WorldWideScience

Sample records for acid oxidase activity

  1. Assays of D-Amino Acid Oxidase Activity

    Directory of Open Access Journals (Sweden)

    Elena Rosini

    2018-01-01

    Full Text Available D-amino acid oxidase (DAAO is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs. Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.

  2. Activation of Recombinantly Expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate

    Directory of Open Access Journals (Sweden)

    Katharina Hahn

    2017-12-01

    Full Text Available l-Amino acid oxidases (l-AAO catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site.

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

    Energy Technology Data Exchange (ETDEWEB)

    Pessati, M.L.; Fontana, J.D.; Guimaraes, M.F. [Federal Univ. of Parana, Curitiba (Brazil)

    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.

  4. D-amino acid oxidase activator gene (DAOA) variation affects cerebrospinal fluid homovanillic acid concentrations in healthy Caucasians

    DEFF Research Database (Denmark)

    Andreou, Dimitrios; Saetre, Peter; Werge, Thomas

    2012-01-01

    The D-amino acid oxidase activator (DAOA) protein regulates the function of D-amino oxidase (DAO), an enzyme that catalyzes the oxidative deamination of D-3,4-dihydroxyphenylalanine (D-DOPA) and D-serine. D-DOPA is converted to L-3,4-DOPA, a precursor of dopamine, whereas D-serine participates...... in glutamatergic transmission. We hypothesized that DAOA polymorphisms are associated with dopamine, serotonin and noradrenaline turnover in the human brain. Four single-nucleotide polymorphisms, previously reported to be associated with schizophrenia, were genotyped. Cerebrospinal fluid (CSF) samples were drawn...... by lumbar puncture, and the concentrations of the major dopamine metabolite homovanillic acid (HVA), the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and the major noradrenaline metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured. Two of the investigated polymorphisms, rs...

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

    Directory of Open Access Journals (Sweden)

    Elaine Hatanaka

    Full Text Available 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.

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

    Science.gov (United States)

    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

  7. Antiglycation, radical scavenging, and semicarbazide-sensitive amine oxidase inhibitory activities of acetohydroxamic acid in vitro

    Directory of Open Access Journals (Sweden)

    Liu YH

    2017-07-01

    Full Text Available Yuh-Hwa Liu,1,2,* Yeh-Lin Lu,3,* Der-Zen Liu,4 Wen-Chi Hou5 1Division of Gastroenterology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; 2Department of General Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 3Department of Pharmacy, Taipei Medical University, Taipei, Taiwan; 4Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, Taipei, Taiwan; 5Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan *These authors contributed equally to this work Abstract: Advanced glycation endproducts (AGEs can promote intracellular reactive oxygen species production, and the levels of AGEs are highly correlated with cardiovascular disease and diabetes complications. Acetohydroxamic acid (acetH is a bacterial urease inhibitor drug used to treat kidney stones and infections in the urinary tract, and hydroxyurea (HU is a drug used for antineoplasm and sickle cell diseases. Both acetH and HU are hydroxamic acid derivatives. It was found that acetH and HU at 2.5 or 5 mM showed anti-AGE formation by lowering the AGEs’ fluorescent intensities and Nε-(carboxymethyllysine formation in bovine serum albumin/galactose models, and both showed better and significant differences (P<0.05 compared to the positive control of aminoguanidine. Regarding radical scavenging activities, the half-inhibition concentrations (IC50 of acetH against α,α-diphenyl-β-picrylhydrazyl radical and hydroxyl radical were 34.86 and 104.42 µM, respectively. The IC50 of acetH against semicarbazide-sensitive amine oxidase was 10.56 µM, and acetH showed noncompetitive inhibition respective to the substrates (benzylamine. The antiglycation, antioxidant, and semicarbazide-sensitive amine oxidase inhibitory activities of acetH prove that it has the potential for treating cardiovascular disease and diabetes complications and it needs further investigation in animal models. Keywords: acetH, AGEs, Nε

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

    Science.gov (United States)

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

    2016-01-01

    Ferulic acid has structural similarity with curcumin which is being reported for its monoamine oxidase (MAO) inhibitory activity. Based on this similarity, we designed a series of ferulic acid amides 6a-m and tested for their inhibitory activity on human MAO (hMAO) isoforms. All the compounds were found to inhibit the hMAO isoforms either selectively or non-selectively. Nine compounds (6a, 6b, 6g-m) were found to inhibit hMAO-B selectively, whereas the other four (6c-f) were found to be non-selective. There is a gradual shift from hMAO-B selectivity (6a,b) to non-selectivity (6c-f) as there is an increase in chain length at the amino terminus. In case of compounds having an aromatic nucleus at the amino terminus, increasing the carbon number between N and the aromatic ring increases the potency as well as selectivity toward hMAO-B. Compounds 6f, 6j, and 6k were subjected to membrane permeability and metabolic stability studies by in vitro assay methods. They were found to have a better pharmacokinetic profile than curcumin, ferulic acid, and selegiline. In order to understand the structural features responsible for the potency and selectivity of 6k, we carried out a molecular docking simulation study. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. A quantitative histochemical study of D-amino acid oxidase activity in rat liver in relationship with feeding conditions

    NARCIS (Netherlands)

    Patel, H. R.; Frederiks, W. M.; Marx, F.; Best, A. J.; van Noorden, C. J.

    1991-01-01

    The histochemical method for the demonstration of D-amino acid oxidase activity in rat liver, based on the use of cerium ions and the diaminobenzidine-cobalt-hydrogen peroxide procedure, was improved by the application of unfixed cryostat sections and a semipermeable membrane interposed between

  10. D-Amino acid oxidase bio-functionalized platforms: Toward an enhanced enzymatic bio-activity

    Science.gov (United States)

    Herrera, Elisa; Valdez Taubas, Javier; Giacomelli, Carla E.

    2015-11-01

    The purpose of this work is to study the adsorption process and surface bio-activity of His-tagged D-amino acid oxidase (DAAO) from Rhodotorula gracilis (His6-RgDAAO) as the first step for the development of an electrochemical bio-functionalized platform. With such a purpose this work comprises: (a) the His6-RgDAAO bio-activity in solution determined by amperometry, (b) the adsorption mechanism of His6-RgDAAO on bare gold and carboxylated modified substrates in the absence (substrate/COO-) and presence of Ni(II) (substrate/COO- + Ni(II)) determined by reflectometry, and (c) the bio-activity of the His6-RgDAAO bio-functionalized platforms determined by amperometry. Comparing the adsorption behavior and bio-activity of His6-RgDAAO on these different solid substrates allows understanding the contribution of the diverse interactions responsible for the platform performance. His6-RgDAAO enzymatic performance in solution is highly improved when compared to the previously used pig kidney (pk) DAAO. His6-RgDAAO exhibits an amperometrically detectable bio-activity at concentrations as low as those expected on a bio-functional platform; hence, it is a viable bio-recognition element of D-amino acids to be coupled to electrochemical platforms. Moreover, His6-RgDAAO bio-functionalized platforms exhibit a higher surface activity than pkDAAO physically adsorbed on gold. The platform built on Ni(II) modified substrates present enhanced bio-activity because the surface complexes histidine-Ni(II) provide with site-oriented, native-like enzymes. The adsorption mechanism responsible of the excellent performance of the bio-functionalized platform takes place in two steps involving electrostatic and bio-affinity interactions whose prevalence depends on the degree of surface coverage.

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

    Science.gov (United States)

    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. © 2013 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  12. Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1.

    Directory of Open Access Journals (Sweden)

    Silvia Schumann

    Full Text Available Mouse aldehyde oxidase (mAOX1 forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Escherichia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.

  13. New derivatives of 3,4-dihydroisoquinoline-3-carboxylic acid with free-radical scavenging, D-amino acid oxidase, acetylcholinesterase and butyrylcholinesterase inhibitory activity.

    Science.gov (United States)

    Solecka, Jolanta; Guśpiel, Adam; Postek, Magdalena; Ziemska, Joanna; Kawęcki, Robert; Lęczycka, Katarzyna; Osior, Agnieszka; Pietrzak, Bartłomiej; Pypowski, Krzysztof; Wyrzykowska, Agata

    2014-09-30

    A series of 3,4-dihydroisoquinoline-3-carboxylic acid derivatives were synthesised and tested for their free-radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS·+), superoxide anion radical (O2·-) and nitric oxide radical (·NO) assays. We also studied d-amino acid oxidase (DAAO), acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity. Almost each of newly synthesised compounds exhibited radical scavenging capabilities. Moreover, several compounds showed moderate inhibitory activities against DAAO, AChE and BuChE. Compounds with significant free-radical scavenging activity may be potential candidates for therapeutics used in oxidative-stress-related diseases.

  14. Gamma radiation affects the anti-Leishmania activity of Bothrops moojeni venom and correlates with L-amino acid oxidase activity

    International Nuclear Information System (INIS)

    Tempone, A.G.; Lourenco, C.O.; Spencer, P.J.; Rogero, J.R.; Nascimento, N.; Andrade Junior, H.F.

    1999-01-01

    Leishmania causes human disfiguring skin disease in endemic areas of Amazon and North Eastern Brazil. Those parasites present a remarkable resistance to most treatments, except those using toxic antimonial salts. We detected a specific anti-Leishmania activity in snake venoms, using an in vitro promastigote assay. In this report, we analyzed the activity of Bothrops moojeni venom against L. Amazonensis, using whole venom or fractions of L-amino acid oxidase (L-AO). Crude venom of B.moojeni, was fractionated by molecular exclusion chromatography. Activity against promastigotes was detected by respiratory oxidative conversion of MTT in a colorimetric assay and L-AO activity was detected by a colorimetric assay with peroxidase and OPD as revealing reagents. Crude venom was irradiated with 500, 1000, and 2000 Gy in a 60 Co gamma radiation source. The venom had an anti-Leishmania activity of 33 pg/promastigote and the active fraction migrates as 100-150 kDa, close to the size described for L-AOs, and also presented L-AO activity. The radiation reduces both the L-AO and anti-Leishmania activity in a dose dependent effect. Those data suggests the anti-Leishmania activity in this venom is closely related to the L-amino acid oxidase activity and also that radiation could be used as a tool to detect specific activities reduction in water solutions, similarly to observed in dry preparations. (author)

  15. Gamma radiation affects the anti-Leishmania activity of Bothrops moojeni venom and correlates with L-amino acid oxidase activity

    Energy Technology Data Exchange (ETDEWEB)

    Tempone, A.G.; Lourenco, C.O.; Spencer, P.J.; Rogero, J.R.; Nascimento, N. [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil). Div. de Radiobiologia; Andrade Junior, H.F. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina. Inst. de Medicina Tropical

    1999-11-01

    Leishmania causes human disfiguring skin disease in endemic areas of Amazon and North Eastern Brazil. Those parasites present a remarkable resistance to most treatments, except those using toxic antimonial salts. We detected a specific anti-Leishmania activity in snake venoms, using an in vitro promastigote assay. In this report, we analyzed the activity of Bothrops moojeni venom against L. Amazonensis, using whole venom or fractions of L-amino acid oxidase (L-AO). Crude venom of B.moojeni, was fractionated by molecular exclusion chromatography. Activity against promastigotes was detected by respiratory oxidative conversion of MTT in a colorimetric assay and L-AO activity was detected by a colorimetric assay with peroxidase and OPD as revealing reagents. Crude venom was irradiated with 500, 1000, and 2000 Gy in a {sup 60} Co gamma radiation source. The venom had an anti-Leishmania activity of 33 pg/promastigote and the active fraction migrates as 100-150 kDa, close to the size described for L-AOs, and also presented L-AO activity. The radiation reduces both the L-AO and anti-Leishmania activity in a dose dependent effect. Those data suggests the anti-Leishmania activity in this venom is closely related to the L-amino acid oxidase activity and also that radiation could be used as a tool to detect specific activities reduction in water solutions, similarly to observed in dry preparations. (author) 13 refs., 3 figs.

  16. Kynurenic acid inhibits intestinal hypermotility and xanthine oxidase activity during experimental colon obstruction in dogs.

    Science.gov (United States)

    Kaszaki, J; Palásthy, Z; Erczes, D; Rácz, A; Torday, C; Varga, G; Vécsei, L; Boros, M

    2008-01-01

    Kynurenic acid (KynA), an endogenous antagonist of N-methyl-d-aspartate (NMDA) glutamate receptors, protects the central nervous system in excitotoxic neurological diseases. We hypothesized that the inhibition of enteric glutamate receptors by KynA may influence dysmotility in the gastrointestinal tract. Group 1 of healthy dogs served as the sham-operated control, in group 2, the animals were treated with KynA, while in groups 3 and 4 mechanical colon obstruction was maintained for 7 h. Group 4 was treated with KynA at the onset of ileus. Hemodynamics and motility changes were monitored, and the activities of xanthine oxidoreductase (XOR) and myeloperoxidase (MPO) were determined from tissue samples. Colon obstruction induced a hyperdynamic circulatory reaction, significantly elevated the motility index and increased the mucosal leucocyte accumulation and the XOR activity. The KynA treatment augmented the tone of the colon, permanently decreased the motility index of the giant colonic contractions and reduced the increases in XOR and MPO activities. These effects were concomitant with the in vitro inhibition of XOR activity. In conclusion, KynA antagonizes the obstruction-induced motility responses and XOR activation in the colon. Inhibition of enteric NMDA receptors may provide an option to influence intestinal hypermotility and inflammatory changes.

  17. Gamma irradiation of mushrooms, preliminary studies: effect on O-diphenyl oxidase activity and amino acid content

    International Nuclear Information System (INIS)

    Bachman, S.; Gebicka, L.

    1992-01-01

    Mushrooms are a valuable food raw materials because of their nutritional and taste values. Post-harvest ripening, chemical composition (94% water) and possible microbial contamination decrease not only organoleptic and nutritional value, but also the shelf-life. As an objective method of evaluation of irradiated mushrooms we adopted activity determination of o-diphenyl oxidase (o-DPO) which is responsible for discoloration of the edible mushrooms and altered qualitative and quantitative content of amino acids. It was observed that doses up to 2 kGy did not cause any increase in the activity of o-DPO; irradiation also did not affect the taste. Mushrooms irradiated with doses up to 4 kGy were of good quality after 5 days of storage at 4 C, while the control samples (unirradiated) after the same time were considerably changed, probably due too post-harvest ripening. Immediately after exposure the activity of o-DPO increased in proportion to the dose used. During subsequent storage, however, no increase in o-DPO activity was observed. Irradiation used in the range from 0.2 to 0.4 kGy did not affect the nutritional value of the raw material. The results are an additional confirmation that radiation can be used for efficient preservation of mushrooms. (author). 14 refs, 6 tabs

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Purification and antibacterial activities of an L-amino acid oxidase from king cobra (Ophiophagus hannah venom

    Directory of Open Access Journals (Sweden)

    CS Phua

    2012-01-01

    Full Text Available Some constituents of snake venom have been found to display a variety of biological activities. The antibacterial property of snake venom, in particular, has gathered increasing scientific interest due to antibiotic resistance. In the present study, king cobra venom was screened against three strains of Staphylococcus aureus [including methicillin-resistant Staphylococcus aureus (MRSA], three other species of gram-positive bacteria and six gram-negative bacteria. King cobra venom was active against all the 12 bacteria tested, and was most effective against Staphylococcus spp. (S. aureus and S. epidermidis. Subsequently, an antibacterial protein from king cobra venom was purified by gel filtration, anion exchange and heparin chromatography. Mass spectrometry analysis confirmed that the protein was king cobra L-amino acid oxidase (Oh-LAAO. SDS-PAGE showed that the protein has an estimated molecular weight of 68 kDa and 70 kDa under reducing and non-reducing conditions, respectively. The minimum inhibitory concentrations (MIC of Oh-LAAO for all the 12 bacteria were obtained using radial diffusion assay method. Oh-LAAO had the lowest MIC value of 7.5 µg/mL against S. aureus ATCC 25923 and ATCC 29213, MRSA ATCC 43300, and S. epidermidis ATCC 12228. Therefore, the LAAO enzyme from king cobra venom may be useful as an antimicrobial agent.

  20. Activation of a peroxisomal Pichia pastoris d-amino acid oxidase, which uses d-alanine as a preferred substrate, depends on pyruvate carboxylase

    NARCIS (Netherlands)

    Klompmaker, Sandra H.; Kilic, Aysun; Baerends, Richard J.; Veenhuis, Marten; van der Klei, Ida J.; Goffeau, André

    d-Amino acid oxidase (DAO) is an important flavo-enzyme that catalyzes the oxidative deamination of d-amino acids into the corresponding alpha-keto acid, ammonia and H(2)O(2). We identified two amino acid oxidases in the methylotrophic yeast Pichia pastoris: Dao1p, which preferentially uses

  1. Variations of L- and D-amino acid levels in the brain of wild-type and mutant mice lacking D-amino acid oxidase activity.

    Science.gov (United States)

    Du, Siqi; Wang, Yadi; Weatherly, Choyce A; Holden, Kylie; Armstrong, Daniel W

    2018-05-01

    D-amino acids are now recognized to be widely present in organisms and play essential roles in biological processes. Some D-amino acids are metabolized by D-amino acid oxidase (DAO), while D-Asp and D-Glu are metabolized by D-aspartate oxidase (DDO). In this study, levels of 22 amino acids and the enantiomeric compositions of the 19 chiral proteogenic entities have been determined in the whole brain of wild-type ddY mice (ddY/DAO +/+ ), mutant mice lacking DAO activity (ddY/DAO -/- ), and the heterozygous mice (ddY/DAO +/- ) using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). No significant differences were observed for L-amino acid levels among the three strains except for L-Trp which was markedly elevated in the DAO +/- and DAO -/- mice. The question arises as to whether this is an unknown effect of DAO inactivity. The three highest levels of L-amino acids were L-Glu, L-Asp, and L-Gln in all the three strains. The lowest L-amino acid level was L-Cys in ddY/DAO +/- and ddY/DAO -/- mice, while L-Trp showed the lowest level in ddY/DAO +/+ mice. The highest concentration of D-amino acid was found to be D-Ser, which also had the highest % D value (~ 25%). D-Glu had the lowest % D value (~ 0.01%) in all the three strains. Significant differences of D-Leu, D-Ala, D-Ser, D-Arg, and D-Ile were observed in ddY/DAO +/- and ddY/DAO -/- mice compared to ddY/DAO +/+ mice. This work provides the most complete baseline analysis of L- and D-amino acids in the brains of ddY/DAO +/+ , ddY/DAO +/- , and ddY/DAO -/- mice yet reported. It also provides the most effective and efficient analytical approach for measuring these analytes in biological samples. This study provides fundamental information on the role of DAO in the brain and may be relevant for future development involving novel drugs for DAO regulation.

  2. The activity of ascorbic acid and catechol oxidase, the rate of photosynthesis and respiration as related to plant organs, stage of development and copper supply

    Directory of Open Access Journals (Sweden)

    St. Łyszcz

    2015-06-01

    Full Text Available Some experiments were performed to investigate the physiological role of copper in oat and sunflower and to recognize some effects of copper deficiency. Oat and sunflower plants were grown in pots on a peat soil under copper deficiency conditions (–Cu or with the optimal copper supply (+Cu. In plants the following measurements were carried out: 1 the activity of ascorbic acid oxidase (AAO and of catechol oxidase (PPO in different plant organs and at different stages of plant development, 2 the activity and the rate of photosynthesis, 3 the activity of RuDP-carboxylase, 4 the intensity of plant respiration. The activity of AAO and of PPO, and also the rate and the activity of photosynthesis were significantly lower under conditions of copper deficiency. The activity of both discussed oxidases depended on: 1 the plant species, 2 plant organs, 3 stage of plant development. Copper deficiency caused decrease of the respiration intensity of sunflower leaves but it increased to some extent the respiration of oat tops. Obtained results are consistent with the earlier suggestion of the authors that the PPO activity in sunflower leaves could be a sensitive indicator of copper supply of the plants, farther experiments are in progress.

  3. Synthesis of Amide and Ester Derivatives of Cinnamic Acid and Its Analogs: Evaluation of Their Free Radical Scavenging and Monoamine Oxidase and Cholinesterase Inhibitory Activities.

    Science.gov (United States)

    Takao, Koichi; Toda, Kazuhiro; Saito, Takayuki; Sugita, Yoshiaki

    2017-01-01

    A series of cinnamic acid derivatives, amides (1-12) and esters (13-22), were synthesized, and structure-activity relationships for antioxidant activity, and monoamine oxidases (MAO) A and B, acetylcholinesterase, and butyrylcholinesterase (BChE) inhibitory activities were analyzed. Among the synthesized compounds, compounds 1-10, 12-18, and rosmarinic acid (23), which contained catechol, o-methoxyphenol or 5-hydroxyindole moieties, showed potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. Compounds 9-11, 15, 17-22 showed potent and selective MAO-B inhibitory activity. Compound 20 was the most potent inhibitor of MAO-B. Compounds 18 and 21 showed moderate BChE inhibitory activity. In addition, compound 18 showed potent antioxidant activity and MAO-B inhibitory activity. In a comparison of the cinnamic acid amides and esters, the amides exhibited more potent DPPH free radical scavenging activity, while the esters showed stronger inhibitory activities against MAO-B and BChE. These results suggested that cinnamic acid derivatives such as compound 18, p-coumaric acid 3,4-dihydroxyphenethyl ester, and compound 20, p-coumaric acid phenethyl ester, may serve as lead compounds for the development of novel MAO-B inhibitors and candidate lead compounds for the prevention or treatment of Alzheimer's disease.

  4. Fructose suppresses uric acid excretion to the intestinal lumen as a result of the induction of oxidative stress by NADPH oxidase activation.

    Science.gov (United States)

    Kaneko, Chihiro; Ogura, Jiro; Sasaki, Shunichi; Okamoto, Keisuke; Kobayashi, Masaki; Kuwayama, Kaori; Narumi, Katsuya; Iseki, Ken

    2017-03-01

    A high intake of fructose increases the risk for hyperuricemia. It has been reported that long-term fructose consumption suppressed renal uric acid excretion and increased serum uric acid level. However, the effect of single administration of fructose on excretion of uric acid has not been clarified. We used male Wistar rats, which were orally administered fructose (5g/kg). Those rats were used in each experiment at 12h after administration. Single administration of fructose suppressed the function of ileal uric acid excretion and had no effect on the function of renal uric acid excretion. Breast cancer resistance protein (BCRP) predominantly contributes to intestinal excretion of uric acid as an active homodimer. Single administration of fructose decreased BCRP homodimer level in the ileum. Moreover, diphenyleneiodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), recovered the suppression of the function of ileal uric acid excretion and the Bcrp homodimer level in the ileum of rats that received single administration of fructose. Single administration of fructose decreases in BCRP homodimer level, resulting in the suppression the function of ileal uric acid excretion. The suppression of the function of ileal uric acid excretion by single administration of fructose is caused by the activation of Nox. The results of our study provide a new insight into the mechanism of fructose-induced hyperuricemia. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum.

    Science.gov (United States)

    Gabler, M; Fischer, L

    1999-08-01

    The fungus Fusarium oxysporum produced a D-amino acid oxidase (EC 1. 4.3.3) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific D-amino acid oxidase activity was increased up to 12.5-fold with various D-amino acids or their corresponding derivatives as inducers. The best inducers were D-alanine (2.7 microkat/g of dry biomass) and D-3-aminobutyric acid (2.6 microkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal D-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric D-amino acid oxidase activity of 17 microkat liter(-1) with D-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 microkat liter(-1) (3,240 U liter(-1)).

  6. Cytotoxic, Anti-Proliferative and Apoptosis Activity of l-Amino Acid Oxidase from Malaysian Cryptelytrops purpureomaculatus (CP-LAAO) Venom on Human Colon Cancer Cells.

    Science.gov (United States)

    Zainal Abidin, Syafiq Asnawi; Rajadurai, Pathmanathan; Hoque Chowdhury, Md Ezharul; Othman, Iekhsan; Naidu, Rakesh

    2018-06-08

    The aim of this study is to investigate the potential anti-cancer activity of l-amino acid oxidase (CP-LAAO) purified from the venom of Cryptelytrops purpureomaculatus on SW480 and SW620 human colon cancer cells. Mass spectrometry guided purification was able to identify and purify CP-LAAO. Amino acid variations identified from the partial protein sequence of CP-LAAO may suggest novel variants of these proteins. The activity of the purified CP-LAAO was confirmed with o-phenyldiamine (OPD)-based spectrophotometric assay. CP-LAAO demonstrated time- and dose-dependent cytotoxic activity and the EC 50 value was determined at 13 µg/mL for both SW480 and SW620 cells. Significant increase of caspase-3 activity, reduction of Bcl-2 levels, as well as morphological changes consistent with apoptosis were demonstrated by CP-LAAO. Overall, these data provide evidence on the potential anti-cancer activity of CP-LAAO from the venom of Malaysian C. purpureomaculatus for therapeutic intervention of human colon cancer.

  7. A new l-amino acid oxidase from Bothrops jararacussu snake venom: Isolation, partial characterization, and assessment of pro-apoptotic and antiprotozoal activities.

    Science.gov (United States)

    Carone, Sante E I; Costa, Tássia R; Burin, Sandra M; Cintra, Adélia C O; Zoccal, Karina F; Bianchini, Francine J; Tucci, Luiz F F; Franco, João J; Torqueti, Maria R; Faccioli, Lúcia H; Albuquerque, Sérgio de; Castro, Fabíola A de; Sampaio, Suely V

    2017-10-01

    A new l-amino acid oxidase (LAAO) from Bothrops jararacussu venom (BjussuLAAO-II) was isolated by using a three-step chromatographic procedure based on molecular exclusion, hydrophobicity, and affinity. BjussuLAAO-II is an acidic enzyme with pI=3.9 and molecular mass=60.36kDa that represents 0.3% of the venom proteins and exhibits high enzymatic activity (4884.53U/mg/mim). We determined part of the primary sequence of BjussuLAAO-II by identifying 96 amino acids, from which 34 compose the N-terminal of the enzyme (ADDRNPLEECFRETDYEEFLEIARNGLSDTDNPK). Multiple alignment of the partial BjussuLAAO-II sequence with LAAOs deposited in the NCBI database revealed high similarity (95-97%) with other LAAOs isolated from Bothrops snake venoms. BjussuLAAO-II exerted a strong antiprotozoal effect against Leishmania amazonensis (IC 50 =4.56μg/mL) and Trypanosoma cruzi (IC 50 =4.85μg/mL). This toxin also induced cytotoxicity (IC 50 =1.80μg/mL) and apoptosis in MCF7 cells (a human breast adenocarcinoma cell line) by activating the intrinsic and extrinsic apoptosis pathways, but were not cytotoxic towards MCF10A cells (a non-tumorigenic human breast epithelial cell line). The results reported herein add important knowledge to the field of Toxinology, especially for the development of new therapeutic agents. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The cytochemical demonstration of catalase and D-amino acid oxidase in the microbodies of teleost kidney cells

    NARCIS (Netherlands)

    Veenhuis, M.; Wendelaar Bonga, S.D.

    1977-01-01

    The distribution of catalase and D-amino acid oxidase, marker enzymes for peroxisomes, was determined cytochemically in the kidney tubules of an euryhaline teleost, the three-spined stickleback. Catalase activity was localized with the diaminobenzidine technique. The presence of D-amino acid oxidase

  9. Plasma diamine oxidase activity in asthmatic children

    Directory of Open Access Journals (Sweden)

    Kyoichiro Toyoshima

    1996-01-01

    Full Text Available Histamine plays an important role in the development of asthmatic symptoms. Diamine oxidase (DAO histaminase, which inactivates histamine, is located in the intestine and kidney and is released into plasma. Plasma DAO activity in asthmatic children was measured by a recently developed high performance liquid chromatographic method using histamine as the DAO substrate. Diamine oxidase activity was higher in severely asthmatic children than in those with mild asthma. A time course study during the acute exacerbation phase revealed that DAO activity rose during acute asthmatic attacks and then decreased gradually over several days. Although the mechanisms of plasma DAO activity increase during acute asthmatic attacks could not be explained, data showed that plasma DAO activity is an important index of histamine metabolism in asthmatics and may relate to some mechanisms of acute exacerbation of airway inflammation. Consequently, fluctuations in plasma DAO can be used as one of various indices of instability in management of asthma.

  10. Response of cytokinin pool and cytokinin oxidase/dehydrogenase activity to abscisic acid exhibits organ specificity in peas

    Czech Academy of Sciences Publication Activity Database

    Vaseva, I.; Todorova, D.; Malbeck, Jiří; Trávníčková, Alena; Macháčková, Ivana

    2008-01-01

    Roč. 30, č. 2 (2008), s. 151-155 ISSN 0137-5881 Institutional research plan: CEZ:AV0Z50380511 Keywords : Abscisic acid * Cytokinins * Cytokinin Subject RIV: EF - Botanics Impact factor: 0.807, year: 2008

  11. Involvement of abscisic acid in regulating antioxidative defense systems and IAA-oxidase activity and improving adventitious rooting in mung bean [Vigna radiata (L.) Wilczek] seedlings under cadmium stress.

    Science.gov (United States)

    Li, Shi-Weng; Leng, Yan; Feng, Lin; Zeng, Xiao-Ying

    2014-01-01

    In vitro experiments were conducted to investigate the effects of abscisic acid (ABA) and Cd on antioxidative defense systems and indole-3-acetic acid (IAA) oxidase during adventitious rooting in mung bean [Vigna radiata (L.) Wilczek] seedlings. The exogenous ABA significantly enhanced the number and fresh weight of the adventitious roots. CdCl2 strongly inhibited adventitious rooting. Pretreatment with 10 μM ABA clearly alleviated the inhibitory effect of Cd on rooting. ABA significantly reduced superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), and catalase (CAT) activities, as well as the levels of glutathione (GSH) and ascorbic acid (ASA) during adventitious rooting. ABA strongly increased IAA-oxidase activity during the induction (0-12 h) and expression (after 48 h) phases and increased the phenols levels. Cd treatment significantly reduced the activities of SOD, APX, POD, and IAA oxidase, as well as GSH level. Cd strongly increased ASA levels. ABA pretreatment counteracted Cd-induced alterations of certain antioxidants and antioxidative enzymes, e.g., remarkably rescued APX and POD activities, reduced the elevated SOD and CAT activities and ASA levels, and recovered the reduced GSH levels, caused by Cd stress. Thus, the physiological effects of the combination of ABA and Cd treatments were opposite of those obtained with Cd treatment alone, suggesting that ABA involved in the regulation of antioxidative defense systems and the alleviation of wounding- and Cd-induced oxidative stress.

  12. Antimutagenic activity of oxidase enzymes

    International Nuclear Information System (INIS)

    Agabeili, R.A.

    1986-01-01

    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

  13. Production of α-keto acids Part I. Immobilized cells ofTrigonopsis variabilis containing D-amino acid oxidase.

    Science.gov (United States)

    Brodelius, P; Nilsson, K; Mosbach, K

    1981-12-01

    Whole cells ofTrigonopsis variabilis were immobilized by entrapment in Ca(2+)-alginate and used for the production of α-keto acids from the corresponding D-amino acids. The D-amino acid oxidase within the immobilized cells has a broad substrate specificity. Hydrogen peroxide formed in the enzymatic reaction was efficiently hydrolyzed by manganese oxide co-immobilized with the cells. The amino acid oxidase activity was assayed with a new method based on reversed-phase HPLC. Oxygen requirements, bead size, concentration of cells in the beads, flow rate, and other factors were investigated in a " trickle-bed " reactor.

  14. Modulation of NADPH oxidase activity by known uraemic retention solutes

    DEFF Research Database (Denmark)

    Schulz, Anna Marta; Terne, Cindy; Jankowski, Vera

    2014-01-01

    chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. RESULTS: Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized......BACKGROUND: Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased...... inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. METHODS: Mononuclear leucocytes...

  15. In vitro oxidation of indoleacetic acid by soluble auxin-oxidases and peroxidases from maize roots

    International Nuclear Information System (INIS)

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

    1990-01-01

    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 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 [ 3 H]indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol

  16. 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.

    Science.gov (United States)

    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-NO 2 ), 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-NO 2 attenuates intracellular oxidative stress is not well investigated. Here, we elucidated the anti-oxidative mechanism of OA-NO 2 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-NO 2 (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-NO 2 promoted nuclear translocation of nuclear factor-E2- related factor-2 (Nrf2) and Nrf2 siRNA partially abrogated these protective effects. In addition, OA-NO 2 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-NO 2 reno-protection involves both inhibition of NADPH oxidase activity and induction of SOD1, Nrf2-dependent HO-1, and GCLM.

  17. Controversial Effects of D-Amino Acid Oxidase Activator (DAOA)/G72 on D-Amino Acid Oxidase (DAO) Activity in Human Neuronal, Astrocyte and Kidney Cell Lines: The N-methyl D-aspartate (NMDA) Receptor Hypofunction Point of View.

    Science.gov (United States)

    Jagannath, Vinita; Brotzakis, Zacharias Faidon; Parrinello, Michele; Walitza, Susanne; Grünblatt, Edna

    2017-01-01

    Dysfunction of D-amino acid oxidase ( DAO ) and DAO activator ( DAOA )/ G72 genes have been linked to neuropsychiatric disorders. The glutamate hypothesis of schizophrenia has proposed that increased DAO activity leads to decreased D-serine, which subsequently may lead to N-methyl-D-aspartate (NMDA) receptor hypofunction. It has been shown that DAOA binds to DAO and increases its activity. However, there are also studies showing DAOA decreases DAO activity. Thus, the effect of DAOA on DAO is controversial. We aimed to understand the effect of DAOA on DAO activity in neuron-like (SH-SY5Y), astrocyte-like (1321N1) and kidney-like (HEK293) human cell lines. DAO activity was measured based on the release of hydrogen peroxide and its interaction with Amplex Red reagent. We found that DAOA increases DAO activity only in HEK293 cells, but has no effect on DAO activity in SH-SY5Y and 1321N1 cells. This might be because of different signaling pathways, or due to lower DAO and DAOA expression in SH-SY5Y and 1321N1 cells compared to HEK293 cells, but also due to different compartmentalization of the proteins. The lower DAO and DAOA expression in neuron-like SH-SY5Y and astrocyte-like 1321N1 cells might be due to tightly regulated expression, as previously reported in the human post-mortem brain. Our simulation experiments to demonstrate the interaction between DAOA and human DAO (hDAO) showed that hDAO holoenzyme [hDAO with flavine adenine dinucleotide (FAD)] becomes more flexible and misfolded in the presence of DAOA, whereas DAOA had no effect on hDAO apoprotein (hDAO without FAD), which indicate that DAOA inactivates hDAO holoenzyme. Furthermore, patch-clamp analysis demonstrated no effect of DAOA on NMDA receptor activity in NR1/NR2A HEK293 cells. In summary, the interaction between DAO and DAOA seems to be cell type and its biochemical characteristics dependent which still needs to be elucidated.

  18. Controversial Effects of D-Amino Acid Oxidase Activator (DAOA/G72 on D-Amino Acid Oxidase (DAO Activity in Human Neuronal, Astrocyte and Kidney Cell Lines: The N-methyl D-aspartate (NMDA Receptor Hypofunction Point of View

    Directory of Open Access Journals (Sweden)

    Vinita Jagannath

    2017-10-01

    Full Text Available Dysfunction of D-amino acid oxidase (DAO and DAO activator (DAOA/G72 genes have been linked to neuropsychiatric disorders. The glutamate hypothesis of schizophrenia has proposed that increased DAO activity leads to decreased D-serine, which subsequently may lead to N-methyl-D-aspartate (NMDA receptor hypofunction. It has been shown that DAOA binds to DAO and increases its activity. However, there are also studies showing DAOA decreases DAO activity. Thus, the effect of DAOA on DAO is controversial. We aimed to understand the effect of DAOA on DAO activity in neuron-like (SH-SY5Y, astrocyte-like (1321N1 and kidney-like (HEK293 human cell lines. DAO activity was measured based on the release of hydrogen peroxide and its interaction with Amplex Red reagent. We found that DAOA increases DAO activity only in HEK293 cells, but has no effect on DAO activity in SH-SY5Y and 1321N1 cells. This might be because of different signaling pathways, or due to lower DAO and DAOA expression in SH-SY5Y and 1321N1 cells compared to HEK293 cells, but also due to different compartmentalization of the proteins. The lower DAO and DAOA expression in neuron-like SH-SY5Y and astrocyte-like 1321N1 cells might be due to tightly regulated expression, as previously reported in the human post-mortem brain. Our simulation experiments to demonstrate the interaction between DAOA and human DAO (hDAO showed that hDAO holoenzyme [hDAO with flavine adenine dinucleotide (FAD] becomes more flexible and misfolded in the presence of DAOA, whereas DAOA had no effect on hDAO apoprotein (hDAO without FAD, which indicate that DAOA inactivates hDAO holoenzyme. Furthermore, patch-clamp analysis demonstrated no effect of DAOA on NMDA receptor activity in NR1/NR2A HEK293 cells. In summary, the interaction between DAO and DAOA seems to be cell type and its biochemical characteristics dependent which still needs to be elucidated.

  19. [Effect of Kaixinsan on monoamine oxidase activity].

    Science.gov (United States)

    Wang, Shi; Dong, Xian-Zhe; Tan, Xiao; Wang, Yu-Ning; Liu, Ping

    2016-05-01

    To observe the effect of antidepressant medicine prescription, Kaixinsan (KXS) on monoamine oxidase (MAO) activity, and explore the mechanism of KXS in elevating the levels of monoamine neurotransmitter from the perspective of metabolism, in vitro enzyme reaction system and C6 neuroglial cells, the effect of KXS at different concentrations on MAO-A and MAO-B activity was observed. In animal studies, the effect of KXS at different concentrations on MAO-A and MAO-B activities of brain mitochondrialin normal rats and solitary chronic unpredictable moderate stress (CMS) model rats after intragastric administration for 1, 2, 3 weeks. Results showed that 10 g•L⁻¹ KXS could significantly reduce the activity of MAO-A and MAO-B in enzyme reaction system; and in C6 cells, KXS within 0.625-10 g•L⁻¹ concentration range had no significant effect on the activity of MAO-A, but had obvious inhibitory effect on the activity of MAO-B in a dose dependent manner. KXS had no significant effect on the activity of MAO-A and MAO-B in brains of normal rats after action for 1, 2, 3 weeks. After 2 and 3 weeks treatment with 338 mg•kg⁻¹ dose KXS, MAO-A activity in the brain of CMS rats was decreased as compared with the model group (PMAO-B activity after 1, 2, 3 weeks of treatment. The results indicated that KXS had certain effect on in vitro MAO-A and MAO-B activity, had no effect on brain MAO-A and MAO-B activity in vivo in normal rats, and had certain inhibitory effect on MAO-A activity in brains of CMS rats. Copyright© by the Chinese Pharmaceutical Association.

  20. Modulation of NADPH oxidase activity by known uraemic retention solutes.

    Science.gov (United States)

    Schulz, Anna Marta; Terne, Cindy; Jankowski, Vera; Cohen, Gerald; Schaefer, Mandy; Boehringer, Falko; Tepel, Martin; Kunkel, Desiree; Zidek, Walter; Jankowski, Joachim

    2014-08-01

    Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. Mononuclear leucocytes isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase activity compared with plasma from healthy subjects. However, this effect was significantly decreased in plasma from patients with CKD-5D after dialysis. The results of this study show that uraemic retention solutes modulated the activity of the NADPH oxidase. The results of this study might be the basis for the development of inhibitors applicable as drug in the situation of increased oxidative stress. © 2014 Stichting European Society for Clinical Investigation Journal Foundation.

  1. Platelet monoamine oxidase: specific activity and turnover number in headache

    International Nuclear Information System (INIS)

    Summers, K.M.; Brown, G.K.; Craig, I.W.; Peatfield, R.; Rose, F.C.

    1982-01-01

    Monoamine oxidase turnover numbers (molecules of substrate converted to product per minute per active site) have been calculated for the human platelet enzyme using [ 3 H]pargyline. Headache patients with high and low monoamine oxidase specific activities relative to controls were found to have turnover numbers very close to those for controls. This finding suggests that their specific activities vary because of differences in the concentration of active monoamine oxidase molecules, rather than differences in the ability of those enzyme molecules to catalyse the deamination reaction. (Auth.)

  2. Xanthine Oxidase Inhibitory Activity of a Plectranthus saccatus aqueous extract

    Directory of Open Access Journals (Sweden)

    Caldeira F

    2016-12-01

    Full Text Available Gout is a disease with high prevalence in developed countries, resulting from the deposition of uric acid crystals in various locations, particularly at the joints. The pharmacotherapeutic approach to chronic gout essentially consists of administration of uric acid-lowering agents. The main mechanism of action of these agents is the inhibition of xanthine oxidase (XO, the enzyme responsible for the formation of uric acid. The therapeutic alternatives available for this purpose are limited, thus justifying the interest of the discovery of potential new uric acidlowering drugs. In this regard, an aqueous extract of the plant Plectranthus saccatus has been studied for its ability to inhibit XO. The composition of the extract was determined by HPLC and rosmarinic acid was identified as the major constituent. Both the extract and rosmarinic acid have demonstrated the ability to inhibit the production of uric acid by interfering with XO activity. The results obtained herein support the continuation of the study of their uric acid-lowering properties in cell-based and in vivo models to further explore their potential in gout therapy.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Koji Sode

    2012-11-01

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

  5. Construction of mutant glucose oxidases with increased dye-mediated dehydrogenase activity.

    Science.gov (United States)

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

    2012-11-02

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

  6. Are colorimetric assays appropriate for measuring phenol oxidase activity in peat soils?

    Science.gov (United States)

    Magdalena M. Wiedermann; Evan S. Kane; Timothy J. Veverica; Erik A. Lilleskov

    2017-01-01

    The activity of extracellular phenol oxidases is believed to play a critical role in decomposition processes in peatlands. The water logged, acidic conditions, and recalcitrant litter from the peatland vegetation, lead to exceptionally high phenolics in the peat. In order to quantify the activity of oxidative enzymes involved in the modification and break down of...

  7. Glucose oxidase variants with improved properities

    OpenAIRE

    Fischer, Rainer; Ostafe, Raluca; Prodanovic, Radivoje

    2014-01-01

    Source: WO14173822A3 [EN] The technology provided herein relates to novel variants of microbial glucose oxidase with improved properties, more specifically to polypeptides having glucose oxidase activity as their major enzymatic activity; to nucleic acid molecules encoding said glucose oxidases; vectors and host cells containing the nucleic acids and methods for producing the glucose oxidase; compositions comprising said glucose oxidase; methods for the preparation and production of such enzy...

  8. Structure and activity of Aspergillus nidulans copper amine oxidase

    DEFF Research Database (Denmark)

    McGrath, Aaron P; Mithieux, Suzanne M; Collyer, Charles A

    2011-01-01

    Aspergillus nidulans amine oxidase (ANAO) has the unusual ability among the family of copper and trihydroxyphenylalanine quinone-containing amine oxidases of being able to oxidize the amine side chains of lysine residues in large peptides and proteins. We show here that in common with the related...... enzyme from the yeast Pichia pastoris, ANAO can promote the cross-linking of tropoelastin and oxidize the lysine residues in α-casein proteins and tropoelastin. The crystal structure of ANAO, the first for a fungal enzyme in this family, has been determined to a resolution of 2.4 Å. The enzyme is a dimer...... with the archetypal fold of a copper-containing amine oxidase. The active site is the most open of any of those of the structurally characterized enzymes in the family and provides a ready explanation for its lysine oxidase-like activity....

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

    Science.gov (United States)

    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.

  10. Serum diamine oxidase activity in patients with histamine intolerance.

    Science.gov (United States)

    Manzotti, G; Breda, D; Di Gioacchino, M; Burastero, S E

    2016-03-01

    Intolerance to various foods, excluding bona fide coeliac disease and lactose intolerance, represents a growing cause of patient visits to allergy clinics.Histamine intolerance is a long-known, multifaceted clinical condition triggered by histamine-rich foods and alcohol and/or by drugs that liberate histamine or block diamine oxidase (DAO), the main enzyme involved in the metabolism of ingested histamine. Histamine limitation diets impose complex, non-standardized restrictions that may severely impact the quality of life of patients. We retrospectively evaluated 14 patients who visited allergy outpatient facilities in northern Italy with a negative diagnosis for IgE-mediated food hypersensitivity, coeliac disease, conditions related to gastric hypersecretion, and systemic nickel hypersensitivity, and who previously underwent a histamine limitation diet with benefits for their main symptoms. Serum diamine oxidase levels and the clinical response to diamine oxidase supplementation were investigated. We found that 10 out of 14 patients had serum DAO activityintolerance. Moreover, 13 out of 14 patients subjectively reported a benefit in at least one of the disturbances related to food intolerances following diamine oxidase supplementation. The mean value (±SD) of diamine oxidase activity in the cohort of patients with histamine intolerance symptoms was 7.04±6.90 U/mL compared to 39.50±18.16 U/mL in 34 healthy controls (P=0.0031). In patients with symptoms triggered by histamine-rich food, measuring the serum diamine oxidase activity can help identify subjects who can benefit from a histamine limitation diet and/or diamine oxidase supplementation.Properly designed, controlled studies investigating histamine intolerance that include histamine provocation are indispensable for providing insights into the area of food intolerances, which are currently primarily managed with non-scientific approaches in Italy. © The Author(s) 2015.

  11. Aristolochic acid and its derivatives as inhibitors of snake venom L-amino acid oxidase.

    Science.gov (United States)

    Bhattacharjee, Payel; Bera, Indrani; Chakraborty, Subhamoy; Ghoshal, Nanda; Bhattacharyya, Debasish

    2017-11-01

    Snake venom L-amino acid oxidase (LAAO) exerts toxicity by inducing hemorrhage, pneumorrhagia, pulmonary edema, cardiac edema, liver cell necrosis etc. Being well conserved, inhibitors of the enzyme may be synthesized using the template of the substrate, substrate binding site and features of the catalytic site of the enzyme. Previous findings showed that aristolochic acid (AA), a major constituent of Aristolochia indica, inhibits Russell's viper venom LAAO enzyme activity since, AA interacts with DNA and causes genotoxicity, derivatives of this compound were synthesized by replacing the nitro group to reduce toxicity while retaining the inhibitory potency. The interactions of AA and its derivatives with LAAO were followed by inhibition kinetics and surface plasmon resonance. Similar interactions with DNA were followed by absorption spectroscopy and atomic force microscopy. LAAO-induced cytotoxicity was evaluated by generation of reactive oxygen species (ROS), cell viability assays, confocal and epifluorescence microscopy. The hydroxyl (AA-OH) and chloro (AA-Cl) derivatives acted as inhibitors of LAAO but did not interact with DNA. The derivatives significantly reduced LAAO-induced ROS generation and cytotoxicity in human embryonic kidney (HEK 293) and hepatoma (HepG2) cell lines. Confocal images indicated that AA, AA-OH and AA-Cl interfered with the binding of LAAO to the cell membrane. AA-OH and AA-Cl significantly inhibited LAAO activity and reduced LAAO-induced cytotoxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. ABTS assay of phenol oxidase activity in soil.

    Science.gov (United States)

    Floch, Carine; Alarcon-Gutiérrez, Enrique; Criquet, Stéven

    2007-12-01

    Phenol oxidases (PO) are involved in degradation of many recalcitrant aromatic compounds and may be sensitive to some pollutants. Hence, their activities may be a useful indicator for evaluating soil quality and health. To this end, the aim of this study was to develop a simple method to assay PO activity directly in bulk samples by spectrophotometric test using 2,2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) as the substrate. Three Mediterranean soils were used as models. For each soil, we studied the kinetic parameters and the effects of certain factors (i.e. amount of soil, pH, temperature, incubation time and substrate concentration) in order to determine the optimum conditions for the ABTS assay. Results showed that PO attain their optimum activities when incubating 0.1 g of soil at 30 degrees C for 5 min with 10 ml of a Modified Universal Buffer (MUB) at pH 2 and 200 microl of a 0.1 M ABTS solution.

  13. Xanthine oxidase activity regulates human embryonic brain cells growth

    Directory of Open Access Journals (Sweden)

    Kevorkian G. A.

    2011-10-01

    Full Text Available Aim. Involvement of Xanthine Oxidase (XO; EC1.1.3.22 in cellular proliferation and differentiation has been suggested by the numerous investigations. We have proposed that XO might have undoubtedly important role during the development, maturation as well as the death of human embryos brain cells. Methods. Human abortion material was utilized for the cultivation of brain cells (E90. XO activity was measured by the formation of uric acid in tissue. Cell death was detected by the utility of Trypan Blue dye. Results. Allopurinol suppressed the XO activity in the brain tissue (0.12 ± 0.02; 0.20 ± 0.03 resp., p < 0.05. On day 12th the number of cells in the culture treated with the Allopurinol at the early stage of development was higher in comparison with the Control (2350.1 ± 199.0 vs 2123 ± 96 and higher in comparison with the late period of treatment (1479.6 ± 103.8, p < < 0.05. In all groups, the number of the dead cells was less than in Control, indicating the protective nature of Allopurinol as an inhibitor of XO. Conclusions. Allopurinol initiates cells proliferation in case of the early treatment of the human brain derived cell culture whereas at the late stages it has an opposite effect.

  14. DNA damage and decrease of cellular oxidase activity in piglet ...

    African Journals Online (AJOL)

    DNA damage and decrease of cellular oxidase activity in piglet sertoli cells exposed to gossypol. Ming Zhang, Hui Yuan, Zuping He, Liyun Yuan, Jine Yi, Sijun Deng, Li Zhu, Chengzhi Guo, Yin Lu, Jing Wu, Lixin Wen, Qiang Wei, Liqun Xue ...

  15. 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

    Science.gov (United States)

    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.

  16. Process technology for the application of d-amino acid oxidases in pharmaceutical intermediate manufacturing

    DEFF Research Database (Denmark)

    Tindal, Stuart; Carr, Reuben; Archer, Ian V. J.

    2011-01-01

    Recent advances in biocatalysis have seen increased interest in the use of D-amino acid oxidase to synthesize optically pure amino acids. However, the creation of a genuine oxidase based platform technology will require suitable process technology as well as an understanding of the challenges...... and opportunities of a wider portfolio of synthetic targets. In this article we address some of the recent progress in process technology to enable the future development of a generic platform technology....

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. Variations in epidermal cytochrome oxidase activity after local irradiation

    International Nuclear Information System (INIS)

    Itoiz, M.E.; Rey, B.M. de; Cabrini, R.L.

    1982-01-01

    Cytochrome oxidase activity was evaluated histochemically as an index of mitochondrial damage after local irradiation with X-rays. It was determined by microphotometry on the tail skin of newly born Wistar rats four days after irradiation with doses ranging from 2 to 16krad. The enzyme activity of the whole epidermis increased after irradiation, the increases being related to the increase in thickness of the epithelium which was observed as a response to irradiation injury. Within the dose range tested, the enzyme concentration (expressed per unit volume of tissue) decreased in relation to the dose applied. At the electron microscopy level, the cytochemical demonstration of cytochrome oxidase revealed an irregular reaction over the cristae, intramitochondrial vacuolization and partial homogenization of the matrix. Positive membrane fragments were seen around lipid droplets. This reaction confirms the mitochondrial origin of these previously observed radiation-induced vacuoles. (author)

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

    Science.gov (United States)

    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 (H 2 O 2 ), 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 (H 2 O 2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H 2 O 2 -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. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  2. Cytochemical localization of catalase and several hydrogen peroxide-producing oxidases in the nucleoids and matrix of rat liver peroxisomes

    NARCIS (Netherlands)

    Veenhuis, M.; Wendelaar Bonga, S.E.

    1979-01-01

    The distribution of catalase, amino acid oxidase, α-hydroxy acid oxidase, urate oxidase and alcohol oxidase was studied cytochemically in rat hepatocytes. The presence of catalase was demonstrated with the conventional diaminobenzidine technique. Oxidase activities were visualized with methods based

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

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei, E-mail: hmliu2004@126.com [Huazhong University of Science and Technology, School of Chemistry and Chemical Engineering (China)

    2016-03-15

    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 Na{sub 2}SeO{sub 3} 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{sub m}) values and maximal reaction velocity (V{sub max}) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

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

    International Nuclear Information System (INIS)

    Guo, Leilei; Huang, Kaixun; Liu, Hongmei

    2016-01-01

    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 Na 2 SeO 3 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.

  5. Sulfite oxidase activity of cytochrome c: Role of hydrogen peroxide

    Directory of Open Access Journals (Sweden)

    Murugesan Velayutham

    2016-03-01

    Full Text Available In humans, sulfite is generated endogenously by the metabolism of sulfur containing amino acids such as methionine and cysteine. Sulfite is also formed from exposure to sulfur dioxide, one of the major environmental pollutants. Sulfite is used as an antioxidant and preservative in dried fruits, vegetables, and beverages such as wine. Sulfite is also used as a stabilizer in many drugs. Sulfite toxicity has been associated with allergic reactions characterized by sulfite sensitivity, asthma, and anaphylactic shock. Sulfite is also toxic to neurons and cardiovascular cells. Recent studies suggest that the cytotoxicity of sulfite is mediated by free radicals; however, molecular mechanisms involved in sulfite toxicity are not fully understood. Cytochrome c (cyt c is known to participate in mitochondrial respiration and has antioxidant and peroxidase activities. Studies were performed to understand the related mechanism of oxidation of sulfite and radical generation by ferric cytochrome c (Fe3+cyt c in the absence and presence of H2O2. Electron paramagnetic resonance (EPR spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO were performed with sulfite, Fe3+cyt c, and H2O2. An EPR spectrum corresponding to the sulfite radical adducts of DMPO (DMPO-SO3- was obtained. The amount of DMPO-SO3- formed from the oxidation of sulfite by the Fe3+cyt c increased with sulfite concentration. In addition, the amount of DMPO-SO3- formed by the peroxidase activity of Fe3+cyt c also increased with sulfite and H2O2 concentration. From these results, we propose a mechanism in which the Fe3+cyt c and its peroxidase activity oxidizes sulfite to sulfite radical. Our results suggest that Fe3+cyt c could have a novel role in the deleterious effects of sulfite in biological systems due to increased production of sulfite radical. It also shows that the increased production of sulfite radical may be responsible for neurotoxicity and some of the injuries which

  6. Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.

    Directory of Open Access Journals (Sweden)

    Martin Mahro

    Full Text Available 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.

  7. Localization of ascorbic acid, ascorbic acid oxidase, and glutathione in roots of Cucurbita maxima L.

    Science.gov (United States)

    Liso, Rosalia; De Tullio, Mario C; Ciraci, Samantha; Balestrini, Raffaella; La Rocca, Nicoletta; Bruno, Leonardo; Chiappetta, Adriana; Bitonti, Maria Beatrice; Bonfante, Paola; Arrigoni, Oreste

    2004-12-01

    To understand the function of ascorbic acid (ASC) in root development, the distribution of ASC, ASC oxidase, and glutathione (GSH) were investigated in cells and tissues of the root apex of Cucubita maxima. ASC was regularly distributed in the cytosol of almost all root cells, with the exception of quiescent centre (QC) cells. ASC also occurred at the surface of the nuclear membrane and correspondingly in the nucleoli. No ASC could be observed in vacuoles. ASC oxidase was detected by immunolocalization mainly in cell walls and vacuoles. This enzyme was particularly abundant in the QC and in differentiating vascular tissues and was absent in lateral root primordia. Administration of the ASC precursor L-galactono-gamma-lactone markedly increased ASC content in all root cells, including the QC. Root treatment with the ASC oxidized product, dehydroascorbic acid (DHA), also increased ASC content, but caused ASC accumulation only in peripheral tissues, where DHA was apparently reduced at the expense of GSH. The different pattern of distribution of ASC in different tissues and cell compartments reflects its possible role in cell metabolism and root morphogenesis.

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

    Science.gov (United States)

    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. Copyright © 2014. Published by Elsevier B.V.

