with the Sp1 or R1 transcription factor on Sp1-binding sites of the MAO A promoter. Androgen also up-regulates MAO A gene expression by direct interaction of androgen receptor with the third glucocorticoid/androgen response element. Androgen receptor indirectly interacts with the Sp1, but not R1 transcription factor, on Sp1-binding sites. This study provides new insights on the differential regulation of MAO A by glucocorticoid and androgen.

  7. Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.

    PubMed

    Bremer, Daniel; Leben, Ruth; Mothes, Ronja; Radbruch, Helena; Niesner, Raluca

    2017-04-03

    Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc.

  8. Induction of mixed function oxidase activity in man by rifapentine (MDL 473), a long-acting rifamycin derivative.

    PubMed Central

    Vital Durand, D; Hampden, C; Boobis, A R; Park, B K; Davies, D S

    1986-01-01

    The effects of rifapentine (MDL 473) administration on hepatic mixed function oxidase activity in man have been investigated in six healthy volunteers. Administration of rifapentine (600 mg 48 h-1) for 10 days resulted in a significant reduction in antipyrine half-life (from 13.2 +/- 1.0 h to 7.7 +/- 0.4 h) and a corresponding increase in its total body clearance (from 41.8 +/- 5.5 ml min-1 to 67.4 +/- 5.6 ml min-1). Twelve days after stopping rifapentine administration, these values had largely returned to base-line. 24-Hour excretion of 6 beta-hydroxycortisol was significantly increased, by approximately three-fold, following administration of rifapentine for 10 days. Again, 12 days after stopping drug administration, 6 beta-hydroxycortisol excretion had returned to pretreatment values. Clearance of antipyrine to its three oxidative metabolites was increased by rifapentine administration, although the increase for 3-hydroxymethylantipyrine was not significant. The greatest increase (+140%) was observed for norantipyrine. Twelve days after the last dose of rifapentine, all values had returned to control levels. It is concluded that, like rifampicin, rifapentine is a potent inducer of mixed function oxidase activity in man and that the possibility of clinically significant drug interactions should be anticipated in the therapeutic use of this compound. PMID:3947503

  9. Down-regulation of acyl-CoA oxidase gene expression and increased NF-kappaB activity in etomoxir-induced cardiac hypertrophy.

    PubMed

    Cabrero, Agatha; Merlos, Manuel; Laguna, Juan C; Carrera, Manuel Vázquez

    2003-02-01

    Activation of nuclear factor-kappaB (NF-kappaB) is required for hypertrophic growth of cardiomyocytes. Etomoxir is an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I) that activates peroxisome proliferator-activated receptor alpha (PPARalpha) and induces cardiac hypertrophy through an unknown mechanism. We studied the mRNA expression of genes involved in fatty acid oxidation in the heart of mice treated for 1 or 10 days with etomoxir (100 mg/kg/day). Etomoxir administration for 1 day significantly increased (4.4-fold induction) the mRNA expression of acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in peroxisomal beta-oxidation. In contrast, etomoxir treatment for 10 days dramatically decreased ACO mRNA levels by 96%. The reduction in ACO expression in the hearts of 10-day etomoxir-treated mice was accompanied by an increase in the mRNA expression of the antioxidant enzyme glutathione peroxidase and the cardiac marker of oxidative stress bax. Moreover, the activity of the redox-regulated transcription factor NF-kappaB was increased in heart after 10 days of etomoxir treatment. Overall, the findings here presented show that etomoxir treatment may induce cardiac hypertrophy via increased cellular oxidative stress and NF-kappaB activation.

  10. Evidence for association and epistasis at the DAOA/G30 and D-amino acid oxidase loci in an Irish schizophrenia sample.

    PubMed

    Corvin, A; McGhee, K A; Murphy, K; Donohoe, G; Nangle, J M; Schwaiger, S; Kenny, N; Clarke, S; Meagher, D; Quinn, J; Scully, P; Baldwin, P; Browne, D; Walsh, C; Waddington, J L; Morris, D W; Gill, M

    2007-10-05

    The D-amino acid oxidase (DAO) signaling pathway has been implicated in schizophrenia pathogenesis. This may be mediated through modulation of NMDA function by DAO, which is in turn activated by DAO activator (DAOA, formerly G72). Chumakov et al. (2002); PNAS 99: 13675-13680, identifying the novel schizophrenia susceptibility gene DAOA/G30 and a number of independent studies have since reported evidence of association between the DAOA and DAO genes and schizophrenia. However, at least two studies have failed to replicate the epistatic interaction between these loci described in the original report and there have been differences in the associated alleles/haplotypes reported at each locus. In this study, we performed association and epistasis analyses of the DAOA/G30 and DAO loci in a sample of 373 cases with DSM-IV schizophrenia/schizoaffective disorder and 812 controls from the Republic of Ireland. Corrected for the number of tests performed, we found evidence for association between markers at both genes and schizophrenia: DAOA/G30 (P = 0.005, OR = 1.34 (1.09, 1.65)) and DAO (P = 0.003, OR = 1.43 (1.12, 1.84). The data suggest that evidence for association at DAO (marker rs2111902) is more consistent than previously realized, particularly in Caucasian schizophrenia populations. We identified evidence for epistatic interaction between the associated SNPs at DAOA and DAO genes in contributing to schizophrenia risk (OR = 9.3 (1.4, 60.5). Based on these data, more systematic investigation of genes involved in DAO signaling is required.

