Purification and partial biochemical characterization of polyphenol oxidase from mango (Mangifera indica cv. Manila).

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Palma-Orozco, Gisela; Marrufo-Hernández, Norma A; Sampedro, José G; Nájera, Hugo

2014-10-08

Polyphenol oxidase (PPO) is an enzyme widely distributed in the plant kingdom that has been detected in most fruits and vegetables. PPO was extracted and purified from Manila mango (Mangifera indica), and its biochemical properties were studied. PPO was purified 216-fold by hydrophobic interaction and ion exchange chromatography. PPO was purified to homogeneity, and the estimated PPO molecular weight (MW) by SDS-PAGE was ≈31.5 kDa. However, a MW of 65 kDa was determined by gel filtration, indicating a dimeric structure for the native PPO. The isolated PPO showed the highest affinity to pyrogallol (Km = 2.77 mM) followed by 4-methylcatechol (Km = 3.14 mM) and catechol (Km = 15.14 mM). The optimum pH for activity was 6.0. PPO was stable in the temperature range of 20-70 °C. PPO activity was completely inhibited by tropolone, ascorbic acid, sodium metabisulfite, and kojic acid at 0.1 mM.

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

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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.

Polyphenol oxidase and peroxidase expression in four pineapple varieties (Ananas comosus L.) after a chilling injury.

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Raimbault, Astrid-Kim; Marie-Alphonsine, Paul-Alex; Horry, Jean-Pierre; Francois-Haugrin, Madlyn; Romuald, Karell; Soler, Alain

2011-01-12

Pineapple internal browning (IB) is a chilling injury that produces enzymatic browning associated with flesh translucency. Pineapple biodiversity allowed the investigation of how polyphenol oxidase (PPO) and peroxidase (POD) activities with their different isoforms are involved in the IB mechanism. Fruits of four varieties that expressed IB symptoms differently, Smooth Cayenne (SCay) and the hybrids MD2, Flhoran 41 (Flh 41), and Flhoran 53 (Flh 53), were stressed by cold. The susceptible varieties showed classical brown spots but different patterns of IB, whereas MD2 and controls showed no IB. Enzymatic activities were measured on fruit protein extracts and PPO and POD isoforms separated on mini-gels (PhastSystem). Only PPO activity was significantly enhanced in the presence of IB. Up to six PPO isoforms were identified in the susceptible varieties. PPO was barely detectable in the nonsusceptible variety MD2 and in controls. The number of PPO isoforms and the total PPO activity after chilling are varietal characteristics.

Impact of high pressure processing on color, bioactive compounds, polyphenol oxidase activity, and microbiological attributes of pumpkin purée.

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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.

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

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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.

Thermal and pH stabilities of partially purified polyphenol oxidase ...

African Journals Online (AJOL)

Dr. Paul Chidoka Chikezie

2012-07-30

Jul 30, 2012 ... indices of the two PPO extracts is summarized in Table. 1. At the end of the .... activity near neutral pH values (Jime´nez-Atie´nzar et al.,. 2004; Dogan and Dogan, 2004). ..... oxidase from white cherry fruit (Starks gold). GIDA.

Impacts on the metabolome of down-regulating polyphenol oxidase in transgenic potato tubers

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Tubers of potato (Solanum tuberosum L. cv. Estima) genetically modified (GM) to reduce polyphenol oxidase (PPO) activity and enzymatic discolouration were assessed for changes in the metabolome using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography (GC)-MS. Metabolome changes ...

Polyphenol Oxidase as a Biochemical Seed Defense Mechanism

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E. Patrick Fuerst

2014-12-01

Full Text Available Seed dormancy and resistance to decay are fundamental survival strategies, which allow a population of seeds to germinate over long periods of time. Seeds have physical, chemical, and biological defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. Here, we hypothesize that seeds also possess enzyme-based biochemical defenses, based on induction of the plant defense enzyme, polyphenol oxidase (PPO, when wild oat (Avena fatua L. caryopses and seeds were challenged with seed-decaying Fusarium fungi. These studies suggest that dormant seeds are capable of mounting a defense response to pathogens. The pathogen-induced PPO activity from wild oat was attributed to a soluble isoform of the enzyme that appeared to result, at least in part, from proteolytic activation of a latent PPO isoform. PPO activity was also induced in wild oat hulls (lemma and palea, non-living tissues that cover and protect the caryopsis. These results are consistent with the hypothesis that seeds possess inducible enzyme-based biochemical defenses arrayed on the exterior of seeds and these defenses represent a fundamental mechanism of seed survival and longevity in the soil. Enzyme-based biochemical defenses may have broader implications since they may apply to other defense enzymes as well as to a diversity of plant species and ecosystems.

Effect of heat treatment on polyphenol oxidase and peroxidase ...

African Journals Online (AJOL)

Effect of heat treatment (55°C/20 min) on polyphenol oxidase (PPO) and peroxidase (POD) activities and total phenolic compounds was investigated in Algerian dates (Deglet Nour variety) at Tamar (fully ripe) stage and in dates stored for 5 months at ambient temperature and in cold storage (10°C). Results obtained ...

Functional Analysis of Polyphenol Oxidases by Antisense/Sense Technology

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Jutharat Attajarusit

2007-07-01

Full Text Available Polyphenol oxidases (PPOs catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum plants with modified PPO expression levels (suppressed PPO and overexpressing PPO. These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F., cotton bollworm (Helicoverpa armigera (Hübner and beet army worm (Spodoptera exigua (Hübner, whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants

Purificação de lacases PPO-I de Botryosphaeria rhodina - DOI: 10.4025/actascibiolsci.v27i3.1317 Purification of laccases PPO-I of fungus Botryosphaeria rhodina - DOI: 10.4025/actascibiolsci.v27i3.1317

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Dalva Tomoe Miyagui

2005-03-01

Full Text Available Lacases são glicoproteínas polifenol oxidases envolvidas na patogenicidade de alguns fungos e úteis em processos biotecnológicos. O ascomiceto ligninolítico Botryosphaeria rhodina tem sido estudado como produtor de exopolissacarídeos e de lacases PPO-I e PPO-II induzidas pelo álcool veratrílico. Como as lacases produzidas ainda não foram isoladas, o objetivo deste trabalho foi purificar lacases PPO-I e identificar os carboidratos constituintes da porção glicosídica. O fungo foi cultivado em meio mínimo de Vogel contendo 1% de glicose e 30,4 mM de álcool veratrílico, a 28C e agitação de 180 rpm durante 4,5 dias. O extrato livre de células apresentou elevada concentração de carboidratos e de PPO-I estáveis a 4ºC e -18ºC durante 40 dias. Técnicas de ultrafiltração, cromatografia em gel Sephadex G-100 e em resina DEAE-Celulose purificaram lacases PPO-I com peso molecular de 113 kDa por eletroforese PAGE-SDS, contendo 40% de proteínas e 60% carboidratos identificados por HPAEC-PAD como fucose, galactose, manose, glucose e glucosaminaLaccases are glycoprotein polyphenol oxidases which are involved in fungal pathogenicity and they are also useful for biotechnological applications. The ligninolytic ascomycete, Botryosphaeria rhodina, has been studied as producer of exopolysaccharide and PPO-I and PPO-II laccases induced by veratryl alcohol. However, as the induced laccases have not been isolated, the aim of this study was to purify the enzyme and to identify the carbohydrates constituents of the glycosidic moiety. The fungus was cultivated on broth Vogel, 1% glucose and 30.4mM veratryl alcohol during 4.5 days at 28°C/180 rpm. The extracellular fluid showed high carbohydrate concentration and the stability of PPO-I laccase under conditions of refrigeration and freezing at 4ºC-18ºC over 40 days. The purification was developed by ultrafiltration using a NMWL 100 and 30 kDa membrane, gelfiltration on Sephadex G-100, and ion

Purification and characterization of polyphenol oxidase from jackfruit ( Artocarpus heterophyllus ) bulbs.

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Tao, Yi-Ming; Yao, Le-Yi; Qin, Qiu-Yan; Shen, Wang

2013-12-26

Polyphenol oxidase (PPO) from jackfruit bulb was purified through acetone precipitation, ion-exchange column, and gel filtration column. PPO was a dimer with the molecular weight of 130 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration. The Km was 8.3 and 18.2 mM using catechol and 4-methylcatechol as substrates, respectively. The optimum pH was 7.0 (catechol as the substrate) or 6.5 (4-methylcatechol as the substrate). The optimum temperature was 8 °C. The enzyme was stable below 40 °C. The activation energy (Ea) of heat inactivation was estimated to be 103.30 kJ/mol. The PPO activity was activated by Mn(2+), SDS, Tween-20, Triton X-100, citric acid, and malic acid but inhibited by K(+), Zn(2+), Mg(2+), Ca(2+), Ba(2+), cetyl trimethyl ammonium bromide (CTAB), kojic acid, tropolone, glutathione (GSH), cysteine (Cys), and ascorbic acid (AA). Cys and AA were effective to reduce browning of jackfruit bulbs during the storage at 8 °C for 15 days.

Polyphenol oxidase activity from three sicilian artichoke [ Cynara cardunculus L. Var. scolymus L. (Fiori)] cultivars: studies and technological application on minimally processed production.

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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).

Isolation and properties of polyphenol oxidase from basidiocarps of Lactarius pergamenus Fr. (Fr. fungi

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M. V. Tsivinska

2015-04-01

Full Text Available Fresh juice of basidiocarps of Lactarius pergamenus Fr. (Fr. fungi was subjected to ion exchange chromatography with used DEAE-toyopearl and CM-cellulose columns, as well as preparative electrophoresis in 7.5% polyacrylamide gels (pH 8.6. Three isoforms of polyphenol oxidase (PPO were discovered and two isoforms (1-1 and 1-2 were purified with a release of protein 0.42 mg/kg and 0.15 mg/kg of basidiocarps, respectively. These isoforms differ in the mobility at disc-electrophoresis in 7.5% PAGE in alkaline buffer system (pH 8.6. Specific activity of isoform 1-2 is 4.8 times higher than that of the isoforms 1-1. The molecular weight determination by gel chromatography on the Toyopearl HW-55 demonstrated that both isoforms 1-1 and 1-2 have the same 64 ± 2 kDa molecular mass. Electrophoresis in 15% PAGE in the presence of sodium dodecylsulphate and β-mercaptoethanol revealed one band with molecular mass of 64 ± 1 kDa which suggests the presence of one polypeptide chain in the molecule of the enzyme. The enzyme has demonstrated the highest activity at pH 6.0 and temperature +10 ºC, and at +70 ºC the enzyme was inactivated. The PPO activity was the highest in young mushrooms and it decreased with their age and positively correlated with the content of the milky juice. Ortho-aminophenol was most effective among all the tested substrates to determine the activity of PPO (o-, m– and p-aminophenol, catechol, tyrosine, resorcinol, phloroglucinol and its relative activity was 129% of the activity of catechol. Ascorbic acid was the most effective inhibitor of the polyphenol oxidase activity which was completely blocked at 1 mM concentration, whereas the same concentration of thiourea and sodium sulphite decreased the enzymatic activity by 40-45%. The PPO in L. pergamenus fungi basidiocarps was mainly localized in the mushroom milky juice where its high activity may be associated with protection of basidiocarps against various pathogens.

Multiple origins of the phenol reaction negative phenotype in foxtail millet, Setaria italica (L.) P. Beauv., were caused by independent loss-of-function mutations of the polyphenol oxidase (Si7PPO) gene during domestication.

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Inoue, Takahiko; Yuo, Takahisa; Ohta, Takeshi; Hitomi, Eriko; Ichitani, Katsuyuki; Kawase, Makoto; Taketa, Shin; Fukunaga, Kenji

2015-08-01

Foxtail millet shows variation in positive phenol color reaction (Phr) and negative Phr in grains, but predominant accessions of this crop are negative reaction type, and the molecular genetic basis of the Phr reaction remains unresolved. In this article, we isolated polyphenol oxidase (PPO) gene responsible for Phr using genome sequence information and investigated molecular genetic basis of negative Phr and crop evolution of foxtail millet. First of all, we searched for PPO gene homologs in a foxtail millet genome database using a rice PPO gene as a query and successfully found three copies of the PPO gene. One of the PPO gene homologs on chromosome 7 showed the highest similarity with PPO genes expressed in hulls (grains) of other cereal species including rice, wheat, and barley and was designated as Si7PPO. Phr phenotypes and Si7PPO genotypes completely co-segregated in a segregating population. We also analyzed the genetic variation conferring negative Phr reaction. Of 480 accessions of the landraces investigated, 87 (18.1 %) showed positive Phr and 393 (81.9 %) showed negative Phr. In the 393 Phr negative accessions, three types of loss-of-function Si7PPO gene were predominant and independently found in various locations. One of them has an SNP in exon 1 resulting in a premature stop codon and was designated as stop codon type, another has an insertion of a transposon (Si7PPO-TE1) in intron 2 and was designated as TE1-insertion type, and the other has a 6-bp duplication in exon 3 resulting in the duplication of 2 amino acids and was designated as 6-bp duplication type. As a rare variant of the stop codon type, one accession additionally has an insertion of a transposon, Si7PPO-TE2, in intron 2 and was designated as "stop codon +TE2 insertion type". The geographical distribution of accessions with positive Phr and those with three major types of negative Phr was also investigated. Accessions with positive Phr were found in subtropical and tropical regions at

Browning in Annona cherimola fruit: role of polyphenol oxidase and characterization of a coding sequence of the enzyme.

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Prieto, Humberto; Utz, Daniella; Castro, Alvaro; Aguirre, Carlos; González-Agüero, Mauricio; Valdés, Héctor; Cifuentes, Nicolas; Defilippi, Bruno G; Zamora, Pablo; Zúñiga, Gustavo; Campos-Vargas, Reinaldo

2007-10-31

Cherimoya (Annona cherimola Mill.) fruit is an attractive candidate for food processing applications as fresh cut. However, along with its desirable delicate taste, cherimoya shows a marked susceptibility to browning. This condition is mainly attributed to polyphenol oxidase activity (PPO). A general lack of knowledge regarding PPO and its role in the oxidative loss of quality in processed cherimoya fruit requires a better understanding of the mechanisms involved. The work carried out included the cloning of a full-length cDNA, an analysis of its properties in the deduced amino sequence, and linkage of its mRNA levels with enzyme activity in mature and ripe fruits after wounding. The results showed one gene different at the nucleotide level when compared with previously reported genes, but a well-conserved protein, either in functional and in structural terms. Cherimoya PPO gene (Ac-ppo, GenBank DQ990911) showed to be present apparently in one copy of the genome, and its transcripts could be significantly detected in leaves and less abundantly in flowers and fruits. Analysis of wounded matured and ripened fruits revealed an inductive behavior for mRNA levels in the flesh of mature cherimoya after 16 h. Although the highest enzymatic activity was observed on rind, a consistent PPO activity was detected on flesh samples. A lack of correlation between PPO mRNA level and PPO activity was observed, especially in flesh tissue. This is probably due to the presence of monophenolic substrates inducing a lag period, enzyme inhibitors and/or diphenolic substrates causing suicide inactivation, and proenzyme or latent isoforms of PPO. To our knowledge this is the first report of a complete PPO sequence in cherimoya. Furthermore, the gene is highly divergent from known nucleotide sequences but shows a well conserved protein in terms of its function, deduced structure, and physiological role.

Forage Polyphenol Oxidase and Ruminant Livestock Nutrition

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Michael Richard F. Lee

2014-12-01

Full Text Available Polyphenol oxidase (PPO is associated with the detrimental effect of browning fruit and vegetables, however interest within PPO containing forage crops has grown since the brownng reaction was associated with reduced nitrogen (N losses in silo and the rumen. The reduction in protein breakdown in silo of red clover (high PPO forage increased the quality of protein, improving N-use efficiency (NUE when fed to ruminants. A further benefit of red clover silage feeding is a significant reduction in lipolysis in silo and an increase in the deposition of beneficial C18 polyunsaturated fatty acid (PUFA in animal products, which has also been linked to PPO activity. PPOs protection of plant protein and glycerol based-PUFA in silo is related to the deactivation of plant proteases and lipases. This deactivation occurs through PPO catalysing the conversion of diphenols to quinones which bind with cellular nucleophiles such as protein reforming a protein-bound phenol (PBP. If the protein is an enzyme the complexing denatures the enzyme. However, PPO is inactive in the anaerobic rumen and therefore any subsequent protection of plant protein and glycerol based-PUFA in the rumen must be as a result of events that occurred to the forage pre-ingestion. Reduced activity of plant proteases and lipases would have little effect on NUE and glycerol based-PUFA in the rumen due to the greater concentration of rumen microbial proteases and lipases. The mechanism for PPOs protection of plant protein in the rumen is a consequence of complexing plant protein, rather than protease deactivation per se. These complexed proteins reduce protein digestibility in the rumen and subsequently increase un-degraded dietary protein flow to the small intestine. The mechanism for protecting glycerol-based PUFA has yet to be fully elucidated but may be associated with entrapment within PBP reducing access to microbial lipases or differences in rumen digestion kinetics of red clover.

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

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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...

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.

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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.

Comparison of content in phenolic compounds, polyphenol oxidase and peroxidase in grains of fifty sorghum cultivars from Burkina Faso.

NARCIS (Netherlands)

Dicko, M.H.; Hilhorst, M.H.; Gruppen, H.; Traore, A.S.; Laane, N.C.M.; Berkel, van W.J.H.; Voragen, A.G.J.

2002-01-01

Analysis of fifty sorghum [Sorghum bicolor (L.) Moench] varieties used in Burkina Faso showed that they have different contents of phenolic compounds, peroxidase (POX), and polyphenol oxidase (PPO). Most of the varieties (82%) had a tannin content less than 0.25% (w/w). POX specific activity was

Will the Amaranthus tuberculatus Resistance Mechanism to PPO-Inhibiting Herbicides Evolve in Other Amaranthus Species?

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Chance W. Riggins

2012-01-01

Full Text Available Resistance to herbicides that inhibit protoporphyrinogen oxidase (PPO has been slow to evolve and, to date, is confirmed for only four weed species. Two of these species are members of the genus Amaranthus L. Previous research has demonstrated that PPO-inhibitor resistance in A. tuberculatus (Moq. Sauer, the first weed to have evolved this type of resistance, involves a unique codon deletion in the PPX2 gene. Our hypothesis is that A. tuberculatus may have been predisposed to evolving this resistance mechanism due to the presence of a repetitive motif at the mutation site and that lack of this motif in other amaranth species is why PPO-inhibitor resistance has not become more common despite strong herbicide selection pressure. Here we investigate inter- and intraspecific variability of the PPX2 gene—specifically exon 9, which includes the mutation site—in ten amaranth species via sequencing and a PCR-RFLP assay. Few polymorphisms were observed in this region of the gene, and intraspecific variation was observed only in A. quitensis. However, sequencing revealed two distinct repeat patterns encompassing the mutation site. Most notably, A. palmeri S. Watson possesses the same repetitive motif found in A. tuberculatus. We thus predict that A. palmeri will evolve resistance to PPO inhibitors via the same PPX2 codon deletion that evolved in A. tuberculatus.

Preferred Provider Organization (PPO) Plans

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... Find & compare doctors, hospitals, & other providers Preferred Provider Organization (PPO) Plans How PPO Plans Work A Medicare ... extra for these benefits. Related Resources Health Maintenance Organization (HMO) Private Fee-for-Service (PFFS) Special Needs ...

Purification and Characterization of Polyphenol Oxidase, Peroxidase and Lipoxygenase from Freshly Cut Lettuce (L. sativa

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Vural Gökmen

2011-01-01

Full Text Available Enzymatic reactions taking place in minimally processed vegetables are considered as a major problem, because they adversely affect sensorial and nutritional quality. Polyphenol oxidase (PPO, peroxidase (POD and lipoxygenase (LOX from lettuce were purified on a column packed with positively charged diethylaminoethyl (DEAE cellulose by applying pH gradient elution from pH=4.0 to 9.0. The main purified fractions (PPO1 and PPO4, POD1 and POD2, LOX1 and LOX2 were characterized for enzyme concentration-reaction rate relationship, thermal stability, pH activity and kinetic parameters. Kinetic properties of each isoform were considerably different. Cysteine was found as the most effective inhibitor of both fractions of PPO. Kinetic parameters of lettuce POD were presented using guaiacol at various H2O2 concentrations. β-carotene directly influences lettuce LOX in the reaction medium available for the catalytic conversion of linoleic acid into hydroperoxides. Ascorbic and oxalic acids appear as effective PPO inhibitors, protecting phenolic compounds against oxidation in lettuce. Understanding the characteristics of deteriorative enzymes becomes important to maintain suitable conditions for fresh-like quality of lettuce. The results can be useful to keep the nutritional quality of minimally processed lettuce during shelf-life.

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel.

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Yang, C P; Fujita, S; Kohno, K; Kusubayashi, A; Ashrafuzzaman, M; Hayashi, N

2001-03-01

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

Combined effect of ultrasound, heat, and pressure on Escherichia coli O157:H7, polyphenol oxidase activity, and anthocyanins in blueberry (Vaccinium corymbosum) juice.

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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.

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.

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

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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

Enzyme characterisation, isolation and cDNA cloning of polyphenol oxidase in the hearts of palm of three commercially important species.

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Shimizu, Milton Massao; Melo, Geraldo Aclécio; Brombini Dos Santos, Adriana; Bottcher, Alexandra; Cesarino, Igor; Araújo, Pedro; Magalhães Silva Moura, Jullyana Cristina; Mazzafera, Paulo

2011-09-01

Heart of palm (palmito) is the edible part of the apical meristem of palms and is considered a gourmet vegetable. Palmitos from the palms Euterpe edulis (Juçara) and Euterpe oleracea (Açaí) oxidise after harvesting, whereas almost no oxidation is observed in palmitos from Bactris gasipaes (Pupunha). Previous investigations showed that oxidation in Juçara and Açaí was mainly attributable to polyphenol oxidase (PPO; EC 1.14.18.1) activity. In this study, we partially purified PPOs from these three palmitos and analysed them for SDS activation, substrate specificity, inhibition by specific inhibitors, thermal stability, optimum pH and temperature conditions, Km and Ki. In addition, the total phenolic content and chlorogenic acid content were determined. Two partial cDNA sequences were isolated and sequenced from Açaí (EoPPO1) and Juçara (EePPO1). Semi-quantitative RT-PCR expression assays showed that Açaí and Juçara PPOs were strongly expressed in palmitos and weakly expressed in leaves. No amplification was observed for Pupunha samples. The lack of oxidation in the palmito Pupunha might be explained by the low PPO expression, low enzyme activity or the phenolic profile, particularly the low content of chlorogenic acid. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

High isostatic pressure and thermal processing of açaí fruit (Euterpe oleracea Martius): Effect on pulp color and inactivation of peroxidase and polyphenol oxidase.

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Jesus, Ana Laura Tibério de; Leite, Thiago Soares; Cristianini, Marcelo

2018-03-01

The present study evaluated the effect of high isostatic pressure (HIP) on the activity of peroxidase (POD) and polyphenol oxidase (PPO) from açaí. Açaí pulp was submitted to several combinations of pressure (400, 500, 600MPa), temperature (25 and 65°C) for 5 and 15min. The combined effect of HIP technology and high temperatures (690MPa by 2 and 5min at 80°C) was also investigated and compared to the conventional thermal treatment (85°C/1min). POD and PPO enzyme activity and instrumental color were examined after processing and after 24h of refrigerated storage. Results showed stability of POD for all pressures at 25°C, which proved to be heat-resistant and baro-resistant at 65°C. For PPO, the inactivation at 65°C was 71.7% for 600MPa after 15min. In general, the increase in temperature from 25°C to 65°C reduced the PPO relative activity with no changes in color. Although the thermal treatment and the HIP (690MPa) along with high temperature (80°C) reduced the PPO relative activity, and relevant darkening was observed in the processed samples. Thus, it can be concluded that POD is more baro-resistant than PPO in açaí pulp subjected to the same HIP processing conditions and processing at 600MPa/65°C for 5min may be an effective alternative for thermal pasteurization treatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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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.

Characterization and purification of polyphenol oxidase from artichoke (Cynara scolymus L.).

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Dogan, Serap; Turan, Yusuf; Ertürk, Hatibe; Arslan, Oktay

2005-02-09

In this study, the polyphenol oxidase (PPO) of artichoke (Cynara scolymus L.) was first purified by a combination of (NH(4))(2)SO(4) precipitation, dialysis, and a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column. At the end of purification, 43-fold purification was achieved. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis indicated that PPO had a 57 kDa molecular mass. Second, the contents of total phenolic and protein of artichoke head extracts were determined. The total phenolic content of artichoke head was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 425 mg 100 g(-1) on a fresh weight basis. Protein content was determined according to Bradford method. Third, the effects of substrate specificity, pH, temperature, and heat inactivation were investigated on the activity of PPO purified from artichoke. The enzyme showed activity to 4-methylcatechol, pyrogallol, catechol, and L-dopa. No activity was detected toward L-tyrosine, resorsinol, and p-cresol. According to V(max)/K(m) values, 4-methylcatechol (1393 EU min(-1) mM(-1)) was the best substrate, followed by pyrogallol (1220 EU min(-1) mM(-1)), catechol (697 EU min(-1) mM(-1)), and L-dopa (102 EU min(-1) mM(-1)). The optimum pH values for PPO were 5.0, 8.0, and 7.0 using 4-methylcatechol, pyrogallol, and catechol as substrate, respectively. It was found that optimum temperatures were dependent on the substrates studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time for 4-methylcatechol and pyrogallol substrates. However, all inactivation experiments for catechol showed that the activity of artichoke PPO increased with mild heating, reached a maximum, and then decreased with time. Finally, inhibition of artichoke PPO was investigated with inhibitors such as L-cysteine, EDTA, ascorbic

Effects of PEO-PPO diblock impurities on the cubic structure of aqueous PEO-PPO-PEO pluronics micelles: fcc and bcc ordered structures in F127

DEFF Research Database (Denmark)

Mortensen, Kell; Pedersen, Walther Batsberg; Hvidt, S.