  9. Snake venom L-amino acid oxidases: an overview on their antitumor effects

    Science.gov (United States)

    2014-01-01

    The L-amino acid oxidases (LAAOs) constitute a major component of snake venoms and have been widely studied due to their widespread presence and various effects, such as apoptosis induction, cytotoxicity, induction and/or inhibition of platelet aggregation, hemorrhage, hemolysis, edema, as well as antimicrobial, antiparasitic and anti-HIV activities. The isolated and characterized snake venom LAAOs have become important research targets due to their potential biotechnological applications in pursuit for new drugs of interest in the scientific and medical fields. The current study discusses the antitumor effects of snake venom LAAOs described in the literature to date, highlighting the mechanisms of apoptosis induction proposed for this class of proteins. PMID:24940304

  10. Purification and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima L. endosperm.

    Science.gov (United States)

    Lange, T

    1994-01-01

    Gibberellin (GA) 20-oxidase was purified to apparent homogeneity from Cucurbita maxima endosperm by fractionated ammonium-sulphate precipitation, gel-filtration chromatography and anion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Average purification after the last step was 55-fold with 3.9% of the activity recovered. The purest single fraction was enriched 101-fold with 0.2% overall recovery. Apparent relative molecular mass of the enzyme was 45 kDa, as determined by gel-filtration HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, indicating that GA 20-oxidase is probably a monomeric enzyme. The purified enzyme degraded on two-dimensional gel electrophoresis, giving two protein spots: a major one corresponding to a molecular mass of 30 kDa and a minor one at 45 kDa. The isoelectric point for both was 5.4. The amino-acid sequences of the amino-terminus of the purified enzyme and of two peptides from a tryptic digest were determined. The purified enzyme catalysed the sequential conversion of [14C]GA12 to [14C]GA15, [14C]GA24 and [14C]GA25, showing that carbon atom 20 was oxidised to the corresponding alcohol, aldehyde and carboxylic acid in three consecutive reactions. [14C]Gibberellin A53 was similarly converted to [14C]GA44, [14C]GA19, [14C]GA17 and small amounts of a fourth product, which was preliminarily identified as [14C]GA20, a C19-gibberellin. All GAs except [14C]GA20 were identified by combined gas chromatography-mass spectrometry. The cofactor requirements in the absence of dithiothreitol were essentially as in its presence (Lange et al., Planta 195, 98-107, 1994), except that ascorbate was essential for enzyme activity and the optimal concentration of catalase was lower.

  11. Ligand complex structures of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change.

    Science.gov (United States)

    Im, Dohyun; Matsui, Daisuke; Arakawa, Takatoshi; Isobe, Kimiyasu; Asano, Yasuhisa; Fushinobu, Shinya

    2018-03-01

    l-Amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 (l-AAO/MOG) catalyzes both the oxidative deamination and oxidative decarboxylation of the α-group of l-Lys to produce a keto acid and amide, respectively. l-AAO/MOG exhibits limited specificity for l-amino acid substrates with a basic side chain. We previously determined its ligand-free crystal structure and identified a key residue for maintaining the dual activities. Here, we determined the structures of l-AAO/MOG complexed with l-Lys, l-ornithine, and l-Arg and revealed its substrate recognition. Asp238 is located at the ceiling of a long hydrophobic pocket and forms a strong interaction with the terminal, positively charged group of the substrates. A mutational analysis on the D238A mutant indicated that the interaction is critical for substrate binding but not for catalytic control between the oxidase/monooxygenase activities. The catalytic activities of the D238E mutant unexpectedly increased, while the D238F mutant exhibited altered substrate specificity to long hydrophobic substrates. In the ligand-free structure, there are two channels connecting the active site and solvent, and a short region located at the dimer interface is disordered. In the l-Lys complex structure, a loop region is displaced to plug the channels. Moreover, the disordered region in the ligand-free structure forms a short helix in the substrate complex structures and creates the second binding site for the substrate. It is assumed that the amino acid substrate enters the active site of l-AAO/MOG through this route. The atomic coordinates and structure factors (codes 5YB6, 5YB7, and 5YB8) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1.4.3.2 (l-amino acid oxidase), 1.13.12.2 (lysine 2-monooxygenase).

  12. Low platelet monoamine oxidase activity in pathological gambling

    International Nuclear Information System (INIS)

    Carrasco, J.L.; Saiz-Ruiz, J.; Hollander, E.; Cesar, J.; Lopez-Ibor, J.J. Jr.

    1994-01-01

    Decreased platelet monoamine oxidase (MAO) activity has been reported in association with sensation-seeking personality type and in some mental disorders associated with a lack of impulse control. Pathological gambling itself has been related with both sensation-seeking and reduced impulse control. Platelet MAO activity was investigated in 15 DSM-III-R pathological gamblers from our outpatient clinic. Gamblers had a significantly lower platelet MAO activity than a group of 25 healthy controls. The range of MAO levels in gamblers was also significantly shorter than in controls. In controls, platelet MAO levels showed the previously described negative correlations with sensation-seeking scores but not in gamblers. The findings are consistent with previous studies showing an association of low platelet MAO activity with impulse control disorders and raise some interesting therapeutic alternatives for pathological gambling. (au) (40 refs.)

  13. Low platelet monoamine oxidase activity in pathological gambling

    Energy Technology Data Exchange (ETDEWEB)

    Carrasco, J.L. [Department of Psychiatry, Centro de Salud Mental, Parla Madrid (Spain); Saiz-Ruiz, J. [Department of Psychiatry and Haematology, Hospital Ramon y Cajal, Madrid (Spain); Hollander, E. [Department of Psychiatry, Mount Sinai School of Medicine, Queens Hospital Center, New York (United States); Cesar, J. [Department of Haematology, Hospital Ramon y Cajal, Madrid (Spain); Lopez-Ibor, J.J. Jr. [Department of Psychiatry, Hospital San Carlos, Complutense University, Madrid (Spain)

    1994-12-01

    Decreased platelet monoamine oxidase (MAO) activity has been reported in association with sensation-seeking personality type and in some mental disorders associated with a lack of impulse control. Pathological gambling itself has been related with both sensation-seeking and reduced impulse control. Platelet MAO activity was investigated in 15 DSM-III-R pathological gamblers from our outpatient clinic. Gamblers had a significantly lower platelet MAO activity than a group of 25 healthy controls. The range of MAO levels in gamblers was also significantly shorter than in controls. In controls, platelet MAO levels showed the previously described negative correlations with sensation-seeking scores but not in gamblers. The findings are consistent with previous studies showing an association of low platelet MAO activity with impulse control disorders and raise some interesting therapeutic alternatives for pathological gambling. (au) (40 refs.).

  14. Stability of glucose oxidase and catalase adsorbed on variously activated 13X zeolite.

    Science.gov (United States)

    Pifferi, P G; Vaccari, A; Ricci, G; Poli, G; Ruggeri, O

    1982-10-01

    The use of 13X zeolite (0.1-0.4-mm granules), treated with 2N and 0.01N HCI, 0.01M citric acid, 0.1M citric-phosphate buffer (pH 3.6), and in untreated form to adsorb glucose oxidase of fungal origin and microbial catalase was examined. Physicochemical analysis of the support demonstrated that its crystalline structure, greatly altered by the HCl and buffer, could be partially maintained with citric acid. The specific adsorption of the enzymes increased with decreasing pH and proved to be considerable for all the supports. The stability with storage at 25 degrees C is strictly correlated with the titrable acidity of the activated zeolite expressed as meq NaOH/g and with pH value of the activation solution. It proved to be lower than 55 h for both enzymes if adsorbed on zeolite treated with 2N HCl, and 15-fold and 30-fold higher for glucose oxidase and catalase adsorbed, respectively, on zeolite treated with the 0.1M citric-phosphate buffer and 0.01M citric acid. The specific adsorption of glucose oxidase and catalase was, respectively, 1840 U/g at pH 3.0 and 6910 U/g at pH 5.0. Their half-life at 25 degrees C with storage at pH 3.5 for the former and at pH 5.0 for the latter was 800 and 1560 h vs. 40 and 110 h for the corresponding free enzymes.

  15. Differences in activity of cytochrome C oxidase in brain between sleep and wakefulness.

    Science.gov (United States)

    Nikonova, Elena V; Vijayasarathy, Camasamudram; Zhang, Lin; Cater, Jacqueline R; Galante, Raymond J; Ward, Stephen E; Avadhani, Narayan G; Pack, Allan I

    2005-01-01

    Increased mRNA level of subunit 1 cytochrome c oxidase (COXI) during wakefulness and after short-term sleep deprivation has been described in brain. We hypothesized that this might contribute to increased activity of cytochrome oxidase (COX) enzyme during wakefulness, as part of the mechanisms to provide sufficient amounts of adenosine triphosphate to meet increased neuronal energy demands. COX activity was measured in isolated mitochondria from different brain regions in groups of rats with 3 hours of spontaneous sleep, 3 hours of spontaneous wake, and 3 hours of sleep deprivation. The group with 3 hours of spontaneous wake was added to delineate the circadian component of changes in the enzyme activity. Northern blot analysis was performed to examine the mRNA levels of 2 subunits of the enzyme COXI and COXIV, encoded by mitochondrial and nuclear DNA, respectively. Laboratory of Biochemistry, Department of Animal Biology, and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania. 2-month-old male Fischer rats (N = 21) implanted for polygraphic recording. For COX activity, there was a main effect by analysis of variance of experimental group (P sleep-deprived groups as compared to the sleep group. A main effect of brain region was also significant (P sleep. There is an increase in COX activity after both 3 hours of spontaneous wake and 3 hours of sleep deprivation as compared with 3 hours of spontaneous sleep in diverse brain regions, which could be, in part, explained by the increased levels of bigenomic transcripts of the enzyme. This likely contributes to increased adenosine triphosphate production during wakefulness. ADP, adenosine diphosphate; ATP, adenosine triphosphate; COXI, cytochrome c oxidase subunit 1 mRNA; COX, cytochrome c oxidase (protein); CREB, cyclic AMP response element binding protein; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; EEG, electroencephalography; EMG, electromyography; GABP, GA binding

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

    Science.gov (United States)

    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...

  17. l-Amino acid oxidase isolated from Calloselasma rhodostoma snake venom induces cytotoxicity and apoptosis in JAK2V617F-positive cell lines

    Directory of Open Access Journals (Sweden)

    Cristiane Tavares

    2016-06-01

    Full Text Available ABSTRACT BACKGROUND: Myeloproliferative neoplasms are Philadelphia chromosome-negative diseases characterized by hyperproliferation of mature myeloid cells, associated or not with the Janus kinase 2 tyrosine kinase mutation, JAK2V617F. As there is no curative therapy, researchers have been investigating new drugs to treat myeloproliferative neoplasms, including l-amino acid oxidase from Calloselasma rhodostoma snake venom (CR-LAAO, which is a toxin capable of eliciting apoptosis in several tumor cell lines. OBJECTIVE: To evaluate the effects of l-amino acid oxidase from C. rhodostoma snake venom in the apoptotic machinery of JAK2-mutated cell lines. METHODS: The HEL 92.1.7 and SET-2 cell lines were cultured with l-amino acid oxidase and catalase for 12 h at 37 °C in 5% carbon dioxide. The cell viability was assessed by the multi-table tournament method, the level of apoptosis was measured by flow cytometry, and the expression of cysteine-dependent aspartate-specific proteases and cleaved Poly(ADP-ribose polymerase were analyzed by Western blotting. RESULTS: l-Amino acid oxidase from C. rhodostoma snake venom was cytotoxic to HEL 92.1.7 and SET-2 cells (50% inhibitory concentration = 0.15 µg/mL and 1.5 µg/mL, respectively and induced apoptosis in a concentration-dependent manner. Cell treatment with catalase mitigated the l-amino acid oxidase toxicity, indicating that hydrogen peroxide is a key component of its cytotoxic effect.The activated caspases 3 and 8 expression and cleaved PARP in HEL 92.1.7 and SET-2 cells confirmed the apoptosis activation by CR-LAAO. CONCLUSIONS: l-Amino acid oxidase from C. rhodostoma snake venom is a potential antineoplastic agent against HEL 92.1.7 and SET-2 JAK2V617F-positive cells as it activates the extrinsic apoptosis pathway.

  18. Activity and functional interaction of alternative oxidase and uncoupling protein in mitochondria from tomato fruit

    Directory of Open Access Journals (Sweden)

    F.E. Sluse

    2000-03-01

    Full Text Available Cyanide-resistant alternative oxidase (AOX is not limited to plant mitochondria and is widespread among several types of protists. The uncoupling protein (UCP is much more widespread than previously believed, not only in tissues of higher animals but also in plants and in an amoeboid protozoan. The redox energy-dissipating pathway (AOX and the proton electrochemical gradient energy-dissipating pathway (UCP lead to the same final effect, i.e., a decrease in ATP synthesis and an increase in heat production. Studies with green tomato fruit mitochondria show that both proteins are present simultaneously in the membrane. This raises the question of a specific physiological role for each energy-dissipating system and of a possible functional connection between them (shared regulation. Linoleic acid, an abundant free fatty acid in plants which activates UCP, strongly inhibits cyanide-resistant respiration mediated by AOX. Moreover, studies of the evolution of AOX and UCP protein expression and of their activities during post-harvest ripening of tomato fruit show that AOX and plant UCP work sequentially: AOX activity decreases in early post-growing stages and UCP activity is decreased in late ripening stages. Electron partitioning between the alternative oxidase and the cytochrome pathway as well as H+ gradient partitioning between ATP synthase and UCP can be evaluated by the ADP/O method. This method facilitates description of the kinetics of energy-dissipating pathways and of ATP synthase when state 3 respiration is decreased by limitation of oxidizable substrate.

  19. Interaction of a snake venom L-amino acid oxidase with different cell types membrane.

    Science.gov (United States)

    Abdelkafi-Koubaa, Zaineb; Aissa, Imen; Morjen, Maram; Kharrat, Nadia; El Ayeb, Mohamed; Gargouri, Youssef; Srairi-Abid, Najet; Marrakchi, Naziha

    2016-01-01

    Snake venom l-amino acid oxidases are multifunctional enzymes that exhibited a wide range of pharmacological activities. Although it has been established that these activities are primarily caused by the H2O2 generated in the enzymatic reaction, the molecular mechanism, however, has not been fully investigated. In this work, LAAO interaction with cytoplasmic membranes using different cell types and Langmuir interfacial monolayers was evaluated. The Cerastes cerastes venom LAAO (CC-LAAO) did not exhibit cytotoxic activities against erythrocytes and peripheral blood mononuclear cells (PBMC). However, CC-LAAO caused cytotoxicity on several cancer cell lines and induced platelet aggregation in dose-dependent manner. Furthermore, the enzyme showed remarkable effect against Gram-positive and Gram-negative bacteria. These activities were inhibited on the addition of catalase or substrate analogs, suggesting that H2O2 liberation× is required for these effects. Binding studies revealed that CC-LAAO binds to the cell surface and enables the production of highly localized concentration of H2O2 in or near the binding interfaces. On another hand, the interaction of CC-LAAO with a mimetic phospholipid film was evaluated, for the first time, using a monomolecular film technique. Results indicated that phospholipid/CC-LAAO interactions are not involved in their binding to membrane and in their pharmacological activities. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. A study of monoamine oxidase activity in fetal membranes.

    Science.gov (United States)

    Sekizawa, A; Ishikawa, H; Morimoto, T; Hirose, K; Suzuki, A; Saito, H; Yanaihara, T; Arai, Y; Oguchi, K

    1996-05-01

    To study the role of decidual monoamine oxidase (MAO)-A and -B activities before delivery, the relationship between MAO activity in fetal membranes and catecholamine (CA) concentration in amniotic fluid (AF) was determined. Fetal membranes and AF were obtained at the time of elective Cesarean section (CS group, n = 11) and Cesarean section due to fetal distress without labor pains (FD group, n = 5). MAO-A and -B activities were radiometrically measured using 14C-5-hydroxytriptamine for MAO-A substrate and 14C-benzylamine for MAO-B substrate. CA concentrations in AF were measured by high performance liquid chromatograph with an electro-chemical detector. Both MAO-A and -B activities in decidua obtained from CS were significantly lower than those obtained from FD. Both norepinephrine (NE) and epinephrine (EP) concentrations were significantly lower in the CS group than the FD group. A significant positive correlation between decidual MAO-A activity and NE concentration in AF was observed. No significant correlation was observed between MAO-B activity and the concentration of NE in AF. There was no correlation between EP concentrations and MAO activities. These results suggest that CA concentration in AF may be related to the activity of MAO in fetal membranes, determined by certain physiological processes during pregnancy. It has been suggested that metabolism of monoamines in fetal membranes also plays an important role in reducing monoamine influx into maternal myometrium from the AF.

  1. Feasibility of converting lactic acid to ethanol in food waste fermentation by immobilized lactate oxidase

    International Nuclear Information System (INIS)

    Ma, Hong-zhi; Xing, Yi; Yu, Miao; Wang, Qunhui

    2014-01-01

    Highlights: • Residue lactic acid in food waste could be converted to pyruvic acid. • Calcium alginate immobilized the lactate oxidase with high pH and thermal stability. • Immobilized enzyme could convert 70% lactic acid to pyruvic acid. • Ethanol yield could be increased by 20% with lactate oxidase added. - Abstract: Adoption of lactic acid bacteria (LAB) into ethanol fermentation from food waste can replace the sterilization process. However, LAB inoculation will convert part of the substrate into lactic acid (LA), not ethanol. This study adopted lactate oxidase to convert the produced LA to pyruvate, and then ethanol fermentation was carried out. The immobilization enzyme was utilized, and corresponding optimum conditions were determined. Results showed that calcium alginate could successfully immobilize the enzyme and improve pH and thermal stability. The optimum pH and temperature were 6.2 and 55 °C, respectively. The utilization of immobilized enzyme with catalytic time of 5 h could convert 70% LA to pyruvate, and the addition of enzyme increased the ethanol yield by 20% more than that of the control. The process could be applied in food waste storage and can help in reducing carbon source consumption

  2. Magnetic field effects on brain monoamine oxidase activity

    Energy Technology Data Exchange (ETDEWEB)

    Borets, V.M.; Ostrovskiy, V.Yu.; Bankovskiy, A.A.; Dudinskaya, T.F.

    1985-03-01

    In view of the increasing use of magnetotherapy, studies were conducted on the effects of 35 mTesla magnetic fields on monoamine oxidase activity in the rat brain. Under in vitro conditions a constant magnetic field in the continuous mode was most effective in inhibiting deamination of dopamine following 1 min exposure, while in vivo studies with 8 min or 10 day exposures showed that inhibition was obtained only with a variable field in the continuous mode. However, inhibition of dopamine deamination was only evident within the first 24 h after exposure was terminated. In addition, in none of the cases was norepinephrine deamination inhibited. The effects of the magnetic fields were, therefore, transient and selective with the CNS as the target system. 9 references.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-04-01

    A series of 2-(substituted benzylamino)-4-methylthiazole-5-carboxylic acid was designed and synthesized as structural analogue of febuxostat. A methylene amine spacer was incorporated between the phenyl ring and thiazole ring in contrast to febuxostat in which the phenyl ring was directly linked with the thiazole moiety. The purpose of incorporating methylene amine was to provide a heteroatom which is expected to favour hydrogen bonding within the active site residues of the enzyme xanthine oxidase. The structure of all the compounds was established by the combined use of FT-IR, NMR and MS spectral data. All the compounds were screened in vitro for their ability to inhibit the enzyme xanthine oxidase as per the reported procedure along with DPPH free radical scavenging assay. Compounds 5j, 5k and 5l demonstrated satisfactory potent xanthine oxidase inhibitory activities with IC50 values, 3.6, 8.1 and 9.9 μm, respectively, whereas compounds 5k, 5n and 5p demonstrated moderate antioxidant activities having IC50 15.3, 17.6 and 19.6 μm, respectively, along with xanthine oxidase inhibitory activity. Compound 5k showed moderate xanthine oxidase inhibitory activity as compared with febuxostat along with antioxidant activity. All the compounds were also studied for their binding affinity in active site of enzyme (PDB ID-1N5X). © 2015 John Wiley & Sons A/S.

  5. [Experimental rationale for the parameters of a rapid method for oxidase activity determination].

    Science.gov (United States)

    Butorina, N N

    2010-01-01

    Experimental rationale is provided for the parameters of a rapid (1-2-min) test to concurrently determine the oxidase activity of all bacteria grown on the membrane filter after water filtration. Oxidase reagents that are the aqueous solutions of tetramethyl-p-phenylenediamine dihydrochloride and demethyl-p-phenylenediamine dihydrochloride have been first ascertained to exert no effect on the viability and enzymatic activity of bacteria after one-hour contact. An algorithm has been improved for the rapid oxidase activity test: the allowable time for bacteria to contact oxidase reagents and procedures for minimizing the effect on bacterial biochemical activity following the contact. An accelerated method based on lactose medium with tergitol 7 and Endo agar has been devised to determine coliform bacteria, by applying the rapid oxidase test: the time of a final response is 18-24 hours. The method has been included into GOST 52426-2005.

  6. Effect of temperature stress on polyphenol oxidase activity in grains of some wheat cultivars

    International Nuclear Information System (INIS)

    Kayani, W.K.

    2011-01-01

    Color is a key quality trait of wheat-based products and polyphenol oxidase (PPO) is implicated to play a significant role in their undesirable darkening. Polyphenol oxidase catalyzes the oxidation of phenols to quinines, which auto oxidize and polymerize with amino acid of cellular proteins resulting brown and black pigmentation propounding reduced nutritional values. In present study, the PPO activity in 50 different Pakistani wheat cultivars was investigated and grouped into three categories viz; low, medium and high PPO activity cultivars. PPO is a heat labile enzyme. To investigate effect of heat stress, nine cultivars from each category were chosen for treatment at 30, 40 and 50 deg. C for 30, 60, and 120 minutes each. A substantial change was experienced in PPO activity as compared to room temperature. Two wheat cultivar Wafaq-2001 and AS-2002 showed a compromising attitude of minimum PPO activity at 30 deg. C for a period of 30 and 60 minutes of incubation. In general, an incubation of 30 deg. C or 60 deg. C (low or high) for a period of 30 minutes can be recommended for suppressing PPO activity. (author)

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

    Science.gov (United States)

    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.

  8. Electrochemical L-Lactic Acid Sensor Based on Immobilized ZnO Nanorods with Lactate Oxidase

    Directory of Open Access Journals (Sweden)

    Kimleang Khun

    2012-02-01

    Full Text Available 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 × 100 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.

  9. Rac1-NADPH oxidase signaling promotes CD36 activation under glucotoxic conditions in pancreatic beta cells.

    Science.gov (United States)

    Elumalai, Suma; Karunakaran, Udayakumar; Lee, In Kyu; Moon, Jun Sung; Won, Kyu Chang

    2017-04-01

    We recently reported that cluster determinant 36 (CD36), a fatty acid transporter, plays a pivotal role in glucotoxicity-induced β-cell dysfunction. However, little is known about how glucotoxicity influences CD36 expression. Emerging evidence suggests that the small GTPase Rac1 is involved in the pathogenesis of beta cell dysfunction in type 2 diabetes (T2D). The primary objective of the current study was to determine the role of Rac1 in CD36 activation and its impact on β-cell dysfunction in diabetes mellitus. To address this question, we subjected INS-1 cells and human beta cells (1.1B4) to high glucose conditions (30mM) in the presence or absence of Rac1 inhibition either by NSC23766 (Rac1 GTPase inhibitor) or small interfering RNA. High glucose exposure in INS-1 and human beta cells (1.1b4) resulted in the activation of Rac1 and induced cell apoptosis. Rac1 activation mediates NADPH oxidase (NOX) activation leading to elevated ROS production in both cells. Activation of the Rac1-NOX complex by high glucose levels enhanced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. The inhibition of Rac1 by NSC23766 inhibited NADPH oxidase activity and ROS generation induced by high glucose concentrations in INS-1 & human 1.1b4 beta cells. Inhibition of Rac1-NOX complex activation by NSC23766 significantly reduced CD36 expression in INS-1 and human 1.1b4 beta cell membrane fractions. In addition, Rac1 inhibition by NSC23766 significantly reduced high glucose-induced mitochondrial dysfunction. Furthermore, NADPH oxidase inhibition by VAS2870 also attenuated high glucose-induced ROS generation and cell apoptosis. These results suggest that Rac1-NADPH oxidase dependent CD36 expression contributes to high glucose-induced beta cell dysfunction and cell death. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Thermal stability of L-ascorbic acid and ascorbic acid oxidase in broccoli (Brassica oleracea var. italica).

    Science.gov (United States)

    Munyaka, Ann Wambui; Makule, Edna Edward; Oey, Indrawati; Van Loey, Ann; Hendrickx, Marc

    2010-05-01

    The thermal stability of vitamin C (including l-ascorbic acid [l-AA] and dehydroascorbic acid [DHAA]) in crushed broccoli was evaluated in the temperature range of 30 to 90 degrees C whereas that of ascorbic acid oxidase (AAO) was evaluated in the temperature range of 20 to 95 degrees C. Thermal treatments (for 15 min) of crushed broccoli at 30 to 60 degrees C resulted in conversion of l-AA to DHAA whereas treatments at 70 to 90 degrees C retained vitamin C as l-AA. These observations indicated that enzymes (for example, AAO) could play a major role in the initial phase (that is, oxidation of l-AA to DHAA) of vitamin C degradation in broccoli. Consequently, a study to evaluate the temperature-time conditions that could result in AAO inactivation in broccoli was carried out. In this study, higher AAO activity was observed in broccoli florets than stalks. During thermal treatments for 10 min, AAO in broccoli florets and stalks was stable until around 50 degrees C. A 10-min thermal treatment at 80 degrees C almost completely inactivated AAO in broccoli. AAO inactivation followed 1st order kinetics in the temperature range of 55 to 65 degrees C. Based on this study, a thermal treatment above 70 degrees C is recommended for crushed vegetable products to prevent oxidation of l-AA to DHAA, the onset of vitamin C degradation. The results reported in this study are applicable for both domestic and industrial processing of vegetables into products such as juices, soups, and purees. In this report, we have demonstrated that processing crushed broccoli in a temperature range of 30 to 60 degrees C could result in the conversion of l-ascorbic acid to dehydroascorbic (DHAA), a very important reaction in regard to vitamin C degradation because DHAA could be easily converted to other compounds that do not have the biological activity of vitamin C.

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

    Directory of Open Access Journals (Sweden)

    Kuo-Chuan Ho

    2009-03-01

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

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

    Science.gov (United States)

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

    2009-01-01

    In this study, use of the covalent enzyme immobilization method was proposed to attach cholesterol oxidase (ChO) on a conducting polymer, poly(3-thiopheneacetic acid), [poly(3-TPAA)]. Three red-orange poly(3-TPAA) films, named electrodes A, B and C, were electropolymerized on a platinum electrode by applying a constant current of 1.5 mA, for 5, 20 and 100 s, respectively. Further, 1-ethyl-3-(3-dimethylamiopropyl)carbodiimide hydrochloride (EDC · HCl) and N-hydroxysuccinimide (NHS) were used to activate the free carboxylic groups of the conducting polymer. Afterwards, the amino groups of the cholesterol oxidase were linked on the activated groups to form peptide bonds. The best sensitivity obtained for electrode B is 4.49 mA M(-1) cm(-2), with a linear concentration ranging from 0 to 8 mM, which is suitable for the analysis of cholesterol in humans. The response time (t(95)) is between 70 and 90 s and the limit of detection is 0.42 mM, based on the signal to noise ratio equal to 3. The interference of species such as ascorbic acid and uric acid increased to 5.2 and 10.3% of the original current response, respectively, based on the current response of cholesterol (100%). With respect to the long-term stability, the sensing response retains 88% of the original current after 13 days.

  13. Amine oxidase from lentil seedlings: energetic domains and effect of temperature on activity.

    Science.gov (United States)

    Moosavi-Nejad, S Z; Rezaei-Tavirani, M; Padiglia, A; Floris, G; Moosavi-Movahedi, A A

    2001-07-01

    Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).

  14. Salicylic Acid Regulation of Respiration in Higher Plants: Alternative Oxidase Expression.

    Science.gov (United States)

    Rhoads, DM; McIntosh, L

    1992-01-01

    Alternative respiratory pathway capacity increases during the development of the thermogenic appendix of a voodoo lily inflorescence. The levels of the alternative oxidase proteins increased dramatically between D-4 (4 days prior to the day of anthesis) and D-3 and continued to increase until the day of anthesis (D-day). The level of salicylic acid (SA) in the appendix is very low early on D-1, but increases to a high level in the evening of D-1. Thermogenesis occurs after a few hours of light on D-day. Therefore, the initial accumulation of the alternative oxidase proteins precedes the increase in SA by 3 days, indicating that other regulators may be involved. A 1.6-kb transcript encoding the alternative oxidase precursor protein accumulated to a high level in the appendix tissue by D-1. Application of SA to immature appendix tissue caused an increase in alternative pathway capacity and a dramatic accumulation of the alternative oxidase proteins and the 1.6-kb transcript. Time course experiments showed that the increase in capacity, protein levels, and transcript level corresponded precisely. The response to SA was blocked by cycloheximide or actinomycin D, indicating that de novo transcription and translation are required. However, nuclear, in vitro transcription assays indicated that the accumulation of the 1.6-kb transcript did not result from a simple increase in the rate of transcription of aox1. PMID:12297672

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

    Science.gov (United States)

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

    2016-02-01

    Fasciolosis is an important cattle and human disease caused by Fasciola hepatica and Fasciola gigantica. One of the possible methods to control this problem is to interrupt the life cycle of Fasciola by killing its larva (redia and cercaria) in host snail. Molecular identification of cercaria larva of F. gigantica was done by comparing the nucleotide sequencing with adult F. gigantica. It was noted that nucleotide sequencing of cercaria larva and adult F. gigantica were 99% same. Every month during the year 2011-2012, in vivo treatment with 60% of 4 h LC50 of phyto cercaricides citral, ferulic acid, umbelliferone, azadirachtin and allicin caused significant inhibition of acetylcholinesterase (AChE) and cytochrome oxidase activity in the treated cercaria larva of F. gigantica. Whereas, activity of both enzymes were not significantly altered in the nervous tissues of vector snail Lymnaea acuminata exposed to same treatments. Maximum reduction in AChE (1.35% of control in month of June) and cytochrome oxidase (3.71% of control in the month of July) activity were noted in the cercaria exposed to 60% of 4 h LC50 of azadirachtin and allicin, respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2009-12-01

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

  17. Neutrophils to the ROScue: Mechanisms of NADPH Oxidase Activation and Bacterial Resistance

    Directory of Open Access Journals (Sweden)

    Giang T. Nguyen

    2017-08-01

    Full Text Available Reactive oxygen species (ROS generated by NADPH oxidase play an important role in antimicrobial host defense and inflammation. Their deficiency in humans results in recurrent and severe bacterial infections, while their unregulated release leads to pathology from excessive inflammation. The release of high concentrations of ROS aids in clearance of invading bacteria. Localization of ROS release to phagosomes containing pathogens limits tissue damage. Host immune cells, like neutrophils, also known as PMNs, will release large amounts of ROS at the site of infection following the activation of surface receptors. The binding of ligands to G-protein-coupled receptors (GPCRs, toll-like receptors, and cytokine receptors can prime PMNs for a more robust response if additional signals are encountered. Meanwhile, activation of Fc and integrin directly induces high levels of ROS production. Additionally, GPCRs that bind to the bacterial-peptide analog fMLP, a neutrophil chemoattractant, can both prime cells and trigger low levels of ROS production. Engagement of these receptors initiates intracellular signaling pathways, resulting in activation of downstream effector proteins, assembly of the NADPH oxidase complex, and ultimately, the production of ROS by this complex. Within PMNs, ROS released by the NADPH oxidase complex can activate granular proteases and induce the formation of neutrophil extracellular traps (NETs. Additionally, ROS can cross the membranes of bacterial pathogens and damage their nucleic acids, proteins, and cell membranes. Consequently, in order to establish infections, bacterial pathogens employ various strategies to prevent restriction by PMN-derived ROS or downstream consequences of ROS production. Some pathogens are able to directly prevent the oxidative burst of phagocytes using secreted effector proteins or toxins that interfere with translocation of the NADPH oxidase complex or signaling pathways needed for its activation

  18. Functional analysis of fructosyl-amino acid oxidases of Aspergillus oryzae.

    Science.gov (United States)

    Akazawa, Shin-Ichi; Karino, Tetsuya; Yoshida, Nobuyuki; Katsuragi, Tohoru; Tani, Yoshiki

    2004-10-01

    Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from Aspergillus oryzae strain RIB40. N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein. FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward N(epsilon)-fructosyl N(alpha)-Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val. The genes encoding the FAOD isozymes (i.e., FAOAo1 and FAOAo2) were cloned by PCR with an FAOD-specific primer set. The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes. The genes was expressed in Escherichia coli and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active. FAOAo2 disruptant was obtained by using ptrA as a selective marker. Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain Delta faoAo2 did not grow. Addition of glucose or (NH(4))(2)SO(4) to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the FAOAo2 gene. Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO(2) as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase. These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in A. oryzae.

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

    Science.gov (United States)

    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.

  20. A structural and functional model for the 1-aminocyclopropane-1-carboxylic acid oxidase.

    Science.gov (United States)

    Sallmann, Madleen; Oldenburg, Fabio; Braun, Beatrice; Réglier, Marius; Simaan, A Jalila; Limberg, Christian

    2015-10-12

    The hitherto most realistic low-molecular-weight analogue for the 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) is reported. The ACCOs 2-His-1-carboxylate iron(II) active site was mimicked by a TpFe moiety, to which the natural substrate ACC could be bound. The resulting complex [Tp(Me,Ph) FeACC] (1), according to X-ray diffraction analysis performed for the nickel analogue, represents an excellent structural model, featuring ACC coordinated in a bidentate fashion-as proposed for the enzymatic substrate complex-as well as a vacant coordination site that forms the basis for the first successful replication also of the ACCO function: 1 is the first known ACC complex that reacts with O2 to produce ethylene. As a FeOOH species had been suggested as intermediate in the catalytic cycle, H2 O2 was tested as the oxidant, too, and indeed evolution of ethylene proceeded even more rapidly to give 65 % yield. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Characterization of active site residues of nitroalkane oxidase.

    Science.gov (United States)

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

    2010-06-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. 2009 Elsevier Inc. All rights reserved.

  2. Periodic variation in bile acids controls circadian changes in uric acid via regulation of xanthine oxidase by the orphan nuclear receptor PPARα.

    Science.gov (United States)

    Kanemitsu, Takumi; Tsurudome, Yuya; Kusunose, Naoki; Oda, Masayuki; Matsunaga, Naoya; Koyanagi, Satoru; Ohdo, Shigehiro

    2017-12-29

    Xanthine oxidase (XOD), also known as xanthine dehydrogenase, is a rate-limiting enzyme in purine nucleotide degradation, which produces uric acid. Uric acid concentrations in the blood and liver exhibit circadian oscillations in both humans and rodents; however, the underlying mechanisms remain unclear. Here, we demonstrate that XOD expression and enzymatic activity exhibit circadian oscillations in the mouse liver. We found that the orphan nuclear receptor peroxisome proliferator-activated receptor-α (PPARα) transcriptionally activated the mouse XOD gene and that bile acids suppressed XOD transactivation. The synthesis of bile acids is known to be under the control of the circadian clock, and we observed that the time-dependent accumulation of bile acids in hepatic cells interfered with the recruitment of the co-transcriptional activator p300 to PPARα, thereby repressing XOD expression. This time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the hepatic expression of XOD, which, in turn, led to circadian alterations in uric acid production. Finally, we also demonstrated that the anti-hyperuricemic effect of the XOD inhibitor febuxostat was enhanced by administering it at the time of day before hepatic XOD activity increased. These results suggest an underlying mechanism for the circadian alterations in uric acid production and also underscore the importance of selecting an appropriate time of day for administering XOD inhibitors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase.

    Science.gov (United States)

    White, Caroline N; Figtree, Gemma A; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Rasmussen, Helge H

    2009-04-01

    The sarcolemmal Na(+)-K(+) pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to epsilon-protein kinase C (epsilonPKC), previously implicated in ANG II-induced Na(+)-K(+) pump inhibition. A role for epsilonPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of epsilonPKC with the receptor for the activated kinase and with the cytosolic p47(phox) subunit of NADPH oxidase. ANG II decreased electrogenic Na(+)-K(+) pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by epsilonPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na(+)-K(+) pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The alpha(1)-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22(phox) and cytosolic p47(phox) NADPH oxidase subunits at baseline. ANG II had no effect on alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na(+)-K(+) pump via PKC-dependent NADPH oxidase activation.

  4. Plasma amine oxidase activities in Norrie disease patients with an X-chromosomal deletion affecting monoamine oxidase.

    Science.gov (United States)

    Murphy, D L; Sims, K B; Karoum, F; Garrick, N A; de la Chapelle, A; Sankila, E M; Norio, R; Breakefield, X O

    1991-01-01

    Two individuals with an X-chromosomal deletion were recently found to lack the genes encoding monoamine oxidase type A (MAO-A) and MAO-B. This abnormality was associated with almost total (90%) reductions in the oxidatively deaminated urinary metabolites of the MAO-A substrate, norepinephrine, and with marked (100-fold) increases in an MAO-B substrate, phenylethylamine, confirming systemic functional consequences of the genetic enzyme deficiency. However, urinary concentrations of the deaminated metabolites of dopamine and serotonin (5-HT) were essentially normal. To investigate other deaminating systems besides MAO-A and MAO-B that might produce these metabolites of dopamine and 5-HT, we examined plasma amine oxidase (AO) activity in these two patients and two additional patients with the same X-chromosomal deletion. Normal plasma AO activity was found in all four Norrie disease-deletion patients, in four patients with classic Norrie disease without a chromosomal deletion, and in family members of patients from both groups. Marked plasma amine metabolite abnormalities and essentially absent platelet MAO-B activity were found in all four Norrie disease-deletion patients, but in none of the other subjects in the two comparison groups. These results indicate that plasma AO is encoded by gene(s) independent of those for MAO-A and MAO-B, and raise the possibility that plasma AO, and perhaps the closely related tissue AO, benzylamine oxidase, as well as other atypical AOs or MAOs encoded independently from MAO-A and MAO-B may contribute to the oxidative deamination of dopamine and 5-HT in humans.

  5. Fluorescent Probes for Analysis and Imaging of Monoamine Oxidase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dokyoung; Jun, Yong Woong; Ahn, Kyo Han [POSTECH, Pohang (Korea, Republic of)

    2014-05-15

    Monoamine oxidases catalyze the oxidative deamination of dietary amines and amine neurotransmitters, and assist in maintaining the homeostasis of the amine neurotransmitters in the brain. Dysfunctions of these enzymes can cause neurological and behavioral disorders including Parkinson's and Alzheimer's diseases. To understand their physiological roles, efficient assay methods for monoamine oxidases are essential. Reviewed in this Perspective are the recent progress in the development of fluorescent probes for monoamine oxidases and their applications to enzyme assays in cells and tissues. It is evident that still there is strong need for a fluorescent probe with desirable substrate selectivity and photophysical properties to challenge the much unsolved issues associated with the enzymes and the diseases.

  6. Cloning, expression, and enzymatic activity evaluation of cholesterol oxidase gene isolated from a native Rhodococcus sp.

    Directory of Open Access Journals (Sweden)

    Hamed Esmaeil Lashgarian

    2016-10-01

    Full Text Available Cholesterol oxidase (CHO is one of the valuable enzymes that play an important role in: measurement of serum cholesterol, food industry as a biocatalyst and agriculture as a biological larvicide. This enzyme was produced by several bacterial strains. Wild type enzyme produced by Rhodococcus sp. secret two forms of CHO enzyme: extra cellular and membrane bound type which its amount is low and unstable. The goal of the study was cloning, expression, and enzymatic activity evaluation of cholesterol oxidase gene isolated from a native Rhodococcus sp. CHO gene was isolated from native bacteria and cloned into pET23a. In the next step, the construct was expressed in E.coli BL21 and induced by different concentration of IPTG ranges from 0.1 - 0.9 mM. This gene contains 1642 bp and encodes a protein consists of 533 amino acids. It has about 96 % homology with CHO gene isolated from Rhodococcus equi. The high expression was obtained in 0.5 mM concentration of IPTG after 4 hour induction. This recombinant enzyme had a molecular weight of 55 kDa, that secretion of intra cellular type is much more than extracellular form. The optimum pH and temperature conditions for the recombinant enzyme were 7.5 and 45°C, respectively. CHO enzyme obtained from Rhodococcus sp. is a cheap enzyme with medical and industrial applications that can be produced easily and purified in large scale with simple methods.

  7. Metavanadate causes cellular accumulation of copper and decreased lysyl oxidase activity

    International Nuclear Information System (INIS)

    Cui, Changtai T.; Uriu-Adams, Janet Y.; Tchaparian, Eskouhie H.; Keen, Carl L.; Rucker, Robert B.

    2004-01-01

    Selected indices of copper metabolism in weanling rats and fibroblast cultures were progressively altered in response to increased levels of sodium metavanadate. In diets, vanadium was added in amounts ranging from 0 to 80 μg V/g of diet, that is, 0-1.6 μmol V/g of diet. In fibroblast cultures, vanadium ranged from 0 to 400 nmol V/ml. The inhibition of P-ATPase-7A activity by metavanadate, important to copper egress from cells, was a primary focus. In skin, and tendon, the copper concentration was increased in response to increased dietary levels of metavanadate, whereas lysyl oxidase activity, a secreted cuproprotein, was reduced. The reduction in lysyl oxidase activity was also accompanied by reduced redox cycling potential of isolated fractions of lysyl oxidase, presumably due to reduced lysyltyrosyl quinone (LTQ) formation at the active site of lysyl oxidase. In contrast, liver copper concentrations and plasma ceruloplasmin activity were not affected by metavanadate exposure. However, semicarbazide-sensitive benzylamine oxidase (SCBO) activity, which was taken as an indirect measure of vascular adhesive protein-1 (VAP-1), was increased. In cultured fibroblasts, cellular copper was also increased and lysyl oxidase decreased in response to metavanadate. Moreover, the steady-state levels of atp7a and lysyl oxidase mRNAs were not affected by addition of metavanadate to culture medium up to 200 nmol/ml. Taken together, these data suggest that pathways involving copper egress and lysyl oxidase activation are particularly sensitive to metavanadate exposure through processes that are predominately posttranslational

  8. Localization of Glucose Oxidase and Catalase Activities in Aspergillus niger

    NARCIS (Netherlands)

    Witteveen, Cor F.B.; Veenhuis, Marten; Visser, Jaap

    The subcellular localization of glucose oxidase (EC 1.1.3.4) in Aspergillus niger N400 (CBS 120.49) was investigated by (immuno)cytochemical methods. By these methods, the bulk of the enzyme was found to be localized in the cell wall. In addition, four different catalases (EC 1.11.1.6) were

  9. MipLAAO, a new L-amino acid oxidase from the redtail coral snake Micrurus mipartitus

    Directory of Open Access Journals (Sweden)

    Paola Rey-Suárez

    2018-06-01

    Full Text Available L-amino acid oxidases (LAAOs are ubiquitous enzymes in nature. Bioactivities described for these enzymes include apoptosis induction, edema formation, induction or inhibition of platelet aggregation, as well as antiviral, antiparasite, and antibacterial actions. With over 80 species, Micrurus snakes are the representatives of the Elapidae family in the New World. Although LAAOs in Micrurus venoms have been predicted by venom gland transcriptomic studies and detected in proteomic studies, no enzymes of this kind have been previously purified from their venoms. Earlier proteomic studies revealed that the venom of M. mipartitus from Colombia contains ∼4% of LAAO. This enzyme, here named MipLAAO, was isolated and biochemically and functionally characterized. The enzyme is found in monomeric form, with an isotope-averaged molecular mass of 59,100.6 Da, as determined by MALDI-TOF. Its oxidase activity shows substrate preference for hydrophobic amino acids, being optimal at pH 8.0. By nucleotide sequencing of venom gland cDNA of mRNA transcripts obtained from a single snake, six isoforms of MipLAAO with minor variations among them were retrieved. The deduced sequences present a mature chain of 483 amino acids, with a predicted pI of 8.9, and theoretical masses between 55,010.9 and 55,121.0 Da. The difference with experimentally observed mass is likely due to glycosylation, in agreement with the finding of three putative N-glycosylation sites in its amino acid sequence. A phylogenetic analysis of MmipLAAO placed this new enzyme within the clade of homologous proteins from elapid snakes, characterized by the conserved Serine at position 223, in contrast to LAAOs from viperids. MmipLAAO showed a potent bactericidal effect on S. aureus (MIC: 2 µg/mL, but not on E. coli. The former activity could be of interest to future studies assessing its potential as antimicrobial agent.

  10. [The X+ chronic granulomatous disease as a fabulous model to study the NADPH oxidase complex activation].

    Science.gov (United States)

    Stasia, Marie-José

    2007-05-01

    Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation.

  11. Quinolinic Carboxylic Acid Derivatives as Potential Multi-target Compounds for Neurodegeneration: Monoamine Oxidase and Cholinesterase Inhibition.

    Science.gov (United States)

    Khan, Nehal A; Khan, Imtiaz; Abid, Syed M A; Zaib, Sumera; Ibrar, Aliya; Andleeb, Hina; Hameed, Shahid; Iqbal, Jamshed

    2018-01-01

    Parkinson's disease (PD), a debilitating and progressive disorder, is among the most challenging and devastating neurodegenerative diseases predominantly affecting the people over 60 years of age. To confront PD, an advanced and operational strategy is to design single chemical functionality able to control more than one target instantaneously. In this endeavor, for the exploration of new and efficient inhibitors of Parkinson's disease, we synthesized a series of quinoline carboxylic acids (3a-j) and evaluated their in vitro monoamine oxidase and cholinesterase inhibitory activities. The molecular docking and in silico studies of the most potent inhibitors were performed to identify the probable binding modes in the active site of the monoamine oxidase enzymes. Moreover, molecular properties were calculated to evaluate the druglikeness of the compounds. The biological evaluation results revealed that the tested compounds were highly potent against monoamine oxidase (A & B), 3c targeted both the isoforms of MAO with IC50 values of 0.51 ± 0.12 and 0.51 ± 0.03 µM, respectively. The tested compounds also demonstrated high and completely selective inhibitory action against acetylcholinesterase (AChE) with IC50 values ranging from 4.36 to 89.24 µM. Among the examined derivatives, 3i was recognized as the most potent inhibitor of AChE with an IC50 value of 4.36 ± 0.12 ±µM. The compounds appear to be promising inhibitors and could be used for the future development of drugs targeting neurodegenerative disorders. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    Science.gov (United States)

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

    2015-11-01

    Chrysin, a bioactive flavonoid, was investigated for its potential to inhibit the activity of xanthine oxidase (XO), a key enzyme catalyzing xanthine to uric acid and finally causing gout. The kinetic analysis showed that chrysin possessed a strong inhibition on XO ability in a reversible competitive manner with IC50 value of (1.26±0.04)×10(-6)molL(-1). The results of fluorescence titrations indicated that chrysin bound to XO with high affinity, and the interaction was predominately driven by hydrogen bonds and van der Waals forces. Analysis of circular dichroism demonstrated that chrysin induced the conformational change of XO with increases in α-helix and β-sheet and reductions in β-turn and random coil structures. Molecular simulation revealed that chrysin interacted with the amino acid residues Leu648, Phe649, Glu802, Leu873, Ser876, Glu879, Arg880, Phe1009, Thr1010, Val1011 and Phe1013 located within the active cavity of XO. The mechanism of chrysin on XO activity may be the insertion of chrysin into the active site occupying the catalytic center of XO to avoid the entrance of xanthine and causing conformational changes in XO. Furthermore, the interaction assays indicated that chrysin and its structural analog apigenin exhibited an additive effect on inhibition of XO. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Antioxidant, xanthine oxidase and lipoxygenase inhibitory activities and phenolics of Bauhinia rufescens Lam. (Caesalpiniaceae).

    Science.gov (United States)

    Compaoré, M; Lamien, C E; Lamien-Meda, A; Vlase, L; Kiendrebeogo, M; Ionescu, C; Nacoulma, O G

    2012-01-01

    An aqueous acetone extract of the stem with the leaves of Bauhinia rufescens and its fractions were analysed for their antioxidant and enzyme-inhibitory activities, as well as their phytochemical composition. For measurement of the antioxidant activities, the 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azinobis(3-ethylbenzoline-6-sulphonate) and the ferric-reducing methods were used. The results indicated that the aqueous acetone, its ethyl acetate and n-butanol fractions possessed considerable antioxidant activity. Further, the xanthine oxidase and lipoxygenase inhibitory assays showed that the n-butanol fraction possessed compounds that can inhibit both these enzymes. In the phytochemical analysis, the ethyl acetate and the n-butanol fractions of the aqueous acetone extract were screened by HPLC-MS for their phenolic content. The results indicated the presence of hyperoside, isoquercitrin, rutin quercetin, quercitrin, p-coumaric and ferulic acids in the non-hydrolysed fractions. In the hydrolysed fractions, kaempferol, p-coumaric and ferulic acids were identified.

  14. NADPH oxidase 4-derived superoxide mediates flow-stimulated NKCC2 activity in thick ascending limbs.

    Science.gov (United States)

    Saez, Fara; Hong, Nancy J; Garvin, Jeffrey L

    2018-05-01

    Luminal flow augments Na + reabsorption in the thick ascending limb more than can be explained by increased ion delivery. This segment reabsorbs 30% of the filtered load of Na + , playing a key role in its homeostasis. Whether flow elevations enhance Na + -K + -2Cl - cotransporter (NKCC2) activity and the second messenger involved are unknown. We hypothesized that raising luminal flow augments NKCC2 activity by enhancing superoxide ([Formula: see text]) production by NADPH oxidase 4 (NOX4). NKCC2 activity was measured in thick ascending limbs perfused at either 5 or 20 nl/min with and without inhibitors of [Formula: see text] production. Raising luminal flow from 5 to 20 nl/min enhanced NKCC2 activity from 4.8 ± 0.9 to 6.3 ± 1.2 arbitrary fluorescent units (AFU)/s. Maintaining flow at 5 nl/min did not alter NKCC2 activity. The superoxide dismutase mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride blunted NKCC2 activity from 3.5 ± 0.4 to 2.5 ± 0.2 AFU/s when flow was 20 nl/min but not 5 nl/min. When flow was 20 nl/min, NKCC2 activity showed no change with time. The selective NOX1/4 inhibitor GKT-137831 blunted NKCC2 activity when thick ascending limbs were perfused at 20 nl/min from 7.2 ± 1.1 to 4.5 ± 0.8 AFU/s but not at 5 nl/min. The inhibitor also prevented luminal flow from elevating [Formula: see text] production. Allopurinol, a xanthine oxidase inhibitor, had no effect on NKCC2 activity when flow was 20 nl/min. Tetanus toxin prevents flow-induced stimulation of NKCC2 activity. We conclude that elevations in luminal flow enhance NaCl reabsorption in thick ascending limbs by stimulating NKCC2 via NOX4 activation and increased [Formula: see text]. NKCC2 activation is primarily the result of insertion of new transporters in the membrane.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Biochemical, biological and molecular characterization of an L-Amino acid oxidase (LAAO) purified from Bothrops pictus Peruvian snake venom.

    Science.gov (United States)

    Lazo, Fanny; Vivas-Ruiz, Dan E; Sandoval, Gustavo A; Rodríguez, Edith F; Kozlova, Edgar E G; Costal-Oliveira, F; Chávez-Olórtegui, Carlos; Severino, Ruperto; Yarlequé, Armando; Sanchez, Eladio F

    2017-12-01

    An L-amino acid oxidase from Peruvian Bothrops pictus (Bpic-LAAO) snake venom was purified using a combination of size-exclusion and ion-exchange chromatography. Bpic-LAAO is a homodimeric glycosylated flavoprotein with molecular mass of ∼65 kDa under reducing conditions and ∼132 kDa in its native form as analyzed by SDS-PAGE and gel filtration chromatography, respectively. N-terminal amino acid sequencing showed highly conserved residues in a glutamine-rich motif related to binding substrate. The enzyme exhibited optimal activity towards L-Leu at pH 8.5, and like other reported SV-LAAOs, it is stable until 55 °C. Kinetic studies showed that the cations Ca 2+ , Mg 2+ and Mn 2+ did not alter Bpic-LAAO activity; however, Zn 2+ is an inhibitor. Some reagents such as β-mercaptoethanol, glutathione and iodoacetate had inhibitory effect on Bpic-LAAO activity, but PMSF, EDTA and glutamic acid did not affect its activity. Regarding the biological activities of Bpic-LAAO, this enzyme induced edema in mice (MED = 7.8 μg), and inhibited human platelet aggregation induced by ADP in a dose-dependent manner and showed antibacterial activity on Gram (+) and Gram (-) bacteria. Bpic-LAAO cDNA of 1494 bp codified a mature protein with 487 amino acid residues comprising a signal peptide of 11 amino acids. Finally, the phylogenetic tree obtained with other sequences of LAAOs, evidenced its similarity to other homologous enzymes, showing two well-established monophyletic groups in Viperidae and Elapidae families. Bpic-LAAO is evolutively close related to LAAOs from B. jararacussu, B. moojeni and B. atrox, and together with the LAAO from B. pauloensis, form a well-defined cluster of the Bothrops genus. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Radio-isotopic determination of platelet monoamine oxidase and regulation of its activity by an indigenous drug

    International Nuclear Information System (INIS)

    Dubey, G.P.; Srivastava, V.K.; Agrawal, A.; Udupa, K.N.

    1988-01-01

    Platelet monoamine oxidase is a mitochondrial enzyme taking part in the deamination reaction of total catecholamine. Recent studies of monoamine oxidase inhibitors have gained its importance in the control of variety of psychosomatic disorders like mental depression, arterial hypertension and anxiety neurosis. 30 apparently normal individuals and 42 diagnosed cases of essential hypertension were selected for the present study. The platelet monoamine oxidase activity was measured by using 14 C-tryptamine bisuccinate. Comparatively low activity of platelet monoamine oxidase was noticed in hypertension cases than in the normal. After oral administration of an indigenous drug 'Geriforte' for three months, a significant rise in platelet monoamine oxidase activity was noticed in hypertension cases. It can be concluded that this indigenous formulation has the capacity to regulate the monoamine oxidase activity, as such, it may provide an alternative remedy in the management of psychosomatic disorders. (author). 11 refs

  18. Genomic sequencing of uric acid metabolizing and clearing genes in relationship to xanthine oxidase inhibitor dose.

    Science.gov (United States)

    Carroll, Matthew B; Smith, Derek M; Shaak, Thomas L

    2017-03-01

    It remains unclear why the dose of xanthine oxidase inhibitors (XOI) allopurinol or febuxostat varies among patients though they reach similar serum uric acid (SUA) goal. We pursued genomic sequencing of XOI metabolism and clearance genes to identify single-nucleotide polymorphisms (SNPs) relate to differences in XOI dose. Subjects with a diagnosis of Gout based on the 1977 American College of Rheumatology Classification Criteria for the disorder, who were on stable doses of a XOI, and who were at their goal SUA level, were enrolled. The primary outcome was relationship between SNPs in any of these genes to XOI dose. The secondary outcome was relationship between SNPs and change in pre- and post-treatment SUA. We enrolled 100 subjects. The average patient age was 68.6 ± 10.6 years old. Over 80% were men and 77% were Caucasian. One SNP was associated with a higher XOI dose: rs75995567 (p = 0.031). Two SNPs were associated with 300 mg daily of allopurinol: rs11678615 (p = 0.022) and rs3731722 on Aldehyde Oxidase (AO) (His1297Arg) (p = 0.001). Two SNPs were associated with a lower dose of allopurinol: rs1884725 (p = 0.033) and rs34650714 (p = 0.006). For the secondary outcome, rs13415401 was the only SNP related to a smaller mean SUA change. Ten SNPs were identified with a larger change in SUA. Though multiple SNPs were identified in the primary and secondary outcomes of this study, rs3731722 is known to alter catalytic function for some aldehyde oxidase substrates.

  19. Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

    International Nuclear Information System (INIS)

    Wang, Yinxi; Liu, Dan; Zhang, Huifeng; Wang, Yixin; Wei, Ling; Liu, Yutong; Liao, Jieying; Gao, Hui-Ming; Zhou, Hui

    2017-01-01

    Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm 2 induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm 2 ) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91 phox , p47 phox and p40 phox ); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47 phox and p67 phox translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to oxidative damage to DA neurons. Our

  20. Ultrafine carbon particles promote rotenone-induced dopamine neuronal loss through activating microglial NADPH oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yinxi; Liu, Dan; Zhang, Huifeng; Wang, Yixin [Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, 100191 (China); Wei, Ling [Beijing Center for Physical & Chemical Analysis, Beijing 100089 (China); Liu, Yutong [School of Life Science, Beijing Normal University, Beijing 100875 (China); Liao, Jieying [Department of Translational Medicine, Xiamen Institute of Rare Earth Materials, Chinese Academy of Sciences, Xiamen 361024 (China); Gao, Hui-Ming [Model Animal Research Center of Nanjing University, Nanjing 211800 (China); Zhou, Hui, E-mail: hardhui@gmail.com [Department of Occupational and Environmental Health Sciences, School of Public Health, Peking University, 100191 (China)

    2017-05-01

    Background: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. Methods: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. Results: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100 μg/cm{sup 2} induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10 μg/cm{sup 2}) and rotenone (2 nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91{sup phox}, p47{sup phox} and p40{sup phox}); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47{sup phox} and p67{sup phox} translocation assembling active NADPH oxidase. Conclusion: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to

  1. Cytochrome oxidase assembly does not require catalytically active cytochrome C.

    Science.gov (United States)

    Barrientos, Antoni; Pierre, Danielle; Lee, Johnson; Tzagoloff, Alexander

    2003-03-14

    Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.