  11. Heredity mode of genetic polymorphism in aldehyde oxidase activity in Donryu strain rats.

    PubMed

    Adachi, M; Itoh, K; Abe, H; Tanaka, Y

    2008-01-01

    Donryu strain rats show genetic polymorphisms in the aldehyde oxidase gene, resulting in the phenotypic expression of ultrarapid metabolizers with homozygous nucleotide sequences (337G, 2604C), extensive metabolizers with heterozygous nucleotide sequences (377G/A, 2604C/T), and poor metabolizers with homozygous nucleotide sequences (377A, 2604T). In the mating experiments the ratio of the number of ultrarapid metabolizers, extensive metabolizers, and poor metabolizers rats in the F1 generation from the heterozygous F0 extensive metabolizers male and female rats was roughly 0.6 : 1.5 : 1, and the ratio converged to approximately 1 : 2 : 1 in the F2 generation from the heterozygous F1 extensive metabolizers male and female rats. On the contrary, all the F2 generation from homozygous F1 ultrarapid metabolizers male and female rats or from homozygous F1 poor metabolizers male and female rats had the ultrarapid metabolizers or the poor metabolizers genotypes and phenotypes. The genotypes completely agreed with the phenotypes in all individuals of F0, F1, and F2 generations. The results indicate that the genetic polymorphism of aldehyde oxidase in Donryu strain rats obeys Mendelian heredity. The reason for a low ratio of the ultrarapid metabolizers rats in the commercially available Donryu strain rats - not more than several per cent - compared with the ratio expected from the Mendelian rule is unknown.

  12. Role of the Rho GTPase Rac in the activation of the phagocyte NADPH oxidase

    PubMed Central

    Pick, Edgar

    2014-01-01

    The superoxide-generating NADPH oxidase of phagocytes consists of the membrane-associated cytochrome b558 (a heterodimer of Nox2 and p22phox) and 4 cytosolic components: p47phox, p67phox, p40phox, and the small GTPase, Rac, in complex with RhoGDI. Superoxide is produced by the NADPH-driven reduction of molecular oxygen, via a redox gradient located in Nox2. Electron flow in Nox2 is initiated by interaction with cytosolic components, which translocate to the membrane, p67phox playing the central role. The participation of Rac is expressed in the following sequence: (1) Translocation of the RacGDP-RhoGDI complex to the membrane; (2) Dissociation of RacGDP from RhoGDI; (3) GDP to GTP exchange on Rac, mediated by a guanine nucleotide exchange factor; (4) Binding of RacGTP to p67phox; (5) Induction of a conformational change in p67phox, promoting interaction with Nox2. The particular involvement of Rac in NADPH oxidase assembly serves as a paradigm for signaling by Rho GTPases, in general. PMID:24598074

  13. 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.

  14. 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

  15. Study of genotoxic, antigenotoxic and antioxidant activities of the digallic acid isolated from Pistacia lentiscus fruits.

    PubMed

    Bhouri, Wissem; Derbel, Safa; Skandrani, Ines; Boubaker, Jihed; Bouhlel, Ines; Sghaier, Mohamed B; Kilani, Soumaya; Mariotte, Anne M; Dijoux-Franca, Marie G; Ghedira, Kamel; Chekir-Ghedira, Leila

    2010-03-01

    The digallic acid obtained from the fruit Pistacia lentiscus exhibits an inhibitory activity against nitrofurantoine and B[a]P induced genotoxicity when tested by the SOS chromotest bacterial assay system in the presence of Escherichia coli PQ37 strain. The antioxidant activity of the tested compound was determined by its ability to scavenge the free radical ABTS(+), to inhibit the xanthine oxidase, involved in the generation of free radicals, and to inhibit the lipid peroxidation induced by H(2)O(2) in the K562 cell line. Our results revealed that digallic acid shows an important free radical scavenging activity towards the ABTS(+) radical (99%) and protection against lipid peroxidation (68%).

  16. Antiprotozoal activity of betulinic acid derivatives.

    PubMed

    Domínguez-Carmona, D B; Escalante-Erosa, F; García-Sosa, K; Ruiz-Pinell, G; Gutierrez-Yapu, D; Chan-Bacab, M J; Giménez-Turba, A; Peña-Rodríguez, L M

    2010-04-01

    Betulinic acid (1), isolated from the crude extract of the leaves of Pentalinon andrieuxii (Apocynaceae), together with betulinic acid acetate (2), betulonic acid (3), betulinic acid methyl ester (4), and betulin (5) were evaluated for their antiprotozoal activity. The results showed that modifying the C-3 position increases leishmanicidal activity while modification of the C-3 and C-28 positions decreases trypanocidal activity.

  17. Quantitative study of the encapsulation of glucose oxidase into multilamellar vesicles and its effect on enzyme activity

    NASA Astrophysics Data System (ADS)

    Olea, David; Faure, Chrystel

    2003-09-01

    The encapsulation of glucose oxidase (GOx) into onion-type multilamellar vesicles is studied and compared to that of GOx into liposomes. The enzyme was shown not to be affected by encapsulation as evidenced by the complete recovery of its activity after being freed. An ˜15% increase of GOx activity was conferred by confinement in onions in the 30-50 °C temperature range. Entrapment of GOx in onions was proved to be effective since a maximum of 10% leak was measured after 45 days of encapsulation. The encapsulation yield, which reaches 80%, and the number of encapsulated enzyme molecules per onion (1000 GOx molecules) were found to be much higher than for liposomes. The effect of onion composition on the encapsulation yield was determined and predicted by a thermodynamic model applied to the lipids-GOx-phosphate buffer system.