2008-01-01

We report on structural properties of PEO-PPO-PEO type of triblock block copolymers (Pluronics F127) with special emphasis on the effect of diblock PEO-PPO impurities on the ordered gel phase. Commercial F127 polymers contain as received roughly 20% PEO-PPO diblock and 80% PEO-PPO-PEO triblock...... copolymers. Aqueous solutions of F127 copolymers used as received form fee ordered micellar structure. Copolymers depleted with respect to the diblock impurity, resulting in a pure PEO-PPO-PEO triblock copolymer system, form bcc ordered micelles within the major parts of the gel phase. However, close...

The polyphenol oxidase gene family in land plants: Lineage-specific duplication and expansion

Directory of Open Access Journals (Sweden)

Tran Lan T

2012-08-01

Full Text Available Abstract Background Plant polyphenol oxidases (PPOs are enzymes that typically use molecular oxygen to oxidize ortho-diphenols to ortho-quinones. These commonly cause browning reactions following tissue damage, and may be important in plant defense. Some PPOs function as hydroxylases or in cross-linking reactions, but in most plants their physiological roles are not known. To better understand the importance of PPOs in the plant kingdom, we surveyed PPO gene families in 25 sequenced genomes from chlorophytes, bryophytes, lycophytes, and flowering plants. The PPO genes were then analyzed in silico for gene structure, phylogenetic relationships, and targeting signals. Results Many previously uncharacterized PPO genes were uncovered. The moss, Physcomitrella patens, contained 13 PPO genes and Selaginella moellendorffii (spike moss and Glycine max (soybean each had 11 genes. Populus trichocarpa (poplar contained a highly diversified gene family with 11 PPO genes, but several flowering plants had only a single PPO gene. By contrast, no PPO-like sequences were identified in several chlorophyte (green algae genomes or Arabidopsis (A. lyrata and A. thaliana. We found that many PPOs contained one or two introns often near the 3’ terminus. Furthermore, N-terminal amino acid sequence analysis using ChloroP and TargetP 1.1 predicted that several putative PPOs are synthesized via the secretory pathway, a unique finding as most PPOs are predicted to be chloroplast proteins. Phylogenetic reconstruction of these sequences revealed that large PPO gene repertoires in some species are mostly a consequence of independent bursts of gene duplication, while the lineage leading to Arabidopsis must have lost all PPO genes. Conclusion Our survey identified PPOs in gene families of varying sizes in all land plants except in the genus Arabidopsis. While we found variation in intron numbers and positions, overall PPO gene structure is congruent with the phylogenetic

Cloning, sequencing, purification, and crystal structure of Grenache (Vitis vinifera) polyphenol oxidase.

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Virador, Victoria M; Reyes Grajeda, Juan P; Blanco-Labra, Alejandro; Mendiola-Olaya, Elizabeth; Smith, Gary M; Moreno, Abel; Whitaker, John R

2010-01-27

The full-length cDNA sequence (P93622_VITVI) of polyphenol oxidase (PPO) cDNA from grape Vitis vinifera L., cv Grenache, was found to encode a translated protein of 607 amino acids with an expected molecular weight of ca. 67 kDa and a predicted pI of 6.83. The translated amino acid sequence was 99%, identical to that of a white grape berry PPO (1) (5 out of 607 amino acid potential sequence differences). The protein was purified from Grenache grape berries by using traditional methods, and it was crystallized with ammonium acetate by the hanging-drop vapor diffusion method. The crystals were orthorhombic, space group C222(1). The structure was obtained at 2.2 A resolution using synchrotron radiation using the 39 kDa isozyme of sweet potato PPO (PDB code: 1BT1 ) as a phase donor. The basic symmetry of the cell parameters (a, b, and c and alpha, beta, and gamma) as well as in the number of asymmetric units in the unit cell of the crystals of PPO, differed between the two proteins. The structures of the two enzymes are quite similar in overall fold, the location of the helix bundles at the core, and the active site in which three histidines bind each of the two catalytic copper ions, and one of the histidines is engaged in a thioether linkage with a cysteine residue. The possibility that the formation of the Cys-His thioether linkage constitutes the activation step is proposed. No evidence of phosphorylation or glycoslyation was found in the electron density map. The mass of the crystallized protein appears to be only 38.4 kDa, and the processing that occurs in the grape berry that leads to this smaller size is discussed.

Comparative study of the banana pulp browning process of 'Giant Dwarf' and FHIA-23 during fruit ripening based on image analysis and the polyphenol oxidase and peroxidase biochemical properties.

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Escalante-Minakata, Pilar; Ibarra-Junquera, Vrani; Ornelas-Paz, José de Jesús; García-Ibáñez, Victoria; Virgen-Ortíz, José J; González-Potes, Apolinar; Pérez-Martínez, Jaime D; Orozco-Santos, Mario

2018-01-01

This work presents a novel method to associate the polyphenol oxidase (PPO) and the peroxidase (POD) activities with the ripening-mediated color changes in banana peel and pulp by computational image analysis. The method was used to follow up the de-greening of peel and browning of homogenized pulp from 'Giant Dwarf' (GD: Musa AAA, subgroup Cavendish) and FHIA-23 (tetraploid hybrid, AAAA) banana cultivars. In both cultivars, the color changes of peel during the ripening process clearly showed four stages, which were used to group the fruit into ripening stages. The PPO and POD were extracted from pulp of fruit at these ripening stages, precipitated, and partially purified by gel filtration chromatography. Moreover, the pulp browning was digitally monitored after homogenization for a span time of up to 120 min. The browning level was higher for GD than FHIA-23 tissues. This fact correlated with an 11.7-fold higher PPO activity in the GD cultivar, as compared with that of FHIA-23. POD activity was 8.1 times higher for GD as compared that that of FHIA-23.

In silico analysis, mapping of regulatory elements and corresponding dna-protein interaction in polyphenol oxidase gene promoter from different rice varieties

International Nuclear Information System (INIS)

Mahmood, T.; Rehman, M.; Aziz, E.

2015-01-01

Polyphenol oxidase (PPO) is an important enzyme that has positive impact regarding plant resistance against different biotic and abiotic stresses. In the present study PPO promoter from six different rice varieties was amplified and then analyzed for cis- and trans-acting elements. The study revealed a total of 79 different cis-acting regulatory elements including 11 elements restricted to only one or other variety. Among six varieties Pakhal-Basmati had highest number (5) of these elements, whereas C-622 and Rachna-Basmati have no such sequences. Rachna-Basmati, IR-36-Basmati and Kashmir- Basmati had 1, 2 and 3 unique elements, respectively. Different elementsrelated to pathogen, salt and water stresses were found, which may be helpful in controlling PPO activity according to changing environment. Moreover, HADDOCK was used to understand molecular mechanism of PPO regulation and it was found that DNA-protein interactions are stabilized by many potential hydrogen bonds. Adenine and arginine were the most reactive residues in DNA and proteins respectively.Structural comparison of different protein-DNA complexes show that even a highly conserved transcriptional factor can adopt different conformations when they contact a different DNA binding sequence, however their stable interactions depend on the number of hydrogen bonds formed and distance. (author)

Characterization of polyphenol oxidase from Cape gooseberry (Physalis peruviana L.) fruit.

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Bravo, Karent; Osorio, Edison

2016-04-15

Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued, however it is a very rich source of polyphenol oxidase (PPO). In this study, Cape gooseberry PPO was isolated and biochemically characterized. The enzyme was extracted and purified using acetone and aqueous two-phase systems. The data indicated that PPO had the highest substrate affinity for chlorogenic acid, 4-methylcatechol and catechol. Chlorogenic acid was the most suitable substrate (Km=0.56±0.07 mM and Vmax=53.15±2.03 UPPO mL(-1) min(-1)). The optimal pH values were 5.5 for catechol and 4-methylcatechol and 5.0 for chlorogenic acid. Optimal temperatures were 40°C for catechol, 25°C for 4-methylcatechol and 20°C for chlorogenic acid. In inhibition tests, the most potent inhibitor was found to be ascorbic acid followed by L-cysteine and quercetin. This study shows possible treatments that can be implemented during the processing of Cape gooseberry fruits to prevent browning. Copyright © 2015 Elsevier Ltd. All rights reserved.

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...

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.

Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.).

Science.gov (United States)

Rahman, Andi Nur Faidah; Ohta, Mayumi; Nakatani, Kazuya; Hayashi, Nobuyuki; Fujita, Shuji

2012-04-11

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).

Study on performance of blended fuel PPO - Diesel at generator

Science.gov (United States)

Prasetyo, Joni; Prasetyo, Dwi Husodo; Murti, S. D. Sumbogo; Adiarso, Priyanto, Unggul

2018-02-01

Bio-energy is renewable energy made from plant. Biomass-based energy sources are potentially CO2 neutral and recycle the same carbon atoms. In order to reduce pollution caused by fossil fuel combustion either for mechanical or electrical energy generation, the performance characteristic of purified palm oil blends are analyzed at various ratios. Bio-energy, Pure Plant Oil, represent a sustainable solution.A generator has been modified due to adapt the viscosity ofblended fuel, PPO - diesel, by pre-heating. Several PPO - diesel composition and injection timing were tested in order to investigate the characteristic of mixed fuel with and without pre-heating. The term biofuel refers to liquid or gaseous fuels for the internal combustion engines that are predominantly produced fro m biomass. Surprising result showed that BSFC of blended PPO - diesel was more efficient when injection timing set more than 15° BTDC. The mixed fuel produced power with less mixed fuel even though the calorie content of diesel is higher than PPO. The most efficient was 20% PPO in diesel with BSFC 296 gr fuel / kwh rather than 100% diesel with BSFC 309 gr fuel / kwh at the same injection timing 18° BTDC with pre-heating. The improvement of BSFC is caused by heating up of mixed fuel which it added calorie in the mixed fuel. Therefore, the heating up of blended PPO - diesel is not only to adapt the viscosity but also improving the efficiency of fuel usage representing by lower BSFC. In addition, torque of the 20% PPO was also as smooth as 100% diesel representing by almost the same torqueat injection timing 15° BTDC. The AIP Proceedings article template has many predefined paragraph styles for you to use/apply as you write your paper. To format your abstract, use the Microsoft Word template style: Abstract. Each paper must include an abstract. Begin the abstract with the word "Abstract" followed by a period in bold font, and then continue with a normal 9 point font.

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.)

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

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

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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.

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

The effect of ultraviolet treatment on enzymatic activity and total phenolic content of minimally processed potato slices.

Science.gov (United States)

Teoh, Li Shing; Lasekan, Ola; Adzahan, Noranizan Mohd; Hashim, Norhashila

2016-07-01

In this work, potato slices were exposed to different doses of UV-C irradiation (i.e. 2.28, 6.84, 11.41, and 13.68 kJ m -2 ) with or without pretreatment [i.e. ascorbic acid and calcium chloride (AACCl) dip] and stored at 4 ± 1 °C. Changes in enzymatic activities of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL), as well as total phenolic content (TPC) were investigated after 0, 3, 7 and 10 days of storage. Results showed that untreated and UV-C treated potato slices at 13.68 kJ m -2 dosage level showed significantly higher PPO, POD and PAL activities. Conversely, untreated potato slices showed the lowest TPC during storage period. Potato slices subjected to AACCl dip plus UV-C at 6.84 kJ m -2 produced lower PPO, POD and PAL activities, as well as maintained a high TPC during storage.

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....

Physical chemical radiation effects in scintillator solutions content PPO and POPOP

International Nuclear Information System (INIS)

Fernandes Neto, Jose Mari; Hamada, Margarida Mizue; Duarte, Celina Lopes; Mesquita, Carlos Henrique de

2005-01-01

Samples containing PPO (1%, g/mL), diluted in toluene, were irradiated in a 60 Co irradiator (6.46 kGy/h) at different doses. The PPO concentration decay bi-exponentially with the dose, generating the degradation products: benzoic acid, benzamide and benzilic alcohol. The liquid scintillator system was not sensitive to the radiation damage until 20 kGy. Otherwise, the pulse height analysis showed that doses between 30 and 40 kGy generated significant loss of quality of the sensor (scintillating liquid) and the light yield was reduced in half with the dose of (34.04 ± 0.80) kGy. This value was confirmed by the photopeak position analysis that resulted in D1/2 = (31.7 ± 1.4) kGy. The transmittance, at 360 nm, of the irradiated solution decreased exponentially. The compartmental model using five compartments (fast decay PPO, slow decay PPO, benzamide, benzoic acid and benzilic alcohol) was satisfactory to explain the decay of the PPO in its degradation products as a function of the dose. The explanation coefficient r2 = 0.985636 demonstrates that the model was capable to explain 98.6% of the experimental variations. The energies involved in the chemical reactions were w = (0.239 ± 0.031) eV/damage (fast decay) and w (1.834 ± 0.301) eV/damage (slow decay). (author)

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.

A structural model of PpoA derived from SAXS-analysis-implications for substrate conversion.

Science.gov (United States)

Koch, Christian; Tria, Giancarlo; Fielding, Alistair J; Brodhun, Florian; Valerius, Oliver; Feussner, Kirstin; Braus, Gerhard H; Svergun, Dmitri I; Bennati, Marina; Feussner, Ivo

2013-09-01

In plants and mammals, oxylipins may be synthesized via multi step processes that consist of dioxygenation and isomerization of the intermediately formed hydroperoxy fatty acid. These processes are typically catalyzed by two distinct enzyme classes: dioxygenases and cytochrome P450 enzymes. In ascomycetes biosynthesis of oxylipins may proceed by a similar two-step pathway. An important difference, however, is that both enzymatic activities may be combined in a single bifunctional enzyme. These types of enzymes are named Psi-factor producing oxygenases (Ppo). Here, the spatial organization of the two domains of PpoA from Aspergillus nidulans was analyzed by small-angle X-ray scattering and the obtained data show that the enzyme exhibits a relatively flat trimeric shape. Atomic structures of the single domains were obtained by template-based structure prediction and docked into the enzyme envelope of the low resolution structure obtained by SAXS. EPR-based distance measurements between the tyrosyl radicals formed in the activated dioxygenase domain of the enzyme supported the trimeric structure obtained from SAXS and the previous assignment of Tyr374 as radical-site in PpoA. Furthermore, two phenylalanine residues in the cytochrome P450 domain were shown to modulate the specificity of hydroperoxy fatty acid rearrangement. Copyright © 2013 Elsevier B.V. All rights reserved.

26 - 31 Bello paid

African Journals Online (AJOL)

DR. AMIN

The effect of pH on the activity and stability of crude polyphenol oxidase (PPO) ... decrease in the activity of the enzyme, and can be a good way of controlling undesirable changes ... diphenol oxidases are present in plants, the ratio of.

Ion implantation induced conducting nano-cluster formation in PPO

International Nuclear Information System (INIS)

Das, A.; Patnaik, A.; Ghosh, G.; Dhara, S.

1997-01-01

Conversion of polymers and non-polymeric organic molecules from insulating to semiconducting materials as an effect of energetic ion implantation is an established fact. Formation of nano-clusters enriched with carbonaceous materials are made responsible for the insulator-semiconductor transition. Conduction in these implanted materials is observed to follow variable range hopping (VRH) mechanism. Poly(2,6-dimethyl phenylene oxide) [PPO] compatible in various proportion with polystyrene is used as a high thermal resistant insulating polymer. PPO has been used for the first time in the ion implantation study

Composition, physicochemical properties and thermal inactivation kinetics of polyphenol oxidase and peroxidase from coconut (Cocos nucifera) water obtained from immature, mature and overly-mature coconut.

Science.gov (United States)

Tan, Thuan-Chew; Cheng, Lai-Hoong; Bhat, Rajeev; Rusul, Gulam; Easa, Azhar Mat

2014-01-01

Composition, physicochemical properties and enzyme inactivation kinetics of coconut water were compared between immature (IMC), mature (MC) and overly-mature coconuts (OMC). Among the samples studied, pH, turbidity and mineral contents for OMC water was the highest, whereas water volume, titratable acidity, total soluble solids and total phenolics content for OMC water were the lowest. Maturity was found to affect sugar contents. Sucrose content was found to increase with maturity, and the reverse trend was observed for fructose and glucose. Enzyme activity assessment showed that polyphenol oxidase (PPO) in all samples was more heat resistant than peroxidase (POD). Compared to IMC and MC, PPO and POD from OMC water showed the lowest thermal resistance, with D83.3°C=243.9s (z=27.9°C), and D83.3°C=129.9s (z=19.5°C), respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

Changes in microbial populations and enzyme activities during the bioremediation of oil-contaminated soil.

Science.gov (United States)

Lin, Xin; Li, Xiaojun; Sun, Tieheng; Li, Peijun; Zhou, Qixing; Sun, Lina; Hu, Xiaojun

2009-10-01

In the process of bioremediation in the soil contaminated by different oil concentrations, the changes in the microbial numbers (bacteria and fungi) and the enzyme (catalase (CAT), polyphenol oxidase (PPO) and lipase) activities were evaluated over a 2-year period. The results showed that the microbial numbers after 2-year bioremediation were one to ten times higher than those in the initial. The changes in the bacterial and the fungal populations were different during the bioremediation, and the highest microbial numbers for bacteria and fungi were 5.51 x 10(9) CFU g(-1) dry soil in treatment 3 (10,000 mg kg(-1)) in the initial and 5.54 x 10(5) CFU g(-1) dry soil in treatment 5 (50,000 mg kg(-1)) after the 2-year bioremediation period, respectively. The CAT and PPO activities in the contaminated soil decreased with increasing oil concentration, while the lipase activity increased. The activities of CAT and PPO improved after the bioremediation, but lipase activity was on the contrary. The CAT activity was more sensible to the oil than others, and could be alternative to monitor the bioremediation process.

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.

Purification and biochemical characterization of ionically unbound polyphenol oxidase from Musa paradisiaca leaf.

Science.gov (United States)

Diwakar, Sanjeev Kumar; Mishra, Sarad Kumar

2011-01-01

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0-9.0) and temperature (30-90°C). From the thermal inactivation studies in the range 60-75°C, the half-life (t(1/2)) values of the enzyme ranged from 17 to 77 min. The inactivation energy (Ea) value of PPO was estimated to be 91.3 kJ mol(-1). It showed higher specificity with catechol (K(m) = 8 mM) as compared to 4-methylcatechol (K(m) = 10 mM). Among metal ions and reagents tested, Cu(2+), Fe(2+), Hg(2+), Mn(2+), Ni(2+), protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K(+), Na(+), Co(2+), kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.

PPO-ethanol system as wavelength shifter for the Cherenkov counting technique using a liquid scintillation counter

International Nuclear Information System (INIS)

Takiue, M.; Fujii, H.; Ishikawa, H.

1984-01-01

2,5-diphenyloxazole (PPO) has been proposed as a wavelength shifter for Cherenkov counting. Since PPO is not incorporated with water, we have introduced the fluor into water in the form of micelle using a PPO-ethanol system. This technique makes it possible to obtain a high Cherenkov counting efficiency under stable sample conditions, attributed to the proper spectrometric features of the PPO. The 32 P Cherenkov counting efficiency (68.4%) obtained from this technique is 1.62 times as large as that measured with a conventional Cherenkov technique. (orig.)

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.

[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.

Characterization of the polyphenol oxidase gene family reveals a novel microRNA involved in posttranscriptional regulation of PPOs in Salvia miltiorrhiza

OpenAIRE

Li, Caili; Li, Dongqiao; Li, Jiang; Shao, Fenjuan; Lu, Shanfa

2017-01-01

Salvia miltiorrhiza is a well-known material of traditional Chinese medicine. Understanding the regulatory mechanisms of phenolic acid biosynthesis and metabolism are important for S. miltiorrhiza quality improvement. We report here that S. miltiorrhiza contains 19 polyphenol oxidases (PPOs), forming the largest PPO gene family in plant species to our knowledge. Analysis of gene structures and sequence features revealed the conservation and divergence of SmPPOs. SmPPOs were differentially exp...

Unintended effects were investigated in antioxidant activity between ...

African Journals Online (AJOL)

Jane

2011-08-22

Aug 22, 2011 ... modified organisms; POD, peroxidase; PPO, polyphenol oxidase; CAT ... the seeds powder were homogenized in 2 ml (for maize) or 3 ml (for canola) 100 mM .... was carried out using the Wiley GC-MS Library. The percent ...

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.

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).

Removal of bisphenol derivatives through quinone oxidation by polyphenol oxidase and subsequent quinone adsorption on chitosan in the heterogeneous system.

Science.gov (United States)

Kimura, Yuji; Takahashi, Ayumi; Kashiwada, Ayumi; Yamada, Kazunori

2015-01-01

In this study, the combined use of a biopolymer chitosan and an oxidoreductase polyphenol oxidase (PPO) was systematically investigated for the removal of bisphenol derivatives from aqueous medium. The process parameters, such as the pH value, temperature, and PPO concentration, were estimated to conduct the enzymatic quinone oxidation of bisphenol derivatives by as little enzyme as possible. Bisphenol derivatives effectively underwent PPO-catalysed quinone oxidation without H2O2 unlike other oxidoreductases, such as peroxidase and tyrosinase, and the optimum conditions were determined to be pH 7.0 and 40°C for bisphenol B, bisphenol E, bisphenol O, and bisphenol Z; pH 7.0 and 30°C for bisphenol C and bisphenol F; and pH 8.0 and 40°C for bisphenol T. They were completely removed through adsorption of enzymatically generated quinone derivatives on chitosan beads or chitosan powders. Quinone adsorption on chitosan beads or chitosan powders in the heterogeneous system was found to be a more effective procedure than generation of aggregates in the homogeneous system with chitosan solution. The removal time was shortened by increasing the amount of chitosan beads or decreasing the size of the chitosan powders.

Purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) pulp.

Science.gov (United States)

Yang, C P; Fujita, S; Ashrafuzzaman, M; Nakamura, N; Hayashi, N

2000-07-01

Polyphenol oxidase (EC 1.10.3.1, PPO) in the pulp of banana (Musa sapientum L.) was purified to 636-fold with a recovery of 3.0%, using dopamine as substrate. The purified enzyme exhibited a clear single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight of the enzyme was estimated to be about 41000 and 42000 by gel filtration and SDS-PAGE, respectively. The enzyme quickly oxidized dopamine, and its K(m) value for dopamine was 2.8 mM. The optimum pH was at 6.5, and the enzyme activity was stable in the range of pH 5-11 at 5 degrees C for 48 h. The enzyme had an optimum temperature of 30 degrees C and was stable even after a heat treatment at 70 degrees C for 30 min. The enzyme activity was completely inhibited by L-ascorbic acid, cysteine, sodium diethyldithiocarbamate, and potassium cyanide. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

Comparative evaluation of oxidative enzyme activities during adventitious rooting in the cuttings of grapevine rootstocks.

Science.gov (United States)

Kose, Cafer; Erdal, Serkan; Kaya, Ozkan; Atici, Okkeş

2011-03-15

This study investigated changes in peroxidase (POX) and polyphenol oxidase (PPO) activities through adventitious rooting in hardwood cuttings of grapevine rootstocks. Three grapevine rootstocks with different propensity to produce adventitious roots were selected: recalcitrant (Ramsey), non-recalcitrant (Rupestris du Lot) and intermediate (99R) cultivars. The averages of root number at 65 days were 96 in Lot, 76 in 99R and 30 in Ramsey. Both enzyme activities characteristically increased before adventitious rooting, regardless of rooting ability of the rootstocks, and then decreased. POX activity increased in Ramsey cuttings at 22 days, in Lot and 99R cuttings at 14 days after planting, and then decreased gradually until 51 days. The highest POX activity was determined in Ramsey rootstock with the highest rooting ability and the lowest activity was determined in the rootstocks with the lowest rooting ability. PPO activity gradually increased in Ramsey rootstock cuttings from 10 days to 22 days, in Lot and 99R cuttings at 14 days, and then decreased until 51 days. A significant correlation was identified between high POX activity and adventitious rooting capability in rootstocks, but the same result was not determined with PPO activity. A recalcitrant rooting variety cannot increase POX activity sufficiently before rooting. Therefore applications that could increase POX activity in stem cuttings during rooting may facilitate increased rooting in such rootstocks. Copyright © 2011 Society of Chemical Industry.