  2. Xanthine oxidase activity and free radical generation in patients with sepsis syndrome

    DEFF Research Database (Denmark)

    Galley, H F; Davies, Michael Jonathan; Webster, N R

    1996-01-01

    OBJECTIVE: To determine xanthine oxidase activity, free radical concentrations, and lipid peroxidation in patients with sepsis syndrome compared with noninfected critically ill patients. DESIGN: A prospective observational study. SETTING: A nine-bed intensive care unit in a university teaching......). CONCLUSIONS: Patients with sepsis have xanthine oxidase activation, high free-radical concentrations, and evidence of free radical damage. The finding that xanthine oxidase activity was lower in those patients who died, coupled with increased lactate concentrations implies more severe ischemia with incomplete...... to the Acute Physiology and Chronic Health Evaluation (APACHE) II score or to the presence of organ dysfunction. The mean ascorbyl radical concentration (arbitrary units) determined by electron paramagnetic resonance following spin trapping was increased in patients compared with healthy subjects (p

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

    Science.gov (United States)

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

    2008-07-01

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

  4. Xanthine oxidase and uric acid as independent predictors of albuminuria in patients with diabetes mellitus type 2.

    Science.gov (United States)

    Klisic, Aleksandra; Kocic, Gordana; Kavaric, Nebojsa; Jovanovic, Milovan; Stanisic, Verica; Ninic, Ana

    2018-05-01

    Xanthine oxidase (XO) is an important enzyme responsible for conversion of purine bases to uric acid and represents the major source of reactive oxygen species (ROS) production in circulation. Since pathophysiological mechanism of the relationship between XO activity and urinary albumin excretion (UAE) rate is not well elucidated, we aimed to investigate this association in patients with diabetes mellitus type 2 (DM2). In addition, we wanted to examine whether uric acid itself plays an independent role in albuminuria onset and progression, or it is only mediated through XO activity. A total of 83 patients with DM2 (of them 56.6% females) were included in this cross-sectional study. Anthropometric, biochemical parameters and blood pressure were obtained. Multivariate logistic regression analysis showed that uric acid and XO were the independent predictors for albuminuria onset in patients with DM2 [odds ratio (OR) 1.015, 95% CI (1.008-1.028), p = 0.026 and OR 1.015, 95% CI (1.006-1.026), p = 0.040, respectively]. Rise in uric acid for 1 µmol/L enhanced the probability for albuminuria by 1.5%. Also, elevation in XO activity for 1 U/L increased the probability for albuminuria for 1.5%. A total of 66.7% of variation in UAE could be explained with this Model. Both XO and uric acid are independently associated with albuminuria in diabetes. Better understanding of pathophysiological relationship between oxidative stress and albuminuria could lead to discoveries of best pharmacological treatment of XO- and/or uric acid-induced ROS, in order to prevent albuminuria onset and progression.

  5. Lead-elevated activity of xanthine oxidase in lead-exposed workers

    Directory of Open Access Journals (Sweden)

    Sławomir Kasperczyk

    2013-04-01

    Full Text Available Background: The aim of the present study was to explore the connection between lead toxicity and the activity of xanthine oxidase (XO. In addition, we indicated the uric acid (UA and creatinine levels and concentration of erythrocyte malondialdehyde (MDA to estimate oxidative stress intensity. Materials and Methods: The examined group consisted of 125 healthy male employees of zinc and lead works. The examined group was divided into tertiles according to blood lead levels. In the collected blood samples, concentrations of lead-exposure indices, UA, creatinine, and MDA as well as activity of XO were measured concomitantly. The control group consisted of 32 healthy male administrative workers who were exposed to lead only environmentally. Results: XO activity and MDA level were significantly elevated in all tertiles compared to the control group. Creatinine level was significantly elevated in the medium and high tertiles. However, the level of UA was significantly elevated in the high tertile, while in the low and medium tertile only a tendency toward higher values was observed. Conclusions: Occupational exposure to lead induces activity of XO. This induction may contribute to the observed simultaneously increased oxidative stress, measured as MDA level, and the increased level of UA. Med Pr 2013;64(2:175–180

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

    Science.gov (United States)

    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.

  7. Antioxidant Activity of Some Plant Extracts Towards Xanthine Oxidase, Lipoxygenase and Tyrosinase

    Directory of Open Access Journals (Sweden)

    Pi-Yu Chen

    2009-08-01

    Full Text Available Natural products have the potential to be developed into new drugs for the treatment of various diseases. The aim of the present study was to screen the antioxidant activities of some common edible fruits, garden plants and medicinal plants indigenous to Taiwan. This was performed by assessing the activities of lipoxygenase, xanthine oxidase and tyrosinase following incubation with extracts from these plants. A further aim was to use HPLC-DAD and tyrosinase to chromatographically identify the antioxidative constituents obtained from an extract exhibiting strong antioxidative properties. The acetone extracts of 27 cultivated plant species from Taiwan were tested for antioxidant activities towards xanthine oxidase, tyrosinase and lipoxygenase using spectrophotometric assays. Koelreuteria henryi, Prunus campanulata, and Rhodiola rosea showed the highest xanthine oxidase inhibitory activities. Camellia sinensis, Rhodiola rosea, and Koelreuteria henryi exhibited good tyrosinase inhibitory activities and potent anti-lipoxygenase activities. As Koelreuteria henryi had notable significant inhibitory activities towards xanthine oxidase, tyrosinase, and lipoxygenase, it was further tested with tyrosinase and HPLC-DAD. The results from this part of the study revealed that the more powerful the antioxidant capability of the extracted component, the greater the decrease in peak height obtained after reacting with tyrosinase. Additional studies are warranted to further characterize the compounds responsible for the antioxidant properties of the examined extracts.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Antibacterial properties of the mammalian L-amino acid oxidase IL4I1.

    Directory of Open Access Journals (Sweden)

    Marie-Line Puiffe

    Full Text Available L-amino acid oxidases (LAAO are flavoproteins that catalyze the oxidative deamination of L-amino acids to a keto-acid along with the production of H₂O₂ and ammonia. Interleukin 4 induced gene 1 (IL4I1 is a secreted LAAO expressed by macrophages and dendritic cells stimulated by microbial derived products or interferons, which is endowed with immunoregulatory properties. It is the first LAAO described in mammalian innate immune cells. In this work, we show that this enzyme blocks the in vitro and in vivo growth of Gram negative and Gram positive bacteria. This antibiotic effect is primarily mediated by H₂O₂ production but is amplified by basification of the medium due to the accumulation of ammonia. The depletion of phenylalanine (the primary amino acid catabolized by IL4I1 may also participate in the in vivo inhibition of staphylococci growth. Thus, IL4I1 plays a distinct role compared to other antibacterial enzymes produced by mononuclear phagocytes.

  11. The Arabidopsis aldehyde oxidase 3 (AA03) gene product catalyzes the final step in abscisic acid biosynthesis in leaves

    NARCIS (Netherlands)

    Seo, M.; Peeters, A.J.M.; Koiwai, H.; Oritani, T.; Marion-Poll, A.; Zeevaart, J.A.D.; Koornneef, M.; Kamiya, Y.; Koshiba, T.

    2000-01-01

    Abscisic acid (ABA) is a plant hormone involved in seed development and germination and in responses to various environmental stresses. The last step of ABA biosynthesis involves oxidation of abscisic aldehyde, and aldehyde oxidase (EC 1.2.3.1) is thought to catalyze this reaction. An aldehyde

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

    Science.gov (United States)

    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. Copyright © 2016

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

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jian-Ching; Rebrin, Igor [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States); Klichko, Vladimir; Orr, William C. [Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275 (United States); Sohal, Rajindar S., E-mail: sohal@usc.edu [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States)

    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.

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

    Science.gov (United States)

    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.

  15. Nitric oxide production and monoamine oxidase activity in cancer patients during interferon-a therapy

    NARCIS (Netherlands)

    D. Fekkes (Durk); A.R. van Gool (Arthur); M. Bannink (Marjolein); S. Sleijfer (Stefan); W.H.J. Kruit (Wim); B. van der Holt (Bronno); A.M.M. Eggermont (Alexander); M.W. Hengeveld (Michiel); G. Stoter (Gerrit)

    2009-01-01

    textabstractAbstract Both increased and decreased nitric oxide (NO) synthesis have been reported in patients treated with interferon-alpha (IFN-alpha). Animal studies showed that IFN-alpha administration results in increased levels of biogenic amines, subsequent activation of monoamine oxidases

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    Research highlights: → Cytochrome c oxidase loses catalytic activity during the aging process. → Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. → 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 2 O 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.

  17. Hierarchical CNFs/MnCo2O4.5 nanofibers as a highly active oxidase mimetic and its application in biosensing

    Science.gov (United States)

    Gao, Mu; Lu, Xiaofeng; Nie, Guangdi; Chi, Maoqiang; Wang, Ce

    2017-12-01

    Recently, much attention has been paid on the nanomaterial-based artificial enzymes due to their tunable catalytic activity, high stability and low cost compared to the natural enzymes. Different from the peroxidase mimics which have been studied for several decades, nanomaterials with oxidase-like property are burgeoning in the recent years. In this paper, hierarchical carbon nanofibers (CNFs)/MnCo2O4.5 nanofibers as efficient oxidase mimics are reported. The products are synthesized by an electrospinning technique and an electrochemcial deposition process in which the CNFs are used as the working electrode where MnCo2O4.5 nanosheets deposit on. The resulting binary metal oxide-based nanocomposites exhibit a good oxidase-like activity toward the oxidations of 3,3‧,5,5‧tetramethylbenzi-dine (TMB), 2,2‧-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) salt and o-phenylenediamine (OPD) without exogenous addition of H2O2. The system of CNFs/MnCo2O4.5-TMB can be used as a candidate to detect sulfite and ascorbic acid via a colorimetric method with a high sensitivity. This work provides the efficient utilization and potential applications of binary metal oxide-based nanocomposites with oxidase activities in biosensors and other biotechnologies.

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

    Science.gov (United States)

    Mitra, Jyotirmoy; Bhattacharyya, Debasish

    2013-01-01

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

  19. Molecular Modeling of Peroxidase and Polyphenol Oxidase: Substrate Specificity and Active Site Comparison

    Directory of Open Access Journals (Sweden)

    Lalida Shank

    2010-09-01

    Full Text Available Peroxidases (POD and polyphenol oxidase (PPO are enzymes that are well known to be involved in the enzymatic browning reaction of fruits and vegetables with different catalytic mechanisms. Both enzymes have some common substrates, but each also has its specific substrates. In our computational study, the amino acid sequence of grape peroxidase (ABX was used for the construction of models employing homology modeling method based on the X-ray structure of cytosolic ascorbate peroxidase from pea (PDB ID:1APX, whereas the model of grape polyphenol oxidase was obtained directly from the available X-ray structure (PDB ID:2P3X. Molecular docking of common substrates of these two enzymes was subsequently studied. It was found that epicatechin and catechin exhibited high affinity with both enzymes, even though POD and PPO have different binding pockets regarding the size and the key amino acids involved in binding. Predicted binding modes of substrates with both enzymes were also compared. The calculated docking interaction energy of trihydroxybenzoic acid related compounds shows high affinity, suggesting specificity and potential use as common inhibitor to grape ascorbate peroxidase and polyphenol oxidase.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Silanikove, Nissim [Ruminant Physiology, Institute of Animal Science, Agricultural Research Organization, Volcani Center, P.O. Box 6, Bet Dagan 50250 (Israel); Shapiro, Fira [Ruminant Physiology, Institute of Animal Science, Agricultural Research Organization, Volcani Center, P.O. Box 6, Bet Dagan 50250 (Israel); Leitner, Gabriel [National Mastitis Reference Center, Kimron Veterinary Institute, Bet Dagan 50250 (Israel)

    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.

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

    International Nuclear Information System (INIS)

    Silanikove, Nissim; Shapiro, Fira; Leitner, Gabriel

    2007-01-01

    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 1/2 ∼ 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

  3. Monoamine oxidase A gene polymorphisms and enzyme activity associated with risk of gout in Taiwan aborigines.

    Science.gov (United States)

    Tu, Hung-Pin; Ko, Albert Min-Shan; Wang, Shu-Jung; Lee, Chien-Hung; Lea, Rod A; Chiang, Shang-Lun; Chiang, Hung-Che; Wang, Tsu-Nai; Huang, Meng-Chuan; Ou, Tsan-Teng; Lin, Gau-Tyan; Ko, Ying-Chin

    2010-02-01

    Taiwanese aborigines have a high prevalence of hyperuricemia and gout. Uric acid levels and urate excretion have correlated with dopamine-induced glomerular filtration response. MAOs represent one of the major renal dopamine metabolic pathways. We aimed to identify the monoamine oxidase A (MAOA, Xp11.3) gene variants and MAO-A enzyme activity associated with gout risk. This study was to investigate the association between gout and the MAOA single-nucleotide polymorphisms (SNPs) rs5953210, rs2283725, and rs1137070 as well as between gout and the COMT SNPs rs4680 Val158Met for 374 gout cases and 604 controls. MAO-A activity was also measured. All three MAOA SNPs were significantly associated with gout. A synonymous MAOA SNP, rs1137070 Asp470Asp, located in exon 14, was associated with the risk of having gout (P = 4.0 x 10(-5), adjusted odds ratio 1.46, 95% confidence intervals [CI]: 1.11-1.91). We also showed that, when compared to individuals with the MAOA GAT haplotype, carriers of the AGC haplotype had a 1.67-fold (95% CI: 1.28-2.17) higher risk of gout. Moreover, we found that MAOA enzyme activity correlated positively with hyperuricemia and gout (P for trend = 2.00 x 10(-3) vs. normal control). We also found that MAOA enzyme activity by rs1137070 allele was associated with hyperuricemia and gout (P for trend = 1.53 x 10(-6) vs. wild-type allele). Thus, our results show that some MAOA alleles, which have a higher enzyme activity, predispose to the development of gout.

  4. Critical role of NADPH oxidase in neuronal oxidative damage and microglia activation following traumatic brain injury.

    Directory of Open Access Journals (Sweden)

    Quan-Guang Zhang

    Full Text Available BACKGROUND: Oxidative stress is known to play an important role in the pathology of traumatic brain injury. Mitochondria are thought to be the major source of the damaging reactive oxygen species (ROS following TBI. However, recent work has revealed that the membrane, via the enzyme NADPH oxidase can also generate the superoxide radical (O(2(-, and thereby potentially contribute to the oxidative stress following TBI. The current study thus addressed the potential role of NADPH oxidase in TBI. METHODOLOGY/PRINCIPAL FINDINGS: The results revealed that NADPH oxidase activity in the cerebral cortex and hippocampal CA1 region increases rapidly following controlled cortical impact in male mice, with an early peak at 1 h, followed by a secondary peak from 24-96 h after TBI. In situ localization using oxidized hydroethidine and the neuronal marker, NeuN, revealed that the O(2(- induction occurred in neurons at 1 h after TBI. Pre- or post-treatment with the NADPH oxidase inhibitor, apocynin markedly inhibited microglial activation and oxidative stress damage. Apocynin also attenuated TBI-induction of the Alzheimer's disease proteins β-amyloid and amyloid precursor protein. Finally, both pre- and post-treatment of apocynin was also shown to induce significant neuroprotection against TBI. In addition, a NOX2-specific inhibitor, gp91ds-tat was also shown to exert neuroprotection against TBI. CONCLUSIONS/SIGNIFICANCE: As a whole, the study demonstrates that NADPH oxidase activity and superoxide production exhibit a biphasic elevation in the hippocampus and cortex following TBI, which contributes significantly to the pathology of TBI via mediation of oxidative stress damage, microglial activation, and AD protein induction in the brain following TBI.

  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).

    Science.gov (United States)

    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. Ethylene biosynthesis by 1-aminocyclopropane-1-carboxylic acid oxidase: a DFT study.

    Science.gov (United States)

    Bassan, Arianna; Borowski, Tomasz; Schofield, Christopher J; Siegbahn, Per E M

    2006-11-24

    The reaction catalyzed by the plant enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) was investigated by using hybrid density functional theory. ACCO belongs to the non-heme iron(II) enzyme superfamily and carries out the bicarbonate-dependent two-electron oxidation of its substrate ACC (1-aminocyclopropane-1-carboxylic acid) concomitant with the reduction of dioxygen and oxidation of a reducing agent probably ascorbate. The reaction gives ethylene, CO(2), cyanide and two water molecules. A model including the mononuclear iron complex with ACC in the first coordination sphere was used to study the details of O-O bond cleavage and cyclopropane ring opening. Calculations imply that this unusual and complex reaction is triggered by a hydrogen atom abstraction step generating a radical on the amino nitrogen of ACC. Subsequently, cyclopropane ring opening followed by O-O bond heterolysis leads to a very reactive iron(IV)-oxo intermediate, which decomposes to ethylene and cyanoformate with very low energy barriers. The reaction is assisted by bicarbonate located in the second coordination sphere of the metal.

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

    Science.gov (United States)

    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 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.

  8. Biophysical and physicochemical methods differentiate highly ligand-efficient human D-amino acid oxidase inhibitors.

    Science.gov (United States)

    Lange, Jos H M; Venhorst, Jennifer; van Dongen, Maria J P; Frankena, Jurjen; Bassissi, Firas; de Bruin, Natasja M W J; den Besten, Cathaline; de Beer, Stephanie B A; Oostenbrink, Chris; Markova, Natalia; Kruse, Chris G

    2011-10-01

    Many early drug research efforts are too reductionist thereby not delivering key parameters such as kinetics and thermodynamics of target-ligand binding. A set of human D-Amino Acid Oxidase (DAAO) inhibitors 1-6 was applied to demonstrate the impact of key biophysical techniques and physicochemical methods in the differentiation of chemical entities that cannot be adequately distinguished on the basis of their normalized potency (ligand efficiency) values. The resulting biophysical and physicochemical data were related to relevant pharmacodynamic and pharmacokinetic properties. Surface Plasmon Resonance data indicated prolonged target-ligand residence times for 5 and 6 as compared to 1-4, based on the observed k(off) values. The Isothermal Titration Calorimetry-derived thermodynamic binding profiles of 1-6 to the DAAO enzyme revealed favorable contributions of both ΔH and ΔS to their ΔG values. Surprisingly, the thermodynamic binding profile of 3 elicited a substantially higher favorable contribution of ΔH to ΔG in comparison with the structurally closely related fused bicyclic acid 4. Molecular dynamics simulations and free energy calculations of 1, 3, and 4 led to novel insights into the thermodynamic properties of the binding process at an atomic level and in the different thermodynamic signatures of 3 and 4. The presented holistic approach is anticipated to facilitate the identification of compounds with best-in-class properties at an early research stage. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  9. Chemical mechanism of D-amino acid oxidase from Rhodotorula gracilis: pH dependence of kinetic parameters.

    Science.gov (United States)

    Ramón, F; Castillón, M; De La Mata, I; Acebal, C

    1998-01-01

    The variation of kinetic parameters of d-amino acid oxidase from Rhodotorula gracilis with pH was used to gain information about the chemical mechanism of the oxidation of D-amino acids catalysed by this flavoenzyme. d-Alanine was the substrate used. The pH dependence of Vmax and Vmax/Km for alanine as substrate showed that a group with a pK value of 6.26-7.95 (pK1) must be unprotonated and a group with a pK of 10.8-9.90 (pK2) must be protonated for activity. The lower pK value corresponded to a group on the enzyme involved in catalysis and whose protonation state was not important for binding. The higher pK value was assumed to be the amino group of the substrate. Profiles of pKi for D-aspartate as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 8.4 becomes unprotonated; this basic group was not detected in Vmax/Km profiles suggesting its involvement in binding of the beta-carboxylic group of the inhibitor. PMID:9461524

  10. Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

    Science.gov (United States)

    Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

    2013-04-10

    Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Interrupted reperfusion reduces the activation of NADPH oxidase after cerebral I/R injury.

    Science.gov (United States)

    Shen, Jia; Bai, Xiao-Yin; Qin, Yuan; Jin, Wei-Wei; Zhou, Jing-Yin; Zhou, Ji-Ping; Yan, Ying-Gang; Wang, Qiong; Bruce, Iain C; Chen, Jiang-Hua; Xia, Qiang

    2011-06-15

    Interrupted reperfusion reduces ischemia/reperfusion (I/R) injury. This study was designed to determine whether NADPH oxidase participates in the neural protection against global I/R injury after interrupted reperfusion. Mice were randomly divided into five groups: sham (sham-operated), I/R (20-min global I/R), RR (I/R+interrupted reperfusion), Apo (I/R+apocynin administration), and RR+Apo. Behavioral tests (pole test, beam walking, and Morris water maze) and Nissl staining were undertaken in all five groups; superoxide levels, expression of gp91(phox) and p47(phox), p47(phox) translocation, and Rac1 activation were measured in the sham, I/R, and RR groups. The motor coordination, bradykinesia, and spatial learning and memory, as well as the neuron survival rates, were better in the RR, Apo, and RR+Apo groups than in the I/R group. The NADPH oxidase-dependent superoxide levels, p47(phox) and gp91(phox) expression, p47(phox) translocation, and Rac1 activation were lower in the RR group than in the I/R group. In conclusion, the neural protective effect of interrupted reperfusion is at least partly mediated by decreasing the expression and assembly of NADPH oxidase and the levels of NADPH oxidase-derived superoxide. The most striking reduction Rac1-GTP in the RR group suggests that interrupted reperfusion also acts on the activation of assembled NADPH oxidase by reducing the availability of Rac1-GTP. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Evaluation of Antimicrobial Activity of Glucose Oxidase from Aspergillus niger EBL-A and Penicillium notatum

    OpenAIRE

    Zia, Muhammad Anjum; Riaz, Ayesha; Rasul, Samreen; Abbas, Rao Zahid

    2013-01-01

    This work aimed to study the production and purification of glucose oxidase by Aspergillus niger and Penicillium notatum using corn steep liquor as the substrate and evaluate its antimicrobial activity for use in pharmaceutical and food industries. The enzyme was purified by ammonium sulfate precipitation (60-85%), DEAE-cellulose ion exchange and Sephadex G-200 size exclusion chromatography. The crude enzyme extracts of A. niger and P. notatum showed 2.32 and 5.53 U mg-1 specific activities, ...

  13. Paraquat and maneb co-exposure induces noradrenergic locus coeruleus neurodegeneration through NADPH oxidase-mediated microglial activation

    International Nuclear Information System (INIS)

    Hou, Liyan; Zhang, Cong; Wang, Ke; Liu, Xiaofang; Wang, Hongwei; Che, Yuning; Sun, Fuqiang; Zhou, Xueying; Zhao, Xiulan; Wang, Qingshan

    2017-01-01

    Highlights: • Microglial activation induced by paraquat and maneb precedes noradrenergic neurodegeneration in locus coeruleus. • NADPH oxidase activation contributes to microglia-mediated neuroinflammation and related noradrenergic neurodegeneration. • Inhibition of NADPH oxidase by apocynin protects noradrenergic neurons against paraquat and maneb-induced toxicity. - Abstract: Co-exposure to paraquat (PQ) and maneb (Mb) has been shown to increase the risk of Parkinson’s disease (PD) and dopaminergic (DA) neurodegeneration in the substantia nigra pars compacta (SNpc) is observed in PQ and Mb-treated experimental animals. The loss of noradrenergic locus coeruleus (LC/NE) neurons in brainstem is a common feature shared by multiple neurodegenerative diseases, including PD. However, whether PQ and Mb is able to damage LC/NE neurons remains undefined. In this study, mice treated with combined PQ and Mb displayed progressive LC/NE neurodegeneration. Time course studies revealed that the activation of microglia preceded LC/NE neurodegeneration. Mechanistically, the activation of NADPH oxidase contributed to microglial activation and subsequent LC/NE neurodegeneration. We found that PQ and Mb co-exposure induced activation of NADPH oxidase as shown by increased superoxide production and membrane translocation of p47 phox , a cytosolic subunit of NADPH oxidase. Inhibition of NADPH oxidase by apocynin, a widely used NADPH oxidase inhibitor, suppressed microglial activation and gene expressions of proinflammatory factors. Furthermore, reduced activation of nuclear factor-κB (NF-κB) pathway was observed in apocynin-treated mice. More importantly, inhibition of NADPH oxidase by apocynin afforded LC/NE neuroprotection against PQ and Mb-induced neurotoxicity. Thus, our findings revealed the critical role NADPH oxidase-mediated microglial activation in driving LC/NE neurodegeneration induced by PQ and Mb, providing new insights into the pathogenesis of environmental

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2013-01-01

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

  16. Improving glyphosate oxidation activity of glycine oxidase from Bacillus cereus by directed evolution.

    Directory of Open Access Journals (Sweden)

    Tao Zhan

    Full Text Available Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO, we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg(51 mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

  17. Isolation, crystallization and preliminary X-ray diffraction analysis of l-amino-acid oxidase from Vipera ammodytes ammodytes venom

    International Nuclear Information System (INIS)

    Georgieva, Dessislava; Kardas, Anna; Buck, Friedrich; Perbandt, Markus; Betzel, Christian

    2008-01-01

    A novel l-amino-acid oxidase was isolated from V. ammodytes ammodytes venom and crystallized. The solution conditions under which the protein sample was monodisperse were optimized using dynamic light scattering prior to crystallization. Preliminary diffraction data were collected to 2.6 Å resolution. l-Amino-acid oxidase from the venom of Vipera ammodytes ammodytes, the most venomous snake in Europe, was isolated and crystallized using the sitting-drop vapour-diffusion method. The solution conditions under which the protein sample was monodisperse were optimized using dynamic light scattering prior to crystallization. The crystals belonged to space group C2, with unit-cell parameters a = 198.37, b = 96.38, c = 109.11 Å, β = 92.56°. Initial diffraction data were collected to 2.6 Å resolution. The calculated Matthews coefficient is approximately 2.6 Å 3 Da −1 assuming the presence of four molecules in the asymmetric unit

  18. Evaluation of Antimicrobial Activity of Glucose Oxidase from Aspergillus niger EBL-A and Penicillium notatum

    Directory of Open Access Journals (Sweden)

    Muhammad Anjum Zia

    2013-12-01

    Full Text Available This work aimed to study the production and purification of glucose oxidase by Aspergillus niger and Penicillium notatum using corn steep liquor as the substrate and evaluate its antimicrobial activity for use in pharmaceutical and food industries. The enzyme was purified by ammonium sulfate precipitation (60-85%, DEAE-cellulose ion exchange and Sephadex G-200 size exclusion chromatography. The crude enzyme extracts of A. niger and P. notatum showed 2.32 and 5.53 U mg-1 specific activities, respectively, which after desalting was 15.52 and 12.05 U mg-1, and after ion exchange and gel filtration chromatography was 29.09 - 62 and 25.72 - 59.37 U mg-1 for A. niger and P. notatum, respectively. The antimicrobial activity was determined by disc diffusion method against selected microbial strains where glucose oxidase from A. niger showed anti-bacterial activity, while no fungicidal effects were shown by both A. niger and P. notatum glucose oxidases.

  19. The HIV-1 Nef protein and phagocyte NADPH oxidase activation

    DEFF Research Database (Denmark)

    Vilhardt, Frederik; Plastre, Olivier; Sawada, Makoto

    2002-01-01

    of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation...

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    Highlights: • We fabricated polyvinyl alcohol/malonic acid nanofibers using electrospinning. • The surface nanofibers were modified by gaseous (air, nitrogen, CO_2 and argon) dielectric barrier discharge. • Among them, air plasma had the most significant effect on glucose oxidase immobilization. • Chemical analysis showed that after modification of nanofibers by air plasma, the carboxyl group increased. • After air plasma treatment, reusability and storage stability of glucose oxidase immobilized on nanofibers improved. - Abstract: 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, CO_2, 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Afshari, Esmail, E-mail: e.afshari@mail.sbu.ac.ir [Laser and Plasma Research Institute, Shahid Beheshti University, Evin, 1983963113 Tehran (Iran, Islamic Republic of); Mazinani, Saeedeh [Amirkabir Nanotechnology Research Institute (ANTRI), Amirkabir University of Technology, 15875-4413, Tehran (Iran, Islamic Republic of); Ranaei-Siadat, Seyed-Omid [Protein Research Center, Shahid Beheshti University, Evin, 1983963113 Tehran (Iran, Islamic Republic of); Ghomi, Hamid [Laser and Plasma Research Institute, Shahid Beheshti University, Evin, 1983963113 Tehran (Iran, Islamic Republic of)

    2016-11-01

    Highlights: • We fabricated polyvinyl alcohol/malonic acid nanofibers using electrospinning. • The surface nanofibers were modified by gaseous (air, nitrogen, CO{sub 2} and argon) dielectric barrier discharge. • Among them, air plasma had the most significant effect on glucose oxidase immobilization. • Chemical analysis showed that after modification of nanofibers by air plasma, the carboxyl group increased. • After air plasma treatment, reusability and storage stability of glucose oxidase immobilized on nanofibers improved. - Abstract: 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, CO{sub 2}, 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.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Ag-doped CdO nanocatalysts: Preparation, characterization and catechol oxidase activity

    Science.gov (United States)

    El-Kemary, Maged; El-Mehasseb, Ibrahim; El-Shamy, Hany

    2018-06-01

    Silver doped cadmium oxide (Ag/CdO) nanoparticles with an average size of 41 nm have been successfully synthesized via thermal decomposition and liquid impregnation technique. The structural characterization has been performed by using several spectroscopic techniques, e.g., X-ray diffraction (XRD), scanning electron microscopy (SEM) and fourier-transform infrared (FT-IR). The catechol oxidase has been studied by UV-visible absorption spectroscopy and fourier-transform infrared as well as the mechanism has been assured by cyclic voltammetry and fluorescence spectroscopy. The results indicate that the oxidation does not occur in the presence of unsupported cadmium oxide particles by silver and in the same time, the catechol oxidase activity of silver doped CdO nanoparticles were improved by about three orders of magnitude than silver ions.

  5. Characterization of three bioenergetically active respiratory terminal oxidases in the cyanobacterium Synechocystis sp. strain PCC 6803.

    Science.gov (United States)

    Pils, D; Schmetterer, G

    2001-09-25

    Synechocystis sp. PCC 6803 contains three respiratory terminal oxidases (RTOs): cytochrome c oxidase (Cox), quinol oxidase (Cyd), and alternate RTO (ARTO). Mutants lacking combinations of the RTOs were used to characterize these key enzymes of respiration. Pentachlorophenol and 2-heptyl-4-hydroxy-quinoline-N-oxide inhibited Cyd completely, but had little effect on electron transport to the other RTOs. KCN inhibited all three RTOs but the in vivo K(I) for Cox and Cyd was quite different (7 vs. 27 microM), as was their affinity for oxygen (K(M) 1.0 vs. 0.35 microM). ARTO has a very low respiratory activity. However, when uptake of 3-O-methylglucose, an active H+ co-transport, was used to monitor energization of the cytoplasmic membrane, ARTO was similarly effective as the other RTOs. As removal of the gene for cytochrome c(553) had the same effects as removal of ARTO genes, we propose that the ARTO might be a second Cox. The possible functions, localization and regulation of the RTOs are discussed.

  6. Barley polyamine oxidase: Characterisation and analysis of the cofactor and the N-terminal amino acid sequence

    DEFF Research Database (Denmark)

    Radova, A.; Sebela, M.; Galuszka, P.

    2001-01-01

    This paper reports the first purification method developed for the isolation of an homogeneous polyamine oxidase (PAO) from etiolated barley seedlings. The crude enzyme preparation was obtained after initial precipitation of the extract with protamine sulphate and ammonium sulphate. The enzyme...... was further confirmed by measuring the fluorescence spectra, Barley PAO is an acidic protein (pI 5.4) containing 3% of neutral sugars: its molecular mass determined by SDS-PAGE was 56 kDa, whilst gel permeation chromatography revealed the higher value of 76 kDa. The N-terminal amino acid sequence of barley...... PAO shows a high degree of similarity to that of maize PAO and to several other flavoprotein oxidases. The polyamines spermine and spermidine were the only two substrates of the enzyme with K-m values 4 x 10(-5) and 3 x 10(-5) M and pH optima of 5.0 and 6.0, respectively. Barley polyamine oxidase...

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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., H 2 O 2 , substrate, and oxidase) due to the aperture limitation of the ZIF-8 shell. H 2 O 2 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 H 2 O 2 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Activation of NADPH oxidase mediates increased endoplasmic reticulum stress and left ventricular remodeling after myocardial infarction in rabbits.

    Science.gov (United States)

    Li, Bao; Tian, Jing; Sun, Yi; Xu, Tao-Rui; Chi, Rui-Fang; Zhang, Xiao-Li; Hu, Xin-Ling; Zhang, Yue-An; Qin, Fu-Zhong; Zhang, Wei-Fang

    2015-05-01

    Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of the NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30 days. The agents were administered beginning at 1 week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expressions were increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. The apocynin treatment prevented increases in the Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. The apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that the activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Identification of the free phenolic profile of Adlay bran by UPLC-QTOF-MS/MS and inhibitory mechanisms of phenolic acids against xanthine oxidase.

    Science.gov (United States)

    Lin, Lianzhu; Yang, Qingyun; Zhao, Kun; Zhao, Mouming

    2018-07-01

    Adlay bran free phenolic extract has been previously demonstrated to possess potent xanthine oxidase (XOD) inhibitory activity. The aims of this study were to characterize the free phenolic profile of adlay bran and investigate the structure-activity relationship, underlying mechanism and interaction of phenolic acids as XOD inhibitors. A total of twenty phenolics including ten phenolic acids, two coumarins, two phenolic aldedhyes and six flavonoids were identified in a phenolic compound-guided separation by UPLC-QTOF-MS/MS. Adlay bran free phenolic extract possessed strong XOD inhibitory activity related to hydroxycinnamic acids with methoxyl groups. The hydrogen bonding and hydrophobic interactions were the main forces in the binding of adlay phenolics to XOD. Sinapic acid, identified in adlay bran for the first time, possessed strong XOD inhibitory activity in a mixed non-competitive manner, and synergistic effects with other adlay phenolic acids at low concentrations, and would be a promising agent for preventing and treating hyperuricemia. Copyright © 2018. Published by Elsevier Ltd.

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

    Science.gov (United States)

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

    2016-06-15

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

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

    Science.gov (United States)

    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. Copyright © 2011 John Wiley & Sons, Ltd.

  13. Expression of novel rice gibberellin 2-oxidase gene is under homeostatic regulation by biologically active gibberellins.

    Science.gov (United States)

    Sakai, Miho; Sakamoto, Tomoaki; Saito, Tamio; Matsuoka, Makoto; Tanaka, Hiroshi; Kobayashi, Masatomo

    2003-04-01

    We have cloned two genes for gibberellin (GA) 2-oxidase from rice ( Oryza sativa L.). Expression of OsGA2ox2 was not observed. The other gene, OsGA2ox3, was expressed in every tissue examined and was enhanced by the application of biologically active GA. Recombinant OsGA2ox3 protein catalyzed the metabolism of GA(1) to GA(8) and GA(20) to GA(29)-catabolite. These results indicate that OsGA2ox3 is involved in the homeostatic regulation of the endogenous level of biologically active GA in rice.

  14. Optimization of Glucose oxidase towards oxygen independency and high mediator activity for amperometric glucose determination in diabetes analytics

    OpenAIRE

    Arango Gutierrez, Erik Uwe

    2015-01-01

    Glucose oxidase is an oxidoreductase exhibiting a high β-D-glucose specificity and high stability which renders glucose oxidase well-suited for applications in diabetes care. Nevertheless, GOx activity is highly oxygen dependent which can lead to inaccuracies in amperometric β-D-glucose determinations. Therefore a directed evolution campaign with two rounds of random mutagenesis (SeSaM followed by epPCR), site saturation mutagenesis studies, and one simultaneous site saturation library (OmniC...

  15. Isolation and genome sequencing of four Arctic marine Psychrobacter strains exhibiting multicopper oxidase activity.

    Science.gov (United States)

    Moghadam, Morteza Shojaei; Albersmeier, Andreas; Winkler, Anika; Cimmino, Lorenzo; Rise, Kjersti; Hohmann-Marriott, Martin Frank; Kalinowski, Jörn; Rückert, Christian; Wentzel, Alexander; Lale, Rahmi

    2016-02-16

    Marine cold-temperature environments are an invaluable source of psychrophilic microbial life for new biodiscoveries. An Arctic marine bacterial strain collection was established consisting of 1448 individual isolates originating from biota, water and sediment samples taken at a various depth in the Barents Sea, North of mainland Norway, with an all year round seawater temperature of 4 °C. The entire collection was subjected to high-throughput screening for detection of extracellular laccase activity with guaiacol as a substrate. In total, 13 laccase-positive isolates were identified, all belonging to the Psychrobacter genus. From the most diverse four strains, based on 16S rRNA gene sequence analysis, all originating from the same Botryllus sp. colonial ascidian tunicate sample, genomic DNA was isolated and genome sequenced using a combined approach of whole genome shotgun and 8 kb mate-pair library sequencing on an Illumina MiSeq platform. The genomes were assembled and revealed genome sizes between 3.29 and 3.52 Mbp with an average G + C content of around 42%, with one to seven plasmids present in the four strains. Bioinformatics based genome mining was performed to describe the metabolic potential of these four strains and to identify gene candidates potentially responsible for the observed laccase-positive phenotype. Up to two different laccase-like multicopper oxidase (LMCO) encoding gene candidates were identified in each of the four strains. Heterologous expression of P11F6-LMCO and P11G5-LMCO2 in Escherichia coli BL21 (DE3) resulted in recombinant proteins exhibiting 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and guaiacol oxidizing activity. Thirteen Psychrobacter species with laccase-positive phenotype were isolated from a collection of Arctic marine bacteria. Four of the isolates were genome sequenced. The overall genome features were similar to other publicly available Psychrobacter genome sequences except for P11G5 harboring seven

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

    International Nuclear Information System (INIS)

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

    1990-01-01

    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 125 I-labeled XO to one rabbit to ascertain whether the observed increase in serum XO was due to dietary or endogenous XO. Isoelectric focusing of sera collected from the test rabbit strongly suggested that at least a portion of the serum XO contained the radioactive label. This is the first direct evidence showing the uptake of dietary active XO from the gut

  17. Spatio-temporal detection of the Thiomonas population and the Thiomonas arsenite oxidase involved in natural arsenite attenuation processes in the Carnoulès Acid Mine Drainage

    Directory of Open Access Journals (Sweden)

    Agnès eHovasse

    2016-02-01

    Full Text Available 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 16S rRNA gene sequence analysis based on pyrosequencing and FISH, 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.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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 2 ·- 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 phox siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91 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 phox , p67 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 phox siRNA. Exposure of MPMVEC obtained from gp91 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 via activation of NADPH oxidase. UFP-induced ROS lead to

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

    Science.gov (United States)

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

    1987-11-01

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

  20. Association of molecular markers with polyphenol oxidase activity in selected wheat genotypes

    International Nuclear Information System (INIS)

    Abbas, Z.; Javad, B.; Majeed, N.; Naqvi, S.

    2016-01-01

    Wheat (Triticum aestivum L.), a major staple food for the people of Pakistan and other Asian countries, is used as bread, chapatti, porridge, noodles and many other. It is established that color quality of wheat products depend on chemical and enzymatic factors especially the polyphenol oxidases (PPOs). These are copper containing enzymes which induce browning in wheat-based products. Various procedures for determining PPO activity available and differences in PPO activity among wheat genotypes have been documented. In present study, an attempt was made to establish the association of molecular markers with polyphenol oxidase activity in wheat genotypes having very high or very low PPO activities. Twelve pairs of markers were used out of which only three primer pairs viz. PPO43, PPO30 and WP2-2 yielded specific pattern discriminating high and low PPO genotypes. Cluster analysis for all 12 markers revealed that all the low PPO lline share the same sub cluster, but high PPO lines were dispersed in different clusters. (author)

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

    Directory of Open Access Journals (Sweden)

    Christian Carpéné

    2016-01-01

    Full Text Available 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.

  2. Intermittent hypoxia-induced cognitive deficits are mediated by NADPH oxidase activity in a murine model of sleep apnea.

    Directory of Open Access Journals (Sweden)

    Deepti Nair

    Full Text Available In rodents, exposure to intermittent hypoxia (IH, a hallmark of obstructive sleep apnea (OSA, is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Excessive NADPH oxidase activity may play a role in IH-induced CNS dysfunction.The effect of IH during light period on two forms of spatial learning in the water maze and well as markers of oxidative stress was assessed in mice lacking NADPH oxidase activity (gp91phox(_/Y and wild-type littermates. On a standard place training task, gp91phox(_/Y displayed normal learning, and were protected from the spatial learning deficits observed in wild-type littermates exposed to IH. Moreover, anxiety levels were increased in wild-type mice exposed to IH as compared to room air (RA controls, while no changes emerged in gp91phox(_/Y mice. Additionally, wild-type mice, but not gp91phox(_/Y mice had significantly elevated levels of NADPH oxidase expression and activity, as well as MDA and 8-OHDG in cortical and hippocampal lysates following IH exposures.The oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by excessive NADPH oxidase activity, and thus pharmacological agents targeting NADPH oxidase may provide a therapeutic strategy in sleep-disordered breathing.

  3. Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by /sup 14/C putrescine method

    Energy Technology Data Exchange (ETDEWEB)

    Fogel, W A [Polish Academy of Sciences, Cracow (Poland). Inst. of Pharmacology; Bieganski, T; Wozniak, J; Maslinski, C

    1978-01-01

    The ..delta../sup 1/ pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi /sup 14/C putrescine test (1961, Archs Biochem. Biophys., vol.95, 242), has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chlorate hydrate and acetaldehyde the level of formation ..delta../sup 1/ pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced ..delta../sup 1/ pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on ..delta../sup 1/ pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seddling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme.

  4. Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by 14C putrescine method

    International Nuclear Information System (INIS)

    Fogel, W.A.; Bieganski, T.; Wozniak, J.; Maslinski, C.

    1978-01-01

    The Δ 1 pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi 14 C putrescine test (1961, Archs Biochem. Biophys., vol.95, 242), has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chlorate hydrate and acetaldehyde the level of formation Δ 1 pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced Δ 1 pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on Δ 1 pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seddling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme. (author)

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

    Science.gov (United States)

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

    2014-05-01

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

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    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.

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

    Science.gov (United States)

    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

  8. Effects of phenylated compounds of methylglyoxal bis(guanylhydrazone) on diamine oxidase activity from rat small intestine.

    Science.gov (United States)

    Balaña-Fouce, R; Pulido, T G; Escudero, D O; Sanz-Sanchez, F

    1986-01-01

    Two phenylated compounds of methylglyoxal bis(guanylhydrazone), potentially inhibitors of diamine oxidase activity, have been synthesized: phenylglyoxal bis(guanylhydrazone) and diphenylglyoxal bis(guanylhydrazone). Their inhibitory capacity was tested: while PGBG was able to reduce the enzyme activity by 50% at 1.3 microM, DPGBG was only able to reduce diamine oxidase activity by less than 2% at a concentration 1000-fold higher. The inhibition of PGBG was non-competitive and the Ki calculated by a Dixon plot was estimated as 1.7 microM.

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

    Science.gov (United States)

    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.

  10. Effect of gamma irradiation on the activity of monoamine oxidase in the hypothalamus of ewes

    International Nuclear Information System (INIS)

    Pastorova, B.; Stanikova, A.

    2008-01-01

    The study investigated changes in monoamine oxidase (MAO) activity in the hypothalamus of ewes in the anoestrous period exposed to a whole body Co-60 irradiation with a total dose of 6.7 Gy for the period of 7 days. The activity of MAO was determined by means of a radiochemical method using C-14 tryptamine as a substrate. Whole body exposure to gamma radiation of total dose of 6.7 Gy increased significantly (P < 0.001) the activity of MAO in the caudal, medial and rostral hypothalamus of the investigated ewes. It may by assumed that an increased degradation of catecholamines caused by MAO is one of the mechanisms responsible for pronounced changes in the level of catecholamines in the hypothalamus of ewes after irradiation. (authors)

  11. Isolation, Identification, and Xanthine Oxidase Inhibition Activity of Alkaloid Compound from Peperomia pellucida

    Science.gov (United States)

    Fachriyah, E.; Ghifari, M. A.; Anam, K.

    2018-04-01

    The research of the isolation and xanthine oxidation inhibition activity of alkaloid compound from Peperomia pellucida has been carried out. Alkaloid extract is isolated by column chromatography and preparative TLC. Alkaloid isolate is identified spectroscopically by UV-Vis spectrophotometer, FT-IR, and LC-MS/MS. Xanthine oxidase inhibition activity is carried out by in vitro assay. The result showed that the alkaloid isolated probably has piperidine basic structure. The alkaloid isolate has N-H, C-H, C = C, C = O, C-N, C-O-C groups and the aromatic ring. The IC50 values of ethanol and alkaloid extract are 71.6658 ppm and 76.3318 ppm, respectively. Alkaloid extract of Peperomia pellucida showed higher activity than ethanol extract.

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

    Science.gov (United States)

    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.

  13. Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress.

    Science.gov (United States)

    Chen, Feng; Qian, Li-Hua; Deng, Bo; Liu, Zhi-Min; Zhao, Ying; Le, Ying-Ying

    2013-09-01

    Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes. © 2013 John Wiley & Sons Ltd.

  14. Structure-Activity Relationship Analysis of 3-phenylcoumarin-Based Monoamine Oxidase B Inhibitors

    Science.gov (United States)

    Rauhamäki, Sanna; Postila, Pekka A.; Niinivehmas, Sanna; Kortet, Sami; Schildt, Emmi; Pasanen, Mira; Manivannan, Elangovan; Ahinko, Mira; Koskimies, Pasi; Nyberg, Niina; Huuskonen, Pasi; Multamäki, Elina; Pasanen, Markku; Juvonen, Risto O.; Raunio, Hannu; Huuskonen, Juhani; Pentikäinen, Olli T.

    2018-03-01

    Monoamine oxidase B (MAO-B) catalyzes deamination of monoamines such as neurotransmitters dopamine and norepinephrine. Accordingly, small-molecule MAO-B inhibitors potentially alleviate the symptoms of dopamine-linked neuropathologies such as depression or Parkinson’s disease. Coumarin with a functionalized 3-phenyl ring system is a promising scaffold for building potent MAO-B inhibitors. Here, a vast set of 3-phenylcoumarin derivatives was designed using virtual combinatorial chemistry or rationally de novo and synthesized using microwave chemistry. The derivatives inhibited the MAO-B at 100 nM - 1 µM. The IC50 value of the most potent derivative 1 was 56 nM. A docking-based structure-activity relationship analysis summarizes the atom-level determinants of the MAO-B inhibition by the derivatives. Finally, the cross-reactivity of the derivatives was tested against monoamine oxidase A and a specific subset of enzymes linked to estradiol metabolism, known to have coumarin-based inhibitors. Overall, the results indicate that the 3-phenylcoumarins, especially derivative 1, present unique pharmacological features worth considering in future drug development.

  15. Active site intermediates in the reduction of O(2) by cytochrome oxidase, and their derivatives.

    Science.gov (United States)

    Wikström, Mårten

    2012-04-01

    The mechanism of dioxygen activation and reduction in cell respiration, as catalysed by cytochrome c oxidase, has a long history. The work by Otto Warburg, David Keilin and Britton Chance defined the dioxygen-binding heme iron centre, viz. das Atmungsferment, or cytochrome a(3). Chance brought the field further in the mid-1970's by ingenious low-temperature studies that for the first time identified the primary enzyme-substrate (ES) Michaelis complex of cell respiration, the dioxygen adduct of heme a(3), which he termed Compound A. Further work using optical, resonance Raman, EPR, and other sophisticated spectroscopic techniques, some of which with microsecond time resolution, has brought us to the situation today, where major principles of how O(2) reduction occurs in respiration are well understood. Nonetheless, some questions have remained open, for example concerning the precise structures, catalytic roles, and spectroscopic properties of the breakdown products of Compound A that have been called P, F (for peroxy and ferryl), and O (oxidised). This nomenclature has been known to be inadequate for some time already, and an alternative will be suggested here. In addition, the multiple forms of P, F and O states have been confusing, a situation that we endeavour to help clarifying. The P and F states formed artificially by reacting cytochrome oxidase with hydrogen peroxide are especially scrutinised, and some novel interpretations will be given that may account for previously unexplained observations. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  17. Novel p47phox-related organizers regulate NADPH oxidase 1 (Nox1) activity and localization

    Science.gov (United States)

    Gianni, Davide; Diaz, Begoña; Taulet, Nicolas; Fowler, Bruce; Courtneidge, Sara A.; Bokoch, Gary M.

    2010-01-01

    The mechanisms that determine localized formation of reactive oxygen species (ROS) via NADPH oxidases (Nox) in nonphagocytic cells are unknown. We show that the c-Src substrate proteins Tks4 and Tks5 are functional members of a p47phox-related organizer superfamily. Tks proteins selectively support Nox1 and Nox3 (vs. Nox2 and Nox4) activity in reconstituted cellular systems, and interact with the NoxA1 activator protein through an SH3-mediated interaction. Endogenous Tks4 is required for Rac GTPase-dependent ROS production by DLD1 colon cancer cells. Tks4 recruits Nox1 to invadopodia that form in DLD1 cells in a Tks- and Nox-dependent fashion. We propose that Tks organizers represent novel members of an organizer superfamily that link Nox to localized ROS formation. PMID:19755710

  18. Blocking Surgically Induced Lysyl Oxidase Activity Reduces the Risk of Lung Metastases

    Directory of Open Access Journals (Sweden)

    Chen Rachman-Tzemah

    2017-04-01

    Full Text Available Surgery remains the most successful curative treatment for cancer. However, some patients with early-stage disease who undergo surgery eventually succumb to distant metastasis. Here, we show that in response to surgery, the lungs become more vulnerable to metastasis due to extracellular matrix remodeling. Mice that undergo surgery or that are preconditioned with plasma from donor mice that underwent surgery succumb to lung metastases earlier than controls. Increased lysyl oxidase (LOX activity and expression, fibrillary collagen crosslinking, and focal adhesion signaling contribute to this effect, with the hypoxic surgical site serving as the source of LOX. Furthermore, the lungs of recipient mice injected with plasma from post-surgical colorectal cancer patients are more prone to metastatic seeding than mice injected with baseline plasma. Downregulation of LOX activity or levels reduces lung metastasis after surgery and increases survival, highlighting the potential of LOX inhibition in reducing the risk of metastasis following surgery.

  19. 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.

    Science.gov (United States)

    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.

  20. Effects of ascorbic acid and glucose oxidase levels on the viability of probiotic bacteria and the physical and sensory characteristics in symbiotic ice-cream

    Directory of Open Access Journals (Sweden)

    M. B. Akın

    2015-05-01

    Full Text Available In this study, the effects of addition of different amounts of ascorbic acid and glucose oxidase on the properties of symbiotic ice cream were investigated. Ice-cream containing inulin (2 % (w/w was produced by mixing fortified milk fermented with probiotic strains with the ice-cream mixes containing different ascorbic acid and glucose oxidase concentrations (0.025, 0.05, 0.1 (w/w. The cultures were grown (37 °C, 12 h in UHT skimmed milk. The fermented milk was added to the ice-cream mix up to a level of 10 % w/w. Increasing the concentration of ascorbic acid stimulated the growth of Lactobacillus acidophilus LA-5 (L. acidophilus and Bifidobacterium animalis subsp. lactis BB-12 (Bifidobacterium BB-12. On contrary, increasing the concentration of glucose oxidase negatively affected the growth of L. acidophilus and Bifidobacterium BB-12. However, both, ascorbic acid and glucose oxidase concentration had no effect on physical and sensory properties of ice cream. The results suggested that the addition of ascorbic acid stimulated the growth of L. acidophilus and Bifidobacterium BB-12 and could be recommended for ice cream production.

  1. An assay of optimal cytochrome c oxidase activity in fish gills.

    Science.gov (United States)

    Hu, Yau-Chung; Chung, Meng-Han; Lee, Tsung-Han

    2018-07-15

    Cytochrome c oxidase (COX) catalyzes the terminal oxidation reaction in the electron transport chain (ETC) of aerobic respiratory systems. COX activity is an important indicator for the evaluation of energy production by aerobic respiration in various tissues. On the basis of the respiratory characteristics of muscle, we established an optimal method for the measurement of maximal COX activity. To validate the measurement of cytochrome c absorbance, different ionic buffer concentrations and tissue homogenate protein concentrations were used to investigate COX activity. The results showed that optimal COX activity is achieved when using 50-100 μg fish gill homogenate in conjunction with 75-100 mM potassium phosphate buffer. Furthermore, we compared branchial COX activities among three species of euryhaline teleost (Chanos chanos, Oreochromis mossambicus, and Oryzias dancena) to investigate differences in aerobic respiration of osmoregulatory organs. COX activities in the gills of these three euryhaline species were compared with COX subunit 4 (COX4) protein levels. COX4 protein abundance and COX activity patterns in the three species occurring in environments with various salinities increased when fish encountered salinity challenges. This COX activity assay therefore provides an effective and accurate means of assessing aerobic metabolism in fish. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Monoamine oxidase inhibitory activity in tobacco particulate matter: Are harman and norharman the only physiologically relevant inhibitors?

    Science.gov (United States)

    Truman, Penelope; Grounds, Peter; Brennan, Katharine A

    2017-03-01

    Monoamine oxidase inhibition is significant in smokers, but it is still unclear how the inhibition that is seen in the brains and bodies of smokers is brought about. Our aim was to test the contribution of the harman and norharman in tobacco smoke to MAO-A inhibition from tobacco smoke preparations, as part of a re-examination of harman and norharman as the cause of the inhibition of MAO-A inhibition in the brain. Tobacco smoke particulate matter and cigarette smoke particulate matter were prepared and the amounts of harman and norharman measured. The results were compared with the total monoamine oxidase-A inhibitory activity. At a nicotine concentration of 0.6μM (a "physiological" concentration in blood) the total monoamine oxidase-A inhibitory activity measured in these samples was sufficient to inhibit the enzyme by approximately 10%. Of this inhibitory activity, only a small proportion of the total was found to be due to harman and norharman. These results show that harman and norharman provide only a moderate contribution to the total monoamine oxidase-A inhibitory activity of tobacco smoke, perhaps under 10%. This suggests that other inhibitors (either known or unknown) may be more significant contributors to total inhibitory activity than has yet been established, and deserve closer examination. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Effect of phenol on germination capacity and polyphenol oxidase, peroxidase and catalase activities in lettuce

    Directory of Open Access Journals (Sweden)

    Tadić Vojin

    2014-01-01

    Full Text Available In this study we examined the activities of polyphenol oxidase (PPO and antioxidant enzymes, peroxidase (POX and catalase (CAT during lettuce seed germination at different concentrations of phenol. Out of eleven varieties of lettuce, four were chosen according to their germination tolerance to phenol as follows: plants exhibiting high (Ljubljanska ledenka - LJL and Nansen - N and low toleranace (Little Gem - LG and Majska kraljica - MK. A decrease in germination efficiency after exposure to LD50 of phenol was determined for these four varieties. The effects of phenol treatment on POX, CAT and PPO activities were determined after 4, 5, 6, 7 and 8 days of growth at LD50 concentrations. A trend of increased peroxidase activity was observed in seeds grown on LD50 of phenol compared to control seeds. A significant increase in CAT activity was observed at the beginning of treatment for MK, LG and N in seeds grown on phenol as well as in control seeds. A trend of increased PPO activity was observed in all control seeds. We also investigated the affinity of PPO for two different substrates that were used for the determination of enzyme activity. Our results show that LJL and N are the varieties most tolerant to growth on phenol. Here we report on the activities of their antioxidant enzymes and PPO during seed germination. [Projekat Ministarstva nauke Republike Srbije, br. ON173017

  4. Bienzymatic Acetylcholinesterase and Choline Oxidase Immobilized Biosensor Based on a Phenyl Carboxylic Acid-Grafted Multiwalled Carbon Nanotube

    Directory of Open Access Journals (Sweden)

    So-Ra Lee

    2014-01-01

    Full Text Available Bienzymatic acetylcholinesterase (AChE and choline oxidase (ChOx immobilized biosensor based on a phenyl carboxylic acid-grafted multiwalled carbon nanotube (MWNT modified glass carbon electrode (GCE and carbon-screen printed electrode (SPE was fabricated for acetylcholine detection in human blood samples. Phenyl carboxylic acid-modified MWNT supports were prepared by electrochemical polymerization of 4-carboxyphenyl diazonium salts, which were synthesized by an amine group and sodium nitrite, on the surface of the MWNT-modified GCE and SPE in 0.1 M PBS. The successful fabrication of the AChE-ChOx-immobilized biosensor was confirmed via scanning electron microscopy (SEM, X-ray photoelectron spectroscopy (XPS, electrochemical impedance spectroscopy (EIS, and cyclic voltammetry (CV. The sensing range of the biosensor based on a GCE and SPE was 1.0~10 μM and 10~100 μM, respectively. The interfering effect of 0.1 M L-ascorbic acid, 0.1 M L-cysteine, and 0.1 M uric acid to 0.1 M acetylcholine was 3.00%, 9.00%, and 3.00%, respectively. Acetylcholine in a human blood sample was detected by the AChE-ChOx-immobilized biosensor.