  18. Identification of an active site cysteine residue in Escherichia coli pyruvate oxidase.

    PubMed

    Koland, J G; Gennis, R B

    1982-06-10

    The cysteine-directed reagent N-ethylmaleimide rapidly and completely inactivates pyruvate oxidase. This inactivation is correlated with the reaction of one cysteine residue per enzyme monomer. In the presence of the cofactor, thiamin pyrophosphate, the enzyme is not inhibited by N-ethylmaleimide. Furthermore, the N-ethylmaleimide-inactivated enzyme exhibits a very low affinity for the cofactor as determined by a fluorescence quenching technique. The presence of a reactive cysteine residue at the thiamin pyrophosphate binding site is therefore indicated. Although N-ethylmaleimide completely inactivates the enzyme, a second sulfhydryl reagent methylmethanethiosulfonate is only partially inhibitory. It is shown that methylmethanethiosulfonate and N-ethylmaleimide react with the same cysteine residue. Thus, the N-ethylmaleimide-sensitive residue is probably not directly involved in catalysis.

  19. Study of the effects of salicylic acid on soybean mitochondrial lipids and respiratory properties using the alternative oxidase as a stress-reporter protein.

    PubMed

    Matos, Ana Rita; Mendes, Ana Teresa; Scotti-Campos, Paula; Arrabaça, João Daniel

    2009-12-01

    Biotic and abiotic stresses can lead to modifications in the lipid composition of cell membranes. Although mitochondria appear to be implicated in stress responses, little is known about the membrane lipid changes that occur in these organelles in plants. Besides cytochrome c oxidase, plant mitochondria have an alternative oxidase (AOX) that accepts electrons directly from ubiquinol, dissipating energy as heat. AOX upregulation occurs under a variety of stresses and its induction by salicylic acid (SA) has been observed in different plant species. AOX was also suggested to be used as a functional marker for cell reprogramming under stress. In the present study, we have used etiolated soybean (Glycine max (L.) Merr. cv Cresir) seedlings to study the effects of SA treatment on the lipid composition and the respiratory properties of hypocotyl mitochondria. AOX expression was studied in detail, as a reporter protein, to evaluate whether modifications in mitochondrial energy metabolism were occurring. In mitochondria extracted from SA-treated seedlings, AOX capacity and protein contents increased. Both AOX1 and AOX2b transcripts accumulated in response to SA, but with different kinetics. A reduction in external NADH oxidation capacity was observed, whereas succinate respiration remained unchanged. The phospholipid composition of mitochondria remained similar in control and SA-treated plants, but a reduction in the relative amount of linolenic acid was observed in phosphatidylcholine, phosphatidylethanolamine and cardiolipin. The possible causes of the fatty acid modifications observed, and the implications for mitochondrial metabolism are discussed.

  20. 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.

  1. Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis).

    PubMed

    Zak, Megan A; Regish, Amy M; McCormick, Stephen D; Manzon, Richard G

    2017-02-14

    Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19°C) or below (8°C) the thermal optimum (13°C) and exposure to exogenous thyroid hormone (60µg T4/g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation.

  2. Tumor necrosis factor receptor-associated protein 1 improves hypoxia-impaired energy production in cardiomyocytes through increasing activity of cytochrome c oxidase subunit II.

    PubMed

    Xiang, Fei; Ma, Si-Yuan; Zhang, Dong-Xia; Zhang, Qiong; Huang, Yue-Sheng

    2016-10-01

    Tumor necrosis factor receptor-associated protein 1 protects cardiomyocytes against hypoxia, but the underlying mechanisms are not completely understood. In the present study, we used gain- and loss-of-function approaches to explore the effects of tumor necrosis factor receptor-associated protein 1 and cytochrome c oxidase subunit II on energy production in hypoxic cardiomyocytes. Hypoxia repressed ATP production in cultured cardiomyocytes, whereas overexpression of tumor necrosis factor receptor-associated protein 1 significantly improved ATP production. Conversely, knockdown of tumor necrosis factor receptor-associated protein 1 facilitated the hypoxia-induced decrease in ATP synthesis. Further investigation revealed that tumor necrosis factor receptor-associated protein 1 induced the expression and activity of cytochrome c oxidase subunit II, a component of cytochrome c oxidase that is important in mitochondrial respiratory chain function. Moreover, lentiviral-mediated overexpression of cytochrome c oxidase subunit II antagonized the decrease in ATP synthesis caused by knockdown of tumor necrosis factor receptor-associated protein 1, whereas knockdown of cytochrome c oxidase subunit II attenuated the increase in ATP synthesis caused by overexpression of tumor necrosis factor receptor-associated protein 1. In addition, inhibition of cytochrome c oxidase subunit II by a specific inhibitor sodium azide suppressed the ATP sy nthesis induced by overexpressed tumor necrosis factor receptor-associated protein 1. Hence, tumor necrosis factor receptor-associated protein 1 protects cardiomyocytes from hypoxia at least partly via potentiation of energy generation, and cytochrome c oxidase subunit II is one of the downstream effectors that mediates the tumor necrosis factor receptor-associated protein 1-mediated energy generation program.