Identification of an extensive gene cluster among a family of PPOs in Trifolium pratense L. (red clover using a large insert BAC library

Directory of Open Access Journals (Sweden)

Thomas Ann

2009-07-01

Full Text Available Abstract Background Polyphenol oxidase (PPO activity in plants is a trait with potential economic, agricultural and environmental impact. In relation to the food industry, PPO-induced browning causes unacceptable discolouration in fruit and vegetables: from an agriculture perspective, PPO can protect plants against pathogens and environmental stress, improve ruminant growth by increasing nitrogen absorption and decreasing nitrogen loss to the environment through the animal's urine. The high PPO legume, red clover, has a significant economic and environmental role in sustaining low-input organic and conventional farms. Molecular markers for a range of important agricultural traits are being developed for red clover and improved knowledge of PPO genes and their structure will facilitate molecular breeding. Results A bacterial artificial chromosome (BAC library comprising 26,016 BAC clones with an average 135 Kb insert size, was constructed from Trifolium pratense L. (red clover, a diploid legume with a haploid genome size of 440–637 Mb. Library coverage of 6–8 genome equivalents ensured good representation of genes: the library was screened for polyphenol oxidase (PPO genes. Two single copy PPO genes, PPO4 and PPO5, were identified to add to a family of three, previously reported, paralogous genes (PPO1–PPO3. Multiple PPO1 copies were identified and characterised revealing a subfamily comprising three variants PPO1/2, PPO1/4 and PPO1/5. Six PPO genes clustered within the genome: four separate BAC clones could be assembled onto a predicted 190–510 Kb single BAC contig. Conclusion A PPO gene family in red clover resides as a cluster of at least 6 genes. Three of these genes have high homology, suggesting a more recent evolutionary event. This PPO cluster covers a longer region of the genome than clusters detected in rice or previously reported in tomato. Full-length coding sequences from PPO4, PPO5, PPO1/5 and PPO1/4 will facilitate

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.

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.

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

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

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

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.

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.

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.

Juglone effects on seedling growth in intact and coatless seeds of ...

African Journals Online (AJOL)

STORAGESEVER

2008-12-17

Dec 17, 2008 ... Key words: Juglone, muskmelon, seedling growth, protein content, polyphenol oxidase activity. INTRODUCTION ... Juglone has been isolated from many plants in the .... In pea seeds, a relation between PPO activity and seed.

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

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

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.

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...

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.

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.

[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.

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.

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)

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.

Polyphenol Oxidases in Crops: Biochemical, Physiological and Genetic Aspects

Directory of Open Access Journals (Sweden)

Francesca Taranto

2017-02-01

Full Text Available Enzymatic browning is a colour reaction occurring in plants, including cereals, fruit and horticultural crops, due to oxidation during postharvest processing and storage. This has a negative impact on the colour, flavour, nutritional properties and shelf life of food products. Browning is usually caused by polyphenol oxidases (PPOs, following cell damage caused by senescence, wounding and the attack of pests and pathogens. Several studies indicated that PPOs play a role in plant immunity, and emerging evidence suggested that PPOs might also be involved in other physiological processes. Genomic investigations ultimately led to the isolation of PPO homologs in several crops, which will be possibly characterized at the functional level in the near future. Here, focusing on the botanic families of Poaceae and Solanaceae, we provide an overview on available scientific literature on PPOs, resulting in useful information on biochemical, physiological and genetic aspects.

Characterization of the polyphenol oxidase gene family reveals a novel microRNA involved in posttranscriptional regulation of PPOs in Salvia miltiorrhiza.

Science.gov (United States)

Li, Caili; Li, Dongqiao; Li, Jiang; Shao, Fenjuan; Lu, Shanfa

2017-03-17

Salvia miltiorrhiza is a well-known material of traditional Chinese medicine. Understanding the regulatory mechanisms of phenolic acid biosynthesis and metabolism are important for S. miltiorrhiza quality improvement. We report here that S. miltiorrhiza contains 19 polyphenol oxidases (PPOs), forming the largest PPO gene family in plant species to our knowledge. Analysis of gene structures and sequence features revealed the conservation and divergence of SmPPOs. SmPPOs were differentially expressed in plant tissues and eight of them were predominantly expressed in phloem and xylem, indicating that some SmPPOs are functionally redundant, whereas the others are associated with different physiological processes. Expression patterns of eighteen SmPPOs were significantly altered under MeJA treatment, and twelve were yeast extract and Ag + -responsive, suggesting the majority of SmPPOs are stress-responsive. Analysis of high-throughput small RNA sequences and degradome data showed that miR1444-mediated regulation of PPOs existing in P. trichocarpa is absent from S. miltiorrhiza. Instead, a subset of SmPPOs was posttranscriptionally regulated by a novel miRNA, termed Smi-miR12112. It indicates the specificity and significance of miRNA-mediated regulation of PPOs. The results shed light on the regulation of SmPPO expression and suggest the complexity of SmPPO-associated phenolic acid biosynthesis and metabolism.

N+ion-target interactions in PPO polymer: A structural characterization

International Nuclear Information System (INIS)

Das, A.; Dhara, S.; Patnaik, A.

1999-01-01

N + ion beam induced effects on the spin coated amorphous poly(2,6-dimethyl phenylene oxide) (PPO) films in terms of chemical structure and electronic and vibrational properties were investigated using Fourier Transform Infrared spectroscopy (FTIR) and Ultraviolet-Visible (UV-VIS) spectroscopy. Both techniques revealed that the stability of PPO was very weak towards 100 keV N + ions revealing the threshold fluence to be 10 14 ions/cm 2 for fragmentation of the polymer. FTIR analysis showed disappearance of all characteristic IR bands at a total fluence of 10 14 ions/cm 2 except for the band C=C at 1608 cm -1 which was found to shift to a lower wave number along with an enhancement in the full width half maximum (FWHM) value with increasing fluence. A new bond appeared due to oxidation as a shoulder at 1680 cm -1 in FTIR spectra indicating the presence of C=O type bond as a result of N + implantation on PPO films. The optical band gap (E g ) deduced from absorption spectra, was observed to decrease from 4.4 to 0.5 eV with fluence. The implantation induced carbonaceous clusters, determined using Robertson's formula for the optical band gap, were found to consist of ∼160 fused hexagonal aromatic rings at the maximum energy fluence. An enhanced absorption coefficient as a function of fluence indicated incorporation of either much larger concentration of charge carriers or their mobility than that of the pristine sample. Calculated band tail width from Urbach band tail region for the implanted samples pointed the band edge sharpness to be strongly dependent on fluence indicating an increased disorder with increasing fluence

Effects of germination on the activities of amylases and phenolic enzymes in sorghum varieties grouped according to food end-use properties

NARCIS (Netherlands)

Dicko, M.H.; Gruppen, H.; Zouzouho, O.C.; Traore, A.S.; Berkel, van W.J.H.; Voragen, A.G.J.

2006-01-01

Fifty sorghum varieties were screened to determine the effects of germination on levels of starch, -amylase, -amylase, phenylalanine ammonia lyase (PAL), peroxidase (POX) and polyphenol oxidase (PPO). Germination decreased starch content, with amylose being more degraded than amylopectin. In

Meest geschikte druivenras-onderstamcombinaties voor verschillende regio's : resultaten van PPO-onderzoek

NARCIS (Netherlands)

Beurskens, Stan; Maas, F.M.

2010-01-01

PPO startte in 2008 een onderzoek om na te gaan hoe verschillende ras-onderstamcombinaties zich ontwikkelen in verschillende bodemtypen in verschillende delen van Nederland. Het doel van het onderzoek is om de verschillende ras-onderstamcombinaties per bodemtype en regio te kunnen rangschikken op

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

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.

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)

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 ...

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.

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.

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.

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

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.

Effect of dose rate of gamma irradiation on biochemical quality and browning of mushrooms Agaricus bisporus

Science.gov (United States)

Beaulieu, M.; D'Aprano, G.; Lacroix, M.

2002-03-01

In order to enhance the shelf-life of edible mature mushrooms Agaricus bisporus, 2 kGy ionising treatments were applied at two different dose rates: 4.5 kGy/h ( I-) and 32 kGy/h ( I+). Both I+ and I- showed 2 and 4 days shelf-life enhancement compared to the control ( C). Before day 9, no significant difference ( p>0.05) in L* value was detected in irradiated mushrooms. However, after day 9, the highest observed L* value (whiteness) was obtained for the mushrooms irradiated in I-. Analyses of phenolic compounds revealed that mushrooms in I- contained more phenols than I+ and C, the latter containing the lower level of phenols. The polyphenol oxidase (PPO) activities of irradiated mushrooms, analysed via catechol oxidase and dopa oxidase substrates, resulted in being significantly lowered ( p⩽0.05) compared to C, with a further decrease in I+. Analyses of the enzymes indicated that PPO activity was lower in I+, contrasting with its lower phenol concentration. Ionising treatments also increased significantly ( p⩽0.05) the phenylalanine ammonia-lyase (PAL) activity. The observation of mushrooms cellular membranes, by electronic microscopy, revealed a better preserved integrity in I- than in I+. It is thus assumed that the browning effect observed in I+ was caused by both the decompartimentation of vacuolar phenol and by the entry of molecular oxygen into the cell cytoplasm. The synergetic effect of the residual active PPO and the molecular oxygen, in contact with the phenols, allowed an increased oxidation rate and, therefore, a more pronounced browning in I+ than in I-.

Effect of dose rate of gamma irradiation on biochemical quality and browning of mushrooms Agaricus bisporus

International Nuclear Information System (INIS)

Beaulieu, M.; D'Aprano, G.; Lacroix, M.

2002-01-01

In order to enhance the shelf-life of edible mature mushrooms Agaricus bisporus, 2 kGy ionising treatments were applied at two different dose rates: 4.5 kGy/h (I - ) and 32 kGy/h (I + ). Both I + and I - showed 2 and 4 days shelf-life enhancement compared to the control (C). Before day 9, no significant difference (p>0.05) in L * value was detected in irradiated mushrooms. However, after day 9, the highest observed L * value (whiteness) was obtained for the mushrooms irradiated in I - . Analyses of phenolic compounds revealed that mushrooms in I - contained more phenols than I + and C, the latter containing the lower level of phenols. The polyphenol oxidase (PPO) activities of irradiated mushrooms, analysed via catechol oxidase and dopa oxidase substrates, resulted in being significantly lowered (p≤0.05) compared to C, with a further decrease in I + . Analyses of the enzymes indicated that PPO activity was lower in I + , contrasting with its lower phenol concentration. Ionising treatments also increased significantly (p≤0.05) the phenylalanine ammonia-lyase (PAL) activity. The observation of mushrooms cellular membranes, by electronic microscopy, revealed a better preserved integrity in I - than in I + . It is thus assumed that the browning effect observed in I + was caused by both the decompartimentation of vacuolar phenol and by the entry of molecular oxygen into the cell cytoplasm. The synergetic effect of the residual active PPO and the molecular oxygen, in contact with the phenols, allowed an increased oxidation rate and, therefore, a more pronounced browning in I + than in I -

Characterization of Plasmid pPO1 from the Hyperacidophile Picrophilus oshimae

Directory of Open Access Journals (Sweden)

Angel Angelov

2011-01-01

Full Text Available Picrophilus oshimae and Picrophilus torridus are free-living, moderately thermophilic and acidophilic organisms from the lineage of Euryarchaeota. With a pH optimum of growth at pH 0.7 and the ability to even withstand molar concentrations of sulphuric acid, these organisms represent the most extreme acidophiles known. So far, nothing is known about plasmid biology in these hyperacidophiles. Also, there are no genetic tools available for this genus. We have mobilized the 7.6 Kbp plasmid from P. oshimae in E. coli by introducing origin-containing transposons and described the plasmid in terms of its nucleotide sequence, copy number in the native host, mode of replication, and transcriptional start sites of the encoded ORFs. Plasmid pPO1 may encode a restriction/modification system in addition to its replication functions. The information gained from the pPO1 plasmid may prove useful in developing a cloning system for this group of extreme acidophiles.

Preparation of biosensors by immobilization of polyphenol oxidase in conducting copolymers and their use in determination of phenolic compounds in red wine.

Science.gov (United States)

Böyükbayram, A Elif; Kiralp, Senem; Toppare, Levent; Yağci, Yusuf

2006-10-01

Electrochemically produced graft copolymers of thiophene capped polytetrahydofuran (TPTHF1 and TPTHF2) and pyrrole were achieved by constant potential electrolysis using sodium dodecylsulfate (SDS) as the supporting electrolyte. Characterizations were based on Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Electrical conductivities were measured by the four-probe technique. Novel biosensors for phenolic compounds were constructed by immobilizing polyphenol oxidase (PPO) into conducting copolymers prepared by electropolymerization of pyrrole with thiophene capped polytetrahydrofuran. Kinetic parameters, maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) and optimum conditions regarding temperature and pH were determined for the immobilized enzyme. Operational stability and shelf-life of the enzyme electrodes were investigated. Enzyme electrodes of polyphenol oxidase were used to determine the amount of phenolic compounds in two brands of Turkish red wines and found very useful owing to their high kinetic parameters and wide pH working range.

Study of the interface solid/solutions containing PEO-PPO block copolymers and asphaltenes by FTIR/ATR; Estudo de solucoes de copolimeros em bloco de PEO-PPO contendo asfaltenos por FTIR/DTA

Energy Technology Data Exchange (ETDEWEB)

Aguiar, Janaina I.S.; Neto, Jessica S.G.; Mansur, Claudia R.E. [Universidade Federal do Rio de Janeiro, Instituto de Macromoleculas, Laboratorio de Macromoleculas e Coloides na Industria de Petroleo, Rio de Janeiro, RJ (Brazil)], E-mails: janaina_333@hotmail.com, kinha_dac_dm@hotmail.com; celias@ima.ufrj.br

2011-07-01

The formation of water/oil emulsions can cause problems in various stages of production, processing and refining of petroleum. In this study, the technique of Fourier transform infrared spectroscopy (FTIR) using the method of attenuated total reflectance (ATR) was applied to study the solid-solutions of block copolymers based on poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO) interface and its interaction in this interface with asphaltenic fractions of petroleum. The solid is the crystal of the ATR. Initially, we determined the critical micelle concentration values of the copolymers, which were consistent those obtained by a tensiometer. Bottle Test was also performed to correlate the efficiency of PEO-PPO copolymers in the breaking of water/oil emulsions with its adsorption at the interfaces solutions. (author)

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.

Design and syntheses of novel N-(benzothiazol-5-yl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione and N-(benzothiazol-5-yl)isoindoline-1,3-dione as potent protoporphyrinogen oxidase inhibitors.

Science.gov (United States)

Jiang, Li-Li; Zuo, Yang; Wang, Zhi-Fang; Tan, Yin; Wu, Qiong-You; Xi, Zhen; Yang, Guang-Fu

2011-06-08

Discovery of protoporphyrinogen oxidase (PPO, EC 1.3.3.4) inhibitors has been one of the hottest research areas in the field of herbicide development for many years. As a continuation of our research work on the development of new PPO-inhibiting herbicides, a series of novel N-(benzothiazol-5-yl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-diones (1a-p) and N-(benzothiazol-5-yl)isoindoline-1,3-diones (2a-h) were designed and synthesized according to the ring-closing strategy of two ortho-substituents. The bioassay results indicated that some newly synthesized compounds exhibited higher PPO inhibition activity than the control of sulfentrazone. Compound 1a, S-(5-(1,3-dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)-6-fluorobenzothiazol-2-yl) O-methyl carbonothioate, was identified as the most potent inhibitor with k(i) value of 0.08 μM, about 9 times higher than that of sulfentrazone (k(i) = 0.72 μM). Further green house assay showed that compound 1b, methyl 2-((5-(1,3-dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)-6-fluorobenzothiazol-2-yl)thio)acetate, exhibited herbicidal activity comparable to that of sulfentrazone even at a concentration of 37.5 g ai/ha. In addition, among six tested crops, wheat exhibited high tolerance to compound 1b even at a dosage of 300 g ai/ha. These results indicated that compound 1b might have the potential to be developed as a new herbicide for weed control of wheat field.

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.

The Effect of Surfactant and Compatibilizer on Inorganic Loading and Properties of PPO-based EPMM Membranes

Science.gov (United States)

Bissadi, Golnaz

Hybrid membranes represent a promising alternative to the limitations of organic and inorganic materials for high productivity and selectivity gas separation membranes. In this study, the previously developed concept of emulsion-polymerized mixed matrix (EPMM) membranes was further advanced by investigating the effects of surfactant and compatibilizer on inorganic loading in poly(2,6-dimethyl-1,4-phenylene oxide) (PPO)-based EPMM membranes, in which inorganic part of the membranes originated from tetraethylorthosilicate (TEOS). The polymerization of TEOS, which consists of hydrolysis of TEOS and condensation of the hydrolyzed TEOS, was carried out as (i) one- and (ii) two-step processes. In the one-step process, the hydrolysis and condensation take place in the same environment of a weak acid provided by the aqueous solution of aluminum hydroxonitrate and sodium carbonate. In the two-step process, the hydrolysis takes place in the environment of a strong acid (solution of hydrochloric acid), whereas the condensation takes place in weak base environment obtained by adding excess of the ammonium hydroxide solution to the acidic solution of the hydrolyzed TEOS. For both one- and two-step processes, the emulsion polymerization of TEOS was carried out in two types of emulsions made of (i) pure trichloroethylene (TCE) solvent, and (ii) 10 w/v% solution of PPO in TCE, using different combinations of the compatibilizer (ethanol) and the surfactant (n-octanol). The experiments with pure TCE, which are referred to as a gravimetric powder method (GPM) allowed assessing the effect of different experimental parameters on the conversion of TEOS. The GPM tests also provided a guide for the synthesis of casting emulsions containing PPO, from which the EPMM membranes were prepared using a spin coating technique. The synthesized EPMM membranes were characterized using 29Si nuclear magnetic resonance (29Si NMR), differential scanning calorimetry (DSC), inductively coupled plasma mass

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.

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.

Analysis of the blackening of green pepper (Piper nigrum Linnaeus) berries.

Science.gov (United States)

Gu, Fenglin; Tan, Lehe; Wu, Huasong; Fang, Yiming; Wang, Qinghuang

2013-06-01

This paper investigates polyphenol oxidase (PPO) activity, reduced weight percentage after sun drying, and the changes in colour and appearance of green pepper (Piper nigrum Linnaeus) berries after blanching and sun drying. The results show that the degree of reduced weight percentage and browning in green pepper berries after blanching for 10 min is greater at 100°C than at 90 and 80°C. Moreover, the samples blanched at 100°C for 10 min had the fastest water loss, but the lowest PPO activity. Thus, the PPO enzymatic oxidation of polyphenols might not be the only reason for the browning of green pepper berries. This result is significantly different from that of Variyar, Pendharkar, Banerjeea, and Bandyopadhyay (1988) and therefore deserves further study. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

'Mastspuit met sensoren brengt middel effectiever aan' (onderzoek praktijkproeven PRI en PPO)

NARCIS (Netherlands)

Engels, A.; PPO BBF Boomkwekerij,; PRI,; Nieuwenhuizen, A.T.

2011-01-01

In mei 2011 starten Plant Research International (PRI), PPO Boomkwekerij en Damcon de eerste praktijkproeven met sensoren op de mastspuit. Dit prototype spuit alleen als het bladeren detecteert. Projectleider Ard Nieuwenhuizen van PRI is ervan overtuigd dat kwekers met zo'n type spuit middelen

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.

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.

C60 and Sc3N@C80(TMB-PPO derivatives as constituents of singlet oxygen generating, thiol-ene polymer nanocomposites

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Ashli R. Toles

2016-07-01

Full Text Available Numerous functionalization methods have been employed to increase the solubility, and therefore, the processability of fullerenes in composite structures, and of these radical addition reactions continue to be an important methodology. C60 and Sc3N@C80 derivatives were prepared via radical addition of the photodecomposition products from the commercial photoinitiator TMB-PPO, yielding C60(TMB-PPO5 and Sc3N@C80(TMB-PPO3 as preferred soluble derivatives obtained in high yields. Characterization of the mixture of isomers using standard techniques suggests an overall 1PPO:6TMB ratio of addends, reflecting the increased reactivity of the carbon radical. Although, a higher percentage of PPO is observed in the Sc3N@C80(TMB-PPO3 population, perhaps due to reverse electronic requirements of the substrate. Visually dispersed thiol-ene nanocomposites with low extractables were prepared using two monomer compositions (PETMP:TTT and TMPMP:TMPDE with increasing fullerene derivative loading to probe network structure-property relationships. Thermal stability of the derivatives and the resulting networks decreased with increased functionality and at high fullerene loadings, respectively. TMPMP:TMPDE composite networks show well-dispersed derivatives via TEM imaging, and increasing Tg’s with fullerene loading, as expected for the incorporation of a more rigid network component. PETMP:TTT composites show phase separation in TEM, which is supported by the observed Tg’s. Singlet oxygen generation of the derivatives decreases with increased functionality; however, this is compensated for by the tremendous increase in solubility in organic solvents and miscibility with monomers. Most importantly, singlet oxygen generation from the composites increased with fullerene derivative loading, with good photostability of the networks.

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.

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

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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

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

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

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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

Isolated sulfite oxidase deficiency.

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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.

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

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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

Extension of the shelf-life of fresh oyster mushrooms ( Pleurotus ...

African Journals Online (AJOL)

The objective of this study was to improve the preservation of fresh oyster mushrooms by modified atmosphere packaging (MAP) and chemical treatments. The MAP effects and treatments on organoleptic quality, weight loss, cell permeability, texture changes and polyphenol oxidase (PPO) activity were studied. In addition ...

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

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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.

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

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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...

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.

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.

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

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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.

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.)

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.).

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

Synthesis and characterizations of anion exchange organic-inorganic hybrid materials based on poly(2,6-dimethyl-1,4-phenylene oxide) (PPO)

International Nuclear Information System (INIS)

Zhang Shaoling; Wu Cuiming; Xu Tongwen; Gong Ming; Xu Xiaolong

2005-01-01

A series of poly(2,6-dimethyl-1,4-phenylene oxide) (PPO)-based organic-inorganic hybrid materials for anion exchange were prepared through sol-gel process of polymer precursors PPO-Si(OCH 3 ) 3 . PPO-Si(OCH 3 ) 3 were obtained from the reaction of bromomethylated PPO with 3-aminopropyl-trimethoxysilane (A1110). These polymer precursors then underwent hydrolysis and condensation with additional A1110 to generate hybrid materials. The reaction to produce polymer precursors was identified by FTIR; while FTIR, TGA, XRD, SEM, as well as conventional ion exchange capacity (IEC) measurements were conducted for the structures and properties of the prepared hybrids. TGA results show that this series of hybrid materials possess high thermal stability; XRD and SEM indicate that the prepared hybrid materials are amorphous and the inorganic and organic contents show good compatibility if the ratio between them is proper. The IEC values of the hybrid materials due to the amine groups range from 1.13 mmol/gBPPO (material i) to 4.80 mmol/gBPPO (material iv)

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.

Tentacle carrier for immobilization of potato phenoloxidase and its application for halogenophenols removal from aqueous solutions

International Nuclear Information System (INIS)

Lončar, Nikola; Vujčić, Zoran

2011-01-01

Highlights: ► We synthesized novel immobilization carrier from reused DEAE-cellulose. ► We used it for immobilization of PPO through coordinative bonding with copper ions. ► Immobilized PPO showed better characteristics than soluble PPO. ► TC-PPO removed over 90% of halogenophenols at concentration of 100 mg/L. - Abstract: Halogenated compounds represent one of the most dangerous environmental pollutants, due to their widespread usage as biocides, fungicides, disinfectants, solvent and other industrial chemicals. Immobilization of a protein through coordinate bonds formed with divalent metal ions is becoming an attractive method due to its reversible nature, since the protein may be easily removed from the support matrix through interruption of the protein–metal bond hence giving inherently cleaner and cheaper technology for wastewater treatment. We have synthesized novel ‘tentacle’ carrier (TC) and used it for immobilization of partially purified potato polyphenol oxidase (PPO). The obtained biocatalyst TC-PPO showed pH optimum at 7.0–8.0 and temperature optimum at 25 °C. Immobilized PPO shows almost 100% of activity at 0 °C. TC-PPO was more resistant to the denaturation induced by sodium dodecyl sulphate (SDS) detergent as compared to its soluble counterpart and was even slightly activated at SDS concentration of 1%. TC-PPO was tested in the batch reactor for 4-chlorophenol and 4-bromophenol removal. More than 90% removal was achieved for both halogenophenols at concentration of 100 mg/L from aqueous solution. For both halogenophenols TC-PPO works with over 90% removal during first three cycles which decrease to 60% removal efficiency after six cycles each of 8 h duration.

PP-O and PP-V, Monascus pigment homologues, production, and phylogenetic analysis in Penicillium purpurogenum.

Science.gov (United States)

Arai, Teppei; Kojima, Ryo; Motegi, Yoshiki; Kato, Jun; Kasumi, Takafumi; Ogihara, Jun

2015-12-01

The production of pigments as secondary metabolites by microbes is known to vary by species and by physiological conditions within a single strain. The fungus strain Penicillium purpurogenum IAM15392 has been found to produce violet pigment (PP-V) and orange pigment (PP-O),Monascus azaphilone pigment homologues, when grown under specific culture conditions. In this study, we analysed PP-V and PP-O production capability in seven strains of P. purpurogenum in addition to strain IAM15392 under specific culture conditions. The pigment production pattern of five strains cultivated in PP-V production medium was similar to that of strain IAM15392, and all violet pigments produced by these five strains were confirmed to be PP-V. Strains that did not produce pigment were also identified. In addition, two strains cultivated in PP-O production medium produced a violet pigment identified as PP-V. The ribosomal DNA (rDNA) internal transcribed spacer (ITS) region sequences from the eight P. purpurogenum strains were sequenced and used to construct a neighbor-joining phylogenetic tree. PP-O and PP-V production of P. purpurogenum was shown to be related to phylogenetic placement based on rDNA ITS sequence. Based on these results, two hypotheses for the alteration of pigment production of P. purpurogenum in evolution were proposed. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Gd2O3:Eu3+/PPO/POPOP/PS composites for digital imaging radiation detectors

International Nuclear Information System (INIS)

Oliveira, J.; Correia, V.; Martins, P.M.; Martins, P.; Lanceros-Mendez, S.; Rocha, J.G.