  5. Dysbindin and d-amino-acid-oxidase gene polymorphisms associated with positive and negative symptoms in schizophrenia

    DEFF Research Database (Denmark)

    Wirgenes, Katrine V; Djurovic, Srdjan; Agartz, Ingrid

    2009-01-01

    -amino-acid-oxidase (DAO) gene, both involved in glutamate receptor function, reported associations with negative symptoms and with anxiety and depression, respectively, when measured with the Positive and Negative Syndrome Scale (PANSS). METHODS: In the present study, the suggested association between dysbindin and DAO...... single nucleotide polymorphisms (SNPs) and PANSS scores was analyzed in 155 Norwegian schizophrenia patients. RESULTS: There was a significant association between the dysbindin SNP rs3213207 and severity of both negative symptoms and total symptom load, as well as between the DAO SNP rs2070587 and total...... symptom score and severity of anxiety and depression. CONCLUSION: The present association of dysbindin SNPs with negative symptoms and DAO SNPs with anxiety and depression is a replication of earlier findings and strengthens the hypothesis of a genetic association. It further indicates involvement...

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

    Science.gov (United States)

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

    2016-11-01

    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. 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. 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. 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. 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. O mecanismo da disfunção vascular induzido pelo consumo de etanol não é totalmente compreendido. Justifica-se, assim a identificação de mecanismos bioquímicos e moleculares que poderiam explicar tais efeitos. Investigar se a ingestão aguda de etanol ativa a via vascular RhoA/Rho quinase

  7. Gold electrode modified with a self-assembled glucose oxidase and 2,6-pyridinedicarboxylic acid as novel glucose bioanode for biofuel cells

    NARCIS (Netherlands)

    Ammam, Malika; Fransaer, Jan

    2014-01-01

    In this study, we have constructed a gold electrode modified with (3-aminopropyl)trimethoxysilane/2,6-pyridinedicarboxylic acid/glucose oxidase (abbreviated as, Au/ATS/PDA/GOx) by sequential chemical adsorption. Au/ATS/PDA/GOx electrode was characterized by Fourier Transform Infrared Spectroscopy

  8. RXR agonists inhibit high glucose-induced upregulation of inflammation by suppressing activation of the NADPH oxidase-nuclear factor-κB pathway in human endothelial cells.

    Science.gov (United States)

    Ning, R B; Zhu, J; Chai, D J; Xu, C S; Xie, H; Lin, X Y; Zeng, J Z; Lin, J X

    2013-12-13

    An inflammatory response induced by high glucose is a cause of endothelial dysfunction in diabetes and is an important contributing link to atherosclerosis. Diabetes is an independent risk factor of atherosclerosis and activation of retinoid X receptor (RXR) has been shown to exert anti-atherogenic effects. In the present study, we examined the effects of the RXR ligands 9-cis-retinoic acid (9-cis-RA) and SR11237 on high glucose-induced inflammation in human umbilical endothelial vein endothelial cells (HUVECs) and explored the potential mechanism. Our results showed that the inflammation induced by high-glucose in HUVECs was mainly mediated by the activation of nuclear factor-B (NF- κB). High glucose-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were in comparison, significantly decreased by treatment with RXR. The effect of RXR agonists was mainly due to the inhibition of NF-κB activation. Using pharmacological inhibitors and siRNA, we confirmed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was an upstream activator of NF-κB. Furthermore, RXR agonists significantly inhibited high glucose-induced activation of NADPH oxidase and significantly decreased the production of reactive oxygen species (ROS). To explore whether the rapid inhibitory effects of RXR agonists were in fact mediated by RXR, we examined the effect of RXR downregulation by RXR siRNA. Our results showed that RXR siRNA largely abrogated the effects of RXR agonists, suggesting the requirement of RXR expression. Therefore, we have shown that RXR is involved in the regulation of NADPH oxidase- NF-κB signal pathway, as the RXR ligands antagonized the inflammatory response in HUVECs induced by high glucose.

  9. Impact of single anaerobic exercise on delayed activation of endothelial xanthine oxidase in men and women.

    Science.gov (United States)

    Wiecek, Magdalena; Maciejczyk, Marcin; Szymura, Jadwiga; Kantorowicz, Malgorzata; Szygula, Zbigniew

    2017-11-01

    The aim of the study was to evaluate the activity of xanthine oxidase (XO) in the blood of men and women during the first hour following a single anaerobic exercise (AN-EX), and after 24 hours of recovery, and to determine whether the changes in XO activity in the blood after AN-EX are dependent on anaerobic performance. Ten men and ten women performed a single AN-EX. Blood was collected before and five times after completion of the AN-EX. The activity of XO was determined. In both groups, a significant (P women (P work performed during the AN-EX and the power decrease. In the first hour after the single AN-EX, XO activity in the blood of women and men did not change, but after 24 hours of recovery, it was significantly higher compared to baseline levels in both sexes. Single AN-EX causes a smaller increase in XO activity in people with higher anaerobic performance.

  10. Radiation effects on diamine oxidase activities in intestine and plasma of the rat

    International Nuclear Information System (INIS)

    Ely, M.J.; Speicher, J.M.; Snyder, S.L.; Catravas, G.N.

    1985-01-01

    Diamine oxidase (DAO; EC 1.4.3.6) activity was measured in plasma and ileal tissue homogenates prepared from male Sprague-Dawley rats sacrificed at 1-15 days after acute whole-body irradiation with 14.5-MeV electrons. Animals irradiated with 1 Gy showed no significant changes in plasma and ileal DAO activities through day 13 relative to nonirradiated controls. Animals irradiated with 5, 10 and 12 Gy displayed marked declines in ileal DAO, with levels reaching a nadir on day 3. This was paralleled by a decrease in plasma DAO activity in all three dose groups. Recovery of ileal and plasma DAO levels was later seen as early as day 4 in animals irradiated with 5 and 10 Gy doses, but animals receiving 12 Gy did not survive beyond day 3. A further study highlights the relationship between radiation dose and levels of plasma and mucosal DAO on day 3, the time of maximum decrease at all doses tested. Mucosal DAO activity decreased almost linearly with doses up to 6 Gy. Plasma DAO levels closely paralleled the dose dependency of the mucosal levels. These data suggest that plasma DAO activity might be useful as a readily measurable marker of intestinal epithelial injury and recovery after acute radiation exposure

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

    Science.gov (United States)

    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 (Pextra 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Minimizing the effects of oxygen interference on l-lactate sensors by a single amino acid mutation in Aerococcus viridansl-lactate oxidase.

    Science.gov (United States)

    Hiraka, Kentaro; Kojima, Katsuhiro; Lin, Chi-En; Tsugawa, Wakako; Asano, Ryutaro; La Belle, Jeffrey T; Sode, Koji

    2018-04-30

    l-lactate biosensors employing l-lactate oxidase (LOx) have been developed mainly to measure l-lactate concentration for clinical diagnostics, sports medicine, and the food industry. Some l-lactate biosensors employ artificial electron mediators, but these can negatively impact the detection of l-lactate by competing with the primary electron acceptor: molecular oxygen. In this paper, a strategic approach to engineering an AvLOx that minimizes the effects of oxygen interference on sensor strips was reported. First, we predicted an oxygen access pathway in Aerococcus viridans LOx (AvLOx) based on its crystal structure. This was subsequently blocked by a bulky amino acid substitution. The resulting Ala96Leu mutant showed a drastic reduction in oxidase activity using molecular oxygen as the electron acceptor and a small increase in dehydrogenase activity employing an artificial electron acceptor. Secondly, the Ala96Leu mutant was immobilized on a screen-printed carbon electrode using glutaraldehyde cross-linking method. Amperometric analysis was performed with potassium ferricyanide as an electron mediator under argon or atmospheric conditions. Under argon condition, the response current increased linearly from 0.05 to 0.5mM l-lactate for both wild-type and Ala96Leu. However, under atmospheric conditions, the response of wild-type AvLOx electrode was suppressed by 9-12% due to oxygen interference. The Ala96Leu mutant maintained 56-69% of the response current at the same l-lactate level and minimized the relative bias error to -19% from -49% of wild-type. This study provided significant insight into the enzymatic reaction mechanism of AvLOx and presented a novel approach to minimize oxygen interference in sensor applications, which will enable accurate detection of l-lactate concentrations. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Changes in the activity of the alternative oxidase in Orobanche seeds during conditioning and their possible physiological function.

    Science.gov (United States)

    Bar Nun, Nurit; Plakhine, Dina; Joel, Daniel M; Mayer, Alfred M

    2003-09-01

    The appearance of the activity of the cyanide insensitive, alternative oxidase (AOX), pathway of oxygen uptake was followed in seeds of Orobanche aegyptiaca during conditioning. The pathway becomes operative during conditioning, up to day three as determined by inhibition of oxygen uptake of the seeds by propyl gallate. At the same time an increasing percentage of oxygen uptake is insensitive to cyanide and an increased oxygen uptake, responsive to propyl gallate, is induced by brief salicylic acid treatment of seeds. By day six of conditioning, these responses decrease and the AOX pathway could not be detected in germinating seeds, after treatment with a germination stimulant. These results were confirmed by following the reaction of extracts of fractions enriched with mitochondria from the conditioned seeds, using a specific antibody against AOX. Treatment of the seeds with inhibitors of AOX during conditioning significantly inhibited their subsequent germination. Addition of hydrogen peroxide after 4 and 7 days of conditioning resulted in reduced germination. In addition treatment of seed with propyl or octyl gallate during conditioning reduced the infection of tomato plants by Orobanche seeds and the development of tubercles of the parasite on the host roots. These results together indicate that the operation of AOX during conditioning has a significant function on the subsequent germination behaviour and pathogenicity of the root parasite. Some potential practical applications of these findings are discussed.

  14. Methadone, monoamine oxidase, and depression: opioid distribution and acute effects on enzyme activity

    International Nuclear Information System (INIS)

    Kaufmann, C.A.; Kreek, M.J.; Raghunath, J.; Arns, P.

    1983-01-01

    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 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

  15. Nitric oxide production and monoamine oxidase activity in cancer patients during interferon-alpha therapy.

    Science.gov (United States)

    Fekkes, Durk; Van Gool, Arthur R; Bannink, Marjolein; Sleijfer, Stefan; Kruit, Wim H J; van der Holt, Bronno; Eggermont, Alexander M M; Hengeveld, Michiel W; Stoter, Gerrit

    2009-10-01

    Both increased and decreased nitric oxide (NO) synthesis have been reported in patients treated with interferon-alpha (IFN-alpha). Animal studies showed that IFN-alpha administration results in increased levels of biogenic amines, subsequent activation of monoamine oxidases (MAOs), and finally in a change in NO production due to the H(2)O(2) generated by MAOs. We examined the potential relationship between NO production in plasma and MAO-B activity in platelets of 43 cancer patients during 8 weeks of treatment with IFN-alpha. NO synthesis was quantitated by measuring both the ratio of citrulline and arginine (CIT/ARG-ratio) and total nitrite/nitrate (NOx) levels. Compared to baseline, MAO activity and NOx increased, while the CIT/ARG-ratio decreased. No associations were found between NOx, MAO and CIT/ARG-ratio. Only few associations were observed between changes in the biochemical parameters and changes in psychopathology induced by IFN-alpha, of which the association between changes in CIT and lassitude was the most consistent. The results suggest that peripheral NO production and MAO activity are unrelated to each other, and that peripheral changes in these biochemical parameters induced by IFN-alpha are unlikely to contribute to definite psychiatric disturbance.

  16. Structure–Activity Relationship of Xanthones as Inhibitors of Xanthine Oxidase

    Directory of Open Access Journals (Sweden)

    Ling-Yun Zhou

    2018-02-01

    Full Text Available Polygala plants contain a large number of xanthones with good physiological activities. In our previous work, 18 xanthones were isolated from Polygala crotalarioides. Extented study of the chemical composition of the other species Polygala sibirica led to the separation of two new xanthones—3-hydroxy-1,2,6,7,8-pentamethoxy xanthone (A and 6-O-β-d-glucopyranosyl-1,7-dimethoxy xanthone (C—together with 14 known xanthones. Among them, some xanthones have a certain xanthine oxidase (XO inhibitory activity. Furthemore, 14 xanthones as XO inhibitors were selected to develop three-dimensional quantitative structure–activity relationship (3D-QSAR using comparative molecular field analysis (CoMFA and comparative molecular similarity indices analysis (CoMSIA models. The CoMFA model predicted a q2 value of 0.613 and an r2 value of 0.997. The best CoMSIA model predicted a q2 value of 0.608 and an r2 value of 0.997 based on a combination of steric, electrostatic, and hydrophobic effects. The analysis of the contour maps from each model provided insight into the structural requirements for the development of more active XO inhibitors.

  17. Methadone, monoamine oxidase, and depression: opioid distribution and acute effects on enzyme activity

    Energy Technology Data Exchange (ETDEWEB)

    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.

  18. Exposure to GSM 900 MHz electromagnetic fields affects cerebral cytochrome c oxidase activity

    International Nuclear Information System (INIS)

    Ammari, Mohamed; Lecomte, Anthony; Sakly, Mohsen; Abdelmelek, Hafedh; Seze, Rene de

    2008-01-01

    The world-wide and rapidly growing use of mobile phones has raised serious concerns about the biological and health-related effects of radio frequency (RF) radiation, particularly concerns about the effects of RFs upon the nervous system. The goal of this study was conducted to measure cytochrome oxidase (CO) levels using histochemical methods in order to evaluate regional brain metabolic activity in rat brain after exposure to a GSM 900 MHz signal for 45 min/day at a brain-averaged specific absorption rate (SAR) of 1.5 W/Kg or for 15 min/day at a SAR of 6 W/Kg over seven days. Compared to the sham and control cage groups, rats exposed to a GSM signal at 6 W/Kg showed decreased CO activity in some areas of the prefrontal and frontal cortex (infralimbic cortex, prelimbic cortex, primary motor cortex, secondary motor cortex, anterior cingulate cortex areas 1 and 2 (Cg1 and Cg2)), the septum (dorsal and ventral parts of the lateral septal nucleus), the hippocampus (dorsal field CA1, CA2 and CA3 of the hippocampus and dental gyrus) and the posterior cortex (retrosplenial agranular cortex, primary and secondary visual cortex, perirhinal cortex and lateral entorhinal cortex). However, the exposure to GSM at 1.5 W/Kg did not affect brain activity. Our results indicate that 6 W/Kg GSM 900 MHz microwaves may affect brain metabolism and neuronal activity in rats

  19. ASIC-like currents in freshly isolated cerebral artery smooth muscle cells are inhibited by endogenous oxidase activity.

    Science.gov (United States)

    Chung, Wen-Shuo; Farley, Jerry M; Drummond, Heather A

    2011-01-01

    The aim of this study was to determine if VSMC ASIC-like currents are regulated by oxidative state. We used whole-cell patch clamp of isolated mouse cerebral VSMCs to determine if 1) reducing agents, such as DTT and GSH, and 2) inhibition of endogenous oxidase activity from NADPH and Xanthine oxidases potentiate active currents and activate electrically silent currents. Pretreatment with 2 mM DTT or GSH, increased the mean peak amplitude of ASIC-like currents evoked by pH 6.0 from 0.4 ± 0.1 to 14.9 ± 3.6 pA/pF, and from 0.9 ± 0.3 to 11.3 ± 2.4 pA/pF, respectively. Pretreatment with apocynin, a NADPH oxidase inhibitor, mimics the effect of the reducing agents, with the mean peak current amplitude increased from 0.9 ± 0.5 to 7.0 ± 2.6 pA/pF and from 0.5 ± 0.2 to 26.4 ± 6.8 pA/pF by 50 and 200 μM apocynin, respectively. Pretreatment with allopurinol, a xanthine oxidase inhibitor, also potentiates the VSMC ASIC-like activity. These findings suggest that VSMC ASIC-like channels are regulated by oxidative state and may be inhibited by basal endogenous oxidative sources such as NADPH and xanthine oxidase. Copyright © 2011 S. Karger AG, Basel.

  20. Evaluation of oxalate decarboxylase and oxalate oxidase for industrial applications.

    Science.gov (United States)

    Cassland, Pierre; Sjöde, Anders; Winestrand, Sandra; Jönsson, Leif J; Nilvebrant, Nils-Olof

    2010-05-01

    Increased recirculation of process water has given rise to problems with formation of calcium oxalate incrusts (scaling) in the pulp and paper industry and in forest biorefineries. The potential in using oxalate decarboxylase from Aspergillus niger for oxalic acid removal in industrial bleaching plant filtrates containing oxalic acid was examined and compared with barley oxalate oxidase. Ten different filtrates from chemical pulping were selected for the evaluation. Oxalate decarboxylase degraded oxalic acid faster than oxalate oxidase in eight of the filtrates, while oxalate oxidase performed better in one filtrate. One of the filtrates inhibited both enzymes. The potential inhibitory effect of selected compounds on the enzymatic activity was tested. Oxalate decarboxylase was more sensitive than oxalate oxidase to hydrogen peroxide. Oxalate decarboxylase was not as sensitive to chlorate and chlorite as oxalate oxidase. Up to 4 mM chlorate ions, the highest concentration tested, had no inhibitory effect on oxalate decarboxylase. Analysis of the filtrates suggests that high concentrations of chlorate present in some of the filtrates were responsible for the higher sensitivity of oxalate oxidase in these filtrates. Oxalate decarboxylase was thus a better choice than oxalate oxidase for treatment of filtrates from chlorine dioxide bleaching.

  1. 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

    OpenAIRE

    Kriaa, Mouna; Ouhibi, Rabeb; Graba, Héla; Besbes, Souhail; Jardak, Mohamed; Kammoun, Radhouane

    2015-01-01

    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. Furthermo...

  2. Carnosine: effect on aging-induced increase in brain regional monoamine oxidase-A activity.

    Science.gov (United States)

    Banerjee, Soumyabrata; Poddar, Mrinal K

    2015-03-01

    Aging is a natural biological process associated with several neurological disorders along with the biochemical changes in brain. Aim of the present investigation is to study the effect of carnosine (0.5-2.5μg/kg/day, i.t. for 21 consecutive days) on aging-induced changes in brain regional (cerebral cortex, hippocampus, hypothalamus and pons-medulla) mitochondrial monoamine oxidase-A (MAO-A) activity with its kinetic parameters. The results of the present study are: (1) The brain regional mitochondrial MAO-A activity and their kinetic parameters (except in Km of pons-medulla) were significantly increased with the increase of age (4-24 months), (2) Aging-induced increase of brain regional MAO-A activity including its Vmax were attenuated with higher dosages of carnosine (1.0-2.5μg/kg/day) and restored toward the activity that observed in young, though its lower dosage (0.5μg/kg/day) were ineffective in these brain regional MAO-A activity, (3) Carnosine at higher dosage in young rats, unlike aged rats significantly inhibited all the brain regional MAO-A activity by reducing their only Vmax excepting cerebral cortex, where Km was also significantly enhanced. These results suggest that carnosine attenuated the aging-induced increase of brain regional MAO-A activity by attenuating its kinetic parameters and restored toward the results of MAO-A activity that observed in corresponding brain regions of young rats. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

  3. Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor

    Science.gov (United States)

    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.

  4. Promising perspectives for ruminal protection of polyunsaturated fatty acids through polyphenol-oxidase-mediated crosslinking of interfacial protein in emulsions.

    Science.gov (United States)

    De Neve, N; Vlaeminck, B; Gadeyne, F; Claeys, E; Van der Meeren, P; Fievez, V

    2018-03-16

    Previously, polyunsaturated fatty acids (PUFA) from linseed oil were effectively protected (>80%) against biohydrogenation through polyphenol-oxidase-mediated protein crosslinking of an emulsion, prepared with polyphenol oxidase (PPO) extract from potato tuber peelings. However, until now, emulsions of only 2 wt% oil have been successfully protected, which implies serious limitations both from a research perspective (e.g. in vivo trials) as well as for further upscaling toward practical applications. Therefore, the aim of this study was to increase the oil/PPO ratio. In the original protocol, the PPO extract served both an emulsifying function as well as a crosslinking function. Here, it was first evaluated whether alternative protein sources could replace the emulsifying function of the PPO extract, with addition of PPO extract and 4-methylcatechol (4MC) to induce crosslinking after emulsion preparation. This approach was then further used to evaluate protection of emulsions with higher oil content. Five candidate emulsifiers (soy glycinin, gelatin, whey protein isolate (WPI), bovine serum albumin and sodium caseinate) were used to prepare 10 wt% oil emulsions, which were diluted five times (w/w) with PPO extract (experiment 1). As a positive control, 2 wt% oil emulsions were prepared directly with PPO extract according to the original protocol. Further, emulsions of 2, 4, 6, 8 and 10 wt% oil were prepared, with 80 wt% PPO extract (experiment 2), or with 90, 80, 70, 60 and 50 wt% PPO extract, respectively (experiment 3) starting from WPI-stabilized emulsions. Enzymatic crosslinking was induced by 24-h incubation with 4MC. Ruminal protection efficiency was evaluated by 24-h in vitro batch simulation of the rumen metabolism. In experiment 1, protection efficiencies were equal or higher than the control (85.5% to 92.5% v. 81.3%). In both experiments 2 and 3, high protection efficiencies (>80%) were achieved, except for emulsions containing 10 wt% oil emulsions

  5. Monoamine Oxidase-A Inhibition and Associated Antioxidant Activity in Plant Extracts with Potential Antidepressant Actions

    Directory of Open Access Journals (Sweden)

    Tomás Herraiz

    2018-01-01

    Full Text Available Monoamine oxidase (MAO catalyzes the oxidative deamination of amines and neurotransmitters and is involved in mood disorders, depression, oxidative stress, and adverse pharmacological reactions. This work studies the inhibition of human MAO-A by Hypericum perforatum, Peganum harmala, and Lepidium meyenii, which are reported to improve and affect mood and mental conditions. Subsequently, the antioxidant activity associated with the inhibition of MAO is determined in plant extracts for the first time. H. perforatum inhibited human MAO-A, and extracts from flowers gave the highest inhibition (IC50 of 63.6 μg/mL. Plant extracts were analyzed by HPLC-DAD-MS and contained pseudohypericin, hypericin, hyperforin, adhyperforin, hyperfirin, and flavonoids. Hyperforin did not inhibit human MAO-A and hypericin was a poor inhibitor of this isoenzyme. Quercetin and flavonoids significantly contributed to MAO-A inhibition. P. harmala seed extracts highly inhibited MAO-A (IC50 of 49.9 μg/L, being a thousand times more potent than H. perforatum extracts owing to its content of β-carboline alkaloids (harmaline and harmine. L. meyenii root (maca extracts did not inhibit MAO-A. These plants may exert protective actions related to antioxidant effects. Results in this work show that P. harmala and H. perforatum extracts exhibit antioxidant activity associated with the inhibition of MAO (i.e., lower production of H2O2.

  6. Novel p47(phox)-related organizers regulate localized NADPH oxidase 1 (Nox1) activity.

    Science.gov (United States)

    Gianni, Davide; Diaz, Begoña; Taulet, Nicolas; Fowler, Bruce; Courtneidge, Sara A; Bokoch, Gary M

    2009-09-15

    The mechanisms that determine localized formation of reactive oxygen species (ROS) through NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (Nox) family members in nonphagocytic cells are unknown. We show that the c-Src substrate proteins Tks4 (tyrosine kinase substrate with four SH3 domains) and Tks5 are functional members of a p47(phox)-related organizer superfamily. Tks proteins selectively support Nox1 and Nox3 (and not Nox2 and Nox4) activity in reconstituted cellular systems and interact with the NoxA1 activator protein through an Src homology 3 domain-mediated interaction. Endogenous Tks4 is required for Rac guanosine triphosphatase- and Nox1-dependent ROS production by DLD1 colon cancer cells. Our results are consistent with the Tks-mediated recruitment of Nox1 to invadopodia that form in DLD1 cells in a Tks- and Nox-dependent fashion. We propose that Tks organizers represent previously unrecognized members of an organizer superfamily that link Nox to localized ROS formation.

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

    Directory of Open Access Journals (Sweden)

    Arthur I. Cederbaum

    2014-01-01

    Full Text Available 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.

  8. Monoamine oxidase B (MAO-B) inhibition by active principles from Uncaria rhynchophylla.

    Science.gov (United States)

    Hou, Wen-Chi; Lin, Rong-Dih; Chen, Cheng-Tang; Lee, Mei-Hsien

    2005-08-22

    Attenuation of monoamine oxidase B (MAO-B) activity may provide protection against oxidative neurodegeneration. For this reason, inhibition of MAO-B activity is used as part of the treatment of Parkinson's and Alzheimer's patients. The hook of Uncaria rhynchophylla (Miq.) Jacks. (Rubiaceae) is a traditional Chinese herbal drug that is generally used to treat convulsive disorders. In this study, the fractionation and purification of Uncaria rhynchophylla extracts using a bioguided assay isolated two known compounds, (+)-catechin and (-)-epicatechin. The compounds inhibited MAO-B, as measured by an assay of rat brain MAO-B separated by electrophoresis on a 7.5% native polyacrylamide gel. The IC(50) values of (+)-catechin and (-)-epicatechin were 88.6 and 58.9 microM, respectively, and inhibition occurred in a dose-dependent manner, as measured by the fluorescence method. The Lineweaver-Burk plot revealed K(i) values for (+)-catechin and (-)-epicatechin of 74 and 21 microM, respectively. This suggests that these two compounds, isolated here for the first time from Uncaria rhynchophylla, might be able to protect against neurodegeneration in vitro, and, therefore, the molecular mechanism deserves further study. This finding may also increase interest in the health benefits of Uncaria rhynchophylla.

  9. Is Xanthine oxidase activity in polycystic ovary syndrome associated with inflammatory and cardiovascular risk factors?

    Science.gov (United States)

    Isık, Hatice; Aynıoglu, Oner; Tımur, Hakan; Sahbaz, Ahmet; Harma, Muge; Can, Murat; Guven, Berrak; Alptekin, Husnu; Kokturk, Furuzan

    2016-08-01

    The aim of this study is to examine women with polycystic ovary syndrome (PCOS) to determine the relationship between xanthine oxidase (XO) and oxidative stress, inflammatory status, and various clinical and biochemical parameters. In this cross-sectional study a total of 83 women including 45 PCOS patients and 38 healthy women were enrolled. We collected blood samples for XO and superoxide dismutase (SOD) activity, hormone levels, cholesterol values, and inflammatory markers. Body mass index (BMI) , waist-to-hip ratio (WHR), and blood pressure were assessed. Blood samples were taken for hormonal levels, cholesterol levels, fasting plasma glucose (FPG), fasting plasma insulin (FPI), homeostatic model assessment-insulin resistance (HOMA-IR) index, quantitative insulin sensitivity check index (QUICKI), C-reactive protein (CRP), white blood cell and neutrophil counts, XO and SOD activities. The basal hormone levels, triglyceride (TG) levels, TG/HDL-C (high density lipoprotein-cholesterol) ratios FPG, FPI and HOMA-IR levels were higher in PCOS patients compared to controls (pPCOS patients (pPCOS patients. Positive correlations between XO and inflammatory markers and cardiovascular disease risk factors suggest that XO plays an important role in the pathogenesis of PCOS and its metabolic complications. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. Improvement of the stability and activity of immobilized glucose oxidase on modified iron oxide magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Abbasi, Mahboube, E-mail: mahbubeabbasi@yahoo.com; Amiri, Razieh, E-mail: razieh.amiri@gmail.com; Bordbar, Abdol-Kalegh, E-mail: bordbar@chem.ui.ac.ir; Ranjbakhsh, Elnaz, E-mail: e.ranjbakhsh@yahoo.com; Khosropour, Ahmad-Reza, E-mail: khosropour@chem.ui.ac.ir

    2016-02-28

    Graphical abstract: - Highlights: • Modified iron oxide magnetic nanoparticles were synthesized by co-precipitation method and characterized by TEM and XRD. • Covalent attachment of GOX to MIMNs was confirmed by FT-IR technique. • Optimization of the reaction time and initial amount of the GOX were carried out. • Improvement of activity and stability of immobilized GOX have been increased in comparison of free GOX. - Abstract: 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.

  11. D-Amino acid oxidase-induced oxidative stress, 3-bromopyruvate and citrate inhibit angiogenesis, exhibiting potent anticancer effects.

    Science.gov (United States)

    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.

  12. Basal levels of metabolic activity are elevated in Genetic Absence Epilepsy Rats from Strasbourg (GAERS): measurement of regional activity of cytochrome oxidase and lactate dehydrogenase by histochemistry.

    Science.gov (United States)

    Dufour, Franck; Koning, Estelle; Nehlig, Astrid

    2003-08-01

    The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are considered an isomorphic, predictive, and homologous model of human generalized absence epilepsy. It is characterized by the expression of spike-and-wave discharges in the thalamus and cortex. In this strain, basal regional rates of cerebral glucose utilization measured by the quantitative autoradiographic [(14)C]2-deoxyglucose technique display a widespread consistent increase compared to a selected strain of genetically nonepileptic rats (NE). In order to verify whether these high rates of glucose metabolism are paralleled by elevated activities of the enzymes of the glycolytic and tricarboxylic acid cycle pathways, we measured by histochemistry the regional activity of the two key enzymes of glucose metabolism, lactate dehydrogenase (LDH) for the anaerobic pathway and cytochrome oxidase (CO) for the aerobic pathway coupled to oxidative phosphorylation. CO and LDH activities were significantly higher in GAERS than in NE rats in 24 and 28 of the 30 brain regions studied, respectively. The differences in CO and LDH activity between both strains were widespread, affected all brain systems studied, and ranged from 12 to 63%. The data of the present study confirm the generalized increase in cerebral glucose metabolism in GAERS, occurring both at the glycolytic and at the oxidative step. However, they still do not allow us to understand why the ubiquitous mutation(s) generates spike-and-wave discharges only in the thalamocortical circuit.

  13. Role of ascorbic acid in the inhibition of polyphenol oxidase and the prevention of browning in different browning-sensitive Lactuca sativa var. capitata (L.) and Eruca sativa (Mill.) stored as fresh-cut produce.

    Science.gov (United States)

    Landi, Marco; Degl'Innocenti, Elena; Guglielminetti, Lorenzo; Guidi, Lucia

    2013-06-01

    Polyphenol oxidase (PPO) and, to a minor extent, peroxidase (POD) represent the key enzymes involved in enzymatic browning, a negative process induced by cutting fresh-cut produce such as lettuce (Lactuca sativa) and rocket salad (Eruca sativa). Although ascorbic acid is frequently utilised as an anti-browning agent, its mechanism in the prevention of the browning phenomenon is not clearly understood. The activity of PPO and POD and their isoforms in lettuce (a high-browning and low-ascorbic acid species) and rocket salad (a low-browning and high-ascorbic species) was characterised. The kinetic parameters of PPO and in vitro ascorbic acid-PPO inhibition were also investigated. In rocket salad, PPO activity was much lower than that in lettuce and cutting induced an increase in PPO activity only in lettuce. Exogenous ascorbic acid (5 mmol L(-1)) reduced PPO activity by about 90% in lettuce. POD did not appear to be closely related to browning in lettuce. PPO is the main enzyme involved in the browning phenomenon; POD appears to play a minor role. The concentration of endogenous ascorbic acid in rocket salad was related to its low-browning sensitivity after cutting. In lettuce, the addition of ascorbic acid directly inhibited PPO activity. The results suggest that the high ascorbic acid content found in rocket salad plays an effective role in reducing PPO activity. © 2012 Society of Chemical Industry.

  14. Oxidative Ce"3"+ sequestration by fungal manganese oxides with an associated Mn(II) oxidase activity

    International Nuclear Information System (INIS)

    Zheng, Haisu; Tani, Yukinori; Naitou, Hirotaka; Miyata, Naoyuki; Tojo, Fuyumi

    2016-01-01

    Sequestration of Ce"3"+ by biogenic manganese oxides (BMOs) formed by a Mn(II)-oxidizing fungus, Acremonium strictum strain KR21-2, was examined at pH 6.0. In anaerobic Ce"3"+ solution, newly formed BMOs exhibited stoichiometric Ce"3"+ oxidation, where the molar ratio of Ce"3"+ sequestered (Ce_s_e_q) relative to Mn"2"+ released (Mn_r_e_l) was maintained at approximately two throughout the reaction. A similar Ce"3"+ sequestration trend was observed in anaerobic treatment of BMOs in which the associated Mn(II) oxidase was completely inactivated by heating at 85 °C for 1 h or by adding 50 mM NaN_3. Aerobic Ce"3"+ treatment of newly formed BMO (enzymatically active) resulted in excessive Ce"3"+ sequestration over Mn"2"+ release, yielding Ce_s_e_q/Mn_r_e_l > 200, whereas heated or poisoned BMOs released a significant amount of Mn"2"+ with lower Ce"3"+ sequestration efficiency. Consequently, self-regeneration by the Mn(II) oxidase in newly formed BMO effectively suppressed Mn"2"+ release and enhanced oxidative Ce"3"+ sequestration under aerobic conditions. Repeated treatments of heated or poisoned BMOs under aerobic conditions confirmed that oxidative Ce"3"+ sequestration continued even after most Mn oxide was released from the solid phase, indicating auto-catalytic Ce"3"+ oxidation at the solid phase produced through primary Ce"3"+ oxidation by BMO. From X-ray diffraction analysis, the resultant solid phases formed through Ce"3"+ oxidation by BMO under both aerobic and anaerobic conditions consisted of cerianite with crystal sizes of 5.00–7.23 Å. Such nano-sized CeO_2 (CeO_2_,_B_M_O) showed faster auto-catalytic Ce"3"+ oxidation than that on well-crystalized cerianite under aerobic conditions, where the normalized pseudo-first order rate constants for auto-catalytic Ce"3"+ oxidation on CeO_2_,_B_M_O was two orders of magnitude higher. Consequently, we concluded that Ce"3"+ contact with BMOs sequesters Ce"3"+ through two oxidation paths: primary Ce"3

  15. Low serum diamine oxidase (DAO) activity levels in patients with migraine.

    Science.gov (United States)

    Izquierdo-Casas, Joan; Comas-Basté, Oriol; Latorre-Moratalla, M Luz; Lorente-Gascón, Marian; Duelo, Adriana; Vidal-Carou, M Carmen; Soler-Singla, Luis

    2018-02-01

    Histamine intolerance is a disorder in the homeostasis of histamine due to a reduced intestinal degradation of this amine, mainly caused by a deficiency in the enzyme diamine oxidase (DAO). Among the several multi-faced symptoms associated with histamine intolerance, headache is one of the most recognized and disabling consequences. The aim of this study was to determine the prevalence of DAO deficiency in patients with a confirmed migraine diagnosis according to the current International Headache Society (IHS) and in non-migraine subjects. DAO activity was assessed in a total of 198 volunteers recruited at the Headache Unit of the Hospital General de Catalunya, 137 in the migraine group and 61 as a control group. DAO enzyme activity in blood samples was determined by ELISA test. Values below 80 HDU/ml (Histamine Degrading Unit/ml) were considered as DAO deficient. Mean value of DAO activity from migraine population (64.5 ± 33.5 HDU/ml) was significantly lower (p < 0.0001) than that obtained from healthy volunteers (91.9 ± 44.3 HDU/ml). DAO deficiency was more prevalent in migraine patients than in the control group. A high incidence rate of DAO deficiency (87%) was observed in the group of patients with migraine. On the other hand, 44% of non-migranous subjects had levels of DAO activity lower than 80 HDU/ml. Despite the multifactorial aetiology of migraine, these results seem to indicate that this enzymatic deficit could be related to the onset of migraine.

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

    Science.gov (United States)

    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

  17. Novel multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative diseases: in vitro studies on antioxidant activity, prevention of lipid peroxide formation and monoamine oxidase inhibition.

    Science.gov (United States)

    Zheng, Hailin; Gal, Shunit; Weiner, Lev M; Bar-Am, Orit; Warshawsky, Abraham; Fridkin, Mati; Youdim, Moussa B H

    2005-10-01

    Iron-dependent oxidative stress, elevated levels of iron and of monoamine oxidase (MAO)-B activity, and depletion of antioxidants in the brain may be major pathogenic factors in Parkinson's disease, Alzheimer's disease and related neurodegenerative diseases. Accordingly, iron chelators, antioxidants and MAO-B inhibitors have shown efficacy in a variety of cellular and animal models of CNS injury. In searching for novel antioxidant iron chelators with potential MAO-B inhibitory activity, a series of new iron chelators has been designed, synthesized and investigated. In this study, the novel chelators were further examined for their activity as antioxidants, MAO-B inhibitors and neuroprotective agents in vitro. Three of the selected chelators (M30, HLA20 and M32) were the most effective in inhibiting iron-dependent lipid peroxidation in rat brain homogenates with IC50 values (12-16 microM), which is comparable with that of desferal, a prototype iron chelator that is not has orally active. Their antioxidant activities were further confirmed using electron paramagnetic resonance spectroscopy. In PC12 cell culture, the three novel chelators at 0.1 microM were able to attenuate cell death induced by serum deprivation and by 6-hydroxydopamine. M30 possessing propargyl, the MAO inhibitory moiety of the anti-Parkinson drug rasagiline, displayed greater neuroprotective potency than that of rasagiline. In addition, in vitro, M30 was a highly potent non-selective MAO-A and MAO-B inhibitor (IC50 < 0.1 microM). However, HLA20 was more selective for MAO-B but had poor MAO inhibition, with an IC50 value of 64.2 microM. The data suggest that M30 and HLA20 might serve as leads in developing drugs with multifunctional activities for the treatment of various neurodegenerative disorders.

  18. Variation of the Phytochemical Constituents and Antioxidant Activities of Zingiber officinale var. rubrum Theilade Associated with Different Drying Methods and Polyphenol Oxidase Activity.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Dietmar Enko

    2016-01-01

    Full Text Available 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.

  20. Improvement of the stability and activity of immobilized glucose oxidase on modified iron oxide magnetic nanoparticles

    Science.gov (United States)

    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.

  1. Synthesis and evaluation of 2-benzylidene-1-tetralone derivatives for monoamine oxidase inhibitory activity.

    Science.gov (United States)

    Amakali, Klaudia T; Legoabe, Lesetja Jan; Petzer, Anel; Petzer, Jacobus P

    2018-05-01

    Chalcone has been identified as a promising lead for the design of monoamine oxidase (MAO) inhibitors. This study attempted to discover potent and selective chalcone-derived MAO inhibitors by synthesising a series consisting of various cyclic chalcone derivatives. The cyclic chalcones were selected based on the possibility that their restricted structures would confer a higher degree of MAO isoform selectivity, and included the following chemical classes: 1-indanone, 1-tetralone, 1-benzosuberone, chromone, thiochromone, 4-chromanone and 4-thiochromanone. The results showed that the cyclic chalcones are in general good potency, and in most instances specific inhibitors of the human MAO-B isoform. Among these compounds, the 4-chromanone derivative was the most potent MAO-B inhibitor with an IC50 value of 0.156 µM. To further investigate the MAO inhibition of cyclic chalcones, a series of twenty-three 2-benzylidene-1-tetralone derivatives were synthesised and evaluated as MAO inhibitors. Most 2-benzylidene-1-tetralones possess good inhibitory activity and specificity for MAO-B with the most potent inhibitor displaying an IC50 value of 0.0064 µM, while the most potent MAO-A inhibitor possessed an IC50 value of 0.754 µM. This study thus shows that certain cyclic chalcones are human MAO-B inhibitors, compounds that could be suitable for the treatment of neurodegenerative disorders such as Parkinson's disease. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Sildenafil promotes eNOS activation and inhibits NADPH oxidase in the transgenic sickle cell mouse penis.

    Science.gov (United States)

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

    2014-02-01

    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. 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. 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. Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse. Continuous treatment with sildenafil reversed (P penis. Sildenafil treatment of WT mice did not affect any of these parameters. 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. © 2013 International Society for Sexual Medicine.

  3. 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

    Energy Technology Data Exchange (ETDEWEB)

    Kajikawa, Takao; Kataoka, Kunishige [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Sakurai, Takeshi, E-mail: tsakurai@se.kanazawa-u.ac.jp [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)

    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.

  4. Evaluation of an antimicrobial L-amino acid oxidase and peptide derivatives from Bothropoides mattogrosensis pitviper venom.

    Directory of Open Access Journals (Sweden)

    Brunna M Okubo

    Full Text Available Healthcare-associated infections (HAIs are causes of mortality and morbidity worldwide. The prevalence of bacterial resistance to common antibiotics has increased in recent years, highlighting the need to develop novel alternatives for controlling these pathogens. Pitviper venoms are composed of a multifaceted mixture of peptides, proteins and inorganic components. L-amino oxidase (LAO is a multifunctional enzyme that is able to develop different activities including antibacterial activity. In this study a novel LAO from Bothrops mattogrosensis (BmLAO was isolated and biochemically characterized. Partial enzyme sequence showed full identity to Bothrops pauloensis LAO. Moreover, LAO here isolated showed remarkable antibacterial activity against Gram-positive and -negative bacteria, clearly suggesting a secondary protective function. Otherwise, no cytotoxic activities against macrophages and erythrocytes were observed. Finally, some LAO fragments (BmLAO-f1, BmLAO-f2 and BmLAO-f3 were synthesized and further evaluated, also showing enhanced antimicrobial activity. Peptide fragments, which are the key residues involved in antimicrobial activity, were also structurally studied by using theoretical models. The fragments reported here may be promising candidates in the rational design of new antibiotics that could be used to control resistant microorganisms.

  5. Contribution of NADPH Oxidase to Membrane CD38 Internalization and Activation in Coronary Arterial Myocytes

    Science.gov (United States)

    Xu, Ming; Li, Xiao-Xue; Ritter, Joseph K.; Abais, Justine M.; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    The CD38-ADP-ribosylcyclase-mediated Ca2+ signaling pathway importantly contributes to the vasomotor response in different arteries. Although there is evidence indicating that the activation of CD38-ADP-ribosylcyclase is associated with CD38 internalization, the molecular mechanism mediating CD38 internalization and consequent activation in response to a variety of physiological and pathological stimuli remains poorly understood. Recent studies have shown that CD38 may sense redox signals and is thereby activated to produce cellular response and that the NADPH oxidase isoform, NOX1, is a major resource to produce superoxide (O2 ·−) in coronary arterial myocytes (CAMs) in response to muscarinic receptor agonist, which uses CD38-ADP-ribosylcyclase signaling pathway to exert its action in these CAMs. These findings led us hypothesize that NOX1-derived O2 ·− serves in an autocrine fashion to enhance CD38 internalization, leading to redox activation of CD38-ADP-ribosylcyclase activity in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2 ·− production and increased CD38-ADP-ribosylcyclase activity in CAMs, while activation of NOX1 by overexpression of Rac1 or Vav2 or administration of exogenous O2 ·− significantly increased CD38 internalization in CAMs. Lastly, ET-1 was found to markedly increase membrane raft clustering as shown by increased colocalization of cholera toxin-B with CD38 and NOX1. Taken together, these results provide direct evidence that Rac1-NOX1-dependent O2 ·− production mediates CD38 internalization in CAMs, which may represent an important mechanism linking receptor activation with CD38 activity in these cells. PMID:23940720

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

    Science.gov (United States)

    Singh, Namrata; Hroudová, Jana; Fišar, Zdeněk

    2017-10-01

    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.

  7. Synthesis and characterization of microparticles based on poly-methacrylic acid with glucose oxidase for biosensor applications.

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. D-amino acid oxidase gene therapy sensitizes glioma cells to the antiglycolytic effect of 3-bromopyruvate.

    Science.gov (United States)

    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.

  9. IAA oxidase activity in relation to adventitious root formation on stem cuttings of some forest tree species. [Salix tetrasperma, Populus Robusta, Hibiscus rosa-sinensis, Eucalyptus citriodora

    Energy Technology Data Exchange (ETDEWEB)

    Bansal, M.P.; Nanda, K.K.

    1981-01-01

    In rooting tests with stem cuttings, IAA oxidase activity was found to be very high in Salix tetrasperma and Populus 'Robusta' both of which rooted profusely, less in Hibiscus rosa-sinensis which rooted but weakly and insignificant in Eucalyptus citriodora, which did not root at all. Proteins extracted from the stem cuttings of E. citriodora inhibited IAA oxidase activity, and also root formation on hypocotyl cuttings of Phaseolus mungo.

  10. A new procedure for the purification of monodisperse highly active cytochrome c oxidase from bovine heart.

    OpenAIRE

    Li, Y; Naqui, A; Frey, T G; Chance, B

    1987-01-01

    A simple and rapid method for the isolation of a large quantity of cytochrome c oxidase from bovine heart mitochondria was developed, based on selective solubilization of mitochondrial protein with first Triton and then lauryl maltoside. Gel filtration shows that the lauryl maltoside-solubilized oxidase preparation is in a hydrodynamically homogeneous state with a Stokes radius of 7.5 +/- 0.2 nm. It contains 8.0 mumol of haem (with an a/a3 ratio of 1)/g of protein. The catalytic constant (max...

  11. ATL9, a RING zinc finger protein with E3 ubiquitin ligase activity implicated in chitin- and NADPH oxidase-mediated defense responses.

    Directory of Open Access Journals (Sweden)

    Marta Berrocal-Lobo

    2010-12-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Jeanne F. Millogo

    2012-06-01

    Full Text Available 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.

  13. Oxidative stress caused by activation of NADPH oxidase 4 promotes contrast-induced acute kidney injury.

    Directory of Open Access Journals (Sweden)

    Bo Young Jeong

    Full Text Available Contrast-induced acute kidney injury (CIAKI is a leading cause of acute kidney injury following radiographic procedures. Intrarenal oxidative stress plays a critical role in CIAKI. Nicotinamide adenine dinucleotide 3-phosphate (NADPH oxidases (Noxs are important sources of reactive oxygen species (ROS. Among the various types of Noxs, Nox4 is expressed predominantly in the kidney in rodents. Here, we evaluated the role of Nox4 and benefit of Nox4 inhibition on CIAKI using in vivo and in vitro models. HK-2 cells were treated with iohexol, with or without Nox4 knockdown, or the most specific Nox1/4 inhibitor (GKT137831. Effects of Nox4 inhibition on CIAKI mice were examined. Expression of Nox4 in HK-2 cells was significantly increased following iohexol exposure. Silencing of Nox4 rescued the production of ROS, downregulated pro-inflammatory markers (particularly phospho-p38 implicated in CIAKI, and reduced Bax and caspase 3/7 activity, which resulted in increased cellular survival in iohexol-treated HK-2 cells. Pretreatment with GKT137831 replicated these effects by decreasing levels of phospho-p38. In a CIAKI mouse model, even though the improvement of plasma blood urea nitrogen was unclear, pretreatment with GKT137831 resulted in preserved structure, reduced expression of 8-hydroxy-2'-deoxyguanosine (8OHdG and kidney injury molecule-1 (KIM-1, and reduced number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. These results suggest Nox4 as a key source of reactive oxygen species responsible for CIAKI and provide a novel potential option for prevention of CIAKI.

  14. Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Jie Hong

    Full Text Available Mechanisms of the progression from Barrett's esophagus (BE to esophageal adenocarcinoma (EA are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

  15. Potato and Mushroom Polyphenol Oxidase Activities Are Differently Modulated by Natural Plant Extracts

    NARCIS (Netherlands)

    Kuijpers, T.F.M.; Herk, van T.; Vincken, J.P.; Janssen, R.H.; Narh, D.L.; Berkel, van W.J.H.; Gruppen, H.

    2014-01-01

    Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the

  16. Validation of tautomeric and protomeric binding modes by free energy calculations. A case study for the structure based optimization of d-amino acid oxidase inhibitors

    Science.gov (United States)

    Orgován, Zoltán; Ferenczy, György G.; Steinbrecher, Thomas; Szilágyi, Bence; Bajusz, Dávid; Keserű, György M.

    2018-02-01

    Optimization of fragment size d-amino acid oxidase (DAAO) inhibitors was investigated using a combination of computational and experimental methods. Retrospective free energy perturbation (FEP) calculations were performed for benzo[d]isoxazole derivatives, a series of known inhibitors with two potential binding modes derived from X-ray structures of other DAAO inhibitors. The good agreement between experimental and computed binding free energies in only one of the hypothesized binding modes strongly support this bioactive conformation. Then, a series of 1-H-indazol-3-ol derivatives formerly not described as DAAO inhibitors was investigated. Binding geometries could be reliably identified by structural similarity to benzo[d]isoxazole and other well characterized series and FEP calculations were performed for several tautomers of the deprotonated and protonated compounds since all these forms are potentially present owing to the experimental pKa values of representative compounds in the series. Deprotonated compounds are proposed to be the most important bound species owing to the significantly better agreement between their calculated and measured affinities compared to the protonated forms. FEP calculations were also used for the prediction of the affinities of compounds not previously tested as DAAO inhibitors and for a comparative structure-activity relationship study of the benzo[d]isoxazole and indazole series. Selected indazole derivatives were synthesized and their measured binding affinity towards DAAO was in good agreement with FEP predictions.

  17. Action of Bothrops moojeni venom and its L-amino acid oxidase fraction, treated with 60Co gamma rays, in Leishmania spp

    International Nuclear Information System (INIS)

    Cardoso, Andre Gustavo Tempone

    1999-01-01

    Bothrops moojeni venom showed an anti leishmania activity in vitro, as determined by a cell viability assay using the reduction of MTT. After venom purification, by chromatography techniques, the fractions with anti leishmania and L-amino acid oxidase activities, eluted in the same positions. The molecular weight of the enzyme was estimated to be 140 kDa by molecular exclusion chromatography, and 69 kDa, by SDS-PAGE, migrating as a single band, with an isoelectric point of 4.8 as determined by isoelectric focusing. The purified LAO from B. moojeni venom, 135-fold more active than crude venom, showed homo dimeric constitution, and was active against Leishmania spp from the New World, with an effective concentration against L(L). amazonensis of 1.80 μg/ml (EC 50 ), L.(V.) panamensis (0.78 |μg/ml) and L.(L.) chagasi (0.63 (μg/ml). Ultrastructural studies of promastigotes affected by LAO demonstrated cell death, with edema in several organelles such as mitochondria and nuclear membrane, before cell disruption and necrosis. The action of LAO was demonstrated to be hydrogen peroxide-dependent. Studies with LLCMK-2 cells, treated with LAO, showed a toxic effect, with an EC 50 of 11|μg/ml. Irradiation of LAO with 6 0C o gamma rays, did not affect its whole oxidative activity, neither detoxified the enzyme. Amastigotes treated with LAO were not affected by its hydrogen peroxide, otherwise, the exogenous product, killed amastigotes with an EC 50 of 0.67mM. These data could be of help in the development of alternative therapeutic approaches to the treatment of leishmaniasis. (author)

  18. Oxidases as Breast Cancer Oncogens

    National Research Council Canada - National Science Library

    Yeldandi, Anjana

    2000-01-01

    ...) in a non-tumorigenic human mammary epithelial cell line to ascertain whether oxidase overexpressing cells undergo transformation when exposed to substrate xanthine for XOX and uric acid for UOX...

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

    Science.gov (United States)

    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.

  20. Acid-activated bentonite

    Indian Academy of Sciences (India)

    In this study, we propose Maghnite-H + , as an ecological, cost-effective and easily renewable catalyst, for the polymerization of decamethylcyclopentasiloxane (D5). The Maghnite is a clay consisting primarily of smectite minerals (montmorillonite group), which can be activated/reactivated through a simple process, ...

  1. NADPH Oxidase Activity in Cerebral Arterioles Is a Key Mediator of Cerebral Small Vessel Disease—Implications for Prevention

    Directory of Open Access Journals (Sweden)

    Mark F. McCarty

    2015-04-01

    Full Text Available Cerebral small vessel disease (SVD, a common feature of brain aging, is characterized by lacunar infarcts, microbleeds, leukoaraiosis, and a leaky blood-brain barrier. Functionally, it is associated with cognitive decline, dementia, depression, gait abnormalities, and increased risk for stroke. Cerebral arterioles in this syndrome tend to hypertrophy and lose their capacity for adaptive vasodilation. Rodent studies strongly suggest that activation of Nox2-dependent NADPH oxidase activity is a crucial driver of these structural and functional derangements of cerebral arterioles, in part owing to impairment of endothelial nitric oxide synthase (eNOS activity. This oxidative stress may also contribute to the breakdown of the blood-brain barrier seen in SVD. Hypertension, aging, metabolic syndrome, smoking, hyperglycemia, and elevated homocysteine may promote activation of NADPH oxidase in cerebral arterioles. Inhibition of NADPH oxidase with phycocyanobilin from spirulina, as well as high-dose statin therapy, may have potential for prevention and control of SVD, and high-potassium diets merit study in this regard. Measures which support effective eNOS activity in other ways—exercise training, supplemental citrulline, certain dietary flavonoids (as in cocoa and green tea, and capsaicin, may also improve the function of cerebral arterioles. Asian epidemiology suggests that increased protein intakes may decrease risk for SVD; conceivably, arginine and/or cysteine—which boosts tissue glutathione synthesis, and can be administered as N-acetylcysteine—mediate this benefit. Ameliorating the risk factors for SVD—including hypertension, metabolic syndrome, hyperglycemia, smoking, and elevated homocysteine—also may help to prevent and control this syndrome, although few clinical trials have addressed this issue to date.

  2. NADPH Oxidase Activity in Cerebral Arterioles Is a Key Mediator of Cerebral Small Vessel Disease-Implications for Prevention.

    Science.gov (United States)

    McCarty, Mark F

    2015-04-15

    Cerebral small vessel disease (SVD), a common feature of brain aging, is characterized by lacunar infarcts, microbleeds, leukoaraiosis, and a leaky blood-brain barrier. Functionally, it is associated with cognitive decline, dementia, depression, gait abnormalities, and increased risk for stroke. Cerebral arterioles in this syndrome tend to hypertrophy and lose their capacity for adaptive vasodilation. Rodent studies strongly suggest that activation of Nox2-dependent NADPH oxidase activity is a crucial driver of these structural and functional derangements of cerebral arterioles, in part owing to impairment of endothelial nitric oxide synthase (eNOS) activity. This oxidative stress may also contribute to the breakdown of the blood-brain barrier seen in SVD. Hypertension, aging, metabolic syndrome, smoking, hyperglycemia, and elevated homocysteine may promote activation of NADPH oxidase in cerebral arterioles. Inhibition of NADPH oxidase with phycocyanobilin from spirulina, as well as high-dose statin therapy, may have potential for prevention and control of SVD, and high-potassium diets merit study in this regard. Measures which support effective eNOS activity in other ways-exercise training, supplemental citrulline, certain dietary flavonoids (as in cocoa and green tea), and capsaicin, may also improve the function of cerebral arterioles. Asian epidemiology suggests that increased protein intakes may decrease risk for SVD; conceivably, arginine and/or cysteine-which boosts tissue glutathione synthesis, and can be administered as N-acetylcysteine-mediate this benefit. Ameliorating the risk factors for SVD-including hypertension, metabolic syndrome, hyperglycemia, smoking, and elevated homocysteine-also may help to prevent and control this syndrome, although few clinical trials have addressed this issue to date.

  3. Characterization and stability of D-amino acid oxidase and catalase ...

    African Journals Online (AJOL)

    ajl yemi

    2011-12-19

    Dec 19, 2011 ... cells was inhibited when treated with sodium azide (0.8 M, 30 min), while no visible effect on the activity and stability of DAAO ... enzyme's persistence against inactivation and facilitate ... components of culture media of this strain were described in a previous ... incubated at different temperature (4 or 30°C).