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

    PubMed

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

    2017-03-01

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

  4. Polyunsaturated fatty acid inhibition of fatty acid synthase transcription is independent of PPAR activation.

    PubMed

    Clarke, S D; Turini, M; Jump, D B; Abraham, S; Reedy, M

    1998-01-01

    Polyunsaturated fatty acids (PUFA) of the (n-6) and (n-3) families inhibit the rate of gene transcription for a number of hepatic lipogenic and glycolytic genes, e.g., fatty acid synthase (FAS). In contrast, saturated and monounsaturated fatty acids have no inhibitory capability. The suppression of gene transcription resulting from the addition of PUFA to a high carbohydrate diet: occurs quickly (< 3 h) after its addition to a high glucose diet; can be recreated with hepatocytes cultured in a serum-free medium containing insulin and glucocorticoids; can be demonstrated in diabetic rats fed fructose; and is independent of glucagon. While the nature of the intracellular PUFA inhibitor is unclear, it appears that delta-6 desaturation is a required step in the process. Recently, the fatty acid activated nuclear factor, peroxisome-proliferator activated receptor (PPAR) was suggested to be the PUFA-response factor. However, the potent PPAR activators ETYA and Wy-14643 did not suppress hepatic expression of FAS, but did induce the PPAR-responsive gene, acyl-CoA oxidase (AOX). Similarly, treating rat hepatocytes with 20:4 (n-6) suppressed FAS expression but had no effect on AOX. Thus, it appears that the PUFA regulation of gene transcription involves a PUFA-response factor that is independent from PPAR.

  5. Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice.

    PubMed

    Liu, Susan B; Ikenaga, Naoki; Peng, Zhen-Wei; Sverdlov, Deanna Y; Greenstein, Andrew; Smith, Victoria; Schuppan, Detlef; Popov, Yury

    2016-04-01

    Collagen stabilization through irreversible cross-linking is thought to promote hepatic fibrosis progression and limit its reversibility. However, the mechanism of this process remains poorly defined. We studied the functional contribution of lysyl oxidase (LOX) to collagen stabilization and hepatic fibrosis progression/reversalin vivousing chronic administration of irreversible LOX inhibitor β-aminopropionitrile (BAPN, or vehicle as control) in C57Bl/6J mice with carbon tetrachloride (CCl4)-induced fibrosis. Fibrotic matrix stability was directly assessed using a stepwise collagen extraction assay and fibrotic septae morphometry. Liver cells and fibrosis were studied by histologic, biochemical methods and quantitative real-time reverse-transcription PCR. During fibrosis progression, BAPN administration suppressed accumulation of cross-linked collagens, and fibrotic septae showed widening and collagen fibrils splitting, reminiscent of remodeling signs observed during fibrosis reversal. LOX inhibition attenuated hepatic stellate cell activation markers and promoted F4/80-positive scar-associated macrophage infiltration without an increase in liver injury. In reversal experiments, BAPN-treated fibrotic mice demonstrated accelerated fibrosis reversal after CCl4withdrawal. Our findings demonstrate for the first time that LOX contributes significantly to collagen stabilization in liver fibrosis, promotes fibrogenic activation of attenuated hepatic stellate cells, and limits fibrosis reversal. Our data support the concept of pharmacologic targeting of LOX pathway to inhibit liver fibrosis and promote its resolution.-Liu, S. B., Ikenaga, N., Peng, Z.-W., Sverdlov, D. Y., Greenstein, A., Smith, V., Schuppan, D., Popov, Y. Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice.

  6. The Use of Cytochrome C Oxidase Enzyme Activity and Immunohistochemistry in Defining Mitochondrial Injury in Kidney Disease.

    PubMed

    Zsengellér, Zsuzsanna K; Rosen, Seymour

    2016-09-01

    The renal biopsy is a dynamic way of looking at renal disease, and tubular elements are an important part of this analysis. The mitochondria in 20 renal biopsies were examined by immunohistochemical (electron transport chain enzyme: cytochrome C oxidase IV [COX IV]) and enzyme histochemical methods (COX), both by light and electron microscopy. The distal convoluted tubules and thick ascending limbs showed the greatest intensity in the COX immunostains and enzyme activity in controls. The degree of mitochondrial COX protein and enzyme activity diminished as the tubules became atrophic. With proximal hypertrophic changes, there was great variation in both COX activity and protein expression. In contrast, in three cases of systemic lupus erythematosus, biopsied for high-grade proteinuria, the activity was consistently upregulated, whereas protein expression remained normal. These unexpected findings of heterogeneous upregulation in hypertrophy and the dyssynchrony of protein expression and activity may indicate mitochondrial dysregulation. Functional electron microscopy showed COX activity delineated by the intense mitochondrial staining in normal or hypertrophic proximal tubules. With atrophic changes, residual small mitochondria with diminished activity could be seen. With mitochondrial size abnormalities (enlargement and irregularity, adefovir toxicity), activity persisted. In the renal biopsy, mitochondrial analysis is feasible utilizing immunohistochemical and enzyme histochemical techniques.

  7. The Structures of the C185S and C185A Mutants of Sulfite Oxidase Reveal Rearrangement of the Active Site

    SciTech Connect

    Qiu, James A.; Wilson, Heather L.; Pushie, M. Jake; Kisker, Caroline; George, Graham N.; Rajagopalan, K.V.