2015-01-01

Polymer-based scintillator composites have been produced by combining polystyrene (PS) and Gd 2 O 3 :Eu 3+ scintillator nanoparticles. Polystyrene has been used since it is a flexible and stable binder matrix, resistant to thermal and light deterioration and with suitable optical properties. Gd 2 O 3 :Eu 3+ has been selected as scintillator material due to its wide band gap, high density and visible light yield. The optical, thermal and electrical characteristics of the composites were studied as a function of filler content, together with their performance as scintillator material. Additionally 1 wt.% of 2,5-diphenyloxazole (PPO) and 0.01 wt.% of 1,4 di[2-(5phenyloxazolyl)]benzene (POPOP) were introduced in the polymer matrix in order to strongly improve light yield, i.e., the measured intensity of the output visible radiation, under X-ray irradiation. Increasing scintillator filler concentration (from 0.25 to 7.5 wt.%) increases scintillator light yield and decreases the optical transparency of the composite. The addition of PPO and POPOP strongly increased the overall transduction performance of the composite due to specific absorption and re-emission processes. It is thus shown that Gd 2 O 3 :Eu 3+ /PPO/POPOP/PS composites with 0.25 wt.% of scintillator content with fluorescence molecules are suitable for the development of innovative large-area X-ray radiation detectors with huge demand from the industries. (orig.)

Production of rabbit antibodies against purified Glucose oxidase

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Muhammad Anjum Zia

2012-02-01

Full Text Available 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 G-200 column for gel filtration chromatography. It was observed that enzyme achieved 59.37 U mg-1of specific activity with 27.08 fold purity and 64.36% recovery. Purified glucose oxidase was injected into rabbits through intravenous route, to raise the glucose oxidase antibodies. After 30 days incubation period, the rabbits were slaughtered and serum was separated from blood. The antibodies were isolated by ammonium sulfate precipitation and confirmed by agar gel precipitation test. This could be a convenient and low cost alternate assay for the estimation of glucose oxidase in biological fluids. Moreover, such antibodies against the said enzyme could be used in various therapeutic and diagnostic applications.

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.

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

Amine oxidases as important agents of pathological processes of rhabdomyolysis in rats.

Science.gov (United States)

Gudkova, O O; Latyshko, N V; Shandrenko, S G

2016-01-01

In this study we have tested an idea on the important role of amine oxidases (semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase) as an additional source of oxidative/carbonyl stress under glycerol-induced rhabdomyolysis, since the enhanced formation of reactive oxygen species and reactive carbonyl species in a variety of tissues is linked to various diseases. In our experiments we used the sensitive fluorescent method devised for estimation of amine oxidases activity in the rat kidney and thymus as targeted organs under rhabdomyolysis. We have found in vivo the multiple rises in activity of semicarbazide-sensitive amine oxidase, diamine oxidase, polyamine oxidase (2-4.5 times) in the corresponding cell fractions, whole cells or their lysates at the 3-6th day after glycerol injection. Aberrant antioxidant activities depended on rhabdomyolysis stage and had organ specificity. Additional treatment of animals with metal chelator ‘Unithiol’ adjusted only the activity of antioxidant enzymes but not amine oxidases in both organs. Furthermore the in vitro experiment showed that Fenton reaction (hydrogen peroxide in the presence of iron) products alone had no effect on semicarbazide-sensitive amine oxidase activity in rat liver cell fraction whereas supplementation with methylglyoxal resulted in its significant 2.5-fold enhancement. Combined action of the both agents had additive effect on semicarbazide-sensitive amine oxidase activity. We can assume that biogenic amine and polyamine catabolism by amine oxidases is upregulated by oxidative and carbonyl stress factors directly under rhabdomyolysis progression, and the increase in catabolic products concentration contributes to tissue damage in glycerol-induced acute renal failure and apoptosis stimulation in thymus.

Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

Science.gov (United States)

Löw, Hans; Crane, Frederick L; Morré, D James

2012-11-01

The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

A preferred provider organization (PPO) case study for mental health and substance abuse.

Science.gov (United States)

Gomillion, I; Self, D R

1988-01-01

The Preferred Provider Organization concept is quickly becoming more popular because of its relative cost-effectiveness and recent successes. Managed care through means of this mental health and substance abuse PPO may well serve as the prototype for the general health care cost containment efforts of the future for the self-insured insurance plans for Alabama state employees and teachers. The first year also revealed several problems in the original proposal especially with respect to the unintended attractiveness of inpatient/residential care. Consequently, copayment plans were added to dissuade unnecessary lengthy stays (see Table 2). Second, a new quality assurance mechanism has been added to further evaluate the need for admissions to facilities, as well as for the need for continued inpatient treatment. The Alabama Quality Assurance Foundation (AQAF) began on January 1, 1988, conducting the preadmission certification on all admissions based upon criteria established jointly by AQAF and the PPO providers. In addition, AQAF will conduct continuing stay reviews at predetermined time periods to ensure that continued treatment in an inpatient setting is indeed necessary.

C₆₀ and Sc₃N@C₈₀(TMB-PPO) derivatives as constituents of singlet oxygen generating, thiol-ene polymer nanocomposites

OpenAIRE

Ashli R. Toles; Janice Paige Buchanan; Kyle A. Guess; Emily M. Barker

2016-01-01

Numerous functionalization methods have been employed to increase the solubility, and therefore, the processability of fullerenes in composite structures, and of these radical addition reactions continue to be an important methodology. C60 and Sc3N@C80 derivatives were prepared via radical addition of the photodecomposition products from the commercial photoinitiator TMB-PPO, yielding C60(TMB-PPO)5 and Sc3N@C80(TMB-PPO)3 as preferred soluble derivatives obtained in high yields. Characterizati...

Tentacle carrier for immobilization of potato phenoloxidase and its application for halogenophenols removal from aqueous solutions

Energy Technology Data Exchange (ETDEWEB)

Loncar, Nikola, E-mail: nloncar@chem.bg.ac.rs [Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade (Serbia); Vujcic, Zoran, E-mail: zvujcic@chem.bg.ac.rs [Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade (Serbia)

2011-11-30

Highlights: Black-Right-Pointing-Pointer We synthesized novel immobilization carrier from reused DEAE-cellulose. Black-Right-Pointing-Pointer We used it for immobilization of PPO through coordinative bonding with copper ions. Black-Right-Pointing-Pointer Immobilized PPO showed better characteristics than soluble PPO. Black-Right-Pointing-Pointer TC-PPO removed over 90% of halogenophenols at concentration of 100 mg/L. - Abstract: Halogenated compounds represent one of the most dangerous environmental pollutants, due to their widespread usage as biocides, fungicides, disinfectants, solvent and other industrial chemicals. Immobilization of a protein through coordinate bonds formed with divalent metal ions is becoming an attractive method due to its reversible nature, since the protein may be easily removed from the support matrix through interruption of the protein-metal bond hence giving inherently cleaner and cheaper technology for wastewater treatment. We have synthesized novel 'tentacle' carrier (TC) and used it for immobilization of partially purified potato polyphenol oxidase (PPO). The obtained biocatalyst TC-PPO showed pH optimum at 7.0-8.0 and temperature optimum at 25 Degree-Sign C. Immobilized PPO shows almost 100% of activity at 0 Degree-Sign C. TC-PPO was more resistant to the denaturation induced by sodium dodecyl sulphate (SDS) detergent as compared to its soluble counterpart and was even slightly activated at SDS concentration of 1%. TC-PPO was tested in the batch reactor for 4-chlorophenol and 4-bromophenol removal. More than 90% removal was achieved for both halogenophenols at concentration of 100 mg/L from aqueous solution. For both halogenophenols TC-PPO works with over 90% removal during first three cycles which decrease to 60% removal efficiency after six cycles each of 8 h duration.

Modeling the Phase Behavior of PEO-PPO-PEO Surfactants in Carbon Dioxide Using the PC-SAFT Equation of State: Application to Dry Decontamination of Solid Substrates

International Nuclear Information System (INIS)

Kleiner, M.; Sadowski, G.; Stoychev, I.; Galy, J.; Fournel, B.; Lacroix-Desmazes, P.

2009-01-01

The phase behavior of several commercially available poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) block copolymers (PEO-PPO-PEO or Pluronics L) in compressed carbon dioxide has been investigated within the framework of dry nuclear decontamination. For this purpose, cloud points have been measured in the pressure and temperature range from P = (10 to 40) MPa and from T = (293 to 338) K, respectively. To find a reliable method for surfactant selection, the perturbed-chain statistical association fluid theory (PC-SAFT) equation of state (EoS) has been applied to model the experimental data. The pure-component and the respective homopolymer + CO 2 binary interaction parameters have been fitted to liquid densities and to homopolymer + CO 2 binary equilibrium data. The phase behavior of Pluronics L copolymers as a function of concentration, molar mass, and copolymer composition has been predicted very accurately using a constant PEO-PPO binary interaction parameter k(PEO-PPO) = 0.007. The P and SAFT model was also successfully applied to Pluronics R copolymers (PPO-PEO-PPO), although a different k(PEO-PPO) = -0.018 was required to match the experimental data. The model predictions have shown that Pluronics L copolymers with molar mass M ≤ 2750 g.mol -1 and a PEO mass fraction in the copolymer of less than 30 % have sufficiently low cloud-point pressures and are therefore the most suitable for the decontamination process. (authors)

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.

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.

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.

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.

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.

Fabrication of a novel hydrogen peroxide biosensor based on Au-(PEO106PPO70PEO106) hairy nanospheres

International Nuclear Information System (INIS)

Ni Yalong; Wang Yan; Zhang Guohui; Shen Jian; Zhao Wenbo; Huang Xiaohua

2012-01-01

In this case, the Au-F127 (triblock copolymer PEO 106 PPO 70 PEO 106 , PEO is short for polyethylene oxide and PPO is the abbreviation of polypropene oxide) hybrid hairy nanospheres were obtained by the redox reaction using pluronic F127 and HAuCl 4 as precursor. A novel hemoglobin (Hb) biosensor was fabricated by immobilizing the Hb onto the Au-F127 film on the surface of glassy carbon electrode (GCE). The Au-F127 nanocomposites and Hb were characterized by transmission electron microscopy (TEM), polycrystalline electron diffraction ring pattern and ultra-violet visible spectra (UV–vis). UV–vis spectra suggested that Hb in the film could keep its native secondary structure. The performances of Hb/(Au-F127) on GCE were characterized with cyclic voltammetry (CV) and typical amperometric response (i–t) measurements. The immobilized Hb maintained its bioactivity and displayed an excellent electrochemical behavior with a formal potential of −339 mV. The resulting electrode showed an electrocatalytic activity to hydrogen peroxide (H 2 O 2 ). The linear response range of the H 2 O 2 biosensor was from 3.0 × 10 −7 to 5.7 × 10 −4 M with a low detection limit of 4.0 × 10 −8 M.

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.

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

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

Eucalyptus ESTs corresponding to the protoporphyrinogen IX oxidase enzyme related to the synthesis of heme, chlorophyll, and to the action of herbicides

Directory of Open Access Journals (Sweden)

Edivaldo Domingues Velini

2005-01-01

Full Text Available This work was aimed at locating Eucalyptus ESTs corresponding to the PROTOX or PPO enzyme (Protoporphyrinogen IX oxidase, E.C. 1.3.3.4 directly related to resistance to herbicides that promote oxidative stress, changing the functionality of this enzyme. PROTOX, which is the site of action of diphenyl-ether (oxyfluorfen, lactofen, fomesafen, oxadiazole (oxadiazon and oxadiargyl, and aryl triazolinone (sulfentrazone and carfentrazone herbicides, acts on the synthesis route of porphyrins which is associated with the production of chlorophyll a, catalases, and peroxidases. One cluster and one single read were located, with e-values better than e-70, associated to PROTOX. The alignment results between amino acid sequences indicated that this enzyme is adequately represented in the ESTs database of the FORESTs project.

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.

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.

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.

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.

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)

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 ...

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.

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.

PPO Glastuinbouw en LEI helpen biologische ketenprojecten op weg: "vernieuwing moet wel uit de biologische hoek komen"

NARCIS (Netherlands)

Staalduinen, van Jan; Poot, E.H.; Wertheim-Heck, S.C.O.

2006-01-01

Het biologische marktsegment is vanwege de hogere kostprijs van het geteelde product een natuurlijke kraamkamer voor productinnovatie. De zoektocht naar tastbaar onderscheid en aansprekende verkoopconcepten vereist multidisciplinaire samenwerking van teelt- en marktonderzoek. LEI en PPO Glastuinbouw

[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.

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...

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.

Biochemical changes in barberries during adventitious root formation: the role of indole-3-butyric acid and hydrogen peroxide

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Ali Tehranifar

2014-03-01

Full Text Available Peroxidase, polyphenol oxidase (PPO, phenolic compounds and total sugars (TS were investigated during root formation in cuttings of Berberis vulgaris var. asperma (BVA and Berberis thunbergii var. atropurpurea (BTA treated with indole-3-butyric acid (IBA and IBA+H2O2. Rooting was observed on BTA cuttings but not on BVA cuttings. The BTA cuttings treated with IBA and IBA+H2O2 showed higher rooting percentages, number of roots, and root length over the control. Those treated with IBA+H2O2 recorded the lowest peroxidase activity after planting. BTA cuttings treated with IBA+H2O2 showed the highest peroxidase activity at 50 d after planting; BVA cuttings under different treatments showed no significant difference for peroxidase activity at planting time or up to 80 d after planting. PPO activity for the BTA cuttings in the control treatment was lower than for other treatments during root formation. The cuttings in the IBA and IBA+H2O2 treatments showed increased PPO activity from 0 to 50 d after planting and a slight decrease in PPO activity from 60 to 80 d after planting. PPO activity for the BVA cuttings was significantly lower than for BTA during root formation. The BTA cuttings treated with IBA and IBA+H2O2 showed the highest phenolic compound content during root formation. The BVA cuttings displayed higher TS than BTA during the initial stage of root formation. A comparison of the anatomical structure of easy-to-root and difficult-to-root cuttings indicated that physical inhibitors did not affect the rooting capacity of BVA.

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.

Activity of the Antioxidant Defense System in a Typical Bioinsecticide-and Synthetic Insecticide-treated Cowpea Storage Beetle F. (Coleoptera: Chrysomelidae

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Ayodele O. Kolawole

2014-01-01

Full Text Available The non-enzymatic and enzymatic antioxidant defense systems play a major role in detoxification of pro-oxidant endobiotics and xenobiotics. The possible involvement of beetle non-enzymatic [α-tocopherol, glutathione (GSH, and ascorbic acid] and enzymatic [catalase (CAT, superoxide dismutase (SOD, peroxidase (POX, and polyphenol oxidase (PPO] antioxidant defense system on the insecticidal activity of synthetic insecticides (cypermethrin, 2,2-dicholorovinyl dimethyl phosphate, and λ-cyhalothrin and ethanolic plant extracts of Tithonia diversifolia, Cyperus rotundus, Hyptis suaveolens leaves , and Jatropha Curcas seeds was investigated. 2,2-Dicholorovinyl dimethyl phosphate (DDVP; 200 ppm, LC 50 = 13.24 ppm and T. diversifolia (20,000 ppm resulted in 100% beetle mortality at 96-hour post-treatment. The post-treatments significantly increased the beetle α-tocopherol and GSH contents. Activities of CAT, SOD, POX, and PPO were modulated by the synthetic insecticides and bioinsecticides to diminish the adverse effect of the chemical stresses. Quantitative and qualitative allelochemical compositions of bioinsecticides and chemical structure of synthetic insecticides possibly account and for modulation of their respective enzyme activities. Altogether, oxidative stress was enormous enough to cause maladaptation in insects. This study established that oxidative imbalance created could be the molecular basis of the efficacy of both insecticides and bio-insecticides. Two, there was development of functional but inadequate antioxidant defense mechanism in the beetle.

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.

Phospholipid alterations in cardiac sarcoplasmic reticulum induced by xanthine oxidase: contamination of commercial preparations of xanthine oxidase by phospholipase A2

International Nuclear Information System (INIS)

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

1986-01-01

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

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

Effect of naphthalene acetic acid on adventitious root development and associated physiological changes in stem cutting of Hemarthria compressa.

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Yan-Hong Yan

Full Text Available In order to find a way to induce rooting on cuttings of Hemarthria compressa cv. Ya'an under controlled conditions, a project was carried out to study the effect of naphthalene acetic acid (NAA on rooting in stem cuttings and related physiological changes during the rooting process of Hemarthria compressa. The cuttings were treated with five concentrations of NAA (0, 100, 200 300, 400 mg/l at three soaking durations (10, 20, 30 minutes, and cuttings without treatment were considered as control. Samples were planted immediately into pots after treatment. IAA-oxidase (IAAO activity, peroxidase (POD activity and polyphenol oxidase (PPO activity were determined after planting. Results showed that NAA had positive effect on rooting at the concentration of 200 mg/l compared to other concentrations at 30 days after planting (DAP. Among the three soaking durations, 20 minutes (min of 200 mg/l NAA resulted in higher percentages of rooting, larger numbers of adventitious roots and heavier root dry weight per cutting. The lowest IAAO activity was obtained when soaked at 200 mg/l NAA for 20 min soaking duration. This was consistent with the best rooting ability, indicating that the lower IAAO activity, the higher POD activity and PPO activity could be used as an indicator of better rooting ability for whip grass cuttings and might serve as a good marker for rooting ability in cuttings.

Activitie of polyphenoloxidase in yellow and purple onion

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Joeliton Alves Calado

2018-01-01

Full Text Available The high perishability of the onion may limit the shelf life of the bulbs, mainly by the biochemical transformations. The objective of this work is to determine and compare polyphenoloxidase activity (PPO in yellow and purple onions and their functional body. The raw material was purchased in the local market , where they were transported to the Food Analysis Laboratory of the, Center for Agro-Food Science and Technology, Federal University of Campina Grande, Campus Pombal, being selected, cleaned, sorted, peeled processed and carried out chemical and physico- chemical analysis and determination of the enzymatic activity of polyphenol oxidase (PPO. The statistical design was completely randomized with two treatments (purple and yellow onion with six repetitions. The purple onion presented levels of soluble solids, flavonoids and anthocyanins, more significant than the white onion. Both onions showed enzymatic activity, without significant differences.

Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

Science.gov (United States)

Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

2001-01-01

Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

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

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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.

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.

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.

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.

Positive effects of temperature and growth conditions on enzymatic and antioxidant status in lettuce plants.

Science.gov (United States)

Boo, Hee-Ock; Heo, Buk-Gu; Gorinstein, Shela; Chon, Sang-Uk

2011-10-01

The contents of two bioactive compounds (polyphenols and flavonoids) and their antioxidant and enzyme activities were determined in the leaves of six lettuce (Latuca sativa L.) cultivars subjected to 4 different day/night temperatures for 6 weeks. The total polyphenol and anthocyanin contents and the corresponding antioxidant activities were the highest at 13/10°C and 20/13°C, followed by 25/20°C and 30/25°C. The enzymatic activities of polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were also the highest at low day/night temperatures, but the peroxidase (POD) activity was decreased at low day/night temperatures and increased at high day/night temperatures. The most significant positive correlation existed between anthocyanin content and PPO activity, total polyphenols and their antioxidant activities. The results showed that at relatively low temperatures, lettuce plants have a high antioxidant and enzymatic status. These results provide additional information for the lettuce growers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: A reevaluation

Energy Technology Data Exchange (ETDEWEB)

Johnson, J.L.; Rajagopalan, K.V. (Duke Univ. Medical Center, Durham, NC (USA)); London, R.E. (National Institute of Environmental Health Science, Research Triangle Park, NC (USA))

1989-09-01

The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase and glucose oxidase. Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and {sup 31}P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well.

Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: A reevaluation

International Nuclear Information System (INIS)

Johnson, J.L.; Rajagopalan, K.V.; London, R.E.

1989-01-01

The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase and glucose oxidase. Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and 31 P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well

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.

The use of cross-linked chitosan beads for nutrients (nitrate and orthophosphate) removal from a mixture of P-PO4, N-NO2 and N-NO3.

Science.gov (United States)

Jóźwiak, Tomasz; Filipkowska, Urszula; Szymczyk, Paula; Kuczajowska-Zadrożna, Małgorzata; Mielcarek, Artur

2017-11-01

A hydrogel chitosan sorbent ionically cross-linked with sodium citrate and covalently cross-linked with epichlorohydrin was used to remove nutrients from an equimolar mixture of P-PO 4 , N-NO 2 and N-NO 3 . The scope of the study included, among other things, determination of the influence of pH on nutrient sorption effectiveness, nutrient sorption kinetics as well as determination of the maximum sorption capacity of cross-linked chitosan sorbents regarding P-PO 4 (H 2 PO 4 - , HPO 4 2- ), N-NO 2 (HNO 2 , NO 2 - ), and N-NO 3 (NO 3 - ). The effect of the type of the cross-linking agent on the affinity of the modified chitosan to each nutrient was studied as well. The kinetics of nutrient sorption on the tested chitosan sorbents was best described with the pseudo-second order model. The model of intramolecular diffusion showed that P-PO 4 , N-NO 2 and N-NO3 sorption on cross-linked hydrogel chitosan beads proceeded in two phases. The best sorbent of nutrients turned out to be chitosan cross-linked covalently with epichlorohydrin; with P-PO 4 , N-NO 2 and N-NO 3 sorption capacity reaching: 1.23, 0.94 and 0.76mmol/g, respectively (total of 2.92mmol/g). For comparison, the sorption capacity of chitosan cross-linked ionically with sodium citrate was: 0.43, 0.39 and 0.39mmol/g for P-PO 4 , N-NO 2 and N-NO 3 , respectively (total of 1.21mmol/g). Copyright © 2017 Elsevier B.V. All rights reserved.

Assays of D-Amino Acid Oxidase Activity

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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.

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.

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.

A multidisciplinary approach providing new insight into fruit flesh browning physiology in apple (Malus x domestica Borkh.).

Science.gov (United States)

Di Guardo, Mario; Tadiello, Alice; Farneti, Brian; Lorenz, Giorgia; Masuero, Domenico; Vrhovsek, Urska; Costa, Guglielmo; Velasco, Riccardo; Costa, Fabrizio

2013-01-01

In terms of the quality of minimally processed fruit, flesh browning is fundamentally important in the development of an aesthetically unpleasant appearance, with consequent off-flavours. The development of browning depends on the enzymatic action of the polyphenol oxidase (PPO). In the 'Golden Delicious' apple genome ten PPO genes were initially identified and located on three main chromosomes (2, 5 and 10). Of these genes, one element in particular, here called Md-PPO, located on chromosome 10, was further investigated and genetically mapped in two apple progenies ('Fuji x Pink Lady' and 'Golden Delicious x Braeburn'). Both linkage maps, made up of 481 and 608 markers respectively, were then employed to find QTL regions associated with fruit flesh browning, allowing the detection of 25 QTLs related to several browning parameters. These were distributed over six linkage groups with LOD values spanning from 3.08 to 4.99 and showed a rate of phenotypic variance from 26.1 to 38.6%. Anchoring of these intervals to the apple genome led to the identification of several genes involved in polyphenol synthesis and cell wall metabolism. Finally, the expression profile of two specific candidate genes, up and downstream of the polyphenolic pathway, namely phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), provided insight into flesh browning physiology. Md-PPO was further analyzed and two haplotypes were characterised and associated with fruit flesh browning in apple.

A Multidisciplinary Approach Providing New Insight into Fruit Flesh Browning Physiology in Apple (Malus x domestica Borkh.)

Science.gov (United States)

Farneti, Brian; Lorenz, Giorgia; Masuero, Domenico; Vrhovsek, Urska; Costa, Guglielmo; Velasco, Riccardo; Costa, Fabrizio

2013-01-01

In terms of the quality of minimally processed fruit, flesh browning is fundamentally important in the development of an aesthetically unpleasant appearance, with consequent off-flavours. The development of browning depends on the enzymatic action of the polyphenol oxidase (PPO). In the ‘Golden Delicious’ apple genome ten PPO genes were initially identified and located on three main chromosomes (2, 5 and 10). Of these genes, one element in particular, here called Md-PPO, located on chromosome 10, was further investigated and genetically mapped in two apple progenies (‘Fuji x Pink Lady’ and ‘Golden Delicious x Braeburn’). Both linkage maps, made up of 481 and 608 markers respectively, were then employed to find QTL regions associated with fruit flesh browning, allowing the detection of 25 QTLs related to several browning parameters. These were distributed over six linkage groups with LOD values spanning from 3.08 to 4.99 and showed a rate of phenotypic variance from 26.1 to 38.6%. Anchoring of these intervals to the apple genome led to the identification of several genes involved in polyphenol synthesis and cell wall metabolism. Finally, the expression profile of two specific candidate genes, up and downstream of the polyphenolic pathway, namely phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), provided insight into flesh browning physiology. Md-PPO was further analyzed and two haplotypes were characterised and associated with fruit flesh browning in apple. PMID:24205065

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.