  4. Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

    Science.gov (United States)

    Jänne, J; Morris, D R

    1984-03-15

    Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylmethionine decarboxylase activity in vitro. The enzyme from both sources was most powerfully inhibited by ethylglyoxal bis(guanylhydrazone). All the diguanidines likewise inhibited diamine oxidase activity in vitro. The maximum intracellular concentrations of the ethyl and dimethylated analogues achieved in activated lymphocytes were only about one-fifth of that of the parent compound. However, both derivatives appeared to utilize the polyamine-carrier system, as indicated by competition experiments with spermidine.

  5. NecroX-7 prevents oxidative stress-induced cardiomyopathy by inhibition of NADPH oxidase activity in rats

    Energy Technology Data Exchange (ETDEWEB)

    Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun; Kim, Hyoung Jin [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Park, Ji-hoon [Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Koo, Sun Young; Kwak, Hyo-Shin; Park, Heui Sul; Kim, Dong Wook; Song, Myoungsub; Yim, Hyeon Joo; Seo, Dong Ook [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of); Kim, Soon Ha, E-mail: shakim@lgls.com [LG Life Sciences Ltd., R and D Park, Daejeon, 305-380 (Korea, Republic of)

    2012-08-15

    Oxidative stress is one of the causes of cardiomyopathy. In the present study, NecroXs, novel class of mitochondrial ROS/RNS scavengers, were evaluated for cardioprotection in in vitro and in vivo model, and the putative mechanism of the cardioprotection of NecroX-7 was investigated by global gene expression profiling and subsequent biochemical analysis. NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced death of H9C2 rat cardiomyocytes at EC{sub 50} = 0.057 μM. In doxorubicin (DOX)-induced cardiomyopathy in rats, NecroX-7 significantly reduced the plasma levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) which were increased by DOX treatment (p < 0.05). Microarray analysis revealed that 21 genes differentially expressed in tBHP-treated H9C2 cells were involved in ‘Production of reactive oxygen species’ (p = 0.022), and they were resolved by concurrent NecroX-7 treatment. Gene-to-gene networking also identified that NecroX-7 relieved cell death through Ncf1/p47phox and Rac2 modulation. In subsequent biochemical analysis, NecroX-7 inhibited NADPH oxidase (NOX) activity by 53.3% (p < 0.001). These findings demonstrate that NecroX-7, in part, provides substantial protection of cardiomyopathy induced by tBHP or DOX via NOX-mediated cell death. -- Highlights: ► NecroX-7 prevented tert-butyl hydroperoxide-induced in vitro cardiac cell death. ► NecroX-7 ameliorated doxorubicin-induced in vivo cardiomyopathy. ► NecroX-7 prevented oxidative stress and necrosis-enriched transcriptional changes. ► NecroX-7 effectively inhibited NADPH oxidase activation. ► Cardioprotection of Necro-7 was brought on by modulation of NADPH oxidase activity.

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

    Science.gov (United States)

    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 k cat /K m and k red for (S)-6-hydroxynicotine, respectively, with larger effects on the k cat /K m value for (S)-6-hydroxynornicotine. The K287M mutation results in ∼10-fold decreases in these parameters and a 6000-fold decrease in the k cat /K m 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 k cat /K m 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.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N 6 -(Δ 2 -isopentenyl)-adenine-2,8- 3 H (i 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 6 -side chain of i 6 Ade

  8. NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)α-mediated cardiovascular effects

    International Nuclear Information System (INIS)

    Newaz, Mohammad; Blanton, Ahmad; Fidelis, Paul; Oyekan, Adebayo

    2005-01-01

    Activation of peroxisome proliferator activated receptor (PPAR)α and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARα ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARα activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARα ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARα knockout (KO) mice compared with its wild type (WT) litter mates (130 ± 10 mmHg versus 107 ± 4 mmHg). L-NAME (100 mg/L p.o.), the inhibitor of NO production abolished the difference between PPARα KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8 ± 1.4 pM/mg versus 8.3 ± 0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46 ± 6%, p 2+ -dependent NOS activity in kidney homogenates of untreated PPARα WT compared with the KO mice. Clofibrate, a PPARα ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19 ± 4%, p < 0.05), increased urinary NO excretion (4.06 ± 0.53-7.07 ± 1.59 μM/24 h; p < 0.05) and reduced plasma 8-isoprostane level (45.8 ± 15 μM versus 31.4 ± 8 μM), and NADP(H) oxidase activity (16 ± 5%). Implantation of DOCA pellet (20 mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193 ± 13 mmHg versus 130 ± 12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARα activation exerts protective actions in hypertension via a mechanism that involves NO production and

  9. Tet1 Oxidase Regulates Neuronal Gene Transcription, Active DNA Hydroxy-methylation, Object Location Memory, and Threat Recognition Memory.

    Science.gov (United States)

    Kumar, Dinesh; Aggarwal, Milan; Kaas, Garrett A; Lewis, John; Wang, Jing; Ross, Daniel L; Zhong, Chun; Kennedy, Andrew; Song, Hongjun; Sweatt, J David

    2015-10-01

    A dynamic equilibrium between DNA methylation and demethylation of neuronal activity-regulated genes is crucial for memory processes. However, the mechanisms underlying this equilibrium remain elusive. Tet1 oxidase has been shown to play a key role in the active DNA demethylation in the CNS. In this study, we used Tet1 gene knockout (Tet1KO) mice to examine the involvement of Tet1 in memory consolidation and storage in the adult brain. We found that Tet1 ablation leads to: altered expression of numerous neuronal activity-regulated genes, compensatory upregulation of active demethylation pathway genes, and upregulation of various epigenetic modifiers. Moreover, Tet1KO mice showed an enhancement in the consolidation and storage of threat recognition (cued and contextual fear conditioning) and object location memories. We conclude that Tet1 plays a critical role in regulating neuronal transcription and in maintaining the epigenetic state of the brain associated with memory consolidation and storage.

  10. Tet1 oxidase regulates neuronal gene transcription, active DNA hydroxymethylation, object location memory, and threat recognition memory

    Directory of Open Access Journals (Sweden)

    Dinesh Kumar

    2015-10-01

    Full Text Available A dynamic equilibrium between DNA methylation and demethylation of neuronal activity-regulated genes is crucial for memory processes. However, the mechanisms underlying this equilibrium remain elusive. Tet1 oxidase has been shown to play a key role in the active DNA demethylation in the central nervous system. In this study, we used Tet1 gene knockout (Tet1KO mice to examine the involvement of Tet1 in memory consolidation and storage in the adult brain. We found that Tet1 ablation leads to altered expression of numerous neuronal activity-regulated genes, compensatory upregulation of active demethylation pathway genes, and upregulation of various epigenetic modifiers. Moreover, Tet1KO mice showed an enhancement in the consolidation and storage of threat recognition (cued and contextual fear conditioning and object location memories. We conclude that Tet1 plays a critical role in regulating neuronal transcription and in maintaining the epigenetic state of the brain associated with memory consolidation and storage.

  11. Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

    Science.gov (United States)

    Aguiló, Juan I; Anel, Alberto; Catalán, Elena; Sebastián, Alvaro; Acín-Pérez, Rebeca; Naval, Javier; Wallich, Reinhard; Simon, Markus M; Pardo, Julián

    2010-07-01

    Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.

  12. Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

    OpenAIRE

    Jänne, J; Morris, D R

    1984-01-01

    Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylm...

  13. Kaempferol modulates pro-inflammatory NF-κB activation by suppressing advanced glycation endproducts-induced NADPH oxidase

    Science.gov (United States)

    Kim, Ji Min; Lee, Eun Kyeong; Kim, Dae Hyun; Yu, Byung Pal

    2010-01-01

    Advanced glycation endproducts (AGE) are oxidative products formed from the reaction between carbohydrates and a free amino group of proteins that are provoked by reactive species (RS). It is also known that AGE enhance the generation of RS and that the binding of AGE to a specific AGE receptor (RAGE) induces the activation of the redox-sensitive, pro-inflammatory transcription factor, nuclear factor-kappa B (NF-ĸB). In this current study, we investigated the anti-oxidative effects of short-term kaempferol supplementation on the age-related formation of AGE and the binding activity of RAGE in aged rat kidney. We further investigated the suppressive action of kaempferol against AGE's ability to stimulate activation of pro-inflammatory NF-ĸB and its molecular mechanisms. For this study, we utilized young (6 months old), old (24 months old), and kaempferol-fed (2 and 4 mg/kg/day for 10 days) old rats. In addition, for the molecular work, the rat endothelial cell line, YPEN-1 was used. The results show that AGE and RAGE were increased during aging and that these increases were blunted by kaempferol. In addition, dietary kaempferol reduced age-related increases in NF-κB activity and NF-ĸB-dependant pro-inflammatory gene activity. The most significant new finding from this study is that kaempferol supplementation prevented age-related NF-κB activation by suppressing AGE-induced nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). Taken together, our results demonstrated that dietary kaempferol exerts its anti-oxidative and anti-inflammatory actions by modulating the age-related NF-κB signaling cascade and its pro-inflammatory genes by suppressing AGE-induced NADPH oxidase activation. Based on these data, dietary kaempferol is proposed as a possible anti-AGE agent that may have the potential for use in anti-inflammation therapies. PMID:20431987

  14. Activation of JNK and c-Jun is involved in glucose oxidase-mediated cell death of human lymphoma cells.

    Science.gov (United States)

    Son, Young-Ok; Jang, Yong-Suk; Shi, Xianglin; Lee, Jeong-Chae

    2009-12-31

    Mitogen-activated protein kinases (MAPK) affect the activation of activator protein-1 (AP-1), which plays an important role in regulating a range of cellular processes. However, the roles of these signaling factors on hydrogen peroxide (H(2)O(2))-induced cell death are unclear. This study examined the effects of H(2)O(2) on the activation of MAPK and AP-1 by exposing the cells to H(2)O(2) generated by either glucose oxidase or a bolus addition. Exposing BJAB or Jurkat cells to H(2)O(2) affected the activities of MAPK differently according to the method of H(2)O(2) exposure. H(2)O(2) increased the AP-1-DNA binding activity in these cells, where continuously generated H(2)O(2) led to an increase in mainly the c-Fos, FosB and c-Jun proteins. The c-Jun-NH(2)-terminal kinase (JNK)-mediated activation of c-Jun was shown to be related to the H(2)O(2)-induced cell death. However, the suppression of H(2)O(2)-induced oxidative stress by either JNK inhibitor or c-Jun specific antisense transfection was temporary in the cells exposed to glucose oxidase but not to a bolus H(2)O(2). This was associated with the disruption of death signaling according to the severe and prolonged depletion of reduced glutathione. Overall, these results suggest that H(2)O(2) may decide differently the mode of cell death by affecting the intracellular redox state of thiol-containing antioxidants, and this depends more closely on the duration exposed to H(2)O(2) than the concentration of this agent.

  15. Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom.

    Science.gov (United States)

    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. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Bothrops pirajai snake venom L-amino acid oxidase: in vitro effects on infection of Toxoplasma gondii in human foreskin fibroblasts

    Directory of Open Access Journals (Sweden)

    Luiz F. M. Izidoro

    2011-06-01

    Full Text Available The effect of an L-amino acid oxidase isolated from Bothrops pirajai snake venom (BpirLAAO-I was investigated on infection of Toxoplasma gondii in human foreskin fibroblasts (HFF. The cytotoxic activity of BpirLAAO-I on HFF cells showed a dose-dependent toxicity with median cytotoxic dose (TD50 of 11.8 µg/mL. BpirLAAO-I induced considerable dose-dependent decrease in the T. gondii infection index under two different conditions, treatment of tachyzoites before infection or treatment of HFF cells after infection. A maximal inhibition of infection (56% was found for treatment before infection, with a median inhibitory dose (ID50 at 1.83 µg/mL and selectivity index (SI at 6.45. For treatment after infection, it was observed a maximal inhibition of infection at 65%, ID50 of 1.20 µg/mL and SI of 9.83. The treatment before infection was also effective to reduce intracellular parasitism up to 62%, although presenting higher values of ID50 (3.14 µg/mL and lower values of SI (3.76. However, treatment after infection was not effective, suggesting that the enzyme seems to have no effect on the parasite intracellular replication for this condition. In conclusion, BpirLAAO-I was more effective to inhibit the infection of neighboring cells and consequently parasite dissemination than primary infection and parasite replication. Thus, the effect of BpirLAAO-I described herein could be taken into account for the development of new synthetic anti-parasite therapeutic agents.

  17. Increased xanthine oxidase during labour--implications for oxidative stress.

    Science.gov (United States)

    Many, A; Roberts, J M

    1997-11-01

    Xanthine dehydrogenase/oxidase (XDH/XO) produces uric acid. When in the oxidase form, this production is coupled with the generation of free radicals. Hypoxia-reperfusion enhances conversion of XDH to XO. Since the placenta is exposed to short periods of hypoxia reperfusion during labour, 17 placentae of pregnancy terminated by elective caesarean section and five placentae of pregnancies terminated by caesarean section during labour were examined for XDH/XO activity. It was found that XO activity was higher in the placentae of labouring women (P = 0.003), which suggests that labour enhances conversion of XDH to XO, facilitating free radical production.

  18. Expression analysis of polyphenol oxidase isozymes by active staining method and tissue browning of head lettuce (Lactuca sativa L.).

    Science.gov (United States)

    Noda, Takahiro; Iimure, Kazuhiko; Okamoto, Shunsuke; Saito, Akira

    2017-08-01

    Browning of plant tissue is generally considered attributable to enzymatic oxidation by polyphenol oxidase (PPO). Electrophoresis followed by activity staining has been used as an effective procedure to visually detect and isolate isozymes; however, it has not been applied for examination of various PPO isozymes in lettuce. Our study demonstrated that different lettuce PPO isozymes could be detected at different pH in active staining, and multiple isozymes were detected only under alkaline conditions. As a result, we concluded that activity staining with approximately pH 8 enabled to detect various PPO isozymes in lettuce. By expression analysis of the PPO isozymes after wounding, PPO isozymes that correlated with time-course of tissue browning were detected. The wound-induced PPO may play a key role in enzymatic browning.

  19. Vicarious trial-and-error behavior and hippocampal cytochrome oxidase activity during Y-maze discrimination learning in the rat.

    Science.gov (United States)

    Hu, Dan; Xu, Xiaojuan; Gonzalez-Lima, Francisco

    2006-03-01

    The present study investigated whether more vicarious trial-and-error (VTE) behavior, defined by head movement from one stimulus to another at a choice point during simultaneous discriminations, led to better visual discrimination learning in a Y-maze, and whether VTE behavior was a function of the hippocampus by measuring regional brain cytochrome oxidase (C.O.) activity, an index of neuronal metabolic activity. The results showed that the more VTEs a rat made, the better the rat learned the visual discrimination. Furthermore, both learning and VTE behavior during learning were correlated to C.O. activity in the hippocampus, suggesting that the hippocampus plays a role in VTE behavior during discrimination learning.

  20. In Vivo Metabolic Trapping Radiotracers for Imaging Monoamine Oxidase-A and –B Enzymatic Activity

    Science.gov (United States)

    Brooks, Allen F.; Shao, Xia; Quesada, Carole A.; Sherman, Phillip; Scott, Peter J.H.; Kilbourn, Michael R.

    2017-01-01

    The isozymes of monoamine oxidase (MAO-A and MAO-B) are important enzymes involved in the metabolism of numerous biogenic amines, including the neurotransmitters serotonin, dopamine and norepinephrine. Recently, changes in concentrations of MAO-B have been proposed as an in vivo marker of neuroinflammation associated with Alzheimer’s disease. Previous developments of in vivo radiotracers for imaging changes in MAO enzyme expression or activity have utilized the irreversible propargylamine-based suicide inhibitors, or high-affinity reversibly-binding inhibitors. As an alternative approach, we have investigated 1-[11C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines as metabolic trapping agents for the monoamine oxidases. MAO-mediated oxidation and spontaneous hydrolysis yields 1-[11C]methyl-2,3-dihydro-4-pyridinone as a hydrophilic metabolite that is trapped within brain tissues. Radiotracers with phenyl, biphenyl and 7-coumarinyl ethers were evaluated using microPET imaging in rat and primate brain. No isozyme selectivity for radiotracer trapping was observed in the rat brain for any compound, but in the monkey brain the phenyl ether demonstrated MAO-A selectivity, and the coumarinyl ether showed MAO-B selectivity. These are lead compounds for further development of 1-[11C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines with optimized brain pharmacokinetics and isozyme selectivity. PMID:26393369

  1. Design, synthesis and inhibitory activities of 8-(substituted styrol-formamido)phenyl-xanthine derivatives on monoamine oxidase B.

    Science.gov (United States)

    Hu, Suwen; Nian, Siyun; Qin, Kuiyou; Xiao, Tong; Li, Lingna; Qi, Xiaolu; Ye, Faqing; Liang, Guang; Hu, Guoxin; He, Jincai; Yu, Yinfei; Song, Bo

    2012-01-01

    The design and synthesis of two series of 8-(substituted styrol-formamido)phenyl-xanthine derivatives are described. Their in vitro monoamine oxidase B (MAO-B) inhibition were tested and the effect of substituents on the N-7, phenyl and the substituted positions are discussed. It was observed that compound 9b displayed significant MAO-B inhibition activity and selectivity, fluorine substitution plays a key role in the selectivity of MAO-B inhibition, and the styrol-formamido group at position-3' may enhance the activity and selectivity of 8-phenyl-xanthine analogues. These results suggest that such compounds may be utilized for the development of new candidate MAO-B inhibitors for treatment of Parkinson's disease.

  2. 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

    Directory of Open Access Journals (Sweden)

    Rogayah Sekeli

    2014-06-01

    Full Text Available 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.

  3. High electro-catalytic activities of glucose oxidase embedded one-dimensional ZnO nanostructures

    International Nuclear Information System (INIS)

    Sarkar, Nirmal K; Bhattacharyya, Swapan K

    2013-01-01

    One-dimensional ZnO nanorods and nanowires are separately synthesized on Zn substrate by simple hydrothermal processes at low temperatures. Electro-catalytic responses of glucose oxidase/ZnO/Zn electrodes using these two synthesized nanostructures of ZnO are reported and compared with others available in literature. It is apparent the Michaelis–Menten constant, K M app , for the present ZnO nanowire, having a greater aspect ratio, is found to be the lowest when compared with others. This sensor shows lower oxidation peak potential with a long detection range of 6.6 μM–380 mM and the highest sensitivity of ∼35.1 μA cm −2 mM −1 , among the reported values in the literature. Enzyme catalytic efficiency and turnover numbers are also found to be remarkably high. (paper)

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

    Science.gov (United States)

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

    2017-01-01

    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 (60 µg 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.

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

    Science.gov (United States)

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

    2017-06-01

    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 T 4 /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. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Isolated sulfite oxidase deficiency.

    Science.gov (United States)

    Rupar, C A; Gillett, J; Gordon, B A; Ramsay, D A; Johnson, J L; Garrett, R M; Rajagopalan, K V; Jung, J H; Bacheyie, G S; Sellers, A R

    1996-12-01

    Isolated sulfite oxidase (SO) deficiency is an autosomal recessively inherited inborn error of sulfur metabolism. In this report of a ninth patient the clinical history, laboratory results, neuropathological findings and a mutation in the sulfite oxidase gene are described. The data from this patient and previously published patients with isolated sulfite oxidase deficiency and molybdenum cofactor deficiency are summarized to characterize this rare disorder. The patient presented neonatally with intractable seizures and did not progress developmentally beyond the neonatal stage. Dislocated lenses were apparent at 2 months. There was increased urine excretion of sulfite and S-sulfocysteine and a decreased concentration of plasma cystine. A lactic acidemia was present for 6 months. Liver sulfite oxidase activity was not detectable but xanthine dehydrogenase activity was normal. The boy died of respiratory failure at 32 months. Neuropathological findings of cortical necrosis and extensive cavitating leukoencephalopathy were reminiscent of those seen in severe perinatal asphyxia suggesting an etiology of energy deficiency. A point mutation that resulted in a truncated protein missing the molybdenum-binding site has been identified.

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

    Science.gov (United States)

    Cai, Lu; Wang, Chao; Huo, Xiao-Kui; 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.

  8. Changes in brain monoamine levels and monoamine oxidase activity in the catfish, Clarias batrachus, during chronic treatments with mercurials

    International Nuclear Information System (INIS)

    Kirubagaran, R.; Joy, K.P.

    1990-01-01

    In mammals, the central nervous system is the primary target for CH 3 Hg poisoning which is clinically known as Minamata disease. Hg is a widely recognized neurotoxin and has been reported to impair brain monoamine neurotransmitter metabolism. Reports on effects of Hg on brain monoamine activity in fishes are scarce. In the present study, therefore, changes in the brain monoamine levels and the degradation enzyme, monoamine oxidase (MAO), are described in the catfish, Clarias batrachus, exposed to sublethal concentrations of mercuric chloride (HgCl 2 -inorganic Hg), methylmercuric chloride (CH 3 HgCl-organic Hg), and a commercial mercurial fungicide formulation, emisan 6 (methoxyethyl Hg-organic Hg) for 45, 90 and 180 d during gonadal recrudescence. These intervals correspond to late preparatory, prespawning and spawning phases, respectively, of the annual reproductive cycle of the catfish

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Immobilization of xanthine oxidase on a polyaniline silicone support.

    Science.gov (United States)

    Nadruz, W; Marques, E T; Azevedo, W M; Lima-Filho, J L; Carvalho, L B

    1996-03-01

    A polyaniline silicone support to immobilize xanthine oxidase is proposed as a reactor coil to monitor the action of xanthine oxidase on hypoxanthine, xanthine and 6-mercaptopurine. A purified xanthine oxidase immobilized on this support lost 80% of the initial activity after 12 min of use. Co-immobilization of superoxide dismutase and catalase increased the stability of immobilized xanthine oxidase so that the derivative maintained 79% of its initial activity after 4.6 h of continuous use in which 1.5 mumol purine bases were converted by the immobilized enzyme system. There is no evidence of either polyaniline or protein leaching from the coil during 3 h of continuous use. When solutions (10 ml) of hypoxanthine, xanthine and 6-mercaptopurine were circulated individually through the xanthine oxidase-superoxide dismutase-catalase-polyaniline coil (1 mm internal diameter and 3 m in length, 3 ml internal volume) activities of 8.12, 11.17 and 1.09 nmol min-1 coil-1, respectively, were obtained. The advantages of the reactor configuration and the redox properties of the polymer, particularly with respect to immobilized oxidoreductases, make this methodology attractive for similar enzyme systems. This immobilized enzyme system using polyaniline-silicone as support converted 6-mercaptopurine to 6-thiouric acid with equal efficiency as resins based on polyacrylamide and polyamide 11.

  11. Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Friis, Martin Barfred; Vorum, Katrine Gribel; Lambert, Ian Henry

    2008-01-01

    Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H(2)O(2) potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19-32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein......+-mobilizing agonist ATP (10 microM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 n......M) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47 phox or p47 phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production...

  12. Add-on treatment of benzoate for schizophrenia: a randomized, double-blind, placebo-controlled trial of D-amino acid oxidase inhibitor.

    Science.gov (United States)

    Lane, Hsien-Yuan; Lin, Ching-Hua; Green, Michael F; Hellemann, Gerhard; Huang, Chih-Chia; Chen, Po-Wei; Tun, Rene; Chang, Yue-Cung; Tsai, Guochuan E

    2013-12-01

    In addition to dopaminergic hyperactivity, hypofunction of the N-methyl-d-aspartate receptor (NMDAR) has an important role in the pathophysiology of schizophrenia. Enhancing NMDAR-mediated neurotransmission is considered a novel treatment approach. To date, several trials on adjuvant NMDA-enhancing agents have revealed beneficial, but limited, efficacy for positive and negative symptoms and cognition. Another method to enhance NMDA function is to raise the levels of d-amino acids by blocking their metabolism. Sodium benzoate is a d-amino acid oxidase inhibitor. To examine the clinical and cognitive efficacy and safety of add-on treatment of sodium benzoate for schizophrenia. A randomized, double-blind, placebo-controlled trial in 2 major medical centers in Taiwan composed of 52 patients with chronic schizophrenia who had been stabilized with antipsychotic medications for 3 months or longer. Six weeks of add-on treatment of 1 g/d of sodium benzoate or placebo. The primary outcome measure was the Positive and Negative Syndrome Scale (PANSS) total score. Clinical efficacy and adverse effects were assessed biweekly. Cognitive functions were measured before and after the add-on treatment. Benzoate produced a 21% improvement in PANSS total score and large effect sizes (range, 1.16-1.69) in the PANSS total and subscales, Scales for the Assessment of Negative Symptoms-20 items, Global Assessment of Function, Quality of Life Scale and Clinical Global Impression and improvement in the neurocognition subtests as recommended by the National Institute of Mental Health's Measurement and Treatment Research to Improve Cognition in Schizophrenia initiative, including the domains of processing speed and visual learning. Benzoate was well tolerated without significant adverse effects. Benzoate adjunctive therapy significantly improved a variety of symptom domains and neurocognition in patients with chronic schizophrenia. The preliminary results show promise for d-amino acid oxidase

  13. CYP714B1 and CYP714B2 encode gibberellin 13-oxidases that reduce gibberellin activity in rice.

    Science.gov (United States)

    Magome, Hiroshi; Nomura, Takahito; Hanada, Atsushi; Takeda-Kamiya, Noriko; Ohnishi, Toshiyuki; Shinma, Yuko; Katsumata, Takumi; Kawaide, Hiroshi; Kamiya, Yuji; Yamaguchi, Shinjiro

    2013-01-29

    Bioactive gibberellins (GAs) control many aspects of growth and development in plants. GA(1) has been the most frequently found bioactive GA in various tissues of flowering plants, but the enzymes responsible for GA(1) biosynthesis have not been fully elucidated due to the enzymes catalyzing the 13-hydroxylation step not being identified. Because of the lack of mutants defective in this enzyme, biological significance of GA 13-hydroxylation has been unknown. Here, we report that two cytochrome P450 genes, CYP714B1 and CYP714B2, encode GA 13-oxidase in rice. Transgenic Arabidopsis plants that overexpress CYP714B1 or CYP714B2 show semidwarfism. There was a trend that the levels of 13-OH GAs including GA(1) were increased in these transgenic plants. Functional analysis using yeast or insect cells shows that recombinant CYP714B1 and CYP714B2 proteins can convert GA(12) into GA(53) (13-OH GA(12)) in vitro. Moreover, the levels of 13-OH GAs including GA(1) were decreased, whereas those of 13-H GAs including GA(4) (which is more active than GA(1)) were increased, in the rice cyp714b1 cyp714b2 double mutant. These results indicate that CYP714B1 and CYP714B2 play a predominant role in GA 13-hydroxylation in rice. The double mutant plants appear phenotypically normal until heading, but show elongated uppermost internode at the heading stage. Moreover, CYP714B1 and CYP714B2 expression was up-regulated by exogenous application of bioactive GAs. Our results suggest that GA 13-oxidases play a role in fine-tuning plant growth by decreasing GA bioactivity in rice and that they also participate in GA homeostasis.

  14. Leptin Induces Oxidative Stress Through Activation of NADPH Oxidase in Renal Tubular Cells: Antioxidant Effect of L-Carnitine.

    Science.gov (United States)

    Blanca, Antonio J; Ruiz-Armenta, María V; Zambrano, Sonia; Salsoso, Rocío; Miguel-Carrasco, José L; Fortuño, Ana; Revilla, Elisa; Mate, Alfonso; Vázquez, Carmen M

    2016-10-01

    Leptin is a protein involved in the regulation of food intake and in the immune and inflammatory responses, among other functions. Evidences demonstrate that obesity is directly associated with high levels of leptin, suggesting that leptin may directly link obesity with the elevated cardiovascular and renal risk associated with increased body weight. Adverse effects of leptin include oxidative stress mediated by activation of NADPH oxidase. The aim of this study was to evaluate the effect of L-carnitine (LC) in rat renal epithelial cells (NRK-52E) exposed to leptin in order to generate a state of oxidative stress characteristic of obesity. Leptin increased superoxide anion (O2 (•) -) generation from NADPH oxidase (via PI3 K/Akt pathway), NOX2 expression and nitrotyrosine levels. On the other hand, NOX4 expression and hydrogen peroxide (H2 O2 ) levels diminished after leptin treatment. Furthermore, the expression of antioxidant enzymes, catalase, and superoxide dismutase, was altered by leptin, and an increase in the mRNA expression of pro-inflammatory factors was also found in leptin-treated cells. LC restored all changes induced by leptin to those levels found in untreated cells. In conclusion, stimulation of NRK-52E cells with leptin induced a state of oxidative stress and inflammation that could be reversed by preincubation with LC. Interestingly, LC induced an upregulation of NOX4 and restored the release of its product, hydrogen peroxide, which suggests a protective role of NOX4 against leptin-induced renal damage. J. Cell. Biochem. 117: 2281-2288, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Class IA phosphoinositide 3-kinase β and δ regulate neutrophil oxidase activation in response to Aspergillus fumigatus hyphae.

    Science.gov (United States)

    Boyle, Keith B; Gyori, David; Sindrilaru, Anca; Scharffetter-Kochanek, Karin; Taylor, Philip R; Mócsai, Attila; Stephens, Len R; Hawkins, Phillip T

    2011-03-01

    An effective immune response to the ubiquitous fungus Aspergillus fumigatus is dependent upon production of reactive oxygen species (ROS) by the NADPH oxidase. This is evidenced by the acute sensitivity of oxidase-deficient humans and mice to invasive aspergillosis. Neutrophils are recruited to the lungs shortly postinfection and respond by phagocytosing conidia and mediating extracellular killing of germinated hyphae in a ROS-dependent manner. However, the signaling mechanisms regulating the generation of ROS in response to hyphae are poorly understood. PI3Ks are important regulators of numerous cellular processes, with much recent work describing unique roles for the different class I PI3K isoforms. We showed by live-cell imaging that the lipid products of class I PI3Ks accumulated at the hyphal-bound neutrophil plasma membrane. Further, we used pharmacological and genetic approaches to demonstrate essential, but overlapping, roles for PI3Kβ and PI3Kδ in the ROS and spreading responses of murine neutrophils to Aspergillus hyphae. Hyphal-induced ROS responses were substantially inhibited by deletion of the common β2-integrin subunit CD18, with only a minor, redundant role for Dectin-1. However, addition of soluble algal glucans plus the genetic deletion of CD18 were required to significantly inhibit activation of the PI3K-effector protein kinase B. Hyphal ROS responses were also totally dependent on the presence of Syk, but not its ITAM-containing adaptor proteins FcRγ or DAP12, and the Vav family of Rac-guanine nucleotide exchange factors. These results start to define the signaling network controlling neutrophil ROS responses to A. fumigatus hyphae.

  16. Mitochondrial Cytochrome c Oxidase Biogenesis Is Regulated by the Redox State of a Heme-Binding Translational Activator.

    Science.gov (United States)

    Soto, Iliana C; Barrientos, Antoni

    2016-02-20

    Mitochondrial cytochrome c oxidase (COX), the last enzyme of the respiratory chain, catalyzes the reduction of oxygen to water and therefore is essential for cell function and viability. COX is a multimeric complex, whose biogenesis is extensively regulated. One type of control targets cytochrome c oxidase subunit 1 (Cox1), a key COX enzymatic core subunit translated on mitochondrial ribosomes. In Saccharomyces cerevisiae, Cox1 synthesis and COX assembly are coordinated through a negative feedback regulatory loop. This coordination is mediated by Mss51, a heme-sensing COX1 mRNA-specific processing factor and translational activator that is also a Cox1 chaperone. In this study, we investigated whether Mss51 hemylation and Mss51-mediated Cox1 synthesis are both modulated by the reduction-oxidation (redox) environment. We report that Cox1 synthesis is attenuated under oxidative stress conditions and have identified one of the underlying mechanisms. We show that in vitro and in vivo exposure to hydrogen peroxide induces the formation of a disulfide bond in Mss51 involving CPX motif heme-coordinating cysteines. Mss51 oxidation results in a heme ligand switch, thereby lowering heme-binding affinity and promoting its release. We demonstrate that in addition to affecting Mss51-dependent heme sensing, oxidative stress compromises Mss51 roles in COX1 mRNA processing and translation. H2O2-induced downregulation of mitochondrial translation has so far not been reported. We show that high H2O2 concentrations induce a global attenuation effect, but milder concentrations specifically affect COX1 mRNA processing and translation in an Mss51-dependent manner. The redox environment modulates Mss51 functions, which are essential for regulation of COX biogenesis and aerobic energy production.

  17. Microwave processing of honey negatively affects honey antibacterial activity by inactivation of bee-derived glucose oxidase and defensin-1.

    Science.gov (United States)

    Bucekova, Marcela; Juricova, Valeria; Monton, Enrique; Martinotti, Simona; Ranzato, Elia; Majtan, Juraj

    2018-02-01

    Microwave (MW) thermal heating has been proposed as an efficient method for honey liquefaction, while maintaining honey quality criteria. However, little is known about the effects of MW thermal heating on honey antibacterial activity. In this study, we aimed to determine the effects of MW heating on the antibacterial activity of raw rapeseed honeys against Pseudomonas aeruginosa and Staphylococcus aureus, with a particular focus on two major bee-derived antibacterial components, defensin-1 and hydrogen peroxide (H 2 O 2 ). Our results demonstrated that MW thermal heating completely abolished honey antibacterial activity whereas conventional thermal treatment at 45 and 55°C did not affect the antibacterial activity of honey samples. A significant decrease in both glucose oxidase activity and H 2 O 2 production as well as defensin-1 amount was observed in MW-treated samples. Given that defensin-1 and H 2 O 2 are regular antibacterial components of all honeys, MW heating may have similar negative effects on every type of crystallized/liquid honey. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Effect of prolonged gamma irradiation (6.7 Gy) on monoamine oxidase activity in ewe hypothalamus in anestral period

    International Nuclear Information System (INIS)

    Pastorova, B.; Arendarcik, J.

    1988-01-01

    Changes were studied of monoamine oxidase (MAO) activity in the hypothalamus and hypophysis of ewes in the anestral period following whole-body 60 Co irradiation for 7 days with a dose of 6.7 Gy. The gamma radiation exposure rate was 0.039 Gy/h. The activity of MAO was determined using the radiochemical method. 14 C-tryptamine was used as the substrate. The highest activity was determined in the rostral hypothalamus (1100 pmol.mg -1 .min -1 ). MAO activity was at its lowest in the caudal region of the hypothalamus (550 pmol). The results show that whole-body exposure to gamma radiation with a total dose of 6.7 Gy makes a statistically significant increase (P<0.001) in MAO activity in the caudal hypothalamus of ewes while remaining at the level of the control group or increasing insignificantly in the rostral and medial hypothalamus. A significant decrease (P<0.05) was recorded in the hypophysis. It may be assumed that the increased degradation of catecholamines caused by MAO is one of the mechanisms responsible for the decreased concentration of catecholamines in the hypothalamus of ewes after irradiation. (author). 1 fig., 22 refs

  19. Modulating effect of new potential antimelanomic agents, spin-labeled triazenes and nitrosoureas on the DOPA-oxidase activity of tyrosinase.

    Science.gov (United States)

    Gadjeva, V; Zheleva, A; Raikova, E

    1999-07-01

    The modulating effect of newly synthesized alkylating spin labeled triazene and spin labeled nitrosourea derivatives on the DOPA-oxidase activity of mushroom tyrosinase has been investigated by Bumett's spectrophotometric method (Burnett et al., 1967). All spin labeled triazenes have exhibited activating effect on DOPA-oxidase activity of tyrosinase, whereas clinically used triazene (DTIC), which does not contain nitroxide moiety, have showed inhibiting effect. At the same experimental conditions the spin labeled aminoacid nitrosoureas have showed dual effect - activating, in the beginning of the enzyme reaction and inhibiting later on. It is deduced that the activating effect of the spin labeled compounds is due to the nitroxide moiety and the inhibiting effect of all compounds depends on their half-life time. This study might contribute to make more clear the mechanism of action of the new compounds and on the other hand would come in quite useful as a preliminary prognosis for their antimelanomic activity.

  20. In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase.

    Science.gov (United States)

    Cao, Gen-Xia; Wu, Xiu-Ming; Dong, Yu-Ming; Li, Zai-Jun; Wang, Guang-Li

    2016-07-09

    In this study, a simple and amplified colorimetric assay is developed for the detection of the enzymatic activity of glucose oxidase (GOx) based on in situ formation of a photoswitchable oxidase mimetic of PO₄(3-)-capped CdS quantum dots (QDs). GOx catalyzes the oxidation of 1-thio-β-d-glucose to give 1-thio-β-d-gluconic acid which spontaneously hydrolyzes to β-d-gluconic acid and H₂S; the generated H₂S instantly reacts with Cd(2+) in the presence of Na₃PO₄ to give PO₄(3-)-stabilized CdS QDs in situ. Under visible-light (λ ≥ 400 nm) stimulation, the PO₄(3-)-capped CdS QDs are a new style of oxidase mimic derived by producing some active species, such as h⁺, (•)OH, O₂(•-) and a little H₂O₂, which can oxidize the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with a color change. Based on the GOx-triggered growth of the oxidase mimetics of PO₄(3-)-capped CdS QDs in situ, we developed a simple and amplified colorimetric assay to probe the enzymatic activity of GOx. The proposed method allowed the detection of the enzymatic activity of GOx over the range from 25 μg/L to 50 mg/L with a low detection limit of 6.6 μg/L. We believe the PO₄(3-)-capped CdS QDs generated in situ with photo-stimulated enzyme-mimicking activity may find wide potential applications in biosensors.

  1. High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhi-wei; Vignaud, Caroline; Jaafar, Adil; Lévy, Bernard; Guéritte, Françoise; Guénard, Daniel; Lederer, Florence; Mathews, F. Scott (CNRS-UMR); (WU-MED)

    2012-05-24

    Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b{sub 2} (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC{sub 50} in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 {angstrom} resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the {approx}100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.

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

    Science.gov (United States)

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

    2015-01-01

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

  3. Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

    Science.gov (United States)

    Kurth, Fabian; Duprez, Wilko; Premkumar, Lakshmanane; Schembri, Mark A.; Fairlie, David P.; Martin, Jennifer L.

    2014-01-01

    The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery. PMID:24831013

  4. Quantitative Collection and Enzymatic Activity of Glucose Oxidase Nanotubes Fabricated by Templated Layer-by-Layer Assembly.

    Science.gov (United States)

    Zhang, Shouwei; Demoustier-Champagne, Sophie; Jonas, Alain M

    2015-08-10

    We report on the fabrication of enzyme nanotubes in nanoporous polycarbonate membranes via the layer-by-layer (LbL) alternate assembly of polyethylenimine (PEI) and glucose oxidase (GOX), followed by dissolution of the sacrificial template in CH2Cl2, collection, and final dispersion in water. An adjuvant-assisted filtration methodology is exploited to extract quantitatively the nanotubes without loss of activity and morphology. Different water-soluble CH2Cl2-insoluble adjuvants are tested for maximal enzyme activity and nanotube stability; whereas NaCl disrupts the tubes by screening electrostatic interactions, the high osmotic pressure created by fructose also contributes to loosening the nanotubular structures. These issues are solved when using neutral, high molar mass dextran. The enzymatic activity of intact free nanotubes in water is then quantitatively compared to membrane-embedded nanotubes, showing that the liberated nanotubes have a higher catalytic activity in proportion to their larger exposed surface. Our study thus discloses a robust and general methodology for the fabrication and quantitative collection of enzymatic nanotubes and shows that LbL assembly provides access to efficient enzyme carriers for use as catalytic swarming agents.

  5. The effect of ultrasound on particle size, color, viscosity and polyphenol oxidase activity of diluted avocado puree.

    Science.gov (United States)

    Bi, Xiufang; Hemar, Yacine; Balaban, Murat O; Liao, Xiaojun

    2015-11-01

    The effect of ultrasound treatment on particle size, color, viscosity, polyphenol oxidase (PPO) activity and microstructure in diluted avocado puree was investigated. The treatments were carried out at 20 kHz (375 W/cm(2)) for 0-10 min. The surface mean diameter (D[3,2]) was reduced to 13.44 μm from an original value of 52.31 μm by ultrasound after 1 min. A higher L(∗) value, ΔE value and lower a(∗) value was observed in ultrasound treated samples. The avocado puree dilution followed pseudoplastic flow behavior, and the viscosity of diluted avocado puree (at 100 s(-1)) after ultrasound treatment for 1 min was 6.0 and 74.4 times higher than the control samples for dilution levels of 1:2 and 1:9, respectively. PPO activity greatly increased under all treatment conditions. A maximum increase of 25.1%, 36.9% and 187.8% in PPO activity was found in samples with dilution ratios of 1:2, 1:5 and 1:9, respectively. The increase in viscosity and measured PPO activity might be related to the decrease in particle size. The microscopy images further confirmed that ultrasound treatment induced disruption of avocado puree structure. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. A benzoxazine derivative induces vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 by elevating NADPH oxidase activity and reactive oxygen species levels.

    Science.gov (United States)

    Zhao, Jing; He, Qiuxia; Cheng, Yizhe; Zhao, Baoxiang; Zhang, Yun; Zhang, Shangli; Miao, Junying

    2009-09-01

    Previously, we found that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO) promoted apoptosis of human umbilical vascular endothelial cells (HUVECs) deprived of growth factors. In this study, we aimed to investigate the effect of DBO and its mechanism of action on angiogenesis and apoptosis of HUVECs in the presence of fibroblast growth factor-2 (FGF-2), which promotes angiogenesis and inhibits apoptosis in vivo and in vitro. DBO significantly inhibited capillary-like tube formation by promoting apoptosis of HUVECs in the presence of FGF-2 in vitro. Furthermore, DBO elevated the levels of reactive oxygen species (ROS) and nitric oxide (NO) and increased the activity of NADPH oxidase and inducible nitric oxide synthase (iNOS) in promoting apoptosis under this condition. Moreover, when NADPH oxidase was inhibited by its specific inhibitor, dibenziodolium chloride (DPI), DBO could not elevate ROS and NO levels in HUVECs. The data suggest that DBO is a new modulator of apoptosis in vitro, and it might function by increasing the activity of NADPH oxidase and iNOS, subsequently elevating the levels of ROS and NO in HUVECs. The findings of this study provide a new small molecule for investigating the FGF-2/NADPH oxidase/iNOS signaling pathway in apoptosis.

  7. Monoamine oxidase-A and B activities in the cerebellum and frontal cortex of children and young adults with autism.

    Science.gov (United States)

    Gu, Feng; Chauhan, Ved; Chauhan, Abha

    2017-10-01

    Monoamine oxidases (MAOs) catalyze the metabolism of monoamine neurotransmitters, such as serotonin, dopamine, and norepinephrine, and are key regulators for brain function. In this study, we analyzed the activities of MAO-A and MAO-B in the cerebellum and frontal cortex from subjects with autism and age-matched control subjects. In the cerebellum, MAO-A activity in subjects with autism (aged 4-38 years) was significantly lower by 20.6% than in controls. When the subjects were divided into children (aged 4-12 years) and young adults (aged 13-38 years) subgroups, a significant decrease by 27.8% in the MAO-A activity was observed only in children with autism compared with controls. When the 95% confidence interval of the control group was taken as a reference range, reduced activity of MAO-A was observed in 70% of children with autism. In the frontal cortex, MAO-A activity in children with autism was also lower by 30% than in the control group, and impaired activity of MAO-A was observed in 55.6% of children with autism, although the difference between the autism and control groups was not significant when all subjects were considered. On the contrary, there was no significant difference in MAO-B activity in both the cerebellum and frontal cortex between children with autism and the control group as well as in adults. These results suggest impaired MAO-A activity in the brain of subjects with autism, especially in children with autism. Decreased activity of MAOs may lead to increased levels of monoaminergic neurotransmitters, such as serotonin, which have been suggested to have a critical role in autism. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  8. Improved production of 2,5-furandicarboxylic acid by overexpression of 5-hydroxymethylfurfural oxidase and 5-hydroxymethylfurfural/furfural oxidoreductase in Raoultella ornithinolytica BF60.

    Science.gov (United States)

    Yuan, Haibo; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Chen, Jian; Shi, Zhongping; Liu, Long

    2018-01-01

    2,5-Furandicarboxylic acid (FDCA) is a promising bio-based building block and can be produced by biotransformation of 5-hydroxymethylfurfural (HMF). To improve the FDCA production, two genes-one encoding HMF oxidase (HMFO; from Methylovorus sp. strain MP688) and another encoding for HMF/Furfural oxidoreductase (HmfH; from Cupriavidus basilensis HMF14)-were introduced into Raoultella ornithinolytica BF60. The FDCA production in the engineered whole-cell biocatalyst increased from 51.0 to 93.6mM, and the molar conversion ratio of HMF to FDCA increased from 51.0 to 93.6%. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Spermine oxidase (SMO) activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm

    International Nuclear Information System (INIS)

    Cervelli, Manuela; Grillo, Rosalba; Woster, Patrick M; Casero, Robert A Jr; Mariottini, Paolo; Bellavia, Gabriella; Fratini, Emiliano; Amendola, Roberto; Polticelli, Fabio; Barba, Marco; Federico, Rodolfo; Signore, Fabrizio; Gucciardo, Giacomo

    2010-01-01

    Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme. BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined. Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors. This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials

  10. Spermine oxidase (SMO activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm

    Directory of Open Access Journals (Sweden)

    Gucciardo Giacomo

    2010-10-01

    Full Text Available Abstract Background Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC. This study investigates the expression of spermine oxidase (SMO and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme. Methods BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined. Results Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors. Conclusions This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials.

  11. Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae*

    Science.gov (United States)

    Nguyen, Don Trinh; Göpfert, Jens Christian; Ikezawa, Nobuhiro; MacNevin, Gillian; Kathiresan, Meena; Conrad, Jürgen; Spring, Otmar; Ro, Dae-Kyun

    2010-01-01

    Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature. PMID:20351109

  12. Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation.

    Science.gov (United States)

    Wang, Su Bin; Jang, Ji Yong; Chae, Yun Hee; Min, Ji Hyun; Baek, Jin Young; Kim, Myunghee; Park, Yunjeong; Hwang, Gwi Seo; Ryu, Jae-Sang; Chang, Tong-Shin

    2015-06-01

    Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47(phox), a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47(phox) and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Reduction of NADPH-oxidase activity ameliorates the cardiovascular phenotype in a mouse model of Williams-Beuren Syndrome.

    Directory of Open Access Journals (Sweden)

    Victoria Campuzano

    2012-02-01

    Full Text Available A hallmark feature of Williams-Beuren Syndrome (WBS is a generalized arteriopathy due to elastin deficiency, presenting as stenoses of medium and large arteries and leading to hypertension and other cardiovascular complications. Deletion of a functional NCF1 gene copy has been shown to protect a proportion of WBS patients against hypertension, likely through reduced NADPH-oxidase (NOX-mediated oxidative stress. DD mice, carrying a 0.67 Mb heterozygous deletion including the Eln gene, presented with a generalized arteriopathy, hypertension, and cardiac hypertrophy, associated with elevated angiotensin II (angII, oxidative stress parameters, and Ncf1 expression. Genetic (by crossing with Ncf1 mutant and/or pharmacological (with ang II type 1 receptor blocker, losartan, or NOX inhibitor apocynin reduction of NOX activity controlled hormonal and biochemical parameters in DD mice, resulting in normalized blood pressure and improved cardiovascular histology. We provide strong evidence for implication of the redox system in the pathophysiology of the cardiovascular disease in a mouse model of WBS. The phenotype of these mice can be ameliorated by either genetic or pharmacological intervention reducing NOX activity, likely through reduced angII-mediated oxidative stress. Therefore, anti-NOX therapy merits evaluation to prevent the potentially serious cardiovascular complications of WBS, as well as in other cardiovascular disorders mediated by similar pathogenic mechanism.

  14. Predicting drunk driving: contribution of alcohol use and related problems, traffic behaviour, personality and platelet monoamine oxidase (MAO) activity.

    Science.gov (United States)

    Eensoo, Diva; Paaver, Marika; Harro, Maarike; Harro, Jaanus

    2005-01-01

    The aim of the study was to characterize the predictive value of socio-economic data, alcohol consumption measures, smoking, platelet monoamine oxidase (MAO) activity, traffic behaviour habits and impulsivity measures for actual drunk driving. Data were collected from 203 male drunk driving offenders and 211 control subjects using self-reported questionnaires, and blood samples were obtained from the two groups. We identified the combination of variables, which predicted correctly, approximately 80% of the subjects' belonging to the drunk driving and control groups. Significant independent discriminators in the final model were, among the health-behaviour measures, alcohol-related problems, frequency of using alcohol, the amount of alcohol consumed and smoking. Predictive traffic behaviour measures were seat belt use and paying for parking. Among the impulsivity measures, dysfunctional impulsivity was the best predictor; platelet MAO activity and age also had an independent predictive value. Our results support the notion that drunk driving is the result of a combination of various behavioural, biological and personality-related risk factors.

  15. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    Energy Technology Data Exchange (ETDEWEB)

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette, E-mail: annette.rompel@univie.ac.at [Universität Wien, Althanstrasse 14, 1090 Wien (Austria)

    2015-05-22

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P2{sub 1}2{sub 1}2{sub 1} and P12{sub 1}1 and diffracted to ∼1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3{sub 1}21. The crystals of latent cgAUS1 belonged to space group P12{sub 1}1 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI)

  16. Role of catechins on ET-1 induced stimulation of PLD and NADPH oxidase activities in pulmonary smooth muscle cells: Determination of the probable mechanism by molecular docking studies.

    Science.gov (United States)

    Chakraborti, Sajal; Sarkar, Jaganmay; Bhuyan, Rajabrata; Chakraborti, Tapati

    2017-12-05

    Treatment of human pulmonary artery smooth muscle cells with ET-1 stimulated PLD and NADPH oxidase activities, which were inhibited upon pretreatment with bosentan (ET-1 receptor antagonist), FIPI (PLD inhibitor), apocynin (NADPH oxidase inhibitor) and EGCG & ECG (catechins having galloyl group), but not EGC & EC (catechins devoid of galloyl group). Herein, we determined the probable mechanism by which the galloyl group containing catechins inhibit ET-1 induced stimulation of PLD activity by molecular docking analyses based on our biochemical studies. ET-1 induced stimulation of PLD activity was inhibited by SecinH3 (inhibitor of cytohesin). Arf-6 and cytohesin-1 were associated in the cell membrane, which was not inhibited by the catechins during ET-1 treatment to the cells. However, EGCG and ECG inhibited binding of GTPγS with Arf-6 even in presence of cytohesin-1. The molecular docking analyses revealed that the galloyl group containing catechins (EGCG/ECG) with cytohesin1-Arf6GDP, but not the non-galloyl-containing catechins (EGC and EC), prevents GDP/GTP exchange in Arf-6 which seems to be an important mechanism for inhibition of ET-1 induced activation of PLD and subsequently increase in NADPH oxidase activities.

  17. Apoptotic action of peroxisome proliferator-activated receptor-gamma activation in human non small-cell lung cancer is mediated via proline oxidase-induced reactive oxygen species formation.

    Science.gov (United States)

    Kim, Ki Young; Ahn, Jin Hee; Cheon, Hyae Gyeong

    2007-09-01

    Peroxisome proliferator-activated receptor (PPAR)-gamma ligands have been shown to inhibit human lung cancers by inducing apoptosis and differentiation. In the present study, we elucidated the apoptotic mechanism of PPARgamma activation in human lung cancers by using a novel PPARgamma agonist, 1-(trans-methylimino-N-oxy)-6-(2-morpholinoethoxy)-3-phenyl-(1H-indene-2-carboxylic acid ethyl ester (KR-62980), and rosiglitazone. PPARgamma activation selectively inhibited cell viability of non-small-cell lung cancer with little effect on small-cell lung cancer and normal lung cells. The cell death induced by PPARgamma activation presented apoptotic features of oligonucleosomal DNA fragmentation in A549 human non-small-cell lung cancer cell line. Reactive oxygen species (ROS) production was accompanied by increased expression of proline oxidase (POX), a redox enzyme expressed in mitochondria, upon incubation with the agonists. POX RNA interference treatment blocked PPARgamma-induced ROS formation and cytotoxicity, suggesting that POX plays a functional role in apoptosis through ROS formation. The apoptotic effects by the agonists were antagonized by bisphenol A diglycidyl ether, a PPARgamma antagonist, and by knockdown of PPARgamma expression, indicating the involvement of PPARgamma in these actions. The results of the present study suggest that PPARgamma activation induces apoptotic cell death in non-small-cell lung carcinoma mainly through ROS formation via POX induction.

  18. A multicopper oxidase is essential for manganese oxidation and laccase-like activity in Pedomicrobium sp. ACM 3067.

    Science.gov (United States)

    Ridge, Justin P; Lin, Marianne; Larsen, Eloise I; Fegan, Mark; McEwan, Alastair G; Sly, Lindsay I

    2007-04-01

    Pedomicrobium sp. ACM 3067 is a budding-hyphal bacterium belonging to the alpha-Proteobacteria which is able to oxidize soluble Mn2+ to insoluble manganese oxide. A cosmid, from a whole-genome library, containing the putative genes responsible for manganese oxidation was identified and a primer-walking approach yielded 4350 bp of novel sequence. Analysis of this sequence showed the presence of a predicted three-gene operon, moxCBA. The moxA gene product showed homology to multicopper oxidases (MCOs) and contained the characteristic four copper-binding motifs (A, B, C and D) common to MCOs. An insertion mutation of moxA showed that this gene was essential for both manganese oxidation and laccase-like activity. The moxB gene product showed homology to a family of outer membrane proteins which are essential for Type I secretion in Gram-negative bacteria. moxBA has not been observed in other manganese-oxidizing bacteria but homologues were identified in the genomes of several bacteria including Sinorhizobium meliloti 1021 and Agrobacterium tumefaciens C58. These results suggest that moxBA and its homologues constitute a family of genes encoding an MCO and a predicted component of the Type I secretion system.

  19. Effects of gamma irradiation on the radiation-resistant bacteria and polyphenol oxidase activity in fresh kale juice

    International Nuclear Information System (INIS)

    Kim, Dongho; Song, Hyunpa; Lim, Sangyong; Yun, Hyejeong; Chung, Jinwoo

    2007-01-01

    Gamma radiation was performed to prolong the shelf life of natural kale juice. The total aerobic bacteria in fresh kale juice, prepared by a general kitchen process, was detected in the range of 10 6 cfu/ml, and about 10 2 cfu/ml of the bacteria survived in the juice in spite of gamma irradiation treatment with a dose of 5 kGy. Two typical radiation-resistant bacteria, Bacillus megaterium and Exiguobacterium acetylicum were isolated and identified from the 5 kGy-irradiated kale juices. The D 10 values of the vegetative cell and endospore of the B. megaterium in peptone water were 0.63±0.05 and 1.52±0.05 kGy, respectively. The D 10 value of the E. acetylicum was calculated as 0.65±0.06 kGy. In the inoculation test, the growth of the surviving B. megaterium and E. acetylicum in the 3-5 kGy-irradiated kale juice retarded and/or decreased significantly during a 3 d post-irradiation storage period. However, there were no significant differences in the residual polyphenol oxidase activity and browning index between the nonirradiated control and the gamma irradiated kale juice during a post-irradiation period

  20. Effects of gamma irradiation on the radiation-resistant bacteria and polyphenol oxidase activity in fresh kale juice

    Science.gov (United States)

    Kim, Dongho; Song, Hyunpa; Lim, Sangyong; Yun, Hyejeong; Chung, Jinwoo

    2007-07-01

    Gamma radiation was performed to prolong the shelf life of natural kale juice. The total aerobic bacteria in fresh kale juice, prepared by a general kitchen process, was detected in the range of 10 6 cfu/ml, and about 10 2 cfu/ml of the bacteria survived in the juice in spite of gamma irradiation treatment with a dose of 5 kGy. Two typical radiation-resistant bacteria, Bacillus megaterium and Exiguobacterium acetylicum were isolated and identified from the 5 kGy-irradiated kale juices. The D10 values of the vegetative cell and endospore of the B. megaterium in peptone water were 0.63±0.05 and 1.52±0.05 kGy, respectively. The D10 value of the E. acetylicum was calculated as 0.65±0.06 kGy. In the inoculation test, the growth of the surviving B. megaterium and E. acetylicum in the 3-5 kGy-irradiated kale juice retarded and/or decreased significantly during a 3 d post-irradiation storage period. However, there were no significant differences in the residual polyphenol oxidase activity and browning index between the nonirradiated control and the gamma irradiated kale juice during a post-irradiation period.

  1. The effect of alloxan diabetes on the activity of some mixed function oxidases in male rats.

    Science.gov (United States)

    Nedjar, A; Stoytchev, T

    1990-01-01

    The effect of alloxan-induced diabetes on the duration of hexobarbital sleep (HB sleep) the activity of ethylmorphine-N-demethylase (EMND), aniline hydroxylase (AH), the content of microsomal cytochrome P-450 and b5, on the activity of ethoxycumarine-0-deethylase (ECOD) and ethoxyresorufine-0-deethylase (EROD) after induction with beta naphthoflavone (beta-NF), as well as the activity of benzphetamine-N-demethylase and pentoxyresorufine-O-dealkylase (PROD) after induction with phenobarbital (PB), was studied in experiments on male Wistar rats. In rats with alloxan diabetes there was a significant prolongation of HB sleep (by 106%) and inhibition of the liver EMND (by 54%), while the AH activity increased by 131%, with a parallel rise in the content of microsomal cytochromes P-450 (by 67%) and b5 (by 113%). In rats with alloxan diabetes the enzyme-inducing effect of beta-NF with respect to the activities of EROD and ECOD is reduced, although diabetes by itself causes a rise in the ECOD activity in untreated animals. When induced with PB, the PROD and benzphetamine-N-demethylase activity in diabetic rats is lower than in the healthy animals. However, if the enzyme activity after the application of inducers is referred to the respective starting enzyme activities of the two groups of animals, it is found that the enzyme-inducing effect of PB is preserved and even slightly potentiated in the diabetic rats compared with the healthy ones: the increases in the benzphetamine-N-demethylase activity is by 60% in the diabetic rats, compared with a rise of 28% in the healthy animals, of the PROD activity 19 times for the diabetic compared with 16 times increase for the healthy rats.

  2. The Effect of MethyI Jasmonate on Ethylene Production, ACC Oxidase Activity and Carbon Dioxide Evolution in the Yellowish-Tangerine Tomato Fruits (Lycopersicon esculentum Mill.

    Directory of Open Access Journals (Sweden)

    Janusz Czapski

    2013-12-01

    Full Text Available The yellowish-tangerine tomato (cv. Bursztyn in the green, light yellow and yellow stages of ripening were treated with 0.1% and 1.0% of methyl jasmonate (JA-Me in lanolin paste and kept for several days and then they were evaluated for production of ethylene, ACC oxidase activity and CO2 evolution. Production of endogenous ethylene in mature green fruits was low and increased during ripening. JA-Me stimulated ethylene production and ACC oxidase activity in all investigated stages of fruit ripening. Slices excised from mature green fruits produced highest amount of carbon dioxide as compared to more advanced stages of ripening. JA-Me in O,1 % and 1,0% concentrations increased significantly CO2 evolution in green fruits, while in light yellow and yellow fruits only higher concentration of JA-Me stimulated carbon dioxide production.

  3. Acyl-coenzyme A oxidases 1 and 3 in brown trout (Salmo trutta f. fario): Can peroxisomal fatty acid β-oxidation be regulated by estrogen signaling?

    Science.gov (United States)

    Madureira, Tânia Vieira; Castro, L Filipe C; Rocha, Eduardo

    2016-02-01

    Acyl-coenzyme A oxidases 1 (Acox1) and 3 (Acox3) are key enzymes in the regulation of lipid homeostasis. Endogenous and exogenous factors can disrupt their normal expression/activity. This study presents for the first time the isolation and characterization of Acox1 and Acox3 in brown trout (Salmo trutta f. fario). Additionally, as previous data point to the existence of a cross-talk between two nuclear receptors, namely peroxisome proliferator-activated receptors and estrogen receptors, it was here evaluated after in vitro exposures of trout hepatocytes the interference caused by ethynylestradiol in the mRNA levels of an inducible (by peroxisome proliferators) and a non-inducible oxidase. The isolated Acox1 and Acox3 show high levels of sequence conservation compared to those of other teleosts. Additionally, it was found that Acox1 has two alternative splicing isoforms, corresponding to 3I and 3II isoforms of exon 3 splicing variants. Both isoforms display tissue specificity, with Acox1-3II presenting a more ubiquitous expression in comparison with Acox1-3I. Acox3 was expressed in almost all brown trout tissues. According to real-time PCR data, the highest estrogenic stimulus was able to cause a down-regulation of Acox1 and an up-regulation of Acox3. So, for Acox1 we found a negative association between an estrogenic input and a directly activated PPARα target gene. In conclusion, changes in hormonal estrogenic stimulus may impact the mobilization of hepatic lipids to the gonads, with ultimate consequences in reproduction. Further studies using in vivo assays will be fundamental to clarify these issues.

  4. Enzymatic activity of Glucose Oxidase from Aspergillus niger IPBCC.08.610 On Modified Carbon Paste Electrode as Glucose Biosensor

    Science.gov (United States)

    Rohmayanti, T.; Ambarsari, L.; Maddu, A.

    2017-03-01

    Glucose oxidase (GOx) has been developed as glucose sensor for measuring blood glucose level because of its specificity to glucose oxidation. This research aimed to determine kinetic parameters of GOx activity voltametrically and further test its potential as a glucose biosensor. GOx, in this research, was produced by local fungi Aspergillus niger IPBCC.08.610 which was isolated from local vine in Tarakan, East Borneo, Indonesia. GOx was immobilized with glutaraldehyde, which cross-linked onto modified carbon paste electrode (MCPE) nanofiber polyaniline. Intracellular GOx activity was higher than extracellular ones. Immobilized GOx used glutaraldehyde 2.5% and dripped on the surface of MCPE nanofiber polyaniline. MCPE have a high conductance in copper with the diameter of 3 mm. The concentration of glucose in the lowest concentration of 0.2 mM generated a current value of 0.413 mA while 2 mM of glucose induced a current of 3,869 mA value. Km and Imax of GOx in MCPE activities polyaniline nanofiber were 2.88 mM and 3.869 mA,respectively, with turnover (Kcat) of 13 s-1. Sensitivity was 1.09 mA/mM and response time to produce a maximum peak current was 25 seconds. Km value was then converted into units of mg/dL and obtained 56.4 mg/dL. GOximmo-IPB|MCPE electrode is potential to be able to detect blood glucose level in a normal condition and hypoglycemia conditions

  5. Increase in activity, glycosylation and expression of cytokinin oxidase/dehydrogenase during the senescence of barley leaf segments in the dark

    Czech Academy of Sciences Publication Activity Database

    Conrad, K.; Motyka, Václav; Schlüter, T.

    2007-01-01

    Roč. 130, č. 4 (2007), s. 572-579 ISSN 0031-9317 R&D Projects: GA ČR GA206/03/0313 Institutional research plan: CEZ:AV0Z50380511 Source of funding: V - iné verejné zdroje Keywords : OXIDASE ACTIVITY * GENE-EXPRESSION * ZEA - MAYS Subject RIV: EF - Botanics Impact factor: 2.192, year: 2007

  6. Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

    Science.gov (United States)

    Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

    2015-12-10

    D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Peroxisome proliferator-activated receptor δ inhibits Porphyromonas gingivalis lipopolysaccharide-induced activation of matrix metalloproteinase-2 by downregulating NADPH oxidase 4 in human gingival fibroblasts.

    Science.gov (United States)

    Yoo, T; Ham, S A; Hwang, J S; Lee, W J; Paek, K S; Oh, J W; Kim, J H; Do, J T; Han, C W; Kim, J H; Seo, H G

    2016-10-01

    We investigated the roles of peroxisome proliferator-activated receptor δ (PPARδ) in Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS)-induced activation of matrix metalloproteinase 2 (MMP-2). In human gingival fibroblasts (HGFs), activation of PPARδ by GW501516, a specific ligand of PPARδ, inhibited Pg-LPS-induced activation of MMP-2 and generation of reactive oxygen species (ROS), which was associated with reduced expression of NADPH oxidase 4 (Nox4). These effects were significantly smaller in the presence of small interfering RNA targeting PPARδ or the specific PPARδ inhibitor GSK0660, indicating that PPARδ is involved in these events. In addition, modulation of Nox4 expression by small interfering RNA influenced the effect of PPARδ on MMP-2 activity, suggesting a mechanism in which Nox4-derived ROS modulates MMP-2 activity. Furthermore, c-Jun N-terminal kinase and p38, but not extracellular signal-regulated kinase, mediated PPARδ-dependent inhibition of MMP-2 activity in HGFs treated with Pg-LPS. Concomitantly, PPARδ-mediated inhibition of MMP-2 activity was associated with the restoration of types I and III collagen to levels approaching those in HGFs not treated with Pg-LPS. These results indicate that PPARδ-mediated downregulation of Nox4 modulates cellular redox status, which in turn plays a critical role in extracellular matrix homeostasis through ROS-dependent regulation of MMP-2 activity. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Graphene-glucose oxidase bioanodes for enzymatic biofuel cells

    DEFF Research Database (Denmark)

    Tang, Jing; Werchmeister, Rebecka Maria Larsen; Engelbrekt, Christian

    2017-01-01

    as supporting material, polyethyleneimine (PEI) as linker and glucose oxidase (GOD) as the chosen enzyme. GOD can catalyze oxidation of glucose to gluconolactone, but needs a mediator to assist electron transfer between the enzyme and electrodes. The redox molecule ferrocene carboxylic acid (Fc...... and systematically investigated. The assembled EBFCs show good reproducibility. EBFCs provide maximum output power density 2.47 μW cm-2 at 35 ℃, indicating the optimized activity of EBFCs fed with glucose....

  9. Correlation of Aqueous Humor Lysyl Oxidase Activity with TGF-ß Levels and LOXL1 Genotype in Pseudoexfoliation.

    Science.gov (United States)

    Gayathri, Ramakrishnan; Coral, Karunakaran; Sharmila, Ferdinamarie; Sripriya, Sarangapani; Sripriya, Krishnamoorthy; Manish, Panday; Shantha, B; Ronnie, George; Vijaya, Lingam; Narayanasamy, Angayarkanni

    2016-10-01

    Pseudoexfoliation (PXF) is a microfibrillopathy involving disordered elastogenesis. Abnormal extracellular matrix (ECM) production underlies the pathophysiology of PXF. The enzyme Lysyl oxidase (LOX) and its isoforms are known to cross-link the elastin and collagen. Though the etiopathogensis of PXF is not well understood, studies report on the genetic risk involving LOXL1 gene. This study aims to screen LOXL1 coding variants rs1048661 and rs3825942 in the South Indian population and the implication of the single nucleotide polymorphism (SNP) with LOX activity. The levels of transforming growth factor β (TGF-β) in aqueous humor and its correlation with the LOX activity were also examined. Blood, plasma, and aqueous aspirates were prospectively collected from PXF cases with and without glaucoma and cataract cases as controls. DNA was extracted from 48 PXF cases without glaucoma, 12 PXF cases with glaucoma, and 40 age-matched cataract-alone controls without PXF/glaucoma for analyzing LOX SNPs. LOX activity was measured in aqueous humor and plasma of 30 PXF cases without glaucoma, 24 age-matched cataract-alone controls without PXF/glaucoma, and 14 PXF cases with glaucoma. Protein levels of LOX, LOXL1, LOXL2, and total TGF-β were estimated in plasma and aqueous humor by ELISA. The specific activity of LOX in aqueous humor was found to be significantly lowered in PXF cases compared with cataract-alone controls (p = 0.014). This decrease in LOX activity in PXF cases was associated with high-risk GG haplotype. However, this was not statistically significant and a larger sample size is warranted. TGF-β1 and TGF-β2 negatively correlated with LOX activity in aqueous humor (p = 0.028; p = 0.046, respectively). The LOXL1 SNPs, rs1048661 and rs3825942, are associated with PXF in the South Indian population correlating with lowered LOX activity in the aqueous humor. The increased level of total TGF-β in the aqueous humor of PXF cases is possibly associated with LOX

  10. Uric acid and allopurinol aggravate absence epileptic activity in Wistar Albino Glaxo Rijswijk rats.

    Science.gov (United States)

    Lakatos, Renáta Krisztina; Dobolyi, Árpád; Kovács, Zsolt

    2018-05-01

    Uric acid has a role in several physiological and pathophysiological processes. For example, uric acid may facilitate seizure generalization while reducing uric acid level may evoke anticonvulsant/antiepileptic effects. Allopurinol blocks the activity of xanthine oxidase, by which allopurinol inhibits catabolism of hypoxanthine to xanthine and uric acid and, as a consequence, decreases the level of uric acid. Although the modulation of serum uric acid level is a widely used strategy in the treatment of certain diseases, our knowledge regarding the effects of uric acid on epileptic activity is far from complete. Thus, the main aim of this study was the investigation of the effect of uric acid on absence epileptic seizures (spike-wave discharges: SWDs) in a model of human absence epilepsy, the Wistar Albino Glaxo/Rijswijk (WAG/Rij) rat. We investigated the influence of intraperitoneally (i.p.) injected uric acid (100 mg/kg and 200 mg/kg), allopurinol (50 mg/kg and 100 mg/kg), a cyclooxygenase 1 and 2 (COX-1 and COX-2) inhibitor indomethacin (10 mg/kg) and inosine (500 mg/kg) alone and the combined application of allopurinol (50 mg/kg) with uric acid (100 mg/kg) or inosine (500 mg/kg) as well as indomethacin (10 mg/kg) with uric acid (100 mg/kg) and inosine (500 mg/kg) with uric acid (100 mg/kg) on absence epileptic activity. We demonstrated that both uric acid and allopurinol alone significantly increased the number of SWDs whereas indomethacin abolished the uric acid-evoked increase in SWD number. Our results suggest that uric acid and allopurinol have proepileptic effects in WAG/Rij rats. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Reducing cytoplasmic polyamine oxidase activity in Arabidopsis increases salt and drought tolerance by reducing reactive oxygen species production and increasing defense gene expression

    Directory of Open Access Journals (Sweden)

    G.H.M. eSagor

    2016-02-01

    Full Text Available The link between polyamine oxidases (PAOs, which function in polyamine catabolism, and stress responses remains elusive. Here, we address this issue using Arabidopsis pao mutants in which the expression of the five PAO genes is knocked-out or knocked-down. As the five single pao mutants and wild type (WT showed similar response to salt stress, we tried to generate the mutants that have either the cytoplasmic PAO pathway (pao1 pao5 or the peroxisomal PAO pathway (pao2 pao3 pao4 silenced. However, the latter triple mutant was not obtained. Thus, in this study, we used two double mutants, pao1 pao5 and pao2 pao4. Of interest, pao1 pao5 mutant was NaCl- and drought-tolerant, whereas pao2 pao4 showed similar sensitivity to those stresses as WT. To reveal the underlying mechanism of salt tolerance, further analyses were performed. Na uptake of the mutant (pao1 pao5 decreased to 75% of WT. PAO activity of the mutant was reduced to 62% of WT. The content of reactive oxygen species (ROS such as hydrogen peroxide, a reaction product of PAO action, and superoxide anion in the mutant became 81% and 72% of the levels in WT upon salt treatment. The mutant contained 2.8-fold higher thermospermine compared to WT. Moreover, the mutant induced the genes of salt overly sensitive-, abscisic acid (ABA-dependent- and ABA-independent- pathways more strongly than WT upon salt treatment. The results suggest that the Arabidopsis plant silencing cytoplasmic PAOs shows salinity tolerance by reducing ROS production and strongly inducing subsets of stress-responsive genes under stress conditions.

  12. Effect of γ-ray Irradiation On the Activities of Monoamine Oxidase in Rat Brain and Liver

    International Nuclear Information System (INIS)

    Kim, Joo Young; Choi, Myung Sun; Choi, Myung Un

    1993-01-01

    In order to evaluate the effects of radiation on mammalian neuronal system, we have examined the effect of gamma-ray radiation on the monoamine oxidase(MAO) activity in monoaminergic neurons. Following the whole body irradiation, MAO activity in the rat brain was measured as well as in the liver for the comparative studies between the neuronal and nonneuronal system. The effects of some radiation protectors and sensitizers were also examined in addition to the O2 effect. The results can be summarized as follows. 1) The MAO activity of rat brain was minimally affected by the radiation dose up to 1,700 cGy. Radiation dose above 2,500 cGy inhibited the brain MAO activity by no less than 10%. MAO-A form was found to be particularly sensitive to radiation. The liver MAO was somewhat inhibited(by about 5%) but hard1y dependent on the dose of radiation. 2) The inhibitory effect on the brain was initiated immediately by the radiation dose of 2,500 cGy. On the contrary, for the liver, the inhibitory effect became apparent only 2 days after irradiation. 3) Two days after a dose of 2,500 cGy, Vmax and Km of the brain mitochondrial MAO decreased. for liver, Vmax decreased while Km increased, which indicates the kinetic patterns for the neuronal and nonneruronal systems are not affected similarly by radiation. 4) The effect of several known radiation protectors and sensitizers on MAO activity was tested but no definite results were obtained. The level of -SH group increased in some degree upon radiation but not by the compounds. 5) MAO activity was not affected by O2 concentration, while an elevated level of lipid peroxidase was found udder the same condition. The results described here indicate that characteristics of MAO, one of the most important central nervous system enzymes, are liable to radiation, which is partially differentiated from the liver MAO. Also indicated are that the -SH groups are hardly related to the effect of radiation but the production of the lipid

  13. Squamosamide derivative FLZ protects dopaminergic neurons against inflammation-mediated neurodegeneration through the inhibition of NADPH oxidase activity

    Directory of Open Access Journals (Sweden)

    Wilson Belinda

    2008-05-01

    Full Text Available Abstract Background Inflammation plays an important role in the pathogenesis of Parkinson's disease (PD through over-activation of microglia, which consequently causes the excessive production of proinflammatory and neurotoxic factors, and impacts surrounding neurons and eventually induces neurodegeneration. Hence, prevention of microglial over-activation has been shown to be a prime target for the development of therapeutic agents for inflammation-mediated neurodegenerative diseases. Methods For in vitro studies, mesencephalic neuron-glia cultures and reconstituted cultures were used to investigate the molecular mechanism by which FLZ, a squamosamide derivative, mediates anti-inflammatory and neuroprotective effects in both lipopolysaccharide-(LPS- and 1-methyl-4-phenylpyridinium-(MPP+-mediated models of PD. For in vivo studies, a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-(MPTP- induced PD mouse model was used. Results FLZ showed potent efficacy in protecting dopaminergic (DA neurons against LPS-induced neurotoxicity, as shown in rat and mouse primary mesencephalic neuronal-glial cultures by DA uptake and tyrosine hydroxylase (TH immunohistochemical results. The neuroprotective effect of FLZ was attributed to a reduction in LPS-induced microglial production of proinflammatory factors such as superoxide, tumor necrosis factor-α (TNF-α, nitric oxide (NO and prostaglandin E2 (PGE2. Mechanistic studies revealed that the anti-inflammatory properties of FLZ were mediated through inhibition of NADPH oxidase (PHOX, the key microglial superoxide-producing enzyme. A critical role for PHOX in FLZ-elicited neuroprotection was further supported by the findings that 1 FLZ's protective effect was reduced in cultures from PHOX-/- mice, and 2 FLZ inhibited LPS-induced translocation of the cytosolic subunit of p47PHOX to the membrane and thus inhibited the activation of PHOX. The neuroprotective effect of FLZ demonstrated in primary neuronal

  14. Action of Bothrops moojeni venom and its L-amino acid oxidase fraction, treated with {sup 60}Co gamma rays, in Leishmania spp; Acao do veneno de Bothrops moojeni e sua fracao L-aminoacido oxidase, submetida ao tratamento com raios gama de {sup 60}Co, em Leishmania spp

    Energy Technology Data Exchange (ETDEWEB)

    Cardoso, Andre Gustavo Tempone

    1999-07-01

    Bothrops moojeni venom showed an anti leishmania activity in vitro, as determined by a cell viability assay using the reduction of MTT. After venom purification, by chromatography techniques, the fractions with anti leishmania and L-amino acid oxidase activities, eluted in the same positions. The molecular weight of the enzyme was estimated to be 140 kDa by molecular exclusion chromatography, and 69 kDa, by SDS-PAGE, migrating as a single band, with an isoelectric point of 4.8 as determined by isoelectric focusing. The purified LAO from B. moojeni venom, 135-fold more active than crude venom, showed homo dimeric constitution, and was active against Leishmania spp from the New World, with an effective concentration against L(L). amazonensis of 1.80 {mu}g/ml (EC{sub 50}), L.(V.) panamensis (0.78 |{mu}g/ml) and L.(L.) chagasi (0.63 ({mu}g/ml). Ultrastructural studies of promastigotes affected by LAO demonstrated cell death, with edema in several organelles such as mitochondria and nuclear membrane, before cell disruption and necrosis. The action of LAO was demonstrated to be hydrogen peroxide-dependent. Studies with LLCMK-2 cells, treated with LAO, showed a toxic effect, with an EC{sub 50} of 11|{mu}g/ml. Irradiation of LAO with 6{sup 0C}o gamma rays, did not affect its whole oxidative activity, neither detoxified the enzyme. Amastigotes treated with LAO were not affected by its hydrogen peroxide, otherwise, the exogenous product, killed amastigotes with an EC{sub 50} of 0.67mM. These data could be of help in the development of alternative therapeutic approaches to the treatment of leishmaniasis. (author)

  15. Bioconversion of α-linolenic acid to n-3 LCPUFA and expression of PPAR-alpha, acyl Coenzyme A oxidase 1 and carnitine acyl transferase I are incremented after feeding rats with α-linolenic acid-rich oils.

    Science.gov (United States)

    González-Mañán, Daniel; Tapia, Gladys; Gormaz, Juan Guillermo; D'Espessailles, Amanda; Espinosa, Alejandra; Masson, Lilia; Varela, Patricia; Valenzuela, Alfonso; Valenzuela, Rodrigo

    2012-07-01

    High dietary intake of n-6 fatty acids in relation to n-3 fatty acids may generate health disorders, such as cardiovascular and other chronic diseases. Fish consumption rich in n-3 fatty acids is low in Latin America, it being necessary to seek other alternatives to provide α-linolenic acid (ALA), precursor of n-3 LCPUFA (EPA and DHA). Two innovative oils were assayed, chia (Salvia hispanica) and rosa mosqueta (Rosa rubiginosa). This study evaluated hepatic bioconversion of ALA to EPA and DHA, expression of PPAR-α, acyl-Coenzyme A oxidase 1 (ACOX1) and carnitine acyltransferase I (CAT-I), and accumulation of EPA and DHA in plasma and adipose tissue in Sprague-Dawley rats. Three experimental groups were fed 21 days: sunflower oil (SFO, control); chia oil (CO); rosa mosqueta oil (RMO). Fatty acid composition of total lipids and phospholipids from plasma, hepatic and adipose tissue was assessed by gas-liquid chromatography and TLC. Expression of PPAR-α (RT-PCR) and ACOX1 and CAT-I (Western blot). CO and RMO increased plasma, hepatic and adipose tissue levels of ALA, EPA and DHA and decreased n-6:n-3 ratio compared to SFO (p oil.

  16. ERK1/2 pathway is involved in renal gluconeogenesis inhibition under conditions of lowered NADPH oxidase activity.

    Science.gov (United States)

    Winiarska, Katarzyna; Jarzyna, Robert; Dzik, Jolanta M; Jagielski, Adam K; Grabowski, Michal; Nowosielska, Agata; Focht, Dorota; Sierakowski, Bartosz

    2015-04-01

    The aim of this study was to elucidate the mechanisms involved in the inhibition of renal gluconeogenesis occurring under conditions of lowered activity of NADPH oxidase (Nox), the enzyme considered to be one of the main sources of reactive oxygen species in kidneys. The in vitro experiments were performed on primary cultures of rat renal proximal tubules, with the use of apocynin, a selective Nox inhibitor, and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a potent superoxide radical scavenger. In the in vivo experiments, Zucker diabetic fatty (ZDF) rats, a well established model of diabetes type 2, were treated with apocynin solution in drinking water. The main in vitro findings are the following: (1) both apocynin and TEMPOL attenuate the rate of gluconeogenesis, inhibiting the step catalyzed by phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of the process; (2) in the presence of the above-noted compounds the expression of PEPCK and the phosphorylation of transcription factor CREB and ERK1/2 kinases are lowered; (3) both U0126 (MEK inhibitor) and 3-(2-aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione (ERK inhibitor) diminish the rate of glucose synthesis via mechanisms similar to those of apocynin and TEMPOL. The observed apocynin in vivo effects include: (1) slight attenuation of hyperglycemia; (2) inhibition of renal gluconeogenesis; (3) a decrease in renal PEPCK activity and content. In view of the results summarized above, it can be concluded that: (1) the lowered activity of the ERK1/2 pathway is of importance for the inhibition of renal gluconeogenesis found under conditions of lowered superoxide radical production by Nox; (2) the mechanism of this phenomenon includes decreased PEPCK expression, resulting from diminished activity of transcription factor CREB; (3) apocynin-evoked inhibition of renal gluconeogenesis contributes to the hypoglycemic action of this compound observed in diabetic animals. Thus, the study has

  17. Regenerative Capacity of Cacti Schlumbergera and Rhipsalidopsis in Relation to Endogenous Phytohormones, Cytokinin Oxidase/Dehydrogenase, and Peroxidase Activities

    Czech Academy of Sciences Publication Activity Database

    Sriskandarajah, S.; Prinsen, E.; Motyka, Václav; Dobrev, Petre; Serek, M.

    2006-01-01

    Roč. 25, č. 1 (2006), s. 79-88 ISSN 0721-7595 R&D Projects: GA ČR GA206/03/0313 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cytokinin dehydrogenase * Cytokinin oxidase * Endogenous phytohormones * In vitro regeneration * Peroxidase * Rhipsalidopsis * Schlumbergera Subject RIV: EF - Botanics Impact factor: 2.107, year: 2006

  18. NADPH oxidase activity in pollen tubes is affected by calcium ions, signaling phospholipids and Rac/Rop GTPases

    Czech Academy of Sciences Publication Activity Database

    Potocký, Martin; Pejchar, Přemysl; Gutkowska, Malgorzata; Jiménez-Quesada, M. J.; Potocká, Andrea; Alché, J.; Kost, B.; Žárský, Viktor

    2012-01-01

    Roč. 169, č. 16 (2012), s. 1654-1663 ISSN 0176-1617 R&D Projects: GA ČR GP522/09/P299 Institutional research plan: CEZ:AV0Z50380511 Keywords : Pollen tube * Tip growth * NADPH oxidase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.699, year: 2012

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

    DEFF Research Database (Denmark)

    Rasmussen, Izabela; Pedersen, Line Hjortshøj; Byg, Luise

    2010-01-01

    Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin d...... dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton....

  20. Theoretical studies of the active-site structure, spectroscopic and thermodynamic properties, and reaction mechanism of multicopper oxidases

    Czech Academy of Sciences Publication Activity Database

    Rulíšek, Lubomír; Ryde, U.

    2013-01-01

    Roč. 257, č. 2 (2013), s. 445-458 ISSN 0010-8545 Institutional support: RVO:61388963 Keywords : multi-copper oxidases * QM/MM calculations * multireference calculations * the oretical spectroscopy Subject RIV: CA - Inorganic Chemistry Impact factor: 12.098, year: 2013

  1. The lipidated peptidomimetic Lau-[(S)-Aoc]-(Lys-βNphe)6-NH2 is a novel formyl peptide receptor 2 agonist that activates both human and mouse neutrophil NADPH-oxidase

    DEFF Research Database (Denmark)

    Holdfeldt, Andre; Skovbakke, Sarah Line; Winther, Malene

    2016-01-01

    Neutrophils expressing formyl peptide receptor 2 (FPR2) play key roles in host defense, immune regulation, and resolution of inflammation. Consequently, the search for FPR2-specific modulators has attracted much attention due to its therapeutic potential. Earlier described agonists......2 (F2M2), showing comparable potency in activating human and mouse neutrophils by inducing a rise in intracellular Ca2+ concentration and assembly of the superoxide-generating NADPH oxidase. This FPR2/Fpr2 agonist contains a headgroup consisting of a 2-aminooctanoic acid (Aoc) residue acylated......2 signaling as well as for development of prophylactic immunomodulatory therapy. This novel class of cross-species FPR2/Fpr2 agonists should enable translation of results obtained with mouse neutrophils (and disease models) into enhanced understanding of human inflammatory and immune diseases....

  2. Better Rooting Procedure to Enhance Survival Rate of Field Grown Malaysian Eksotika Papaya Transformed with 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Gene

    Science.gov (United States)

    Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom

    2013-01-01

    A high survival rate for transformed papaya plants when transferred to the field is useful in the quest for improving the commercial quality traits. We report in this paper an improved rooting method for the production of transformed Malaysian Eksotika papaya with high survival rate when transferred to the field. Shoots were regenerated from embryogenic calli transformed with antisense and RNAi constructs of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes using the Agrobacterium tumefaciens-mediated transformation method. Regenerated transformed shoots, each measuring approximately 3-4 cm in height, were cultured in liquid half-strength Murashige and Skoog (MS) medium or sterile distilled water, and with either perlite or vermiculite supplementation. All the culturing processes were conducted either under sterile or nonsterile condition. The results showed that rooting under sterile condition was better. Shoots cultured in half-strength MS medium supplemented with vermiculite exhibited a 92.5% rooting efficiency while perlite showed 77.5%. The survival rate of the vermiculite-grown transformed papaya plantlets after transfer into soil, contained in polybags, was 94%, and the rate after transfer into the ground was 92%. Morpho-histological analyses revealed that the tap roots were more compact, which might have contributed to the high survival rates of the plantlets. PMID:25969786

  3. Better rooting procedure to enhance survival rate of field grown malaysian eksotika papaya transformed with 1-aminocyclopropane-1-carboxylic Acid oxidase gene.

    Science.gov (United States)

    Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom

    2013-01-01

    A high survival rate for transformed papaya plants when transferred to the field is useful in the quest for improving the commercial quality traits. We report in this paper an improved rooting method for the production of transformed Malaysian Eksotika papaya with high survival rate when transferred to the field. Shoots were regenerated from embryogenic calli transformed with antisense and RNAi constructs of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes using the Agrobacterium tumefaciens-mediated transformation method. Regenerated transformed shoots, each measuring approximately 3-4 cm in height, were cultured in liquid half-strength Murashige and Skoog (MS) medium or sterile distilled water, and with either perlite or vermiculite supplementation. All the culturing processes were conducted either under sterile or nonsterile condition. The results showed that rooting under sterile condition was better. Shoots cultured in half-strength MS medium supplemented with vermiculite exhibited a 92.5% rooting efficiency while perlite showed 77.5%. The survival rate of the vermiculite-grown transformed papaya plantlets after transfer into soil, contained in polybags, was 94%, and the rate after transfer into the ground was 92%. Morpho-histological analyses revealed that the tap roots were more compact, which might have contributed to the high survival rates of the plantlets.

  4. Overexpression of Arabidopsis thaliana gibberellic acid 20 oxidase (AtGA20ox) gene enhance the vegetative growth and fiber quality in kenaf (Hibiscus cannabinus L.) plants

    Science.gov (United States)

    Withanage, Samanthi Priyanka; Hossain, Md Aktar; Kumar M., Sures; Roslan, Hairul Azman B; Abdullah, Mohammad Puad; Napis, Suhaimi B.; Shukor, Nor Aini Ab.

    2015-01-01

    Kenaf (Hibiscus cannabinus L.; Family: Malvaceae), is multipurpose crop, one of the potential alternatives of natural fiber for biocomposite materials. Longer fiber and higher cellulose contents are required for good quality biocomposite materials. However, average length of kenaf fiber (2.6 mm in bast and 1.28 mm in whole plant) is below the critical length (4 mm) for biocomposite production. Present study describes whether fiber length and cellulose content of kenaf plants could be enhanced by increasing GA biosynthesis in plants by overexpressing Arabidopsis thaliana Gibberellic Acid 20 oxidase (AtGA20ox) gene. AtGA20ox gene with intron was overexpressed in kenaf plants under the control of double CaMV 35S promoter, followed by in planta transformation into V36 and G4 varieties of kenaf. The lines with higher levels of bioactive GA (0.3–1.52 ng g−1 fresh weight) were further characterized for their morphological and biochemical traits including vegetative and reproductive growth, fiber dimension and chemical composition. Positive impact of increased gibberellins on biochemical composition, fiber dimension and their derivative values were demonstrated in some lines of transgenic kenaf including increased cellulose content (91%), fiber length and quality but it still requires further study to confirm the critical level of this particular bioactive GA in transgenic plants. PMID:26175614

  5. Pyruvate Oxidase Influences the Sugar Utilization Pattern and Capsule Production in Streptococcus pneumoniae

    NARCIS (Netherlands)

    Carvalho, Sandra M.; Farshchi Andisi, Vahid; Gradstedt, Henrik; Neef, Jolanda; Kuipers, Oscar P.; Neves, Ana R.; Bijlsma, Jetta J. E.

    2013-01-01

    Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence,

  6. LEACH ARCILLAS ACTIVATED PARTIAL ACID SULFURICO

    OpenAIRE

    Romero Y Otiniano, P.; Pizarra Cabrera, R.

    2014-01-01

    This work is concerned with the activation of calcium bentonite from Junín- Perú (with a moisture content of 24.1% and an average of particle size 40 µ ) with sulphuric acid. The parameters studied are the ratio of bentonite to acid solution, acid concentration and reaction time to boiling temperatura of the mixture. The optimum conditions obtained are the following: 0.47 kg. of bentonite/kg. of acid solution to 4.8 N, 4 h of treatment to 104 ºC and the conversion of 45.6% alumina and 73.5% o...

  7. An amperometric enzyme electrode and its biofuel cell based on a glucose oxidase-poly(3-anilineboronic acid)-Pd nanoparticles bionanocomposite for glucose biosensing.

    Science.gov (United States)

    Sun, Lingen; Ma, Yixuan; Zhang, Pei; Chao, Long; Huang, Ting; Xie, Qingji; Chen, Chao; Yao, Shouzhuo

    2015-06-01

    A new amperometric enzyme electrode and its biofuel cell were fabricated based on a glucose oxidase (GOx)-poly(3-anilineboronic acid) (PABA)-Pd nanoparticles (PdNPs) bionanocomposite for biosensing of glucose. Briefly, Pd was electroplated on a multiwalled carbon nanotubes (MWCNTs)-modified Au electrode, and the GOx-PABA-PdNPs bionanocomposite was prepared on the Pd(plate)/MWCNTs/Au electrode through the chemical oxidation of a GOx-3-anilineboronic acid adduct by Na2PdCl4, followed by electrode-modification with an outer-layer chitosan (CS) film. The thus-prepared CS/GOx-PABA-PdNPs/Pd(plate)/MWCNTs/Au electrode exhibited a linear amperometric response to glucose concentration from 2.0 μM to 4.5 mM with a sensitivity of 160 μA/mM/cm(2), sub-μM detection limit, and excellent operation/storage stability in the first-generation biosensing mode, as well as excellent analytical performance in the second-generation biosensing mode. The good recoveries of glucose obtained from spiked urine samples revealed the application potential of our amperometric enzyme electrode. In addition, a glucose/O2 biofuel cell was constructed using this enzyme electrode as the anode and a Pt/MWCNTs/Au electrode as the cathode, and this biofuel cell as a self-powered biosensing device showed a linear voltage response to glucose concentration from 100 μM to 13.5 mM with a sensitivity of 43.5 mV/mM/cm(2) and excellent operation/storage stability. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Oral administration of D-alanine in monkeys robustly increases plasma and cerebrospinal fluid levels but experimental D-amino acid oxidase inhibitors had minimal effect.

    Science.gov (United States)

    Rojas, Camilo; Alt, Jesse; Ator, Nancy A; Wilmoth, Heather; Rais, Rana; Hin, Niyada; DeVivo, Michael; Popiolek, Michael; Tsukamoto, Takashi; Slusher, Barbara S

    2016-09-01

    Hypofunction of the N-methyl-d-aspartate (NMDA) receptor is thought to exacerbate psychosis in patients diagnosed with schizophrenia. Consistent with this hypothesis, D-alanine, a co-agonist at the glycine site of the NMDA receptor, was shown to improve positive and cognitive symptoms when used as add-on therapy for schizophrenia treatment. However, D-alanine had to be administered at high doses (~7 g) to observe clinical effects. One possible reason for the high dose is that D-alanine could be undergoing oxidation by D-amino acid oxidase (DAAO) before it reaches the brain. If this is the case, the dose could be reduced by co-administration of D-alanine with a DAAO inhibitor (DAAOi). Early studies with rodents showed that co-administration of D-alanine with 5-chloro-benzo[d]isoxazol-3-ol (CBIO), a prototype DAAOi, significantly enhanced the levels of extracellular D-alanine in the frontal cortex compared with D-alanine alone. Further, the use of CBIO reduced the dose of D-alanine needed to attenuate prepulse inhibition deficits induced by dizocilpine. The objective of the work reported herein was to confirm the hypothesis that DAAO inhibition can enhance D-alanine exposure in a species closer to humans: non-human primates. We report that while oral D-alanine administration to baboons (10 mg/kg) enhanced D-alanine plasma and CSF levels over 20-fold versus endogenous levels, addition of experimental DAAOi to the regimen exhibited a 2.2-fold enhancement in plasma and no measurable effect on CSF levels. The results provide caution regarding the utility of DAAO inhibition to increase D-amino acid levels as treatment for patients with schizophrenia. © The Author(s) 2016.

  9. The peroxidase and oxidase-like activity of NiCo{sub 2}O{sub 4} mesoporous spheres: Mechanistic understanding and colorimetric biosensing

    Energy Technology Data Exchange (ETDEWEB)

    Su, Li, E-mail: suli@htu.edu.cn [Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007 (China); Henan Key Laboratory of Green Chemical Media and Reactions, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007 (China); Dong, Wenpei; Wu, Chengke; Gong, Yijun; Zhang, Yan; Li, Ling; Mao, Guojiang; Feng, Suling [Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007 (China); Henan Key Laboratory of Green Chemical Media and Reactions, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007 (China)

    2017-01-25

    The synthesized NiCo{sub 2}O{sub 4} mesoporous spheres (MS) displayed intrinsic peroxidase and oxidase-like activity were firstly reported. The catalytic mechanism of the oxidase-like activity of NiCo{sub 2}O{sub 4} MS was analyzed in detail using the electron spin resonance (ESR) method. It is found that NiCo{sub 2}O{sub 4} MS could directly oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) but did not produce {sup 1}O{sub 2} and ·OH. And the mechanism of the peroxidase-like activity of NiCo{sub 2}O{sub 4} MS was also verified that the oxidation of TMB stemmed from not only ·OH but also {sup 1}O{sub 2}. Based on the NiCo{sub 2}O{sub 4} MS showed excellent peroxidase-like activity over a broad temperature range, especially at normal body temperature, a detection tool was designed for glucose determination in diabetics' serum samples. And this detection method based on NiCo{sub 2}O{sub 4} MS gave a lower limit of detection than the method using Co{sub 3}O{sub 4} NPs and NiO NPs, as the single-component oxides of NiCo{sub 2}O{sub 4}. Our study may open up the possibility to make a great influence on the next generation of enzyme mimetics system. - Highlights: • NiCo{sub 2}O{sub 4} MS were found to possess the peroxidase and oxidase-like activity. • The peroxidase-like activity of NiCo{sub 2}O{sub 4} MS was stemmed from not only ·OH but also {sup 1}O{sub 2}. • The oxidase-like activity may stem from NiCo{sub 2}O{sub 4} MS′ oxidation rather than ·OH and {sup 1}O{sub 2}. • A colorimetric detection tool is designed for glucose determination in serum samples.

  10. Optimization of glucose oxidase production by Aspergillus niger

    African Journals Online (AJOL)

    user

    2011-02-28

    Feb 28, 2011 ... manganese, cobalt, thioglycolic acid, and gluconic acid according to (Liu et al., .... In this experiment duplicate media of glucose 10% were adjusted at different ... Glucose oxidase as a pharmaceutical anti oxidant Drug. Devt. ... Plush KS, Hellmuth K, Rinas U (1996). kinetics of glucose oxidase excretion by ...

  11. Immunological and molecular comparison of polyphenol oxidase in Rosaceae fruit trees.

    Science.gov (United States)

    Haruta, M; Murata, M; Kadokura, H; Homma, S

    1999-03-01

    An antibody raised against apple polyphenol oxidase (PPO) cross-reacted with PPOs from Japanese pear (Pyrus pyrifolia), pear (Pyrus communis), peach (Prunus persica), Chinese quince (Pseudocydonia sinensis) and Japanese loquat (Eriobotrya japonica). Core fragments (681 bp) of the corresponding PPO genes were amplified and characterized. The deduced protein sequences showed identities of 85.3 to 97.5%. Chlorogenic acid oxidase activity of these PPOs showed higher activities when assayed at pH 4 than at pH 6. These results indicate that PPOs in Rosaceae plants are structurally and enzymatically similar.

  12. Safrole oxide induces neuronal apoptosis through inhibition of integrin beta4/SOD activity and elevation of ROS/NADPH oxidase activity.

    Science.gov (United States)

    Su, Le; Zhao, BaoXiang; Lv, Xin; Wang, Nan; Zhao, Jing; Zhang, ShangLi; Miao, JunYing

    2007-02-20

    Neuronal apoptosis is a very important event in the development of the central nervous system (CNS), but the underlying mechanisms remain to be elucidated. We have previously shown that safrole oxide, a small molecule, induces integrin beta4 expression and promotes apoptosis in vascular endothelial cells. In this study, the effects of safrole oxide on cell growth and apoptosis have been examined in primary cultures of mouse neurons. Safrole oxide was found to significantly inhibit neuronal cell growth and to induce apoptosis. The inhibitory and apoptotic activities of safrole oxide followed a dose- and time-dependent manner. Interestingly, the expression of integrin beta4 was significantly inhibited with safrole oxide treatment. Furthermore, safrole oxide dramatically increases the level of intracellular reactive oxygen species (ROS) and the activity of NADPH oxidase. Moreover, manganese-dependent superoxide dismutase (MnSOD) activity was decreased significantly with safrole oxide treatment. Our study thus demonstrates that safrole oxide induces neuronal apoptosis through integrin beta4, ROS, NADPH, and MnSOD.

  13. Amyloid β induces NLRP3 inflammasome activation in retinal pigment epithelial cells via NADPH oxidase- and mitochondria-dependent ROS production.

    Science.gov (United States)

    Wang, Ke; Yao, Yong; Zhu, Xue; Zhang, Kai; Zhou, Fanfan; Zhu, Ling

    2017-06-01

    Amyloid β (Aβ)-induced chronic inflammation is believed to be a key pathogenic process in early-stage age-related macular degeneration (AMD). Nucleotide oligomerization domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation triggered by Aβ is responsible for retinal pigment epithelium (RPE) dysfunction in the onset of AMD; however, the detailed molecular mechanism remains unclear. In this study, we investigated the involvement of NADPH oxidase- and mitochondria-derived reactive oxygen species (ROS) in the process of Aβ 1-40 -induced NLRP3 inflammasome activation in LPS-primed ARPE-19 cells. The results showed that Aβ 1-40 could induce excessive ROS generation, MAPK/NF-κB signaling activation and subsequently NLRP3 inflammasome activation in LPS-primed ARPE-19 cells. Furthermore, the inductive effect of Aβ 1-40 on NLRP3 inflammasome activation was mediated in a manner dependent on NADPH oxidase- and mitochondria-derived ROS. Our findings may provide a novel insight into the molecular mechanism by which Aβ contributes to the early-stage AMD. © 2016 Wiley Periodicals, Inc.

  14. Cardiovascular activity of rasagiline, a selective and potent inhibitor of mitochondrial monoamine oxidase B: comparison with selegiline

    OpenAIRE

    Abassi, Zaid A; Binah, Ofer; Youdim, Moussa B H

    2004-01-01

    Selegiline is used for treating Parkinson's disease. Despite its efficacy, the clinical use of selegiline in combination with L-dihydroxphenylalanine in Parkinsonian patients is hampered by cardiovascular complications, such as hypotension. This study was designed to compare in rats the cardiovascular effects of selegiline and rasagiline, their metabolites L-methamphetamine and aminoindan (TVP-136), respectively, and the second rasagiline metabolite non-monoamine oxidase (MAO) inhibitor TVP-1...

  15. Comparison of endogenous cytokinins and cytokinin oxidase/dehydrogenase activity in germinating and thermoinhibited Tagetes minuta achenes

    Czech Academy of Sciences Publication Activity Database

    Stirk, W. A.; Novák, Ondřej; Žižková, Eva; Motyka, Václav; Strnad, Miroslav; van Staden, J.

    2012-01-01

    Roč. 169, č. 7 (2012), s. 696-703 ISSN 0176-1617 R&D Projects: GA MŠk(CZ) LC06034; GA ČR(CZ) GAP506/11/0774 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinin biosynthesis * cytokinin oxidase/dehydrogenase * deactivation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.699, year: 2012

  16. Cytochrome oxidase activity in the preoptic area correlates with differences in sexual behavior of intact and castrated male leopard geckos (Eublepharis macularius).

    Science.gov (United States)

    Sakata, J T; Crews, D

    2004-08-01

    Although the utility of analyzing behavioral experience effects on neural cytochrome oxidase (CO) activity is well recognized, the behavioral correlates of endogenous differences in CO activity have rarely been explored. In male leopard geckos (Eublepharis macularius), the incubation temperature experienced during embryogenesis (IncT) and age affect CO activity in the preoptic area (POA), an area that modulates copulatory behavior. In this study, the authors assessed whether differences in POA CO activity correlate with differences in sexual behavior in intact and castrated geckos. Males with IncT- and age-dependent increases in POA CO activity mounted females with shorter latencies while intact and after castration and ejaculated more frequently after castration. The authors discuss the predictive value of CO activity and propose similar parallels in other species.

  17. Role of TLR4/NADPH oxidase/ROS-activated p38 MAPK in VCAM-1 expression induced by lipopolysaccharide in human renal mesangial cells

    Directory of Open Access Journals (Sweden)

    Lee I-Ta

    2012-11-01

    Full Text Available Abstract Background In bacteria-induced glomerulonephritis, Toll-like receptor 4 (TLR4 activation by lipopolysaccharide (LPS, a key component of the outer membranes of Gram-negative bacteria can increase oxidative stress and the expression of vascular cell adhesion molecule-1 (VCAM-1, which recruits leukocytes to the glomerular mesangium. However, the mechanisms underlying VCAM-1 expression induced by LPS are still unclear in human renal mesangial cells (HRMCs. Results We demonstrated that LPS induced VCAM-1 mRNA and protein levels associated with an increase in the promoter activity of VCAM-1, determined by Western blot, RT-PCR, and promoter assay. LPS-induced responses were inhibited by transfection with siRNAs of TLR4, myeloid differentiation factor 88 (MyD88, Nox2, Nox4, p47phox, c-Src, p38 MAPK, activating transcription factor 2 (ATF2, and p300 or pretreatment with the inhibitors of reactive oxygen species (ROS, edaravone, NADPH oxidase [apocynin (APO or diphenyleneiodonium chloride (DPI], c-Src (PP1, p38 MAPK (SB202190, and p300 (GR343. LPS induced NADPH oxidase activation, ROS production, and p47phox translocation from the cytosol to the membrane, which were reduced by PP1 or c-Src siRNA. We observed that LPS induced TLR4, MyD88, c-Src, and p47phox complex formation determined by co-immunoprecipitation and Western blot. We further demonstrated that LPS stimulated ATF2 and p300 phosphorylation and complex formation via a c-Src/NADPH oxidase/ROS/p38 MAPK pathway. Up-regulation of VCAM-1 led to enhancing monocyte adhesion to HRMCs challenged with LPS, which was inhibited by siRNAs of c-Src, p47phox, p38 MAPK, ATF2, and p300 or pretreatment with an anti-VCAM-1 neutralizing antibody. Conclusions In HRMCs, LPS-induced VCAM-1 expression was, at least in part, mediated through a TLR4/MyD88/ c-Src/NADPH oxidase/ROS/p38 MAPK-dependent p300 and ATF2 pathway associated with recruitment of monocyte adhesion to kidney. Blockade of these pathways may

  18. Electrochemical pretreatment of amino-carbon nanotubes on graphene support as a novel platform for bilirubin oxidase with improved bioelectrocatalytic activity towards oxygen reduction.

    Science.gov (United States)

    Navaee, Aso; Salimi, Abdollah; Jafari, Fereydoon

    2015-03-23

    The electrochemical conditioning of amino-carbon nanotubes (CNTs) on a graphene support in an alkaline solution is used to produce -NHOH as hydrophilic functional groups for the efficient immobilization of bilirubin oxidase enzyme. The application of the immobilized enzyme for the direct electrocatalytic reduction of O2 is investigated. The onset potential of 0.81 V versus NHE and peak current density of 2.3 mA cm(-2) for rotating modified electrode at 1250 rpm, indicate improved biocatalytic activity of the proposed system for O2 reduction. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. IN VITRO INHIBITION OF CELERY (Apium graveolens L. EXTRACT ON THE ACTIVITY OF XANTHINE OXIDASE AND DETERMINATION OF ITS ACTIVE COMPOUND

    Directory of Open Access Journals (Sweden)

    Dyah Iswantini

    2012-12-01

    Full Text Available The objective of this study was to determine the inhibition effect of celery extracts toward xanthine oxidase by in vitro method, and its active compounds. Roots and herb of celery were extracted using water and ethanol solvents. Results indicated that the herbal ethanol extract had the highest inhibition effect (91.40% at 1400 ppm. The components contained in the herbal ethanol extract were then separated by column chromatography using the best eluent (chloroform : ethyl acetate at 7:3. All of the fractions had inhibition effect greater than 50%. The fraction number 4 was the one with the highest inhibition effect followed by fraction 5 with inhibition percentage of both fractions at 200 ppm were 88.62 and 85.44%, respectively. The analysis of the ultraviolet spectrum of fraction 4 showed the presence of π-π* transition which was resulted by the aromatic C=C, -OH, and C-O chromophores, and also showing the n-σ* transition which was given by -C=O chromophore. The infrared spectrum analysis indicated the presence of aromatic -C=C, -OH, and C=O functional groups. Based on the phytochemical assay and both instrumental spectrums, it was thought that the active compounds of fraction 4 and 5 were in the flavonoid group.

  20. Effect of pulsed electric field treatment on enzyme kinetics and thermostability of endogenous ascorbic acid oxidase in carrots (Daucus carota cv. Nantes).

    Science.gov (United States)

    Leong, Sze Ying; Oey, Indrawati

    2014-03-01

    The objective of this research was to study the enzyme kinetics and thermostability of endogenous ascorbic acid oxidase (AAO) in carrot purée (Daucus carota cv. Nantes) after being treated with pulsed electric field (PEF) processing. Various PEF treatments using electric field strength between 0.2 and 1.2kV/cm and pulsed electrical energy between 1 and 520kJ/kg were conducted. The enzyme kinetics and the kinetics of AAO thermal inactivation (55-70°C) were described using Michaelis-Menten model and first order reaction model, respectively. Overall, the estimated Vmax and KM values were situated in the same order of magnitude as the untreated carrot purée after being exposed to pulsed electrical energy between 1 and 400kJ/kg, but slightly changed at pulsed electrical energy above 500kJ/kg. However, AAO presented different thermostability depending on the electric field strength applied. After PEF treatment at the electric field strength between 0.2 and 0.5kV/cm, AAO became thermolabile (i.e. increase in inactivation rate (k value) at reference temperature) but the temperature dependence of k value (Ea value) for AAO inactivation in carrot purée decreased, indicating that the changes in k values were less temperature dependent. It is obvious that PEF treatment affects the temperature stability of endogenous AAO. The changes in enzyme kinetics and thermostability of AAO in carrot purée could be related to the resulting carrot purée composition, alteration in intracellular environment and the effective concentration of AAO released after being subjected to PEF treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. ATP mediates NADPH oxidase/ROS generation and COX-2/PGE2 expression in A549 cells: role of P2 receptor-dependent STAT3 activation.

    Directory of Open Access Journals (Sweden)

    Shin-Ei Cheng

    Full Text Available BACKGROUND: Up-regulation of cyclooxygenase (COX-2 and its metabolite prostaglandin E(2 (PGE(2 are frequently implicated in lung inflammation. Extracellular nucleotides, such as ATP have been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases, such as lung inflammation. However, the mechanisms underlying ATP-induced COX-2 expression and PGE(2 release remain unclear. PRINCIPAL FINDINGS: Here, we showed that ATPγS induced COX-2 expression in A549 cells revealed by western blot and real-time PCR. Pretreatment with the inhibitors of P2 receptor (PPADS and suramin, PKC (Gö6983, Gö6976, Ro318220, and Rottlerin, ROS (Edaravone, NADPH oxidase [diphenyleneiodonium chloride (DPI and apocynin], Jak2 (AG490, and STAT3 [cucurbitacin E (CBE] and transfection with siRNAs of PKCα, PKCι, PKCμ, p47(phox, Jak2, STAT3, and cPLA(2 markedly reduced ATPγS-induced COX-2 expression and PGE(2 production. In addition, pretreatment with the inhibitors of P2 receptor attenuated PKCs translocation from the cytosol to the membrane in response to ATPγS. Moreover, ATPγS-induced ROS generation and p47(phox translocation was also reduced by pretreatment with the inhibitors of P2 receptor, PKC, and NADPH oxidase. On the other hand, ATPγS stimulated Jak2 and STAT3 activation which were inhibited by pretreatment with PPADS, suramin, Gö6983, Gö6976, Ro318220, GF109203X, Rottlerin, Edaravone, DPI, and apocynin in A549 cells. SIGNIFICANCE: Taken together, these results showed that ATPγS induced COX-2 expression and PGE(2 production via a P2 receptor/PKC/NADPH oxidase/ROS/Jak2/STAT3/cPLA(2 signaling pathway in A549 cells. Increased understanding of signal transduction mechanisms underlying COX-2 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.

  2. An update on potential molecular mechanisms underlying the actions of snake venom L-amino acid oxidases (LAAOs).

    Science.gov (United States)

    Paloschi, Mauro Valentino; Pontes, Adriana Silva; Soares, Andreimar Martins; Zuliani, Juliana Pavan

    2017-11-08

    LAAOs (EC 1.4.3.2) are found in concentrations that vary according to each species of snakes; Viperidae, Crotalidae and Elapidae contain 1-9% of this enzyme in their venoms. This review focuses on an update on molecular mechanisms, platelet activities, antimicrobial, antiprotozoal, induction of apoptosis and inflammatory potential underlying the actions of SV-LAAOs. Snake venom LAAOs (SV-LAAOs) have become an interesting subject for pharmacological, structural and molecular studies. Although the mechanisms of action of these enzymes are not well understood they are a subject of a variety of studies, because LAAOs are multifunctional enzymes exhibiting a wide range of pharmacological effects, including the inhibition or induction of platelet aggregation, hemolysis and hemorrhage, in addition to the stimulation of apoptosis, the activation of leukocytes and the formation of edema. Moreover, SV-LAAOs play an important role in bactericidal, cytotoxic, anti-parasitic, anti-tumor, and antiviral activities. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Catabolism of caffeine and purification of a xanthine oxidade responsible for methyluric acids production in Pseudomonas putida L Catabolismo de cafeína e purificação de xantina oxidase responsável pela produção de ácidos metilúricos em Pseudomonas putida L

    Directory of Open Access Journals (Sweden)

    Dirce Mithico Yamaoka-Yano

    1999-01-01

    Full Text Available Caffeine catabolism and a xanthine oxidase involved in the alkaloid breakdown were studied in Pseudomonas putida L, a strain displaying high ability to grow on this substrate. Cells cultured with unlabelled caffeine and 14C labeled caffeine and xanthine showed that this alkaloid was broken-down via theobromine/paraxanthine -> 7-methylxanthine -> xanthine -> uric acid -> allantoin -> allantoic acid. Methyluric acids were formed from the oxidation of theobromine, paraxanthine and 7-methylxanthine, although no bacterial growth was observed on these compounds, indicating that this might be due to a wide substrate specificity of xanthine oxidase. This was confirmed by activity staining in PAGE where activity was observed with theophylline and 3-methylxanthine, which are not involved in the alkaloid breakdown. A single band of activity was detected without addition of NAD+, showing an oxidase form of the enzyme. The enzyme optimum temperature and pH were 30oC and 7.0, respectively. The determined Km was 169 µM, and the pI 3.1 - 4.0. The molecular weight determined by side by side comparison of activity staining of the enzyme in PAGE and PAGE of BSA was 192 kDa, which was coincident with the sum (198.4 kDa of three subunits (71, 65.6 and 61.8 kDa of the purified protein.O catabolismo de cafeína e a enzima xantina oxidase, envolvida na sua degradação, foram estudados em Pseudomonas putida L, uma linhagem com alta capacidade para utilizar este substrato como fonte de energia. Células crescidas na presença de cafeína e xantina marcadas com 14C, e cafeína não marcada, mostraram que este alcalóide foi degradado via teobromina/paraxantina -> 7-metilxantina -> xantina -> ácido úrico -> alantoína -> ácido alantóico. Ácidos metilúricos foram formados a partir de teobromina, paraxantina e 7-metilxantina, embora nenhum crescimento bacteriano ter sido observado quando estes compostos foram usados como substratos, indicando que a xantina oxidase

  4. Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice (Oryza sativa L.).