    2010-11-03

    Sulfite oxidase (SO) catalyzes the physiologically critical conversion of sulfite to sulfate. Enzymatic activity is dependent on the presence of the metal molybdenum complexed with a pyranopterin-dithiolene cofactor termed molybdopterin. Comparison of the amino acid sequences of SOs from a variety of sources has identified a single conserved Cys residue essential for catalytic activity. The crystal structure of chicken liver sulfite oxidase indicated that this residue, Cys185 in chicken SO, coordinates the Mo atom in the active site. To improve our understanding of the role of this residue in the catalytic mechanism of sulfite oxidase, serine and alanine variants at position 185 of recombinant chicken SO were generated. Spectroscopic and kinetic studies indicate that neither variant is capable of sulfite oxidation. The crystal structure of the C185S variant was determined to 1.9 {angstrom} resolution and to 2.4 {angstrom} resolution in the presence of sulfite, and the C185A variant to 2.8 {angstrom} resolution. The structures of the C185S and C185A variants revealed that neither the Ser or Ala side chains appeared to closely interact with the Mo atom and that a third oxo group replaced the usual cysteine sulfur ligand at the Mo center, confirming earlier extended X-ray absorption fine structure spectroscopy (EXAFS) work on the human C207S mutant. An unexpected result was that in the C185S variant, in the absence of sulfite, the active site residue Tyr322 became disordered as did the loop region flanking it. In the C185S variant crystallized in the presence of sulfite, the Tyr322 residue relocalized to the active site. The C185A variant structure also indicated the presence of a third oxygen ligand; however, Tyr322 remained in the active site. EXAFS studies of the Mo coordination environment indicate the Mo atom is in the oxidized Mo{sup VI} state in both the C185S and C185A variants of chicken SO and show the expected trioxodithiolene active site. Density

  8. Azachalcones: a new class of potent polyphenol oxidase inhibitors.

    PubMed

    Radhakrishnan, Sini Karanayil; Shimmon, Ronald Gibrial; Conn, Costa; Baker, Anthony T

    2015-04-15

    A library of potent inhibitors of polyphenol oxidase and their structure activity relationships are described. Azachalcone derivatives were synthesized and tested for their tyrosinase inhibitory activity. Their inhibitory activities on mushroom tyrosinase using l-DOPA as a substrate were investigated. Two compounds that are the reduction congeners of the pyridinyl azachalcones strongly inhibited the enzyme activity and were more potent than the positive control kojic acid.

  9. 5,5-Dithiobis(2-nitrobenzoic acid) pyrene derivative-carbon nanotube electrodes for NADH electrooxidation and oriented immobilization of multicopper oxidases for the development of glucose/O2 biofuel cells.

    PubMed

    Giroud, Fabien; Sawada, Koichi; Taya, Masahito; Cosnier, Serge

    2017-01-15

    We report the functionalization of multi-walled carbon nanotubes (MWCNTs) electrodes by a bifunctional nitroaromatic molecule accomplished via π-π interactions of a pyrene derivative. DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) has the particularity to possess both electroactivable nitro groups and negatively charged carboxylic groups. The integration of the DTNB-modified MWCNTs was evaluated for different bioelectrocatalytic systems. The immobilized DTNB-based electrodes showed electrocatalytic activity toward the oxidation of the reduced form of nicotinamide adenine dinucleotide (NADH) with low overpotential of -0.09V vs Ag/AgCl at neutral pH. Glucose dehydrogenase was successfully immobilized at the surface of DTNB-based electrodes and, in the presence of NAD(+), the resulting bioelectrode achieved efficient glucose oxidation with high current densities of 2.03mAcm(-2). On the other hand, the aromatic structure and the negatively charged nature of the DTNB provoked orientation of both laccase and bilirubin oxidase onto the electrode, which enhanced their ability to undergo a direct electron transfer for oxygen reduction. Due to the proper orientation, low overpotentials were obtained (ca. 0.6V vs Ag/AgCl) and high electrocatalytic currents of about 3.5mAcm(-)(2) were recorded at neutral pH in O2 saturated conditions for bilirubin oxidase electrodes. The combination of these bioanodes and bilirubin oxidase biocathodes provided glucose/O2 enzymatic biofuel cells (EBFC) exhibiting an open-circuit potential of 0.640V, with an associated maximum current density of 2.10mAcm(-)(2). Moreover, the fuel cell delivered a maximum power density of 0.50mWcm(-)(2) at 0.36 V.

  10. Effect of Alkaloids Isolated from Phyllodium pulchellum on Monoamine Levels and Monoamine Oxidase Activity in Rat Brain

    PubMed Central

    Cai, Lu; Wang, Chao; Dong, Pei-pei; Zhang, Bao-jing; Zhang, Hou-Li; Huang, Shan-shan; Zhang, Bo; Yu, Sheng-ming; Zhong, Ming; Ma, Xiao-Chi

    2016-01-01

    Phyllodium pulchellum (P. pulchellum) is a folk medicine with a significant number of bioactivities. The aim of this study was to investigate the effects displayed by alkaloids fractions, isolated from the roots of P. pulchellum, on neurotransmitters monoamine levels and on monoamine oxidase (MAO) activity. Six alkaloids, which had indolealkylamine or β-carboline skeleton, were obtained by chromatographic technologies and identified by spectroscopic methods such as NMR and MS. After treatment with alkaloids of P. pulchellum, the reduction of DA levels (54.55%) and 5-HT levels (35.01%) in rat brain was observed by HPLC-FLD. The effect of alkaloids on the monoamines metabolism was mainly related to MAO inhibition, characterized by IC50 values of 37.35 ± 6.41 and 126.53 ± 5.39 μg/mL for MAO-A and MAO-B, respectively. The acute toxicity indicated that P. pulchellum extract was nontoxic. PMID:27195015

  11. Changes of alternative oxidase activity, capacity and protein content in leaves of Cucumis sativus wild-type and MSC16 mutant grown under different light intensities.