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.

Molecular Basis for Converting (2S-Methylsuccinyl-CoA Dehydrogenase into an Oxidase

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Simon Burgener

2017-12-01

Full Text Available Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases.

Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability.

Science.gov (United States)

Vuong, Thu V; Foumani, Maryam; MacCormick, Benjamin; Kwan, Rachel; Master, Emma R

2016-11-21

Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H 2 O 2 . By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H 2 O 2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H 2 O 2 , the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H 2 O 2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.

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, ...

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.

Effect of Infrared Blanching on Enzyme Activity and Retention of β-Carotene and Vitamin C in Dried Mango.

Science.gov (United States)

Guiamba, Isabel R F; Svanberg, Ulf; Ahrné, Lilia

2015-06-01

The objectives of this work were to evaluate infrared (IR) dry blanching in comparison with conventional water blanching prior to hot air drying of mango to inactivate polyphenol oxidase (PPO) and ascorbic acid oxidase (AAO) enzymes, and to study its effect on color change and retention of vitamin C and β-carotene. Mango cylinders were blanched under similar temperature-time conditions either by IR heating or by immersion in a water bath during 2 min at 90 °C (high-temperature-short-time-HTST) or for 10 min at 65 °C (low-temperature-long-time-LTLT). After blanching mango was hot air dried at 70 °C. PPO was completely inactivated during the blanching treatments, but AAO had a moderate remaining activity after LTLT treatment (∼30%) and a low remaining activity after HTST treatment (9% to 15%). A higher retention of vitamin C was observed in mango subjected to IR dry blanching, 88.3 ± 1.0% (HTST) and 69.2 ± 2.9% (LTLT), compared with water blanching, 61.4 ± 5.3% (HTST) and 50.7 ± 9.6% (LTLT). All-trans-β-carotene retention was significantly higher in water blanched dried mango, 93.2 ± 5.2% (LTLT) and 91.4 ± 5.1% (HTST), compared with IR dry blanched, 73.6 ± 3.6% (LTLT) and 76.9 ± 2.9% (HTST). Increased levels of 13-cis-β-carotene isomer were detected only in IR dry blanched mango, and the corresponding dried mango also had a slightly darker color. IR blanching of mango prior to drying can improve the retention of vitamin C, but not the retention of carotenoids, which showed to be more dependent on the temperature than the blanching process. A reduction of drying time was observed in LTLT IR-blanching mango. © 2015 Institute of Food Technologists®

Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

Energy Technology Data Exchange (ETDEWEB)

Saad, Fawzy A. [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Torres, Marie [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Wang, Hao [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Graham, Lila, E-mail: lilagraham@cs.com [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)

2010-06-11

LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

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.

Structure–Activity Relationship of Xanthones as Inhibitors of Xanthine Oxidase

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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.

Enzymatic browning in avocado (Persea americana) revisited: History, advances, and future perspectives.

Science.gov (United States)

Toledo, Lea; Aguirre, Carolina

2017-12-12

Considering nearly 80 years of research regarding one of the enzymes responsible for catalyzing the formation of pigments in higher animals, plants, fungi and bacteria, this review will focus on collecting and categorizing the existing information about polyphenol oxidase (PPO) in fruits, with particular emphasis on the information in relation to avocado, which is one of the hardiest species in terms of inactivation, has documented dual activity (EC 1.14.18.1/EC 1.10.3.1), and represents one of the oldest challenges for food science research and fruit processors. It is expected that this review will contribute to the further development of the field by highlighting the questions that have arisen during the characterization of PPO, the progress that has been made and the questions that remain today, in addition to new methodologies that are being applied to study this system. Holistic methodologies offer unexplored potential for advancing our understanding of the complex phenomena that govern PPO activity in fruits, because these methodologies will enable the characterization of this family of enzymes in all of its complexity. Subsequently, it will be possible to develop better techniques for controlling enzymatic browning in this valuable fruit.

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.

Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications.

Science.gov (United States)

Barlow, J N; Zhang, Z; John, P; Baldwin, J E; Schofield, C J

1997-03-25

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final step in the biosynthesis of the plant signaling molecule ethylene. It is a member of the ferrous iron dependent family of oxidases and dioxygenases and is unusual in that it displays a very short half-life under catalytic conditions, typically less than 20 min, and a requirement for CO2 as an activator. The rates of inactivation of purified, recombinant ACC oxidase from tomato under various combinations of substrates and cofactors were measured. Inactivation was relatively slow in the presence of buffer alone (t1/2 > 1 h), but fast in the presence of ferrous iron and ascorbate (t1/2 approximately 10 min). The rate of iron/ascorbate-mediated inactivation was increased by the addition of ACC, unaffected by the addition of CO2 at saturation (supplied as bicarbonate) but decreased by the addition of catalase or ACC + CO2 at saturation (supplied as bicarbonate). Iron/ascorbate-mediated inactivation was accompanied by partial proteolysis as observed by SDS-PAGE analysis. The fragmentation pattern was altered when ACC was also included, suggesting that ACC can bind to ACC oxidase in the absence of bicarbonate. N-terminal sequencing of fragments resulted in identification of an internal cleavage site which we propose is proximate to active-site bound iron. Thus, ACC oxidase inactivates via relatively slow partial unfolding of the catalytically active conformation, oxidative damage mediated via hydrogen peroxide which is catalase protectable and oxidative damage to the active site which results in partial proteolysis and is not catalase protectable.

Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

International Nuclear Information System (INIS)

Pan, Heng-Yen; Whittaker, Mei M.; Bouveret, Romaric; Berna, Anne; Bernier, Francois; Whittaker, James W.

2007-01-01

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an α-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4 x 10 4 U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions

Herbivore Oral Secreted Bacteria Trigger Distinct Defense Responses in Preferred and Non-Preferred Host Plants.

Science.gov (United States)

Wang, Jie; Chung, Seung Ho; Peiffer, Michelle; Rosa, Cristina; Hoover, Kelli; Zeng, Rensen; Felton, Gary W

2016-06-01

Insect symbiotic bacteria affect host physiology and mediate plant-insect interactions, yet there are few clear examples of symbiotic bacteria regulating defense responses in different host plants. We hypothesized that plants would induce distinct defense responses to herbivore- associated bacteria. We evaluated whether preferred hosts (horsenettle) or non-preferred hosts (tomato) respond similarly to oral secretions (OS) from the false potato beetle (FPB, Leptinotarsa juncta), and whether the induced defense triggered by OS was due to the presence of symbiotic bacteria in OS. Both horsenettle and tomato damaged by antibiotic (AB) treated larvae showed higher polyphenol oxidase (PPO) activity than those damaged by non-AB treated larvae. In addition, application of OS from AB treated larvae induced higher PPO activity compared with OS from non-AB treated larvae or water treatment. False potato beetles harbor bacteria that may provide abundant cues that can be recognized by plants and thus mediate corresponding defense responses. Among all tested bacterial isolates, the genera Pantoea, Acinetobacter, Enterobacter, and Serratia were found to suppress PPO activity in tomato, while only Pantoea sp. among these four isolates was observed to suppress PPO activity in horsenettle. The distinct PPO suppression caused by symbiotic bacteria in different plants was similar to the pattern of induced defense-related gene expression. Pantoea inoculated FPB suppressed JA-responsive genes and triggered a SA-responsive gene in both tomato and horsenettle. However, Enterobacter inoculated FPB eliminated JA-regulated gene expression and elevated SA-regulated gene expression in tomato, but did not show evident effects on the expression levels of horsenettle defense-related genes. These results indicate that suppression of plant defenses by the bacteria found in the oral secretions of herbivores may be a more widespread phenomenon than previously indicated.

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

Effect of temperature on enzymatic and physiological factors related to chilling injury in carambola fruit (Averrhoa carambola L.).

Science.gov (United States)

Pérez-Tello, G O; Silva-Espinoza, B A; Vargas-Arispuro, I; Briceño-Torres, B O; Martinez-Tellez, M A

2001-10-05

Three groups of carambola fruits (Averrhoa carambola L.) were stored at 2 and 10 degrees C (85-90% relative humidity). The major physicochemical, physiological, and enzymatic responses of fruit were measured in each group over a 30-day period: chilling injury index (CII), decay (%), intracuticular waxes, cuticle permeability, pulp firmness, weight loss, sucrose, fructose and glucose contents, ion electrolyte leakage in pulp (%), ethylene and carbon dioxide production rates, and the activities of peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) enzymes. CII values were statistically different at 2 and 10 degrees C, showing high significance with respect to sucrose content and weight loss (P < 0.05). Chilling injury included darkened ribs and skin desiccation. According to the CI symptom development, a possible relationship of POD and PPO activities was found at 2 degrees C. A significant sucrose content increase was observed at 10 degrees C. CI symptoms were associated with POD and PAL activities. Copyright 2001 Academic Press.

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.

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.

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

How arbuscular mycorrhizal fungi influence the defense system of sunflower during different abiotic stresses.

Science.gov (United States)

Mayer, Zoltán; Duc, Nguyen Hong; Sasvári, Zita; Posta, Katalin

2017-12-01

The association between terrestrial plants and arbuscular mycorrhizal (AM) fungi is one of the most common and widespread mutualistic plant-fungi interaction. AM fungi are of beneficial effects on the water and nutrient uptake of plants and increase plant defense mechanisms to alleviate different stresses. The aim of this study was to determine the level of polyphenol oxidase (PPO), guaiacol peroxidase (POX) and glutathione S-transferase (GST) enzyme activities and to track the expression of glutathione S-transferase (GST) gene in plant-arbuscular mycorrhizal system under temperature- and mechanical stress conditions. Our results suggest that induced tolerance of mycorrhizal sunflower to high temperature may be attributed to the induction of GST, POX and PPO enzyme activities as well as to the elevated expression of GST. However, the degree of tolerance of the plant is significantly influenced by the age which is probably justified by the energy considerations.

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.

Biphenyl Modulates the Expression and Function of Respiratory Oxidases in the Polychlorinated-Biphenyls Degrader Pseudomonas pseudoalcaligenes KF707

Directory of Open Access Journals (Sweden)

Federica Sandri

2017-06-01

Full Text Available Pseudomonas pseudoalcaligenes KF707 is a soil bacterium which is known for its capacity to aerobically degrade harmful organic compounds such as polychlorinated biphenyls (PCBs using biphenyl as co-metabolite. Here we provide the first genetic and functional analysis of the KF707 respiratory terminal oxidases in cells grown with two different carbon sources: glucose and biphenyl. We identified five terminal oxidases in KF707: two c(caa3 type oxidases (Caa3 and Ccaa3, two cbb3 type oxidases (Cbb31 and Cbb32, and one bd type cyanide-insensitive quinol oxidase (CIO. While the activity and expression of both Cbb31 and Cbb32 oxidases was prevalent in glucose grown cells as compared to the other oxidases, the activity and expression of the Caa3 oxidase increased considerably only when biphenyl was used as carbon source in contrast to the Cbb32 oxidase which was repressed. Further, the respiratory activity and expression of CIO was up-regulated in a Cbb31 deletion strain as compared to W.T. whereas the CIO up-regulation was not present in Cbb32 and C(caa3 deletion mutants. These results, together, reveal that both function and expression of cbb3 and caa3 type oxidases in KF707 are modulated by biphenyl which is the co-metabolite needed for the activation of the PCBs-degradation pathway.

Blockade of TGF-β 1 Signalling Inhibits Cardiac NADPH Oxidase Overactivity in Hypertensive Rats

Directory of Open Access Journals (Sweden)

José Luis Miguel-Carrasco

2012-01-01

Full Text Available NADPH oxidases constitute a major source of superoxide anion (⋅O2 - in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-β 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-β 1 receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-β 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-β 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-β 1.

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.

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.

Allelic variations of functional markers for polyphenol oxidase (PPO)

Indian Academy of Sciences (India)

genes in Indian bread wheat (Triticum aestivum L.) cultivars. Rajender Singh, Umesh Goutam, R. K. Gupta, G. C. Pandey, Jag Shoran and Ratan Tiwari. J. Genet. 88, 325–329. Figure 1. Phenol colour reaction of kernels. Kernels without treatment by phenol solution are shown as controls. Phenol colour reaction of kernels; ...

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.

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.

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 ...

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

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)).

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

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

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

Acid-Labile Amphiphilic PEO-b-PPO-b-PEO Copolymers: Degradable Poloxamer Analogs.

Science.gov (United States)

Worm, Matthias; Kang, Biao; Dingels, Carsten; Wurm, Frederik R; Frey, Holger

2016-05-01

Poly ((ethylene oxide)-b-(propylene oxide)-b-(ethylene oxide)) triblock copolymers commonly known as poloxamers or Pluronics constitute an important class of nonionic, biocompatible surfactants. Here, a method is reported to incorporate two acid-labile acetal moieties in the backbone of poloxamers to generate acid-cleavable nonionic surfactants. Poly(propylene oxide) is functionalized by means of an acetate-protected vinyl ether to introduce acetal units. Three cleavable PEO-PPO-PEO triblock copolymers (Mn,total = 6600, 8000, 9150 g·mol(-1) ; Mn,PEO = 2200, 3600, 4750 g·mol(-1) ) have been synthesized using anionic ring-opening polymerization. The amphiphilic copolymers exhibit narrow molecular weight distributions (Ð = 1.06-1.08). Surface tension measurements reveal surface-active behavior in aqueous solution comparable to established noncleavable poloxamers. Complete hydrolysis of the labile junctions after acidic treatment is verified by size exclusion chromatography. The block copolymers have been employed as surfactants in a miniemulsion polymerization to generate polystyrene (PS) nanoparticles with mean diameters of ≈200 nm and narrow size distribution, as determined by dynamic light scattering and scanning electron microscopy. Acid-triggered precipitation facilitates removal of surfactant fragments from the nanoparticles, which simplifies purification and enables nanoparticle precipitation "on demand." © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Oxidative enzymes activity in sugarcane juice as a function of the planting system

Directory of Open Access Journals (Sweden)

Tadeu Alcides Marques

2013-03-01

Full Text Available In Brazil, the largest producer of sugarcane in the world, the industrial process transforms this crop into ethanol and/or granulated sugar. Some cultivars exhibit enzymatic browning in the extracted sugarcane juice at levels harmful to the manufacturing process of white granulated sugar. The objective of this study was to assess the effect of sugarcane straw used as soil coverage, the use of different planting systems, and treatments with hydrogel polymer on enzymatic activity. The cultivar RB 86 7515 was sampled for 8 months; the first sample was obtained by cutting the upper portion of the stalk at the internode, which was taken to the laboratory for determination of the enzymatic activity of polyphenoloxidase (PPO and peroxidase (POD. The soil coverage with different forms of straw as well as the planting systems did not change the enzymatic activity of polyphenoloxidase (PPO and peroxidase (POD. The polyphenoloxidase (PPO activity increased with the use of a polymer due to increased polyphenoloxidase (PPO activity in the groove system. The enzymes studied showed changes in activity during the experimental period. The production of sugar at the end of the season (August to November avoids the periods of highest enzymatic activity.

Peroxisomal Polyamine Oxidase and NADPH-Oxidase cross-talk for ROS homeostasis which affects respiration rate in Arabidopsis thaliana

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Efthimios A. Andronis

2014-04-01

Full Text Available Homeostasis of reactive oxygen species (ROS in the intracellular compartments is of critical importance as ROS have been linked with nearly all cellular processes and more importantly with diseases and aging. PAs are nitrogenous molecules with an evolutionary conserved role in the regulation of metabolic and energetic status of cells. Recent evidence also suggests that polyamines (PA are major regulators of ROS homeostasis. In Arabidopsis the backconversion of the PAs spermidine (Spd and spermine (Spm to putrescine (Put and Spd, respectively is catalyzed by two peroxisomal PA oxidases (AtPAO. However, the physiological role of this pathway remains largely elusive. Here we explore the role of peroxisomal PA backconversion and in particular that catalyzed by the highly expressed AtPAO3 in the regulation of ROS homeostasis and mitochondrial respiratory burst. Exogenous PAs exert an NADPH-oxidase dependent stimulation of oxygen consumption, with Spd exerting the strongest effect. This increase is attenuated by treatment with the NADPH-oxidase blocker diphenyleneiodonium iodide (DPI. Loss-of-function of AtPAO3 gene results to increased NADPH-oxidase-dependent production of superoxide anions (O2.-, but not H2O2, which activate the mitochondrial alternative oxidase pathway (AOX. On the contrary, overexpression of AtPAO3 results to an increased but balanced production of both H2O2 and O2.-. These results suggest that the ratio of O2.-/H2O2 regulates respiratory chain in mitochondria, with PA-dependent production of O2.- by NADPH-oxidase tilting the balance of electron transfer chain in favor of the AOX pathway. In addition, AtPAO3 seems to be an important component in the regulating module of ROS homeostasis, while a conserved role for PA backconversion and ROS across kingdoms is discussed.

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...

Surface reconstitution of glucose oxidase onto a norbornylogous bridge self-assembled monolayer

International Nuclear Information System (INIS)

Liu Jingquan; Paddon-Row, Michael N.; Gooding, J. Justin

2006-01-01

An electrode construct was fabricated in which a self-assembled monolayer containing a novel norbornylogous bridge was covalently attached to flavin adenine dinucleotide (FAD), the redox active centre of several oxidase enzymes. The electrochemistry of the construct was investigated before and after the reconstitution of glucose oxidase around the surface bound FAD. Rapid rates of electron transfer were observed both before and after the reconstitution of biocatalytically active enzyme. However, no biocatalytic activity was observed under anaerobic conditions suggesting the a lack of enzyme turnover through direct electron transfer. It is proposed that a decrease in the electronic coupling between the redox active FAD and the electrode following reconstitution of the glucose oxidase - a probable consequence of the FAD being immersed in a protein environment - was responsible for the inability of the enzyme to be turned over under anaerobic conditions

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.

Effect of halide salts on development of surface browning on fresh-cut 'Granny Smith' (Malus × domestica Borkh) apple slices during storage at low temperature.

Science.gov (United States)

Li, Yongxin; Wills, Ron B H; Golding, John B; Huque, Roksana

2015-03-30

The postharvest life of fresh-cut apple slices is limited by browning on cut surfaces. Dipping in halide salt solutions was examined for their inhibition of surface browning on 'Granny Smith' apple slices and the effects on biochemical factors associated with browning. Delay in browning by salts was greatest with chloride = phosphate > sulfate > nitrate with no difference between sodium, potassium and calcium ions. The effectiveness of sodium halides on browning was fluoride > chloride = bromide > iodide = control. Polyphenol oxidase (PPO) activity of tissue extracted from chloride- and fluoride-treated slices was not different to control but when added into the assay solution, NaF > NaCl both showed lower PPO activity at pH 3-5 compared to control buffer. The level of polyphenols in treated slices was NaF > NaCl > control. Addition of chlorogenic acid to slices enhanced browning but NaCl and NaF counteracted this effect. There was no effect of either halide salt on respiration, ethylene production, ion leakage, and antioxidant activity. Dipping apple slices in NaCl is a low cost treatment with few impediments to commercial use and could be a replacement for other anti-browning additives. The mode of action of NaCl and NaF is through decreasing PPO activity resulting in reduced oxidation of polyphenols. © 2014 Society of Chemical Industry.

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.

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.

Role of salicylic acid in induction of plant defense system in chickpea (Cicer arietinum L.).

Science.gov (United States)

War, Abdul Rashid; Paulraj, Michael Gabriel; War, Mohd Yousf; Ignacimuthu, Savarimuthu

2011-11-01

Salicylic acid (SA), a plant hormone plays an important role in induction of plant defense against a variety of biotic and abiotic stresses through morphological, physiological and biochemical mechanisms. A series of experiments were carried out to evaluate the biochemical response of the chickpea (Cicer arietinum L.) plants to a range of SA concentrations (1, 1.5, and 2 mM). Water treated plants were maintained as control. Activities of peroxidase (POD) and polyphenol oxidase (PPO) were evaluated and amounts of total phenols, hydrogen peroxide (H2O2), and proteins were calculated after 96 h of treatment. Plants responded very quickly to SA at 1.5 mM and showed higher induction of POD and PPO activities, besides the higher accumulation of phenols, H2O2 and proteins. Plants treated with SA at 2 mM showed phytotoxic symptoms. These results suggest that SA at 1.5 mM is safe to these plants and could be utilized for the induction of plant defense.

Effect of irradiation on physiological and biochemical properties of Bt rice seedlings

International Nuclear Information System (INIS)

Wang Zhonghua; Chen Xiaojian; Bao Xusheng; Chen Yuling; Gu Qinqin

2011-01-01

The seeds of two varieties of Bt rice were treated by 60 Co γ-rays at the doses of 50, 100, 150, 250 and 350 Gy, respectively, their original parent was used as control material. The seedlings cultured from above seeds were used to detect the root activity, seedling growth, chlorophyll content,activities of phenylalanine ammonialyase (PAL), polyphenol oxidase (PPO), catalase(CAT), superoxide dismutase (SOD) and amylase to investigate the effect of irradiation treatment on the physiological and biochemical properties of Bt rice. The results showed that root activity, chlorophyll content, activities of PAL, PPO, CAT, SOD of Bt rice seedlings and amylase of germinating seeds were lower than those of the control group after irradiation treatment of < 250 Gy, but the differences were not significant, which was similar to those of original parent. Meanwhile, it was found that with dose increasing, the seedling height was increased, suggesting that irradiation treatment could stimulate the seedling growth. Therefore, Bt transgene can not change the irradiation sensitivity of rice and the conventional method of rice can be used in Bt rice irradiation mutation breeding. (authors)

Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase.

Science.gov (United States)

Thrower, J S; Blalock, R; Klinman, J P

2001-08-14

1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.

Functional expression of amine oxidase from Aspergillus niger (AO-I) in Saccharomyces cerevisiae.

Science.gov (United States)

Kolaríková, Katerina; Galuszka, Petr; Sedlárová, Iva; Sebela, Marek; Frébort, Ivo

2009-01-01

The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.

Characterization of Two VAO-Type Flavoprotein Oxidases from Myceliophthora thermophila

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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.

Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

Science.gov (United States)

Souabni, Hajer; Ezzine, Aymen; Bizouarn, Tania; Baciou, Laura

2017-01-01

Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b 558 (Cytb 558 ), constituted by the tight association of the gp91 phox (also named Nox2) and p22 phox proteins. The Cytb 558 is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47 phox , p67 phox , p40 phox , Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.

Identification and statistical optimization of fermentation conditions for a newly isolated extracellular cholesterol oxidase-producing Streptomyces cavourensis strain NEAE-42

OpenAIRE

El-Naggar, Noura El-Ahmady; El-Shweihy, Nancy M.; El-Ewasy, Sara M.

2016-01-01

Background Due to broad range of clinical and industrial applications of cholesterol oxidase, isolation and screening of bacterial strains producing extracellular form of cholesterol oxidase is of great importance. Results One hundred and thirty actinomycete isolates were screened for their cholesterol oxidase activity. Among them, a potential culture, strain NEAE-42 is displayed the highest extracellular cholesterol oxidase activity. It was selected and identified as Streptomyces cavourensis...

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.

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...

Cytochrome oxidase as an indicator of ice storage and frozen storage

DEFF Research Database (Denmark)

Godiksen, Helene; Jessen, Flemming

2001-01-01

in different cods was 21%, and the coefficient of variation of different analyses on the same homogenate was 5%. It was shown that ice storage of muscle samples before they were frozen and thawed resulted in a major freezing-induced activation of cytochrome oxidase activity. The enzyme may therefore be used...... as an indicator of frozen fish to determine if the fish has been stored on ice before freezing. Cytochrome oxidase activity showed also potential as an indicator of frozen storage, as it was possible to distinguish between the frozen storage temperatures -9, -20, and -40 degreesC....

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

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

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

Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

Directory of Open Access Journals (Sweden)

Sluse F.E.

1998-01-01

Full Text Available Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression or short-term (post-translational modification, allosteric activation regulated. Electron distribution (partitioning between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach. Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon and with harmful reactive oxygen species formation.