    Science.gov (United States)

    Huang, Jian; Tang, Ding; Shen, Yi; Qin, Baoxiang; Hong, Lilan; You, Aiqing; Li, Ming; Wang, Xin; Yu, Hengxiu; Gu, Minghong; Cheng, Zhukuan

    2010-01-01

    Gibberellin (GA) 2-oxidase plays a key role in the GA catabolic pathway through 2beta-hydroxylation. In the present study, we isolated a CaMV 35S-enhancer activation tagged mutant, H032. This mutant exhibited a dominant dwarf and GA-deficient phenotype, with a final stature that was less than half of its wild-type counterpart. The endogenous bioactive GAs are markedly decreased in the H032 mutant, and application of bioactive GAs (GA(3) or GA(4)) can reverse the dwarf phenotype. The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR. An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant, which might be due to the enhancer role of the CaMV 35S promoter. RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene. BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases, which is a novel type of GA2ox that uses C20-GAs (GA(12) and/or GA(53)) as the substrates. Interestingly, we found that a GA biosynthesis inhibitor, paclobutrazol, positively regulated the OsGA2ox6 gene. Unlike the over-expression of OsGA2ox1, which led to a high rate of seed abortion, the H032 mutant retained normal flowering and seed production. These results indicate that OsGA2ox6 mainly affects plant stature, and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding. Copyright 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  5. Calcineurin Aβ regulates NADPH oxidase (Nox) expression and activity via nuclear factor of activated T cells (NFAT) in response to high glucose.

    Science.gov (United States)

    Williams, Clintoria R; Gooch, Jennifer L

    2014-02-21

    Hypertrophy is an adaptive response that enables organs to appropriately meet increased functional demands. Previously, we reported that calcineurin (Cn) is required for glomerular and whole kidney hypertrophy in diabetic rodents (Gooch, J. L., Barnes, J. L., Garcia, S., and Abboud, H. E. (2003). Calcineurin is activated in diabetes and is required for glomerular hypertrophy and ECM accumulation. Am. J. Physiol. Renal Physiol. 284, F144-F154; Reddy, R. N., Knotts, T. L., Roberts, B. R., Molkentin, J. D., Price, S. R., and Gooch, J. L. (2011). Calcineurin Aβ is required for hypertrophy but not matrix expansion in the diabetic kidney. J. Cell Mol. Med. 15, 414-422). Because studies have also implicated the reactive oxygen species-generating enzymes NADPH oxidases (Nox) in diabetic kidney responses, we tested the hypothesis that Nox and Cn cooperate in a common signaling pathway. First, we examined the role of the two main isoforms of Cn in hypertrophic signaling. Using primary kidney cells lacking a catalytic subunit of Cn (CnAα(-/-) or CnAβ(-/-)), we found that high glucose selectively activates CnAβ, whereas CnAα is constitutively active. Furthermore, CnAβ but not CnAα mediates hypertrophy. Next, we found that chronic reactive oxygen species generation in response to high glucose is attenuated in CnAβ(-/-) cells, suggesting that Cn is upstream of Nox. Consistent with this, loss of CnAβ reduces basal expression and blocks high glucose induction of Nox2 and Nox4. Inhibition of nuclear factor of activated T cells (NFAT), a CnAβ-regulated transcription factor, decreases Nox2 and Nox4 expression, whereas NFAT overexpression increases Nox2 and Nox4, indicating that the CnAβ/NFAT pathway modulates Nox. These data reveal that the CnAβ/NFAT pathway regulates Nox and plays an important role in high glucose-mediated hypertrophic responses in the kidney.

  6. Chemoenzymatic combination of glucose oxidase with titanium silicalite -1

    DEFF Research Database (Denmark)

    Vennestrøm, Peter Nicolai Ravnborg; Taarning, Esben; Christensen, Claus H.

    2010-01-01

    Zeozymes: A proof-of-concept is presented for the chemoenzymatic combination of titanium silicalite-1 zeolite with glucose oxidase. In this combination, glucose is oxidized to gluconic acid and the H2O2 byproduct formed in situ is used for the simultaneous oxidation of chemical substrates. Both...... a soluble glucose oxidase and a truly integrated heterogeneous combination whereby the oxidase enzyme is anchored onto the zeolite surface are reported....

  7. Cytokinin-induced upregulation of cytokinin oxidase activity in tobacco includes changes in enzyme glycosylation and secretion

    Czech Academy of Sciences Publication Activity Database

    Motyka, Václav; Vaňková, Radomíra; Čapková, Věra; Petrášek, Jan; Kamínek, Miroslav; Schmülling, T.

    2003-01-01

    Roč. 117, č. 1 (2003), s. 11-21 ISSN 0031-9317 R&D Projects: GA ČR GA522/00/1346; GA ČR GA206/02/0967; GA ČR GA522/99/1130; GA AV ČR IAA6038002 Grant - others:Volkswagen Stiftung(DE) I/72076 Institutional research plan: CEZ:AV0Z5038910 Keywords : cytokinin oxidase * Nicotiana tabacum * N6-benzylaminopurine Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.767, year: 2003

  8. Eicosapentaenoic Acid Modulates Trichomonas vaginalis Activity.

    Science.gov (United States)

    Korosh, Travis; Jordan, Kelsey D; Wu, Ja-Shin; Yarlett, Nigel; Upmacis, Rita K

    2016-01-01

    Trichomonas vaginalis is a sexually transmitted parasite and, while it is often asymptomatic in males, the parasite is associated with disease in both sexes. Metronidazole is an effective treatment for trichomoniasis, but resistant strains have evolved and, thus, it has become necessary to investigate other possible therapies. In this study, we examined the effects of native and oxidized forms of the sodium salts of eicosapentaenoic, docosahexaenoic, and arachidonic acids on T. vaginalis activity. Eicosapentaenoic acid was the most toxic with 190 and 380 μM causing approximately 90% cell death in Casu2 and ATCC 50142 strains, respectively. In contrast, oxidized eicosapentaenoic acid was the least toxic, requiring > 3 mM to inhibit activity, while low levels (10 μM) were associated with increased parasite density. Mass spectrometric analysis of oxidized eicosapentaenoic acid revealed C20 products containing one to six additional oxygen atoms and various degrees of bond saturation. These results indicate that eicosapentaenoic acid has different effects on T. vaginalis survival, depending on whether it is present in the native or oxidized form. A better understanding of lipid metabolism in T. vaginalis may facilitate the design of synthetic fatty acids that are effective for the treatment of metronidazole-resistant T. vaginalis. © 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.

  9. Expression Studies of Gibberellin Oxidases in Developing Pumpkin Seeds1

    Science.gov (United States)

    Frisse, Andrea; Pimenta, Maria João; Lange, Theo

    2003-01-01

    Two cDNA clones, 3-ox and 2-ox, have been isolated from developing pumpkin (Cucurbita maxima) embryos that show significant amino acid homology to gibberellin (GA) 3-oxidases and 2-oxidases, respectively. Recombinant fusion protein of clone 3-ox converted GA12-aldehyde, GA12, GA15, GA24, GA25, and GA9 to GA14-aldehyde, GA14, GA37, GA36, GA13, and GA4, respectively. Recombinant 2-ox protein oxidized GA9, GA4, and GA1 to GA51, GA34, and GA8, respectively. Previously cloned GA 7-oxidase revealed additional 3β-hydroxylation activity of GA12. Transcripts of this gene were identified in endosperm and embryo of the developing seed by quantitative reverse transcriptase-polymerase chain reaction and localized in protoderm, root apical meristem, and quiescent center by in situ hybridization. mRNA of the previously cloned GA 20-oxidase from pumpkin seeds was localized in endosperm and in tissues of protoderm, ground meristem, and cotyledons of the embryo. However, transcripts of the recently cloned GA 20-oxidase from pumpkin seedlings were found all over the embryo, and in tissues of the inner seed coat at the micropylar end. Previously cloned GA 2β,3β-hydroxylase mRNA molecules were specifically identified in endosperm tissue. Finally, mRNA molecules of the 3-ox and 2-ox genes were found in the embryo only. 3-ox transcripts were localized in tissues of cotyledons, protoderm, and inner cell layers of the root apical meristem, and 2-ox transcripts were found in all tissues of the embryo except the root tips. These results indicate tissue-specific GA-biosynthetic pathways operating within the developing seed. PMID:12644672

  10. The influence of local glucose oxidase activity on the potential/current distribution on stainless steel: A study by the wire beam electrode method

    Energy Technology Data Exchange (ETDEWEB)

    Wang Wei [Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, College of Chemistry and Chemical Engineering, Qingdao, 266100 (China)], E-mail: wwei@ouc.edu.cn; Zhang Xia [Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, College of Chemistry and Chemical Engineering, Qingdao, 266100 (China); Wang Jia [Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, College of Chemistry and Chemical Engineering, Qingdao, 266100 (China); State Key Laboratory for Corrosion and Protection, Shenyang, 110016 (China)

    2009-09-30

    The wire beam electrode (WBE) method was first used to study the activity of local glucose oxidase (GOD) on stainless steel surface in seawater. Glucose oxidase was immobilized in calcium alginate gel capsules, which were embedded in a layer of artificial biofilm (calcium alginate gel) on the WBE surface. The potential/current distributions on the WBE surface were mapped using a newly developed device for the WBE method in our lab. The results demonstrated that the catalysis of H{sub 2}O{sub 2} formation by GOD can produce local noble potential peaks and cathodic current zones on the stainless steel surface. An interesting fluctuant current distribution around cathodic zones was observed the first time. The potential and current maps showed that the enzyme heterogeneity of the artificial biofilm caused a corresponding electrochemical heterogeneity at the biofilm/metal interface. The application of the WBE method to ennoblement study enables us to observe the heterogeneous electrochemistry at biofilm/stainless steel interface directly, providing us with a powerful tool to investigate other biofilm-related processes such as microbially influenced corrosion (MIC)

  11. The influence of local glucose oxidase activity on the potential/current distribution on stainless steel: A study by the wire beam electrode method

    International Nuclear Information System (INIS)

    Wang Wei; Zhang Xia; Wang Jia

    2009-01-01

    The wire beam electrode (WBE) method was first used to study the activity of local glucose oxidase (GOD) on stainless steel surface in seawater. Glucose oxidase was immobilized in calcium alginate gel capsules, which were embedded in a layer of artificial biofilm (calcium alginate gel) on the WBE surface. The potential/current distributions on the WBE surface were mapped using a newly developed device for the WBE method in our lab. The results demonstrated that the catalysis of H 2 O 2 formation by GOD can produce local noble potential peaks and cathodic current zones on the stainless steel surface. An interesting fluctuant current distribution around cathodic zones was observed the first time. The potential and current maps showed that the enzyme heterogeneity of the artificial biofilm caused a corresponding electrochemical heterogeneity at the biofilm/metal interface. The application of the WBE method to ennoblement study enables us to observe the heterogeneous electrochemistry at biofilm/stainless steel interface directly, providing us with a powerful tool to investigate other biofilm-related processes such as microbially influenced corrosion (MIC).

  12. A role for NADPH oxidase in antigen presentation

    Directory of Open Access Journals (Sweden)

    Gail J Gardiner

    2013-09-01

    Full Text Available The nicotinamide adenine dinucleotide phosphate (NADPH oxidase expressed in phagocytes is a multi-subunit enzyme complex that generates superoxide (O2.-. This radical is an important precursor of hydrogen peroxide (H2O2 and other reactive oxygen species (ROS needed for microbicidal activity during innate immune responses. Inherited defects in NADPH oxidase give rise to chronic granulomatous disease (CGD, a primary immunodeficiency characterized by recurrent infections and granulomatous inflammation. Interestingly, CGD, CGD carrier status, and oxidase gene polymorphisms have all been associated with autoinflammatory and autoimmune disorders, suggesting a potential role for NADPH oxidase in regulating adaptive immune responses. Here, NADPH oxidase function in antigen processing and presentation is reviewed. NADPH oxidase influences dendritic cell (DC crosspresentation by major histocompatibility complex class I molecules (MHC-I through regulation of the phagosomal microenvironment, while in B lymphocytes, NADPH oxidase alters epitope selection by major histocompatibility complex class II molecules (MHC-II.

  13. Immobilization of glucose oxidase onto a novel platform based on modified TiO{sub 2} and graphene oxide, direct electrochemistry, catalytic and photocatalytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Haghighi, Nasibeh, E-mail: Haghighi.nasibeh@yahoo.com [Department of Chemistry, University of Kurdistan, P.O. Box 416, Sanandaj (Iran, Islamic Republic of); Hallaj, Rahman, E-mail: Rhallaj@uok.ac.ir [Department of Chemistry, University of Kurdistan, P.O. Box 416, Sanandaj (Iran, Islamic Republic of); Nanotechnology Research Center, University of Kurdistan, P.O. Box 416, Sanandaj (Iran, Islamic Republic of); Salimi, Abdollah [Department of Chemistry, University of Kurdistan, P.O. Box 416, Sanandaj (Iran, Islamic Republic of); Nanotechnology Research Center, University of Kurdistan, P.O. Box 416, Sanandaj (Iran, Islamic Republic of)

    2017-04-01

    In this work a new organic–inorganic nanocomposite has been introduced for enzyme immobilization. The composite consisting of graphene oxide (GO) and titanium oxide nanoparticles (TiO{sub 2}) modified with 2, 2′-dithioxo-3, 3′-bis (3-(triethoxysilyl) propyl)-2H, 2′H-[5, 5′-bithiazolylidene]-4, 4′(3H, 3′H)-dione as Organic-Inorganic Supporting Ligand (OISL). The OISL was covalently attached to TiO{sub 2} nanoparticles and employed for obtaining a suitable solid surface to enzyme attachment. The glucose oxidase (GOD) was irreversibly loaded on the GC/GO/TiO{sub 2}-OISL using consecutive cyclic voltammetry. The enzyme immobilization and the enzymatic activity were determined by electrochemical methods. The cyclic voltammogram displayed a pair of well-defined and nearly symmetric redox peaks with a formal potential of − 0.465 V and an apparent electron transfer rate constant of 1.74 s{sup −1}. The GO/TiO{sub 2}-OISL can catalyze the electroreduction and electrooxidation of hydrogen peroxide. The GC/GO/TiO{sub 2}-OISL/GOD electrode was used in the hydrogen peroxide determination. The fabricated nanobiocomposite shows dramatic photoelectrocatalytic activity which evaluated by studying the electrocatalytic activity of the fabricated electrode toward hydrogen peroxide in darkness and in the presences of light. - Highlights: • In this work a novel platform used to successful immobilization of glucose oxidase. Due to its large functional group this modified nanoparticles load enzyme (GOD) and remain enzyme whit out denaturation for a long time. • The loaded enzyme shows direct electron transfer and excellent charge transfer kinetic. Also the fabricated nano-bio-composite has good catalytic activity toward hydrogen peroxide during electrooxidation and electro reduction process. • The nano-bio-composite shows excellent photocatalytic activity.

  14. Immobilization of glucose oxidase onto a novel platform based on modified TiO2 and graphene oxide, direct electrochemistry, catalytic and photocatalytic activity

    International Nuclear Information System (INIS)

    Haghighi, Nasibeh; Hallaj, Rahman; Salimi, Abdollah

    2017-01-01

    In this work a new organic–inorganic nanocomposite has been introduced for enzyme immobilization. The composite consisting of graphene oxide (GO) and titanium oxide nanoparticles (TiO 2 ) modified with 2, 2′-dithioxo-3, 3′-bis (3-(triethoxysilyl) propyl)-2H, 2′H-[5, 5′-bithiazolylidene]-4, 4′(3H, 3′H)-dione as Organic-Inorganic Supporting Ligand (OISL). The OISL was covalently attached to TiO 2 nanoparticles and employed for obtaining a suitable solid surface to enzyme attachment. The glucose oxidase (GOD) was irreversibly loaded on the GC/GO/TiO 2 -OISL using consecutive cyclic voltammetry. The enzyme immobilization and the enzymatic activity were determined by electrochemical methods. The cyclic voltammogram displayed a pair of well-defined and nearly symmetric redox peaks with a formal potential of − 0.465 V and an apparent electron transfer rate constant of 1.74 s −1 . The GO/TiO 2 -OISL can catalyze the electroreduction and electrooxidation of hydrogen peroxide. The GC/GO/TiO 2 -OISL/GOD electrode was used in the hydrogen peroxide determination. The fabricated nanobiocomposite shows dramatic photoelectrocatalytic activity which evaluated by studying the electrocatalytic activity of the fabricated electrode toward hydrogen peroxide in darkness and in the presences of light. - Highlights: • In this work a novel platform used to successful immobilization of glucose oxidase. Due to its large functional group this modified nanoparticles load enzyme (GOD) and remain enzyme whit out denaturation for a long time. • The loaded enzyme shows direct electron transfer and excellent charge transfer kinetic. Also the fabricated nano-bio-composite has good catalytic activity toward hydrogen peroxide during electrooxidation and electro reduction process. • The nano-bio-composite shows excellent photocatalytic activity.

  15. Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase ACOX2 in breast carcinomas

    International Nuclear Information System (INIS)

    Bjørklund, Sunniva Stordal; Kristensen, Vessela N.; Seiler, Michael; Kumar, Surendra; Alnæs, Grethe I. Grenaker; Ming, Yao; Kerrigan, John; Naume, Bjørn; Sachidanandam, Ravi; Bhanot, Gyan; Børresen-Dale, Anne-Lise; Ganesan, Shridar

    2015-01-01

    Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype. Ribonucleic acid-sequencing (RNA-seq) data from breast cancer samples was used to identify an intronic start variant transcript of Acyl-CoA oxidase 2, ACOX2 (ACOX2-i9). Difference in expression between Estrogen Receptor (ER) positive and ER negative patients was assessed by the Wilcoxon rank sum test, and the findings validated in The Cancer Genome Atlas (TCGA) breast cancer dataset (BRCA). ACOX2-i9 expression was also assessed in cell lines using both quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis. Knock down by short hairpin RNA (shRNA) and colony formation assays were used to determine whether ACOX2-i9 expression would influence cellular fitness. The effect of ACOX2-i9 expression on patient survival was assessed by the Kaplan-Meier survival function, and association to clinical parameters was analyzed using a Fisher exact test. The expression and translation of ACOX2-i9 into a 25 kDa protein was demonstrated in HepG2 cells as well as in several breast cancer cell lines. shRNA knock down of the ACOX2-i9 variant resulted in decreased cell viability of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 was shown to be estrogen regulated, being induced by propyl pyrazoletriol and inhibited by tamoxifen and fulvestrant in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript showed expression predominantly in ER-positive breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA breast cancer dataset, and expression was associated with better outcome in ER positive patients. ACOX2-i9 is specifically enriched in ER+ breast

  16. Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase ACOX2 in breast carcinomas.

    Science.gov (United States)

    Bjørklund, Sunniva Stordal; Kristensen, Vessela N; Seiler, Michael; Kumar, Surendra; Alnæs, Grethe I Grenaker; Ming, Yao; Kerrigan, John; Naume, Bjørn; Sachidanandam, Ravi; Bhanot, Gyan; Børresen-Dale, Anne-Lise; Ganesan, Shridar

    2015-07-17

    Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype. Ribonucleic acid-sequencing (RNA-seq) data from breast cancer samples was used to identify an intronic start variant transcript of Acyl-CoA oxidase 2, ACOX2 (ACOX2-i9). Difference in expression between Estrogen Receptor (ER) positive and ER negative patients was assessed by the Wilcoxon rank sum test, and the findings validated in The Cancer Genome Atlas (TCGA) breast cancer dataset (BRCA). ACOX2-i9 expression was also assessed in cell lines using both quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis. Knock down by short hairpin RNA (shRNA) and colony formation assays were used to determine whether ACOX2-i9 expression would influence cellular fitness. The effect of ACOX2-i9 expression on patient survival was assessed by the Kaplan-Meier survival function, and association to clinical parameters was analyzed using a Fisher exact test. The expression and translation of ACOX2-i9 into a 25 kDa protein was demonstrated in HepG2 cells as well as in several breast cancer cell lines. shRNA knock down of the ACOX2-i9 variant resulted in decreased cell viability of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 was shown to be estrogen regulated, being induced by propyl pyrazoletriol and inhibited by tamoxifen and fulvestrant in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript showed expression predominantly in ER-positive breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA breast cancer dataset, and expression was associated with better outcome in ER positive patients. ACOX2-i9 is specifically enriched in ER+ breast

  17. Characterization of Two VAO-Type Flavoprotein Oxidases from Myceliophthora thermophila

    Directory of Open Access Journals (Sweden)

    Alessandro R. Ferrari

    2018-01-01

    Full Text Available The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615 and XP_003665713 (MtVAO713, were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid.

  18. Cytochemical Localization of Glucose Oxidase in Peroxisomes of Aspergillus niger

    NARCIS (Netherlands)

    Veenhuis, Marten; Dijken, Johannes Pieter van

    1980-01-01

    The subcellular localization of glucose oxidase (E.C. 1.1.3.4) in mycelia of Aspergillus niger has been investigated using cytochemical staining techniques. Mycelia from fermenter cultures, which produced gluconic acid from glucose, contained elevated levels of glucose oxidase and catalase. Both

  19. Association between plasma activities of semicarbazide-sensitive amine oxidase and angiotensin-converting enzyme in patients with type 1 diabetes mellitus

    DEFF Research Database (Denmark)

    Boomsma, F; Pedersen-Bjergaard, U; Agerholm-Larsen, Birgit

    2005-01-01

    AIMS/HYPOTHESIS: Plasma semicarbazide-sensitive amine oxidase (SSAO) is elevated in patients with type 1 and type 2 diabetes and has been implicated in the pathophysiology of diabetic late complications. The regulation of SSAO production remains unknown. We studied correlations between plasma SSAO...... activity and parameters associated with diabetic late complications. METHODS: Plasma SSAO was measured in a well-characterised group of 287 patients with type 1 diabetes. Standard statistical methods were used to investigate correlations with clinical parameters and components of the renin.......001, r=0.27) was found between plasma SSAO and serum ACE activity in patients untreated with ACE inhibitors or angiotensin II receptor antagonists (n=221), but plasma SSAO did not differ by ACE I/D genotype. Plasma SSAO correlated positively with duration of diabetes, HbA(1)c and plasma renin...

  20. Design and synthesis of a tetradentate '3-amine-1-carboxylate' ligand to mimic the metal binding environment at the non-heme iron(II) oxidase active site.

    Science.gov (United States)

    Dungan, Victoria J; Ortin, Yannick; Mueller-Bunz, Helge; Rutledge, Peter J

    2010-04-07

    Non-heme iron(II) oxidases (NHIOs) catalyse a diverse array of oxidative chemistry in Nature. As part of ongoing efforts to realize biomimetic, iron-mediated C-H activation, we report the synthesis of a new 'three-amine-one-carboxylate' ligand designed to complex with iron(II) and mimic the NHIO active site. The tetradentate ligand has been prepared as a single enantiomer in nine synthetic steps from N-Cbz-L-alanine, pyridine-2,6-dimethanol and diphenylamine, using Seebach oxazolidinone chemistry to control the stereochemistry. X-Ray crystal structures are reported for two important intermediates, along with variable temperature NMR experiments to probe the hindered interconversion of conformational isomers of several key intermediates, 2,6-disubstituted pyridine derivatives. The target ligand and an N-Cbz-protected precursor were each then complexed with iron(II) and tested for their ability to promote alkene dihydroxylation, using hydrogen peroxide as the oxidant.

  1. Antidiabetic Activity from Gallic Acid Encapsulated Nanochitosan

    Science.gov (United States)

    Purbowatiningrum; Ngadiwiyana; Ismiyarto; Fachriyah, E.; Eviana, I.; Eldiana, O.; Amaliyah, N.; Sektianingrum, A. N.

    2017-02-01

    Diabetes mellitus (DM) has become a health problem in the world because it causes death. One of the phenolic compounds that have antidiabetic activity is gallic acid. However, the use of this compound still provides unsatisfactory results due to its degradation during the absorption process. The solution offered to solve the problem is by encapsulated it within chitosan nanoparticles that serve to protect the bioactive compound from degradation, increases of solubility and delivery of a bioactive compound to the target site by using freeze-drying technique. The result of chitosan nanoparticle’s Scanning Electron Microscopy (SEM) showed that chitosan nanoparticle’s size is uniform and it is smaller than chitosan. The value of encapsulation efficiency (EE) of gallic acid which encapsulated within chitosan nanoparticles is about 50.76%. Inhibition test result showed that gallic acid-chitosan nanoparticles at 50 ppm could inhibite α-glucosidase activity in 28.87% with 54.94 in IC50. So it can be concluded that gallic acid can be encapsulated in nanoparticles of chitosan and proved that it could inhibit α-glucosidase.

  2. Protection from cyanide-induced brain injury by the Nrf2 transcriptional activator carnosic acid.

    Science.gov (United States)

    Zhang, Dongxian; Lee, Brian; Nutter, Anthony; Song, Paul; Dolatabadi, Nima; Parker, James; Sanz-Blasco, Sara; Newmeyer, Traci; Ambasudhan, Rajesh; McKercher, Scott R; Masliah, Eliezer; Lipton, Stuart A

    2015-06-01

    Cyanide is a life-threatening, bioterrorist agent, preventing cellular respiration by inhibiting cytochrome c oxidase, resulting in cardiopulmonary failure, hypoxic brain injury, and death within minutes. However, even after treatment with various antidotes to protect cytochrome oxidase, cyanide intoxication in humans can induce a delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Additional mechanisms are thought to underlie cyanide-induced neuronal damage, including generation of reactive oxygen species. This may account for the fact that antioxidants prevent some aspects of cyanide-induced neuronal damage. Here, as a potential preemptive countermeasure against a bioterrorist attack with cyanide, we tested the CNS protective effect of carnosic acid (CA), a pro-electrophilic compound found in the herb rosemary. CA crosses the blood-brain barrier to up-regulate endogenous antioxidant enzymes via activation of the Nrf2 transcriptional pathway. We demonstrate that CA exerts neuroprotective effects on cyanide-induced brain damage in cultured rodent and human-induced pluripotent stem cell-derived neurons in vitro, and in vivo in various brain areas of a non-Swiss albino mouse model of cyanide poisoning that simulates damage observed in the human brain. Cyanide, a potential bioterrorist agent, can produce a chronic delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Here, cyanide poisoning treated with the proelectrophillic compound carnosic acid, results in reduced neuronal cell death in both in vitro and in vivo models through activation of the Nrf2/ARE transcriptional pathway. Carnosic acid is therefore a potential treatment for the toxic central nervous system (CNS) effects of cyanide poisoning. ARE, antioxidant responsive element; Nrf2 (NFE2L2, Nuclear factor (erythroid-derived 2)-like 2). © 2015 International Society for Neurochemistry.

  3. Engineering glucose oxidase to minimize the influence of oxygen on sensor response

    International Nuclear Information System (INIS)

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

    2014-01-01

    Glucose oxidase (GOx) is an important industrial enzyme and is recognized as the gold standard for monitoring blood glucose. However, due to its inherent oxidase property, the presence of oxygen affects electrochemical measurements of venous blood glucose employing artificial electron mediators. We therefore attempted to engineer Penicillium amagasakiense-derived GOx into a dehydrogenase by focusing on the amino acid residues predicted to interact with oxygen. Our rational amino acid substitution approach resulted in the construction of the Ser114Ala/Phe355Leu mutant, which has an 11-fold decrease in oxidase activity and 2.8-fold increase in dehydrogenase activity compared with wild-type GOx. As a result, the dehydrogenase/oxidase activity ratio of the engineered enzyme was 32-fold greater than that of the wild-type enzyme. The enzyme sensor constructed with Ser114Ala/Phe355Leu was considerably less affected by oxygen than the wild-type GOx-based sensor at lower glucose concentrations

  4. Synthesis and anticonvulsant activity of novel bicyclic acidic amino acids

    DEFF Research Database (Denmark)

    Conti, Paola; De Amici, Marco; Joppolo Di Ventimiglia, Samuele

    2003-01-01

    Bicyclic acidic amino acids (+/-)-6 and (+/-)-7, which are conformationally constrained homologues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested toward ionotropic and metabotropic glutamate receptor subtypes; both of them...

  5. Antiviral Activity of Polyacrylic and Polymethacrylic Acids

    Science.gov (United States)

    De Somer, P.; De Clercq, E.; Billiau, A.; Schonne, E.; Claesen, M.

    1968-01-01

    Polyacrylic acid (PAA) and polymethacrylic acid (PMAA) were investigated for their antiviral properties in tissue culture. Compared to other related polyanions, as dextran sulfate, polystyrene sulfonate, polyvinyl sulfate, and polyphloroglucinol phosphate, PAA and PMAA were found to be significantly more antivirally active and less cytotoxic. PMAA added 24 hr prior to virus inoculation inhibited viral growth most efficiently but it was still effective when added 3 hr after infection. Neither a direct irreversible action on the virus nor inhibition of virus penetration into the cell could explain the antiviral activity of PMAA. PMAA inhibited the adsorption of the virus to the host cell and suppressed the one-cycle viral synthesis in tissue cultures inoculated with infectious RNA. PMID:4302187

  6. Polyphenol oxidase activity from three sicilian artichoke [ Cynara cardunculus L. Var. scolymus L. (Fiori)] cultivars: studies and technological application on minimally processed production.

    Science.gov (United States)

    Todaro, Aldo; Peluso, Orazio; Catalano, Anna Eghle; Mauromicale, Giovanni; Spagna, Giovanni

    2010-02-10

    Several papers helped with the development of more methods to control browning, or study thermal polyphenol oxidase (PPO) inactivation, but did not provide any solutions to technological process problems and food process improvement. Artichokes [ Cynara cardunculus L. var. scolymus L. (Fiori)] are susceptible to browning; this alteration could affect and reduce the suitability for its use, fresh or processed. Within this study, the catecholase and cresolase activities of PPO from three different Sicilian artichokes cultivar were characterized with regard to substrate specificity and enzyme kinetics, optimum pH and temperature, temperature and pH stability, and inhibitor test; all of the results were used for technological purposes, particularly to optimize minimally processed productions (ready-to-eat and cook-chilled artichokes).

  7. Construction of a catalytically inactive cholesterol oxidase mutant: investigation of the interplay between active site-residues glutamate 361 and histidine 447.

    Science.gov (United States)

    Yin, Ye; Liu, Pingsheng; Anderson, Richard G W; Sampson, Nicole S

    2002-06-15

    Cholesterol oxidase catalyzes the oxidation of cholesterol to cholest-5-en-3-one and its subsequent isomerization into cholest-4-en-3-one. Two active-site residues, His447 and Glu361, are important for catalyzing the oxidation and isomerization reactions, respectively. Double-mutants were constructed to test the interplay between these residues in catalysis. We observed that the k(cat) of oxidation for the H447Q/E361Q mutant was 3-fold less than that for H447Q and that the k(cat) of oxidation for the H447E/E361Q mutant was 10-fold slower than that for H447E. Because both doubles-mutants do not have a carboxylate at position 361, they do not catalyze isomerization of the reaction intermediate cholest-5-en-3-one to cholest-4-en-3-one. These results suggest that Glu361 can compensate for the loss of histidine at position 447 by acting as a general base catalyst for oxidation of cholesterol. Importantly, the construction of the double-mutant H447E/E361Q yields an enzyme that is 31,000-fold slower than wild type in k(cat) for oxidation. The H447E/E361Q mutant is folded like native enzyme and still associates with model membranes. Thus, this mutant may be used to study the effects of membrane binding in the absence of catalytic activity. It is demonstrated that in assays with caveolae membrane fractions, the wild-type enzyme uncouples platelet-derived growth factor receptor beta (PDGFRbeta) autophosphorylation from tyrosine phosphorylation of neighboring proteins, and the H447E/E361Q mutant does not. Thus maintenance of membrane structure by cholesterol is important for PDGFRbeta-mediated signaling. The cholesterol oxidase mutant probe described will be generally useful for investigating the role of membrane structure in signal transduction pathways in addition to the PDGFRbeta-dependent pathway tested.

  8. A novel amperometric alcohol biosensor developed in a 3rd generation bioelectrode platform using peroxidase coupled ferrocene activated alcohol oxidase as biorecognition system.

    Science.gov (United States)

    Chinnadayyala, Somasekhar R; Kakoti, Ankana; Santhosh, Mallesh; Goswami, Pranab

    2014-05-15

    Alcohol oxidase (AOx) with a two-fold increase in efficiency (Kcat/Km) was achieved by physical entrapment of the activator ferrocene in the protein matrix through a simple microwave based partial unfolding technique and was used to develop a 3rd generation biosensor for improved detection of alcohol in liquid samples. The ferrocene molecules were stably entrapped in the AOx protein matrix in a molar ratio of ~3:1 through electrostatic interaction with the Trp residues involved in the functional activity of the enzyme as demonstrated by advanced analytical techniques. The sensor was fabricated by immobilizing ferrocene entrapped alcohol oxidase (FcAOx) and sol-gel chitosan film coated horseradish peroxidase (HRP) on a multi-walled carbon nanotube (MWCNT) modified glassy carbon electrode through layer-by-layer technique. The bioelectrode reactions involved the formation of H2O2 by FcAOx biocatalysis of substrate alcohol followed by HRP-catalyzed reduction of the liberated H2O2 through MWCNT supported direct electron transfer mechanism. The amperometric biosensor exhibited a linear response to alcohol in the range of 5.0 × 10(-6) to 30 × 10(-4)mol L(-1) with a detection limit of 2.3 × 10(-6) mol L(-1), and a sensitivity of 150 µA mM(-1) cm(-2). The biosensor response was steady for 28 successive measurements completed in a period of 5h and retained ~90% of the original response even after four weeks when stored at 4 °C. The biosensor was successfully applied for the determination of alcohol in commercial samples and its performance was validated by comparing with the data obtained by GC analyses of the samples. © 2013 Published by Elsevier B.V.

  9. Monoamine Oxidase Inhibitors (MAOIs)

    Science.gov (United States)

    ... health-medications/index.shtml. Accessed May 16, 2016. Hirsch M, et al. Monoamine oxidase inhibitors (MAOIs) for ... www.uptodate.com/home. Accessed May 16, 2016. Hirsch M, et al. Discontinuing antidepressant medications in adults. ...

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

    Directory of Open Access Journals (Sweden)

    Ana Maria Pandolfo Feoli

    2014-01-01

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

  11. Xanthine oxidase activity is associated with risk factors for cardiovascular disease and inflammatory and oxidative status markers in metabolic syndrome: effects of a single exercise session.

    Science.gov (United States)

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

    2014-01-01

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

  12. Effects of high pressure homogenization on the activity, stability, kinetics and three-dimensional conformation of a glucose oxidase produced by Aspergillus niger.

    Directory of Open Access Journals (Sweden)

    Alline Artigiani Lima Tribst

    Full Text Available High pressure homogenization (HPH is a non-thermal method, which has been employed to change the activity and stability of biotechnologically relevant enzymes. This work investigated how HPH affects the structural and functional characteristics of a glucose oxidase (GO from Aspergillus niger. The enzyme was homogenized at 75 and 150 MPa and the effects were evaluated with respect to the enzyme activity, stability, kinetic parameters and molecular structure. The enzyme showed a pH-dependent response to the HPH treatment, with reduction or maintenance of activity at pH 4.5-6.0 and a remarkable activity increase (30-300% at pH 6.5 in all tested temperatures (15, 50 and 75°C. The enzyme thermal tolerance was reduced due to HPH treatment and the storage for 24 h at high temperatures (50 and 75°C also caused a reduction of activity. Interestingly, at lower temperatures (15°C the activity levels were slightly higher than that observed for native enzyme or at least maintained. These effects of HPH treatment on function and stability of GO were further investigated by spectroscopic methods. Both fluorescence and circular dichroism revealed conformational changes in the molecular structure of the enzyme that might be associated with the distinct functional and stability behavior of GO.

  13. Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

    Science.gov (United States)

    Göttlicher, M; Widmark, E; Li, Q; Gustafsson, J A

    1992-01-01

    Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. Testing of compounds related to lipid metabolism or peroxisomal proliferation revealed that 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activate the receptor chimera. In addition, saturated fatty acids induce the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. In conclusion, the present results indicate that fatty acids can regulate gene expression mediated by a member of the steroid nuclear receptor superfamily. Images PMID:1316614

  14. Anticholinesterase activity of fluorochloronitroacetic acid esters

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, Yu.Ya.; Brel, V.K. Martynov, I.V.

    1984-11-01

    Results are presented from pharmacologic and biochemical experiments leading to the conclusion that fluorochloronitroacetic acid esters have anticholinesterase activity. Since the esters caused muscular weakness in mice, experiments were performed on isolated tissue preparation. The biochemical experiments consisted of finding the biomolecular constants of irreversible inhibition of acetylcholinesterase by the esters, using acetylcholinesterase from human erythrocytes, as well as horse serum cholinesterase. The ethyl and n-propyl esters of halogen nitroacetic acid were used in all experiments. It was found that the propyl ester caused an increase in the force of individual contractions in the isolated muscle specimens, plus an inability of the muscle to retain tetanus. The substances were determined to have an anticholinesterase effect. The mechanism of cholinesterase inhibition is not yet known. It is probable that the substances acylate the serine hydroxyl of the esterase center of the cholinestersase. 7 references, 1 figure.

  15. Gene cloning and characterization of NADH oxidase from ...

    African Journals Online (AJOL)

    use

    2011-12-07

    Dec 7, 2011 ... potent inhibitors of NADH oxidases, silver nitrate and potassium cyanide did not show any significant ... anaerobes, a class of organisms that have not been ... DNA and amino acid sequence analyses were performed using.

  16. Lactic acid bacteria from chicken carcasses with inhibitory activity against Salmonella spp. and Listeria monocytogenes.

    Science.gov (United States)

    Sakaridis, I; Soultos, N; Dovas, C I; Papavergou, E; Ambrosiadis, I; Koidis, P

    2012-02-01

    This study was conducted to isolate psychrotrophic lactic acid bacteria (LAB) from chicken carcasses with inhibitory activity against strains of Salmonella spp. and Listeria monocytogenes. A total of 100 broiler samples were examined for the presence of LAB. Ninety-two LAB isolates that showed antimicrobial effects against Salmonella spp. and L. monocytogenes were further analysed to examine their LAB (Gram-positive, catalase negative, oxidase negative) and psychrotrophic characteristics (ability to grow at 7 °C). Fifty isolates were further selected and identified initially using standard biochemical tests in miniature (Micro-kits API CH 50) and then by sequencing of the 16s-23s rRNA gene boundary region (Intergenic Spacer Region). By molecular identification, these isolates were classified into 5 different LAB species: Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus johnsonii, Pediococcus acidilactici, and Lactobacillus paralimentarius. None of the isolates produced tyramine or histamine. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Cardiovascular activity of rasagiline, a selective and potent inhibitor of mitochondrial monoamine oxidase B: comparison with selegiline.

    Science.gov (United States)

    Abassi, Zaid A; Binah, Ofer; Youdim, Moussa B H

    2004-10-01

    Selegiline is used for treating Parkinson's disease. Despite its efficacy, the clinical use of selegiline in combination with l-dihydroxphenylalanine in Parkinsonian patients is hampered by cardiovascular complications, such as hypotension. This study was designed to compare in rats the cardiovascular effects of selegiline and rasagiline, their metabolites l-methamphetamine and aminoindan (TVP-136), respectively, and the second rasagiline metabolite non-monoamine oxidase (MAO) inhibitor TVP-1022 (N-propargyl-1S(-)aminoindan). Intravenous (i.v.) administration of selegiline and rasagiline (1 mg kg(-1)) to anaesthetized rats (thiobutabarbital, 100 mg kg(-1), i.p.) did not affect mean arterial pressure (MAP), carotid blood flow (CBF) or carotid vascular resistance (CVR). Selegiline (10 mg kg(-1), i.v.) decreased MAP, CBF and increased CVR. In contrast, rasagiline (10 mg kg(-1), i.v.) caused a small transient decrease in MAP, while CBF and CVR were unchanged. l-methamphetamine (1 mg kg(-1), i.v.) administration provoked a dramatic and long-lasting depressor response, decreased CBF and increased CVR. In contrast, injection of aminoindan or TVP-1022 at a similar dose produced gradual nonsignificant decreases in MAP and CBF. Chronic oral treatment (21 days) of awake rats with selegiline at 10 mg kg(-1) decreased systolic blood pressure (SBP), diastolic blood pressure (DBP), and MAP, whereas heart rate was unaffected. Since the effective MAO-B inhibitory and clinical dose of rasagiline is about one-tenth that of selegiline, administration of 1 mg kg(-1) day(-1) rasagiline resulted in moderate decreases in SBP, DBP, and MAP, which were significantly lower than those caused by the 10 mg kg(-1) day(-1) dose of selegiline. These findings indicate that rasagiline, when given at doses equivalent to selegiline, is less likely to be hypotensive.

  18. Impact of high pressure processing on color, bioactive compounds, polyphenol oxidase activity, and microbiological attributes of pumpkin purée.

    Science.gov (United States)

    González-Cebrino, Francisco; Durán, Rocío; Delgado-Adámez, Jonathan; Contador, Rebeca; Bernabé, Rosario Ramírez

    2016-04-01

    Physicochemical parameters, bioactive compounds' content (carotenoids and total phenols), total antioxidant activity, and enzymatic activity of polyphenol oxidase (PPO) were evaluated after high pressure processing (HPP) on a pumpkin purée (cv. 'Butternut'). Three pressure levels (400, 500, and 600 MPa) were combined with three holding times (200, 400, and 600 s). The applied treatments reduced the levels of total aerobic mesophilic (TAM), total psychrophilic and psychrotrophic bacteria (TPP), and molds and yeasts (M&Y). All applied treatments did not affect enzymatic activity of PPO. Pressure level increased CIE L* values, which could enhance the lightness perception of high pressure (HP)-treated purées. No differences were found between the untreated and HP-treated purées regarding total phenols and carotenoids content (lutein, α-carotene, and β-carotene) and total antioxidant activity. HPP did not affect most quality parameters and maintained the levels of bioactive compounds. However, it did not achieve the complete inhibition of PPO, which could reduce the shelf-life of the pumpkin purée. © The Author(s) 2015.

  19. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

    International Nuclear Information System (INIS)

    Yang, Chuen-Mao; Lee, I-Ta; Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der

    2013-01-01

    TNF-α plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-α in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-α-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-α induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-κB (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47 phox , p42, p38, JNK1, p65, or PYK2. Moreover, TNF-α markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-α-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-α-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-α-stimulated MAPKs and NF-κB activation. Thus, in H9c2 cells, we are the first to show that TNF-α-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-κB cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-α-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-α on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-α induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-α induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF-α induces

  20. Triboelectrification of active pharmaceutical ingredients: week acids and their salts.

    Science.gov (United States)

    Fujinuma, Kenta; Ishii, Yuji; Yashihashi, Yasuo; Yonemochi, Estuo; Sugano, Kiyohiko; Tarada, Katsuhide

    2015-09-30

    The effect of salt formulation on the electrostatic property of active pharmaceutical ingredients was investigated. The electrostatic property of weak acids (carboxylic acids and amide-enole type acid) and their sodium salts was evaluated by a suction-type Faraday cage meter. Free carboxylic acids showed negative chargeability, whereas their sodium salts showed more positive chargeability than the free acids. However, no such trend was observed for amide-enole type acids. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Visual expression analysis of the responses of the alternative oxidase gene (aox1) to heat shock, oxidative, and osmotic stresses in conidia of citric acid-producing Aspergillus niger.

    Science.gov (United States)

    Honda, Yuki; Hattori, Takasumi; Kirimura, Kohtaro

    2012-03-01

    The citric acid-producing filamentous fungus Aspergillus niger WU-2223L shows cyanide-insensitive respiration catalyzed by alternative oxidase in addition to the cytochrome pathway. Sequence analysis of the 5' flanking region of the alternative oxidase gene (aox1) revealed a potential heat shock element (HSE) and a stress response element (STRE). We have previously confirmed aox1 expression in conidia. In this study, to confirm whether the upstream region of aox1 responds to various stresses, we used a visual expression analysis system for single-cell conidia of the A. niger strain AOXEGFP-1. This strain harbored a fusion gene comprising aox1 and egfp, which encodes the enhanced green fluorescent protein (EGFP). The fluorescence intensity of EGFP increased in conidia of A. niger AOXEGFP-1 that were subjected to heat shock at 35-45 °C, oxidative stress by exposure to 5mM paraquat or 1 mM t-butylhydroperoxide, or osmotic stresses by exposure to 0.5 M KCl or 1.0 M mannitol. These results indicate that the putative HSE and STRE in the upstream region of aox1 directly or indirectly respond to heat shock, oxidative, and osmotic stresses. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Influence of iodinated contrast media on the activities of histamine inactivating enzymes diamine oxidase and histamine N-methyltransferase in vitro.

    Science.gov (United States)

    Kuefner, M A; Feurle, J; Petersen, J; Uder, M; Schwelberger, H G

    2014-01-01

    Iodinated contrast media can cause pseudoallergic reactions associated with histamine release in significant numbers of patients. To clarify whether these adverse reactions may be aggravated by a compromised histamine catabolism we asked if radiographic contrast agents in vitro inhibit the histamine inactivating enzymes diamine oxidase (DAO) and histamine N-methyltransferase (HMT). Nine iodinated contrast agents were tested in vitro. Following pre-incubation of purified porcine kidney DAO and recombinant human HMT with 0.1-10mM of the respective contrast medium (H2O and specific inhibitors of DAO and HMT as controls) enzyme activities were determined by using radiometric micro assays. None of the contrast media irrespective of their structure showed significant inhibition of the activities of DAO and HMT. Pre-incubation of the enzymes with specific inhibitors led to complete inhibition of the respective enzymatic activity. The iodinated contrast media tested in vitro did not exhibit inhibition of histamine converting enzymes at physiologically relevant concentrations. However due to the in vitro character of this study these results do not directly reflect the in vivo situation. Copyright © 2012 SEICAP. Published by Elsevier Espana. All rights reserved.

  3. Hydroxysafflor Yellow A Inhibits LPS-Induced NLRP3 Inflammasome Activation via Binding to Xanthine Oxidase in Mouse RAW264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Xiaolong Xu

    2016-01-01

    Full Text Available Hydroxysafflor yellow A (HSYA is an effective therapeutic agent for inflammatory diseases and autoimmune disorders; however, its regulatory effect on NLRP3 inflammasome activation in macrophages has not been investigated. In this study, we predicted the potential interaction between HSYA and xanthine oxidase (XO via PharmMapper inverse docking and confirmed the binding inhibition via inhibitory test (IC50 = 40.04 μM. Computation docking illustrated that, in this HSYA-XO complex, HSYA was surrounded by Leu 648, Leu 712, His 875, Leu 873, Ser 876, Glu 879, Phe 649, and Asn 650 with a binding energy of −5.77 kcal/M and formed hydrogen bonds with the hydroxyl groups of HSYA at Glu 879, Asn 650, and His 875. We then found that HSYA significantly decreased the activity of XO in RAW264.7 macrophages and suppressed LPS-induced ROS generation. Moreover, we proved that HSYA markedly inhibited LPS-induced cleaved caspase-1 activation via suppressing the sensitization of NLRP3 inflammasome and prevented the mature IL-1β formation from pro-IL-1β form. These findings suggest that XO may be a potential target of HSYA via direct binding inhibition and the combination of HSYA-XO suppresses LPS-induced ROS generation, contributing to the depression of NLRP3 inflammasome and inhibition of IL-1β secretion in macrophages.

  4. Activation of peroxisome proliferator-activated receptor-α (PPARα) suppresses postprandial lipidemia through fatty acid oxidation in enterocytes

    International Nuclear Information System (INIS)

    Kimura, Rino; Takahashi, Nobuyuki; Murota, Kaeko; Yamada, Yuko; Niiya, Saori; Kanzaki, Noriyuki; Murakami, Yoko; Moriyama, Tatsuya; Goto, Tsuyoshi; Kawada, Teruo

    2011-01-01

    Highlights: → PPARα activation increased mRNA expression levels of fatty acid oxidation-related genes in human intestinal epithelial Caco-2 cells. → PPARα activation also increased oxygen consumption rate and CO 2 production and decreased secretion of triglyceride and ApoB from Caco-2 cells. → Orally administration of bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and CO 2 production in small intestinal epithelial cells. → Treatment with bezafibrate decreased postprandial serum concentration of triglyceride after oral injection of olive oil in mice. → It suggested that intestinal lipid metabolism regulated by PPARα activation suppresses postprandial lipidemia. -- Abstract: Activation of peroxisome proliferator-activated receptor (PPAR)-α which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPARα activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPARα activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPARα agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and production of CO 2 and acid soluble metabolites in enterocytes. Moreover

  5. Circadian profiling reveals higher histamine plasma levels and lower diamine oxidase serum activities in 24% of patients with suspected histamine intolerance compared to food allergy and controls.

    Science.gov (United States)

    Pinzer, T C; Tietz, E; Waldmann, E; Schink, M; Neurath, M F; Zopf, Y

    2018-04-01

    Histamine intolerance is thought to trigger manifold clinical symptoms after ingesting histamine-rich food due to reduced activity of diamine oxidase (DAO). No study has hitherto systematically assessed daily fluctuations of histamine levels and DAO activities in symptomatic patients. The aim of the study was to investigate the presence of histamine intolerance, to therefore establish day profiles of histamine levels and DAO activities, and to compare the results between patients with suspected histamine intolerance, food allergy and healthy controls. We determined day profiles of histamine plasma levels and DAO serum activities in 33 patients with suspected histamine intolerance, in 21 patients with proven food allergy and in 10 healthy control patients. Clinical symptoms, food intolerances and further clinical and laboratory chemical parameters were evaluated. Twenty-four percent (8 of 33) suspected histamine-intolerant patients showed elevated histamine levels during the day. That might be caused by constantly and significantly reduced DAO activities in these patients compared to food-allergic and control patients. The remaining 25 patients presented normal histamine levels and DAO activities, but an increased prevalence of multiple food intolerances compared to the other subgroup of suspected histamine-intolerants. There was no correlation between subjective complaints and serological histamine parameters in patients with suspected histamine intolerance. We determined by daily profiling that decreased DAO activities correlated with elevated histamine levels in a subgroup of suspected histamine-intolerants. This finding discriminates these patients from food intolerant individuals with similar clinical symptoms and strongly suggests the presence of histamine intolerance. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

  6. "JCE" Classroom Activity #109: My Acid Can Beat Up Your Acid!

    Science.gov (United States)

    Putti, Alice

    2011-01-01

    In this guided-inquiry activity, students investigate the ionization of strong and weak acids. Bead models are used to study acid ionization on a particulate level. Students analyze seven strong and weak acid models and make generalizations about the relationship between acid strength and dissociation. (Contains 1 table and 2 figures.)

  7. The antioxidant properties, cytotoxicity and monoamine oxidase ...

    African Journals Online (AJOL)

    Tarchonanthus camphoratus (camphor bush) has been widely used for numerous medicinal purposes. The aim of the present study was to evaluate the antioxidant properties, cytotoxicity and monoamine oxidase inhibition activities of the crude dichloromethane leaf extract of T. camphoratus. The antioxidant activities were ...

  8. Functional Analysis of the Trichoderma harzianum nox1 Gene, Encoding an NADPH Oxidase, Relates Production of Reactive Oxygen Species to Specific Biocontrol Activity against Pythium ultimum▿†

    Science.gov (United States)

    Montero-Barrientos, M.; Hermosa, R.; Cardoza, R. E.; Gutiérrez, S.; Monte, E.

    2011-01-01

    The synthesis of reactive oxygen species (ROS) is one of the first events following pathogenic interactions in eukaryotic cells, and NADPH oxidases are involved in the formation of such ROS. The nox1 gene of Trichoderma harzianum was cloned, and its role in antagonism against phytopathogens was analyzed in nox1-overexpressed transformants. The increased levels of nox1 expression in these transformants were accompanied by an increase in ROS production during their direct confrontation with Pythium ultimum. The transformants displayed an increased hydrolytic pattern, as determined by comparing protease, cellulase, and chitinase activities with those for the wild type. In confrontation assays against P. ultimum the nox1-overexpressed transformants were more effective than the wild type, but not in assays against Botrytis cinerea or Rhizoctonia solani. A transcriptomic analysis using a Trichoderma high-density oligonucleotide (HDO) microarray also showed that, compared to gene expression for the interaction of wild-type T. harzianum and P. ultimum, genes related to protease, cellulase, and chitinase activities were differentially upregulated in the interaction of a nox1-overexpressed transformant with this pathogen. Our results show that nox1 is involved in T. harzianum ROS production and antagonism against P. ultimum. PMID:21421791

  9. An LC-MS/MS-Based Method for the Quantification of Pyridox(am)ine 5'-Phosphate Oxidase Activity in Dried Blood Spots from Patients with Epilepsy.

    Science.gov (United States)

    Wilson, Matthew P; Footitt, Emma J; Papandreou, Apostolos; Uudelepp, Mari-Liis; Pressler, Ronit; Stevenson, Danielle C; Gabriel, Camila; McSweeney, Mel; Baggot, Matthew; Burke, Derek; Stödberg, Tommy; Riney, Kate; Schiff, Manuel; Heales, Simon J R; Mills, Kevin A; Gissen, Paul; Clayton, Peter T; Mills, Philippa B

    2017-09-05

    We report the development of a rapid, simple, and robust LC-MS/MS-based enzyme assay using dried blood spots (DBS) for the diagnosis of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (OMIM 610090). PNPO deficiency leads to potentially fatal early infantile epileptic encephalopathy, severe developmental delay, and other features of neurological dysfunction. However, upon prompt treatment with high doses of vitamin B 6 , affected patients can have a normal developmental outcome. Prognosis of these patients is therefore reliant upon a rapid diagnosis. PNPO activity was quantified by measuring pyridoxal 5'-phosphate (PLP) concentrations in a DBS before and after a 30 min incubation with pyridoxine 5'-phosphate (PNP). Samples from 18 PNPO deficient patients (1 day-25 years), 13 children with other seizure disorders receiving B 6 supplementation (1 month-16 years), and 37 child hospital controls (5 days-15 years) were analyzed. DBS from the PNPO-deficient samples showed enzyme activity levels lower than all samples from these two other groups as well as seven adult controls; no false positives or negatives were identified. The method was fully validated and is suitable for translation into the clinical diagnostic arena.

  10. Oral Administration of Semicarbazide Limits Weight Gain together with Inhibition of Fat Deposition and of Primary Amine Oxidase Activity in Adipose Tissue

    Directory of Open Access Journals (Sweden)

    Josep Mercader

    2011-01-01

    Full Text Available An enzyme hitherto named semicarbazide-sensitive amine oxidase (SSAO, involved in the oxidation of primary amines, is abundantly expressed in adipocytes. Although SSAO physiological functions remain unclear, several molecules inhibiting its activity have been described to limit fat accumulation in preadipocyte cultures or to reduce body weight gain in obese rodents. Here, we studied whether oral administration of semicarbazide, a prototypical SSAO inhibitor, limits fat deposition in mice. Prolonged treatment with semicarbazide at 0.125% in drinking water limited food and water consumption, hampered weight gain, and deeply impaired fat deposition. The adiposomatic index was reduced by 31%, while body mass was reduced by 15%. Such treatment completely inhibited SSAO, but did not alter MAO activity in white adipose tissue. Consequently, the insulin-like action of the SSAO substrate benzylamine on glucose transport was abolished in adipocytes from semicarbazide-drinking mice, while their insulin sensitivity was not altered. Although semicarbazide is currently considered as a food contaminant with deleterious effects, the SSAO inhibition it induces appears as a novel concept to modulate adipose tissue development, which is promising for antiobesity drug discovery.