    PubMed

    Florez-Sarasa, Igor; Ostaszewska, Monika; Galle, Alexander; Flexas, Jaume; Rychter, Anna M; Ribas-Carbo, Miquel

    2009-12-01

    In vitro studies demonstrated that alternative oxidase (AOX) is biochemically regulated by a sulfhydryl-disulfide system, interaction with alpha-ketoacids, ubiquinone pool redox state and protein content among others. However, there is still scarce information about the in vivo regulation of the AOX. Cucumis sativus wild-type (WT) and MSC16 mutant plants were grown under two different light intensities and were used to analyze the relationship between the amount of leaf AOX protein and its in vivo capacity and activity at night and day periods. WT and MSC16 plants presented lower total respiration (V(t)), cytochrome oxidase pathway (COP) activity (v(cyt)) and alternative oxidase pathway (AOP) activity (v(alt)) when grown at low light (LL), although growth light intensity did not change the amount of cytochrome oxidase (COX) nor AOX protein. Changes of v(cyt) related to growing light conditions suggested a substrate availability and energy demand control. On the other hand, the total amount of AOX protein present in the tissue does not play a role in the regulation neither of the capacity nor of the activity of the AOP in vivo. Soluble carbohydrates were directly related to the activity of the AOP. However, although differences in soluble sugar contents mostly regulate the capacity of the AOP at different growth light intensities, additional regulatory mechanisms are necessary to fully explain the observed results.

  12. Mixed-function oxidase enzyme activity and oxidative stress in lake trout (Salvelinus namaycush) exposed to 3,3{prime},4,4{prime}5-pentachlorobiphenyl (PCB-126)

    SciTech Connect

    Palace, V.P.; Klaverkamp, J.F.; Lockhart, W.L. |; Metner, D.A.; Muir, D.C.G.; Brown, S.B.

    1996-06-01

    Juvenile lake trout were intraperitoneally injected with corn oil containing nominal concentrations of 0, 0.6, 6.3, or 25 {micro}g [{sup 14}C]-3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB-126) per gram of body weight. The PCB-126 accumulated in liver in a dose-dependent manner to a sustained concentration by 6 weeks and remained elevated for the 30-week experimental period. Mixed-function oxidase (MFO) enzyme activity was elevated in the two highest dose groups relative to the control group, but not in the low-dose group throughout the 30 weeks. Oxidative stress, measured by the thiobarbituric acid reactive substances test, was correlated with ethoxyresorufin O-deethylase and was elevated in liver of the two highest PCB dose groups but not the low-dose group. The activities of the enzymatic antioxidants superoxide dismutase, catalase, and glutathione peroxidase were unaffected by PCB-126 exposure. The nonenzymatic antioxidants superoxide dismutase, catalase, and glutathione peroxidase were unaffected by PCB-126 exposure. The nonenzymatic antioxidant tocopherol was depleted to approximately 75% of the control concentration in liver of all three PCB-dosed groups. Hepatic ascorbic acid levels were not different in any of the treatment groups. Retinol was depleted by greater than an order of magnitude in liver of the two highest dose groups but not in the los-dose group. This study demonstrates a correlation between hepatic MFO activity and oxidative stress in PCB-exposed lake trout. Tocopherol and retinol may be important mediators of oxidative stress but additional study is required to confirm the antioxidant activity of retinol.

  13. Determination of total creatine kinase activity in blood serum using an amperometric biosensor based on glucose oxidase and hexokinase.

    PubMed

    Kucherenko, I S; Soldatkin, O O; Lagarde, F; Jaffrezic-Renault, N; Dzyadevych, S V; Soldatkin, A P

    2015-11-01

    Creatine kinase (CK: adenosine-5-triphosphate-creatine phosphotransferase) is an important enzyme of muscle cells; the presence of a large amount of the enzyme in blood serum is a biomarker of muscular injuries, such as acute myocardial infarction. This work describes a bi-enzyme (glucose oxidase and hexokinase based) biosensor for rapid and convenient determination of CK activity by measuring the rate of ATP production by this enzyme. Simultaneously the biosensor determines glucose concentration in the sample. Platinum disk electrodes were used as amperometric transducers. Glucose oxidase and hexokinase were co-immobilized via cross-linking with BSA by glutaraldehyde and served as a biorecognition element of the biosensor. The biosensor work at different concentrations of CK substrates (ADP and creatine phosphate) was investigated; optimal concentration of ADP was 1mM, and creatine phosphate - 10 mM. The reproducibility of the biosensor responses to glucose, ATP and CK during a day was tested (relative standard deviation of 15 responses to glucose was 2%, to ATP - 6%, to CK - 7-18% depending on concentration of the CK). Total time of CK analysis was 10 min. The measurements of creatine kinase in blood serum samples were carried out (at 20-fold sample dilution). Twentyfold dilution of serum samples was chosen as optimal for CK determination. The biosensor could distinguish healthy and ill people and evaluate the level of CK increase. Thus, the biosensor can be used as a test-system for CK analysis in blood serum or serve as a component of multibiosensors for determination of important blood substances. Determination of activity of other kinases by the developed biosensor is also possible for research purposes.