IMPACT OF FOLIAR APPLICATION OF ASCORBIC ACID AND α-TOCOPHEROL ON ANTIOXIDANT ACTIVITY AND SOME BIOCHEMICAL ASPECTS OF FLAX CULTIVARS UNDER SALINITY STRESS

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Hala M.S. El-Bassiouny

2015-05-01

Full Text Available ABSTRACT The interactive effects of saline water (2000, 4000 and 6000 mg/l and foliar application of 400 mg/l of ascorbic acid (Asc or α – tocopherol (α-Toco on three flax cultivars (Sakha 3, Giza 8 and Ariane were conducted during two successive seasons (2011 and 2012. The results showed that, total soluble carbohydrates, free amino acids, proline contents were significantly increased with increasing salinity levels in all three tested cultivars except free amino acid content of Giza 8 which showed a non significant decrease. While, nucleic acids (DNA and RNA showed significant decreases compared with the corresponding controls. Moreover, applications of vitamins (Asc or α-Toco as foliar spraying increased all mentioned contents compared to the corresponding salinity levels. On the other hand, lipid peroxidation, and activity levels of polyphenol oxidase (PPO, peroxidase (POX and catalase (CAT enzymes showed progressive significant increases with increasing salinity levels of all tested three cultivars, while the behaviour of superoxide dismutase (SOD activity showed an opposite response as compared with the control in Sakha 3 and Giza 8. Treatments with Asc or α-Toco induced significant reduction in lipid peroxidation and activities of PPO and POX of all three tested cultivars. Meanwhile, SOD increased in all three cultivars, and CAT activities increased only in Sakha 3 cultivar under salt stress as compared with reference controls. Some modifications are observed in protein patterns hence some proteins were disappeared, while certain other proteins were selectively increased and synthesis of a new set of proteins were induced, some of these responses were observed under treatments and salinity, while others were induced by either treatments or salinity.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases

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Tom A. Ewing

2018-01-01

Full Text Available Vanillyl alcohol oxidase (VAO and eugenol oxidase (EUGO are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol and a EUGO variant (V436I with increased activity towards chavicol (4-allylphenol and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

Science.gov (United States)

Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

2018-01-14

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen

International Nuclear Information System (INIS)

Pasqualini, Stefania; Tedeschini, Emma; Frenguelli, Giuseppe; Wopfner, Nicole; Ferreira, Fatima; D'Amato, Gennaro; Ederli, Luisa

2011-01-01

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O 3 ) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O 3 fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O 3 fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O 3 , determined from the mRNA levels of the major allergens. We conclude that O 3 can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. - Highlights: → O 3 reduces the viability of ragweed pollen. → ROS and allergens of ragweed pollen were not affected by O 3 exposure. → O 3 enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. → O 3 increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation. - This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.

Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen

Energy Technology Data Exchange (ETDEWEB)

Pasqualini, Stefania, E-mail: spas@unipg.it [Department of Applied Biology, University of Perugia, Perugia (Italy); Tedeschini, Emma; Frenguelli, Giuseppe [Department of Applied Biology, University of Perugia, Perugia (Italy); Wopfner, Nicole; Ferreira, Fatima [Department of Molecular Biology, CD Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg (Austria); D' Amato, Gennaro [Division of Respiratory and Allergic Diseases, ' A. Cardarelli' High Speciality Hospital, Naples (Italy); Ederli, Luisa [Department of Applied Biology, University of Perugia, Perugia (Italy)

2011-10-15

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O{sub 3}) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O{sub 3} fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O{sub 3} fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O{sub 3}, determined from the mRNA levels of the major allergens. We conclude that O{sub 3} can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase. - Highlights: > O{sub 3} reduces the viability of ragweed pollen. > ROS and allergens of ragweed pollen were not affected by O{sub 3} exposure. > O{sub 3} enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. > O{sub 3} increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation. - This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.

Biosynthesis of hydroxycinnamoyl esters and amides in legume species

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In forage crops, protein that is degraded following harvest is poorly utilized by ruminant animals, resulting in both economic and environmental consequences. In red clover, secondary reactions of quinones resulting from polyphenol oxidase (PPO)-mediated oxidation of the caffeic acid derivatives pha...

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

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)

Xanthine oxidase activity regulates human embryonic brain cells growth

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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.

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...

Multi-Copper Oxidases and Human Iron Metabolism

Science.gov (United States)

Vashchenko, Ganna; MacGillivray, Ross T. A.

2013-01-01

Multi-copper oxidases (MCOs) are a small group of enzymes that oxidize their substrate with the concomitant reduction of dioxygen to two water molecules. Generally, multi-copper oxidases are promiscuous with regards to their reducing substrates and are capable of performing various functions in different species. To date, three multi-copper oxidases have been detected in humans—ceruloplasmin, hephaestin and zyklopen. Each of these enzymes has a high specificity towards iron with the resulting ferroxidase activity being associated with ferroportin, the only known iron exporter protein in humans. Ferroportin exports iron as Fe2+, but transferrin, the major iron transporter protein of blood, can bind only Fe3+ effectively. Iron oxidation in enterocytes is mediated mainly by hephaestin thus allowing dietary iron to enter the bloodstream. Zyklopen is involved in iron efflux from placental trophoblasts during iron transfer from mother to fetus. Release of iron from the liver relies on ferroportin and the ferroxidase activity of ceruloplasmin which is found in blood in a soluble form. Ceruloplasmin, hephaestin and zyklopen show distinctive expression patterns and have unique mechanisms for regulating their expression. These features of human multi-copper ferroxidases can serve as a basis for the precise control of iron efflux in different tissues. In this manuscript, we review the biochemical and biological properties of the three human MCOs and discuss their potential roles in human iron homeostasis. PMID:23807651

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

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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.

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

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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.

Effects of a high O2 dynamic-controlled atmosphere technology on the browning of postharvest white mushroom ( Agaricus bisporus) in relation to energy metabolism.

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Li, Ling; Sun, Han; Kitazawa, Hiroaki; Wang, Xiangyou

2017-07-01

Browning is one of the main problems in senescence of mushrooms, and it is also one of the most important attributes accounting for the loss of the quality and reduction in market value. In order to study the relationship between the energy metabolism and the browning of white mushroom under high O 2 dynamic-controlled atmosphere (HO-DCA), mushrooms were stored in 100% O 2 (SCA1), 80% O 2  + 20% CO 2 (SCA2), 100% O 2 for three days and then transferred into the treatment of 80% O 2  + 20% CO 2 (HO-DCA) at 2 ± 1 ℃ and air as control. In this study, adenosine triphosphate (ATP) content, energy charge level, sensory evaluation, browning of surface and flesh, cell membrane integrity, exogenous ATP, polyphenol oxidase (PPO) and peroxidase (POD) activity and genes encoding PPO of the white mushroom were investigated. These were all closely related to the browning of products. The optimal storage condition of the HO-DCA treatment could delay the browning of pericarp and flesh tissues of the mushrooms, inhibit PPO activity and reduce the relative expression levels of the three genes encoding PPO. Meanwhile, it maintained moderate POD activity, good sensory properties and cell membrane integrity in a certain extent and thus slowed down the senescence of mushrooms. Results indicated that there was a positive correlation between the ATP content and whitening index ( r = 0.901). In addition, HO-DCA maintained a higher ATP level, prolonged the storage time to 28 days and it might be an ideal strategy for preserving the quality of mushroom during storage.

in Escherichia coli with native cholesterol oxidase expressed

African Journals Online (AJOL)

The structure and bio-activity of an endogenous cholesterol oxidase from Brevibacterium sp. was compared to the same enzyme exogenously expressed in Escherichia coli BL21 (DE3) with and without N- or C-terminal his-tags. The different proteins were purified with affinity and subtractive protocols. The specific activity of ...

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

Moulded interconnect device fabrication by two shot molding and lasert induced selective activation

DEFF Research Database (Denmark)

Sun, Jie; Hansen, Hans Nørgaard

material combinations such as PEI (GE Ultem 1000) +PPO (GTX 810) and PEEK (Victrex 150GL30) +PPO (GTX 810) were investgated which can be selected electroless plating for metallization. Several plastics such as PC (GE Lexan 500R) and PEEK (Victrex 150GL30) were applied to the laser induced activation...

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

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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.

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.

Biofilm formed from organic-inorganic hybrid tri-ureasil PPO for transdermal drug delivery system

Energy Technology Data Exchange (ETDEWEB)

Molina, Eduardo F.; Jesus, Natana Aparecida; Oliveira, Pollyana Francielli; Furtado, Ricardo A.; Tavares, Denise Crispim, E-mail: eduardo.molina@unifran.edu.br [Universidade de Franca (UNIFRAN), SP (Brazil)

2016-07-01

Full text: In this work we evaluated the viability of the tri-ureasil PPO hybrid as biofilm forming for release of active substances such as lignans. The samples were characterized by X-ray diffraction (XRD) and infrared (FTIR). The swelling degree and the influence of the catalyst on time of formation of a hybrid biofilm were evaluated. The cytotoxicity of the materials were evaluated using the XTT colorimetric assay where GM07492A strain was treated with different concentrations of the hybrid. The time of film formation depends on the quantity of the catalyst used in the synthesis. By varying the catalyst quantity during the synthesis, a good flexible film can be obtained, which is easy to be coated on the skin surface and in situ formed a very thin and comfortable film with an aesthetical appearance. Moreover, the hybrid films were colorless and transparent. The toxicity/viability of all samples has also been studied using normal human cells for future applications. The hybrid matrices did not significantly reduce cell viability, demonstrating that siloxane-polyether materials were biocompatible. All the materials presenting a amorphous structure (XRD) and the characteristic bands of vibrations (FTIR) of the polymer chain do not change after incorporation of lignans. (author)

Biofilm formed from organic-inorganic hybrid tri-ureasil PPO for transdermal drug delivery system

International Nuclear Information System (INIS)

Molina, Eduardo F.; Jesus, Natana Aparecida; Oliveira, Pollyana Francielli; Furtado, Ricardo A.; Tavares, Denise Crispim

2016-01-01

Full text: In this work we evaluated the viability of the tri-ureasil PPO hybrid as biofilm forming for release of active substances such as lignans. The samples were characterized by X-ray diffraction (XRD) and infrared (FTIR). The swelling degree and the influence of the catalyst on time of formation of a hybrid biofilm were evaluated. The cytotoxicity of the materials were evaluated using the XTT colorimetric assay where GM07492A strain was treated with different concentrations of the hybrid. The time of film formation depends on the quantity of the catalyst used in the synthesis. By varying the catalyst quantity during the synthesis, a good flexible film can be obtained, which is easy to be coated on the skin surface and in situ formed a very thin and comfortable film with an aesthetical appearance. Moreover, the hybrid films were colorless and transparent. The toxicity/viability of all samples has also been studied using normal human cells for future applications. The hybrid matrices did not significantly reduce cell viability, demonstrating that siloxane-polyether materials were biocompatible. All the materials presenting a amorphous structure (XRD) and the characteristic bands of vibrations (FTIR) of the polymer chain do not change after incorporation of lignans. (author)

Lysyl Oxidase and the Tumor Microenvironment

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Tong-Hong Wang

2016-12-01

Full Text Available The lysyl oxidase (LOX family of oxidases contains a group of extracellular copper-dependent enzymes that catalyze the cross-linking of collagen and elastin by oxidation, thus maintaining the rigidity and structural stability of the extracellular matrix (ECM. Aberrant expression or activation of LOX alters the cellular microenvironment, leading to many diseases, including atherosclerosis, tissue fibrosis, and cancer. Recently, a number of studies have shown that LOX is overexpressed in most cancers and that it is involved in the regulation of tumor progression and metastasis. In contrast, a few reports have also indicated the tumor-suppressing role of LOX. In this short review, we discuss recent research on the correlations between LOX and cancer. Further, the role of LOX in tumor microenvironment remodeling, tumorigenesis, and metastasis and the underlying mechanisms have also been elucidated.

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

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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.

Shelf-life extension of Pacific white shrimp using algae extracts during refrigerated storage.

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Li, Yingchang; Yang, Zhongyan; Li, Jianrong

2017-01-01

Shrimp is a low-fat, high-protein aquatic product, and is susceptible to spoilage during storage. To establish an effective method for the quality control of Pacific white shrimp, the effects of polyphenols (PP) and polysaccharides (PS) from Porphyra yezoensis on the quality of Pacific white shrimp were assessed during refrigerated storage. Pacific white shrimp samples were treated with 5 g L -1 polyphenols, and 8 g L -1 polysaccharides, then stored at 4 ± 1 °C for 8 days. All samples were subjected to measurement of total viable count (TVC), pH, total volatile basic nitrogen (TVB-N), K-value, thiobarbituric acid (TBA), polyphenol oxidase (PPO) activity, and were also assessed by sensory evaluation. The results showed that PP, PS, and the mixture of polyphenols and polysaccharides (PP+PS) could inhibit the increase of total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA) and K-value, and reduce total viable count (TVC) compared with the control group. PP could also inhibit polyphenol oxidase (PPO) activity. Sensory evaluation proved the efficacy of PP and PS by maintaining the overall quality of Pacific white shrimp during refrigerated storage. Moreover, PP+PS could extend the shelf-life of shrimp by 3-4 days compared with the control group. PP+PS could more effectively maintain quality and extend shelf-life during refrigerated storage. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Analysis of Chlorogenic Acid Oxidation Pathway in Simulated ...

African Journals Online (AJOL)

Purpose: To investigate the pathways involved in the oxidation of chlorogenic acid (CA) and phenol metabolism in honeysuckle buds. Methods: A model that mimics CA oxidation by honeysuckle polyphenol oxidase (PPO) by controlling the reaction temperature or reaction duration was employed, and the resulting products ...

Biochemical responses during the pathogenesis of Sclerotium rolfsii ...

African Journals Online (AJOL)

Subhadfip Nandi

a variety of novel proteins and secondary metabolites. This study aimed to examine the induction of different stress related enzymes like phenyl alanineammonia lyase (PAL), chitinase, β-1,3 glucanase, oxidative enzymes like peroxidases (POD), poly phenol oxidases (PPO) and phenolics after inoculation of Sclerotium ...

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.

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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.

Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli

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Molla Gianluca

2010-04-01

Full Text Available Abstract Background Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO. Results Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant E. coli cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from ~500 up to ~25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies. Conclusions Comparison of our results with those published on non-covalent (type I COs expressed in recombinant form (either in E. coli or Streptomyces spp., shows that the fully active type II BCO can be produced in E. coli at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.

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.

Exploring flavin-containing carbohydrate oxidases

NARCIS (Netherlands)

Ferrari, Alessandro Renato

2017-01-01

Oxidases are enzymes capable of removing one or more electrons from their substrate and transfer them to molecular oxygen, forming hydrogen peroxide. Due to their high regio- and enantioselectivity, their use is preferred over traditional organic chemistry methods. Among the oxidases, flavoprotein

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

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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.

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.

Effect of carrot puree edible films on quality preservation of fresh-cut carrots

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Wang X.

2015-06-01

Full Text Available The effect of edible films based on carrot puree, chitosan, corn starch, gelatin, glycerol and cinnamaldehyde on fresh-cut carrots was studied during storage. Several parameters, such as firmness, colour, weight loss, total carotenoids, total phenols, polyphenol oxidase (PPO activity and peroxidase (POD activity in coated carrots were determined at regular intervals and then compared with the uncoated carrots throughout the storage period. Significant and expected changes were observed in all carrot samples that were compared. The coating treatment significantly (P < 0.05 delayed the senescence, reduced the deterioration of exterior quality and retained total carotenoids well compared with control (P < 0.05. In addition, significant inhibition of PPO activity (P < 0.05 and POD activity (P < 0.05 as well as reduced accumulation of polyphenols (P < 0.05 were observed for all coated samples. All of these favourable responses induced by coating treatment on minimally processed fresh-cut carrots showed beneficial physiological effects, which would give some useful references to the fresh-cut fruit and vegetable processing industry and satisfy people’s requirements allowing for extending product shelf life without negatively affecting the sensory quality or acceptability.

Xanthine Oxidase Inhibitory Activity of a Plectranthus saccatus aqueous extract

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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.

Effect of Harvest Time and L-Cysteine as an Antioxidant on Flesh Browning of Fresh-Cut Cherimoya (Annona cherimola Mill. Efecto del Momento de Cosecha y L-Cisteína como un Antioxidante en el Pardeamiento de Pulpa de Chirimoya (Annona cherimola Mill. Precortada

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Reinaldo Campos-Vargas

2008-09-01

Full Text Available Browning development is the most important factor limiting the quality of fresh-cut cherimoya (Annona cherimola Mill.. However, there is little information available about its causes and methods of control. The effectiveness of L-cysteine (0.125, 0.25 and 0.5% in fresh-cut cherimoya harvested on two occasions (October and November and stored for 6 and 12 days at 0 °C was studied. In order to understand the biological basis of browning, polyphenol oxidase (PPO enzyme activity and total phenolic content in fresh-cut pieces were measured. Quality measurements and sensory analysis indicated that 0.5% L-cysteine was somewhat effective in reducing browning development, without affecting other quality attributes. In terms of physiological parameters, PPO activity did not show differences between mature (at harvest and ripe fruit (at processing in both harvest times, but cherimoya fruits picked in November presented lower PPO activity than fruit from October. In general, PPO activity and total phenolic content of L-cysteine treated fruits did not show consistent differences with untreated fruit at 6 or 12 days at 0 °C. PPO activity analyses demonstrated that PPO activity was higher in the outer part of cherimoya flesh compared to the middle or inner sector. These results would support the possibility of using L-cysteine as a postharvest treatment to reduce browning development in fresh-cut cherimoya.El desarrollo de pardeamiento es uno de los factores limitantes en la calidad de chirimoya (Annona cherimola Mill. precortada (fresh-cut. No obstante, existe poca información disponible en relación a sus causas y métodos de control en esta fruta. Se estudió la efectividad de L-cisteína (0,125; 0,25 y 0,5% en chirimoya precortada cosechada en dos oportunidades (octubre y noviembre y almacenada por 6 y 12 días a 0 ºC. Con el objetivo de conocer las bases biológicas del pardeamiento, se estudió la actividad de polifenol oxidasa (PPO y contenido de

Influence of thermal treatment on color, enzyme activities, and antioxidant capacity of innovative pastelike parsley products.

Science.gov (United States)

Kaiser, Andrea; Brinkmann, Maike; Carle, Reinhold; Kammerer, Dietmar R

2012-03-28

Conventional spice powders are often characterized by low sensory quality and high microbial loads. Furthermore, genuine enzymes are only inhibited but not entirely inactivated upon drying, so that they may regain their activity upon rehydration of dried foods. To overcome these problems, initial heating was applied in the present study as the first process step for the production of innovative pastelike parsley products. For this purpose, fresh parsley was blanched (80, 90, and 100 °C for 1-10 min) and subsequently comminuted to form a paste. Alternatively, mincing was carried out prior to heat treatment. Regardless of temperature, the color of the latter product did not show any change after heating for 1 min. With progressing exposure time the green color turned to olive hues due to marked pheophytin formation. Inactivation of genuine peroxidase (POD) and polyphenol oxidase (PPO) was achieved at all temperature-time regimes applied. In contrast, the parsley products obtained after immediate water-blanching were characterized by brighter green colors and enhanced pigment retention. With the exception of the variants water-blanched at 80 °C, POD and PPO were completely inactivated at any of the thermal treatments. Furthermore, in water-blanched samples, antioxidant capacities as determined by the TEAC and FRAP assays were even enhanced compared to unheated parsley, whereas a decrease of phenolic contents could not be prevented. Consequently, the innovative process presented in this study allows the production of novel herb and spice products characterized by improved sensory quality as compared to conventional spice products.

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

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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.

Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger

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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.

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

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.

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

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.

Role of pH in oxidase variability of Aeromonas hydrophila.

OpenAIRE

Hunt, L K; Overman, T L; Otero, R B

1981-01-01

Some strains of Aeromonas hydrophila may be oxidase negative or only weakly oxidase positive by the Kovacs method taken from the surface of a differential medium, such as MacConkey agar. Six strains of A. hydrophila, two oxidase variable, one oxidase constant, and three weakly oxidase positive on MacConkey agar, were studied to determine the cause of oxidase variability. The bacteriostatic dyes in MacConkey agar were considered possible inhibitors of the oxidase reaction. The concentration of...

Structure of choline oxidase in complex with the reaction product glycine betaine.

Science.gov (United States)

Salvi, Francesca; Wang, Yuan-Fang; Weber, Irene T; Gadda, Giovanni

2014-02-01

Choline oxidase from Arthrobacter globiformis, which is involved in the biosynthesis of glycine betaine from choline, has been extensively characterized in its mechanistic and structural properties. Despite the knowledge gained on the enzyme, the details of substrate access to the active site are not fully understood. The `loop-and-lid' mechanism described for the glucose-methanol-choline enzyme superfamily has not been confirmed for choline oxidase. Instead, a hydrophobic cluster on the solvent-accessible surface of the enzyme has been proposed by molecular dynamics to control substrate access to the active site. Here, the crystal structure of the enzyme was solved in complex with glycine betaine at pH 6.0 at 1.95 Å resolution, allowing a structural description of the ligand-enzyme interactions in the active site. This structure is the first of choline oxidase in complex with a physiologically relevant ligand. The protein structures with and without ligand are virtually identical, with the exception of a loop at the dimer interface, which assumes two distinct conformations. The different conformations of loop 250-255 define different accessibilities of the proposed active-site entrance delimited by the hydrophobic cluster on the other subunit of the dimer, suggesting a role in regulating substrate access to the active site.

Boundary Lubrication of PEO-PPO-PEO Triblock Copolymer Physisorbed on Polypropylene, Polyethylene, and Cellulose Surfaces

KAUST Repository

Li, Yangyang

2012-02-22

In situ lateral force microscopy (LFM) and X-ray photoelectron spectroscopy (XPS) were used to probe the lubrication behavior of an aqueous solution of poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) (PEO-PPO-PEO) symmetric triblock copolymer on thin films of polypropylene (PP), polyethylene (PE), and cellulose. LFM experiments were carried out while the substrates were immersed in water and in solutions of the copolymer. The friction coefficient on PP and PE was reduced after adsorption from the PEO-PPO-PEO aqueous solution while the opposite effect was observed for cellulose surfaces. A critical normal loading force, at which the friction coefficient of the lubricated and unlubricated surfaces is equal, was identified and related to the affinity of the polymer with the substrate. Further experiments were performed to mimic practical operations involving lubricant addition during manufacturing and postprocessing removal. XPS was used to verify the presence of the lubricant on the polymeric substrates and to evaluate its removal by water washing. The lubricant layer was easily removed by water from the PP and cellulose surfaces while a durable layer was found on PE. The XPS results were in agreement with the highest critical normal loading force measured for PE (52 nN for PE in contrast to a minimum of 10 nN for cellulose). While several reports exist on lubrication on hard surfaces, friction behavior on soft surfaces is still not well documented as the substrates usually deform under loading pressure. Therefore, we also propose a simple lubrication model for PP, PE, and cellulose and the use of critical normal loading force as a parameter to predict lubricity and durability of adsorbed nonionic block copolymers. © 2012 American Chemical Society.

Crosstalk between HDAC6 and Nox2-based NADPH oxidase mediates HIV-1 Tat-induced pro-inflammatory responses in astrocytes

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Gi Soo Youn

2017-08-01

Full Text Available Histone deacetylase 6 (HDAC6 likely is important in inflammatory diseases. However, how HDAC6 exerts its effect on inflammatory processes remains unclear. HIV-1 transactivator of transcription (Tat activates NADPH oxidase resulting in generation of reactive oxygen species (ROS, leading to extensive neuro-inflammation in the central nervous system. We investigated the correlation of HDAC6 and NADPH oxidase in HIV-1 Tat-stimulated astrocytes. HDAC6 knockdown attenuated HIV-1 Tat-induced ROS generation and NADPH oxidase activation. HDAC6 knockdown suppressed HIV-1 Tat-induced expression of NADPH oxidase subunits, such as Nox2, p47phox, and p22phox. Specific inhibition of HDAC6 using tubastatin A suppressed HIV-1 Tat-induced ROS generation and activation of NADPH oxidase. N-acetyl cysteine, diphenyl iodonium, and apocynin suppressed HIV-1 Tat-induced expression of HDAC6 and the pro-inflammatory chemokines CCL2, CXCL8, and CXCL10. Nox2 knockdown attenuated HIV-1 Tat-induced HDAC6 expression and subsequent expression of chemokines. The collective results point to the potential crosstalk between HDAC6 and NADPH oxidase, which could be a combined therapeutic target for relief of HIV-1 Tat-mediated neuro-inflammation. Keywords: HIV-1 Tat, HDAC6, NADPH oxidase, ROS, Inflammation, Astrocytes

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

Inhibition of xanthine oxidase reduces oxidative stress and improves skeletal muscle function in response to electrically stimulated isometric contractions in aged mice

Science.gov (United States)

Ryan, Michael J.; Jackson, Janna R.; Hao, Yanlei; Leonard, Stephen S.; Alway, Stephen E.

2012-01-01

Oxidative stress is a putative factor responsible for reducing function and increasing apoptotic signaling in skeletal muscle with aging. This study examined the contribution and functional significance of the xanthine oxidase enzyme as a potential source of oxidant production in aged skeletal muscle during repetitive in situ electrically stimulated isometric contractions. Xanthine oxidase activity was inhibited in young adult and aged mice via a subcutaneously placed time release (2.5 mg/day) allopurinol pellet, 7 days prior to the start of in situ electrically stimulated isometric contractions. Gastrocnemius muscles were electrically activated with 20 maximal contractions for three consecutive days. Xanthine oxidase activity was 65% greater in the gastrocnemius muscle of aged mice compared to young mice. Xanthine oxidase activity also increased after in situ electrically stimulated isometric contractions in muscles from both young (33%) and aged (28%) mice, relative to contralateral non-contracted muscles. Allopurinol attenuated the exercise-induced increase in oxidative stress, but it did not affect the elevated basal levels of oxidative stress that was associated with aging. In addition, inhibition of xanthine oxidase activity decreased caspase 3 activity, but it had no effect on other markers of mitochondrial associated apoptosis. Our results show that compared to control conditions, suppression of xanthine oxidase activity by allopurinol reduced xanthine oxidase activity, H2O2 levels, lipid peroxidation and caspase-3 activity, prevented the in situ electrically stimulated isometric contraction-induced loss of glutathione, prevented the increase of catalase and copper-zinc superoxide dismutase activities, and increased maximal isometric force in the plantar flexor muscles of aged mice after repetitive electrically evoked contractions. PMID:21530649

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.