  11. Antioxidant and cyclooxygenase activities of fatty acids found in food.

    Science.gov (United States)

    Henry, Geneive E; Momin, Rafikali A; Nair, Muraleedharan G; Dewitt, David L

    2002-04-10

    Several commercially available C-8 to C-24 saturated and unsaturated fatty acids (1-29) were assayed for cyclooxygenase-I (COX-I) and cyclooxygenase-II (COX-II) inhibitory and antioxidant activities. Among the saturated fatty acids tested at 60 microg mL(-1), there was an increase in antioxidant activity with increasing chain length from octanoic acid to myristic acid (C-8-C-14) and a decrease thereafter. All unsaturated fatty acids tested at 60 microg mL(-1) showed good antioxidant activity except for undecylenic acid (12), cis-5-dodecenoic acid (13), and nervonic acid (29). The highest inhibitory activities among the saturated fatty acids tested on cyclooxygenase enzymes COX-I and COX-II were observed for decanoic acid to lauric acid (3-5) at 100 microg mL(-1). Similarly, among the unsaturated fatty acids tested, the highest activities were observed for cis-8,11,14-eicosatrienoic acid (25) and cis-13,16-docosadienoic acid (27) at 100 microg mL(-1).

  12. Glucose oxidase-functionalized fluorescent gold nanoclusters as probes for glucose

    International Nuclear Information System (INIS)

    Xia, Xiaodong; Long, Yunfei; Wang, Jianxiu

    2013-01-01

    Highlights: ► A glucose oxidase/gold nanocluster conjugates formed by etching chemistry. ► Integration of the bioactivities and fluorescence properties within a single unit. ► These conjugates serve as novel fluorescent probe for glucose. -- Abstract: Creation and application of noble metal nanoclusters have received continuous attention. By integrating enzyme activity and fluorescence for potential applications, enzyme-capped metal clusters are more desirable. This work demonstrated a glucose oxidase (an enzyme for glucose)-functionalized gold cluster as probe for glucose. Under physiological conditions, such bioconjugate was successfully prepared by an etching reaction, where tetrakis (hydroxylmethyl) phosphonium-protected gold nanoparticle and thioctic acid-modified glucose oxidase were used as precursor and etchant, respectively. These bioconjugates showed unique fluorescence spectra (λ em max = 650 nm, λ ex max = 507 nm) with an acceptable quantum yield (ca. 7%). Moreover, the conjugated glucose oxidase remained active and catalyzed reaction of glucose and dissolved O 2 to produce H 2 O 2 , which quenched quantitatively the fluorescence of gold clusters and laid a foundation of glucose detection. A linear range of 2.0 × 10 −6 –140 × 10 −6 M and a detection limit of 0.7 × 10 −6 M (S/N = 3) were obtained. Also, another horseradish peroxidase/gold cluster bioconjugate was produced by such general synthesis method. Such enzyme/metal cluster bioconjugates represented a promising class of biosensors for biologically important targets in organelles or cells

  13. Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Tamayo-Ramos Juan Antonio

    2012-12-01

    Full Text Available Abstract Background Laccase-like multicopper oxidases have been reported in several Aspergillus species but they remain uncharacterized. The biocatalytic potential of the Aspergillus niger fungal pigment multicopper oxidases McoA and McoB and ascomycete laccase McoG was investigated. Results The laccase-like multicopper oxidases McoA, McoB and McoG from the commonly used cell factory Aspergillus niger were homologously expressed, purified and analyzed for their biocatalytic potential. All three recombinant enzymes were monomers with apparent molecular masses ranging from 80 to 110 kDa. McoA and McoG resulted to be blue, whereas McoB was yellow. The newly obtained oxidases displayed strongly different activities towards aromatic compounds and synthetic dyes. McoB exhibited high catalytic efficiency with N,N-dimethyl-p-phenylenediamine (DMPPDA and 2,2-azino-di(3-ethylbenzthiazoline sulfonic acid (ABTS, and appeared to be a promising biocatalyst. Besides oxidizing a variety of phenolic compounds, McoB catalyzed successfully the decolorization and detoxification of the widely used textile dye malachite green. Conclusions The A. niger McoA, McoB, and McoG enzymes showed clearly different catalytic properties. Yellow McoB showed broad substrate specificity, catalyzing the oxidation of several phenolic compounds commonly present in different industrial effluents. It also harbored high decolorization and detoxification activity with the synthetic dye malachite green, showing to have an interesting potential as a new industrial biocatalyst.

  14. Suspended biofilm carrier and activated sludge removal of acidic pharmaceuticals

    DEFF Research Database (Denmark)

    Falås, Per; Baillon-Dhumez, Aude; Andersen, Henrik Rasmus

    2012-01-01

    Removal of seven active pharmaceutical substances (ibuprofen, ketoprofen, naproxen, diclofenac, clofibric acid, mefenamic acid, and gemfibrozil) was assessed by batch experiments, with suspended biofilm carriers and activated sludge from several full-scale wastewater treatment plants. A distinct...... and attached solids for the carriers) of diclofenac, ketoprofen, gemfibrozil, clofibric acid and mefenamic acid compared to the sludges. Among the target pharmaceuticals, only ibuprofen and naproxen showed similar removal rates per unit biomass for the sludges and biofilm carriers. In contrast...

  15. The use of glucose oxidase and catalase for the enzymatic reduction of the potential ethanol content in wine.

    Science.gov (United States)

    Röcker, Jessica; Schmitt, Matthias; Pasch, Ludwig; Ebert, Kristin; Grossmann, Manfred

    2016-11-01

    Due to the increase of sugar levels in wine grapes as one of the impacts of climate change, alcohol reduction in wines becomes a major focus of interest. This study combines the use of glucose oxidase and catalase activities with the aim of rapid conversion of glucose into non-fermentable gluconic acid. The H2O2 hydrolysing activity of purified catalase is necessary in order to stabilize glucose oxidase activity. After establishing the adequate enzyme ratio, the procedure was applied in large-scale trials (16L- and 220L-scale) of which one was conducted in a winery under industrial wine making conditions. Both enzyme activity and wine flavour were clearly influenced by the obligatory aeration in the different trials. With the enzyme treatment an alcohol reduction of 2%vol. was achieved after 30h of aeration. However the enzyme treated wines were significantly more acidic and less typical. Copyright © 2016. Published by Elsevier Ltd.

  16. Combined effect of ultrasound, heat, and pressure on Escherichia coli O157:H7, polyphenol oxidase activity, and anthocyanins in blueberry (Vaccinium corymbosum) juice.

    Science.gov (United States)

    Zhu, Jinyan; Wang, Yuehua; Li, Xinghe; Li, Bin; Liu, Suwen; Chang, Nan; Jie, Ding; Ning, Chong; Gao, Haiyan; Meng, Xianjun

    2017-07-01

    The objective of this study was to evaluate the effect of different treatments-heat treatment (HT), sonication (SC), thermosonication (TS), manosonication (MS), manothermal (MT), and manothermosonication (MTS) on Escherichia coli O157:H7, polyphenol oxidase (PPO), and anthocyanin content in blueberry juice. First, samples were treated at different temperatures (30, 40, 50, 60, 70, and 80°C) and power intensities (280, 420, 560, and 700W) for 10min. Subsequently, samples were treated using combinations of power intensity and mild temperature for 10min. For further study, samples were treated using HT (80°C), TS (40°C, 560W), MT (350MPa, 40°C), MS (560W, 5min/350MPa), or MTS (560W, 5min, 40°C/350MPa, 40°C) for 5, 10, 15, 20min for each treatment, and the results compared between treatments. HT significantly reduced PPO activation (2.05% residual activity after only 5min), and resulted in a 2.00-log reduction in E. coli O157:H7 and an 85.25% retention of anthocyanin. Escherichia coli O157:H7 was slightly inactivated by TS after 5min (0.17-log reduction), while residual PPO activity was 23.36% and anthocyanin retention was 98.48%. However, Escherichia coli O157:H7 was rapidly inactivated by MTS (5.85-log reduction) after 5min, while anthocyanin retention was 97.49% and residual PPO activity dropped to 10.91%. The destruction of E. coli cells as a result of these treatments were confirmed using SEM and TEM. Therefore, a combination of sonication, high pressure, and mild heat allows the safety of blueberry juice to be maintained without compromising the retention of desirable antioxidant compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients

    Energy Technology Data Exchange (ETDEWEB)

    Nunes, S.F.; Figueiredo, I.V. [Laboratório de Farmacologia, Faculdade de Farmácia, Universidade de Coimbra, Coimbra (Portugal); Pereira, J.S. [Instituto Português de Oncologia de Coimbra, Coimbra (Portugal); Lopes, M.C.; Caramona, M.M. [Laboratório de Farmacologia, Faculdade de Farmácia, Universidade de Coimbra, Coimbra (Portugal)

    2011-11-25

    The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: K{sub m} (r = 0.612, P = 0.034) and V{sub max} (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: K{sub m} (r = -0.625, P = 0.029) and V{sub max} (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

  18. Exercise training protects against atherosclerotic risk factors through vascular NADPH oxidase, extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/c-Jun N-terminal kinase downregulation in obese rats.

    Science.gov (United States)

    Touati, Sabeur; Montezano, Augusto C I; Meziri, Fayçal; Riva, Catherine; Touyz, Rhian M; Laurant, Pascal

    2015-02-01

    Exercise training reverses atherosclerotic risk factors associated with metabolic syndrome and obesity. The aim of the present study was to determine the molecular anti-inflammatory, anti-oxidative and anti-atherogenic effects in aorta from rats with high-fat diet-induced obesity. Male Sprague-Dawley rats were placed on a high-fat (HFD) or control (CD) diet for 12 weeks. The HFD rats were then divided into four groups: (i) sedentary HFD-fed rats (HFD-S); (ii) exercise trained (motor treadmill 5 days/week, 60 min/day, 12 weeks) HFD-fed rats (HFD-Ex); (iii) modified diet (HFD to CD) sedentary rats (HF/CD-S); and (iv) an exercise-trained modified diet group (HF/CD-Ex). Tissue levels of NADPH oxidase (activity and expression), NADPH oxidase (Nox) 1, Nox2, Nox4, p47(phox) , superoxide dismutase (SOD)-1, angiotensin AT1 and AT2 receptors, phosphorylated mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK)) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the aorta. Plasma cytokines (tumour necrosis factor (TNF)-α and interleukin (IL)-6) levels were also measured. Obesity was accompanied by increases in NADPH oxidase activity, p47(phox) translocation, Nox4 and VCAM-1 protein expression, MAPK (ERK1/2, SAPK/JNK) phosphorylation and plasma TNF-α and IL-6 levels. Exercise training and switching from the HFD to CD reversed almost all these molecular changes. In addition, training increased aortic SOD-1 protein expression and decreased ERK1/2 phosphorylation. These findings suggest that protective effects of exercise training on atherosclerotic risk factors induced by obesity are associated with downregulation of NADPH oxidase, ERK1/2 and SAPK/JNK activity and increased SOD-1 expression. © 2014 Wiley Publishing Asia Pty Ltd.

  19. Synthesis, X-ray crystallography, spectroscopic characterization and spectroscopic/electrochemical evidence of formation of phenoxy free radical in active center analogs of galactose oxidase - [Cu(Salgly)H₂O] and [Cu(Salphenylalanine)H₂O].

    Science.gov (United States)

    Das, Biva; Medhi, Okhil K

    2013-03-01

    The formation of phenolate free radical is the factor of high turnover for catalytic activity of galactose oxidase (GO) compared to that by inorganic complexes. A new active center analog of GO, [Cu(II)(Salphenylalanine)H(2)O] have been synthesized and its single crystal X-ray analysis was done. In aqueous surfactant micellar solution chemical oxidation as well as electrochemical oxidation of structural models of galactose oxidase - [Cu(II)Salgly·H(2)O] and [Cu(II)(Salphenylalanine)·H(2)O], have been found to generate free radical originating at the phenolate group. Formation of the free radical have been proved by electron paramagnetic resonance spectroscopy, electronic spectroscopy and electrochemistry. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Gallic Acid Content in Taiwanese Teas at Different Degrees of Fermentation and Its Antioxidant Activity by Inhibiting PKCδ Activation: In Vitro and in Silico Studies.

    Science.gov (United States)

    Kongpichitchoke, Teeradate; Chiu, Ming-Tzu; Huang, Tzou-Chi; Hsu, Jue-Liang

    2016-10-12

    Teas can be classified according to their degree of fermentation, which has been reported to affect both the bioactive components in the teas and their antioxidative activity. In this study, four kinds of commercial Taiwanese tea at different degrees of fermentation, which include green (non-fermented), oolong (semi-fermented), black (fully fermented), and Pu-erh (post-fermented) tea, were profiled for catechin levels by using high performance liquid chromatography (HPLC). The result indicated that the gallic acid content in tea was directly proportional to the degree of fermentation in which the lowest and highest gallic acid content were 1.67 and 21.98 mg/g from green and Pu-erh tea, respectively. The antioxidative mechanism of the gallic acid was further determined by in vitro and in silico analyses. In vitro assays included the use of phorbol ester-induced macrophage RAW264.7 cell model for determining the inhibition of reactive oxygen species (ROS) production, and PKCδ and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit (p47) activations. The results showed that only at a concentration of 5.00 μM could gallic acid significantly ( p gallic acid could block PKCδ activation by occupying the phorbol ester binding sites of the protein.

  1. Alterations of nitric-oxide synthase and xanthine-oxidase activities of human keratinocytes by ultraviolet-B radiation -potential role for peroxynitrite in skin inflammation

    Energy Technology Data Exchange (ETDEWEB)

    Deliconstantinos, G.; Villiotou, V.; Stavrides, J.C. [Athens Univ. (Greece). School of Medicine

    1996-06-28

    In the present study, we demonstrated that NO synthase (cNOS) and xanthine oxidase (XO) of human keratinocytes can be activated to release NO, superoxide (O-2(-)) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation. We defined that this photo induced response may be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human keratinocytes with UVB (290-320 nm) radiation (up to 200 mJ/cm(2)) resulted in a dose-dependent increase in NO and ONOO-release that was inhibited by N-monomethyl-L-arginine (L-NMMA). NO and ONOO- release from keratinocytes was accompanied by an increase in intracellular cGMP levels. Treatment of human keratinocyte cytosol with various doses of UVB (up to 100 mJ/cm(2)) resulted in an increase in XO activity that was inhibited by oxypurinol. In in vivo experiments, when experimental animals were subjected to UVB radiation, a protection factor (PF) of 6.5 {+-} 1.8 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA) (2%) and L-NMMA (2%) was applied to their skin. The present study indicates that UVB radiation acts as a potent stimulator of cNOS and XO activities in human keratinocytes. NO and ONOO- may exert cytotoxic effects in keratinocytes themselves, as well as in their neighbouring endothelial and smooth muscle cells. This may be a major part of the integrated response leading to erythema production and the inflammation process. (UK).

  2. Alterations of nitric-oxide synthase and xanthine-oxidase activities of human keratinocytes by ultraviolet-B radiation -potential role for peroxynitrite in skin inflammation

    International Nuclear Information System (INIS)

    Deliconstantinos, G.; Villiotou, V.; Stavrides, J.C.

    1996-01-01

    In the present study, we demonstrated that NO synthase (cNOS) and xanthine oxidase (XO) of human keratinocytes can be activated to release NO, superoxide (O-2(-)) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation. We defined that this photo induced response may be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human keratinocytes with UVB (290-320 nm) radiation (up to 200 mJ/cm(2)) resulted in a dose-dependent increase in NO and ONOO-release that was inhibited by N-monomethyl-L-arginine (L-NMMA). NO and ONOO- release from keratinocytes was accompanied by an increase in intracellular cGMP levels. Treatment of human keratinocyte cytosol with various doses of UVB (up to 100 mJ/cm(2)) resulted in an increase in XO activity that was inhibited by oxypurinol. In in vivo experiments, when experimental animals were subjected to UVB radiation, a protection factor (PF) of 6.5 ± 1.8 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA) (2%) and L-NMMA (2%) was applied to their skin. The present study indicates that UVB radiation acts as a potent stimulator of cNOS and XO activities in human keratinocytes. NO and ONOO- may exert cytotoxic effects in keratinocytes themselves, as well as in their neighbouring endothelial and smooth muscle cells. This may be a major part of the integrated response leading to erythema production and the inflammation process. (UK)

  3. Production of rabbit antibodies against purified Glucose oxidase

    OpenAIRE

    Zia,Muhammad Anjum; Ain,Qurat-ul; Iftikhar,Tehreema; Abbas,Rao Zahid; Rahman,Khalil-ur

    2012-01-01

    Glucose oxidase is an active oxygen species generating enzyme produced from Aspergillus niger grown in submerged fermentation. Disintegration of the mycelium resulted in high glucose oxidase activity that was subjected to ammonium sulfate precipitation at 60-85% saturation rates that resulted to 6.14 U mg -1 specific activity. Purification of enzyme by anion exchange column (DEAE-Cellulose) resulted into 22.53 U mg-1 specific activity and 10.27 fold purification. This was applied to sephadex ...

  4. Depletion of rat cortical norepinephrine and the inhibition of [3H]norepinephrine uptake by xylamine does not require monoamine oxidase activity

    International Nuclear Information System (INIS)

    Dudley, M.W.

    1988-01-01

    Inhibition of monoamine oxidase A through pretreatment of rats with clorgyline or the pro-drug MDL 72,394 did not block the amine-depleting action of xylamine. Xylamine treatment resulted in a loss of approximately 60% of the control level of norepinephrine in the cerebral cortex. A 1-hr pretreatment, but not a 24-hr pretreatment, with the monoamine oxidase B inhibitor, L-deprenyl, prevented the depletion of norepinephrine by xylamine. In addition, pretreatment with MDL 72,974, a monoamine oxidase B inhibitor without amine-releasing or uptake - inhibiting effects, did not prevent cortical norepinephrine levels. Inhibition of monoamine oxidase by either MDL 72,974 or MDL 72,394 did not prevent the inhibition of [ 3 H]norepinephrine uptake into rat cortical synaptosomes by xylamine. These data indicate that monoamine oxidase does not mediate the amine-releasing or uptake inhibiting properties of xylamine. The protection afforded by L-deprenyl following a 1-hr pretreatment most probably was due to accumulation of its metabolite, L-amphetamine, which would inhibit the uptake carrier. A functional carrier is required for depletion since desipramine administered 1 hr prior to xylamine, was also able to prevent depletion of norepinephrine

  5. Changes of oxidase and hydrolase activities in pecan leaves elicited by black pecan aphid (Hemiptera: Aphididae) feeding.

    Science.gov (United States)

    Chen, Yigen; Ni, Xinzhi; Cottrell, Ted E; Wood, Bruce W; Buntin, G David

    2009-06-01

    The black pecan aphid, Melanocallis caryaefoliae (Davis) (Hemiptera: Aphididae), is a foliar feeder of pecan, Carya illinoinensis (Wangenh.) K. Koch (Juglandaceae). The pest causes chlorosis of leaflet lamina, physiological damage to foliage and trees, and commonly limits the profitability of commercial pecan orchard enterprises. However, key aspects of this host-pest interaction are poorly understood. We report here the effects of M. caryaefoliae feeding on the foliar activity of oxidative (i.e., catalase, lipoxygenase [LOX]-1 and 3, and peroxidase) and hydrolytic (i.e., esterase) enzymes in relation to the degree of aphid resistance among pecan varieties. The 2-yr study showed that M. caryaefoliae-infested foliage exhibited elevated peroxidase activity only in susceptible ('Desirable', 'Sumner', and 'Schley'), but not in resistant ('Cape Fear', 'Gloria Grande', and 'Money Maker') genotypes. Susceptible genotypes also exhibited more severe leaf chlorosis in response to M. caryaefoliae feeding than the resistant genotypes; however, the aphid feeding did not influence catalase or esterase activity in all varieties, except the increase of esterase activity in Desirable and Gloria Grande. Melanocallis caryaefoliae feeding also influences activity of two lipoxygenase isozymes, with LOX3 being more frequently induced than LOX1. Foliar LOX3 activity was more frequently induced by M. caryaefoliae feeding in the moderately resistant 'Oconee' and highly resistant Money Maker and Cape Fear than in the susceptible genotypes. Therefore, the elevation of peroxidase is likely to be associated with aphid susceptibility and contributed to the severe leaf chlorosis, whereas the increase of LOX3 activity might be associated with aphid resistance in pecan. These findings contribute to our understanding of the etiology of M. caryaefoliae-elicited leaf chlorosis on pecan foliage. Such information may also be used to develop enzyme markers for identifying black pecan aphid resistance

  6. A sensitive two-photon probe to selectively detect monoamine oxidase B activity in Parkinson’s disease models

    Science.gov (United States)

    Li, Lin; Zhang, Cheng-Wu; Chen, Grace Y. J.; Zhu, Biwei; Chai, Chou; Xu, Qing-Hua; Tan, Eng-King; Zhu, Qing; Lim, Kah-Leong; Yao, Shao Q.

    2014-02-01

    The unusually high MAO-B activity consistently observed in Parkinson’s disease (PD) patients has been proposed as a biomarker; however, this has not been realized due to the lack of probes suitable for MAO-B-specific detection in live cells/tissues. Here we report the first two-photon, small molecule fluorogenic probe (U1) that enables highly sensitive/specific and real-time imaging of endogenous MAO-B activities across biological samples. We also used U1 to confirm the reported inverse relationship between parkin and MAO-B in PD models. With no apparent toxicity, U1 may be used to monitor MAO-B activities in small animals during disease development. In clinical samples, we find elevated MAO-B activities only in B lymphocytes (not in fibroblasts), hinting that MAO-B activity in peripheral blood cells might be an accessible biomarker for rapid detection of PD. Our results provide important starting points for using small molecule imaging techniques to explore MAO-B at the organism level.

  7. Activity of earthworm in Latosol under simulated acid rain stress

    Science.gov (United States)

    Jia-En Zhang; Jiayu Yu; Ying Ouyang

    2015-01-01

    Acid rain is still an issue of environmental concerns. This study investigated the impacts of simulated acid rain (SAR) upon earthworm activity from the Latosol (acidic red soil). Laboratory experiment was performed by leaching the soil columns grown with earthworms (Eisenia fetida) at the SAR pH levels ranged from 2.0 to 6.5 over a 34-day period....

  8. Inhibitory effects of drugs on the metabolic activity of mouse and human aldehyde oxidases and influence on drug-drug interactions.

    Science.gov (United States)

    Takaoka, Naoki; Sanoh, Seigo; Okuda, Katsuhiro; Kotake, Yaichiro; Sugahara, Go; Yanagi, Ami; Ishida, Yuji; Tateno, Chise; Tayama, Yoshitaka; Sugihara, Kazumi; Kitamura, Shigeyuki; Kurosaki, Mami; Terao, Mineko; Garattini, Enrico; Ohta, Shigeru

    2018-04-17

    As aldehyde oxidase (AOX) plays an emerging role in drug metabolism, understanding its significance for drug-drug interactions (DDI) is important. Therefore, we tested 10 compounds for species-specific and substrate-dependent differences in the inhibitory effect of AOX activity using genetically engineered HEK293 cells over-expressing human AOX1, mouse AOX1 or mouse AOX3. The IC 50 values of 10 potential inhibitors of the three AOX enzymes were determined using phthalazine and O 6 -benzylguanine as substrates. 17β-Estradiol, menadione, norharmane and raloxifene exhibited marked differences in inhibitory effects between the human and mouse AOX isoforms when the phthalazine substrate was used. Some of the compounds tested exhibited substrate-dependent differences in their inhibitory effects. Docking simulations with human AOX1 and mouse AOX3 were conducted for six representative inhibitors. The rank order of the minimum binding energy reflected the order of the corresponding IC 50 values. We also evaluated the potential DDI between an AOX substrate (O 6 -benzylguanine) and an inhibitor (hydralazine) using chimeric mice with humanized livers. Pretreatment of hydralazine increased the maximum plasma concentration (C max ) and the area under the plasma concentration-time curve (AUC 0-24 ) of O 6 -benzylguanine compared to single administration. Our in vitro data indicate species-specific and substrate-dependent differences in the inhibitory effects on AOX activity. Our in vivo data demonstrate the existence of a DDI which may be of relevance in the clinical context. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Bilirubin Oxidase from Myrothecium verrucaria Physically Absorbed on Graphite Electrodes. Insights into the Alternative Resting Form and the Sources of Activity Loss.

    Directory of Open Access Journals (Sweden)

    Federico Tasca

    Full Text Available The oxygen reduction reaction is one of the most important chemical processes in energy converting systems and living organisms. Mediator-less, direct electro-catalytic reduction of oxygen to water was achieved on spectrographite electrodes modified by physical adsorption of bilirubin oxidases from Myrothecium verrucaria. The existence of an alternative resting form of the enzyme is validated. The effect on the catalytic cycle of temperature, pH and the presence of halogens in the buffer was investigated. Previous results on the electrochemistry of bilirubin oxidase and on the impact of the presence of halogens are reviewed and reinterpreted.

  10. Identification of aldehyde oxidase 1 and aldehyde oxidase homologue 1 as dioxin-inducible genes

    International Nuclear Information System (INIS)

    Rivera, Steven P.; Choi, Hyun Ho; Chapman, Brett; Whitekus, Michael J.; Terao, Mineko; Garattini, Enrico; Hankinson, Oliver

    2005-01-01

    Aldehyde oxidases are a family of highly related molybdo-flavoenzymes acting upon a variety of compounds of industrial and medical importance. We have identified aldehyde oxidase 1 (AOX1) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) inducible gene in the mouse hepatoma cell line Hepa-1. AOX1 mRNA levels were not increased by dioxin in mutant derivatives of the Hepa-1 cell line lacking either functional aryl hydrocarbon receptor (AHR) or aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, thus demonstrating that transcriptional induction of AOX1 in response to dioxin occurs through the AHR pathway. Dioxin induction of AOX1 mRNA was also observed in mouse liver. In addition, levels of AOX1 protein as well as those of aldehyde oxidase homologue 1 (AOH1), a recently identified homolog of AOX1, were elevated in mouse liver in response to dioxin. Employing an aldehyde oxidase specific substrate, AOX1/AOH1 activity was shown to be induced by dioxin in mouse liver. This activity was inhibited by a known inhibitor of aldehyde oxidases, and eliminated by including tungstate in the mouse diet, which is known to lead to inactivation of molybdoflavoenzymes, thus confirming that the enzymatic activity was attributable to AOX1/AOH1. Our observations thus identify two additional xenobiotic metabolizing enzymes induced by dioxin

  11. Quantitation of immunoadsorbed flavoprotein oxidases by luminol-mediated chemiluminescence.

    Science.gov (United States)

    Hinkkanen, A; Maly, F E; Decker, K

    1983-04-01

    The detection of the flavoenzymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase at the sub-femtomol level was achieved by coupling the reaction of the immunoadsorbed proteins to the peroxidase-catalysed oxidation of luminol. The H2O2-producing oxidases retained their full activity when bound to the respective immobilized antibodies. This fact allowed the concentration of the enzymes from very dilute solutions and the quantitative assay of their activities in the microU range. Due to strict stereoselectivity and the absence of immunological cross-reactivity, the two flavoproteins could be determined in the same solution. This method was used to measure the 6-hydroxy-D-nicotine oxidase and 6-hydroxy-L-nicotine oxidase activities in Escherichia coli RR1 and different Arthrobacter strains cultured under non-inducing conditions. The same activity ratio of 6-hydroxy-L-nicotine oxidase/6-hydroxy-D-nicotine oxidase as in D L-nicotine-induced cells of A. oxidans was observed in non-induced wild type and in riboflavin-requiring (rf-) mutant cells of this aerob.

  12. Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics.

    Science.gov (United States)

    Arango Gutierrez, Erik; Mundhada, Hemanshu; Meier, Thomas; Duefel, Hartmut; Bocola, Marco; Schwaneberg, Ulrich

    2013-12-15

    Glucose oxidase is an oxidoreductase exhibiting a high β-D-glucose specificity and high stability which renders glucose oxidase well-suited for applications in diabetes care. Nevertheless, GOx activity is highly oxygen dependent which can lead to inaccuracies in amperometric β-D-glucose determinations. Therefore a directed evolution campaign with two rounds of random mutagenesis (SeSaM followed by epPCR), site saturation mutagenesis studies on individual positions, and one simultaneous site saturation library (OmniChange; 4 positions) was performed. A diabetes care well suited mediator (quinone diimine) was selected and the GOx variant (T30V I94V) served as starting point. For directed GOx evolution a microtiter plate detection system based on the quinone diimine mediator was developed and the well-known ABTS-assay was applied in microtiter plate format to validate oxygen independency of improved GOx variants. Two iterative rounds of random diversity generation and screening yielded to two subsets of amino acid positions which mainly improved activity (A173, A332) and oxygen independency (F414, V560). Simultaneous site saturation of all four positions with a reduced subset of amino acids using the OmniChange method yielded finally variant V7 with a 37-fold decreased oxygen dependency (mediator activity: 7.4 U/mg WT, 47.5 U/mg V7; oxygen activity: 172.3 U/mg WT, 30.1 U/mg V7). V7 is still highly β-D-glucose specific, highly active with the quinone diimine mediator and thermal resistance is retained (prerequisite for GOx coating of diabetes test stripes). The latter properties and V7's oxygen insensitivity make V7 a very promising candidate to replace standard GOx in diabetes care applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae

    Directory of Open Access Journals (Sweden)

    Faccio Greta

    2010-08-01

    Full Text Available Abstract Background Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. Results In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Conclusions Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

  14. Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae.

    Science.gov (United States)

    Faccio, Greta; Kruus, Kristiina; Buchert, Johanna; Saloheimo, Markku

    2010-08-20

    Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

  15. Studies on the acid activation of Brazilian smectitic clays

    Directory of Open Access Journals (Sweden)

    Valenzuela Díaz Francisco R.

    2001-01-01

    Full Text Available Fuller's earth and acid activated smectitic clays are largely used as bleaching earth for the industrial processing of vegetable, animal and mineral oils and waxes. The paper comments about the nomenclature used for these materials, the nature of the acid activation of smectitic clays (bentonites, activation laboratory procedures and presents a review of the acid activation of bentonites from 20 deposits from several regions of Brazil. The activated clays were tested and show good decolorizing power for soybean, castor, cottonseed, corn and sunflower oils.

  16. Characterization of polyphenol oxidase from blueberry (Vaccinium corymbosum L.).

    Science.gov (United States)

    Siddiq, M; Dolan, K D

    2017-03-01

    Polyphenol oxidase (PPO) was extracted and characterized from high-bush blueberries. PPO showed an optimum activity at pH 6.1-6.3 and 35°C, with the enzyme showing significant activity over a wide temperature range (25-60°C). Catechol was the most readily oxidized substrate followed by 4-methylcatechol, DL-DOPA, and dopamine. Blueberry PPO showed a K m of 15mM and V max of 2.57 ΔA 420 nm/min×10 -1 , determined with catechol. PPO was completely inactivated in 20min at 85°C, however, after 30minat 75°C it showed about 10% residual activity. Thermal treatment at 55 and 65°C for 30min resulted in the partial inactivation of PPO. Ascorbic acid, sodium diethyldithiocarbamic acid, L-cysteine, and sodium metabisulfite were effective inhibitors of PPO at 1.0mM. Benzoic acid and cinnamic acid series inhibitors showed relatively weak inhibition of PPO (21.8-27.6%), even at as high as 2.0mM concentration. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Lysyl oxidase activates cancer stromal cells and promotes gastric cancer progression: quantum dot-based identification of biomarkers in cancer stromal cells

    Directory of Open Access Journals (Sweden)

    Peng CW

    2017-12-01

    Full Text Available Chunwei Peng,1 Jiuyang Liu,1 Guifang Yang,2 Yan Li3 1Department of Gastrointestinal Surgery, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors & Hubei Cancer Clinical Study Center, 2Department of Pathology, Zhongnan Hospital of Wuhan University, Wuchang District, Wuhan, 3Department of Peritoneal Cancer Surgery, Cancer Center of Beijing Shijitan Hospital Affiliated to the Capital Medical University, Yangfangdian, Beijing, People’s Republic of China Purpose: Semiconductor quantum dots (QDs are a promising alternative to organic fluorescent dyes for multiplexed molecular imaging of cancer stroma, which have great advantages in holistically analyzing the complex interactions among cancer stromal components in situ.Patients and methods: A QD probe-based multiplexed spectral molecular imaging method was established for simultaneous imaging. Three tissue microarrays (TMAs including 184 gastric cancer (GC tissues were constructed for the study. Multispectral analyses were performed for quantifying stromal biomarkers, such as lysyl oxidase (LOX. The stromal status including infiltrating of immune cells (high density of macrophages, angiogenesis (high density of microvessel density [MVD], low neovessel maturation and extracellular matrix (ECM remodeling (low density of type IV collagen, intense expression of matrix metalloproteinase 9 [MMP-9] was evaluated.Results: This study compared the imaging features of the QD probe-based single molecular imaging method, immunohistochemistry, and organic dye-based immunofluorescent methods, and showed the advantages of the QD probe-based multiple molecular imaging method for simultaneously visualizing complex components of cancer stroma. The risk of macrophages in high density, high MVD, low neomicrovessel maturation, MMP-9 expression and low type IV collagen was significantly increased for the expression of LOX. With the advantages of the established QD probe

  18. Tumour suppressor protein p53 regulates the stress activated bilirubin oxidase cytochrome P450 2A6

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Hao, E-mail: hao.hu1@uqconnect.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Yu, Ting, E-mail: t.yu2@uq.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Arpiainen, Satu, E-mail: Satu.Juhila@orion.fi [Institute of Biomedicine, Department of Pharmacology and Toxicology and Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu (Finland); Lang, Matti A., E-mail: m.lang@uq.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Hakkola, Jukka, E-mail: Jukka.hakkola@oulu.fi [Institute of Biomedicine, Department of Pharmacology and Toxicology and Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu (Finland); Abu-Bakar, A' edah, E-mail: a.abubakar@uq.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia)

    2015-11-15

    Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3 kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5′-Luc constructs – down to − 160 bp from the TSS – showed p53 responsiveness in p53 overexpressed C3A cells. However, a further deletion from − 160 to − 74 bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene – a well-known p53 activator – increased the expression of the p53 responsive positive control and the CYP2A6-5′-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5′-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6. - Highlights: • CYP2A6 is an immediate target gene of p53. • Six putative p53REs located on 3 kb proximate CYP2A6 promoter region. • The region − 160 bp from TSS is highly homologous with the p53 consensus sequence. • P53 specifically bind to the p53RE on the − 160 bp region. • HNF4

  19. Acid-base characteristics of powdered-activated-carbon surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Reed, B.E. (West Virginia Univ., Morgantown (United States)); Jensen, J.N.; Matsumoto, M.R. (State Univ. of New York, Buffalo (United States))

    Adsorption of heavy metals onto activated carbon has been described using the surface-complex-formation (SCF) model, a chemical equilibrium model. The SCF model requires a knowledge of the amphoteric nature of activated carbon prior to metal adsorption modeling. In the past, a single-diprotic-acid-site model had been employed to describe the amphoteric nature of activated-carbon surfaces. During this study, the amphoteric nature of two powdered activated carbons were investigated, and a three-monoprotic site surface model was found to be a plausible alternative. The single-diprotic-acid-site and two-monoprotic-site models did not describe the acid-base behavior of the two carbons studied adequately. The two-diprotic site was acceptable for only one of the study carbons. The acid-base behavior of activated carbon surfaces seem to be best modeled as a series of weak monoprotic acids.

  20. Natural Cinnamic Acids, Synthetic Derivatives and Hybrids with Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Juan David Guzman

    2014-11-01

    Full Text Available Antimicrobial natural preparations involving cinnamon, storax and propolis have been long used topically for treating infections. Cinnamic acids and related molecules are partly responsible for the therapeutic effects observed in these preparations. Most of the cinnamic acids, their esters, amides, aldehydes and alcohols, show significant growth inhibition against one or several bacterial and fungal species. Of particular interest is the potent antitubercular activity observed for some of these cinnamic derivatives, which may be amenable as future drugs for treating tuberculosis. This review intends to summarize the literature data on the antimicrobial activity of the natural cinnamic acids and related derivatives. In addition, selected hybrids between cinnamic acids and biologically active scaffolds with antimicrobial activity were also included. A comprehensive literature search was performed collating the minimum inhibitory concentration (MIC of each cinnamic acid or derivative against the reported microorganisms. The MIC data allows the relative comparison between series of molecules and the derivation of structure-activity relationships.

  1. A quantitative histochemical procedure for the demonstration of purine nucleoside phosphorylase activity in rat and human liver using Tetranitro BT and xanthine oxidase as auxiliary enzyme

    NARCIS (Netherlands)

    Frederiks, W. M.; Bosch, K. S.; van Gulik, T.

    1993-01-01

    A quantitative histochemical procedure was developed for the demonstration of purine nucleoside phosphorylase in rat liver using unfixed cryostat sections and the auxiliary enzyme xanthine oxidase. The optimum incubation medium contained 18% (w/v) poly(vinyl alcohol), 100 mM phosphate buffer, pH

  2. Characterization and antioxidant activity of gallic acid derivative

    Science.gov (United States)

    Malinda, Krissan; Sutanto, Hery; Darmawan, Akhmad

    2017-11-01

    Peroxidase enzyme was used to catalyze the dimerization process of gallic acid. The structure of the dimerization product was characterized by 1H NMR and LC-MS-MS. The mechanism of gallic acid dimerization was also discussed. It was proposed that ellagic acid was formed through an oxidative coupling mechanism that lead to the formation of a C-C bond and followed by an intramolecular Fischer esterification mechanism that lead to the formation of two C-O bonds. Moreover, the antioxidant activity of gallic acid and ellagic acid were also studied. Gallic acid and ellagic acid exhibited the DPPH radical scavenging activity with IC50 values of 13.2 μM and 15.9 μM, respectively.

  3. Potent in vitro antifungal activities of naturally occurring acetylenic acids.

    Science.gov (United States)

    Li, Xing-Cong; Jacob, Melissa R; Khan, Shabana I; Ashfaq, M Khalid; Babu, K Suresh; Agarwal, Ameeta K; Elsohly, Hala N; Manly, Susan P; Clark, Alice M

    2008-07-01

    Our continuing effort in antifungal natural product discovery has led to the identification of five 6-acetylenic acids with chain lengths from C(16) to C(20): 6-hexadecynoic acid (compound 1), 6-heptadecynoic acid (compound 2), 6-octadecynoic acid (compound 3), 6-nonadecynoic acid (compound 4), and 6-icosynoic acid (compound 5) from the plant Sommera sabiceoides. Compounds 2 and 5 represent newly isolated fatty acids. The five acetylenic acids were evaluated for their in vitro antifungal activities against Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Trichophyton mentagrophytes, and Trichophyton rubrum by comparison with the positive control drugs amphotericin B, fluconazole, ketoconazole, caspofungin, terbinafine, and undecylenic acid. The compounds showed various degrees of antifungal activity against the 21 tested strains. Compound 4 was the most active, in particular against the dermatophytes T. mentagrophytes and T. rubrum and the opportunistic pathogens C. albicans and A. fumigatus, with MICs comparable to several control drugs. Inclusion of two commercially available acetylenic acids, 9-octadecynoic acid (compound 6) and 5,8,11,14-eicosatetraynoic acid (compound 7), in the in vitro antifungal testing further demonstrated that the antifungal activities of the acetylenic acids were associated with their chain lengths and positional triple bonds. In vitro toxicity testing against mammalian cell lines indicated that compounds 1 to 5 were not toxic at concentrations up to 32 muM. Furthermore, compounds 3 and 4 did not produce obvious toxic effects in mice at a dose of 34 mumol/kg of body weight when administered intraperitoneally. Taking into account the low in vitro and in vivo toxicities and significant antifungal potencies, these 6-acetylenic acids may be excellent leads for further preclinical studies.

  4. Acid activation of natural clays aiming their application in adsorption

    International Nuclear Information System (INIS)

    Silva, M.M. da; Sousa, A.K.F. de; Lima, W.S.; Vasconcelos, P.N.M. de; Rodrigues, M. G.F.

    2012-01-01

    Clays of smectite type have wide application in industrial, mainly due to their adsorption properties. However, it is necessary to subject them to chemical treatments to optimize their potential. This study aimed to analyze the effects of acid activation on the clay Brasgel fresh. In the acid activation was used concentrated hydrochloric acid at different concentrations (3M, 4.5 M and 6 M) at a temperature of 70 ° C for 30 minutes. The samples fresh and activated technique were characterized by X-ray Diffraction (XRD). The results show that the properties of clay after activation are improved, it could be used as adsorbents in the treatment of wastewater. (author)

  5. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    Science.gov (United States)

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2016-10-25

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacylethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings. The subject matter disclosed herein relates to enhancers of amidohydrolase activity.

  6. Hypochlorous Acid - Analytical Methods and Antimicrobial Activity ...

    African Journals Online (AJOL)

    Hypochlorous acid (HOCl) is produced by the human body's immune cells to fight infections. It is effective against a broad range of microorganisms. It is non-toxic, non-irritant and non-corrosive at proper usage concentrations. There are some available commercial products that contain HOCl. However, its low storage ...

  7. Endothelial NLRP3 inflammasome activation and arterial neointima formation associated with acid sphingomyelinase during hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    Saisudha Koka

    2017-10-01

    Full Text Available The NLRP3 inflammasome has been reported to be activated by atherogenic factors, whereby endothelial injury and consequent atherosclerotic lesions are triggered in the arterial wall. However, the mechanisms activating and regulating NLRP3 inflammasomes remain poorly understood. The present study tested whether acid sphingomyelinase (ASM and ceramide associated membrane raft (MR signaling platforms contribute to the activation of NLRP3 inflammasomes and atherosclerotic lesions during hypercholesterolemia. We found that 7-ketocholesterol (7-Keto or cholesterol crystal (ChC markedly increased the formation and activation of NLRP3 inflammasomes in mouse carotid arterial endothelial cells (CAECs, as shown by increased colocalization of NLRP3 with ASC or caspase-1, enhanced caspase-1 activity and elevated IL-1β levels, which were markedly attenuated by mouse Asm siRNA, ASM inhibitor- amitriptyline, and deletion of mouse Asm gene. In CAECs with NLRP3 inflammasome formation, membrane raft (MR clustering with NADPH oxidase subunits was found remarkably increased as shown by CTXB (MR marker and gp91phox aggregation indicating the formation of MR redox signaling platforms. This MR clustering was blocked by MR disruptor (MCD, ROS scavenger (Tempol and TXNIP inhibitor (verapamil, accompanied by attenuation of 7-Keto or ChC-induced increase in caspase-1 activity. In animal experiments, Western diet fed mice with partially ligated left carotid artery (PLCA were found to have significantly increased neointimal formation, which was associated with increased NLRP3 inflammasome formation and IL-1β production in the intima of Asm+/+ mice but not in Asm-/- mice. These results suggest that Asm gene and ceramide associated MR clustering are essential to endothelial inflammasome activation and dysfunction in the carotid arteries, ultimately determining the extent of atherosclerotic lesions.

  8. Synthesis and biological activity of amino acid conjugates of abscisic acid.

    Science.gov (United States)

    Todoroki, Yasushi; Narita, Kenta; Muramatsu, Taku; Shimomura, Hajime; Ohnishi, Toshiyuki; Mizutani, Masaharu; Ueno, Kotomi; Hirai, Nobuhiro

    2011-03-01

    We prepared 19 amino acid conjugates of the plant hormone abscisic acid (ABA) and investigated their biological activity, enzymatic hydrolysis by a recombinant Arabidopsis amidohydrolases GST-ILR1 and GST-IAR3, and metabolic fate in rice seedlings. Different sets of ABA-amino acids induced ABA-like responses in different plants. Some ABA-amino acids, including some that were active in bioassays, were hydrolyzed by recombinant Arabidopsis GST-IAR3, although GST-ILR1 did not show hydrolysis activity for any of the ABA-amino acids. ABA-L-Ala, which was active in all the bioassays, an Arabidopsis seed germination, spinach seed germination, and rice seedling elongation assays, except in a lettuce seed germination assay and was hydrolyzed by GST-IAR3, was hydrolyzed to free ABA in rice seedlings. These findings suggest that some plant amidohydrolases hydrolyze some ABA-amino acid conjugates. Because our study indicates the possibility that different plants have hydrolyzing activity toward different ABA-amino acids, an ABA-amino acid may function as a species-selective pro-hormone of ABA. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Strong activation of bile acid-sensitive ion channel (BASIC) by ursodeoxycholic acid

    Science.gov (United States)

    Wiemuth, Dominik; Sahin, Hacer; Lefèvre, Cathérine M.T.; Wasmuth, Hermann E.; Gründer, Stefan

    2013-01-01

    Bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC gene family of unknown function. Rat BASIC (rBASIC) is inactive at rest. We have recently shown that cholangiocytes, the epithelial cells lining the bile ducts, are the main site of BASIC expression in the liver and identified bile acids, in particular hyo- and chenodeoxycholic acid, as agonists of rBASIC. Moreover, it seems that extracellular divalent cations stabilize the resting state of rBASIC, because removal of extracellular divalent cations opens the channel. In this addendum, we demonstrate that removal of extracellular divalent cations potentiates the activation of rBASIC by bile acids, suggesting an allosteric mechanism. Furthermore, we show that rBASIC is strongly activated by the anticholestatic bile acid ursodeoxycholic acid (UDCA), suggesting that BASIC might mediate part of the therapeutic effects of UDCA. PMID:23064163

  10. Enzymatic biosensor based on entrapment of d-amino acid oxidase on gold nanofilm/MWCNTs nanocomposite modified glassy carbon electrode by sol-gel network: Analytical applications for d-alanine in human serum.

    Science.gov (United States)

    Shoja, Yalda; Rafati, Amir Abbas; Ghodsi, Javad

    2017-05-01

    Sensing and determination of d-alanine is studied by using an enzymatic biosensor which was constructed on the basis of d-amino acid oxidase (DAAO) immobilization by sol-gel film onto glassy carbon electrode surface modified with nanocomposite of gold nanofilm (Au-NF) and multiwalled carbon nanotubes (MWCNTs). The Au-NF/MWCNT nanocomposite was prepared by applying the potentiostatic technique for electrodeposition of Au-NF on the MWCNT immobilized on glassy carbon electrode surface. The modified electrode is investigated by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), linear sweep voltammetry (LSV) and cyclic voltammetry(CV) techniques. The linear sweep voltammetry was used for determination of d-alanine and the results showed an excellent linear relationship between biosensor response and d-alanine concentration ranging from 0.25μM to 4.5μM with correction coefficient of 0.999 (n=20). Detection limit for the fabricated sensor was calculated about 20nM (for S/N=3) and sensitivity was about 56.1μAμM -1 cm -2 . The developed biosensor exhibited rapid and accurate response to d-alanine, a good stability (4 weeks) and an average recovery of 98.9% in human serum samples. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Oxidase-based biocatalytic processes

    DEFF Research Database (Denmark)

    Ramesh, Hemalata; Woodley, John; Krühne, Ulrich

    interestingbiocatalystsbecause they use a mild oxidant (oxygen) as a substrateas opposed to their chemical counterparts which use strong oxidants such as permanganates. A class of oxidases calledmonoamine oxidases has been used as the central case study for the thesis. The rationale for choosing thissystemis that it has been...

  12. Transient accumulation of cis- and trans-zeatin type cytokinins and its relation to cytokinin oxidase activity during cell cycle of synchronized tobacco BY-2 cells

    Czech Academy of Sciences Publication Activity Database

    Dobrev, Petre; Motyka, Václav; Gaudinová, Alena; Malbeck, Jiří; Trávníčková, Alena; Kamínek, Miroslav; Vaňková, Radomíra

    2002-01-01

    Roč. 40, - (2002), s. 333-337 ISSN 0981-9428 R&D Projects: GA ČR GA522/99/1130; GA ČR GV206/96/K188; GA AV ČR IAA6038002 Institutional research plan: CEZ:AV0Z5038910 Keywords : Cis zeatin * Cytokinins * Cytokinin oxidase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.582, year: 2002

  13. Effect of exogenous cytokinins, auxins and adenine on cytokinin N-glucosylation and cytokinin oxidase/dehydrogenase activity in de-rooted radish seedlings

    Czech Academy of Sciences Publication Activity Database

    Blagoeva, Elitsa; Dobrev, Petre; Malbeck, Jiří; Motyka, Václav; Gaudinová, Alena; Vaňková, Radomíra

    2004-01-01

    Roč. 44, č. 1 (2004), s. 15-23 ISSN 0167-6903 R&D Projects: GA ČR GA522/99/1130; GA AV ČR IAA6038002; GA MŠk LN00A081; GA MŠk ME 505 Institutional research plan: CEZ:AV0Z5038910 Keywords : Auxin * Cytokinin * Cytokinin oxidase/dehydrogenase Subject RIV: GE - Plant Breeding Impact factor: 0.693, year: 2004

  14. Non-Acidic Free Fatty Acid Receptor 4 Agonists with Antidiabetic Activity

    DEFF Research Database (Denmark)

    Goncalves de Azavedo, Carlos M. B. P.; Watterson, Kenneth R; Wargent, Ed T

    2016-01-01

    The free fatty acid receptor 4 (FFA4 or GPR120) has appeared as an interesting potential target for the treatment of metabolic disorders. At present, most FFA4 ligands are carboxylic acids that are assumed to mimic the endogenous long-chain fatty acid agonists. Here, we report preliminary structure......-activity relationship studies of a previously disclosed non-acidic sulfonamide FFA4 agonist. Mutagenesis studies indicate that the compounds are orthosteric agonists despite the absence of a carboxylate function. The preferred compounds showed full agonist activity on FFA4 and complete selectivity over FFA1, although...... a significant fraction of these non-carboxylic acids also showed partial antagonistic activity on FFA1. Studies in normal and diet-induced obese (DIO) mice with the preferred compound 34 showed improved glucose tolerance after oral dosing in an oral glucose tolerance test. Chronic dosing of 34 in DIO mice...

  15. Polylactic acid (PLA)/Silver-NP/VitaminE bionanocomposite electrospun nanofibers with antibacterial and antioxidant activity

    Science.gov (United States)

    Munteanu, Bogdanel Silvestru; Aytac, Zeynep; Pricope, Gina M.; Uyar, Tamer; Vasile, Cornelia

    2014-10-01

    The antibacterial property of silver nanoparticles (Ag-NPs) and the antioxidant activity of Vitamin E have been combined by incorporation of these two active components within polylactic acid (PLA) nanofibers via electrospinning (PLA/Ag-NP/VitaminE nanofibers). The morphological and structural characterizations of PLA/Ag-NP/VitaminE nanofibers were performed by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy and X-ray diffraction. The average fiber diameter was 140 ± 60 nm, and the size of the Ag-NP was 2.7 ± 1.5 nm. PLA/Ag-NP/VitaminE nanofibers inhibited growth of Escherichia coli, Listeria monocytogenes and Salmonella typhymurium up to 100 %. The amount of released Ag ions from the nanofibers immersed in aqueous solution was determined by Inductively Coupled Plasma Mass Spectrometry, and it has been observed that the release of Ag ions was kept approximately constant after 10 days of immersion. The antioxidant activity of PLA/Ag-NP/VitaminE nanofibers was evaluated according to DPPH (2,2-diphenyl-1-picrylhydrazyl) method and determined as 94 %. The results of the tests on fresh apple and apple juice indicated that the PLA/Ag/VitaminE nanofiber membrane actively reduced the polyphenol oxidase activity. The multifunctional electrospun PLA nanofibers incorporating Ag-NP and Vitamin E may be quite applicable in food packaging due to the extremely large surface area of nanofibers along with antibacterial and antioxidant activities. These materials could find application in food industry as a potential preservative packaging for fruits and juices.

  16. Correlation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity with blood-brain barrier monoamine oxidase activity

    International Nuclear Information System (INIS)

    Kalaria, R.N.; Mitchell, M.J.; Harik, S.I.

    1987-01-01

    Systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes parkinsonism in humans and subhuman primates, but not in rats and many other laboratory animals; mice are intermediate in their susceptibility. Since MPTP causes selective dopaminergic neurotoxicity when infused directly into rat substantia nigra, the authors hypothesized that systemic MPTP may be metabolized by monoamine oxidase and/or other enzymes in rat brain capillaries and possibly other peripheral organs and thus prevented from reaching its neuronal sites of toxicity. They tested this hypothesis by assessing monoamine oxidase in isolated cerebral microvessels of humans, rats, and mice by measuring the specific binding of [ 3 H]pargyline, an irreversible monoamine oxidase inhibitor, and by estimating the rates of MPTP and benzylamine oxidation. [ 3 H]Pargyline binding to rat cerebral microvessels was about 10-fold higher than to human or mouse microvessels. Also, MPTP oxidation by rat brain microvessels was about 30-fold greater than by human microvessels; mouse microvessels yielded intermediate values. These results may explain, at least in part, the marked species differences in susceptibility to systemic MPTP. They also suggest the potential importance of enzyme barriers at the blood-brain interface that can metabolize toxins not excluded by structural barriers, and may provide biological bases for developing therapeutic strategies for the prevention of MPTP-induced neurotoxicity and other neurotoxic conditions including, possibly, Parkinson's disease

  17. Diversity of Two-Domain Laccase-Like Multicopper Oxidase Genes in Streptomyces spp.: Identification of Genes Potentially Involved in Extracellular Activities and Lignocellulose Degradation during Composting of Agricultural Waste

    Science.gov (United States)

    Lu, Lunhui; Zhang, Jiachao; Chen, Anwei; Chen, Ming; Jiang, Min; Yuan, Yujie; Wu, Haipeng; Lai, Mingyong; He, Yibin

    2014-01-01

    Traditional three-domain fungal and bacterial laccases have been extensively studied for their significance in various biotechnological applications. Growing molecular evidence points to a wide occurrence of more recently recognized two-domain laccase-like multicopper oxidase (LMCO) genes in Streptomyces spp. However, the current knowledge about their ecological role and distribution in natural or artificial ecosystems is insufficient. The aim of this study was to investigate the diversity and composition of Streptomyces two-domain LMCO genes in agricultural waste composting, which will contribute to the understanding of the ecological function of Streptomyces two-domain LMCOs with potential extracellular activity and ligninolytic capacity. A new specific PCR primer pair was designed to target the two conserved copper binding regions of Streptomyces two-domain LMCO genes. The obtained sequences mainly clustered with Streptomyces coelicolor, Streptomyces violaceusniger, and Streptomyces griseus. Gene libraries retrieved from six composting samples revealed high diversity and a rapid succession of Streptomyces two-domain LMCO genes during composting. The obtained sequence types cluster in 8 distinct clades, most of which are homologous with Streptomyces two-domain LMCO genes, but the sequences of clades III and VIII do not match with any reference sequence of known streptomycetes. Both lignocellulose degradation rates and phenol oxidase activity at pH 8.0 in the composting process were found to be positively associated with the abundance of Streptomyces two-domain LMCO genes. These observations provide important clues that Streptomyces two-domain LMCOs are potentially involved in bacterial extracellular phenol oxidase activities and lignocellulose breakdown during agricultural waste composting. PMID:24657870

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

    Science.gov (United States)

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

    2009-07-01

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

  19. Synthesis and antiplatelet activity of thioaryloxyacids analogues of clofibric acid.

    Science.gov (United States)

    Ammazzalorso, Alessandra; Amoroso, Rosa; Baraldi, Mario; Bettoni, Giancarlo; Braghiroli, Daniela; De Filippis, Barbara; Giampietro, Letizia; Tricca, Maria L; Vezzalini, Francesca

    2005-09-01

    The thiophene-, benzothiazole- and pyridine-thioaryloxyacids analogues of clofibric acid were synthesized and their antiplatelet activity was screened. Some compounds exhibited antiaggregating properties. The platelet-related haemostasis was measured on a PFA-100 analyzer using bull blood.

  20. Antimicrobial activities of lactic acid bacteria isolated from akamu ...

    African Journals Online (AJOL)

    The partially purified inhibitory compounds were screened by agar spot assay method for antagonistic ... The partially purified compounds exhibited strong activity against ... Keywords: Bacteriocins, lactic acid bacteria (LAB), target organisms, ...

  1. Synthesis and antituberculosis activity of new fatty acid amides.

    Science.gov (United States)

    D'Oca, Caroline Da Ros Montes; Coelho, Tatiane; Marinho, Tamara Germani; Hack, Carolina Rosa Lopes; Duarte, Rodrigo da Costa; da Silva, Pedro Almeida; D'Oca, Marcelo Gonçalves Montes

    2010-09-01

    This work reports the synthesis of new fatty acid amides from C16:0, 18:0, 18:1, 18:1 (OH), and 18:2 fatty acids families with cyclic and acyclic amines and demonstrate for the first time the activity of these compounds as antituberculosis agents against Mycobacterium tuberculosis H(37)Rv, M. tuberculosis rifampicin resistance (ATCC 35338), and M. tuberculosis isoniazid resistance (ATCC 35822). The fatty acid amides derivate from ricinoleic acid were the most potent one among a series of tested compounds, with a MIC 6.25 microg/mL for resistance strains. Copyright 2010 Elsevier Ltd. All rights reserved.

  2. Method for enhancing amidohydrolase activity of fatty acid