  14. The Promotion of Indole-3-acetic Acid Oxidation in Pea Buds by Gibberellic Acid and Treatment 1

    PubMed Central

    Ockerse, Ralph; Waber, Jack

    1970-01-01

    Terminal buds of dark-grown pea (Pisum sativum) seedlings have an indole-3-acetic acid oxidase which does not require Mn2+ and 2,4-dichlorophenol as cofactors. Oxidase activity is at least 50 times higher in buds of tall peas than in dwarf seedlings. Administration of gibberellic acid to dwarf peas stimulates both growth and indoleacetic acid oxidase activity to the same levels as in tall seedlings. By contrast, indoleacetic acid oxidation assayed in the presence of Mn2+ and 2,4-dichlorophenol proceeds at similar rates regardless of gibberellin application. Treatment of tall peas with the growth retardant AMO-1618 reduces growth and oxidase activity. Such treated seedlings are indistinguishably dwarf. The enzyme does not appear to be polyphenol oxidase, nor do the results suggest that reduced activity in dwarf buds is due to higher levels of a dialyzable inhibitor. The peroxidative nature of the oxidase is probable. PMID:5500209

  15. RNA interference of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1 and ACO2) genes expression prolongs the shelf life of Eksotika (Carica papaya L.) papaya fruit.

    PubMed

    Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom; Yeong, Wee Chien; Pillai, Vilasini

    2014-06-19

    The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.

  16. Comparison of CYP106A1 and CYP106A2 from Bacillus megaterium - identification of a novel 11-oxidase activity.

    PubMed

    Kiss, Flora Marta; Schmitz, Daniela; Zapp, Josef; Dier, Tobias K F; Volmer, Dietrich A; Bernhardt, Rita

    2015-10-01

    The CYP106A subfamily hydroxylates steroids, diterpenes, and triterpenes in a regioselective and stereoselective manner, which is a challenging task for synthetic chemistry. The well-studied CYP106A2 enzyme, from the Bacillus megaterium strain ATCC 13368, is a highly promising candidate for the pharmaceutical industry. It shares 63 % amino acid sequence identity with CYP106A1 from B. megaterium DSM319, which was recently characterized. A focused steroid library was screened with both CYP106A1 and CYP106A2. Out of the 23 tested steroids, 19 were successfully converted by both enzymes during in vitro and in vivo reactions. Thirteen new substrates were identified for CYP106A1, while the substrate spectrum of CYP106A2 was extended by seven new members. Finally, six chosen steroids were further studied on a preparative scale employing a recombinant B. megaterium MS941 whole-cell system, yielding sufficient amounts of product for structure characterization by nuclear magnetic resonance. The hydroxylase activity was confirmed at positons 6β, 7β, 9α, and 15β. In addition, the CYP106A subfamily showed unprecedented 11-oxidase activity, converting 11β-hydroxysteroids to their 11-keto derivatives. This novel reaction and the diverse hydroxylation positions on pharmaceutically relevant compounds underline the role of the CYP106A subfamily in drug development and production.

  17. Succinate oxidase in Neurospora.

    PubMed

    West, D J; Woodward, D O

    1973-02-01

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

  18. Effects of environmental enrichment on anxiety responses, spatial memory and cytochrome c oxidase activity in adult rats.

    PubMed

    Sampedro-Piquero, P; Zancada-Menendez, C; Begega, A; Rubio, S; Arias, J L

    2013-09-01

    We have studied the effect of an environmental enrichment (EE) protocol in adult Wistar rats on the activity in the elevated zero-maze (EZM), performance in the radial-arm water maze (RAWM) and we have also examined the changes in the neuronal metabolic activity of several brain regions related to anxiety response and spatial memory through cytochrome c oxidase histochemistry (COx). Our EE protocol had anxiolytic effect in the EZM; the animals spent more time and made more entries into the open quadrants, they had lower latency to enter into the open quadrant and lower levels of defecation. Also, the EE group showed fewer working memory and reference memory errors, as well as lesser distance travelled in the first day of the spatial training. In relation to the neuronal metabolic activity, EE reduced the COx activity in brain regions related to anxiety response, such as the infralimbic cortex, the paraventricular thalamic and hypothalamic nucleus, the basolateral amygdala, and the ventral hippocampus. Interestingly, there were no significant differences between groups in the dorsal hippocampus, more related to spatial cognition. These results suggest a beneficial effect of EE on spatial memory as a result of reducing anxiety levels and the COx activity in brain regions involved in anxiety response. We also found a differential pattern of activation inside the hippocampus, suggesting that the dorsal hippocampus has a preferential involvement in spatial learning and memory, whereas the ventral hippocampus has a role in anxiety response.