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

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

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase

NARCIS (Netherlands)

van Kuilenburg, A. B.; Gorren, A. C.; Dekker, H. L.; Nieboer, P.; van Gelder, B. F.; Muijsers, A. O.

1992-01-01

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of

Influence of Laccase and Tyrosinase on the Antioxidant Capacity of Selected Phenolic Compounds on Human Cell Lines

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Matthias Riebel

2015-09-01

Full Text Available Polyphenolic compounds affect the color, odor and taste of numerous food products of plant origin. In addition to the visual and gustatory properties, they serve as radical scavengers and have antioxidant effects. Polyphenols, especially resveratrol in red wine, have gained increasing scientific and public interest due to their presumptive beneficial impact on human health. Enzymatic oxidation of phenolic compounds takes place under the influence of polyphenol oxidases (PPO, including tyrosinase and laccase. Several studies have demonstrated the radical scavenger effect of plants, food products and individual polyphenols in vitro, but, apart from resveratrol, such impact has not been proved in physiological test systems. Furthermore, only a few data exist on the antioxidant capacities of the enzymatic oxidation products of phenolic compounds generated by PPO. We report here first results about the antioxidant effects of phenolic substances, before and after oxidation by fungal model tyrosinase and laccase. In general, the common chemical 2,2-diphenyl-1-picrylhydrazyl assay and the biological tests using two different types of cell cultures (monocytes and endothelial cells delivered similar results. The phenols tested showed significant differences with respect to their antioxidant activity in all test systems. Their antioxidant capacities after enzymatic conversion decreased or increased depending on the individual PPO used.

Glucose oxidase-functionalized fluorescent gold nanoclusters as probes for glucose

Energy Technology Data Exchange (ETDEWEB)

Xia, Xiaodong [College of Chemistry and Chemical Engineering, Central South University, Changsha 410083 (China); School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201 (China); Long, Yunfei, E-mail: l_yunfei927@163.com [School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201 (China); Wang, Jianxiu, E-mail: jxiuwang@csu.edu.cn [College of Chemistry and Chemical Engineering, Central South University, Changsha 410083 (China)

2013-04-15

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{sub em} {sub max} = 650 nm, λ{sub ex} {sub 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{sub 2} to produce H{sub 2}O{sub 2}, which quenched quantitatively the fluorescence of gold clusters and laid a foundation of glucose detection. A linear range of 2.0 × 10{sup −6}–140 × 10{sup −6} M and a detection limit of 0.7 × 10{sup −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.

Stress responses of duckweed (Lemna minor L.) and water velvet (Azolla filiculoides Lam.) to anionic surfactant sodium-dodecyl-sulphate (SDS).

Science.gov (United States)

Forni, C; Braglia, R; Harren, F J M; Cristescu, S M

2012-04-01

Surfactants are used for several purposes and recently they have attracted the attention for their ability to modify the behavior of other preexistent or co-disposed contaminants, although their use or discharge in wastewaters can represent a real or potential risk for the environment. Lemna minor L. and Azolla filiculoides Lam. are floating aquatic macrophytes, very effective in accumulating several pollutants including sodium dodecyl sulphate (SDS). In this work we evaluated the effects of SDS on these species by determining the stress ethylene production via laser-based trace gas detection, and the activities of enzymes involved in stress response, such as guaiacol peroxidase (G-POD), phenylalanine ammonia-lyase (PAL) and polyphenol-oxidase (PPO). Phenolics content was also determined. The macrophytes were treated with different concentrations of SDS for one week. SDS affected duckweed enzymatic activities and phenol content. While in the fern phenolics amount, PAL, G-POD and PPO activities were not affected by SDS except for 100 ppm SDS, the only concentration that was taken up and not completely degraded. Stress ethylene production was induced only in the fern treated with 50 and 100 ppm SDS. Copyright © 2011 Elsevier B.V. All rights reserved.

Role of TLR4/NADPH oxidase/ROS-activated p38 MAPK in VCAM-1 expression induced by lipopolysaccharide in human renal mesangial cells

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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

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.

NADPH oxidases as novel pharmacologic targets against influenza A virus infection.

Science.gov (United States)

Vlahos, Ross; Selemidis, Stavros

2014-12-01

Influenza A viruses represent a major global health care challenge, with imminent pandemics, emerging antiviral resistance, and long lag times for vaccine development, raising a pressing need for novel pharmacologic strategies that ideally target the pathology irrespective of the infecting strain. Reactive oxygen species (ROS) pervade all facets of cell biology with both detrimental and protective properties. Indeed, there is compelling evidence that activation of the NADPH oxidase 2 (NOX2) isoform of the NADPH oxidase family of ROS-producing enzymes promotes lung oxidative stress, inflammation, injury, and dysfunction resulting from influenza A viruses of low to high pathogenicity, as well as impeding virus clearance. By contrast, the dual oxidase isoforms produce ROS that provide vital protective antiviral effects for the host. In this review, we propose that inhibitors of NOX2 are better alternatives than broad-spectrum antioxidant approaches for treatment of influenza pathologies, for which clinical efficacy may have been limited owing to poor bioavailability and inadvertent removal of beneficial ROS. Finally, we briefly describe the current suite of NADPH oxidase inhibitors and the molecular features of the NADPH oxidase enzymes that could be exploited by drug discovery for development of more specific and novel inhibitors to prevent or treat disease caused by influenza. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

A multicopper oxidase-related protein is essential for insect viability, longevity and ovary development.

Science.gov (United States)

Peng, Zeyu; Green, Peter G; Arakane, Yasuyuki; Kanost, Michael R; Gorman, Maureen J

2014-01-01

Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms. These copper ions are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence similarity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We predicted that MCORPs would lack oxidase activity due to the absence of copper-coordinating residues. To test this prediction, we purified recombinant Tribolium castaneum (red flour beetle) MCORP and analyzed its enzymatic activity using a variety of substrates. As expected, no oxidase activity was detected. To study MCORP function in vivo, we analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP, and assessed RNAi-mediated knockdown phenotypes. We found that both MCORPs are constitutively expressed at a low level in all of the tissues we analyzed. Injection of TcMCORP dsRNA into larvae resulted in 100% mortality prior to adult eclosion, with death occurring mainly during the pharate pupal stage or late pharate adult stage. Injection of TcMCORP dsRNA into pharate pupae resulted in the death of approximately 20% of the treated insects during the pupal to adult transition and a greatly shortened life span for the remaining insects. In addition, knockdown of TcMCORP in females prevented oocyte maturation and, thus, greatly decreased the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests.

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.

Systemic Manifestations in Pyridox(am)ine 5'-Phosphate Oxidase Deficiency.

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Guerriero, Réjean M; Patel, Archana A; Walsh, Brian; Baumer, Fiona M; Shah, Ankoor S; Peters, Jurriaan M; Rodan, Lance H; Agrawal, Pankaj B; Pearl, Phillip L; Takeoka, Masanori

2017-11-01

Pyridoxine is converted to its biologically active form pyridoxal-5-phosphate (P5P) by the enzyme pyridox(am)ine 5'-phosphate oxidase and serves as a cofactor in nearly 200 reactions in the central nervous system. Pyridox(am)ine 5'-phosphate oxidase deficiency leads to P5P dependent epilepsy, typically a neonatal- or infantile-onset epileptic encephalopathy treatable with P5P or in some cases, pyridoxine. Following identification of retinopathy in a patient with pyridox(am)ine 5'-phosphate oxidase deficiency that was reversible with P5P therapy, we describe the systemic manifestations of pyridox(am)ine 5'-phosphate oxidase deficiency. A series of six patients with homozygous mutations of PNPO, the gene coding pyridox(am)ine 5'-phosphate oxidase, were evaluated in our center over the course of two years for phenotyping of neurological and systemic manifestations. Five of six were born prematurely, three had anemia and failure to thrive, and two had elevated alkaline phosphatase. A movement disorder was observed in two children, and a reversible retinopathy was observed in the most severely affected infant. All patients had neonatal-onset epilepsy and were on a continuum of developmental delay to profound encephalopathy. Electroencephalographic features included background slowing and disorganization, absent sleep features, and multifocal and generalized epileptiform discharges. All the affected probands carried a homozygous PNPO mutation (c.674 G>T, c.686 G>A and c.352G>A). In addition to the well-described epileptic encephalopathy, pyridox(am)ine 5'-phosphate oxidase deficiency causes a range of neurological and systemic manifestations. A movement disorder, developmental delay, and encephalopathy, as well as retinopathy, anemia, and failure to thrive add to the broadening clinical spectrum of P5P dependent epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

Biodegradation of phenolic compounds with oxidases from sorghum and non-defined mixed bacterium media

International Nuclear Information System (INIS)

Obame, C. E. L.; Savadogo, P. W.; Mamoudou, D. H.; Dembele, R. H.; Traore, A. S.

2009-01-01

The biodegradation of the phenolic compounds is performed using oxidative enzymes, e. g. polyphenol oxidases (PPOs) and peroxidases (POXs). These oxidases displaying a wide spectrum for the oxidation of phenolic compounds were isolated either from sorghum or mixed bacteria. Spectrophotometric methods were used to assess the monophenolase and diphenolase activities of PPOs as well as the hydrogen-dependant oxidation of POXs. (Author)

Biodegradation of phenolic compounds with oxidases from sorghum and non-defined mixed bacterium media

Energy Technology Data Exchange (ETDEWEB)

Obame, C. E. L.; Savadogo, P. W.; Mamoudou, D. H.; Dembele, R. H.; Traore, A. S.

2009-07-01

The biodegradation of the phenolic compounds is performed using oxidative enzymes, e. g. polyphenol oxidases (PPOs) and peroxidases (POXs). These oxidases displaying a wide spectrum for the oxidation of phenolic compounds were isolated either from sorghum or mixed bacteria. Spectrophotometric methods were used to assess the monophenolase and diphenolase activities of PPOs as well as the hydrogen-dependant oxidation of POXs. (Author)

Determination of the structure of the organized phase of the block copolymer PEO-PPO-PEO in aqueous solutions under flow by small-angle neutron scattering

International Nuclear Information System (INIS)

Perreur, Christelle; Habas, Jean-Pierre; Francois, Jeanne; Peyrelasse, Jean; Lapp, Alain

2002-01-01

The organization of Tetronic 908 registered (T908), a star copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks, has been examined. Above critical conditions of temperature and concentration, the micelles formed by the aggregation of PPO units self-organize into particular structures. While small-angle neutron scattering (SANS) characterizations performed with static conditions demonstrate the organization of the medium, the experimental results do not allow us to make a distinction between simple cubic and body-centered-cubic structures. However, SANS measurements realized under shear produce characteristic diffraction diagrams. In this paper, an accurate methodology is proposed to identify, without ambiguity, the exact nature of the organized phase. Applied to our system, indexing of the diffraction pattern spots reveals that the organization of T908 is of bcc type oriented with the [111] direction parallel to the direction of flow, but the crystals can present any orientation about this direction. The lattice size has been estimated and compared to previous published results

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.

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.

The microglial NADPH oxidase complex as a source of oxidative stress in Alzheimer's disease

Directory of Open Access Journals (Sweden)

Landreth Gary E

2006-11-01

Full Text Available Abstract Alzheimer's disease is the most common cause of dementia in the elderly, and manifests as progressive cognitive decline and profound neuronal loss. The principal neuropathological hallmarks of Alzheimer's disease are the senile plaques and the neurofibrillary tangles. The senile plaques are surrounded by activated microglia, which are largely responsible for the proinflammatory environment within the diseased brain. Microglia are the resident innate immune cells in the brain. In response to contact with fibrillar beta-amyloid, microglia secrete a diverse array of proinflammatory molecules. Evidence suggests that oxidative stress emanating from activated microglia contribute to the neuronal loss characteristic of this disease. The source of fibrillar beta-amyloid induced reactive oxygen species is primarily the microglial nicotinamide adenine dinucleotide phosphate (NADPH oxidase. The NADPH oxidase is a multicomponent enzyme complex that, upon activation, produces the highly reactive free radical superoxide. The cascade of intracellular signaling events leading to NADPH oxidase assembly and the subsequent release of superoxide in fibrillar beta-amyloid stimulated microglia has recently been elucidated. The induction of reactive oxygen species, as well as nitric oxide, from activated microglia can enhance the production of more potent free radicals such as peroxynitrite. The formation of peroxynitrite causes protein oxidation, lipid peroxidation and DNA damage, which ultimately lead to neuronal cell death. The elimination of beta-amyloid-induced oxidative damage through the inhibition of the NADPH oxidase represents an attractive therapeutic target for the treatment of Alzheimer's disease.

A study of overproduction and enhanced secretion of enzymes. Quarterly report

Energy Technology Data Exchange (ETDEWEB)

Dashek, W.V.

1993-09-01

Wood decay within forests, a significant renewable photosynthetic energy resource, is caused primarily by Basidiomycetous fungi, e.g., white rot fungi. These organisms possess the ability to degrade lignin, cellulose and hemicellulose, the main organic polymers of wood. In the case of the white rot fungi, e.g., Coriolus versicolor, the capacity results from the fungus` ability to elaborate extracellular cellulolytic and ligninolytic enzymes. With regard to the latter, at least one of the enzymes, polyphenol oxidase (PPO) appears within a defined growth medium. This proposal focuses on the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. There are two major sections to the proposal: (1) overproduction of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electro microscopical techniques and (2) the biochemical/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO enzymes.

Regulation of the NADPH Oxidase RBOHD During Plant Immunity.

Science.gov (United States)

Kadota, Yasuhiro; Shirasu, Ken; Zipfel, Cyril

2015-08-01

Pathogen recognition induces the production of reactive oxygen species (ROS) by NADPH oxidases in both plants and animals. ROS have direct antimicrobial properties, but also serve as signaling molecules to activate further immune outputs. However, ROS production has to be tightly controlled to avoid detrimental effects on host cells, but yet must be produced in the right amount, at the right place and at the right time upon pathogen perception. Plant NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family, which contains 10 members in the model plant Arabidopsis thaliana. The perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) leads to a rapid, specific and strong production of ROS, which is dependent on RBOHD. RBOHD is mainly controlled by Ca(2+) via direct binding to EF-hand motifs and phosphorylation by Ca(2+)-dependent protein kinases. Recent studies have, however, revealed a critical role for a Ca(2+)-independent regulation of RBOHD. The plasma membrane-associated cytoplasmic kinase BIK1 (BOTRYTIS-INDUCED KINASE1), which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. Impairment of these phosphorylation events completely abolishes the function of RBOHD in immunity. These results suggest that RBOHD activity is tightly controlled by multilayered regulations. In this review, we summarize recent advances in our understanding of the regulatory mechanisms controlling RBOHD activation. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

NADPH oxidase is involved in regulation of gene expression and ROS overproduction in soybean (Glycine max L. seedlings exposed to cadmium

Directory of Open Access Journals (Sweden)

Jagna Chmielowska-Bąk

2017-06-01

Full Text Available Cadmium-induced oxidative burst is partially mediated by NADPH oxidase. The aim of the present research was to evaluate the role of NADPH oxidase in soybeans’ response to short-term cadmium stress. The application of an NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI, affected expression of two Cd-inducible genes, encoding DOF1 and MYBZ2 transcription factors. This effect was observed after 3 h of treatment. Interestingly, Cd-dependent increases in NADPH oxidase activity occurred only after a period of time ranging from 6 and 24 h of stress. Stimulation of the enzyme correlated in time with a significant accumulation of reactive oxygen species (ROS. Further analysis revealed that pharmacological inhibition of NADPH oxidase activity during 24 h of Cd stress does not affect Cd uptake, seedling growth, or the level of lipid peroxidation. The role of NADPH oxidase in the response of soybean seedlings to short-term Cd exposure is discussed.

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

Enhanced production of glucose oxidase from UVmutant of ...

African Journals Online (AJOL)

UV rays were used as mutagen in wild type strain of Aspergillus niger for enhanced production of glucose oxidase. After mutangenization and selection, mutant A. niger strains, resistant to 2-deoxy-Dglucose were obtained. The mutants showed 1.57 and 1.98 fold increase in activities of extra and intra cellular glucose ...

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

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.

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae

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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.

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.

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.

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.

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.

H2O2 and NADPH oxidases involve in regulation of 2-(2-phenylethyl)chromones accumulation during salt stress in Aquilaria sinensis calli.

Science.gov (United States)

Wang, Xiaohui; Dong, Xianjuan; Feng, Yingying; Liu, Xiao; Wang, Jinling; Zhang, Zhongxiu; Li, Jun; Zhao, Yunfang; Shi, Shepo; Tu, Pengfei

2018-04-01

2-(2-Phenylethyl)chromones are the main compounds responsible for the quality of agarwood, which is widely used in traditional medicines, incenses and perfumes. H 2 O 2 and NADPH oxidases (also known as respiratory burst oxidase homologs, Rbohs) mediate diverse physiological and biochemical processes in environmental stress responses. However, little is known about the function of H 2 O 2 and NADPH oxidases in 2-(2-phenylethyl)chromones accumulation. In this study, we found that salt stress induced a transient increase in content of H 2 O 2 and 2-(2-phenylethyl)chromones accumulation in Aquilaria sinensis calli. Exogenous H 2 O 2 remarkably decreased the production of 2-(2-phenylethyl)chromones, while dimethylthiourea (DMTU), a scavenger of H 2 O 2 , significantly increased 2-(2-phenylethyl)chromones accumulation in salt treated calli. Three new H 2 O 2 -generating genes, named AsRbohA-C, were isolated and characterized from A. sinensis. Salt stress also induced a transient increase in AsRbohA-C expression and NADPH oxidase activity. Furthermore, exogenous H 2 O 2 increased AsRbohA-C expression and NADPH oxidase activity, while DMTU inhibited AsRbohA-C expression and NADPH oxidase activity under salt stress. Moreover, diphenylene iodonium (DPI), the inhibitor of NADPH oxidases, reduced AsRbohA-C expression and NADPH oxidase activity, but significantly induced 2-(2-phenylethyl)chromones accumulation during salt stress. These results clearly demonstrated the central role of H 2 O 2 and NADPH oxidases in regulation of salt-induced 2-(2-phenylethyl)chromones accumulation in A. sinensis calli. Copyright © 2018 Elsevier B.V. All rights reserved.

Polyphenol Oxidase Enzyme and Inactivation Methods

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Leman Yılmaz

2018-03-01

Full Text Available Polyphenol oxidase enzyme is found in vegetables and fruits, as well as in some animal organs and microorganisms. Polyphenol oxidase enzyme responsible for enzymatic browning is a group of copper proteins that catalyses the oxidation of phenolic compounds to quinones, which produce brown pigments, commonly found in fruits and vegetables. During the industrial preparation of fruits and vegetables, results of catalytic effect of polyphenol oxidase causes enzymatic browning. Enzymatic browning impairs the appearance of products containing phenolic compounds along with undesirable colour, odor and taste formation and significant loss of nutritional value of the products. This affects the acceptability of the products by the consumers and causes economic losses. In this review, some characteristics of polyphenol oxidase enzyme in different fruits and vegetables have been reviewed and information about chemical antibrowning agents, thermal applications, irradiation applications and alternative methods such as high pressure processing, pulse electric field, supercritical carbon dioxide and ultrasound applications to inactivate this enzyme has been presented.

Control of wilt and rot pathogens of tomato by antagonistic pink pigmented facultative methylotrophic Delftia lacustris and Bacillus spp.

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Veeranan Janahiraman

2016-11-01

Full Text Available The studies on the biocontrol potential of pink pigmented facultative methylotrophic (PPFM bacteria other than the genus Methylobacterium are scarce. In the present study, we report three facultative methylotrophic isolates; PPO-1, PPT-1 and PPB-1, respectively identified as Delftia lacustris, Bacillus subtilis and Bacillus cereus by 16S rRNA gene sequence analysis. Hemolytic activity was tested to investigate the potential pathogenicity of isolates to plants and humans, the results indicates that the isolates PPO-1, PPT-1 and PPB-1 are not pathogenic strains. Under in vitro conditions, D. lacustris PPO-1, B. subtilis PPT-1 and B. cereus PPB-1 showed direct antagonistic effect by inhibiting the mycelial growth of fungal pathogens; Fusarium oxysporum f. sp. lycopersici (2.15, 2.05 and 1.95 cm, Sclerotium rolfsii (2.14, 2.04 and1.94 cm, Pythium ultimum (2.12, 2.02 and 1.92cm, and Rhizoctonia solani (2.18, 2.08 and 1.98 cm and also produced volatile inhibitory compounds. Under plant growth chamber condition methylotrophic bacterial isolates; D. lacustris PPO-1, B. subtilis PPT-1 and B. cereus PPB-1 significantly reduced the disease incidence of tomato. Under greenhouse condition, D. lacustris PPO-1, B. subtilis PPT-1 and B. cereus PPB-1 inoculated tomato plants, when challenged with F. oxysporum f. sp. lycopersici, S. rolfsii, P. ultimum and R. solani, increased the pathogenesis related proteins (β-1, 3-glucanase and chitinase and defense enzymes (phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, and catalase on day 5 after inoculation. In the current study, we first report the facultative methylotrophy in pink pigmented Delftia lacustris, B. subtilis, and B. cereus and their antagonistic potential against fungal pathogens. Direct antagonistic and ISR effects of these isolates against fungal pathogens of tomato evidenced their possible use as a biocontrol agent.

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...

The Cytochrome bd Oxidase of Porphyromonas gingivalis Contributes to Oxidative Stress Resistance and Dioxygen Tolerance.

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Julia Leclerc

Full Text Available Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans. The disease is associated with the formation of a mixed oral biofilm which is exposed to oxygen and environmental stress, such as oxidative stress. To investigate possible roles for cytochrome bd oxidase in the growth and persistence of this anaerobic bacterium inside the oral biofilm, mutant strains deficient in cytochrome bd oxidase activity were characterized. This study demonstrated that the cytochrome bd oxidase of Porphyromonas gingivalis, encoded by cydAB, was able to catalyse O2 consumption and was involved in peroxide and superoxide resistance, and dioxygen tolerance.

Permeability and Selectivity of PPO/Graphene Composites as Mixed Matrix Membranes for CO2 Capture and Gas Separation

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Riccardo Rea

2018-01-01

Full Text Available We fabricated novel composite (mixed matrix membranes based on a permeable glassy polymer, Poly(2,6-dimethyl-1,4-phenylene oxide (PPO, and variable loadings of few-layer graphene, to test their potential in gas separation and CO2 capture applications. The permeability, selectivity and diffusivity of different gases as a function of graphene loading, from 0.3 to 15 wt %, was measured at 35 and 65 °C. Samples with small loadings of graphene show a higher permeability and He/CO2 selectivity than pure PPO, due to a favorable effect of the nanofillers on the polymer morphology. Higher amounts of graphene lower the permeability of the polymer, due to the prevailing effect of increased tortuosity of the gas molecules in the membrane. Graphene also allows dramatically reducing the increase of permeability with temperature, acting as a “stabilizer” for the polymer matrix. Such effect reduces the temperature-induced loss of size-selectivity for He/N2 and CO2/N2, and enhances the temperature-induced increase of selectivity for He/CO2. The study confirms that, as observed in the case of other graphene-based mixed matrix glassy membranes, the optimal concentration of graphene in the polymer is below 1 wt %. Below such threshold, the morphology of the nanoscopic filler added in solution affects positively the glassy chains packing, enhancing permeability and selectivity, and improving the selectivity of the membrane at increasing temperatures. These results suggest that small additions of graphene to polymers can enhance their permselectivity and stabilize their properties.

The terminal oxidases of Paracoccus denitrificans

NARCIS (Netherlands)

de Gier, J.-W.; Lübben, M; Reijnders, W N; Tipker, C A; Slotboom, D.J.; van Spanning, R J; Stouthamer, A.H.; van der Oost, J.

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding subunit I of the aa3-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to

Functional heterogeneity of NADPH oxidase-mediated contractions to endothelin with vascular aging.

Science.gov (United States)

Meyer, Matthias R; Barton, Matthias; Prossnitz, Eric R

2014-11-24

Aging, a physiological process and main risk factor for cardiovascular and renal diseases, is associated with endothelial cell dysfunction partly resulting from NADPH oxidase-dependent oxidative stress. Because increased formation of endothelium-derived endothelin-1 (ET-1) may contribute to vascular aging, we studied the role of NADPH oxidase function in age-dependent contractions to ET-1. Renal arteries and abdominal aortas from young and old C57BL6 mice (4 and 24 months of age) were prepared for isometric force measurements. Contractions to ET-1 (0.1-100 nmol/L) were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat (3 μmol/L). To exclude age-dependent differential effects of NO bioactivity between vascular beds, all experiments were conducted in the presence of the NO synthase inhibitor L-NAME (300 μmol/L). In young animals, ET-1-induced contractions were 6-fold stronger in the renal artery than in the aorta (prenal artery and aorta, respectively (pAging had no effect on NADPH oxidase-dependent and -independent contractions to ET-1 in the renal artery. In contrast, contractions to ET-1 were markedly reduced in the aged aorta (5-fold, page-dependent heterogeneity of NADPH oxidase-mediated vascular contractions to ET-1, demonstrating an inherent resistance to functional changes in the renal artery but not in the aorta with aging. Thus, local activity of NADPH oxidase differentially modulates responses to ET-1 with aging in distinct vascular beds. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Cyanobacterial Lactate Oxidases Serve as Essential Partners in N2 Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants[w

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Hackenberg, Claudia; Kern, Ramona; Hüge, Jan; Stal, Lucas J.; Tsuji, Yoshinori; Kopka, Joachim; Shiraiwa, Yoshihiro; Bauwe, Hermann; Hagemann, Martin

2011-01-01

Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N2-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high l-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N2-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N2 fixation was more sensitive to O2 in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O2-scavenging enzyme to protect nitrogenase in extant N2-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration. PMID:21828292

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

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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.

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

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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.

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.

Mediation of glycosylated and partially-deglycosylated glucose oxidase of Aspergillus niger by a ferrocene-derivatised detergent.

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Fraser, D M; Zakeeruddin, S M; Grätzel, M

1992-01-30

A ferrocene-derivatised detergent, (11-ferrocenylundecyl) trimethylammonium bromide (FTMAB), when oxidised to the corresponding ferricinium ion, was found by electrochemical studies to be an effective electron acceptor for reduced glucose oxidase of Aspergillus niger (EC 1.13.4) and thus acts as a electron-transfer mediator between glucose oxidase and a working electrode held at a potential sufficiently positive to reoxidise reduced FTMAB. An increase in mediating activity was produced when FTMAB was present in concentrations above its critical micelle concentration. An 'enzyme electrode' was formed by adsorption of glucose oxidase and FTMAB surfactant on a graphite rod. The electrode functioned as an amperometric biosensor for glucose in phosphate-buffered saline solution. A mixed micelle of glucose oxidase and FTMAB, probably adsorbed on the electrode surface, appears to be advantageous for the amperometric determination of glucose. Additionally, glucose oxidase was treated with alpha-mannosidase. When this partially-deglycosylated glucose oxidase was incorporated in an enzyme electrode, a 100-fold increase in the second-order rate constant (k) for electron transfer between the enzyme and FTMAB was observed, together with increased current densities, with respect to the equivalent values for FTMAB and commercial glucose oxidase. The use of deglycosylated enzymes in biosensors is suggested.

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

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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.

Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis.

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Musicki, Biljana; Liu, Tongyun; Sezen, Sena F; Burnett, Arthur L

2012-08-01

Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Relative to hemi mice, SCD increased (Ppenis. Apocynin treatment of sickle mice reversed (P0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis. © 2012 International Society for Sexual Medicine.

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.

Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals

DEFF Research Database (Denmark)

Galuszka, P.; Frebort, I.; Sebela, M.

2001-01-01

An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purifi...

Thermal Characterization of Purified Glucose Oxidase from A Newly Isolated Aspergillus Niger UAF-1

Science.gov (United States)

Anjum Zia, Muhammad; Khalil-ur-Rahman; K. Saeed, Muhammad; Andaleeb, Fozia; I. Rajoka, Muhammad; A. Sheikh, Munir; A. Khan, Iftikhar; I. Khan, Azeem

2007-01-01

An intracellular glucose oxidase was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger UAF-1. The enzyme was purified to a yield of 28.43% and specific activity of 135 U mg−1 through ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The enzyme showed high affinity for D-glucose with a Km value of 2.56 mM. The enzyme exhibited optimum catalytic activity at pH 5.5. Temperature optimum for glucose oxidase, catalyzed D-glucose oxidation was 40°C. The enzyme showed a high thermostability having a half-life 30 min, enthalpy of denaturation 99.66 kJ mol−1 and free energy of denaturation 103.63 kJ mol−1. These characteristics suggest the use of glucose oxidase from Aspergillus niger UAF-1 as an analytical reagent and in the design of biosensors for clinical, biochemical and diagnostic assays. PMID:18193107

Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans

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Coppée Jean-Yves

2010-02-01

Full Text Available Abstract Background Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III to As(V as a detoxification mechanism. Results In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III. To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (σ54 of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE and a putative -12/-24 σ54-dependent promoter motif was identified upstream of aoxAB coding sequences. Conclusion These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III in this microorganism.

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.

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.

Confirmation of a blocked amino terminus of sulfhydryl oxidase

International Nuclear Information System (INIS)

Janolino, V.G.; Morrison-Rowe, S.J.; Swaisgood, H.E.

1990-01-01

The isolation of sulfhydryl oxidase from bovine milk in a suitably pure form for sequencing was carried out by transient covalent affinity chromatography of diafiltered whey using cysteinylsuccinamidopropyl-glass as matrix. The glutathione-eluted proteins were separated by SDS-PAGE. By radiolabeling the affinity chromatography-purified enzyme with [ 14 C]iodoacetate before subjecting to SDS-PAGE, the sulfhydryl oxidase band was identified, because sulfhydryl oxidase is known to be inactivated by alkylation of one sulfhydryl group per mole. The results confirmed that sulfhydryl oxidase corresponds to the 85 (± 5)-kDa band observed on SDS-PAGE. The protein band corresponding to radiolabeled sulfhydryl oxidase was recovered from SDS-PAGE gels by electrophoretic elution and by electroblotting on polyvinylidene difluoride membrane and subjected to gas phase sequencing. Precautions were taken during electrophoretic elution to prevent reactions that result in N-terminal blocking. Both methods of protein recovery yielded negative results when subjected to sequence analysis indicating that the N-terminus of sulfhydryl oxidase is blocked

Immobilization of oxidases and their analytical applications

International Nuclear Information System (INIS)

Yasinzai, M.

2007-01-01

Immobilized enzymes are replacing their soluble counter-parts in nearly every field of application. These enzyme modifications have evolved from a research curiosity into an entire branch of Biotechnology. An immobilization method for flavin containing oxidases and their use in flow injection system is described. An electrochemical detector for H/sub 2/O/sub 2/ is assembled which is used effectively for the determination of glucose using more common glucose oxidase and the simultaneous determination of sugars. The combination of oxidases with hydrolases have been used for the determination of maltose and starch. (author)

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.

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.

NADPH Oxidases: Progress and Opportunities

OpenAIRE

San Martin, Alejandra; Griendling, Kathy K.

2014-01-01

From the initial discovery in 1999 that NADPH oxidases comprise a family of enzymes to our current focus on drug development to treat multiple pathologies related to this enzyme family, progress has been swift and impressive. We have expanded our understanding of the extent of the family, the basic enzymatic biochemistry, the multiple cellular functions controlled by NADPH oxidases, and their varied roles in physiology and diseases. We have developed numerous cell culture tools, animal models...

Expression and Characterization of Glucose Oxidase from Aspergillus niger in Yarrowia lipolytica.

Science.gov (United States)

Khadivi Derakshan, Fatemeh; Darvishi, Farshad; Dezfulian, Mehrouz; Madzak, Catherine

2017-08-01

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.

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.

The Evolution of Total Phenolic Compounds and Antioxidant Activities during Ripening of Grapes (Vitis vinifera L., cv. Tempranillo Grown in Semiarid Region: Effects of Cluster Thinning and Water Deficit

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Inmaculada Garrido

2016-11-01

Full Text Available A study was made of how water status (rainfed vs. irrigated and crop load (no cluster thinning vs. cluster thinning can together affect the grapes of Vitis vinifera cv. Tempranillo vines growing in a semiarid zone of Extremadura (Spain. The grapes were monitored at different stages of ripening, measuring the peroxidase (POX and superoxide dismutase (SOD antioxidant activities and the phenolic content (flavonoids and phenylpropanoids, together with other parameters. The irrigation regime was adjusted to provide 100% of crop evapotranspiration (ETc. The findings confirmed previous results that both thinning and water deficit advance ripening, while irrigation and high crop load (no thinning lengthen the growth cycle. The SOD activity remained practically constant throughout ripening in the thinned treatments and was always lower than in the unthinned treatments, an aspect which could have been the cause of the observed greater level of lipid peroxidation in the water deficit, thinned treatment. The nonspecific peroxidase activity was very low, especially in the thinned treatments. The effect of thinning was enhanced when combined with water deficit, inducing increases in phenylpropanoids and, above all, flavonoids at the harvest stage of ripening, while leaving the polyphenol oxidase activity (PPO unaffected.

Effect of gamma irradiation on aspergillus niger for enhanced production of glucose oxidase

International Nuclear Information System (INIS)

Zia, M.A.; Rasul, S.

2012-01-01

Developing countries have a high prevalence of diabetes and their populations are suffering from associated adverse factors. Such a frequency requires more effective diagnosis, mostly achieved by glucose diagnostic kits. Although high priced kits are available in market but local production of such kits can be highly cost effective and may confer the decline in incidence of the disease. Glucose oxidase is the key enzyme for the determination of glucose in such analytical tools. Enhanced production of glucose oxidase was performed by mutagenesis of Aspergillus niger by gamma irradiation. A dose of 80 krad was found as optimum for derivation of positive mutant strains. Following the screening by triton X-100 and 2-deoxy-D-glucose, the selected strains A. niger G-80-A, A. niger G-80-B and A. niger G-80-C showed 27.5, 23.20 and 20.55 UmL/sub -1/ glucose oxidase activity in enzyme diffusion zone test; which is much higher to parental strain (7.5 UmL/sup -1/). A. niger G-80-A was subjected to submerged fermentation and obtained highest yields after 36 h, at CSL 2%, pH 6.5, 30 degree C, KH/sub 2/PO/sub 4/ 0.8% and urea 0.3%. Partial purification by ammonium sulfate resulted in 175 UmL/sup -1/ of glucose oxidase activity after dialysis. Kinetic parameters like optimum pH, temperature, K/sub m/ and V/sub max/ were found to be 6.0 (180 +- 2 UmL/sup -1/), 30 degree C (185 +- 0.5 UmL/sup -1/), 5.26 mM and 400 U mL/sup -1/, respectively. Active inhibition of the enzyme by increasing concentration of PLP in reaction mixture confirmed the presence of functional lysyl residue on the active site of enzyme. (author)

Bioelectrocatalytic mediatorless dioxygen reduction at carbon ceramic electrodes modified with bilirubin oxidase

International Nuclear Information System (INIS)

Nogala, Wojciech; Celebanska, Anna; Szot, Katarzyna; Wittstock, Gunther; Opallo, Marcin

2010-01-01

Carbon ceramic electrodes were prepared by sol-gel processing of a hydrophobic precursor - methyltrimethoxysilane (MTMOS) - together with dispersed graphite microparticles according to a literature procedure. Bilirubin oxidase (BOx) was adsorbed on this electrode from buffer solution and this process was followed by atomic force microscopy (AFM). The electrodes exhibited efficient mediatorless electrocatalytic activity towards dioxygen reduction. The activity depends on the time of adsorption of the enzyme and the pH. The electrode remains active in neutral solution. The bioelectrocatalytic activity is further increased when a fraction of the carbon microparticles is replaced by sulfonated carbon nanoparticles (CNPs). This additive enhances the electrical communication between the enzyme and the electronic conductor. At pH 7 the carbon ceramic electrode modified with bilirubin oxidase retains ca. half of its highest activity. The role of the modified nanoparticles is confirmed by experiments in which a film embedded in a hydrophobic silicate matrix also exhibited efficient mediatorless biocatalytic dioxygen reduction. Scanning electrochemical microscopy (SECM) of the studied electrodes indicated a rather even distribution of the catalytic activity over the electrode surface.

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

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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.

Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process.

Science.gov (United States)

Jia, Yuying; Yao, Xingdong; Zhao, Mingzhe; Zhao, Qiang; Du, Yanli; Yu, Cuimei; Xie, Futi

2015-08-07

The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert) with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman), which showed a relatively weak susceptibility. Gibberellin (GA) levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA). Higher zeatin riboside (ZR) content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA) content, polyphenol oxidase (PPO) and peroxidase (POD) activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.

Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process

Directory of Open Access Journals (Sweden)

Yuying Jia

2015-08-01

Full Text Available The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman, which showed a relatively weak susceptibility. Gibberellin (GA levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA. Higher zeatin riboside (ZR content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA content, polyphenol oxidase (PPO and peroxidase (POD activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.

Layer-by-Layer Assembly of Glucose Oxidase on Carbon Nanotube Modified Electrodes.

Science.gov (United States)

Suroviec, Alice H

2017-01-01

The use of enzymatically modified electrodes for the detection of glucose or other non-electrochemically active analytes is becoming increasingly common. Direct heterogeneous electron transfer to glucose oxidase has been shown to be kinetically difficult, which is why electron transfer mediators or indirect detection is usually used for monitoring glucose with electrochemical sensors. It has been found, however, that electrodes modified with single or multi-walled carbon nanotubes (CNTs) demonstrate fast heterogeneous electron transfer kinetics as compared to that found for traditional electrodes. Incorporating CNTs into the assembly of electrochemical glucose sensors, therefore, affords the possibility of facile electron transfer to glucose oxidase, and a more direct determination of glucose. This chapter describes the methods used to use CNTs in a layer-by-layer structure along with glucose oxidase to produce an enzymatically modified electrode with high turnover rates, increased stability and shelf-life.

Gravity Responsive NADH Oxidase of the Plasma Membrane

Science.gov (United States)

Morre, D. James (Inventor)

2002-01-01

A method and apparatus for sensing gravity using an NADH oxidase of the plasma membrane which has been found to respond to unit gravity and low centrifugal g forces. The oxidation rate of NADH supplied to the NADH oxidase is measured and translated to represent the relative gravitational force exerted on the protein. The NADH oxidase of the plasma membrane may be obtained from plant or animal sources or may be produced recombinantly.

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

Luminescent properties of composite scintillators based on PPO and o-POPOP doped SiO{sub 2} xerogel matrices

Energy Technology Data Exchange (ETDEWEB)

Viagin, O., E-mail: viagin@isma.kharkov.ua [Institute for Scintillation Materials of NAS of Ukraine, 60 Science Ave., 61001 Kharkiv (Ukraine); Masalov, A.; Bespalova, I.; Zelenskaya, O.; Tarasov, V.; Seminko, V.; Voloshina, L. [Institute for Scintillation Materials of NAS of Ukraine, 60 Science Ave., 61001 Kharkiv (Ukraine); Zorenko, Yu. [Institute of Physics of Kazimierz Wielki University of Bydgoszcz, 2 Powstańców Wielkopolskich str., 85-090 Bydgoszcz (Poland); Malyukin, Yu. [Institute for Scintillation Materials of NAS of Ukraine, 60 Science Ave., 61001 Kharkiv (Ukraine)

2016-11-15

New composite scintillation detectors were obtained by incorporation of PPO and o-POPOP organic scintillators into porous sol–gel silica matrices. Composites possess high photoluminescence intensity and decay time in nanosecond range. The absolute light yield of composite scintillators at excitation by alpha-radiation is about 4000–5000 photons/MeV and the pulse–height resolution is about 30%. The investigations of time-resolved luminescence of composites performed under excitation by synchrotron radiation in the 3.7–25 eV range have shown that the non-radiative energy transfer between host matrix and dopant molecules occurs via singlet states of SiO{sub 2} oxygen-deficient centers.

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.

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.

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.

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

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

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

Calcium transport in vesicles energized by cytochrome oxidase

Energy Technology Data Exchange (ETDEWEB)

Rosier, Randy N. [Univ. of Rochester, NY (United States)

1979-01-01

Experiments on the reconstitution of cytochrome oxidase into phospholipid vesicles were carried out using techniques of selectivity energizing the suspensions with ascorbate and cytochrome c or ascorbate, PMS, and internally trapped cytochrome c. It was found that the K⁺ selective ionophore valinomycin stimulated the rate of respiration of cytochrome oxidase vesicles regardless of the direction of the K⁺ flux across the vesicle membranes. The stimulation occurred in the presence of protonophoric uncouplers and in the complete absence of potassium or in detergent-lysed suspensions. Gramicidin had similar effects and it was determined that the ionophores acted by specific interaction with cytochrome oxidase rather than by the previously assumed collapse of membrane potentials. When hydrophobic proteins and appropriate coupling factors were incorporated into the cytochrome oxidase, vesicles phosphorylation of ADP could be coupled to the oxidation reaction of cytochrome oxidase. Relatively low P:O, representing poor coupling of the system, were problematical and precluded measurements of protonmotive force. However the system was used to study ion translocation.

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

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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.

Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells.

Science.gov (United States)

Kim, Tae Hwan; Kim, Ju Sung; Kim, Zoo Haye; Huang, Ren Bin; Chae, Young Lye; Wang, Ren Sheng

2014-07-10

Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an

A Plastid Terminal Oxidase Associated with Carotenoid Desaturation during Chromoplast Differentiation1

Science.gov (United States)

Josse, Eve-Marie; Simkin, Andrew J.; Gaffé, Joël; Labouré, Anne-Marie; Kuntz, Marcel; Carol, Pierre

2000-01-01

The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and ζ-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development. PMID:10938359

Lysyl oxidase in colorectal cancer

DEFF Research Database (Denmark)

Cox, Thomas R; Erler, Janine T

2013-01-01

Colorectal cancer is the third most prevalent form of cancer worldwide and fourth-leading cause of cancer-related mortality, leading to ~600,000 deaths annually, predominantly affecting the developed world. Lysyl oxidase is a secreted, extracellular matrix-modifying enzyme previously suggested...... to act as a tumor suppressor in colorectal cancer. However, emerging evidence has rapidly implicated lysyl oxidase in promoting metastasis of solid tumors and in particular colorectal cancer at multiple stages, affecting tumor cell proliferation, invasion, and angiogenesis. This emerging research has...... advancements in the field of colorectal cancer....

Crystallization and preliminary X-ray analysis of Aspergillus oryzae catechol oxidase

International Nuclear Information System (INIS)

Kaljunen, Heidi; Gasparetti, Chiara; Kruus, Kristiina; Rouvinen, Juha; Hakulinen, Nina

2011-01-01

Catechol oxidase from A. oryzae was crystallized by the hanging-drop vapour-diffusion method. Catechol oxidase is an enzyme that catalyzes the oxidation of o-diphenols to the corresponding o-quinones. It is a copper-containing enzyme with a binuclear copper active site. Here, the crystallization and multiple-wavelength anomalous dispersion data collection of catechol oxidase from the mould fungus Aspergillus oryzae are described. During the purification, three forms of the enzyme (39.3, 40.5 and 44.3 kDa) were obtained. A mixture of these three forms was initially crystallized and gave crystals that diffracted to 2.5 Å resolution and belonged to space group P3 2 21, with unit-cell parameters a = b = 118.9, c = 84.5 Å, α = β = 90, γ = 120°. A preparation containing only the shorter form (39.3 kDa) produced crystals that diffracted to 2.9 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 51.8, b = 95.3, c = 139.5 Å, α = β = γ = 90°

Effect of retinal impulse blockage on cytochrome oxidase-poor interpuffs in the macaque striate cortex: quantitative EM analysis of neurons.

Science.gov (United States)

Wong-Riley, M T; Trusk, T C; Kaboord, W; Huang, Z

1994-09-01

One of the hallmarks of the primate striate cortex is the presence of cytochrome oxidase-rich puffs in its supragranular layers. Neurons in puffs have been classified as type A, B, and C in ascending order of cytochrome oxidase content, with type C cells being the most vulnerable to retinal impulse blockade. The present study aimed at analysing cytochrome oxidase-poor interpuffs with reference to their metabolic cell types and the effect of intraretinal tetrodotoxin treatment. The same three metabolic types were found in interpuffs, except that type B and C neurons were smaller and less cytochrome oxidase-reactive in interpuffs than in puffs. Type A neurons had small perikarya, low levels of cytochrome oxidase, and received exclusively symmetric axosomatic synapses. The largest neurons were pyramidal, type B cells with moderate cytochrome oxidase activity and were also contacted exclusively by symmetric axosomatic synapses. Type C cells medium-sized with a rich supply of large, darkly reactive mitochondria and possessed all the characteristics of GABAergic neurons. They were the only cell type that received both symmetric and asymmetric axosomatic synapses. Two weeks of monocular tetrodotoxin blockade in adult monkeys caused all three major cell types in deprived interpuffs to suffer a significant downward shift in the size and cytochrome oxidase reactivity of their mitochondria, but the effects were more severe in type B and C neurons. In nondeprived interpuffs, all three cell types gained both in size and absolute number of mitochondria, and type A cells also had an elevated level of cytochrome oxidase, indicating that they might be functioning at a competitive advantage over cells in deprived columns. However, type B and C neurons showed a net loss of darkly reactive mitochondria, indicating that these cells became less active. Thus, mature interpuff neurons remained vulnerable to retinal impulse blockade and the metabolic capacity of these cells remains tightly

A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen.

Science.gov (United States)

Jeong, Eunjoo; Houn, Thavrak; Kuk, Yongin; Kim, Eun-Seon; Chandru, Hema Kumar; Baik, Myunggi; Back, Kyoungwhan; Guh, Ja-Ock; Han, Oksoo

2003-10-01

In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.

Isolation and Characterization of a Catabolite Repression-Insensitive Mutant of a Methanol Yeast, Candida boidinii A5, Producing Alcohol Oxidase in Glucose-Containing Medium

OpenAIRE

Sakai, Yasuyoshi; Sawai, Tohru; Tani, Yoshiki

1987-01-01

Mutants exhibiting alcohol oxidase (EC 1.1.3.13) activity when grown on glucose in the presence of methanol were found among 2-deoxyglucose-resistant mutants derived from a methanol yeast, Candida boidinii A5. One of these mutants, strain ADU-15, showed the highest alcohol oxidase activity in glucose-containing medium. The growth characteristics and also the induction and degradation of alcohol oxidase were compared with the parent strain and mutant strain ADU-15. In the parent strain, initia...

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

Erv2p: characterization of the redox behavior of a yeast sulfhydryl oxidase

DEFF Research Database (Denmark)

Wang, Wenzhong; Winther, Jakob R; Thorpe, Colin

2007-01-01

centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter...... and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative......The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox...

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.

fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils

Directory of Open Access Journals (Sweden)

María A. Hidalgo

2015-01-01

Full Text Available N-Formyl-methionyl-leucyl-phenylalanine (fMLP and platelet-activating factor (PAF induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8 release and nicotinamide adenine dinucleotide phosphate reduced (NADPH oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP, diphenyleneiodonium (DPI, and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

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.

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....

In vitro analysis of the role of glucose oxidase from Talaromyces flavus in biocontrol of the plant pathogen Verticillium dahliae.

OpenAIRE

Stosz, S K; Fravel, D R; Roberts, D P

1996-01-01

Culture filtrates from Talaromyces flavus grown on glucose contained high levels of glucose oxidase activity, while culture filtrates from T. flavus grown on xylan contained negligible glucose oxidase activity. Culture filtrates from T-flavus grown on both media contained complex protein profiles. However, only culture filtrates from T. flavus grown on glucose inhibited germination of microsclerotia of Verticillium dahliae in in vitro inhibition assays. A polyclonal antiserum preparation, pAB...

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.

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.

Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin

NARCIS (Netherlands)

Heuvel, van den R.H.H.; Berg, van den W.A.M.; Rovida, S.; Berkel, van W.J.H.

2004-01-01

The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol

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.

Unique role of NADPH oxidase 5 in oxidative stress in human renal proximal tubule cells

Directory of Open Access Journals (Sweden)

Peiying Yu

2014-01-01

Full Text Available NADPH oxidases are the major sources of reactive oxygen species in cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5 gene is present in humans but not rodents. Because Nox isoforms in renal proximal tubules (RPTs are involved in the pathogenesis of hypertension, we tested the hypothesis that NOX5 is differentially expressed in RPT cells from normotensive (NT and hypertensive subjects (HT. We found that NOX5 mRNA, total NOX5 protein, and apical membrane NOX5 protein were 4.2±0.7-fold, 5.2±0.7-fold, and 2.8±0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was 4.5±0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2±0.2-fold greater in HT than NT (P=<0.001, n=6–14/group. Ionomycin increased total NOX and NOX5 activities in RPT cells from HT (P<0.01, n=4, ANOVA, effects that were abrogated by pre-treatment of the RPT cells with diphenylene-iodonium or superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase activity (−45.1±3.2% vs. mock-siRNA, n=6–8 in HT. D1-like receptor stimulation decreased NADPH oxidase activity to a greater extent in NT (−32.5±1.8% than HT (−14.8±1.8. In contrast to the marked increase in expression and activity of NOX5 in HT, NOX1 mRNA and protein were minimally increased in HT, relative to NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT, relative to NT, was much less than those observed with NOX5. Thus, we demonstrate, for the first time, that NOX5 is expressed in human RPT cells and to greater extent than the other Nox isoforms in HT than NT. We suggest that the increased expression of NOX5, which may be responsible for the increased oxidative stress in RPT cells in human essential hypertension, is caused, in part, by a defective renal dopaminergic system.

Cloning and Functional Analysis of the Promoter of an Ascorbate Oxidase Gene from Gossypium hirsutum

OpenAIRE

Xin, Shan; Tao, Chengcheng; Li, Hongbin

2016-01-01

Apoplastic ascorbate oxidase (AO) plays significant roles in plant cell growth. However, the mechanism of underlying the transcriptional regulation of AO in Gossypium hirsutum remains unclear. Here, we obtained a 1,920-bp promoter sequence from the Gossypium hirsutum ascorbate oxidase (GhAO1) gene, and this GhAO1 promoter included a number of known cis-elements. Promoter activity analysis in overexpressing pGhAO1::GFP-GUS tobacco (Nicotiana benthamiana) showed that the GhAO1 promoter exhibite...

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.

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

Monoamine Oxidase Inhibitors (MAOIs)