  19. Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells*

    PubMed Central

    Herchenhan, Andreas; Uhlenbrock, Franziska; Eliasson, Pernilla; Weis, MaryAnn; Eyre, David; Kadler, Karl E.; Magnusson, S. Peter; Kjaer, Michael

    2015-01-01

    Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation. PMID:25979340

  20. Cardiac Cytochrome c Oxidase Activity and Contents of Submits 1 and 4 are Altered in Offspring by Low Prenatal Intake by Rat Dams

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It has been reported previously that the offspring of rat dams consuming low dietary copper (Cu) during pregnancy and lactation experience a deficiency in cardiac cytochrome c oxidase (CCO) characterized by reduced catalytic activity and mitochondrial- and nuclear-subunit content after postnatal day...

  1. Effects of N-Acetylcysteine on Nicotinamide Dinucleotide Phosphate Oxidase Activation and Antioxidant Status in Heart, Lung, Liver and Kidney in Streptozotocin-Induced Diabetic Rats

    PubMed Central

    Lei, Shaoqing; Liu, Yanan; Liu, Huimin; Yu, Hong; Wang, Hui

    2012-01-01

    Purpose Hyperglycemia increases reactive oxygen species (ROS) and the resulting oxidative stress plays a key role in the pathogenesis of diabetic complications. Nicotinamide dinucleotide phosphate (NADPH) oxidase is one of the major sources of ROS production in diabetes. We, therefore, examined the possibility that NADPH oxidase activation is increased in various tissues, and that the antioxidant N-acetylcysteine (NAC) may have tissue specific effects on NADPH oxidase and tissue antioxidant status in diabetes. Materials and Methods Control (C) and streptozotocin-induced diabetic (D) rats were treated either with NAC (1.5 g/kg/day) orally or placebo for 4 weeks. The plasma, heart, lung, liver, kidney were harvested immediately and stored for biochemical or immunoblot analysis. Results levels of free 15-F2t-isoprostane were increased in plasma, heart, lung, liver and kidney tissues in diabetic rats, accompanied with significantly increased membrane translocation of the NADPH oxidase subunit p67phox in all tissues and increased expression of the membrane-bound subunit p22phox in heart, lung and kidney. The tissue antioxidant activity in lung, liver and kidney was decreased in diabetic rats, while it was increased in heart tissue. NAC reduced the expression of p22phox and p67phox, suppressed p67phox membrane translocation, and reduced free 15-F2t-isoprostane levels in all tissues. NAC increased antioxidant activity in liver and lung, but did not significantly affect antioxidant activity in heart and kidney. Conclusion The current study shows that NAC inhibits NADPH oxidase activation in diabetes and attenuates tissue oxidative damage in all organs, even though its effects on antioxidant activity are tissue specific. PMID:22318816

  2. Effect of exchange of the cysteine molybdenum ligand with selenocysteine on the structure and function of the active site in human sulfite oxidase.

    PubMed

    Reschke, Stefan; Niks, Dimitri; Wilson, Heather; Sigfridsson, Kajsa G V; Haumann, Michael; Rajagopalan, K V; Hille, Russ; Leimkühler, Silke

    2013-11-19

    Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite oxidizing activity (kcat/KM) of SeSOMD4Ser was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD4Ser and SOMD4Cys(wt). X-ray absorption spectroscopy revealed a Mo(VI)-Se bond length of 2.51 Å, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se(Cys)O2Mo(VI)S2(MPT) site structures in the three constructs. The low-pH form of the Mo(V) electron paramagnetic resonance (EPR) signal of SeSOMD4Ser was altered compared to those of SOMD4Ser and SOMD4Cys(wt), with g1 in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo-Se bond.

  3. Covalent attachment of cholesterol oxidase and horseradish peroxidase on perlite through silanization: activity, stability and co-immobilization.

    PubMed

    Torabi, Seyed-Fakhreddin; Khajeh, Khosro; Ghasempur, Salehe; Ghaemi, Nasser; Siadat, Seyed-Omid Ranaei

    2007-08-31

    In the present work, co-immobilization of cholesterol oxidase (COD) and horseradish peroxidase (POD) on perlite surface was attempted. The surface of perlite were activated by 3-aminopropyltriethoxysilane and covalently bonded with COD and POD via glutaraldehyde. Enzymes activities have been assayed by spectrophotometric technique. The stabilities of immobilized COD and POD to pH were higher than those of soluble enzymes and immobilization shifted optimum pH of enzymes to the lower pH. Heat inactivation studies showed improved thermostability of the immobilized COD for more than two times, but immobilized POD was less thermostable than soluble POD. Also activity recovery of immobilized COD was about 50% since for immobilized POD was 11%. The K(m) of immobilized enzymes was found slightly lower than that of soluble enzymes. Immobilized COD showed inhibition in its activity at high cholesterol concentration which was not reported for soluble COD before. Co-immobilized enzymes retained 65% of its initial activity after 20 consecutive reactor batch cycles.

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

    PubMed

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

    2014-01-01

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

  5. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity

    PubMed Central

    Deep, Gagan; Kumar, Rahul; Jain, Anil K.; Dhar, Deepanshi; Panigrahi, Gati K.; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P.; Oberlies, Nicholas H.; Agarwal, Rajesh

    2016-01-01

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1–5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47phox). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity. PMID:26979487

  6. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity.

    PubMed

    Deep, Gagan; Kumar, Rahul; Jain, Anil K; Dhar, Deepanshi; Panigrahi, Gati K; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P; Oberlies, Nicholas H; Agarwal, Rajesh

    2016-03-16

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagn