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Sample records for oxidizing enzymes elucidating

  1. Enzymes of respiratory iron oxidation

    SciTech Connect

    Blake, R. II.

    1991-01-01

    This report focuses on the progress made in three areas of research concerned with enzymes involved in respiratory iron oxidation. The three areas are as follows: development of an improved procedure for the routine large scale culture of iron oxidizing chemolithotrophs based on the in-situ electrolysis of the soluble iron in the growth medium; to perform iron oxidation kinetic studies on whole cells using the oxygen electrode; and to identify, separate, purify, and characterize the individual cellular components.

  2. Elucidating nitric oxide synthase domain interactions by molecular dynamics.

    PubMed

    Hollingsworth, Scott A; Holden, Jeffrey K; Li, Huiying; Poulos, Thomas L

    2016-02-01

    Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing L-Arg to NO and L-citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH-derived electrons are transferred to the heme-containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN-to-heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme-FMN-calmodulin NOS complex based on the recent biophysical studies using a 105-ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis.

  3. Mechanism elucidation of the radical SAM enzyme spore photoproduct lyase (SPL)

    PubMed Central

    Li, Lei

    2011-01-01

    Spore photoproduct lyase (SPL) repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct or SP at the bacterial early germination phase. SP is the exclusive DNA photo-damage product in bacterial endospores; its generation and swift repair by SPL are responsible for the spores’ extremely high UV resistance. The early in vivo studies suggested that SPL utilizes a direct reversal strategy to repair the SP in the absence of light. The research in the past decade further established SPL as a radical SAM enzyme, which utilizes a tri-cysteine CXXXCXXC motif to harbor a [4Fe-4S] cluster. At the 1+ oxidation state, the cluster provides an electron to the S-adenosylmethionine (SAM), which binds to the cluster in a bidentate manner as the fourth and fifth ligands, to reductively cleave the C-S bond associated with the sulfonium ion in SAM, generating a reactive 5′-deoxyadenosyl (5′-dA) radical. This 5′-dA radical abstracts the proR hydrogen atom from the C6 carbon of SP to initiate the repair process; the resulting SP radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired TpT. SAM is suggested to be regenerated at the end of each catalytic cycle; and only a catalytic amount of SAM is needed in the SPL reaction. The H atom source for the back donation step is suggested to be a cysteine residue (C141 in B. subtilis SPL), and the H-atom transfer reaction leaves a thiyl radical behind on the protein. This thiyl radical thus must participate in the SAM regeneration process; however how the thiyl radical abstracts an H atom from the 5′-dA to regenerate SAM is unknown. This paper reviews and discusses the history and the latest progress in the mechanistic elucidation of SPL. Despite some recent breakthroughs, more questions are raised in the mechanistic understanding of this intriguing DNA repair enzyme. PMID:22197590

  4. Bacterial sulfite-oxidizing enzymes.

    PubMed

    Kappler, Ulrike

    2011-01-01

    Enzymes belonging to the Sulfite Oxidase (SO) enzyme family are found in virtually all forms of life, and are especially abundant in prokaryotes as shown by analysis of available genome data. Despite this fact, only a limited number of bacterial SO family enzymes has been characterized in detail to date, and these appear to be involved in very different metabolic processes such as energy generation from sulfur compounds, host colonization, sulfite detoxification and organosulfonate degradation. The few characterized bacterial SO family enzymes also show an intriguing range of structural conformations, including monomeric, dimeric and heterodimeric enzymes with varying numbers and types of redox centres. Some of the bacterial enzymes even catalyze novel reactions such as dimethylsulfoxide reduction that previously had been thought not to be catalyzed by SO family enzymes. Classification of the SO family enzymes based on the structure of their Mo domain clearly shows that three distinct groups of enzymes belong to this family, and that almost all SOEs characterized to date are representatives of the same group. The widespread occurrence and obvious structural and functional plasticity of the bacterial SO family enzymes make this an exciting field for further study, in particular the unraveling of the metabolic roles of the three enzyme groups, some of which appear to be associated almost exclusively with pathogenic microorganisms. Copyright © 2010 Elsevier B.V. All rights reserved.

  5. The Mismetallation of Enzymes during Oxidative Stress*

    PubMed Central

    Imlay, James A.

    2014-01-01

    Mononuclear iron enzymes can tightly bind non-activating metals. How do cells avoid mismetallation? The model bacterium Escherichia coli may control its metal pools so that thermodynamics favor the correct metallation of each enzyme. This system is disrupted, however, by superoxide and hydrogen peroxide. These species oxidize ferrous iron and thereby displace it from many iron-dependent mononuclear enzymes. Ultimately, zinc binds in its place, confers little activity, and imposes metabolic bottlenecks. Data suggest that E. coli compensates by using thiols to extract the zinc and by importing manganese to replace the catalytic iron atom. Manganese resists oxidants and provides substantial activity. PMID:25160623

  6. Enzymes of respiratory iron oxidation

    SciTech Connect

    Blake, R. II.

    1992-01-01

    This report describes experimental progress in characterizing and identifying redox proteins in a number of iron-oxidizing bacteria. Sections of the paper are entitled (1) In Situ electrolysis was explored to achieve enhanced yields of iron-oxidizing bacteria, (2)Structure/function studies were performed on redox-active biomolecules from Thiobacillus ferrooxidans, (3) Novel redox-active biomolecules were demonstrated in other iron autotrophs, and (4) New probes of metalloprotein electron-transfer reactions were synthesized and characterized.

  7. Oxidative Reactions with Nonaqueous Enzymes

    SciTech Connect

    Jonathan S. Dordick; Douglas Clark; Brian H Davison; Alexander Klibanov

    2001-12-30

    The objective of this work is to demonstrate a proof-of-concept of enzymatic oxidative processing in nonaqueous media using alkene epoxidation and phenolic polymerization as relevant targets. This project will provide both the fundamental and applied investigations necessary to initiate the implementation of oxidative biocatalysts as commercially relevant alternatives to chemical processing in general, and to phenolic polymerizations and alkene epoxidation specifically. Thus, this work will address the Bioprocessing Solicitation Area to: (1) makes major improvements to phenolic polymerization and alkene epoxidation technologies; (2) is expected to be cost competitive with competing conventional processes; and (3) produces higher yields with less waste.

  8. Untargeted Analysis of Mass Spectrometry Data for Elucidation of Metabolites and Function of Enzymes

    PubMed Central

    Sanchez-Ponce, Raymundo; Guengerich, F. Peter

    2012-01-01

    A Matlab®-based computer program termed “Discovery of General Endo- and Xenobiotics” (DoGEX) was developed, which uses wavelets and morphological analysis to process liquid chromatography-mass spectrometry (LC-MS) data. The output of the program is a list of integration areas as a function of retention time and molecular mass. A feature of the computer program is spectral filtering to facilitate the detection of chromatographic peaks with a particular isotopic ratio. The program DoGEX was used to automatically select oxidation products formed from felodipine (i.e. two chlorine atoms) and bromocriptine (one bromine atom) with cytochrome P450 3A4. The recognized isotope ratio can be changed to permit a natural or artificial mixture of isotopes to be monitored for selections. This computer program can be used to analyze LC-MS data for untargeted metabolic profiling experiments, e.g. to assign endogenous functions to newly characterized cytochrome P450 enzymes. In a representative example, an incubation of testosterone, NADPH, and a 1:1 16O2:18O2 mixture yielded products with M and M+2 ions resembling bromine doublets. Another use of the program is the subtraction of one set of tR, m/z data from another, e.g. in comparisons of changes in patterns during enzyme reactions. PMID:17408243

  9. Biogeochemistry of nitrous oxide in Lake Kizaki, Japan, elucidated by nitrous oxide isotopomer analysis

    NASA Astrophysics Data System (ADS)

    Sasaki, Y.; Koba, K.; Yamamoto, M.; Makabe, A.; Ueno, Y.; Nakagawa, M.; Toyoda, S.; Yoshida, N.; Yoh, M.

    2011-12-01

    The biogeochemistry of nitrous oxide (N2O) was investigated in Lake Kizaki, Japan, where accumulation of N2O in the water column has been observed. The N2O concentration profile showed weak accumulation in the oxic zone, although much higher and much lower N2O concentrations were observed in the deeper oxygen-deficient zone. Intramolecular partitioning of 15N (site preference) of N2O within the oxic zone increased concomitantly with increased N2O concentration. The site preference of the newly produced N2O in the oxic zone was estimated as 33.6‰. This high site preference strongly suggests that this N2O was produced by hydroxylamine oxidation. In regions of the oxygen-deficient zone, the nitrate (NO3-) concentration decreased rapidly, concomitantly with increased nitrogen ? and oxygen ? isotope ratios, indicating denitrification. The high site preference and nitrogen isotope ratio of N2O (δ15Nbulk), combined with isotopic data of NO3-, strongly suggest denitrification as the main N2O source. Moreover, site preference and δ15Nbulk data suggest that the existing N2O in the oxygen-deficient zone was already strongly reduced (more than 75%) to N2. Results of this study demonstrate the feasibility of using isotope and isotopomer analyses of N compounds to elucidate the complex biogeochemistry of N2O in an intact ecosystem.

  10. Constructing Kinetic Network Models to Elucidate Mechanisms of Functional Conformational Changes of Enzymes and Their Recognition with Ligands.

    PubMed

    Zhang, L; Jiang, H; Sheong, F K; Pardo-Avila, F; Cheung, P P-H; Huang, X

    2016-01-01

    Enzymes are biological macromolecules that catalyze complex reactions in life. In order to perform their functions effectively and efficiently, enzymes undergo conformational changes between different functional states. Therefore, elucidating the dynamics between these states is essential to understand the molecular mechanisms of enzymes. Although experimental methods such as X-ray crystallography and cryoelectron microscopy can produce high-resolution structures, the detailed conformational dynamics of many enzymes still remain obscure. While molecular dynamics (MD) simulations are able to complement the experiments by providing structure-based dynamics at atomic resolution, it is usually difficult for them to reach the biologically relevant timescales (hundreds of microseconds or longer). Kinetic network models (KNMs), in particular Markov state models (MSMs), hold great promise to overcome this challenge because they can bridge the timescale gap between MD simulations and experimental observations. In this chapter, we review the procedure of constructing KNMs to elucidate the molecular mechanisms of enzymes. First, we will give a general introduction of MSMs, including the methods to construct and validate MSMs. Second, we will present the applications of KNMs to study two important enzymes: the human Argonaute protein and the RNA polymerase II. We conclude by discussing the future perspectives regarding the potential of KNMs to investigate the dynamics of enzymes' functional conformational changes.

  11. Bleaching with lignin-oxidizing enzymes.

    PubMed

    Bajpai, Pratima; Anand, Aradhna; Bajpai, Pramod K

    2006-01-01

    General concern about the environmental impact of chlorine bleaching effluents has led to a trend towards elementary chlorine-free or totally chlorine free bleaching methods. Considerable interest has been focused on the use of biotechnology in pulp bleaching, as large number of microbes and the enzymes produced by them are known to be capable of preferential degradation of native lignin and complete degradation of wood. Enzymes of the hemicellulolytic type, particularly xylan-attacking enzymes xylanases are now used commercially in the mills for pulp treatment and subsequent incorporation into bleach sequences. Certain white-rot fungi can delignify Kraft pulps increasing their brightness and their responsiveness to brightening with chemicals. The fungal treatments are too slow but the enzymes produced from the fungi can also delignify pulps and these enzymatic processes are likely to be easier to optimize and apply than the fungal treatments. This article presents an overview of the developments in the application of lignin-oxidizing enzymes in bleaching of chemical pulps. The present knowledge of the mechanisms on the action of enzymes as well as the practical results and advantages obtained on the laboratory and industrial scale are discussed.

  12. [Oxidative inactivation of angiotensin-converting enzyme].

    PubMed

    Sakharov, I Iu; Dukhanina, E A; Puchnina, E A; Danilov, S M; Muzykantov, V R

    1991-01-01

    Hydrogen peroxide inactivates the purified human angiotensin-converting enzyme (ACE) in vitro; the inactivating effect of H2O2 is eliminated by an addition of catalase. The lung and kidney ACE are equally sensitive to the effect of hydrogen peroxide. After addition of oxidants (H2O2 alone or H2O2 + ascorbate or H2O2 + Fe2+ mixtures) to the membranes or homogenates of the lung, the inactivation of membrane-bound ACE is far less pronounced despite the large-scale accumulation of lipid peroxidation products. The marked inactivation of ACE in the membrane fraction (up to 55% of original activity) was observed during ACE incubation with a glucose:glucose oxidase:Fe2+ mixture. Presumably the oxidative potential of H2O2 in tissues in consumed, predominantly, for the oxidation of other components of the membrane (e.g., lipids) rather than for ACE inactivation.

  13. Biogeochemistry of nitrous oxide in groundwater in a forested ecosystem elucidated by nitrous oxide isotopomer measurements

    NASA Astrophysics Data System (ADS)

    Koba, K.; Osaka, K.; Tobari, Y.; Toyoda, S.; Ohte, N.; Katsuyama, M.; Suzuki, N.; Itoh, M.; Yamagishi, H.; Kawasaki, M.; Kim, S. J.; Yoshida, N.; Nakajima, T.

    2009-06-01

    The biological and physical controls on microbial processes that produce and consume N 2O in soils are highly complex. Isotopomer ratios of N 2O, with abundance of 14N 15N 16O, 15N 14N 16O, and 14N 14N 18O relative to 14N 14N 16O, are promising for elucidation of N 2O biogeochemistry in an intact ecosystem. Site preference, the nitrogen isotope ratio of the central nitrogen atom minus that of the terminal nitrogen atom, is useful to distinguish between N 2O via hydroxylamine oxidation and N 2O via nitrite reduction. We applied this isotopomer analysis to a groundwater system in a temperate coniferous-forested ecosystem. Results of a previous study at this location showed that the N 2O concentration in groundwater varied greatly according to groundwater chemistry, i.e. NO 3-, DOC, and DO, although apportionment of N 2O production to nitrification or denitrification was ambiguous. Our isotopic analysis (δ 15N and δ 18O) of NO 3- and N 2O implies that denitrification is the dominant production process of N 2O, but definitive information is not derived from δ 15N and δ 18O analysis because of large variations in isotopic fractionations during production and consumption of N 2O. However, the N 2O site preference and the difference in δ 15N between NO 3- and N 2O indicate that nitrification contributes to total N 2O production and that most measured N 2O has been subjected to further N 2O reduction to N 2. The implications of N 2O biogeochemistry derived from isotope and isotopomer data differ entirely from those derived from conventional concentration data of DO, NO 3-, and N 2O. That difference underscores the need to reconsider our understanding of the N cycle in the oxic-anoxic interface.

  14. Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes

    NASA Astrophysics Data System (ADS)

    Carlson, Jacob C.; Li, Shengying; Gunatilleke, Shamila S.; Anzai, Yojiro; Burr, Douglas A.; Podust, Larissa M.; Sherman, David H.

    2011-08-01

    Elucidation of natural product biosynthetic pathways provides important insights into the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here, we show full reconstitution, in vitro, of an unusual multi-step oxidative cascade for post-assembly-line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and a flavin adenine dinucleotide-dependent oxidase (TamL), which act co-dependently through the repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a previously unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C10 oxidation of TirE.

  15. Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes.

    PubMed

    Carlson, Jacob C; Li, Shengying; Gunatilleke, Shamila S; Anzai, Yojiro; Burr, Douglas A; Podust, Larissa M; Sherman, David H

    2011-07-17

    Elucidation of natural product biosynthetic pathways provides important insights into the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here, we show full reconstitution, in vitro, of an unusual multi-step oxidative cascade for post-assembly-line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and a flavin adenine dinucleotide-dependent oxidase (TamL), which act co-dependently through the repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a previously unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C10 oxidation of TirE.

  16. Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity

    PubMed Central

    Funk, Michael A.; Judd, Evan T.; Marsh, E. Neil G.; Elliott, Sean J.; Drennan, Catherine L.

    2014-01-01

    Anaerobic degradation of the environmental pollutant toluene is initiated by the glycyl radical enzyme benzylsuccinate synthase (BSS), which catalyzes the radical addition of toluene to fumarate, forming benzylsuccinate. We have determined crystal structures of the catalytic α-subunit of BSS with its accessory subunits β and γ, which both bind a [4Fe-4S] cluster and are essential for BSS activity in vivo. We find that BSSα has the common glycyl radical enzyme fold, a 10-stranded β/α-barrel that surrounds the glycyl radical cofactor and active site. Both accessory subunits β and γ display folds related to high potential iron–sulfur proteins but differ substantially from each other in how they interact with the α-subunit. BSSγ binds distally to the active site, burying a hydrophobic region of BSSα, whereas BSSβ binds to a hydrophilic surface of BSSα that is proximal to the active site. To further investigate the function of BSSβ, we determined the structure of a BSSαγ complex. Remarkably, we find that the barrel partially opens, allowing the C-terminal region of BSSα that houses the glycyl radical to shift within the barrel toward an exit pathway. The structural changes that we observe in the BSSαγ complex center around the crucial glycyl radical domain, thus suggesting a role for BSSβ in modulating the conformational dynamics required for enzyme activity. Accompanying proteolysis experiments support these structural observations. PMID:24982148

  17. Thymidine kinase 2 enzyme kinetics elucidate the mechanism of thymidine-induced mitochondrial DNA depletion.

    PubMed

    Sun, Ren; Wang, Liya

    2014-10-07

    Mitochondrial thymidine kinase 2 (TK2) is a nuclear gene-encoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis-Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in late-onset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.

  18. Isolation, structural elucidation, and biological evaluation of a 5-hydroxymethyl-2-furfural derivative, asfural, from enzyme-treated asparagus extract.

    PubMed

    Ito, Tomohiro; Sato, Atsuya; Ono, Tomoko; Goto, Kazunori; Maeda, Takahiro; Takanari, Jun; Nishioka, Hiroshi; Komatsu, Kenichi; Matsuura, Hideyuki

    2013-09-25

    A novel 5-hydroxymethyl-2-furfural (HMF; 1) derivative, which is named asfural (compound 2), was isolated from enzyme-treated asparagus extract (ETAS) along with HMF (1) as a heat shock protein 70 (HSP70) inducible compound. The structure of compound 2 was elucidated on the basis of its spectroscopic data from HREIMS and NMR, whereas the absolute configuration was determined using chiral HPLC analysis, compared to two synthesized compounds, (S)- and (R)-asfural. As a result, compound 2 derived from ETAS was assigned as (S)-(2-formylfuran-5-yl)methyl 5-oxopyrrolidine-2-carboxylate. When compound 2, synthesized (S)- and (R)-asfural, and HMF (1) were evaluated in terms of HSP70 mRNA expression-enhancing activity in HL-60 cells, compound 2 and (S)-asfural significantly increased the expression level in a concentration-dependent manner. HMF (1) also showed significant activity at 0.25 mg/mL.

  19. Foot of the Wave Analysis for Mechanistic Elucidation and Benchmarking Applications in Molecular Water Oxidation Catalysis.

    PubMed

    Matheu, Roc; Neudeck, Sven; Meyer, Franc; Sala, Xavier; Llobet, Antoni

    2016-12-08

    The description of the foot of the wave analysis (FOWA) applied to the electrocatalytic oxidation of water to dioxygen is reported for cases where the rate determining step is first order and second order with regard to catalyst concentration, coinciding mechanistically with the so-called water nucleophilic attack (WNA) and the interaction of two M-O units (I2M, where M represents the metal center of the catalyst), respectively. The newly adapted equations are applied to a range of relevant molecular catalysts, both in homogeneous and heterogeneous phase, and the kinetic parameters are determined, including apparent rate constants and turnover frequencies. In this respect, the application of FOWA at different catalyst concentrations allows elucidation of the reaction mechanism that operates in each case. In addition, catalytic Tafel plots are used for assessing the performance of several molecular water oxidation catalysts (WOCs) as a function of overpotential under analogous conditions, and thus can be used for benchmarking purposes. This analysis was carried out earlier for oxide-based WOCs; however, this is the first report using molecular WOCs. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Elucidation of insulin degrading enzyme catalyzed site specific hydrolytic cleavage of amyloid beta peptide: a comparative density functional theory study.

    PubMed

    Bora, Ram Prasad; Ozbil, Mehmet; Prabhakar, Rajeev

    2010-05-01

    In this B3LYP study, the catalytic mechanisms for the hydrolysis of the three different peptide bonds (Lys28-Gly29, Phe19-Phe20, and His14-Gln15) of Alzheimer amyloid beta (Abeta) peptide by insulin-degrading enzyme (IDE) have been elucidated. For all these peptides, the nature of the substrate was found to influence the structure of the active enzyme-substrate complex. The catalytic mechanism is proposed to proceed through the following three steps: (1) activation of the metal-bound water molecule, (2) formation of the gem-diol intermediate, and (3) cleavage of the peptide bond. With the computed barrier of 14.3, 18.8, and 22.3 kcal/mol for the Lys28-Gly29, Phe19-Phe20, and His14-Gln15 substrates, respectively, the process of water activation was found to be the rate-determining step for all three substrates. The computed energetics show that IDE is the most efficient in hydrolyzing the Lys28-Gly29 (basic polar-neutral nonpolar) peptide bond followed by the Phe19-Phe20 (neutral nonpolar-neutral nonpolar) and His14-Gln15 (basic polar-neutral polar) bonds of the Abeta substrate.

  1. Iron Oxide Nanozyme: A Multifunctional Enzyme Mimetic for Biomedical Applications

    PubMed Central

    Gao, Lizeng; Fan, Kelong; Yan, Xiyun

    2017-01-01

    Iron oxide nanoparticles have been widely used in many important fields due to their excellent nanoscale physical properties, such as magnetism/superparamagnetism. They are usually assumed to be biologically inert in biomedical applications. However, iron oxide nanoparticles were recently found to also possess intrinsic enzyme-like activities, and are now regarded as novel enzyme mimetics. A special term, “Nanozyme”, has thus been coined to highlight the intrinsic enzymatic properties of such nanomaterials. Since then, iron oxide nanoparticles have been used as nanozymes to facilitate biomedical applications. In this review, we will introduce the enzymatic features of iron oxide nanozyme (IONzyme), and summarize its novel applications in biomedicine. PMID:28900505

  2. Chemical reactivity of graphene oxide towards amines elucidated by solid-state NMR

    NASA Astrophysics Data System (ADS)

    Vacchi, Isabella A.; Spinato, Cinzia; Raya, Jésus; Bianco, Alberto; Ménard-Moyon, Cécilia

    2016-07-01

    Graphene oxide (GO) is an attractive nanomaterial for many applications. Controlling the functionalization of GO is essential for the design of graphene-based conjugates with novel properties. But, the chemical composition of GO has not been fully elucidated yet. Due to the high reactivity of the oxygenated moieties, mainly epoxy, hydroxyl and carboxyl groups, several derivatization reactions may occur concomitantly. The reactivity of GO with amine derivatives has been exploited in the literature to design graphene-based conjugates, mainly through amidation. However, in this study we undoubtedly demonstrate using magic angle spinning (MAS) solid-state NMR that the reaction between GO and amine functions occurs via ring opening of the epoxides, and not by amidation. We also prove that there is a negligible amount of carboxylic acid groups in two GO samples obtained by a different synthesis process, hence eliminating the possibility of amidation reactions with amine derivatives. This work brings additional insights into the chemical reactivity of GO, which is fundamental to control its functionalization, and highlights the major role of MAS NMR spectroscopy for a comprehensive characterization of derivatized GO.Graphene oxide (GO) is an attractive nanomaterial for many applications. Controlling the functionalization of GO is essential for the design of graphene-based conjugates with novel properties. But, the chemical composition of GO has not been fully elucidated yet. Due to the high reactivity of the oxygenated moieties, mainly epoxy, hydroxyl and carboxyl groups, several derivatization reactions may occur concomitantly. The reactivity of GO with amine derivatives has been exploited in the literature to design graphene-based conjugates, mainly through amidation. However, in this study we undoubtedly demonstrate using magic angle spinning (MAS) solid-state NMR that the reaction between GO and amine functions occurs via ring opening of the epoxides, and not by

  3. Glycosylation of resveratrol protects it from enzymic oxidation.

    PubMed Central

    Regev-Shoshani, Gilly; Shoseyov, Oded; Bilkis, Itzhak; Kerem, Zohar

    2003-01-01

    Plant polyphenols, including dietary polyphenols such as resveratrol, are important components in the plant antioxidant and defence systems. They are also known to exert beneficial effects on human health through diet. As they are produced, these polyphenols may be subjected to deleterious enzymic oxidation by the plant polyphenol oxidases. They are generally synthesized as glycosides like 5,4'-dihydroxystilbene-3-O-beta-D-glucopyranoside, the 3-glucoside of resveratrol. The effects of the glycosylation and methylation of the parent resveratrol on its enzymic oxidation were studied. Methyl and glucosyl derivatives were synthesized using simple one-step methodologies. The kinetics of their enzymic oxidation by tyrosinases were defined. Substitution at the p-hydroxy group, by either glucose or methyl, abolished enzymic oxidation by both mushroom and grape tyrosinases. Substitution at the m-hydroxy group with methyl had a small effect, but substitution with glucose resulted in a much lower affinity of the enzymes for the glycoside. We suggest that glycosylation of polyphenols in nature helps to protect these vital molecules from enzymic oxidation, extending their half-life in the cell and maintaining their beneficial antioxidant capacity and biological properties. PMID:12697026

  4. Chemical reactivity of graphene oxide towards amines elucidated by solid-state NMR.

    PubMed

    Vacchi, Isabella A; Spinato, Cinzia; Raya, Jésus; Bianco, Alberto; Ménard-Moyon, Cécilia

    2016-07-14

    Graphene oxide (GO) is an attractive nanomaterial for many applications. Controlling the functionalization of GO is essential for the design of graphene-based conjugates with novel properties. But, the chemical composition of GO has not been fully elucidated yet. Due to the high reactivity of the oxygenated moieties, mainly epoxy, hydroxyl and carboxyl groups, several derivatization reactions may occur concomitantly. The reactivity of GO with amine derivatives has been exploited in the literature to design graphene-based conjugates, mainly through amidation. However, in this study we undoubtedly demonstrate using magic angle spinning (MAS) solid-state NMR that the reaction between GO and amine functions occurs via ring opening of the epoxides, and not by amidation. We also prove that there is a negligible amount of carboxylic acid groups in two GO samples obtained by a different synthesis process, hence eliminating the possibility of amidation reactions with amine derivatives. This work brings additional insights into the chemical reactivity of GO, which is fundamental to control its functionalization, and highlights the major role of MAS NMR spectroscopy for a comprehensive characterization of derivatized GO.

  5. Genetic Elucidation of Nitric Oxide Signaling in Incompatible Plant-Pathogen Interactions[w

    PubMed Central

    Zeier, Jürgen; Delledonne, Massimo; Mishina, Tatiana; Severi, Emmanuele; Sonoda, Masatoshi; Lamb, Chris

    2004-01-01

    Recent experiments indicate that nitric oxide (NO) plays a pivotal role in disease resistance and several other physiological processes in plants. However, most of the current information about the function of NO in plants is based on pharmacological studies, and additional approaches are therefore required to ascertain the role of NO as an important signaling molecule in plants. We have expressed a bacterial nitric oxide dioxygenase (NOD) in Arabidopsis plants and/or avirulent Pseudomonas syringae pv tomato to study incompatible plant-pathogen interactions impaired in NO signaling. NOD expression in transgenic Arabidopsis resulted in decreased NO levels in planta and attenuated a pathogen-induced NO burst. Moreover, NOD expression in plant cells had very similar effects on plant defenses compared to NOD expression in avirulent Pseudomonas. The defense responses most affected by NO reduction during the incompatible interaction were decreased H2O2 levels during the oxidative burst and a blockage of Phe ammonia lyase expression, the key enzyme in the general phenylpropanoid pathway. Expression of the NOD furthermore blocked UV light-induced Phe ammonia lyase and chalcone synthase gene expression, indicating a general signaling function of NO in the activation of the phenylpropanoid pathway. NO possibly functions in incompatible plant-pathogen interactions by inhibiting the plant antioxidative machinery, and thereby ensuring locally prolonged H2O2 levels. Additionally, albeit to a lesser extent, we observed decreases in salicylic acid production, a diminished development of hypersensitive cell death, and a delay in pathogenesis-related protein 1 expression during these NO-deficient plant-pathogen interactions. Therefore, this genetic approach confirms that NO is an important regulatory component in the signaling network of plant defense responses. PMID:15347797

  6. pH-Controlled Oxidation of an Aromatic Ketone: Structural Elucidation of the Products of Two Green Chemical Reactions

    ERIC Educational Resources Information Center

    Ballard, C. Eric

    2010-01-01

    A laboratory experiment emphasizing the structural elucidation of organic compounds has been developed as a discovery exercise. The "unknown" compounds are the products of the pH-controlled oxidation of 4'-methoxyacetophenone with bleach. The chemoselectivity of this reaction is highly dependent on the pH of the reaction media: under basic…

  7. pH-Controlled Oxidation of an Aromatic Ketone: Structural Elucidation of the Products of Two Green Chemical Reactions

    ERIC Educational Resources Information Center

    Ballard, C. Eric

    2010-01-01

    A laboratory experiment emphasizing the structural elucidation of organic compounds has been developed as a discovery exercise. The "unknown" compounds are the products of the pH-controlled oxidation of 4'-methoxyacetophenone with bleach. The chemoselectivity of this reaction is highly dependent on the pH of the reaction media: under basic…

  8. Ferulic Acid Orchestrates Anti-Oxidative Properties of Danggui Buxue Tang, an Ancient Herbal Decoction: Elucidation by Chemical Knock-Out Approach

    PubMed Central

    Gong, Amy G. W.; Huang, Vincent Y.; Wang, Huai Y.; Lin, Huang Q.; Dong, Tina T. X.; Tsim, Karl W. K.

    2016-01-01

    Ferulic acid, a phenolic acid derived mainly from a Chinese herb Angelica Sinensis Radix (ASR), was reported to reduce the formation of free radicals. Danggui Buxue Tang (DBT), a herbal decoction composing of Astragali Radix (AR) and ASR, has been utilized for more than 800 years in China having known anti-oxidative property. Ferulic acid is a major active ingredient in DBT; however, the role of ferulic acid within the herbal mixture has not been resolved. In order to elucidate the function of ferulic acid within this herbal decoction, a ferulic acid-depleted herbal decoction was created and named as DBTΔfa. The anti-oxidative properties of chemically modified DBT decoction were systemically compared in cultured H9C2 rat cardiomyoblast cell line. The application of DBT and DBTΔfa into the cultures showed functions in (i) decreasing the reactive oxygen species (ROS) formation, detected by laser confocal; (ii) increasing of the activation of Akt; (iii) increasing the transcriptional activity of anti-oxidant response element (ARE); and (iv) increasing the expressions of anti-oxidant enzymes, i.e. NQO1 and GCLM. In all scenario, the aforementioned anti-oxidative properties of DBTΔfa in H9C2 cells were significantly reduced, as compared to authentic DBT. Thus, ferulic acid could be an indispensable chemical in DBT to orchestrate multi-components of DBT as to achieve maximal anti-oxidative functions. PMID:27824860

  9. 99Tc and Re incorporated into metal oxide polyoxometalates: oxidation state stability elucidated by electrochemistry and theory.

    PubMed

    McGregor, Donna; Burton-Pye, Benjamin P; Mbomekalle, Israel M; Aparicio, Pablo A; Romo, Susanna; López, Xavier; Poblet, Josep M; Francesconi, Lynn C

    2012-08-20

    The radioactive element technetium-99 ((99)Tc, half-life = 2.1 × 10(5) years, β(-) of 253 keV), is a major byproduct of (235)U fission in the nuclear fuel cycle. (99)Tc is also found in radioactive waste tanks and in the environment at National Lab sites and fuel reprocessing centers. Separation and storage of the long-lived (99)Tc in an appropriate and stable waste-form is an important issue that needs to be addressed. Considering metal oxide solid-state materials as potential storage matrixes for Tc, we are examining the redox speciation of Tc on the molecular level using polyoxometalates (POMs) as models. In this study we investigate the electrochemistry of Tc complexes of the monovacant Wells-Dawson isomers, α(1)-P(2)W(17)O(61)(10-) (α1) and α(2)-P(2)W(17)O(61)(10-) (α2) to identify features of metal oxide materials that can stabilize the immobile Tc(IV) oxidation state accessed from the synthesized Tc(V)O species and to interrogate other possible oxidation states available to Tc within these materials. The experimental results are consistent with density functional theory (DFT) calculations. Electrochemistry of K(7-n)H(n)[Tc(V)O(α(1)-P(2)W(17)O(61))] (Tc(V)O-α1), K(7-n)H(n)[Tc(V)O(α(2)-P(2)W(17)O(61))] (Tc(V)O-α2) and their rhenium analogues as a function of pH show that the Tc-containing derivatives are always more readily reduced than their Re analogues. Both Tc and Re are reduced more readily in the lacunary α1 site as compared to the α2 site. The DFT calculations elucidate that the highest oxidation state attainable for Re is VII while, under the same electrochemistry conditions, the highest oxidation state for Tc is VI. The M(V)→ M(IV) reduction processes for Tc(V)O-α1 are not pH dependent or only slightly pH dependent suggesting that protonation does not accompany reduction of this species unlike the M(V)O-α2 (M = (99)Tc, Re) and Re(V)O-α1 where M(V/IV) reduction process must occur hand in hand with protonation of the terminal M═O to

  10. The Cytochemical Localization of Oxidative Enzymes

    PubMed Central

    Scarpelli, D. G.; Hess, R.; Pearse, A. G. E.

    1958-01-01

    Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 µ in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases. PMID:13610939

  11. Graphene oxide immobilized enzymes show high thermal and solvent stability

    NASA Astrophysics Data System (ADS)

    Hermanová, Soňa; Zarevúcká, Marie; Bouša, Daniel; Pumera, Martin; Sofer, Zdeněk

    2015-03-01

    The thermal and solvent tolerance of enzymes is highly important for their industrial use. We show here that the enzyme lipase from Rhizopus oryzae exhibits exceptionally high thermal stability and high solvent tolerance and even increased activity in acetone when immobilized onto a graphene oxide (GO) nanosupport prepared by Staudenmaier and Brodie methods. We studied various forms of immobilization of the enzyme: by physical adsorption, covalent attachment, and additional crosslinking. The activity recovery was shown to be dependent on the support type, enzyme loading and immobilization procedure. Covalently immobilized lipase showed significantly better resistance to heat inactivation (the activity recovery was 65% at 70 °C) in comparison with the soluble counterpart (the activity recovery was 65% at 40 °C). Physically adsorbed lipase achieved over 100% of the initial activity in a series of organic solvents. These findings, showing enhanced thermal stability and solvent tolerance of graphene oxide immobilized enzyme, will have a profound impact on practical industrial scale uses of enzymes for the conversion of lipids into fuels.The thermal and solvent tolerance of enzymes is highly important for their industrial use. We show here that the enzyme lipase from Rhizopus oryzae exhibits exceptionally high thermal stability and high solvent tolerance and even increased activity in acetone when immobilized onto a graphene oxide (GO) nanosupport prepared by Staudenmaier and Brodie methods. We studied various forms of immobilization of the enzyme: by physical adsorption, covalent attachment, and additional crosslinking. The activity recovery was shown to be dependent on the support type, enzyme loading and immobilization procedure. Covalently immobilized lipase showed significantly better resistance to heat inactivation (the activity recovery was 65% at 70 °C) in comparison with the soluble counterpart (the activity recovery was 65% at 40 °C). Physically adsorbed

  12. Enzymic oxidation of monoclonal antibodies by soluble and immobilized bifunctional enzyme complexes.

    PubMed

    Solomon, B; Koppel, R; Schwartz, F; Fleminger, G

    1990-06-27

    Site-specific modification of monoclonal antibodies was achieved by oxidation of the carbohydrate moieties of antibodies which are located remote from the antigen binding sites. Sialic acid and galactose are terminal sugars of these carbohydrate chains. Concomitant treatment of the antibodies with neuraminidase and galactose oxidase generated aldehyde groups in the oligosaccharide moieties of immunoglobulins which reacted selectively with amino or hydrazide groups of the matrix. Subsequent immobilization of neuraminidase and galactose oxidase on Eupergit C-adipic dihydrazide proved to be an efficient and selective system for the enzymic oxidation of the monoclonal antibodies without impairing their immunological activity. Oriented immobilization of enzymically oxidized monoclonal antibodies on hydrazide or amino Eupergit C derivatives thus leads to the formation of antibody matrix conjugates which possess high antigen-binding activities.

  13. Isolation, structure elucidation and enzyme inhibition studies of a new hydroxy ester and other compounds from Berberis jaeschkeana Schneid stem.

    PubMed

    Alamzeb, Muhammad; Khan, M Rafiullah; Mamoon-Ur-Rashid; Ali, Saqib; Khan, Ashfaq Ahmad

    2015-01-01

    Bioassay-guided isolation and fractionation of Berberis jaeschkeana Schneid var. jaeschkeana stem resulted in the isolation and characterisation of a new long chain hydroxy ester named as berberinol (1) along with six known compounds (2-7). All the structures were established from 1D and 2D spectroscopic data. Crude extract, sub-fractions and all the isolated compounds were evaluated for their anti-fungal and urease enzyme inhibition properties. All of the sub-fractions and compounds showed good anti-fungal and urease enzyme inhibition properties. Minimum inhibitory concentrations (MICs) were calculated for all active samples in case of urease enzyme inhibition. MICs values were found to be in the range of 39.03-49.78 μg/mL for urease enzyme inhibition.

  14. The Bremner factor for elucidation of nitrous oxide evolution from soils

    USDA-ARS?s Scientific Manuscript database

    It is well established that agricultural emissions of nitrous oxide are an important source of atmospheric green house gas loading implicated in global climate change. The atmospheric concentration of nitrous oxide has increased by 17% since the 1800’s and continues to increase at an exponential r...

  15. Enzyme kinetics of oxidative metabolism: cytochromes P450.

    PubMed

    Korzekwa, Ken

    2014-01-01

    The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 3 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapter 4 ). Models and equations that can result in these kinetic profiles will be presented and discussed.

  16. Theoretical elucidation of the competitive electro-oxidation mechanisms of formic acid on Pt(111).

    PubMed

    Gao, Wang; Keith, John A; Anton, Josef; Jacob, Timo

    2010-12-29

    The mechanisms of formic acid (HCOOH) oxidation on Pt(111) under electrochemical conditions have been studied using density functional theory and then compared with the analogous gas-phase reaction. Results show that HCOOH oxidation under a water-covered surface behaves substantially differently than in the gas phase or using a solvation model involving only a few water molecules. Using these models, we evaluated the detailed reaction process, including energies and geometric structures of intermediates and transition states under the influence of different solvation models and electrode potentials. Our calculations indicate that this potential-dependent electrochemical oxidation proceeds via a multipath mechanism (involving both the adsorbed HCOOH and HCOO intermediates), a result succinctly rationalizing conflicting experimental observations. Moreover, this study highlights how subtle changes in electrochemical reaction environments can influence (electro)catalysis.

  17. The Degradation Chemistry of Farglitazar and Elucidation of the Oxidative Degradation Mechanisms.

    PubMed

    Reynolds, Dan W; Campbell, John M; Johnson, Byron S; Joshi, Biren K; Facchine, Kevin L; Long, Stacey; O'Connell, Thomas M; Paulus, Iris V; Sides, Scott L; Kraft, Eric S; Wolters, Andrew M

    2017-04-01

    The chemical degradation of farglitazar (1) was investigated using a series of controlled stress testing experiments. Farglitazar drug substance was stressed under acidic, natural pH, basic, and oxidative conditions in solution. In the solid state, the drug substance was stressed with heat, high humidity, and light. Farglitazar was found to be most labile toward oxidative stress. A series of mechanistic experiments are described in which the use of (18)O-labelled oxygen demonstrated that oxidative degradation of farglitazar is caused primarily by singlet oxygen formed under thermal conditions. Major degradation products were isolated and fully characterized. Mechanisms for the formation of degradation products are proposed. Drug product tablets were also stressed in the solid state with heat, high humidity, and light. Stressed tablets afforded many of the same degradation products observed during drug substance stress testing, with oxidation again being the predominant degradation pathway. Evidence for the activity of singlet oxygen, formed during thermal stress testing of the solid oral dosage form, is presented. The degradation pathways observed during stress testing matched those observed during long-term stability trials of the drug product. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  18. Electrochemical oxidation of xanthosine. Isolation and structure elucidation of a new dimeric xanthine nucleoside

    SciTech Connect

    Xinhua Ji; Subramanian, P.; van der Helm, D.; Dryhurst, G. )

    1990-02-16

    Electrochemical oxidation of xanthosine (2) at pH 2 at a pyrolytic graphite electrode generates an electrophilic radical cation intermediate. Nucleophilic attack by 2 on this radical results, ultimately, in formation of 3-(8-xanthosyl)xanthosine (7). Hydrolytic cleavage of one ribose residue from 7 in acidic solution leads to 3-(8-xanthosyl)xanthine (8). The structure of 8, a new dimeric xanthine nucleoside, has been established using spectral and X-ray diffraction methods.

  19. Network Inference Algorithms Elucidate Nrf2 Regulation of Mouse Lung Oxidative Stress

    PubMed Central

    Singhal, Mudita; Malhotra, Deepti; Biswal, Shyam

    2008-01-01

    A variety of cardiovascular, neurological, and neoplastic conditions have been associated with oxidative stress, i.e., conditions under which levels of reactive oxygen species (ROS) are elevated over significant periods. Nuclear factor erythroid 2-related factor (Nrf2) regulates the transcription of several gene products involved in the protective response to oxidative stress. The transcriptional regulatory and signaling relationships linking gene products involved in the response to oxidative stress are, currently, only partially resolved. Microarray data constitute RNA abundance measures representing gene expression patterns. In some cases, these patterns can identify the molecular interactions of gene products. They can be, in effect, proxies for protein–protein and protein–DNA interactions. Traditional techniques used for clustering coregulated genes on high-throughput gene arrays are rarely capable of distinguishing between direct transcriptional regulatory interactions and indirect ones. In this study, newly developed information-theoretic algorithms that employ the concept of mutual information were used: the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE), and Context Likelihood of Relatedness (CLR). These algorithms captured dependencies in the gene expression profiles of the mouse lung, allowing the regulatory effect of Nrf2 in response to oxidative stress to be determined more precisely. In addition, a characterization of promoter sequences of Nrf2 regulatory targets was conducted using a Support Vector Machine classification algorithm to corroborate ARACNE and CLR predictions. Inferred networks were analyzed, compared, and integrated using the Collective Analysis of Biological Interaction Networks (CABIN) plug-in of Cytoscape. Using the two network inference algorithms and one machine learning algorithm, a number of both previously known and novel targets of Nrf2 transcriptional activation were identified. Genes predicted as

  20. Cyclobutyl methyl ketone as a model compound for pinonic acid to elucidate oxidation mechanisms

    NASA Astrophysics Data System (ADS)

    Praplan, A. P.; Barmet, P.; Dommen, J.; Baltensperger, U.

    2012-04-01

    3-Methyl-1,2,3-tricarboxylic acid (MBTCA), terpenylic acid and diaterpenylic acid acetate were identified in secondary organic aerosol (SOA) from α-pinene photooxidation or ozonolysis. These compounds display interesting structural features: MBTCA has a high oxygen to carbon ratio, terpenylic acid contains a lactone ring in its structure and diaterpenylic acid acetate possesses an ester functional group. The reaction mechanisms leading to these products are still unknown, but it was demonstrated experimentally in earlier studies that MBTCA is formed from pinonic acid, a primary ozonolysis product of α-pinene. Because the direct observation of pinonic acid oxidation in a smog chamber would be difficult due to its relatively low volatility, a model compound possessing the substructure of interest was used instead: cyclobutyl methyl ketone (CMK). From its oxidation, several organic acids could be measured with ion chromatography (IC) coupled to a mass spectrometer (MS). Succinic acid, the analogous product of MBTCA is formed at molar yields of 2 to 5%. Butyrolactone is detected as butanoic acid, due to hydrolysis in the sampling device. A monocarboxylic acid with nominal mass 146 was detected in the absence of nitrogen oxides (NOx) and could be the analogous product of diaterpenylic acid acetate. However, due to a lack of available standards, the exact structure of this compound remains unelucidated. Finally, 4-oxobutanoic acid could also be measured and two structures of its expected analogous compound from pinonic acid oxidation are proposed. Because these compounds are primary products of the CMK oxidation, reaction mechanisms capable of adding one or two carboxylic functional groups without formation of stable intermediate products needs to be formulated. Such a formation mechanism of MBTCA from pinonic acid was found in the literature; however, it includes a hydrogen atom migration to an acyloxy radical, which is expected to loose carbon dioxide (CO2) very

  1. Supercomplex organization of the oxidative phosphorylation enzymes in yeast mitochondria.

    PubMed

    Stuart, Rosemary A

    2008-10-01

    Accumulating evidence indicates that the enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) are co-assembled into higher-ordered supercomplexes within the mitochondrial inner membrane. This review will focus largely on the OXPHOS supercomplexes of the yeast Saccharomyces cerevisiae. The recent evidence to indicate that diversity in the populations of the cytochrome bc (1)-COX supercomplexes exist shall be outlined. In addition, the existence of dimeric/oligomeric F(1)F(o)-ATP synthase complexes and their proposed role in establishment of the cristae architecture of the inner mitochondrial membrane shall also be discussed.

  2. The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics

    PubMed Central

    Biebricher, Andreas S.; Heller, Iddo; Roijmans, Roel F. H.; Hoekstra, Tjalle P.; Peterman, Erwin J. G.; Wuite, Gijs J. L.

    2015-01-01

    DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro. It is well known that they perturb DNA structure and stability, which can in turn influence DNA-processing by proteins. Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3. We show that their DNA-binding affinity is mainly governed by a strongly tension-dependent dissociation rate. These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength. We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and maximal efficacy. PMID:26084388

  3. Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition.

    PubMed

    Davis, Katherine M; Schramma, Kelsey R; Hansen, William A; Bacik, John P; Khare, Sagar D; Seyedsayamdost, Mohammad R; Ando, Nozomi

    2017-09-26

    Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules known as RiPPs (ribosomally synthesized and posttranslationally modified peptides). Although the leader sequence of the precursor peptide is often required for turnover, the exact mode of recognition by the modifying enzymes remains unclear for many members of this class of natural products. Here, we have used X-ray crystallography and computational modeling to examine the role of the leader peptide in the biosynthesis of a homolog of streptide, a recently identified peptide natural product with an intramolecular lysine-tryptophan cross-link, which is installed by the radical S-adenosylmethionine (SAM) enzyme, StrB. We present crystal structures of SuiB, a close ortholog of StrB, in various forms, including apo SuiB, SAM-bound SuiB, and a complex of SuiB with SAM and its peptide substrate, SuiA. Although the N-terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel rather than the N-terminal domain. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross-linking residues of the precursor peptide within the active site. Together the results shed light onto binding of the precursor peptide and the associated conformational changes needed for the formation of the unique carbon-carbon cross-link in the streptide family of natural products.

  4. An XPS analytical approach for elucidating the microbially mediated enargite oxidative dissolution.

    PubMed

    Fantauzzi, M; Rossi, G; Elsener, B; Loi, G; Atzei, D; Rossi, A

    2009-04-01

    In this work, the microbe-mediated oxidative dissolution of enargite surfaces (Cu(3)AsS(4)) was studied on powdered samples exposed to 9K nutrient solution (pH 2.3) inoculated by Acidithiobacillus ferrooxidans initially adapted to arsenopyrite. These conditions simulate the acid mine environment. The redox potential of the inoculated solutions increased up to +0.72 V vs normal hydrogen electrode (NHE), indicating the increase of the Fe(3+) to Fe(2+) ratio, and correspondingly the pH decreased to values as low as 1.9. In the sterile 9K control, the redox potential and pH remained constant at +0.52 V NHE and 2.34, respectively. Solution analyses showed that in inoculated medium Cu and As dissolved stoichiometrically with a dissolution rate of about three to five times higher compared to the sterile control. For the first time, X-ray photoelectron spectroscopy (XPS) was carried out on the bioleached enargite powder with the aim of clarifying the role of the microorganisms in the dissolution process. XPS results provide evidence of the formation of a thin oxidized layer on the mineral surface. Nitrogen was also detected on the bioleached surfaces and was attributed to the presence of an extracellular polymer substance layer supporting a mechanism of bacteria attachment via the formation of a biofilm a few nanometers thick, commonly known as nanobiofilm.

  5. Elucidating the Chemical Complexity of Organic Aerosol Constituents Measured During the Southeastern Oxidant and Aerosol Study (SOAS)

    NASA Astrophysics Data System (ADS)

    Yee, L.; Isaacman, G. A.; Spielman, S. R.; Worton, D. R.; Zhang, H.; Kreisberg, N. M.; Wilson, K. R.; Hering, S. V.; Goldstein, A. H.

    2013-12-01

    Thousands of volatile organic compounds are uniquely created in the atmosphere, many of which undergo chemical transformations that result in more highly-oxidized and often lower vapor pressure species. These species can contribute to secondary organic aerosol, a complex mixture of organic compounds that is still not chemically well-resolved. Organic aerosol collected on filters taken during the Southeastern Oxidant and Aerosol Study (SOAS) constitute hundreds of unique chemical compounds. Some of these include known anthropogenic and biogenic tracers characterized using standardized analytical techniques (e.g. GC-MS, UPLC, LC-MS), but the majority of the chemical diversity has yet to be explored. By employing analytical techniques involving sample derivatization and comprehensive two-dimensional gas chromatography (GC x GC) with high-resolution-time-of-flight mass spectrometry (HR-ToF-MS), we elucidate the chemical complexity of the organic aerosol matrix along the volatility and polarity grids. Further, by utilizing both electron impact (EI) and novel soft vacuum ultraviolet (VUV) ionization mass spectrometry, a greater fraction of the organic mass is fully speciated. The GC x GC-HR-ToF-MS with EI/VUV technique efficiently provides an unprecedented level of speciation for complex ambient samples. We present an extensive chemical characterization and quantification of organic species that goes beyond typical atmospheric tracers in the SOAS samples. We further demonstrate that complex organic mixtures can be chemically deconvoluted by elucidation of chemical formulae, volatility, functionality, and polarity. These parameters provide insight into the sources (anthropogenic vs. biogenic), chemical processes (oxidation pathways), and environmental factors (temperature, humidity), controlling organic aerosol growth in the Southeastern United States.

  6. Isolation and structure elucidation of new radical oxidation products of 5-hydroxy steroids.

    PubMed

    Khripach, Vladimir A; Zhabinskii, Vladimir N; Kuchto, Anna I; Zhiburtovich, Yuliya Y; Lyakhov, Alexander S; Govorova, Alla A; Groen, Marinus B; van der Louw, Jaap; de Groot, Aede

    2006-06-01

    Three new products have been isolated from the lead-tetraacetate version of the hypoiodite oxidation of 3beta,17beta-diacetoxy-5-hydroxy-5 alpha-androstane. Along with the expected 1(10)-unsaturated 5,10-seco steroidal 5-ketones, the fragmentation reaction gave two epimeric C-4 iodides. Their structural assignment was based on X-ray data of one of them ((4R,10S)-4-iodo-3beta,17beta-diacetoxy-5,10-secoandrostan-5-one). The third new product was found to be the 5 beta,6 beta-epoxide resulting from the dehydration of the tertiary alcohol followed by epoxidation of the intermediate Delta(5)-olefin.

  7. Elucidating Quantum Confinement in Graphene Oxide Dots Based On Excitation-Wavelength-Independent Photoluminescence.

    PubMed

    Yeh, Te-Fu; Huang, Wei-Lun; Chung, Chung-Jen; Chiang, I-Ting; Chen, Liang-Che; Chang, Hsin-Yu; Su, Wu-Chou; Cheng, Ching; Chen, Shean-Jen; Teng, Hsisheng

    2016-06-02

    Investigating quantum confinement in graphene under ambient conditions remains a challenge. In this study, we present graphene oxide quantum dots (GOQDs) that show excitation-wavelength-independent photoluminescence. The luminescence color varies from orange-red to blue as the GOQD size is reduced from 8 to 1 nm. The photoluminescence of each GOQD specimen is associated with electron transitions from the antibonding π (π*) to oxygen nonbonding (n-state) orbitals. The observed quantum confinement is ascribed to a size change in the sp(2) domains, which leads to a change in the π*-π gap; the n-state levels remain unaffected by the size change. The electronic properties and mechanisms involved in quantum-confined photoluminescence can serve as the foundation for the application of oxygenated graphene in electronics, photonics, and biology.

  8. Role of oxidative stress and antioxidant enzymes in Crohn's disease.

    PubMed

    Iborra, Marisa; Moret, Inés; Rausell, Francisco; Bastida, Guillermo; Aguas, Mariam; Cerrillo, Elena; Nos, Pilar; Beltrán, Belén

    2011-08-01

    There is increasing interest in oxidative stress being a potential aetiological factor and/or a triggering factor in Crohn's disease, rather than a concomitant occurrence during the pathogenesis of the disease. Recent research has shown that the immune mononuclear cells of Crohn's disease patients are induced to produce hydrogen peroxide (H2O2). Similarly, the regulation of antioxidant enzymes during disease in these cells has been unravelled, showing that SOD (superoxide dismutase) activity and GPx (glutathione peroxidase) activity is increased during active disease and returns to normal in remission phases. However, catalase remains constantly inhibited which supports the idea that catalase is not a redox-sensitive enzyme, but a regulator of cellular processes. ROS (reactive oxygen species) can be produced under the stimulus of different cytokines such as TNFα (tumour necrosis factor α). It has been shown in different experimental models that they are also able to regulate apoptosis and other cellular processes. The status of oxidative stress elements in Crohn's disease and their possible implications in regulating cellular processes are reviewed in the present paper.

  9. Oxidative enzyme changes in sorghum infested by shoot fly.

    PubMed

    Padmaja, P G; Shwetha, B L; Swetha, G; Patil, J V

    2014-01-01

    This research investigated the role of oxidative enzymes in the defense response of sorghum, Sorghum bicolor (L.) Moench (Poales: Poaceae), to the sorghum shoot fly, Atherigona soccata Rondani (Diptera: Muscidae). Changes in polyphenol oxidase and peroxidase activity and total protein content were observed in resistant and susceptible sorghum genotypes in response to A. soccata feeding. Resistant plants exhibited higher levels of peroxidase and polyphenol oxidase activities and total protein content compared with susceptible plants. Peroxidase and polyphenol oxidase activities and total protein content in the infested resistant and susceptible genotypes were higher when compared with their control plants, respectively. These findings suggest that resistant genotypes may be able to tolerate shoot fly feeding by increasing their peroxidase and polyphenol oxidase activities. Among the enzymes examined, differences in isozyme profiles for peroxidase and polyphenol oxidase were detected between control and infested IS 18551, M35-1, 296B, SSV 84, and DJ 6514 plants. Differences in protein profiles were observed between A. soccata infested and their respective uninfested controls of all the genotypes. In conclusion, this study revealed that these defense enzymes and proteins might attribute to the resistance mechanisms in sorghum plants against A. soccata infestation.

  10. Elucidation of hydrocarbon structure in an enzyme-catalyzed benzo[a]pyrene-poly (G) covalent complex.

    PubMed Central

    Meehan, T; Straub, K; Calvin, M

    1976-01-01

    The carcinogen, benzo[a]pyrene, was covalently attached to poly (G) by liver microsomes from rats pretreated with 3-methylcholanthrene. The complex was hydrolyzed with enzymes or base and products were isolated by Sephadex chromatography. Absorbance and fluorescence spectra of the products fit that of red-shifted pyrene aromatic system and suggest that metabolism has occurred at the 7-, 8-, 9-, and 10-positions of the hydrocarbon. Benzanthracene or chrysene fluorescence were not observed in these preparations. Benzo[a]pyrene derivatives were synthesized and purified by high-pressure liquid chromatography. Dehydration of 7,8-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene resulted in the formation of small amounts of 7-oxo-7,8,9,10-tetrahydrobenzoa[a]pyrene. A 7-keto species was also observed after similar treatment of the hydrocarbon-poly(G) hydrolysis products. Evidence of dehydration at the 9,10-positions was not observed. The hydrocarbon covalently bound to poly(G) is, therefore, a derivative of 7,8-dihydroxy-7,8,9,10-tetrahydrobenzol[a]pyrene with nucleic acid substitution at C-10 or 9. PMID:1064016

  11. Elucidation of Xylem-Specific Transcription Factors and Absolute Quantification of Enzymes Regulating Cellulose Biosynthesis in Populus trichocarpa.

    PubMed

    Loziuk, Philip L; Parker, Jennifer; Li, Wei; Lin, Chien-Yuan; Wang, Jack P; Li, Quanzi; Sederoff, Ronald R; Chiang, Vincent L; Muddiman, David C

    2015-10-02

    Cellulose, the main chemical polymer of wood, is the most abundant polysaccharide in nature.1 The ability to perturb the abundance and structure of cellulose microfibrils is of critical importance to the pulp and paper industry as well as for the textile, wood products, and liquid biofuels industries. Although much has been learned at the transcript level about the biosynthesis of cellulose, a quantitative understanding at the proteome level has yet to be established. The study described herein sought to identify the proteins directly involved in cellulose biosynthesis during wood formation in Populus trichocarpa along with known xylem-specific transcription factors involved in regulating these key proteins. Development of an effective discovery proteomic strategy through a combination of subcellular fractionation of stem differentiating xylem tissue (SDX) with recently optimized FASP digestion protocols, StageTip fractionation, as well as optimized instrument parameters for global proteomic analysis using the quadrupole-orbitrap mass spectrometer resulted in the deepest proteomic coverage of SDX protein from P. trichocarpa with 9,146 protein groups being identified (1% FDR). Of these, 20 cellulosic/hemicellulosic enzymes and 43 xylem-specific transcription factor groups were identified. Finally, selection of surrogate peptides led to an assay for absolute quantification of 14 cellulosic proteins in SDX of P. trichocarpa.

  12. Extended friction elucidates the breakdown of fast water transport in graphene oxide membranes

    NASA Astrophysics Data System (ADS)

    Montessori, A.; Amadei, C. A.; Falcucci, G.; Sega, M.; Vecitis, C. D.; Succi, S.

    2016-12-01

    The understanding of water transport in graphene oxide (GO) membranes stands out as a major theoretical problem in graphene research. Notwithstanding the intense efforts devoted to the subject in the recent years, a consolidated picture of water transport in GO membranes is yet to emerge. By performing mesoscale simulations of water transport in ultrathin GO membranes, we show that even small amounts of oxygen functionalities can lead to a dramatic drop of the GO permeability, in line with experimental findings. The coexistence of bulk viscous dissipation and spatially extended molecular friction results in a major decrease of both slip and bulk flow, thereby suppressing the fast water transport regime observed in pristine graphene nanochannels. Inspection of the flow structure reveals an inverted curvature in the near-wall region, which connects smoothly with a parabolic profile in the bulk region. Such inverted curvature is a distinctive signature of the coexistence between single-particle zero-temperature (noiseless) Langevin friction and collective hydrodynamics. The present mesoscopic model with spatially extended friction may offer a computationally efficient tool for future simulations of water transport in nanomaterials.

  13. Elucidation of stability determinants of cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Colwellia maris, by construction of chimeric enzymes.

    PubMed

    Watanabe, Seiya; Yasutake, Yoshiaki; Tanaka, Isao; Takada, Yasuhiro

    2005-04-01

    To elucidate determinants of differences in thermostability between mesophilic and psychrophilic monomeric isocitrate dehydrogenases (IDHs) from Azotobacter vinelandii (AvIDH) and Colwellia maris (CmIDH), respectively, chimeric enzymes derived from the two IDHs were constructed based on the recently resolved three-dimensional structure of AvIDH, and several characteristics of the two wild-type and six chimeric IDHs were examined. These characteristics were then compared with those of dimeric IDH from Escherichia coli (EcIDH). All recombinant enzymes with a (His)(6)-tag attached to the N-terminal were overexpressed in the E. coli cells and purified by Ni(2+)-affinity chromatography. The catalytic activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) of the wild-type AvIDH and CmIDH were higher than those of EcIDH, implying that an improved catalytic rate more than compensates for the loss of a catalytic site in the former two IDHs due to monomerization. Analyses of the thermostability and kinetic parameters of the chimeric enzymes indicated that region 2, corresponding to domain II, and particularly region 3 located in the C-terminal part of domain I, are involved in the thermolability of CmIDH, and that the corresponding two regions of AvIDH are important for exhibiting higher catalytic activity and affinity for isocitrate than CmIDH. The relationships between the stability, catalytic activity and structural characteristics of AvIDH and CmIDH are discussed.

  14. A mitochondrial enzyme degrades carotenoids and protects against oxidative stress

    PubMed Central

    Amengual, Jaume; Lobo, Glenn P.; Golczak, Marcin; Li, Hua Nan M.; Klimova, Tatyana; Hoppel, Charles L.; Wyss, Adrian; Palczewski, Krzysztof; von Lintig, Johannes

    2011-01-01

    Carotenoids are the precursors for vitamin A and are proposed to prevent oxidative damage to cells. Mammalian genomes encode a family of structurally related nonheme iron oxygenases that modify double bonds of these compounds by oxidative cleavage and cis-to-trans isomerization. The roles of the family members BCMO1 and RPE65 for vitamin A production and vision have been well established. Surprisingly, we found that the third family member, β,β-carotene-9′,10′-oxygenase (BCDO2), is a mitochondrial carotenoid-oxygenase with broad substrate specificity. In BCDO2-deficient mice, carotenoid homeostasis was abrogated, and carotenoids accumulated in several tissues. In hepatic mitochondria, accumulated carotenoids induced key markers of mitochondrial dysfunction, such as manganese superoxide dismutase (9-fold), and reduced rates of ADP-dependent respiration by 30%. This impairment was associated with an 8- to 9-fold induction of phosphor-MAP kinase and phosphor-AKT, markers of cell signaling pathways related to oxidative stress and disease. Administration of carotenoids to human HepG2 cells depolarized mitochondrial membranes and resulted in the production of reactive oxygen species. Thus, our studies in BCDO2-deficient mice and human cell cultures indicate that carotenoids can impair respiration and induce oxidative stress. Mammalian cells thus express a mitochondrial carotenoid-oxygenase that degrades carotenoids to protect these vital organelles.—Amengual, J., Lobo, G. P., Golczak, M., Li, H. N. M., Klimova, T., Hoppel, C. L., Wyss, A., Palczewski, K., von Lintig, J. A mitochondrial enzyme degrades carotenoids and protects against oxidative stress. PMID:21106934

  15. Effects of sodium tungstate on oxidative stress enzymes in rats.

    PubMed

    Sachdeva, Sherry; Kushwaha, Pramod; Flora, S J S

    2013-09-01

    Tungsten, due to its distinguished physical properties, has wide industrial and military applications. Environmental exposure to tungsten, which mainly occurs through various sources like food, water, soil, etc., is of growing concern as various toxic effects have recently been reported. In this study, we investigated the effects of oral and intraperitoneal (i.p.) administration of sodium tungstate on various biochemical variables indicative of oxidative stress in erythrocytes and soft tissue damage in rats. Male rats were administered to 119 mg, 238 mg/kg of sodium tungstate orally or 20 mg and 41 mg/kg through i.p. route, for 14 consecutive days. The results demonstrated a significant increase in Reactive Oxygen Species (ROS) and an increase in catalase and glutathione peroxidase antioxidant enzymes activities in erythrocytes. Erythrocyte glutathione-S-transferase (GST) activity showed significant inhibition, while tissue ROS and thiobarbituric acid reactive substance levels increased accompanied by a decreased reduced glutathione, oxidized glutathione (GSH:GSSG) ratio. These changes were supported by an increase in plasma transaminases activities, creatinine, and urea levels, suggesting hepatic and renal injury. These biochemical alterations were prominent in rats intraperitoneally administrated with sodium tungstate than oral administration, suggesting more pronounced toxicity. The study also suggests oxidative stress as one of the major mechanism involved in the toxic manifestations of sodium tungstate.

  16. Modulation of angiotensin-converting enzyme by nitric oxide

    PubMed Central

    Ackermann, A; Fernández-Alfonso, M S; Sánchez de Rojas, R; Ortega, T; Paul, M; González, C

    1998-01-01

    The aim of the present study was to determine the effect of nitric oxide (NO) on angiotensin-converting enzyme (ACE) activity.A biochemical study was performed in order to analyse the effect of the NO-donors, SIN-1 and diethylamine/NO (DEA/NO), and of an aqueous solution of nitric oxide on the ACE activity in plasma from 3-month old male Sprague-Dawley rats and on ACE purified from rabbit lung. SIN-1 significantly inhibited the activity of both enzymes in a concentration-dependent way between 1 and 100 μM. DEA/NO inhibited the activity of purified ACE from 0.1 μM to 10 μM and plasma ACE, with a lower potency, between 1 and 100 μM. An aqueous solution of NO (100 and 150 μM) also inhibited significantly the activity of both enzymes. Lineweaver-Burk plots indicated an apparent competitive inhibition of Hip-His-Leu hydrolysis by NO-donors.Modulation of ACE activity by NO was also assessed in the rat carotid artery by comparing contractions elicited by angiotensin I (AI) and AII. Concentration-response curves to both peptides were performed in arteries with endothelium in the presence of the guanylyl cyclase inhibitor, ODQ (10 μM), and the inhibitor of NO formation, L-NAME (0.1 mM). NO, which is still released from endothelium in the presence of 10 μM ODQ, elicited a significant inhibition of AI contractions at low concentrations (1 and 5 nM). In the absence of endothelium, 1 μM SIN-1 plus 10 μM ODQ, as well as 10 μM DEA/NO plus 10 μM ODQ induced a significant inhibition on AI-induced contractions at 1 and 5 nM and at 1–100 nM, respectively.In conclusion, we demonstrated that (i) NO and NO-releasing compounds inhibit ACE activity in a concentration-dependent and competitive way and that (ii) NO release from endothelium physiologically reduces conversion of AI to AII. PMID:9641545

  17. Alkane Oxidation: Methane Monooxygenases, Related Enzymes, and Their Biomimetics.

    PubMed

    Wang, Vincent C-C; Maji, Suman; Chen, Peter P-Y; Lee, Hung Kay; Yu, Steve S-F; Chan, Sunney I

    2017-02-16

    Methane monooxygenases (MMOs) mediate the facile conversion of methane into methanol in methanotrophic bacteria with high efficiency under ambient conditions. Because the selective oxidation of methane is extremely challenging, there is considerable interest in understanding how these enzymes carry out this difficult chemistry. The impetus of these efforts is to learn from the microbes to develop a biomimetic catalyst to accomplish the same chemical transformation. Here, we review the progress made over the past two to three decades toward delineating the structures and functions of the catalytic sites in two MMOs: soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO). sMMO is a water-soluble three-component protein complex consisting of a hydroxylase with a nonheme diiron catalytic site; pMMO is a membrane-bound metalloenzyme with a unique tricopper cluster as the site of hydroxylation. The metal cluster in each of these MMOs harnesses O2 to functionalize the C-H bond using different chemistry. We highlight some of the common basic principles that they share. Finally, the development of functional models of the catalytic sites of MMOs is described. These efforts have culminated in the first successful biomimetic catalyst capable of efficient methane oxidation without overoxidation at room temperature.

  18. Nickel oxide hollow microsphere for non-enzyme glucose detection.

    PubMed

    Ci, Suqin; Huang, Taizhong; Wen, Zhenhai; Cui, Shumao; Mao, Shun; Steeber, Douglas A; Chen, Junhong

    2014-04-15

    A facile strategy has been developed to fabricate nickel oxide hollow microspheres (NiO-HMSs) through a solvothermal method by using a mixed solvent of ethanol and water with the assistance of sodium dodecyl sulfate (SDS). Various techniques, including transmission electron microscopy (TEM), scanning electron microscopy (SEM), and powder X-ray diffraction (XRD), were used to characterize the morphology and the structure of as-prepared samples. It was confirmed that the products possess a hollow microsphere structure that is constructed by interconnecting porous nanoplate framework. Electrochemical studies indicate that the NiO-HMS exhibits excellent stability and high catalytic activity for electrocatalytic oxidation of glucose in alkaline solutions, which enables the NiO-HMS to be used in enzyme-free amperometric sensors for glucose determination. It was demonstrated that the NiO-HMS-based glucose biosensor offers a variety of merits, such as a wide linear response window for glucose concentrations of 1.67 μM-6.87 mM, short response time (3 s), a lower detection limit of 0.53 μM (S/N=3), high sensitivity (~2.39 mA mM(-1) cm(-2)) as well as good stability and repeatability. © 2013 Published by Elsevier B.V.

  19. Oxidative phenols in forage crops containing polyphenol oxidase enzymes.

    PubMed

    Parveen, Ifat; Threadgill, Michael D; Moorby, Jon M; Winters, Ana

    2010-02-10

    Polyphenol oxidases (PPOs) are copper-containing enzymes that catalyze oxidation of endogenous monophenols to ortho-dihydroxyaryl compounds and of ortho-dihydroxyaryl compounds to ortho-quinones. Subsequent nucleophilic addition reactions of phenols, amino acids, and proteins with the electrophilic ortho-quinones form brown-, black-, or red-colored secondary products associated with the undesired discolouration of fruit and vegetables. Several important forage plants also exhibit significant PPO activity, and a link with improved efficiency of ruminant production has been established. In ruminant animals, extensive degradation of forage proteins, following consumption, can result in high rates of excretion of nitrogen, which contributes to point-source and diffuse pollution. Reaction of quinones with forage proteins leads to the formation of protein-phenol complexes that are resistant to proteolytic activity during ensilage and during rumen fermentation. Thus, PPO in red clover (Trifolium pratense) has been shown to improve protein utilization by ruminants. While PPO activity has been demonstrated in a number of forage crops, little work has been carried out to identify substrates of PPO, knowledge of which would be beneficial for characterizing this trait in these forages. In general, a wide range of 1,2-dihydroxyarenes can serve as PPO substrates because these are readily oxidized because of the ortho positioning of the hydroxy groups. Naturally occurring phenols isolated from forage crops with PPO activity are reviewed. A large number of phenols, which may be directly or indirectly oxidized as a consequence of PPO activity, have been identified in several forage grass, legume, cereal, and brassica species; these include hydroxybenzoic acids, hydroxycinnamates, and flavonoids. In conclusion, a number of compounds are known or postulated to enable PPO activity in important PPO-expressing forage crops. Targeting the matching of these compounds with PPO activity

  20. Applications of pulsed EPR spectroscopy to structural studies of sulfite oxidizing enzymes

    SciTech Connect

    Klein, Eric L.; Astashkin, Andrei V.; Raitsimring, Arnold M.; Enemark, John H.

    2013-01-01

    Sulfite oxidizing enzymes (SOEs), including sulfite oxidase (SO) and bacterial sulfite dehydrogenase (SDH), catalyze the oxidation of sulfite (SO32-) to sulfate (SO42-). The active sites of SO and SDH are nearly identical, each having a 5-coordinate, pseudo-square-pyramidal Mo with an axial oxo ligand and three equatorial sulfur donor atoms. One sulfur is from a conserved Cys residue and two are from a pyranopterindithiolene (molybdopterin, MPT) cofactor. The identity of the remaining equatorial ligand, which is solvent-exposed, varies during the catalytic cycle. Numerous in vitro studies, particularly those involving electron paramagnetic resonance (EPR) spectroscopy of the Mo(V) states of SOEs, have shown that the identity and orientation of this exchangeable equatorial ligand depends on the buffer pH, the presence and concentration of certain anions in the buffer, as well as specific point mutations in the protein. Until very recently, however, EPR has not been a practical technique for directly probing specific structures in which the solvent-exposed, exchangeable ligand is an O, OH-, H2O, SO32-, or SO42- group, because the primary O and S isotopes (16O and 32S) are magnetically silent (I = 0). This review focuses on the recent advances in the use of isotopic labeling, variable-frequency high resolution pulsed EPR spectroscopy, synthetic model compounds, and DFT calculations to elucidate the roles of various anions, point mutations, and steric factors in the formation, stabilization, and transformation of SOE active site structures.

  1. Proteomic Analyses of Transgenic LQT1 and LQT2 Rabbit Hearts Elucidate an Increase in Expression and Activity of Energy Producing Enzymes

    PubMed Central

    Jindal, Hitesh K.; Merchant, Elisabeth; Balschi, James A.; Zhangand, Yajie; Koren, Gideon

    2012-01-01

    Various biochemical and genomic mechanisms are considered to be hallmark of metabolic remodeling in the stressed heart, including in the hypertrophied and failing heart. In this study, we used quantitative proteomic 2-D Fluorescence Difference In-Gel Electrophoresis (2-D DIGE) in conjunction with mass spectrometry to demonstrate differential protein expression in the hearts of transgenic rabbit models of Long QT Syndrome 1 (LQT1) and Long QT Syndrome 2 (LQT2) as compared to littermate controls (LMC). The results of our proteomic analysis revealed upregulation of key metabolic enzymes involved in all pathways associated with ATP generation, including creatine kinase in both LQT1 and LQT2 rabbit hearts. Additionally, the expression of lamin-A protein was increased in both LQT1 and LQT2 rabbit hearts as was the expression of mitochondrial aldehyde dehydrogenase and desmoplakin in LQT1 and LQT 2 rabbit hearts, respectively. Results of the proteomic analysis also demonstrated down regulation in the expression of protein disulfide-isomerase A3 precuorsor and dynamin-like 120 kDa protein (mitochondrial) in LQT1, and of alpha-actinin 2 in LQT2 rabbit hearts. Up regulation of the expression of the enzymes associated with ATP generation was substantiated by the results of selective enzyme assays in LQT1 and LQT2 hearts, as compared to LMC, which revealed increases in the activities of glycogen phosphorylase (+50%, +65%, respectively), lactate dehydrogenase (+25%, +25%) pyruvate dehydrogenase (+31%, +22%), and succinate dehydrogenase (+32%, +60%). The activity of cytochrome c-oxidase, a marker for the mitochondrial function was also found to be significantly elevated (+80%) in LQT1 rabbit hearts as compared with LMC. Western blot analysis in LQT1 and LQT2 hearts compared to LMC revealed an increase in the expression of very-long chain-specific acyl-CoA dehydrogenase (+35%, +33%), a rate-limiting enzymes in β-oxidation of fatty acids. Collectively, our results demonstrate

  2. Oxidative Stress and Anti-Oxidant Enzyme Activities in the Trophocytes and Fat Cells of Queen Honeybees (Apis mellifera)

    PubMed Central

    Hsieh, Yu-Shan

    2013-01-01

    Abstract Trophocytes and fat cells of queen honeybees have been used for delayed cellular senescence studies, but their oxidative stress and anti-oxidant enzyme activities with advancing age are unknown. In this study, we assayed reactive oxygen species (ROS) and anti-oxidant enzymes in the trophocytes and fat cells of young and old queens. Young queens had lower ROS levels, lower superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, and higher thioredoxin reductase (TR) activity compared to old queens. These results show that oxidative stress and anti-oxidant enzyme activities in trophocytes and fat cells increase with advancing age in queens and suggest that an increase in oxidative stress and a consequent increase in stress defense mechanisms are associated with the longevity of queen honeybees. PMID:23738955

  3. Effect of deuterium oxide on neutrophil oxidative metabolism, phagocytosis, and lysosomal enzyme release

    SciTech Connect

    Tsan, M.F.; Turkall, R.M.

    1982-12-01

    We have previously shown that deuterium oxide (D/sub 2/O) enhances the oxidation of methionine, a myeloperoxidase (MPO) -mediated reaction, by human neutrophils during phagocytosis. However, D/sub 2/O has no effect on the oxidation of methionine by the purified MPO-H/sub 2/O/sub 2/-Cl- system. To explain this observation, we studied the effect of D/sub 2/O on the oxidative metabolism, phagocytosis, and lysosomal enzyme release by human neutrophils. D/sub 2/O stimulated the hexose monophosphate shunt (HMS) activity of resting neutrophils in a dose-response fashion. In the presence of latex particles or phorbol myristate acetate (PMA), D/sub 2/O brought about an exaggerated stimulation of the HMS activity. This enhancement of the HMS activity by D/sub 2/O was markedly reduced when neutrophils form two patients with X-linked chronic granulomatous disease (CGD) were used, either in the presence or absence of latex particles or PMA. Superoxide and H/sub 2/O/sub 2/ production by neutrophils in the presence of latex particles or PMA were also stimulated by D/sub 2/O. In contrast, D/sub 2/O inhibited the ingestion of latex particles. D/sub 2/O enhanced the extracellular release of MPO, but not lactate dehydrogenase, by neutrophils only in the simultaneous presence of cytochalasin B and latex particles. The enhancement of HMS activity and MPO release by D/sub 2/O was partially inhibited by colchicine. Our results suggest that enhancement of neutrophil oxidative metabolism by D/sub 2/O may in part explain the stimulation of methionine oxidation by phagocytosing neutrophils.

  4. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  5. Clinical consequences of urea cycle enzyme deficiencies and potential links to arginine and nitric oxide metabolism.

    PubMed

    Scaglia, Fernando; Brunetti-Pierri, Nicola; Kleppe, Soledad; Marini, Juan; Carter, Susan; Garlick, Peter; Jahoor, Farook; O'Brien, William; Lee, Brendan

    2004-10-01

    Urea cycle disorders (UCD) are human conditions caused by the dysregulation of nitrogen transfer from ammonia nitrogen into urea. The biochemistry and the genetics of these disorders were well elucidated. Earlier diagnosis and improved treatments led to an emerging, longer-lived cohort of patients. The natural history of some of these disorders began to point to pathophysiological processes that may be unrelated to the primary cause of acute morbidity and mortality, i.e., hyperammonemia. Carbamyl phosphate synthetase I single nucleotide polymorphisms may be associated with altered vascular resistance that becomes clinically relevant when specific environmental stressors are present. Patients with argininosuccinic aciduria due to a deficiency of argininosuccinic acid lyase are uniquely prone to chronic hepatitis, potentially leading to cirrhosis. Moreover, our recent observations suggest that there may be an increased prevalence of essential hypertension. In contrast, hyperargininemia found in patients with arginase 1 deficiency is associated with pyramidal tract findings and spasticity, without significant hyperammonemia. An intriguing potential pathophysiological link is the dysregulation of intracellular arginine availability and its potential effect on nitric oxide (NO) metabolism. By combining detailed natural history studies with the development of tissue-specific null mouse models for urea cycle enzymes and measurement of nitrogen flux through the cycle to urea and NO in UCD patients, we may begin to dissect the contribution of different sources of arginine to NO production and the consequences on both rare genetic and common multifactorial diseases.

  6. Nitric oxide and antioxidant enzyme levels in blood of respiratory distress syndrome--Egyptian preterms and their mothers.

    PubMed

    El-Masry, Hosny M A; Nasr, Ahmed A M; Al Kabeer, Ashraf M; Amin, H H; Eldeeb, Hussein M H

    2015-01-01

    The mechanisms of tissue injury in respiratory distress syndrome (RDS) have not been completely elucidated but the involvement of oxidative damage due to reactive oxygen species (ROS) is important in the pathogenesis of preterm labor and RDS. To assess the oxidative status in preterms with and without RDS and in their mothers. Measuring and comparing concentrations of serum nitric oxide (NO), and erythrocytic activities of some antioxidant enzymes in blood of 20 preterms with RDS and 20 preterms without and their mothers, respectively. Our results confirm significant elevation of mean levels of serum NO and reduced mean levels of erythrocytic activities of antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), in RDS preterms and their mothers compared with controls and their mothers. A significant positive correlations were found between maternal and preterm serum NO (r=0.3 & 0.6) and erythrocytic activity of SOD (r=0.5 & 0.4) for RDS and control groups, respectively. In addition, a significant negative correlations were found between NO and SOD in all groups. The existence of oxygen metabolites and lipid peroxidation was significantly more obvious in RDS preterms and in their mothers than those without.

  7. Fe-O stable isotope pairs elucidate a high-temperature origin of Chilean iron oxide-apatite deposits

    NASA Astrophysics Data System (ADS)

    Bilenker, Laura D.; Simon, Adam C.; Reich, Martin; Lundstrom, Craig C.; Gajos, Norbert; Bindeman, Ilya; Barra, Fernando; Munizaga, Rodrigo

    2016-03-01

    Iron oxide-apatite (IOA) ore deposits occur globally and can host millions to billions of tons of Fe in addition to economic reserves of other metals such as rare earth elements, which are critical for the expected growth of technology and renewable energy resources. In this study, we pair the stable Fe and O isotope compositions of magnetite samples from several IOA deposits to constrain the source reservoir of these elements in IOAs. Since magnetite constitutes up to 90 modal% of many IOAs, identifying the source of Fe and O within the magnetite may elucidate high-temperature and/or lower-temperature processes responsible for their formation. Here, we focus on the world-class Los Colorados IOA in the Chilean iron belt (CIB), and present data for magnetite from other Fe oxide deposits in the CIB (El Laco, Mariela). We also report Fe and O isotopic values for other IOA deposits, including Mineville, New York (USA) and the type locale, Kiruna (Sweden). The ranges of Fe isotopic composition (δ56Fe, 56Fe/54Fe relative to IRMM-14) of magnetite from the Chilean deposits are: Los Colorados, δ56Fe (±2σ) = 0.08 ± 0.03‰ to 0.24 ± 0.08‰; El Laco, δ56Fe = 0.20 ± 0.03‰ to 0.53 ± 0.03‰; Mariela, δ56Fe = 0.13 ± 0.03‰. The O isotopic composition (δ18O, 18O/16O relative to VSMOW) of the same Chilean magnetite samples are: Los Colorados, δ18O (±2σ) = 1.92 ± 0.08‰ to 3.17 ± 0.03‰; El Laco, δ18O = 4.00 ± 0.10‰ to 4.34 ± 0.10‰; Mariela, δ18O = (1.48 ± 0.04‰). The δ18O and δ56Fe values for Kiruna magnetite yield an average of 1.76 ± 0.25‰ and 0.16 ± 0.07‰, respectively. The Fe and O isotope data from the Chilean IOAs fit unequivocally within the range of magnetite formed by high-temperature magmatic or magmatic-hydrothermal processes (i.e., δ56Fe 0.06-0.49‰ and δ18O = 1.0-4.5‰), consistent with a high-temperature origin for Chilean IOA deposits. Additionally, minimum formation temperatures calculated by using the measured Δ18O

  8. Angiotensin-converting enzyme inhibitors attenuate propofol-induced pro-oxidative and antifibrinolytic effect in human endothelial cells.

    PubMed

    Wojewodzka-Zelezniakowicz, Marzena; Gromotowicz-Poplawska, Anna; Kisiel, Wioleta; Konarzewska, Emilia; Szemraj, Janusz; Ladny, Jerzy Robert; Chabielska, Ewa

    2017-01-01

    The aim of this study was to investigate the effects of plasma and tissue angiotensin-converting enzyme inhibitors (ACE-Is) against propofol-induced endothelial dysfunction and to elucidate the involved mechanisms in vitro. We examined the effects of propofol (50 μM), quinaprilat and enalaprilat (10(-5) M) on fibrinolysis (t-PA, PAI-1, TAFI antigen levels), oxidative stress parameters (H2O2 and MDA antigen levels and SOD and NADPH oxidase mRNA levels) and nitric oxide bioavailability (NO2/NO3 concentration and NOS expression at the level of mRNA) in human umbilical vein endothelial cells (HUVECs). We found that both ACE-Is promoted similar endothelial fibrinolytic properties and decreased oxidative stress in vitro. Propofol alone increased the release of antifibrinolytic and pro-oxidative factors from the endothelium and increased mRNA iNOS expression. We also found that the incubation of HUVECs in the presence of propofol following ACE-Is pre-incubation caused weakness of the antifibrinolytic and pro-oxidative potential of propofol and this effect was similar after both ACE-Is. This observation suggests that the studied ACE-Is exerted protective effects against endothelial cell dysfunction caused by propofol, independently of hemodynamics.

  9. Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion.

    PubMed

    Lawton, Thomas J; Rosenzweig, Amy C

    2016-08-03

    Biological conversion of natural gas to liquids (Bio-GTL) represents an immense economic opportunity. In nature, aerobic methanotrophic bacteria and anaerobic archaea are able to selectively oxidize methane using methane monooxygenase (MMO) and methyl coenzyme M reductase (MCR) enzymes. Although significant progress has been made toward genetically manipulating these organisms for biotechnological applications, the enzymes themselves are slow, complex, and not recombinantly tractable in traditional industrial hosts. With turnover numbers of 0.16-13 s(-1), these enzymes pose a considerable upstream problem in the biological production of fuels or chemicals from methane. Methane oxidation enzymes will need to be engineered to be faster to enable high volumetric productivities; however, efforts to do so and to engineer simpler enzymes have been minimally successful. Moreover, known methane-oxidizing enzymes have different expression levels, carbon and energy efficiencies, require auxiliary systems for biosynthesis and function, and vary considerably in terms of complexity and reductant requirements. The pros and cons of using each methane-oxidizing enzyme for Bio-GTL are considered in detail. The future for these enzymes is bright, but a renewed focus on studying them will be critical to the successful development of biological processes that utilize methane as a feedstock.

  10. Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion

    PubMed Central

    Lawton, Thomas J.; Rosenzweig, Amy C.

    2017-01-01

    Biological conversion of natural gas to liquids (Bio-GTL) represents an immense economic opportunity. In nature, aerobic methanotrophic bacteria and anaerobic archaea are able to selectively oxidize methane using methane monooxygenase (MMO) and methyl coenzyme M reductase (MCR) enzymes. Although significant progress has been made toward genetically manipulating these organisms for biotechnological applications, the enzymes themselves are slow, complex, and not recombinantly tractable in traditional industrial hosts. With turnover numbers of 0.16–13 s−1, these enzymes pose a considerable upstream problem in the biological production of fuels or chemicals from methane. Methane oxidation enzymes will need to be engineered to be faster to enable high volumetric productivities; however, efforts to do so and to engineer simpler enzymes have been minimally successful. Moreover, known methane-oxidizing enzymes have different expression levels, carbon and energy efficiencies, require auxiliary systems for biosynthesis and function, and vary considerably in terms of complexity and reductant requirements. The pros and cons of using each methane-oxidizing enzyme for Bio-GTL are considered in detail. The future for these enzymes is bright, but a renewed focus on studying them will be critical to the successful development of biological processes that utilize methane as a feedstock. PMID:27366961

  11. Radiation effects on rat testes. IX. Studies on oxidative enzymes after partial body gamma irradiation.

    PubMed

    Gupta, G S; Bawa, S R

    1975-08-01

    Oxidative enzymes in the rat testes have been studied after gamma irradiation. The role of these enzymes in relation to spermatogenesis and steroidogenesis after radiation injury to testis has been discussed. Loss of succinic dehydrogenase and sorbitol dehydrogenase reflects the losts of germ cell population. Malic enzyme and malic dehydrogenase seem to the related to the deficiency of steroid hormones, whereas increase in glucose-6-phosphate dehydrogenase and NADP isocitric dehydrogenase is due to secondary stimulation of pituitary.

  12. Enzyme phylogenies as markers for the oxidation state of the environment: the case of respiratory arsenate reductase and related enzymes.

    PubMed

    Duval, Simon; Ducluzeau, Anne-Lise; Nitschke, Wolfgang; Schoepp-Cothenet, Barbara

    2008-07-16

    Phylogenies of certain bioenergetic enzymes have proved to be useful tools for deducing evolutionary ancestry of bioenergetic pathways and their relationship to geochemical parameters of the environment. Our previous phylogenetic analysis of arsenite oxidase, the molybdopterin enzyme responsible for the biological oxidation of arsenite to arsenate, indicated its probable emergence prior to the Archaea/Bacteria split more than 3 billion years ago, in line with the geochemical fact that arsenite was present in biological habitats on the early Earth. Respiratory arsenate reductase (Arr), another molybdopterin enzyme involved in microbial arsenic metabolism, serves as terminal oxidase, and is thus situated at the opposite end of bioenergetic electron transfer chains as compared to arsenite oxidase. The evolutionary history of the Arr-enzyme has not been studied in detail so far. We performed a genomic search of genes related to arrA coding for the molybdopterin subunit. The multiple alignment of the retrieved sequences served to reconstruct a neighbor-joining phylogeny of Arr and closely related enzymes. Our analysis confirmed the previously proposed proximity of Arr to the cluster of polysulfide/thiosulfate reductases but also unravels a hitherto unrecognized clade even more closely related to Arr. The obtained phylogeny strongly suggests that Arr originated after the Bacteria/Archaea divergence in the domain Bacteria, and was subsequently laterally distributed within this domain. It further more indicates that, as a result of accumulation of arsenate in the environment, an enzyme related to polysulfide reductase and not to arsenite oxidase has evolved into Arr. These findings are paleogeochemically rationalized by the fact that the accumulation of arsenate over arsenite required the increase in oxidation state of the environment brought about by oxygenic photosynthesis.

  13. Elucidation of the reaction mechanism during the removal of copper oxide by halogen surfactant at the surface of copper plate

    NASA Astrophysics Data System (ADS)

    Yokoyama, Shun; Takahashi, Hideyuki; Itoh, Takashi; Motomiya, Kenichi; Tohji, Kazuyuki

    2013-01-01

    Although copper nanoparticles have various attractive properties, electrical applications of these was not achieved because of its surface oxide layer which prohibited electrical conduction. Thus, it can be considered that a new elimination method of the oxide on Cu surface, which simultaneously provide the resistance to re-oxidized, should be developed. In this study, the reaction between the metal oxide on Cu plate surface and halogen surfactant was introduced into development as a new elimination method of surface oxide layer. Since electrochemical and surface analysis are effective for analyzing the reaction mechanism which expected to be the reduction reaction of the oxide on metal surface, Cu electrode, which represented material of Cu nanoparticles surface, was used for the reaction mechanism analysis. The oxide is removed by controlling the temperature and selecting the optimal combination of solvents and the halogen surfactant (TIC). Results of electrochemical measurements strongly suggest that the chemical reaction between the oxides on the surface with the halogen surfactant is a substitution reaction which converts Cu oxide to Cu bromide, and continuously formed Cu bromide was dissolved into solvent. Totally, the oxide on the Cu surface was successfully eliminated.

  14. Molecular co-evolution of a protease and its substrate elucidated by analysis of the activity of predicted ancestral hatching enzyme

    PubMed Central

    2013-01-01

    Background Hatching enzyme is a protease that digests the egg envelope, enabling hatching of the embryo. We have comprehensively studied the molecular mechanisms of the enzyme action to its substrate egg envelope, and determined the gene/protein structure and phylogenetic relationships. Because the hatching enzyme must have evolved while maintaining its ability to digest the egg envelope, the hatching enzyme-egg envelope protein pair is a good model for studying molecular co-evolution of a protease and its substrate. Results Hatching enzymes from medaka (Oryzias latipes) and killifish (Fundulus heteroclitus) showed species-specific egg envelope digestion. We found that by introducing four medaka-type residue amino acid substitutions into recombinant killifish hatching enzyme, the mutant killifish hatching enzyme could digest medaka egg envelope. Further, we studied the participation of the cleavage site of the substrate in the species-specificity of hatching enzyme. A P2-site single amino acid substitution was responsible for the species-specificity. Estimation of the activity of the predicted ancestral enzymes towards various types of cleavage sites along with prediction of the evolutionary timing of substitutions allowed prediction of a possible evolutionary pathway, as follows: ancestral hatching enzyme, which had relatively strict substrate specificity, developed broader specificity as a result of four amino acid substitutions in the active site cleft of the enzyme. Subsequently, a single substitution occurred within the cleavage site of the substrate, and the recent feature of species-specificity was established in the hatching enzyme-egg envelope system. Conclusions The present study clearly provides an ideal model for protease-substrate co-evolution. The evolutionary process giving rise to species-specific egg envelope digestion of hatching enzyme was initiated by amino acid substitutions in the enzyme, resulting in altered substrate specificity, which later

  15. Anti-oxidant enzyme activities and expression and oxidative damage in patients with non-immediate reactions to drugs

    PubMed Central

    Cornejo-Garcia, J A; Mayorga, C; Torres, M J; Fernandez, T D; R-Pena, R; Bravo, I; Mates, J M; Blanca, M

    2006-01-01

    Adverse drug reactions with an immunological basis (ADRIB) may involve activation of other concomitant, non-specific mechanisms, amplifying the specific response and contributing to the severity and duration. One concomitant mechanism could be the generation of reactive oxygen species (ROS) and/or their detoxification by anti-oxidants, including anti-oxidant enzymes. We analysed the activity of the anti-oxidant enzymes Cu/Zn-superoxide dismutase (SOD), catalase (CAT) and cellular glutathione peroxidase (GPX), as well as certain markers of oxidative damage (thiobarbituric acid reactive substances (TBARS) and carbonyl content) in peripheral blood mononuclear cells from patients with non-immediate ADRIB using spectrophotometric methods and the anti-oxidant enzymes expression by quantitative real-time reverse transcription–polymerase chain reaction. SOD activity and expression were increased in all types of non-immediate reactions (urticaria, maculopapular exanthema and toxic epidermal necrolysis). Regarding oxidative damage, TBARS were increased in urticaria and maculopapular exanthema, and carbonyl groups in all types of reactions. Our observations indicate that oxidative damage occurs in non-immediate reactions. Carbonyl stress and the inadequacy of the anti-oxidant defences are probable causes. PMID:16879248

  16. Anti-oxidant enzyme activities and expression and oxidative damage in patients with non-immediate reactions to drugs.

    PubMed

    Cornejo-Garcia, J A; Mayorga, C; Torres, M J; Fernandez, T D; R-Pena, R; Bravo, I; Mates, J M; Blanca, M

    2006-08-01

    Adverse drug reactions with an immunological basis (ADRIB) may involve activation of other concomitant, non-specific mechanisms, amplifying the specific response and contributing to the severity and duration. One concomitant mechanism could be the generation of reactive oxygen species (ROS) and/or their detoxification by anti-oxidants, including anti-oxidant enzymes. We analysed the activity of the anti-oxidant enzymes Cu/Zn-superoxide dismutase (SOD), catalase (CAT) and cellular glutathione peroxidase (GPX), as well as certain markers of oxidative damage (thiobarbituric acid reactive substances (TBARS) and carbonyl content) in peripheral blood mononuclear cells from patients with non-immediate ADRIB using spectrophotometric methods and the anti-oxidant enzymes expression by quantitative real-time reverse transcription-polymerase chain reaction. SOD activity and expression were increased in all types of non-immediate reactions (urticaria, maculopapular exanthema and toxic epidermal necrolysis). Regarding oxidative damage, TBARS were increased in urticaria and maculopapular exanthema, and carbonyl groups in all types of reactions. Our observations indicate that oxidative damage occurs in non-immediate reactions. Carbonyl stress and the inadequacy of the anti-oxidant defences are probable causes.

  17. Enzymic oxidation of some alkylbenzenes in insects and vertebrates.

    PubMed

    Chakroborty, J; Smith, J N

    1967-02-01

    1. Oxidation rates of alkylbenzenes have been measured in 10000g supernatants of vertebrate livers, locust fat bodies and housefly abdomens. 2. Activity per g. of insect was greater in fly than locust preparations but both were of the same order as a range of vertebrate species. 3. Methyl groups of toluene and p-nitrotoluene were oxidized more rapidly than the side chains of higher homologues. 4. In the higher homologues hydroxylation occurred most readily at the alpha-methylene group and less readily at penultimate methylene and terminal methyl groups. 5. Oxidations in both vertebrates and insects were inhibited by piperonylbutoxide and similar synergists. 6. Oxidation activity was stimulated by pretreatment of rats, but not locusts, with phenobarbitone or 3,4-benzopyrene.

  18. Peroxisomal. beta. -oxidation enzyme proteins in adrenoleukodystrophy: distinction between x-linked adrenoleukodystrophy and neonatal adrenoleukodystrophy

    SciTech Connect

    Chen, W.W.; Watkins, P.A.; Osumi, T.; Hashimoto, T.; Moser, H.W.

    1987-03-01

    Very long chain fatty acids, which accumulate in plasma and tissues in x-linked adrenoleukodystrophy (ALD), neonatal ALD, and the Zellweger cerebrohepatorenal syndrome, are degraded by the peroxisomal ..beta..-oxidation pathway, consisting of acyl-CoA oxidase, the bifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, and ..beta..-ketothiolase. A marked deficiency of all three enzyme proteins was reported in livers from patients with the Zellweger syndrome, a disorder in which peroxisomes are decreased or absent. Peroxisomes are not as markedly decreased in neonatal ALD and appear normal in x-linked ALD. Immunoblot analysis of the peroxisomal ..beta..-oxidation enzymes revealed an almost complete lack of the bifunctional enzymes in neonatal ALD liver, similar to the finding in Zellweger tissues. In contrast, acyl-CoA oxidase and ..beta..-ketothiolase were present in neonatal ALD liver, although the thiolase appeared to be in precursor form (2-3 kDa larger than the mature enzyme) in neonatal ALD. Unlike either neonatal ALD or Zellweger syndrome, all three peroxisomal ..beta..-oxidation enzymes were present in x-linked ALD liver. Despite the absence in neonatal ALD liver of bifunctional enzyme protein, its mRNA was detected by RNA blot analysis in fibroblasts from these patients. These observations suggest that lack of bifunctional enzyme protein in neonatal ALD results from either abnormal translation of the mRNA or degradation of the enzyme prior to its entry into peroxisomes.

  19. Enzymes of respiratory iron oxidation. Progress report, March 1990--November 1991

    SciTech Connect

    Blake, R. II

    1991-12-31

    This report focuses on the progress made in three areas of research concerned with enzymes involved in respiratory iron oxidation. The three areas are as follows: development of an improved procedure for the routine large scale culture of iron oxidizing chemolithotrophs based on the in-situ electrolysis of the soluble iron in the growth medium; to perform iron oxidation kinetic studies on whole cells using the oxygen electrode; and to identify, separate, purify, and characterize the individual cellular components.

  20. Nitric Oxide Measurement from Purified Enzymes and Estimation of Scavenging Activity by Gas Phase Chemiluminescence Method.

    PubMed

    Kumari, Aprajita; Gupta, Alok Kumar; Mishra, Sonal; Wany, Aakanksha; Gupta, Kapuganti Jagadis

    2016-01-01

    In plants, nitrate reductase (NR) is a key enzyme that produces nitric oxide (NO) using nitrite as a substrate. Lower plants such as algae are shown to have nitric oxide synthase enzyme and higher plants contain NOS activity but enzyme responsible for NO production in higher plants is subjected to debate. In plant nitric oxide research, it is very important to measure NO very precisely in order to determine its functional role. A significant amount of NO is being scavenged by various cell components. The net NO production depends in production minus scavenging. Here, we describe methods to measure NO from purified NR and inducible nitric oxide synthase from mouse (iNOS), we also describe a method of measure NO scavenging by tobacco cell suspensions and mitochondria from roots.

  1. Oxidative bioelectrocatalysis: From natural metabolic pathways to synthetic metabolons and minimal enzyme cascades.

    PubMed

    Minteer, Shelley D

    2016-05-01

    Anodic bioelectrodes for biofuel cells are more complex than cathodic bioelectrodes for biofuel cells, because laccase and bilirubin oxidase can individually catalyze four electron reduction of oxygen to water, whereas most anodic enzymes only do a single two electron oxidation of a complex fuel (i.e. glucose oxidase oxidizing glucose to gluconolactone while generating 2 electrons of the total 24 electrons), so enzyme cascades are typically needed for complete oxidation of the fuel. This review article will discuss the lessons learned from natural metabolic pathways about multi-step oxidation and how those lessons have been applied to minimal or artificial enzyme cascades. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

  2. CP12-mediated protection of Calvin-Benson cycle enzymes from oxidative stress.

    PubMed

    Marri, Lucia; Thieulin-Pardo, Gabriel; Lebrun, Régine; Puppo, Rémy; Zaffagnini, Mirko; Trost, Paolo; Gontero, Brigitte; Sparla, Francesca

    2014-02-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) are two energy-consuming enzymes of the Calvin-Benson cycle, whose regulation is crucial for the global balance of the photosynthetic process under different environmental conditions. In oxygen phototrophs, GAPDH and PRK regulation involves the redox-sensitive protein CP12. In the dark, oxidized chloroplast thioredoxins trigger the formation of a GAPDH/CP12/PRK complex in which both enzyme activities are down-regulated. In this report, we show that free GAPDH (A4-isoform) and PRK are also inhibited by oxidants like H2O2, GSSG and GSNO. Both in the land plant Arabidopsis thaliana and in the green microalga Chlamydomonas reinhardtii, both enzymes can be glutathionylated as shown by biotinylated-GSSG assay and MALDI-ToF mass spectrometry. CP12 is not glutathionylated but homodisulfides are formed upon oxidant treatments. In Arabidopsis but not in Chlamydomonas, the interaction between oxidized CP12 and GAPDH provides full protection from oxidative damage. In both organisms, preformed GAPDH/CP12/PRK complexes are protected from GSSG or GSNO oxidation, and in Arabidopsis also from H2O2 treatment. Overall, the results suggest that the role of CP12 in oxygen phototrophs needs to be extended beyond light/dark regulation, and include protection of enzymes belonging to Calvin-Benson cycle from oxidative stress. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  3. Modeling nitrous oxide production and reduction in soil through explicit representation of denitrification enzyme kinetics.

    PubMed

    Zheng, Jianqiu; Doskey, Paul V

    2015-02-17

    An enzyme-explicit denitrification model with representations for pre- and de novo synthesized enzymes was developed to improve predictions of nitrous oxide (N2O) accumulations in soil and emissions from the surface. The metabolic model of denitrification is based on dual-substrate utilization and Monod growth kinetics. Enzyme synthesis/activation was incorporated into each sequential reduction step of denitrification to regulate dynamics of the denitrifier population and the active enzyme pool, which controlled the rate function. Parameterizations were developed from observations of the dynamics of N2O production and reduction in soil incubation experiments. The model successfully reproduced the dynamics of N2O and N2 accumulation in the incubations and revealed an important regulatory effect of denitrification enzyme kinetics on the accumulation of denitrification products. Pre-synthesized denitrification enzymes contributed 20, 13, 43, and 62% of N2O that accumulated in 48 h incubations of soil collected from depths of 0-5, 5-10, 10-15, and 15-25 cm, respectively. An enzyme activity function (E) was defined to estimate the relative concentration of active enzymes and variation in response to environmental conditions. The value of E allows for activities of pre-synthesized denitrification enzymes to be differentiated from de novo synthesized enzymes. Incorporating explicit representations of denitrification enzyme kinetics into biogeochemical models is a promising approach for accurately simulating dynamics of the production and reduction of N2O in soils.

  4. Enzymic oxidation of unconjugated bilirubin by rat liver.

    PubMed Central

    Cardenas-Vazquez, R; Yokosuka, O; Billing, B H

    1986-01-01

    The presence of the enzyme bilirubin oxidase, which degrades bilirubin in vitro, was demonstrated in the liver. Subcellular-fractionation experiments indicate that bilirubin oxidase is located in both the inner and outer membranes of the mitochondria. The mean rate of the reaction is 1.57 +/- 0.38 (S.D.) nmol of bilirubin degraded/min per mg of mitochondrial protein (munits/mg of protein). With respect to the overall breakdown of bilirubin, the enzyme has a Km' of 136 microM-bilirubin and a Vmax.' of 9.13 munits/mg of protein. Its activity is influenced by the ionic strength of the media and is inhibited by KCN, thiol reagents, NADH and albumin. The enzyme is aerobic, and between 1 and 1.5 mol of O2 are consumed per mol of bilirubin degraded. The products of the reaction include propentdyopents. The hepatic bilirubin oxidase activity of the jaundiced Gunn-rat liver is not significantly different from that of the Sprague-Dawley rat, and it is not induced by beta-naphthoflavone. PMID:3790083

  5. Go it alone: four-electron oxidations by mononuclear non-heme iron enzymes.

    PubMed

    Peck, Spencer C; van der Donk, Wilfred A

    2017-04-01

    This review discusses the current mechanistic understanding of a group of mononuclear non-heme iron-dependent enzymes that catalyze four-electron oxidation of their organic substrates without the use of any cofactors or cosubstrates. One set of enzymes acts on α-ketoacid-containing substrates, coupling decarboxylation to oxygen activation. This group includes 4-hydroxyphenylpyruvate dioxygenase, 4-hydroxymandelate synthase, and CloR involved in clorobiocin biosynthesis. A second set of enzymes acts on substrates containing a thiol group that coordinates to the iron. This group is comprised of isopenicillin N synthase, thiol dioxygenases, and enzymes involved in the biosynthesis of ergothioneine and ovothiol. The final group of enzymes includes HEPD and MPnS that both carry out the oxidative cleavage of the carbon-carbon bond of 2-hydroxyethylphosphonate but generate different products. Commonalities amongst many of these enzymes are discussed and include the initial substrate oxidation by a ferric-superoxo-intermediate and a second oxidation by a ferryl species.

  6. Is Liver Enzyme Release Really Associated with Cell Necrosis Induced by Oxidant Stress?

    PubMed

    Contreras-Zentella, Martha Lucinda; Hernández-Muñoz, Rolando

    2016-01-01

    Hepatic diseases are a major concern worldwide. Increased specific plasma enzyme activities are considered diagnostic features for liver diseases, since enzymes are released into the blood compartment following the deterioration of the organ. Release of liver mitochondrial enzymes is considered strong evidence for hepatic necrosis, which is associated with an increased production of ROS, often leading to greater hepatic lipid peroxidation. Lipotoxic mediators and intracellular signals activated Kupffer cells, which provides evidence strongly suggesting the participation of oxidant stress in acute liver damage, inducing the progression of liver injury to chronic liver damage. Elevated transaminase activities are considered as an index marker of hepatotoxicity, linked to oxidant stress. However, a drastic increase of serum activities of liver enzyme markers ought not necessarily to reflect liver cell death. In fact, increased serum levels of cytoplasmic enzymes have readily been observed after partial hepatectomy (PH) in the regenerating liver of rats. In this regard, we are now showing that in vitro modifications of the oxidant status affect differentially the release of liver enzymes, indicating that this release is a strictly controlled event and not directly related to the onset of oxidant stress of the liver.

  7. Is Liver Enzyme Release Really Associated with Cell Necrosis Induced by Oxidant Stress?

    PubMed Central

    Contreras-Zentella, Martha Lucinda; Hernández-Muñoz, Rolando

    2016-01-01

    Hepatic diseases are a major concern worldwide. Increased specific plasma enzyme activities are considered diagnostic features for liver diseases, since enzymes are released into the blood compartment following the deterioration of the organ. Release of liver mitochondrial enzymes is considered strong evidence for hepatic necrosis, which is associated with an increased production of ROS, often leading to greater hepatic lipid peroxidation. Lipotoxic mediators and intracellular signals activated Kupffer cells, which provides evidence strongly suggesting the participation of oxidant stress in acute liver damage, inducing the progression of liver injury to chronic liver damage. Elevated transaminase activities are considered as an index marker of hepatotoxicity, linked to oxidant stress. However, a drastic increase of serum activities of liver enzyme markers ought not necessarily to reflect liver cell death. In fact, increased serum levels of cytoplasmic enzymes have readily been observed after partial hepatectomy (PH) in the regenerating liver of rats. In this regard, we are now showing that in vitro modifications of the oxidant status affect differentially the release of liver enzymes, indicating that this release is a strictly controlled event and not directly related to the onset of oxidant stress of the liver. PMID:26798419

  8. Detoxification of sulfur mustard by enzyme-catalyzed oxidation using chloroperoxidase.

    PubMed

    Popiel, S; Nawała, J

    2013-10-10

    One of the most interesting methods for the detoxification of sulfur mustard is enzyme-catalyzed oxidation. This study examined the oxidative destruction of a sulfur mustard by the enzyme chloroperoxidase (EC 1.11.1.10). Chloroperoxidase (CPO) belongs to a group of enzymes that catalyze the oxidation of various organic compounds by peroxide in the presence of a halide ion. The enzymatic oxidation reaction is affected by several factors: pH, presence or absence of chloride ion, temperature, the concentrations of hydrogen peroxide and enzyme and aqueous solubility of the substrate. The optimum reaction conditions were determined by analyzing the effects of all factors, and the following conditions were selected: solvent, Britton-Robinson buffer (pH=3) with tert-butanol (70:30 v/v); CPO concentration, 16U/mL; hydrogen peroxide concentration, 40mmol/L; sodium chloride concentration, 20mmol/L. Under these reaction conditions, the rate constant for the reaction is 0.006s(-1). The Michaelis constant, a measure of the affinity of an enzyme for a particular substrate, is 1.87×10(-3)M for this system. The Michaelis constant for enzymes with a high affinity for their substrate is in the range of 10(-5) to 10(-4)M, so this value indicates that CPO does not have a very high affinity for sulfur mustard. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. A mitochondrial enzyme degrades carotenoids and protects against oxidative stress.

    PubMed

    Amengual, Jaume; Lobo, Glenn P; Golczak, Marcin; Li, Hua Nan M; Klimova, Tatyana; Hoppel, Charles L; Wyss, Adrian; Palczewski, Krzysztof; von Lintig, Johannes

    2011-03-01

    Carotenoids are the precursors for vitamin A and are proposed to prevent oxidative damage to cells. Mammalian genomes encode a family of structurally related nonheme iron oxygenases that modify double bonds of these compounds by oxidative cleavage and cis-to-trans isomerization. The roles of the family members BCMO1 and RPE65 for vitamin A production and vision have been well established. Surprisingly, we found that the third family member, β,β-carotene-9',10'-oxygenase (BCDO2), is a mitochondrial carotenoid-oxygenase with broad substrate specificity. In BCDO2-deficient mice, carotenoid homeostasis was abrogated, and carotenoids accumulated in several tissues. In hepatic mitochondria, accumulated carotenoids induced key markers of mitochondrial dysfunction, such as manganese superoxide dismutase (9-fold), and reduced rates of ADP-dependent respiration by 30%. This impairment was associated with an 8- to 9-fold induction of phosphor-MAP kinase and phosphor-AKT, markers of cell signaling pathways related to oxidative stress and disease. Administration of carotenoids to human HepG2 cells depolarized mitochondrial membranes and resulted in the production of reactive oxygen species. Thus, our studies in BCDO2-deficient mice and human cell cultures indicate that carotenoids can impair respiration and induce oxidative stress. Mammalian cells thus express a mitochondrial carotenoid-oxygenase that degrades carotenoids to protect these vital organelles.

  10. Technical decision making with higher order structure data: utilization of differential scanning calorimetry to elucidate critical protein structural changes resulting from oxidation.

    PubMed

    Arthur, Kelly K; Dinh, Nikita; Gabrielson, John P

    2015-04-01

    Differential scanning calorimetry (DSC) is a useful tool for monitoring thermal stability of the molecular conformation of proteins. Here, we present an example of the sensitivity of DSC to changes in stability arising from a common chemical degradation pathway, oxidation. This Note is part of a series of industry case studies demonstrating the application of higher order structure data for technical decision making. For this study, six protein products from three structural classes were evaluated at multiple levels of oxidation. For each protein, the melting temperature (Tm ) decreased linearly as a function of oxidation; however, differences in the rate of change in Tm , as well as differences in domain Tm stability were observed across and within structural classes. For one protein, analysis of the impact of oxidation on protein function was also performed. For this protein, DSC was shown to be a leading indicator of decreased antigen binding suggesting a subtle conformation change may be underway that can be detected using DSC prior to any observable impact on product potency. Detectable changes in oxidized methionine by mass spectrometry (MS) occurred at oxidation levels below those with a detectable conformational or functional impact. Therefore, by using MS, DSC, and relative potency methods in concert, the intricate relationship between a primary structural modification, changes in conformational stability, and functional impact can be elucidated.

  11. The reduction of graphene oxide with hydrazine: elucidating its reductive capability based on a reaction-model approach.

    PubMed

    Chua, Chun Kiang; Pumera, Martin

    2016-01-04

    We have performed an experimental investigation on the effects of hydrazine treatment on graphene oxide via a reaction-model approach. Hydrazine was reacted with small conjugated aromatic compounds containing various oxygen functional groups to mimic the structure of graphene oxide. The hydroxyl and carboxylic groups were not readily removed while carbonyl groups reacted with hydrazine to form the corresponding hydrazone complexes. In the presence of adjacent hydroxyl groups, carboxyl groups underwent thermal decarboxylation.

  12. Nitric oxide dioxygenase: An enzymic function for flavohemoglobin

    PubMed Central

    Gardner, Paul R.; Gardner, Anne M.; Martin, Lori A.; Salzman, Andrew L.

    1998-01-01

    Nitric oxide (NO•) is a toxin, and various life forms appear to have evolved strategies for its detoxification. NO•-resistant mutants of Escherichia coli were isolated that rapidly consumed NO•. An NO•-converting activity was reconstituted in extracts that required NADPH, FAD, and O2, was cyanide-sensitive, and produced NO3−. This nitric oxide dioxygenase (NOD) contained 19 of 20 N-terminal amino acids identical to those of the E. coli flavohemoglobin. Furthermore, NOD activity was produced by the flavohemoglobin gene and was inducible by NO•. Flavohemoglobin/NOD-deficient mutants were also sensitive to growth inhibition by gaseous NO•. The results identify a function for the evolutionarily conserved flavohemoglobins and, moreover, suggest that NO• detoxification may be a more ancient function for the widely distributed hemoglobins, and associated methemoglobin reductases, than dioxygen transport and storage. PMID:9724711

  13. Mutagenicity screening of reaction products from the enzyme-catalyzed oxidation of phenolic pollutants

    SciTech Connect

    Massey, I.J.; Aitken, M.D.; Ball, L.M.; Heck, P.E. . Dept. of Environmental Sciences and Engineering)

    1994-11-01

    Phenol-oxidizing enzymes such as peroxidases, laccases, and mushroom polyphenol oxidase are capable of catalyzing the oxidation of a wide range of phenolic pollutants. Although the use of these enzymes in waste-treatment applications has been proposed by a number of investigators, little information exists on the toxicological characteristics of the oxidation products. The enzymes chloroperoxidase, horseradish peroxidase, lignin peroxidase, and mushroom polyphenol oxidase were used in this study to catalyze the oxidation of phenol, several mono-substituted phenols, and pentachlorophenol. Seventeen reaction mixtures representing selected combinations of enzyme and parent phenol were subjected to mutagenicity screening using the Ames Salmonella typhimurium plate incorporation assay; five selected mixtures were also incubated with the S9 microsomal preparation to detect the possible presence of promutagens. The majority of reaction mixtures tested were not directly mutagenic, and none of those tested with S9 gave a positive response. Such lack of mutagenicity of enzymatic oxidation products provides encouragement for establishing the feasibility of enzyme-catalyzed oxidation as a waste-treatment process. The only positive responses were obtained with reaction products from the lignin peroxidase-catalyzed oxidation of 2-nitrophenol and 4-nitrophenol. Clear positive responses were observed when strain TA100 was incubated with 2-nitrophenol reaction-product mixtures, and when strain TA98 was incubated with the 4-nitrophenol reaction mixture. Additionally, 2,4-dinitrophenol was identified as a reaction product from 4-nitrophenol, and preliminary evidence indicates that both 2,4- and 2,6-dinitrophenol are produced from the oxidation of 2-nitrophenol. Possible mechanism by which these nitration reactions occur are discussed.

  14. Regio and Stereodivergent Antibiotic Oxidative Carbocyclizations Catalyzed by Rieske Oxygenase-Like Enzymes

    PubMed Central

    Sydor, Paulina K.; Barry, Sarah M.; Odulate, Olanipekun M.; Barona-Gomez, Francisco; Haynes, Stuart W.; Corre, Christophe; Song, Lijiang; Challis, Gregory L.

    2011-01-01

    Oxidative cyclizations, exemplified by the biosynthetic assembly of the penicillin nucleus from a tripeptide precursor, are arguably the most synthetically-powerful implementation of C-H activation reactions in Nature. Here we show that Rieske oxygenase-like enzymes mediate regio and stereodivergent oxidative cyclizations to form 10- and 12-membered carbocyclic rings in the key steps of the biosynthesis of the antibiotics streptorubin B and metacycloprodigiosin, respectively. These reactions represent the first examples of oxidative carbocyclizations catalyzed by non-heme iron-dependent oxidases and define a novel type of catalytic activity for Rieske enzymes. A better understanding of how these enzymes achieve such remarkable regio and stereocontrol in the functionalization of unactivated hydrocarbon chains will greatly facilitate the development of selective manmade C-H activation catalysts. PMID:21505498

  15. Regio- and stereodivergent antibiotic oxidative carbocyclizations catalysed by Rieske oxygenase-like enzymes.

    PubMed

    Sydor, Paulina K; Barry, Sarah M; Odulate, Olanipekun M; Barona-Gomez, Francisco; Haynes, Stuart W; Corre, Christophe; Song, Lijiang; Challis, Gregory L

    2011-05-01

    Oxidative cyclizations, exemplified by the biosynthetic assembly of the penicillin nucleus from a tripeptide precursor, are arguably the most synthetically powerful implementation of C-H activation reactions in nature. Here, we show that Rieske oxygenase-like enzymes mediate regio- and stereodivergent oxidative cyclizations to form 10- and 12-membered carbocyclic rings in the key steps of the biosynthesis of the antibiotics streptorubin B and metacycloprodigiosin, respectively. These reactions represent the first examples of oxidative carbocyclizations catalysed by non-haem iron-dependent oxidases and define a novel type of catalytic activity for Rieske enzymes. A better understanding of how these enzymes achieve such remarkable regio- and stereocontrol in the functionalization of unactivated hydrocarbon chains will greatly facilitate the development of selective man-made C-H activation catalysts.

  16. Integrated structural biology and molecular ecology of N-cycling enzymes from ammonia-oxidizing archaea

    DOE PAGES

    Tolar, Bradley B.; Herrmann, Jonathan; Bargar, John R.; ...

    2017-06-01

    In this paper, knowledge of the molecular ecology and environmental determinants of ammonia-oxidizing organisms is critical to understanding and predicting the global nitrogen (N) and carbon cycles, but an incomplete biochemical picture hinders in vitro studies of N-cycling enzymes. Although an integrative structural and dynamic characterization at the atomic scale would advance our understanding of function tremendously, structural knowlede of key N-cycling enzymes from ecologically-relevant ammonia oxidizers is unfortunately extremely limited. Here, we discuss the challenges and opportunities for examining the ecology of ammonia-oxidizing organisms, particularly uncultivated Thaumarchaeota, though (meta)genome-driven structural biology of the enzymes ammonia monooxygenase (AMO) andmore » nitrite reductase (NirK).« less

  17. Regio- and stereodivergent antibiotic oxidative carbocyclizations catalysed by Rieske oxygenase-like enzymes

    NASA Astrophysics Data System (ADS)

    Sydor, Paulina K.; Barry, Sarah M.; Odulate, Olanipekun M.; Barona-Gomez, Francisco; Haynes, Stuart W.; Corre, Christophe; Song, Lijiang; Challis, Gregory L.

    2011-05-01

    Oxidative cyclizations, exemplified by the biosynthetic assembly of the penicillin nucleus from a tripeptide precursor, are arguably the most synthetically powerful implementation of C-H activation reactions in nature. Here, we show that Rieske oxygenase-like enzymes mediate regio- and stereodivergent oxidative cyclizations to form 10- and 12-membered carbocyclic rings in the key steps of the biosynthesis of the antibiotics streptorubin B and metacycloprodigiosin, respectively. These reactions represent the first examples of oxidative carbocyclizations catalysed by non-haem iron-dependent oxidases and define a novel type of catalytic activity for Rieske enzymes. A better understanding of how these enzymes achieve such remarkable regio- and stereocontrol in the functionalization of unactivated hydrocarbon chains will greatly facilitate the development of selective man-made C-H activation catalysts.

  18. Elucidating the mechanism of Cr(VI) formation upon the interaction with metal oxides during coal oxy-fuel combustion.

    PubMed

    Chen, Juan; Jiao, Facun; Zhang, Lian; Yao, Hong; Ninomiya, Yoshihiko

    2013-10-15

    The thermodynamics underpinning the interaction of Cr-bearing species with basic metal oxides, i.e. K2O, Fe2O3, MgO and CaO, during the air and oxy-fuel combustion of coal have been examined. The synchrotron-based X-ray adsorption near-edge spectroscopy (XANES) was used for Cr speciation. For the oxides tested, Cr(VI) formation is dominated by the reduction potential of the metals. The oxides of Ca(2+) with high reduction potential favored the oxidation of Cr(III), same for K(+). The other two basic metals, Fe2O3 and MgO with lower reduction potentials reacted with Cr(III) to form the corresponding chromites at the temperatures above 600°C. Coal combustion experiments in drop-tube furnace have confirmed the rapid capture of Cr vapors, either trivalent or hexavalent, by CaO into solid ash. The existence of HCl in flue gas favored the vaporization of Cr as CrO2Cl2, which was in turn captured by CaO into chromate. Both Fe2O3 and MgO exhibited less capability on scavenging the Cr(VI) vapor. Particularly, MgO alone exhibited a low capability for capturing the vaporized Cr(III) vapors. However, its co-existence with CaO in the furnace inhibited the Cr(VI) formation. This is beneficial for minimizing the toxicity of Cr in the coal combustion-derived fly ash.

  19. Functionalized Anodic Aluminum Oxide Membrane–Electrode System for Enzyme Immobilization

    PubMed Central

    2015-01-01

    A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions—a one-step “reverse” sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6–20 min–1 on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min–1. UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process. PMID:25025628

  20. Functionalized anodic aluminum oxide membrane-electrode system for enzyme immobilization.

    PubMed

    Chen, Zhiqiang; Zhang, Jianjun; Singh, Shanteri; Peltier-Pain, Pauline; Thorson, Jon S; Hinds, Bruce J

    2014-08-26

    A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions—a one-step “reverse” sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6–20 min(–1) on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min(–1). UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process.

  1. Aerobic oxidation of β-isophorone catalyzed by N-hydroxyphthalimide: the key features and mechanism elucidated.

    PubMed

    Chen, Kexian; Sun, Yong; Wang, Congmin; Yao, Jia; Chen, Zhirong; Li, Haoran

    2012-09-21

    Due to the insufficient understanding of the selective oxidation mechanism of α/β-isophorones (α/β-IP) to ketoisophorone (KIP), the key features in the β-IP oxidation catalyzed by N-hydroxyphthalimide (NHPI) have been explored via theoretical calculations. β-IP is more favourable to being activated by phthalimide-N-oxyl radical (PINO˙) and peroxyl radical (ROO˙) than α-IP owing to the different C-H strengths at their reactive sites, thereby exhibiting selective product distributions. It was found that NHPI accelerates β-IP activation due to the higher reactivity of PINO˙ than ROO˙ and the equilibrium reaction between them, yielding considerable hydroperoxide (ROOH) and ROO˙. In addition, the ROOH decomposition is more favourable viaα-H abstraction by radicals than its self-dehydration and thermal dissociation. The strong exothermicity of this α-H abstraction, along with that from H-abstraction by co-yielded hot HO˙, is in favor of the straightforward formation of KIP, simultaneously leading to the isomerization of a few β-IP to α-IP and production of 4-hydroxyisophorone (HIP) and water. The proposed mechanisms, consistent with the experimental observations, allow for the deeper understanding and effective design of oxidation systems involving similar substrates or NHPI analogues that are of industrial importance.

  2. Status of oxidant, antioxidantand serum enzymes in thalassaemic children receiving multiple blood transfusions.

    PubMed

    Asif, Mahmood; Manzoor, Zahid; Farooq, Muhammad Shehzad; Munawar, Shaukat Hussain; Aziz, Abdul; Khan, Imran Ahmad

    2015-08-01

    To determine the levels of oxidant, antioxidant and serum enzymes in thalassaemic children receiving multiple blood transfusions. The case-control study was done from February to August 2012, and comprised thalassaemic children receiving multiple blood transfusions at Allied Hospital, Ali Zeb Foundation, and the Thalassaemia Centre in Hilal-e-Ahmar Hospital, Faisalabad, Pakistan. Healthy subjects were also screened for any related disease condition that could prejudice the results. Blood samples were analysed for the values of total oxidant status, total antioxidant capacity, serum malondialdehyde, catalase, paraoxonase, arylesterase, glutathione peroxidase and ceruloplasmin. There were 180 children in the study; 90(50%) cases and 90(50%) controls. Of the cases, 48(53.3%) were under-weight while the weight of 42(46.7%) was in the normal range. The values of total oxidant status and total antioxidant capacity were significantly (p<0.01) higher in thalassaemic children compared to normal values. Serum malondialdehyde and catalase levels were also considerably elevated (p<0.05), suggesting the increased activity of these enzymes. However, the concentrations of serum paraoxonase, arylesterase, glutathione peroxidase were significantly (p<0.01) lower in cases than the controls, displaying diminished activities during multiple blood transfusions in these patients. Multiple blood transfusions disconcert the levels of oxidants, antioxidants and serum enzymes of thalassaemic children. Oxidative damage is seen because of the increased iron overload in these patients. Hence, regular evaluation of oxidant and antioxidant status should be monitored in thalassaemic patients during initial few years of life.

  3. Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

    PubMed

    Kitta, Ryo; Kuwamoto, Marina; Yamahama, Yumi; Mase, Keisuke; Sawada, Hiroshi

    2016-12-01

    To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori.

  4. Differential expression of genes encoding anti-oxidant enzymes in Sydney rock oysters, Saccostrea glomerata (Gould) selected for disease resistance.

    PubMed

    Green, Timothy J; Dixon, Tom J; Devic, Emilie; Adlard, Robert D; Barnes, Andrew C

    2009-05-01

    Sydney rock oysters (Saccostrea glomerata) selectively bred for disease resistance (R) and wild-caught control oysters (W) were exposed to a field infection of disseminating neoplasia. Cumulative mortality of W oysters (31.7%) was significantly greater than R oysters (0.0%) over the 118 days of the experiment. In an attempt to understand the biochemical and molecular pathways involved in disease resistance, differentially expressed sequence tags (ESTs) between R and W S. glomerata hemocytes were identified using the PCR technique, suppression subtractive hybridisation (SSH). Sequencing of 300 clones from two SSH libraries revealed 183 distinct sequences of which 113 shared high similarity to sequences in the public databases. Putative function could be assigned to 64 of the sequences. Expression of nine ESTs homologous to genes previously shown to be involved in bivalve immunity was further studied using quantitative reverse-transcriptase PCR (qRT-PCR). The base-line expression of an extracellular superoxide dismutase (ecSOD) and a small heat shock protein (sHsP) were significantly increased, whilst peroxiredoxin 6 (Prx6) and interferon inhibiting cytokine factor (IK) were significantly decreased in R oysters. From these results it was hypothesised that R oysters would be able to generate the anti-parasitic compound, hydrogen peroxide (H(2)O(2)) faster and to higher concentrations during respiratory burst due to the differential expression of genes for the two anti-oxidant enzymes of ecSOD and Prx6. To investigate this hypothesis, protein extracts from hemolymph were analysed for oxidative burst enzyme activity. Analysis of the cell free hemolymph proteins separated by native-polyacrylamide gel electrophoresis (PAGE) failed to detect true superoxide dismutase (SOD) activity by assaying dismutation of superoxide anion in zymograms. However, the ecSOD enzyme appears to generate hydrogen peroxide, presumably via another process, which is yet to be elucidated. This

  5. Structure of an archaeal PCNA1–PCNA2–FEN1 complex: elucidating PCNA subunit and client enzyme specificity

    PubMed Central

    Doré, Andrew S.; Kilkenny, Mairi L.; Jones, Sarah A.; Oliver, Antony W.; Roe, S. Mark; Bell, Stephen D.; Pearl, Laurence H.

    2006-01-01

    The archaeal/eukaryotic proliferating cell nuclear antigen (PCNA) toroidal clamp interacts with a host of DNA modifying enzymes, providing a stable anchorage and enhancing their respective processivities. Given the broad range of enzymes with which PCNA has been shown to interact, relatively little is known about the mode of assembly of functionally meaningful combinations of enzymes on the PCNA clamp. We have determined the X-ray crystal structure of the Sulfolobus solfataricus PCNA1–PCNA2 heterodimer, bound to a single copy of the flap endonuclease FEN1 at 2.9 Å resolution. We demonstrate the specificity of interaction of the PCNA subunits to form the PCNA1–PCNA2–PCNA3 heterotrimer, as well as providing a rationale for the specific interaction of the C-terminal PIP-box motif of FEN1 for the PCNA1 subunit. The structure explains the specificity of the individual archaeal PCNA subunits for selected repair enzyme ‘clients’, and provides insights into the co-ordinated assembly of sequential enzymatic steps in PCNA-scaffolded DNA repair cascades. PMID:16945955

  6. Mitochondrial targeting of bilirubin regulatory enzymes: An adaptive response to oxidative stress

    SciTech Connect

    Muhsain, Siti Nur Fadzilah; Lang, Matti A.; Abu-Bakar, A'edah

    2015-01-01

    The intracellular level of bilirubin (BR), an endogenous antioxidant that is cytotoxic at high concentrations, is tightly controlled within the optimal therapeutic range. We have recently described a concerted intracellular BR regulation by two microsomal enzymes: heme oxygenase 1 (HMOX1), essential for BR production and cytochrome P450 2A5 (CYP2A5), a BR oxidase. Herein, we describe targeting of these enzymes to hepatic mitochondria during oxidative stress. The kinetics of microsomal and mitochondrial BR oxidation were compared. Treatment of DBA/2J mice with 200 mg pyrazole/kg/day for 3 days increased hepatic intracellular protein carbonyl content and induced nucleo-translocation of Nrf2. HMOX1 and CYP2A5 proteins and activities were elevated in microsomes and mitoplasts but not the UGT1A1, a catalyst of BR glucuronidation. A CYP2A5 antibody inhibited 75% of microsomal BR oxidation. The inhibition was absent in control mitoplasts but elevated to 50% after treatment. An adrenodoxin reductase antibody did not inhibit microsomal BR oxidation but inhibited 50% of mitochondrial BR oxidation. Ascorbic acid inhibited 5% and 22% of the reaction in control and treated microsomes, respectively. In control mitoplasts the inhibition was 100%, which was reduced to 50% after treatment. Bilirubin affinity to mitochondrial and microsomal CYP2A5 enzyme is equally high. Lastly, the treatment neither released cytochrome c into cytoplasm nor dissipated membrane potential, indicating the absence of mitochondrial membrane damage. Collectively, the observations suggest that BR regulatory enzymes are recruited to mitochondria during oxidative stress and BR oxidation by mitochondrial CYP2A5 is supported by mitochondrial mono-oxygenase system. The induced recruitment potentially confers membrane protection. - Highlights: • Pyrazole induces oxidative stress in the mouse liver. • Pyrazole-induced oxidative stress induces mitochondrial targeting of key bilirubin regulatory enzymes, HMOX1

  7. Evidence for the transport function of uricase, an oxidative enzyme

    SciTech Connect

    Pordy, W.T.; Lipkowitz, M.S.; Abramson, R.G. )

    1987-10-01

    (2-{sup 14}C)urate uptake was examined in proteoliposomes prepared with phosphatidylcholine and either pig liver uricase or albumin, and in protein-free liposomes. Urate uptake was only evident in proteoliposomes that contained active uricase. Uptakes were indistinguishable in the presence and absence of inwardly directed gradients of sodium, potassium, or choline chloride or outwardly directed hydroxyl gradients. Both urate and allantoin accumulated within proteoliposomes during urate uptake; however, (2-{sup 14}C)allantoin was not taken up by proteoliposomes. Urate uptake was accelerated in the presence of unlabeled urate in the trans position, saturable, and competitively inhibited by oxonate, findings consistent with carrier-mediated transport. Finally, the kinetics of urate uptake and oxidation were virtually identical, implying that the transporter is uricase. Thus, these studies provide evidence that uricase can function as a transport protein for urate when inserted in a lipid bilayer: transport via uricase is neither cation dependent (not a cotransporter) nor dependent on an exchangeable anion (not a urate/anion exchanger). Additionally, these studies demonstrate that neither urate nor allantoin cross lipid bilayers by simple or nonionic diffusion.

  8. Grape seed procyanidins prevent oxidative injury by modulating the expression of antioxidant enzyme systems.

    PubMed

    Puiggros, Francesc; Llópiz, Niurka; Ardévol, Anna; Bladé, Cinta; Arola, Lluís; Salvadó, M Josepa

    2005-07-27

    In the present paper, we report the effect of a grape seed procyanidin extract (GSPE) on antioxidant enzyme systems (AOEs). Gene expression was tested using the hepatocarcinoma cell line HepG2 by exposing it to several GSPE doses between 0 and 100 mg/L for 24 h. We evaluated mRNA expression and enzyme activity levels using real time RT-PCR and spectrophotometry. The results suggested a transcriptional GSPE regulation of glutathione related enzymes caused by an increase both in mRNA and in enzyme activity levels overall at 15 mg/L. We also assessed the GSPE effect on AOEs in cells submitted to oxidative stress. Under oxidative conditions (1 mM H(2)O(2), 1 h), we found a decrease in GSH content and an increase in MDA, and we suggested a posttranslational regulation of GPx/GR mRNAs and a transcriptional enhancement of GST mRNA. The GSPE pretreatment (15 mg/L, 23 h) before HepG2 submission to H(2)O(2) (1 mM, 1 h) showed an increase of the mRNA of GPx/GR with respect to the H(2)O(2) group, whereas the GSH content was similar to the control group. However, the GPx/GR enzyme activities were not increased. We hypothesize that GSPE probably improves the cellular redox status via glutathione synthesis pathways instead of regulation of the GPx and/or GR activities protecting against oxidative damage.

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

    SciTech Connect

    Murata, Kenichi; Nakamura, Nobuhumi Ohno, Hiroyuki

    2008-03-07

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

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

    PubMed

    Murata, Kenichi; Nakamura, Nobuhumi; Ohno, Hiroyuki

    2008-03-07

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

  11. Serum prolidase enzyme activity in obese subjects and its relationship with oxidative stress markers.

    PubMed

    Aslan, Mehmet; Duzenli, Ufuk; Esen, Ramazan; Soyoral, Yasemin Usul

    2017-10-01

    The relationship between increased serum enzyme activity of prolidase and increased rate of collagen turnover in the arterial wall has been asserted in previous studies. Collagen reflects much of the strength to the connective tissue involved in the arterial wall. Atherosclerosis is very common vessel disease and oxidative stress plays a pivotal role in the etiopathogenesis. Our objective was to examine the serum enzyme activity of prolidase and its possible relationships with oxidative stress parameters in obese subjects. Our present study was conducted 27 obese subjects and 26 age-matched healthy control subjects. The serum enzyme activity of prolidase in all study population was evaluated spectrophotometrically. Oxidative stress levels in obese subjects were analyzed with total antioxidant capacity (TAC) and total oxidant status (TOS) as well as oxidative stress index (OSI). Obese subjects have higher serum TOS and OSI indicators as well as prolidase activity than those in control subjects (for all; p<0.001). Moreover, obese subjects have lower levels of TAC than in those in healthy subjects (p<0.001). In the Pearson's correlation analysis, enzyme activity of prolidase was positively related with TOS (p<0.001, r=0.529) and OSI (p<0.001, r=0.519) as well as BMI (p<0.001, r=0.692) and inversely related with TAC (p<0.05, r=-0.405) in obese subjects. Increased serum prolidase activity and decreased antioxidant levels are likely to be a results of increased of oxidative stress levels in obese subjects. The significantly correlation between increased oxidative stress and increased prolidase activity may play a pivotal role in etiopathogenesis of atherosclerotic cardiovascular diseases in obese subjects. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Mitochondrial targeting of bilirubin regulatory enzymes: An adaptive response to oxidative stress.

    PubMed

    Muhsain, Siti Nur Fadzilah; Lang, Matti A; Abu-Bakar, A'edah

    2015-01-01

    The intracellular level of bilirubin (BR), an endogenous antioxidant that is cytotoxic at high concentrations, is tightly controlled within the optimal therapeutic range. We have recently described a concerted intracellular BR regulation by two microsomal enzymes: heme oxygenase 1 (HMOX1), essential for BR production and cytochrome P450 2A5 (CYP2A5), a BR oxidase. Herein, we describe targeting of these enzymes to hepatic mitochondria during oxidative stress. The kinetics of microsomal and mitochondrial BR oxidation were compared. Treatment of DBA/2J mice with 200mgpyrazole/kg/day for 3days increased hepatic intracellular protein carbonyl content and induced nucleo-translocation of Nrf2. HMOX1 and CYP2A5 proteins and activities were elevated in microsomes and mitoplasts but not the UGT1A1, a catalyst of BR glucuronidation. A CYP2A5 antibody inhibited 75% of microsomal BR oxidation. The inhibition was absent in control mitoplasts but elevated to 50% after treatment. An adrenodoxin reductase antibody did not inhibit microsomal BR oxidation but inhibited 50% of mitochondrial BR oxidation. Ascorbic acid inhibited 5% and 22% of the reaction in control and treated microsomes, respectively. In control mitoplasts the inhibition was 100%, which was reduced to 50% after treatment. Bilirubin affinity to mitochondrial and microsomal CYP2A5 enzyme is equally high. Lastly, the treatment neither released cytochrome c into cytoplasm nor dissipated membrane potential, indicating the absence of mitochondrial membrane damage. Collectively, the observations suggest that BR regulatory enzymes are recruited to mitochondria during oxidative stress and BR oxidation by mitochondrial CYP2A5 is supported by mitochondrial mono-oxygenase system. The induced recruitment potentially confers membrane protection. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

  13. Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida.

    PubMed Central

    Irie, S; Doi, S; Yorifuji, T; Takagi, M; Yano, K

    1987-01-01

    The nucleotide sequence of the genes from Pseudomonas putida encoding oxidation of benzene to catechol was determined. Five open reading frames were found in the sequence. Four corresponding protein molecules were detected by a DNA-directed in vitro translation system. Escherichia coli cells containing the fragment with the four open reading frames transformed benzene to cis-benzene glycol, which is an intermediate of the oxidation of benzene to catechol. The relation between the product of each cistron and the components of the benzene oxidation enzyme system is discussed. Images PMID:3667527

  14. [Photodynamic reaction and oxidative stress - influence of the photodynamic effect on the activity antioxidant enzymes].

    PubMed

    Romiszewska, Anna; Nowak-Stępniowska, Agata

    2014-01-01

    The interaction of light with a photosensitizer, accumulated in a tissue in the presence of oxygen, leads to formation of reactive oxygen species, mainly of singlet oxygen and free radicals. These factors react with biomolecules producing their oxidized states. Reactive oxygen species, such as singlet oxygen and free radicals are able to damage membranes, DNA, enzymes, structural peptides and other cellular structures leading to cell death. An antioxidant protection of cell is formed by enzymes belonging to the family of oxidoreductases: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR). Photodynamic therapy leads to the increased production of oxidizing toxic forms. It is important to analyze impact of PDT on the activity of antioxidant enzymes, such as SOD, CAT, GPx. The activity of antioxidant enzymes during the photodynamic effect is influenced by both the light energy dose and the concentration of photosensitizer. The presence only of the photosensitizer or only the light energy may also result in changes in the activity of these enzymes. The differences in changes in the activity of these enzymes depend on the type of used photosensitizer. A phenomenon of selective accumulation of photosensitizer in tumor tissues is used in the photodynamic method of tumor diagnosis and treatment.

  15. Elucidation of the chemistry of enzyme-bound thiamin diphosphate prior to substrate binding: defining internal equilibria among tautomeric and ionization states.

    PubMed

    Nemeria, Natalia; Korotchkina, Lioubov; McLeish, Michael J; Kenyon, George L; Patel, Mulchand S; Jordan, Frank

    2007-09-18

    Both solution and crystallographic studies suggest that the 4'-aminopyrimidine ring of the thiamin diphosphate coenzyme participates in catalysis, likely as an intramolecular general acid-base catalyst via the unusual 1',4'-iminopyrimidine tautomer. It is indeed uncommon for a coenzyme to be identified in its rare tautomeric form on its reaction pathways, yet this has been possible with thiamin diphosphate, in some cases even in the absence of substrate [Nemeria, N., Chakraborty, S., Baykal, A., Korotchkina, L., Patel, M. S., and Jordan, F. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 78-82.]. The ability to detect both the aminopyrimidine and iminopyrimidine tautomeric forms of thiamin diphosphate on enzymes has enabled us to assign the predominant tautomeric form present in individual intermediates on the pathway. Herein, we report the pH dependence of these tautomeric forms providing the first data for the internal thermodynamic equilibria on thiamin diphosphate enzymes for the various ionization and tautomeric forms of this coenzyme on four enzymes: benzaldehyde lyase, benzoylformate decarboxylase, pyruvate oxidase, and the E1 component of the human pyruvate dehydrogenase multienzyme complex. Evidence is provided for an important function of the enzyme environment in altering both the ionization and tautomeric equilibria on the coenzyme even prior to addition of substrate. The pKa for the 4'-aminopyrimidinium moiety coincides with the pH for optimum activity thereby ensuring that all ionization states and tautomeric states are accessible during the catalytic cycle. The dramatic influence of the protein on the internal equilibria also points to conditions under which the long-elusive ylide intermediate could be stabilized.

  16. Structure and mechanism of ORF36, an Aminosugar Oxidizing Enzyme in Everninomicin Biosynthesis†

    PubMed Central

    Vey, Jessica L.; Al-Mestarihi, Ahmad; Hu, Yunfeng; Funk, Michael A.; Bachmann, Brian O.; Iverson, T. M.

    2010-01-01

    Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of antibiotics and possesses potent gram-positive antibiotic activity, including broad-spectrum efficacy against multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitrosugar, l-evernitrose, analogs of which decorate a variety of natural products. Recently, we identified a nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the flavin-dependent double oxidation of synthetically-generated thymidine diphosphate (TDP)-l-epi-vancosamine to the corresponding nitroso sugar. Herein, we utilize a five enzyme in vitro pathway both to verify that ORF36 catalyzes oxidation of biogenic TDP-l-epi-vancosamine and to determine whether ORF36 exhibits catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. Progenitors solely undergo single oxidation reactions and terminate in the hydroxylamine oxidation state. Performing the in vitro reactions in the presence of 18O2 establishes that molecular oxygen, rather than oxygen from water, is incorporated into ORF36-generated intermediates and products, and identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The 3.15 Å resolution x-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-coA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3. However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase mechanism for amino sugar oxidation by ORF36. PMID:20866105

  17. Structure and Mechanism of ORF36, an Amino Sugar Oxidizing Enzyme in Everninomicin Biosynthesis

    SciTech Connect

    Vey, Jessica L.; Al-Mestarihi, Ahmad; Hu, Yunfeng; Funk, Michael A.; Bachmann, Brian O.; Iverson, T.M.

    2010-12-07

    Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of antibiotics and possesses potent Gram-positive antibiotic activity, including broad-spectrum efficacy against multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitro sugar, L-evernitrose, analogues of which decorate a variety of natural products. Recently, we identified a nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the flavin-dependent double oxidation of synthetically generated thymidine diphosphate (TDP)-L-epi-vancosamine to the corresponding nitroso sugar. Herein, we utilize a five-enzyme in vitro pathway both to verify that ORF36 catalyzes oxidation of biogenic TDP-L-epi-vancosamine and to determine whether ORF36 exhibits catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. Progenitors solely undergo single-oxidation reactions and terminate in the hydroxylamine oxidation state. Performing the in vitro reactions in the presence of {sup 18}O{sub 2} establishes that molecular oxygen, rather than oxygen from water, is incorporated into ORF36-generated intermediates and products and identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The 3.15 {angstrom} resolution X-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-CoA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3. However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase mechanism for amino sugar oxidation by ORF36.

  18. Purine utilization by Klebsiella oxytoca M5al: genes for ring-oxidizing and -opening enzymes.

    PubMed

    Pope, Scott D; Chen, Li-Ling; Stewart, Valley

    2009-02-01

    The enterobacterium Klebsiella oxytoca uses a variety of inorganic and organic nitrogen sources, including purines, nitrogen-rich compounds that are widespread in the biosphere. We have identified a 23-gene cluster that encodes the enzymes for utilizing purines as the sole nitrogen source. Growth and complementation tests with insertion mutants, combined with sequence comparisons, reveal functions for the products of these genes. Here, we report our characterization of 12 genes, one encoding guanine deaminase and the others encoding enzymes for converting (hypo)xanthine to allantoate. Conventionally, xanthine dehydrogenase, a broadly distributed molybdoflavoenzyme, catalyzes sequential hydroxylation reactions to convert hypoxanthine via xanthine to urate. Our results show that these reactions in K. oxytoca are catalyzed by a two-component oxygenase (HpxE-HpxD enzyme) homologous to Rieske nonheme iron aromatic-ring-hydroxylating systems, such as phthalate dioxygenase. Our results also reveal previously undescribed enzymes involved in urate oxidation to allantoin, catalyzed by a flavoprotein monooxygenase (HpxO enzyme), and in allantoin conversion to allantoate, which involves allantoin racemase (HpxA enzyme). The pathway also includes the recently described PuuE allantoinase (HpxB enzyme). The HpxE-HpxD and HpxO enzymes were discovered independently by de la Riva et al. (L. de la Riva, J. Badia, J. Aguilar, R. A. Bender, and L. Baldoma, J. Bacteriol. 190:7892-7903, 2008). Thus, several enzymes in this K. oxytoca purine utilization pathway differ from those in other microorganisms. Isofunctional homologs of these enzymes apparently are encoded by other species, including Acinetobacter, Burkholderia, Pseudomonas, Saccharomyces, and Xanthomonas.

  19. Oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays

    NASA Technical Reports Server (NTRS)

    Reinecke, D. M.; Bandurski, R. S.

    1988-01-01

    Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.

  20. Oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays

    NASA Technical Reports Server (NTRS)

    Reinecke, D. M.; Bandurski, R. S.

    1988-01-01

    Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.

  1. Elucidating the interactions and phytotoxicity of zinc oxide nanoparticles with agriculturally beneficial bacteria and selected crop plants.

    PubMed

    Boddupalli, Anuraag; Tiwari, Rameshwar; Sharma, Anamika; Singh, Surender; Prasanna, Radha; Nain, Lata

    2017-05-01

    There is a growing interest in the use of bioinoculants to assist mineral fertilizers in improving crop production and yield. Azotobacter and Pseudomonas are two agriculturally relevant strains of bacteria which have been established as efficient bioinoculants. An experiment involving addition of graded concentrations of zinc oxide (ZnO) nanoparticles was undertaken using log phase cultures of Azotobacter and Pseudomonas. Growth kinetics revealed a clear trend of gradual decrease with Pseudomonas; however, Azotobacter exhibited a twofold enhancement in growth with increase in the concentration of ZnO concentration. Scanning electron microscopy (SEM), supported by energy-dispersive X-ray (EDX) analyses, illustrated the significant effect of ZnO nanoparticles on Azotobacter by the enhancement in the abundance of globular biofilm-like structures and the intracellular presence of ZnO, with the increase in its concentration. It can be surmised that extracellular mucilage production in Azotobacter may be providing a barrier to the nanoparticles. Further experiments with Azotobacter by inoculation of wheat and tomato seeds with ZnO nanoparticles alone or bacteria grown on ZnO-infused growth medium revealed interesting results. Vigour index of wheat seeds reduced by 40-50% in the presence of different concentrations of ZnO nanoparticles alone, which was alleviated by 15-20%, when ZnO and Azotobacter were present together. However, a drastic 50-60% decrease in vigour indices of tomato seeds was recorded, irrespective of Azotobacter inoculation.

  2. Mutational and oxidative stress analysis in patients with mucopolysaccharidosis type I undergoing enzyme replacement therapy.

    PubMed

    Pereira, Vanessa Gonçalves; Martins, Ana Maria; Micheletti, Cecília; D'Almeida, Vânia

    2008-01-01

    Mucopolysaccharidosis type I (MPS I) patients present a wide range of clinical manifestations, which could be due to the high molecular heterogeneity of the IDUA gene and to pathological events besides the enzyme deficiency. The aim of this study was to identify the most common MPS I causing mutations and to evaluate some oxidative stress markers in Brazilian patients. 3 common mutations in the IDUA gene were searched in 11 MPS I patients by PCR-RFLP. Activities of antioxidant enzymes catalase and superoxide dismutase, and levels of total glutathione and thiobarbituric acid reactive substances were evaluated by spectrophotometric and colorimetric methods, during different periods of enzyme replacement therapy. The most common mutations were P533R and W402X, with allelic frequencies of 33.33% and 27.8% respectively. MPS I patients presented high levels of lipid peroxidation and enzyme replacement therapy led to an increase of catalase and a decrease of superoxide dismutase activities. P533R and W402X accounted for more than 60% of the alleles, but no genotype-phenotype correlation could be established. The alterations in antioxidant enzyme activities suggest that oxidative stress may be an important event among MPS I patients, which could contribute to the physiopathology of the disease.

  3. Non-P450 aldehyde oxidizing enzymes: the aldehyde dehydrogenase superfamily

    PubMed Central

    Marchitti, Satori A; Brocker, Chad; Stagos, Dimitrios; Vasiliou, Vasilis

    2009-01-01

    Background Aldehydes are highly reactive molecules. While several non-P450 enzyme systems participate in their metabolism, one of the most important is the aldehyde dehydrogenase (ALDH) superfamily, composed of NAD(P)+-dependent enzymes that catalyze aldehyde oxidation. Objective This article presents a review of what is currently known about each member of the human ALDH superfamily including the pathophysiological significance of these enzymes. Methods Relevant literature involving all members of the human ALDH family was extensively reviewed, with the primary focus on recent and novel findings. Conclusion To date, 19 ALDH genes have been identified in the human genome and mutations in these genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases, including Sjögren-Larsson syndrome, type II hyperprolinemia, γ-hydroxybutyric aciduria and pyridoxine-dependent seizures. ALDH enzymes also play important roles in embryogenesis and development, neurotransmission, oxidative stress and cancer. Finally, ALDH enzymes display multiple catalytic and non-catalytic functions including ester hydrolysis, antioxidant properties, xenobiotic bioactivation and UV light absorption. PMID:18611112

  4. Effect of acute vs chronic H2O2-induced oxidative stress on antioxidant enzyme activities.

    PubMed

    Miguel, Fernanda; Augusto, Amanda C; Gurgueira, Sonia A

    2009-04-01

    H2O2 can freely crosses membranes and in the presence of Fe2+ (or Cu+) it is prone to participate in Fenton reaction. This study evaluated the concentration and time-dependent effects of H2O2-induced oxidative stress on MnSOD, Se:GPx and catalase and on aconitase. Acute and chronic H2O2 treatments were able to induce oxidative stress in HeLa cells as they significantly decreased aconitase activity and also caused a very significant decrease on antioxidant enzyme activities. The inhibition of enzyme activities was time- and concentration-dependent. Chronic treatment with 5 microM H2O2/h after 24 h was able to decrease all enzyme activities almost at the same level as the acute treatment. Acute and chronic treatments on antioxidant enzyme activities were prevented by cell treatment with ascorbic acid or N-acetylcysteine. These results indicate that antioxidant enzymes can also be affected by the same ROS they produce or neutralize if the time of exposure is long enough.

  5. Oxidative stress in patients with mucopolysaccharidosis type II before and during enzyme replacement therapy.

    PubMed

    Filippon, Letícia; Vanzin, Camila S; Biancini, Giovana B; Pereira, Izabela N; Manfredini, Vanusa; Sitta, Angela; Peralba, Maria do Carmo R; Schwartz, Ida V D; Giugliani, Roberto; Vargas, Carmen R

    2011-06-01

    Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficiency of the enzyme iduronate-2-sulfatase, responsible for the degradation of glycosaminoglycans dermatan and heparan sulfate. Once the generation of free radicals is involved in the pathogenesis of many diseases, including some inborn errors of metabolism, the aim of this study was to evaluate blood oxidative stress parameters in MPS II patients, before and during 6 months of enzyme replacement therapy. We found significantly increased levels of malondialdehyde and carbonyl groups in plasma as well as erythrocyte catalase activity in patients before treatment compared to the control group. Plasma sulfhydryl group content and total antioxidant status were significantly reduced before treatment, while superoxide dismutase enzyme was not altered at this time when compared to controls. During enzyme replacement therapy, there was a significant reduction in levels of malondialdehyde when compared to pretreatment. Sulfhydryl groups were significantly increased until three months of treatment in MPS II patients in comparison to pretreatment. There were no significant alterations in plasma total antioxidant status and carbonyl groups as well as in catalase and superoxide dismutase activities during treatment in relation to pretreatment. The results indicate that MPS II patients are subject to lipid and protein oxidative damage and present reduction in non-enzymatic antioxidants, suggesting a possible involvement of free radicals in the pathophysiology of this disease. Also, the results may suggest that enzyme replacement therapy seems to protect against lipid peroxidation and protein damage in these patients.

  6. Elevated DNA Oxidation and DNA Repair Enzyme Expression in Brain White Matter in Major Depressive Disorder.

    PubMed

    Szebeni, Attila; Szebeni, Katalin; DiPeri, Timothy P; Johnson, Luke A; Stockmeier, Craig A; Crawford, Jessica D; Chandley, Michelle J; Hernandez, Liza J; Burgess, Katherine C; Brown, Russell W; Ordway, Gregory A

    2017-05-01

    Pathology of white matter in brains of patients with major depressive disorder (MDD) is well-documented, but the cellular and molecular basis of this pathology are poorly understood. Levels of DNA oxidation and gene expression of DNA damage repair enzymes were measured in Brodmann area 10 (BA10) and/or amygdala (uncinate fasciculus) white matter tissue from brains of MDD (n=10) and psychiatrically normal control donors (n=13). DNA oxidation was also measured in BA10 white matter of schizophrenia donors (n=10) and in prefrontal cortical white matter from control rats (n=8) and rats with repeated stress-induced anhedonia (n=8). DNA oxidation in BA10 white matter was robustly elevated in MDD as compared to control donors, with a smaller elevation occurring in schizophrenia donors. DNA oxidation levels in psychiatrically affected donors that died by suicide did not significantly differ from DNA oxidation levels in psychiatrically affected donors dying by other causes (non-suicide). Gene expression levels of two base excision repair enzymes, PARP1 and OGG1, were robustly elevated in oligodendrocytes laser captured from BA10 and amygdala white matter of MDD donors, with smaller but significant elevations of these gene expressions in astrocytes. In rats, repeated stress-induced anhedonia, as measured by a reduction in sucrose preference, was associated with increased DNA oxidation in white, but not gray, matter. Cellular residents of brain white matter demonstrate markers of oxidative damage in MDD. Medications that interfere with oxidative damage or pathways activated by oxidative damage have potential to improve treatment for MDD.

  7. Reaction of trichloroethylene and trichloroethylene oxide with cytochrome P450 enzymes: inactivation and sites of modification.

    PubMed

    Cai, H; Guengerich, F P

    2001-04-01

    Trichloroethylene (TCE) has been shown to be toxic to experimental animals and humans. TCE oxide is a reactive electrophile formed during TCE oxidation and rearranges to acylating intermediates [Cai, H., and Guengerich, F. P. (1999) J. Am. Chem. Soc. 121, 11656-11663], which may be related to the toxicity. Mice treated with TCE have been reported to contain N(6)-dichloroacetylLys residues in P450 2E1, as detected by immunochemical methods. TCE can be oxidized by both P450 2E1 and (rat) 2B1. In this work, direct reaction of TCE oxide with either human P450 2E1, P450 2B1, or NADPH-P450 reductase was shown to lead to enzyme inactivation, and no recovery of the activity of either enzyme occurred, consistent with the view of inactivation reactions with Lys groups and not hydroxyls or Cys. Furthermore, Lys adducts were detected in the reaction of TCE oxide with both P450 2E1 and NADPH-P450 reductase, with a larger amount of N(6)-formylLys observed compared to N(6)-dichloroacetylLys in both cases. Inactivation of P450 2E1 during NADPH-dependent TCE oxidation was not observed, compared to control experiments. However, inactivation of P450 2B1 during NADPH-dependent TCE oxidation was detected. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of tryptic peptides indicated that the direct reaction of TCE oxide with human P450 2E1 resulted in the modification of peptides containing Lys87 (AVKEALLDYK), Lys251 (VKEHHQSLDPNCPR), and Lys487 (YKLCVIPR), with either a formyl or dichloroacetyl group attached. Lys87 and Lys487 of human P450 2E1 appear to be modified during the oxidation of TCE, using the same approach. The results are considered in the context of comparison of species and P450s.

  8. Prometryne-induced oxidative stress and impact on antioxidant enzymes in wheat.

    PubMed

    Jiang, Lei; Yang, Hong

    2009-09-01

    Prometryne is one of the herbicides widely used for controlling weed/grass in agricultural practice. However, whether it has an adverse effect on crops is unknown. In this study, we investigated prometryne-induced oxidative stress in wheat (Triticum aestivum). Wheat plants were grown in soils with prometryne at 0-24 mgkg(-1) soil. The growth of wheat treated with prometryne was inhibited. Chlorophyll content significantly decreased even at the low level of prometryne (4 mgkg(-1) soil). Accumulation of thiobarbituric acid reactive substances (TBARS), an indicator of cellular peroxidation, increased, suggesting oxidative damage to the plants. The prometryne-induced oxidative stress triggered significant changes in activities of a variety of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and glutathione S-transferase (GST). Activities of the enzymes showed a general increase at low prometryne concentrations but a decrease at high levels. Analysis of non-denaturing polyacrylamide gel electrophoresis (PAGE) confirmed the results. To get an insight into the molecular response, a qRT-PCR-based assay was performed to analyze the transcript abundance of Cu/Zn-SOD and GST with prometryne exposure. Our analysis revealed that both genes displayed up-regulated expression patterns similar to the activities of the two enzymes. These data imply that prometryne-induced oxidative stress was responsible for the disturbance of the growth and antioxidant defensive systems in wheat plants.

  9. Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects cardiomyocytes against oxidant stress.

    PubMed

    Reuland, Danielle J; Khademi, Shadi; Castle, Christopher J; Irwin, David C; McCord, Joe M; Miller, Benjamin F; Hamilton, Karyn L

    2013-03-01

    Increased production of reactive oxygen species has been implicated in the pathogenesis of cardiovascular disease (CVD), and enhanced endogenous antioxidants have been proposed as a mechanism for regulating redox balance. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcriptional regulator of phase II antioxidant enzymes, and activation of Nrf2 has been suggested to be an important step in attenuating oxidative stress associated with CVD. A well-defined combination of five widely studied medicinal plants derived from botanical sources (Bacopa monniera, Silybum marianum (milk thistle), Withania somnifera (Ashwagandha), Camellia sinensis (green tea), and Curcuma longa (turmeric)) has been shown to activate Nrf2 and induce phase II enzymes through the antioxidant response element. The purpose of these experiments was to determine if treatment of cardiomyocytes with this phytochemical composition, marketed as Protandim, activates Nrf2, induces phase II detoxification enzymes, and protects cardiomyocytes from oxidant-induced apoptosis in a Nrf2-dependent manner. In cultured HL-1 cardiomyocytes, phytochemical treatment was associated with nuclear accumulation of Nrf2, significant induction of phase II enzymes, and concomitant protection against hydrogen peroxide-induced apoptosis. The protection against oxidant stress was abolished when Nrf2 was silenced by shRNA, suggesting that our phytochemical treatment worked through the Nrf2 pathway. Interestingly, phytochemical treatment was found to be a more robust activator of Nrf2 than oxidant treatment, supporting the use of the phytochemicals as a potential treatment to increase antioxidant defenses and protect heart cells against an oxidative challenge. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Crystal structures of protein phosphatase-1 bound to motuporin and dihydromicrocystin-LA: elucidation of the mechanism of enzyme inhibition by cyanobacterial toxins.

    PubMed

    Maynes, Jason T; Luu, Hue A; Cherney, Maia M; Andersen, Raymond J; Williams, David; Holmes, Charles F B; James, Michael N G

    2006-02-10

    The microcystins and nodularins are tumour promoting hepatotoxins that are responsible for global adverse human health effects and wildlife fatalities in countries where drinking water supplies contain cyanobacteria. The toxins function by inhibiting broad specificity Ser/Thr protein phosphatases in the host cells, thereby disrupting signal transduction pathways. A previous crystal structure of a microcystin bound to the catalytic subunit of protein phosphatase-1 (PP-1c) showed distinct changes in the active site region when compared with protein phosphatase-1 structures bound to other toxins. We have elucidated the crystal structures of the cyanotoxins, motuporin (nodularin-V) and dihydromicrocystin-LA bound to human protein phosphatase-1c (gamma isoform). The atomic structures of these complexes reveal the structural basis for inhibition of protein phosphatases by these toxins. Comparisons of the structures of the cyanobacterial toxin:phosphatase complexes explain the biochemical mechanism by which microcystins but not nodularins permanently modify their protein phosphatase targets by covalent addition to an active site cysteine residue.

  11. Enzyme induction and histopathology elucidate aryl hydrocarbon receptor-mediated versus non-aryl hydrocarbon receptor-mediated effects of Aroclor 1268 in American mink (Neovison vison).

    PubMed

    Folland, William R; Newsted, John L; Fitzgerald, Scott D; Fuchsman, Phyllis C; Bradley, Patrick W; Kern, John; Kannan, Kurunthachalam; Zwiernik, Matthew J

    2016-03-01

    Polychlorinated biphenyl (PCB) concentrations reported in preferred prey and blubber of bottlenose dolphins from the Turtle-Brunswick River estuary (Georgia, USA) suggest the potential for adverse effects. However, PCBs in Turtle-Brunswick River estuary dolphins are primarily derived from Aroclor 1268, and predicting toxic effects of Aroclor 1268 is uncertain because of the mixture's unique composition and associated physiochemical characteristics. These differences suggest that toxicity benchmarks for other PCB mixtures may not be relevant to dolphins exposed to Aroclor 1268. American mink (Neovison vison) were used as a surrogate model for cetaceans to characterize mechanisms of action associated with Aroclor 1268 exposure. Mink share similarities in phylogeny and life history with cetaceans and are characteristically sensitive to PCBs, making them an attractive surrogate species for marine mammals in ecotoxicity studies. Adult female mink and a subsequent F1 generation were exposed to Aroclor 1268 through diet, and effects on enzyme induction, histopathology, thyroid hormone regulation, hematology, organ weights, and body condition index were compared to a negative control and a 3,3',4,4',5-pentachlorobiphenyl (PCB 126)-positive control. Aroclor 1268 dietary exposure concentrations ranged from 1.8 µg/g wet weight to 29 µg/g wet weight. Anemia, hypothyroidism, and hepatomegaly were observed in mink exposed to Aroclor 1268 beyond various dietary thresholds. Cytochrome P450 induction and squamous epithelial proliferation jaw lesions were low in Aroclor 1268 treatments relative to the positive control. Differences in enzyme induction and the development of squamous epithelial proliferation jaw lesions between Aroclor 1268 treatments and the positive control, coupled with effects observed in Aroclor 1268 treatments not observed in the positive control, indicate that mechanisms additional to the aryl hydrocarbon receptor-mediated pathway are associated with

  12. Screening of Enzyme Biomarker for Nanotoxicity of Zinc Oxide in OREOCHROMIS MOSSAMBICUS

    NASA Astrophysics Data System (ADS)

    Subramanian, Periasamy; Bupesh, Giridharan

    2011-06-01

    Experiments were conducted to determine the effects of Zinc oxide (ZnO) nanoparticles (NPs) on fish models. Oreochromis mossambicus was orally administered with ZnO NPs (50-100 nm) once and its effects at five different concentrations (60 ppm-100 ppm) were observed for 12 days. Enzymatic assays were performed at every three days interval in the vital tissues of liver, gill, muscle and kidney. The defense enzymes, ethoxyresorufin O-deethylase (EROD) and glutathione S transferase (GST) exerted a dose dependent elevation up to 6 days. This hike then declines in higher concentrations and extended duration. Whereas the tissue damaging enzymes, glutamate oxaloacetic transaminase (GOT), glutamate pyruvic transaminase (GPT) and alkaline phosphatase (ALP) as well as the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) exhibited a dose and duration dependent increase until the end of the experiment. Among these enzymes, the antioxidant enzymes response to ZnO NP toxicity on fish showed notable continuous induction. This study demonstrates that antioxidant enzymes responses in O. mossambicus could be used as a biomarker for the early detection of nanotoxicity.

  13. Bromo-oxidation reaction in enzyme-entrapped alginate hollow microfibers.

    PubMed

    Asthana, Amit; Lee, Kwang Ho; Shin, Su-Jung; Perumal, Jayakumar; Butler, Lauren; Lee, Sang-Hoon; Kim, Dong-Pyo

    2011-06-01

    In this article, the authors present the fabrication of an enzyme-entrapped alginate hollow fiber using a microfluidic device. Further use of enzyme-entrapped alginate hollow fibers as a biocatalytic microchemical reactor for chemical synthesis is also deliberated in this article. To ensure that there is no enzyme leaching from the fiber, fiber surfaces were coated with chitosan. To confine the mobility of reactants and products within the porous hollow fibers the entire fibers were embedded into a transparent polydimethylsiloxane (PDMS) matrix which also works as a support matrix. A vanadium-containing bromoperoxidase enzyme isolated from Corallina confusa was used as a model enzyme to demonstrate the use of these alginate hollow-fiber reactors in bromo-oxidation of phenol red to bromophenol blue at different dye flow rates. Stability of the entrapped enzyme at different temperatures and the effect of the chitosan coating on the reaction conversion were also studied. It was observed that molecules as big as 27 kDa can be retained in the matrix after coating with chitosan while molecules with molecular-weight of around 378 Da can still diffuse in and out of the matrix. The kinetic conversion rate in this microfluidic bioreactor was more than 41-fold faster when compared with the standard test-tube procedure.

  14. Bromo-oxidation reaction in enzyme-entrapped alginate hollow microfibers

    PubMed Central

    Asthana, Amit; Lee, Kwang Ho; Shin, Su-Jung; Perumal, Jayakumar; Butler, Lauren; Lee, Sang-Hoon; Kim, Dong-Pyo

    2011-01-01

    In this article, the authors present the fabrication of an enzyme-entrapped alginate hollow fiber using a microfluidic device. Further use of enzyme-entrapped alginate hollow fibers as a biocatalytic microchemical reactor for chemical synthesis is also deliberated in this article. To ensure that there is no enzyme leaching from the fiber, fiber surfaces were coated with chitosan. To confine the mobility of reactants and products within the porous hollow fibers the entire fibers were embedded into a transparent polydimethylsiloxane (PDMS) matrix which also works as a support matrix. A vanadium-containing bromoperoxidase enzyme isolated from Corallina confusa was used as a model enzyme to demonstrate the use of these alginate hollow-fiber reactors in bromo-oxidation of phenol red to bromophenol blue at different dye flow rates. Stability of the entrapped enzyme at different temperatures and the effect of the chitosan coating on the reaction conversion were also studied. It was observed that molecules as big as 27 kDa can be retained in the matrix after coating with chitosan while molecules with molecular-weight of around 378 Da can still diffuse in and out of the matrix. The kinetic conversion rate in this microfluidic bioreactor was more than 41-fold faster when compared with the standard test-tube procedure. PMID:21799723

  15. Relationship of hemoxygenase-1 and prolidase enzyme activity with oxidative stress in papillary thyroid cancer

    PubMed Central

    Terzioglu, D; Teksoz, S; Arikan, AE; Uslu, E; Yılmaz, E; Eren, B

    2016-01-01

    Aim: Recent studies associate thyroid cancer with oxidative stress. We aim to clarify the relation between papillary thyroid cancer, oxidative stress, hemoxygenase-1, prolidase enzymes and investigate the availability of these enzymes as markers for diagnosis, success of treatment, and follow-up. Methods: Thirty-one patients with papillary thyroid carcinoma and 25 healthy control subjects were included in this study. Hemoxygenase-1, prolidase (oxidant stress indicator), malondialdehyde, protein carbonyl, and superoxide dismutase (an indicator of antioxidant defense system) were measured pre-operatively and 30 days after thyroidectomy. Results: There was a significant decrease in serum levels of malondialdehyde and superoxide dismutase (p <0.001 for both) after thyroidectomy in papillary thyroid carcinoma group. In addition, there was a significant difference in the postoperative serum levels of prolidase, malondialdehyde, protein carbonyl, and superoxide dismutase between papillary thyroid carcinoma and control groups (p =0.024, p <0.001, p =0.002, and p =0.016, respectively) beside significant difference of malondialdehyde, protein carbonyl, hemoxygenase-1, and superoxide dismutase pre-operative serum levels (p <0.001, p =0.003, p =0.006, and p =0.025, respectively). Conclusion: When the unquestionable role of oxidative stress in the pathogenesis of cancer is considered, in the future it is expected to associate parametric changes in the serum of patients caused by oxidative stress to papillary thyroid cancer. Hippokratia 2016, 20(1): 55-59 PMID:27895444

  16. Ocular non-P450 oxidative, reductive, hydrolytic, and conjugative drug metabolizing enzymes.

    PubMed

    Argikar, Upendra A; Dumouchel, Jennifer L; Dunne, Christine E; Bushee, Andrea J

    2017-08-01

    Metabolism in the eye for any species, laboratory animals or human, is gaining rapid interest as pharmaceutical scientists aim to treat a wide range of so-called incurable ocular diseases. Over a period of decades, reports of metabolic activity toward various drugs and biochemical markers have emerged in select ocular tissues of animals and humans. Ocular cytochrome P450 (P450) enzymes and transporters have been recently reviewed. However, there is a dearth of collated information on non-P450 drug metabolizing enzymes in eyes of various preclinical species and humans in health and disease. In an effort to complement ocular P450s and transporters, which have been well reviewed in the literature, this review is aimed at presenting collective information on non-P450 oxidative, hydrolytic, and conjugative ocular drug metabolizing enzymes. Herein, we also present a list of xenobiotics or drugs that have been reported to be metabolized in the eye.

  17. Nanoporous aluminum oxide as a novel support material for enzyme biosensors.

    PubMed

    Heilmann, A; Teuscher, N; Kiesow, A; Janasek, D; Spohn, U

    2003-10-01

    To construct novel amperometric sensors for the detection of hydrogen peroxide and pyruvate, peroxidase and pyruvate oxidase were immobilized in self-supporting nanoporous alumina membranes those made by anodic oxidation. Pyruvate oxidase and other enzymes were enclosed in poly(carbamoylsulfonate) hydrogel and sucked into the nanoporous alumina structure before polymerization. The alumina membranes were investigated by scanning electron microscopy before and after the enzyme immobilization. In an amperometric flow detector cell, pyruvate and hydrogen peroxide were detected under flow injection analysis conditions in concentration ranges from 1 microM to 100 microM and 5 microM to 500 microM, respectively. The achieved operational stability showed that alumina membranes can be used to construct enzyme-modified electrodes.

  18. The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants☆

    PubMed Central

    Tsoumpra, Maria K.; Muniz, Joao R.; Barnett, Bobby L.; Kwaasi, Aaron A.; Pilka, Ewa S.; Kavanagh, Kathryn L.; Evdokimov, Artem; Walter, Richard L.; Von Delft, Frank; Ebetino, Frank H.; Oppermann, Udo; Russell, R. Graham G.; Dunford, James E.

    2015-01-01

    Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding. PMID:26318908

  19. An oxidative N-demethylase reveals PAS transition from ubiquitous sensor to enzyme.

    PubMed

    Ortmayer, Mary; Lafite, Pierre; Menon, Binuraj R K; Tralau, Tewes; Fisher, Karl; Denkhaus, Lukas; Scrutton, Nigel S; Rigby, Stephen E J; Munro, Andrew W; Hay, Sam; Leys, David

    2016-11-24

    The universal Per-ARNT-Sim (PAS) domain functions as a signal transduction module involved in sensing diverse stimuli such as small molecules, light, redox state and gases. The highly evolvable PAS scaffold can bind a broad range of ligands, including haem, flavins and metal ions. However, although these ligands can support catalytic activity, to our knowledge no enzymatic PAS domain has been found. Here we report characterization of the first PAS enzyme: a haem-dependent oxidative N-demethylase. Unrelated to other amine oxidases, this enzyme contains haem, flavin mononucleotide, 2Fe-2S and tetrahydrofolic acid cofactors, and specifically catalyses the NADPH-dependent oxidation of dimethylamine. The structure of the α subunit reveals that it is a haem-binding PAS domain, similar in structure to PAS gas sensors. The dimethylamine substrate forms part of a highly polarized oxygen-binding site, and directly assists oxygen activation by acting as both an electron and proton donor. Our data reveal that the ubiquitous PAS domain can make the transition from sensor to enzyme, suggesting that the PAS scaffold can support the development of artificial enzymes.

  20. Facet Energy versus Enzyme-like Activities: The Unexpected Protection of Palladium Nanocrystals against Oxidative Damage.

    PubMed

    Ge, Cuicui; Fang, Ge; Shen, Xiaomei; Chong, Yu; Wamer, Wayne G; Gao, Xingfa; Chai, Zhifang; Chen, Chunying; Yin, Jun-Jie

    2016-11-22

    To develop nanomaterials as artificial enzymes, it is necessary to better understand how their physicochemical properties affect their enzyme-like activities. Although prior research has demonstrated that nanomaterials exhibit tunable enzyme-like activities depending on their size, structure, and composition, few studies have examined the effect of surface facets, which determine surface energy or surface reactivity. Here, we use electron spin-resonance spectroscopy to report that lower surface energy {111}-faceted Pd octahedrons have greater intrinsic antioxidant enzyme-like activity than higher surface energy {100}-faceted Pd nanocubes. Our in vitro experiments found that those same Pd octahedrons are more effective than Pd nanocubes at scavenging reactive oxygen species (ROS). Those reductions in ROS preserve the homogeneity of mitochondrial membrane potential and attenuate damage to important biomolecules, thereby allowing a substantially higher number of cells to survive oxidative challenges. Our computations of molecular mechanisms for the antioxidant activities of {111}- and {100}-faceted Pd nanocrystals, as well as their activity order, agree well with experimental observations. These findings can guide the design of antioxidant-mimicking nanomaterials, which could have therapeutic or preventative potential against oxidative stress related diseases.

  1. Investigating the oxidation of alkenes by non-heme iron enzyme mimics.

    PubMed

    Barry, Sarah M; Mueller-Bunz, Helge; Rutledge, Peter J

    2012-09-28

    Iron is emerging as a key player in the search for efficient and environmentally benign methods for the functionalisation of C-H bonds. Non-heme iron enzymes catalyse a diverse array of oxidative chemistry in nature, and small-molecule complexes designed to mimic the non-heme iron active site have great potential as C-H activation catalysts. Herein we report the synthesis of a series of organic ligands that incorporate key features of the non-heme iron active site. Iron(II) complexes of these ligands have been generated in situ and their ability to promote hydrocarbon oxidation has been investigated. Several of these systems promote the biomimetic dihydroxylation of cyclohexene at low levels, when hydrogen peroxide is used as the oxidant; allylic oxidation products are also observed. An investigation of ligand stability reveals formation of several breakdown products under the conditions of the oxidative turnover reactions. These products arise via oxidative decarboxylation, dehydration and deamination reactions. Taken together these results indicate that competing mechanisms are at play with these systems: biomimetic hydroxylation involving high-valent iron species, and allylic oxidation via Fenton chemistry and Haber-Weiss radical pathways.

  2. Nigella sativa fixed and essential oil modulates glutathione redox enzymes in potassium bromate induced oxidative stress.

    PubMed

    Sultan, Muhammad Tauseef; Butt, Masood Sadiq; Karim, Roselina; Ahmed, Waqas; Kaka, Ubedullah; Ahmad, Shakeel; Dewanjee, Saikat; Jaafar, Hawa Z E; Zia-Ul-Haq, M

    2015-09-18

    Nigella sativa is an important component of several traditional herbal preparations in various countries. It finds its applications in improving overall health and boosting immunity. The current study evaluated the role of fixed and essential oil of Nigella sativa against potassium bromate induced oxidative stress with special reference to modulation of glutathione redox enzymes and myeloperoxidase. Animals; 30 rats (Sprague Dawley) were divided in three groups and oxidative stress was induced using mild dose of potassium bromate. The groups were on their respective diets (iso-caloric diets for a period of 56 days) i.e. control and two experimental diets containing N. sativa fixed (4%) and essential (0.3%) oils. The activities of enzymes involved in glutathione redox system and myeloperoxidase (MPO) were analyzed. The experimental diets modulated the activities of enzymes i.e. glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) positively. Indices of antioxidant status like tocopherols and glutathione were in linear relationship with that of GPx, GR and GST (P<0.01). MPO activities were in negative correlation with GST (P<0.01) but positive correlation with some other parameters. Our results indicated that both Nigella sativa fixed and essential oil are effective in improving the antioxidant indices against potassium bromate induced oxidative stress.

  3. Antioxidant enzymes in Spodoptera littoralis (Boisduval): are they enhanced to protect gut tissues during oxidative stress?

    PubMed

    Krishnan, Natraj; Kodrík, Dalibor

    2006-01-01

    The Egyptian armyworm Spodoptera littoralis is a polyphagous insect attacking a number of plant species including those belonging to the Solanaceae and Cruciferaceae families. Its digestive physiology must therefore adapt to the food plant to ensure maximum extraction of nutrients with minimum trade-off in terms of growth retardation by pro-oxidant allelochemicals. To investigate this, the caterpillars of S. littoralis were fed on a semi-artificial diet (Manduca Premix-Heliothis Premix) and for 24 h on potato plants (Solanum tuberosum), respectively, at the mature 6th instar, and the levels of oxidative radicals and antioxidant enzymes in their guts were compared. The gut pH, standard redox potential (Eh) and electron availability (pe) revealed that oxidizing conditions prevail which promote oxidation of pro-oxidant allelochemicals in foliage. Oxidative stress in the foregut and midgut tissue and the gut contents was assessed from the generation of superoxide radical, total peroxide content and protein carbonyl content. Antioxidant defense was measured by the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX) and glutathione S-transferase peroxidase (GSTpx). A significant (p < 0.001) increase in the superoxide radical production (in foregut tissue, foregut and midgut contents), concomitant with an increase in total peroxide (in foregut contents) and protein carbonyl levels (in foregut and midgut tissue) were noted in larvae fed on the plants in contrast to those fed the semi-artificial diet. Similarly, a significant up-regulation of antioxidant enzymes SOD (in midgut tissues), CAT (in foregut, midgut tissue and contents), APOX (in foregut contents, midgut tissue and contents) and GSTpx (in foregut tissues) was recorded on the plant diet in comparison to the semi-artificial diet. The pro-oxidant allelochemicals in the plant diet are thus eliminated by the insect at the expense of up-regulation of antioxidative enzymes in response

  4. Immunological detection of enzymes for sulfate reduction in anaerobic methane-oxidizing consortia.

    PubMed

    Milucka, Jana; Widdel, Friedrich; Shima, Seigo

    2013-05-01

    Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) at marine gas seeps is performed by archaeal-bacterial consortia that have so far not been cultivated in axenic binary or pure cultures. Knowledge about possible biochemical reactions in AOM consortia is based on metagenomic retrieval of genes related to those in archaeal methanogenesis and bacterial sulfate reduction, and identification of a few catabolic enzymes in protein extracts. Whereas the possible enzyme for methane activation (a variant of methyl-coenzyme M reductase, Mcr) was shown to be harboured by the archaea, enzymes for sulfate activation and reduction have not been localized so far. We adopted a novel approach of fluorescent immunolabelling on semi-thin (0.3-0.5 μm) cryosections to localize two enzymes of the SR pathway, adenylyl : sulfate transferase (Sat; ATP sulfurylase) and dissimilatory sulfite reductase (Dsr) in microbial consortia from Black Sea methane seeps. Both Sat and Dsr were exclusively found in an abundant microbial morphotype (c. 50% of all cells), which was tentatively identified as Desulfosarcina/Desulfococcus-related bacteria. These results show that ANME-2 archaea in the Black Sea AOM consortia did not express bacterial enzymes of the canonical sulfate reduction pathway and thus, in contrast to previous suggestions, most likely cannot perform canonical sulfate reduction. Moreover, our results show that fluorescent immunolabelling on semi-thin cryosections which to our knowledge has been so far only applied on cell tissues, is a powerful tool for intracellular protein detection in natural microbial associations.

  5. Electrocatalytic tuning of biosensing response through electrostatic or hydrophobic enzyme-graphene oxide interactions.

    PubMed

    Baptista-Pires, Luis; Pérez-López, Briza; Mayorga-Martinez, Carmen C; Morales-Narváez, Eden; Domingo, Neus; Esplandiu, Maria Jose; Alzina, Francesc; Sotomayor-Torres, Clivia M; Merkoçi, Arben

    2014-11-15

    The effect of graphene oxidative grades upon the conductivity and hydrophobicity and consequently the influence on an enzymatic biosensing response is presented. The electrochemical responses of reduced graphene oxide (rGO) have been compared with the responses obtained from the oxide form (oGO) and their performances have been accordingly discussed with various evidences obtained by optical techniques. We used tyrosinase enzyme as a proof of concept receptor with interest for phenolic compounds detection through its direct adsorption onto a screen-printed carbon electrode previously modified with oGO or rGO with a carbon-oxygen ratio of 1.07 or 1.53 respectively. Different levels of oGO directly affect the (bio)conjugation properties of the biosensor due to changes at enzyme/graphene oxide interface coming from the various electrostatic or hydrophobic interactions with biomolecules. The developed biosensor was capable of reaching a limit of detection of 0.01 nM catechol. This tuning capability of the biosensor response can be of interest for building several other biosensors, including immunosensors and DNA sensors for various applications.

  6. [Effect of acute phosgene inhalation on antioxidant enzymes, nitric oxide and nitric-oxide synthase in rats].

    PubMed

    Qin, Xu-jun; Hai, Chun-xu; Liang, Xin; Wang, Peng; Chen, Hong-li; Liu, Rui

    2004-06-01

    To study the effect of acute phosgene inhalation on the antioxidant enzymes, nitric oxide (NO) and nitric oxide synthase (NOS) in rats. Phosgene was produced by decomposing bis (trichdomethyl) carbonate in the presence of N,N-dimethyl formamide. SD rats were randomly divided into two groups: control and phosgene exposure groups. In a special experimental device with equipment modulating the gas flow, phosgene exposed rats inhaled phosgene quantitatively for five minutes. Two hours later, all the rats were sacrificed and the ratio of wet weight to dried weight of lung (WW/DW) was calculated. Peripheral blood, serum and liver were collected to examine the activities of antioxidant enzymes including glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), NOS, and NO level. The total content of proteins were also determined. The WW/DW ratio of lung in phosgene exposure group was much higher than that in control group (P < 0.01). The activities of GST in serum and liver of phosgene exposure group increased significantly (P < 0.05). The activities of SOD, CAT, GSHPx and NOS in serum or blood and liver of phosgene exposure group were also increased significantly (P < 0.05). But the content of NO was significantly decreased (P < 0.01). Acute phosgene inhalation may cause a dramatically changes of several antioxidant enzyme activities, and acute injury of liver to some extent in rats. The latter is related to reactive oxygen species. But the elevation of antioxidant enzyme activities suggests that antioxidative treatment for acute phosgene poisoning should not be considered first.

  7. Pyrazolate-based cobalt(II)-containing metal-organic frameworks in heterogeneous catalytic oxidation reactions: elucidating the role of entatic states for biomimetic oxidation processes.

    PubMed

    Tonigold, Markus; Lu, Ying; Mavrandonakis, Andreas; Puls, Angela; Staudt, Reiner; Möllmer, Jens; Sauer, Joachim; Volkmer, Dirk

    2011-07-25

    Crystal structures of two metal-organic frameworks (MFU-1 and MFU-2) are presented, both of which contain redox-active Co(II) centres coordinated by linear 1,4-bis[(3,5-dimethyl)pyrazol-4-yl] ligands. In contrast to many MOFs reported previously, these compounds show excellent stability against hydrolytic decomposition. Catalytic turnover is achieved in oxidation reactions by employing tert-butyl hydroperoxide and the solid catalysts are easily recovered from the reaction mixture. Whereas heterogeneous catalysis is unambiguously demonstrated for MFU-1, MFU-2 shows catalytic activity due to slow metal leaching, emphasising the need for a deeper understanding of structure-reactivity relationships in the future design of redox-active metal-organic frameworks. Mechanistic details for oxidation reactions employing tert-butyl hydroperoxide are studied by UV/Vis and IR spectroscopy and XRPD measurements. The catalytic process accompanying changes of redox states and structural changes were investigated by means of cobalt K-edge X-ray absorption spectroscopy. To probe the putative binding modes of molecular oxygen, the isosteric heats of adsorption of O(2) were determined and compared with models from DFT calculations. The stabilities of the frameworks in an oxygen atmosphere as a reactive gas were examined by temperature-programmed oxidation (TPO). Solution impregnation of MFU-1 with a co-catalyst (N-hydroxyphthalimide) led to NHPI@MFU-1, which oxidised a range of organic substrates under ambient conditions by employing molecular oxygen from air. The catalytic reaction involved a biomimetic reaction cascade based on free radicals. The concept of an entatic state of the cobalt centres is proposed and its relevance for sustained catalytic activity is briefly discussed. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Resveratrol regulates oxidative biomarkers and antioxidant enzymes in the brain of streptozotocin-induced diabetic rats.

    PubMed

    Sadi, Gökhan; Konat, Dilan

    2016-07-01

    Oxidative stress has been implicated in the progression of pathogenesis in diabetes mellitus and leads to a variety of deformations in the central nervous system. Recent studies have provided several insights on therapeutic uses of resveratrol in diabetic complications. The present study determines if resveratrol ameliorates oxidative stress and molecular changes in the brain frontal cortex of streptozotocin-induced diabetic rats. Rats were divided into four groups: control, diabetic, resveratrol-treated control, and resveratrol-treated diabetic. After diabetes induction, resveratrol (20 mg/kg) was given intraperitoneally once daily for 4 weeks. In addition to enzymatic activities, gene and protein expression of brain antioxidant enzymes were utilized by qRT-PCR and Western blot, respectively. The results indicated a significant elevation in total oxidant species (1.22-fold) and malonedialdehyde (1.38-fold) contents in diabetic rat brain cortex tissues. In addition, significant augmentation in the activities of catalase (1.38-fold) and superoxide dismutase (3-fold) was witnessed with the gene and protein expression levels reflecting a transcriptional regulation. Resveratrol treatment significantly normalized diabetic malonedialdehyde and oxidized glutathione levels and strengthens the action of all antioxidant enzymes. Recovery of the diabetes-associated changes reflects the reduction of oxidative conditions by resveratrol and reveals the decrease in the requirement for the activation of antioxidant defense systems in the brain tissues of diabetic rats. Potent antioxidant and neuroprotective properties of resveratrol against diabetes-induced oxidative damage were demonstrated and the results support the conduct of new studies searching for the molecular mechanism of diabetes-induced changes in brain tissues.

  9. Lycium chinensis Mill attenuates glutamate induced oxidative toxicity in PC12 cells by increasing antioxidant defense enzymes and down regulating ROS and Ca(2+) generation.

    PubMed

    Olatunji, Opeyemi J; Chen, Hongxia; Zhou, Yifeng

    2016-03-11

    Lycium chinensis Mill is a famous traditional Chinese medicine which displays several medicinal activities including antioxidant and neuroprotective activities. However, the mechanism of action towards the neuroprotective action has not been fully elucidated. This work was aimed at investigating the neuroprotective effects of L. chinensis Mill against glutamate-induced oxidative neurotoxicity in PC12 cells. Oxidative cell death was induced with 5mM glutamate in PC12 cells. Cell viability, LDH release, intracellular Ca(2+) concentration, reactive oxygen species (ROS) accumulation, GSH-Px, CAT and SOD antioxidant enzyme levels were measured. Our results indicated that pretreatment of PC12 cells with L. chinensis Mill extracts markedly attenuated the loss of cell viability, the release of lactate dehydrogenase (LDH), Ca(2+) overload, ROS generation, and cell apoptosis induced by glutamate toxicity. Furthermore, L. chinensis Mill extracts also significantly increased the levels of innate antioxidant enzymes GSH-Px, SOD and CAT in glutamate-induced PC12 cells. Conclusively, our results provided substantial evidence that L. chinensis Mill protected PC12 cells against glutamate-induced cell death by attenuating ROS generation, Ca(2+) influx, and increased the antioxidant defense capacity of PC12 cells against oxidative stress damages, suggesting the possible potential of extracts from the plant as sources of bioactive molecules in the treatment of neurodegenerative disorders. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. Confining a bi-enzyme inside the nanochannels of a porous aluminum oxide membrane for accelerating the enzymatic reactions.

    PubMed

    Shangguan, Li; Wei, Yuanqing; Liu, Xu; Yu, Jiachao; Liu, Songqin

    2017-02-28

    An artificial metabolon with high conversion efficiency was constructed by confining a bi-enzyme into porous aluminum oxide nanochannels, which accelerated enzymatic reactions by minimizing the diffusion loss of intermediate species.

  11. Crystal structures of nitric oxide reductases provide key insights into functional conversion of respiratory enzymes.

    PubMed

    Tosha, Takehiko; Shiro, Yoshitsugu

    2013-03-01

    Respiration is an essential biological process to get bioenergy, ATP, for all kingdoms of life. Cytochrome c oxidase (COX) plays central role in aerobic respiration, catalyzing the reduction of O(2) coupled with pumping proton across the biological membrane. Nitric oxide reductase (NOR) involved in anaerobic nitrate respiration is suggested to be evolutionary related to COX and share the same progenitor with COX, on the basis of the amino acid sequence homology. Contrary to COX, NOR catalyzes the reduction of nitric oxide and shows no proton pumping ability. Thus, the respiratory enzyme acquires (or loses) proton pumping ability in addition to the conversion of the catalytic property along with the environmental change on earth. Recently, we solved the structures of two types of NORs, which provides novel insights into the functional conversion of the respiratory enzymes. In this review, we focus on the structural similarities and differences between COXs and NORs and discuss possible mechanism for the functional conversion of these enzymes during molecular evolution. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  12. DNA damage in fish (Anguilla anguilla) exposed to a glyphosate-based herbicide -- elucidation of organ-specificity and the role of oxidative stress.

    PubMed

    Guilherme, S; Gaivão, I; Santos, M A; Pacheco, M

    2012-03-18

    Organophosphate herbicides are among the most dangerous agrochemicals for the aquatic environment. In this context, Roundup(®), a glyphosate-based herbicide, has been widely detected in natural water bodies, representing a potential threat to non-target organisms, namely fish. Thus, the main goal of the present study was to evaluate the genotoxic potential of Roundup(®) in the teleost fish Anguilla anguilla, addressing the possible causative involvement of oxidative stress. Fish were exposed to environmentally realistic concentrations of this herbicide (58 and 116 μgL(-1)) during one or three days. The standard procedure of the comet assay was applied to gill and liver cells in order to determine organ-specific genetic damage. Since liver is a central organ in xenobiotic metabolism, nucleoids of hepatic cells were also incubated with a lesion-specific repair enzyme (formamidopyrimidine DNA glycosylase - FPG), in order to recognise oxidised purines. Antioxidants were determined in both organs as indicators of pro-oxidant state. In general, both organs displayed an increase in DNA damage for the two Roundup(®) concentrations and exposure times, although liver showed to be less susceptible to the lower concentration. The enzyme-modified comet assay showed the occurrence of FPG-sensitive sites in liver only after a 3-day exposure to the higher Roundup(®) concentration. The antioxidant defences were in general unresponsive, despite a single increment of catalase activity in gills (116 μgL(-1), 3-day) and a decrease of superoxide dismutase activity in liver (58 μgL(-1), 3-day). Overall, the mechanisms involved in Roundup(®)-induced DNA strand-breaks showed to be similar in both organs. Nevertheless, it was demonstrated that the type of DNA damage varies with the concentration and exposure duration. Hence, after 1-day exposure, an increase on pro-oxidant state is not a necessary condition for the induction of DNA-damaging effects of Roundup(®). By increasing the

  13. Effect of spaceflight on oxidative and antioxidant enzyme activity in rat diaphragm and intercostal muscles

    NASA Technical Reports Server (NTRS)

    Lee, Mona D.; Tuttle, Ronald; Girten, Beverly

    1995-01-01

    There are limited data regarding changes in oxidative and antioxidant enzymes induced by simulated or actual weightlessness, and any additional information would provide insight into potential mechanisms involving other changes observed in muscles from animals previously flown in space. Thus, the NASA Biospecimen Sharing Program was an opportunity to collect valuable information. Oxidative and antioxidant enzyme levels, as well as lipid peroxidation, were measured in respiratory muscles from rates flown on board Space Shuttle mission STS-54. The results indicated that there was an increasing trend in citrate synthase activity in the flight diaphragm when compared to ground based controls, and there were no significant changes observed in the intercostal muscles for any of the parameters. However, the lipid peroxidation was significantly (p less than 0.05) decreased in the flight diaphragm. These results indicate that 6 day exposure to microgravity may have a different effect on oxidative and antioxidant activity in rat respiratory muscles when compared to data from previous 14 day hindlimb suspension studies.

  14. Effect of spaceflight on oxidative and antioxidant enzyme activity in rat diaphragm and intercostal muscles

    NASA Technical Reports Server (NTRS)

    Lee, Mona D.; Tuttle, Ronald; Girten, Beverly

    1995-01-01

    There are limited data regarding changes in oxidative and antioxidant enzymes induced by simulated or actual weightlessness, and any additional information would provide insight into potential mechanisms involving other changes observed in muscles from animals previously flown in space. Thus, the NASA Biospecimen Sharing Program was an opportunity to collect valuable information. Oxidative and antioxidant enzyme levels, as well as lipid peroxidation, were measured in respiratory muscles from rates flown on board Space Shuttle mission STS-54. The results indicated that there was an increasing trend in citrate synthase activity in the flight diaphragm when compared to ground based controls, and there were no significant changes observed in the intercostal muscles for any of the parameters. However, the lipid peroxidation was significantly (p less than 0.05) decreased in the flight diaphragm. These results indicate that 6 day exposure to microgravity may have a different effect on oxidative and antioxidant activity in rat respiratory muscles when compared to data from previous 14 day hindlimb suspension studies.

  15. Vascular peroxidase 1: a novel enzyme in promoting oxidative stress in cardiovascular system.

    PubMed

    Ma, Qi-Lin; Zhang, Guo-Gang; Peng, Jun

    2013-07-01

    Vascular peroxidase 1 (VPO1) is a recently identified novel family member of peroxidases in cardiovascular system. As an enzyme that is downstream of NADPH oxidases (NOX), VPO1 functions to utilize NOX - derived hydrogen peroxide (H2O2) to produce hypochlorous acid (HOCl), a strong oxidant which is believed to greatly promote oxidative stress. Under multiple conditions, NOX is activated concomitantly with an increase in superoxide anion (O2(.-)) and H2O2 production. The latter is converted to HOCl by VPO1. In this process (O2(.-) → H2O2 → HOCl), the oxidant reactivities of reactive oxygen species (ROS) are significantly increased and therefore the oxidative stress is dramatically amplified. Several lines of evidence suggest that the NOX/VPO1 pathway - mediated oxidative stress plays an important role in myocardial ischemia-reperfusion injury, endothelial cell apoptosis and/or smooth muscle cell proliferation. In addition, VPO1 can be secreted into the extracellular space to participate in extracellular matrix formation, suggesting that VPO1 may also play a role in cardiovascular remodeling (such as fibrosis). This function is independent of the peroxidase activity of VPO1. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Evaluation of gender-related differences in various oxidative stress enzymes in mice.

    PubMed

    Chen, Ying; Ji, Li-Li; Liu, Tian-Yu; Wang, Zheng-Tao

    2011-12-31

    Oxidative stress caused by redundant free radical, lipid oxygen and peroxide usually results in the pathogenesis of various diseases, which can be alleviated by cellular antioxidant enzymes. According to statistics, there are different incidence rates of some diseases depending on the gender. The present study aimed to investigate potential gender-related differences of antioxidant enzymes in mice. The activities of glutamate-cysteine ligase (GCL), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) in the kidney, brain, lung and heart of both male and female mice were determined. Our results showed that GPx and GCL activities were higher in female kidney and brain than those in male. On the other hand, the activities of SOD were higher in female brain and lung than those in male. Moreover, female kidney appeared to show higher activities of CAT than the male kidney. But the activities of GCL and GPx were higher in male heart than those in female. Taken together, our results demonstrate that there are gender-related differences in the activities of cellular antioxidant enzymes in various important organs in mice. Variations in such enzymes may be the explanation for some gender-related diseases.

  17. Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation

    PubMed Central

    Zhou, Yan-Ting; Shimabukuro, Michio; Koyama, Kazunori; Lee, Young; Wang, May-Yun; Trieu, Falguni; Newgard, Christopher B.; Unger, Roger H.

    1997-01-01

    We have studied mechanisms by which leptin overexpression, which reduces body weight via anorexic and thermogenic actions, induces triglyceride depletion in adipocytes and nonadipocytes. Here we show that leptin alters in pancreatic islets the mRNA of the genes encoding enzymes of free fatty acid metabolism and uncoupling protein-2 (UCP-2). In animals infused with a recombinant adenovirus containing the leptin cDNA, the levels of mRNAs encoding enzymes of mitochondrial and peroxisomal oxidation rose 2- to 3-fold, whereas mRNA encoding an enzyme of esterification declined in islets from hyperleptinemic rats. Islet UCP-2 mRNA rose 6-fold. All in vivo changes occurred in vitro in normal islets cultured with recombinant leptin, indicating direct extraneural effects. Leptin overexpression increased UCP-2 mRNA by more than 10-fold in epididymal, retroperitoneal, and subcutaneous fat tissue of normal, but not of leptin–receptor-defective obese rats. By directly regulating the expression of enzymes of free fatty acid metabolism and of UCP-2, leptin controls intracellular triglyceride content of certain nonadipocytes, as well as adipocytes. PMID:9177227

  18. Inhibition effect of graphene oxide on the catalytic activity of acetylcholinesterase enzyme.

    PubMed

    Wang, Yong; Gu, Yao; Ni, Yongnian; Kokot, Serge

    2015-11-01

    Variations in the enzyme activity of acetylcholinesterase (AChE) in the presence of the nano-material, graphene oxide (GO), were investigated with the use of molecular spectroscopy UV-visible and fluorescence methods. From these studies, important kinetic parameters of the enzyme were extracted; these were the maximum reaction rate, Vm , and the Michaelis constant, Km . A comparison of these parameters indicated that GO inhibited the catalytic activity of the AChE because of the presence of the AChE-GO complex. The formation of this complex was confirmed with the use of fluorescence data, which was resolved with the use of the MCR-ALS chemometrics method. Furthermore, it was found that the resonance light-scattering (RLS) intensity of AChE changed in the presence of GO. On this basis, it was demonstrated that the relationship between AChE and GO was linear and such models were used for quantitative analyses of GO.

  19. A novel glutamine biosensor based on zinc oxide nanorod and glutaminase enzyme from Hypocria jecorina.

    PubMed

    Albayrak, Dilruba; Karakuş, Emine

    2016-01-01

    A novel biosensor for determination of L-glutamine in pharmaceutical glutamine powder was developed via immobilizing our produced glutaminase enzyme from Hypocria jecorina onto our prepared zinc oxide (ZnO) nanorod and chitosan. ZnO nanorods were prepared as surface-dependent and surface-independent and both were used. The biosensor is specific for L-glutamine and the peculiar analytical properties (linearity range, reproducibility, and accuracy) of it were experimentally determined. The optimum operating conditions of the biosensor such as buffer concentration, buffer pH, and medium temperature effect on the response of biosensor were studied. Km and Vmax values for the our-producing glutaminase enzyme from Hypocria jecorina immobilized on the biosensor were also determined as 0.29 mM and 208.33 mV/min., respectively, from Lineweaver-Burk plot. The biosensor was then used for the determination of glutamine contained in pharmaceutical formulations.

  20. Protective effects of zinc on oxidative stress enzymes in liver of protein-deficient rats.

    PubMed

    Sidhu, Pardeep; Garg, M L; Dhawan, D K

    2005-01-01

    Persons afflicted with protein malnutrition are generally deficient in a variety of essential micronutrients like zinc, copper, iron, and selenium, which in turn affects number of metabolic processes in the body. To evaluate the protective effects of zinc on the enzymes involved in oxidative stress induced in liver of protein-deficient rats, the current study was designed. Zinc sulfate at a dose level of 227 mg/L zinc in drinking water was administered to female Sprague-Dawley normal control as well as protein-deficient rats for a total duration of 8 weeks. The effects of zinc treatment in conditions of protein deficiency were studied on rat liver antioxidant enzymes, which included catalase, glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), glutathione reduced (GSH), and glutathione-S-transferase (GST). Protein deficiency in normal rats resulted in a significant increase in hepatic activities of catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase and the levels of lipid peroxidation. A significant inhibition in the levels of reduced glutathione and the enzyme activity of superoxide dismutase has been observed after protein deficiency in normal rats. Interestingly, Zn treatment to protein-deficient animals lowered already raised activity catalase, glutathione peroxidase, and glutathione-S-transferase and levels of lipid peroxidation to significant levels when compared to protein-deficient animals. Also, Zn treatment to the protein-deficient animals resulted in a significant elevation in the levels of GSH and SOD activity as compared to their respective controls, thereby indicating its effectiveness in regulating their levels in adverse conditions. It has also been observed that concentrations of zinc, copper, iron, and selenium were found to be decreased significantly in protein-deficient animals. However, the levels of these elements came back to within normal limits when zinc was administrated

  1. Temporal variations in the gene expression levels of cyanobacterial anti-oxidant enzymes through geological history: implications for biological evolution during the Great Oxidation Event

    NASA Astrophysics Data System (ADS)

    Harada, M.; Furukawa, R.; Yokobori, S. I.; Tajika, E.; Yamagishi, A.

    2016-12-01

    A significant rise in atmospheric O2 levels during the GOE (Great Oxidation Event), ca. 2.45-2.0 Ga, must have caused a great stress to biosphere, enforcing life to adapt to oxic conditions. Cyanobacteria, oxygenic photosynthetic bacteria that had been responsible for the GOE, are at the same time one of the organisms that would have been greatly affected by the rise of O2 level in the surface environments. Knowledge on the evolution of cyanobacteria is not only important to elucidate the cause of the GOE, but also helps us to better understand the adaptive evolution of life in response to the GOE. Here we performed phylogenetic analysis of an anti-oxidant enzyme Fe-SOD (iron superoxide dismutase) of cyanobacteria, to assess the adaptive evolution of life under the GOE. The rise of O2 level must have increased the level of toxic reactive oxygen species in cyanobacterial cells, thus forced them to change activities or the gene expression levels of Fe-SOD. In the present study, we focus on the change in the gene expression levels of the enzyme, which can be estimated from the promoter sequences of the gene. Promoters are DNA sequences found upstream of protein encoding regions, where RNA polymerase binds and initiates transcription. "Strong" promoters that efficiently interact with RNA polymerase induce high rates of transcription, leading to high levels of gene expression. Thus, from the temporal changes in the promoter sequences, we can estimate the variations in the gene expression levels during the geological time. Promoter sequences of Fe-SOD at each ancestral node of cyanobacteria were predicted from phylogenetic analysis, and the ancestral promoter sequences were compared to the promoters of known highly expressed genes. The similarity was low at the time of the emergence of cyanobacteria; however, increased at the branching nodes diverged 2.4 billon years ago. This roughly coincided with the onset of the GOE, implying that the transition from low to high gene

  2. Dynamic electrochemical investigations of hydrogen oxidation and production by enzymes and implications for future technology.

    PubMed

    Armstrong, Fraser A; Belsey, Natalie A; Cracknell, James A; Goldet, Gabrielle; Parkin, Alison; Reisner, Erwin; Vincent, Kylie A; Wait, Annemarie F

    2009-01-01

    This tutorial review describes studies of hydrogen production and oxidation by biological catalysts--metalloenzymes known as hydrogenases--attached to electrodes. It explains how the electrocatalytic properties of hydrogenases are studied using specialised electrochemical techniques and how the data are interpreted to allow assessments of catalytic rates and performance under different conditions, including the presence of O2, CO and H2S. It concludes by drawing some comparisons between the enzyme active sites and platinum catalysts and describing some novel proof-of-concept applications that demonstrate the high activities and selectivities of these 'alternative' catalysts for promoting H2 as a fuel.

  3. Glutathione, glutathione-related enzymes, and oxidative stress in individuals with subacute occupational exposure to lead.

    PubMed

    Dobrakowski, Michał; Pawlas, Natalia; Hudziec, Edyta; Kozłowska, Agnieszka; Mikołajczyk, Agnieszka; Birkner, Ewa; Kasperczyk, Sławomir

    2016-07-01

    The aim of the study was to investigate the influence of subacute exposure to lead on the glutathione-related antioxidant defense and oxidative stress parameters in 36 males occupationally exposed to lead for 40±3.2days. Blood lead level in the examined population increased significantly by 359% due to lead exposure. Simultaneously, erythrocyte glutathione level decreased by 16%, whereas the activity of glutathione-6-phosphate dehydrogenase in erythrocytes and leukocytes decreased by 28% and 10%, respectively. Similarly, the activity of glutathione-S-transferase in erythrocytes decreased by 45%. However, the activity of glutathione reductase in erythrocytes and leukocytes increased by 26% and 6%, respectively, whereas the total oxidant status value in leukocytes increased by 37%. Subacute exposure to lead results in glutathione pool depletion and accumulation of lipid peroxidation products; however, it does not cause DNA damage. Besides, subacute exposure to lead modifies the activity of glutathione-related enzymes.

  4. Thin-film antimony-antimony-oxide enzyme electrode for penicillin determination.

    PubMed

    Flanagan, M T; Carroll, N J

    1986-07-01

    A potentiometric penicillinase electrode is reported in which the base pH transducer is a thin-film antimony-antimony-oxide electrode deposited by vacuum evaporation. Several enzyme immobilization procedures have been examined and a crosslinked protein film found to be the most appropriate to this type of sensor. The use of an adjacent antimony-antimony-oxide track as a pseudoreference electrode was successfully demonstrated. The overall response was shown to be independent of the stirring rate above 100 rpm, but the kinetics of the response were found to depend markedly on the stirring rate. The intrinsic linear response range was 3 x 10(-4)M to 7 x 10(-3)M penicillin G. Linearizing transforms that extend the useful range were examined.

  5. Removal of hydantoin products of 8-oxoguanine oxidation by the Escherichia coli DNA repair enzyme, FPG.

    PubMed

    Leipold, M D; Muller, J G; Burrows, C J; David, S S

    2000-12-05

    An intriguing feature of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is that it is highly reactive toward further oxidation. Indeed, OG has been shown to be a "hot spot" for oxidative damage and susceptible to oxidation by a variety of cellular oxidants. Recent work has identified two new DNA lesions, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), resulting from one-electron oxidation of OG. The presence of Gh and Sp lesions in DNA templates has been shown to result in misinsertion of G and A by DNA polymerases, and therefore, both are potentially mutagenic DNA lesions. The base excision repair (BER) glycosylases Fpg and MutY serve to prevent mutations associated with OG in Escherichia coli, and therefore, we have investigated the ability of these two enzymes to process DNA duplex substrates containing the further oxidized OG lesions, Gh and Sp. The Fpg protein, which removes OG and a variety of other oxidized purine base lesions, was found to remove Gh and Sp efficiently opposite all four of the natural DNA bases. The intrinsic rate of damaged base excision by Fpg was measured under single-turnover conditions and was found to be highly dependent upon the identity of the base opposite the OG, Gh, or Sp lesion; as expected, OG is removed more readily from an OG:C- than an OG:A-containing substrate. However, when adenine is paired with Gh or Sp, the rate of removal of these damaged lesions by Fpg was significantly increased relative to the rate of removal of OG from an OG:A mismatch. The adenine glycosylase MutY, which removes misincorporated A residues from OG:A mismatches, is unable to remove A paired with Gh or Sp. Thus, the activity of Fpg on Gh and Sp lesions may dramatically influence their mutagenic potential. This work suggests that, in addition to OG, oxidative products resulting from further oxidation of OG should be considered when evaluating oxidative DNA damage and its associated effects on DNA mutagenesis.

  6. Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria

    NASA Astrophysics Data System (ADS)

    Yamazaki, T.; Hozuki, T.; Arai, K.; Toyoda, S.; Koba, K.; Fujiwara, T.; Yoshida, N.

    2014-05-01

    Nitrous oxide (N2O) is a potent greenhouse gas and produced in denitrification and nitrification by various microorganisms. Site preference (SP) of 15N in N2O, which is defined as the difference in the natural abundance of isotopomers 14N15NO and 15N14NO relative to 14N14NO, has been reported to be a useful tool to quantitatively distinguish N2O production pathways. To determine representative SP values for each microbial process, we firstly measured SP of N2O produced in the enzyme reaction of hydroxylamine oxidoreductase (HAO) purified from two species of ammonia oxidizing bacteria (AOB), Nitrosomonas europaea and Nitrosococcus oceani, and that of nitric oxide reductase (NOR) from Paracoccus denitrificans. The SP value for NOR reaction (-5.9 ± 2.1‰) showed nearly the same value as that reported for N2O produced by P. denitrificans in pure culture. In contrast, SP value for HAO reaction (36.3 ± 2.3‰) was a little higher than the values reported for N2O produced by AOB in aerobic pure culture. Using the SP values obtained by HAO and NOR reactions, we calculated relative contribution of the nitrite (NO2-) reduction (which is followed by NO reduction) to N2O production by N. oceani incubated under different O2 availability. Our calculations revealed that previous in vivo studies might have underestimated the SP value for the NH2OH oxidation pathway possibly due to a small contribution of NO2- reduction pathway. Further evaluation of isotopomer signatures of N2O using common enzymes of other processes related to N2O would improve the isotopomer analysis of N2O in various environments.

  7. Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria

    NASA Astrophysics Data System (ADS)

    Yamazaki, T.; Hozuki, T.; Arai, K.; Toyoda, S.; Koba, K.; Fujiwara, T.; Yoshida, N.

    2013-10-01

    Nitrous oxide (N2O) is a potent greenhouse gas and produced in denitrification and nitrification in environmental nitrogen cycle by various microorganism. Site preference (SP) of 15N in N2O, which is defined as the difference in the natural abundance of isotopomers 14N15NO and 15N14NO relative to 14N14NO, has been reported to be a useful tool to quantitatively distinguish N2O production pathway. To determine representative SP value for each microbial process, we firstly measured SP of N2O produced in the enzyme reaction of hydroxylamine oxidoreductase (HAO) purified from two species of ammonia oxidizing bacteria (AOB), Nitrosomonas europaea and Nitrosococcus oceani, and that of nitric oxide reductase (NOR) from Paracoccus denitrificans, respectively. The SP value for NOR reaction (-5.9 ± 2.1‰) showed nearly the same value as that reported for N2O produced by P. denitrificans in pure culture. In contrast, SP value for HAO reaction (36.3 ± 2.3‰) was a little higher than the values reported for N2O produced by AOB in aerobic pure culture. Using the SP values obtained by HAO and NOR reactions, we calculated relative contribution of the nitrite (NO2-) reduction (which is followed by NO reduction) to N2O production by N. oceani incubated under different O2 availability. Our calculations revealed that previous in vivo studies might have underestimated the SP value for NH2OH oxidation pathway possibly due to a small contribution of NO2- reduction pathway. Further evaluation of isotopomer signatures of N2O using common enzymes of other processes related to N2O would improve the isotopomer analysis of N2O in various environments.

  8. Brown propolis attenuates cerebral ischemia-induced oxidative damage via affecting antioxidant enzyme system in mice.

    PubMed

    Bazmandegan, Gholamreza; Boroushaki, Mohammad Taher; Shamsizadeh, Ali; Ayoobi, Fatemeh; Hakimizadeh, Elham; Allahtavakoli, Mohammad

    2017-01-01

    Oxidative stress plays a critical role in ischemic brain injury. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) are the enzymes underlying the endogenous antioxidant mechanisms affected by stroke and are considered as oxidative stress biomarkers. Brown propolis (BP) is a bioactive natural product with a set of biological activities that in turn may differ depending on the area from which the substance is originated. The aim of this study was to investigate the effect of water-extracted brown propolis (WEBPs), from two regions of Iran, against cerebral ischemia-induced oxidative injury in a mouse model of stroke. Experimentally, the chemical characterization and total polyphenol content were determined using GC/MS and Folin-Ciocalteu assay respectively. Seventy-two adult male mice were randomly divided into the surgical sham group, control group (treated with vehicle), and four groups of WEBPs-treated animals. The WEBPs were administered at the doses of 100 and 200mg/kg IP, during four different time points. Oxidative stress biomarkers (SOD and GPx activity, SOD/GPx ratio), lipid peroxidation (LPO) index (malondialdehyde content) and infarct volume were measured 48h post stroke. Behavioral tests were evaluated 24 and 48h after stroke. WEBPs treatment resulted in significant restoration of antioxidant enzymes activity and a subsequent decrease in LPO as well as the infarct volume compared to the control group. Sensory-motor impairment and neurological deficits were improved significantly as well. These results indicate that Iranian BP confers neuroprotection on the stroke-induced neuronal damage via an antioxidant mechanism which seems to be mediated by the endogenous antioxidant system. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  9. Phase 2 enzyme induction by conjugated linoleic acid improves lupus-associated oxidative stress.

    PubMed

    Bergamo, Paolo; Maurano, Francesco; Rossi, Mauro

    2007-07-01

    Conjugated linoleic acid (CLA) exhibits anticancer and anti-inflammatory properties. Its ability to increase total GSH (GSH+GSSG) amount and gamma-glutamylcysteine ligase (gammaGCL) protein expression was recently associated with the inhibition of typical pathological signs in MRL/MpJ-Fas(lpr) mice (MRL/lpr). In the present study the ability of CLA to modulate oxidative stress and phase 2 enzyme activity in the same animal model was investigated. Disease severity was associated with age-dependent production of anti-double-stranded DNA antibodies (anti-dsDNA IgGs) and with enhanced extent of oxidative stress markers: reduced total GSH, increased protein 3-nitrotyrosines (3-NT), and protein-bound carbonyl (PC) amounts. To examine the effect of CLA on antioxidant status, CLA or olive oil (as control) was administered to pregnant MRL/lpr mice. Significantly higher total GSH and Trolox equivalent antioxidant capacity (TEAC) levels were measured in serum of CLA-treated dams (and their pups), as compared with controls. Finally, the antioxidant and chemopreventive properties of CLA were investigated in old MRL/lpr mice. Sera of CLA-treated mice contained higher concentrations of total GSH which were negatively correlated with the levels of oxidative stress markers. Moreover, increased GSH, gammaGCL, glutathione S-transferase (GSTs), and NAD(P)H:quinone oxidoreductase (NQO1) activities were measured in liver and spleen of CLA-treated animals. In conclusion our data indicate that the activation of detoxifying enzymes may be one of the mechanisms whereby dietary CLA down-regulates oxidative stress in MRL/lpr mice.

  10. The effect of β-N-methylamino-L-alanine (BMAA) on oxidative stress response enzymes of the macrophyte Ceratophyllum demersum.

    PubMed

    Esterhuizen-Londt, M; Pflugmacher, S; Downing, T G

    2011-04-01

    Cyanobacteria are known to produce bioactive secondary metabolites such as hepatotoxins, cytotoxins and neurotoxins. The newly recognized neurotoxin β-N-methylamino-L-alanine (BMAA) is a naturally occurring non-protein amino acid found in the majority of cyanobacterial genera tested. Evidence that exists for implication of BMAA in neurodegenerative disorders relies on bioaccumulation and biomagnification from symbiotic cyanobacteria. Uptake and accumulation of free BMAA by various non-symbiotic organisms, including aquatic macrophytes, has been documented but to date limited evidence of ecotoxicology exists. We therefore investigated the effect of BMAA on the oxidative stress responses of the macrophyte, Ceratophyllum demersum. Markers for oxidative stress in this study are the antioxidative enzymes superoxide dismutase, catalase, guaiacol peroxidase, glutathione peroxidase and glutathione reductase. We found that BMAA had an inhibitory effect on all the oxidative stress response enzymes tested in plants exposed to BMAA. However enzymes not related to oxidative stress response were not affected by BMAA in in vitro experiments. Binding studies in the presence of BMAA showed reduced enzyme specific activity over time compared to the control. This study shows that BMAA causes oxidative stress indirectly as it inhibits antioxidant enzymes required to combat reactive oxygen species that cause damage to cells. Further investigations are required to fully understand the inhibitory effect of BMAA on these enzymes. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Use of chemical auxiliaries to control p450 enzymes for predictable oxidations at unactivated C-h bonds of substrates.

    PubMed

    Auclair, Karine; Polic, Vanja

    2015-01-01

    Cytochrome P450 enzymes (P450s) have the ability to oxidize unactivated C-H bonds of substrates with remarkable regio- and stereoselectivity. Comparable selectivity for chemical oxidizing agents is typically difficult to achieve. Hence, there is an interest in exploiting P450s as potential biocatalysts. Despite their impressive attributes, the current use of P450s as biocatalysts is limited. While bacterial P450 enzymes typically show higher activity, they tend to be highly selective for one or a few substrates. On the other hand, mammalian P450s, especially the drug-metabolizing enzymes, display astonishing substrate promiscuity. However, product prediction continues to be challenging. This review discusses the use of small molecules for controlling P450 substrate specificity and product selectivity. The focus will be on two approaches in the area: (1) the use of decoy molecules, and (2) the application of substrate engineering to control oxidation by the enzyme.

  12. Effect of ageing and oxidative stress on antioxidant enzyme activity in different regions of the rat kidney.

    PubMed

    Thiab, Noor Riyadh; King, Nicola; Jones, Graham L

    2015-10-01

    Oxidative stress has been implicated in ageing and the pathogenesis of chronic kidney disease. We examined levels of antioxidant enzymes glutathione peroxidase, glutathione reductase, glutathione S-transferase, catalase and superoxide dismutase as modulated by age and oxidative stress in different regions of the kidney. Antioxidant enzymes were examined in different regions of the kidney in male Wistar rats. Kidneys from rats of different ages (5, 12, 36 and 60 weeks) were dissected into cortex, outer medulla and inner medulla. Tissues were incubated for 30 min with or without 0.2 mM H2O2 to induce oxidative stress. Antioxidant enzyme activities progressively decreased with age under both control and stress conditions (P < 0.05) after peaking at 12 weeks. Antioxidant enzyme activities were greater in the cortex (P < 0.05) by comparison with the outer and inner medulla, respectively.

  13. Use of Chemical Auxiliaries to Control P450 Enzymes for Predictable Oxidations at Unactivated C–H Bonds of Substrates

    PubMed Central

    Auclair, Karine; Polic, Vanja

    2016-01-01

    Cytochrome P450 enzymes (P450s) have the ability to oxidize unactivated C-H bonds of substrates with remarkable regio- and stereoselectivity. Comparable selectivity for chemical oxidizing agents is typically difficult to achieve. Hence, there is an interest in exploiting P450s as potential biocatalysts. Despite their impressive attributes, the current use of P450s as biocatalysts is limited. While bacterial P450 enzymes typically show higher activity, they tend to be highly selective for one or a few substrates. On the other hand, mammalian P450s, especially the drug-metabolizing enzymes, display astonishing substrate promiscuity. However, product prediction continues to be challenging. This review discusses the use of small molecules for controlling P450 substrate specificity and product selectivity. The focus will be on two approaches in the area: (1) the use of decoy molecules, and (2) the application of substrate engineering to control oxidation by the enzyme. PMID:26002737

  14. Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

    PubMed Central

    Yui, Daishi; Nishida, Yoichiro; Nishina, Tomoko; Mogushi, Kaoru; Tajiri, Mio; Ishibashi, Satoru; Ajioka, Itsuki; Ishikawa, Kinya; Mizusawa, Hidehiro; Murayama, Shigeo; Yokota, Takanori

    2015-01-01

    Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa-/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa-/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa-/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD. PMID:26637123

  15. Impaired energy metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes

    PubMed Central

    Baraibar, Martín A.; Hyzewicz, Janek; Rogowska-Wrzesinska, Adelina; Bulteau, Anne-Laure; Prip-Buus, Carina; Butler-Browne, Gillian; Friguet, Bertrand

    2016-01-01

    Accumulation of oxidized proteins is a hallmark of cellular and organismal aging. Adult muscle stem cell (or satellite cell) replication and differentiation is compromised with age contributing to sarcopenia. However, the molecular events related to satellite cell dysfunction during aging are not completely understood. In the present study we have addressed the potential impact of oxidatively modified proteins on the altered metabolism of senescent human satellite cells. By using a modified proteomics analysis we have found that proteins involved in protein quality control and glycolytic enzymes are the main targets of oxidation (carbonylation) and modification with advanced glycation/lipid peroxidation end products during the replicative senescence of satellite cells. Inactivation of the proteasome appeared to be a likely contributor to the accumulation of such damaged proteins. Metabolic and functional analyses revealed an impaired glucose metabolism in senescent cells. A metabolic shift leading to increased mobilization of non-carbohydrate substrates such as branched chain amino acids or long chain fatty acids was observed. Increased levels of acyl-carnitines indicated an increased turnover of storage and membrane lipids for energy production. Taken together, these results support a link between oxidative protein modifications and the altered cellular metabolism associated with the senescent phenotype of human myoblasts. PMID:27922824

  16. Impaired energy metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes.

    PubMed

    Baraibar, Martín A; Hyzewicz, Janek; Rogowska-Wrzesinska, Adelina; Bulteau, Anne-Laure; Prip-Buus, Carina; Butler-Browne, Gillian; Friguet, Bertrand

    2016-12-04

    Accumulation of oxidized proteins is a hallmark of cellular and organismal aging. Adult muscle stem cell (or satellite cell) replication and differentiation is compromised with age contributing to sarcopenia. However, the molecular events related to satellite cell dysfunction during aging are not completely understood. In the present study we have addressed the potential impact of oxidatively modified proteins on the altered metabolism of senescent human satellite cells. By using a modified proteomics analysis we have found that proteins involved in protein quality control and glycolytic enzymes are the main targets of oxidation (carbonylation) and modification with advanced glycation/lipid peroxidation end products during the replicative senescence of satellite cells. Inactivation of the proteasome appeared to be a likely contributor to the accumulation of such damaged proteins. Metabolic and functional analyses revealed an impaired glucose metabolism in senescent cells. A metabolic shift leading to increased mobilization of non-carbohydrate substrates such as branched chain amino acids or long chain fatty acids was observed. Increased levels of acyl-carnitines indicated an increased turnover of storage and membrane lipids for energy production. Taken together, these results support a link between oxidative protein modifications and the altered cellular metabolism associated with the senescent phenotype of human myoblasts.

  17. Active paraplegics are protected against exercise-induced oxidative damage through the induction of antioxidant enzymes.

    PubMed

    Inglés, M; Serra-Añó, P; Gambini, J; Abu-Sharif, F; Dromant, M; Garcia-Valles, R; Pareja-Galeano, H; Garcia-Lucerga, C; Gomez-Cabrera, M C

    2016-10-01

    Experimental study. Exercise improves functional capacity in spinal cord injury (SCI). However, exhaustive exercise, especially when sporadic, is linked to the production of reactive oxygen species that may have a detrimental effect on SCI. We aimed to study the effect of a single bout of exhaustive exercise on systemic oxidative stress parameters and on the expression of antioxidant enzymes in individuals with paraplegia. The study was conducted in the Physical Therapy department and the Physical Education and Sports department of the University of Valencia. Sixteen paraplegic subjects were submitted to a graded exercise test (GET) until volitional exhaustion. They were divided into active or non-active groups. Blood samples were drawn immediately, 1 and 2 h after the GET. We determined plasma malondialdehyde (MDA) and protein carbonylation as markers of oxidative damage. Antioxidant gene expression (catalase and glutathione peroxidase-GPx) was determined in peripheral blood mononuclear cells. We found a significant increase in plasma MDA and protein carbonyls immediately after the GET (P<0.05). This increment correlated significantly with the lactate levels. Active paraplegics showed lower levels of exercise-induced oxidative damage (P<0.05) and higher exercise-induced catalase (P<0.01) and GPx (P<0.05) gene expression after the GET. These results suggest that exercise training may be useful in SCI patients to develop systemic antioxidant defenses that may protect them against exercise-induced oxidative damage.

  18. Determination of oxidative stress and activities of antioxidant enzymes in guinea pigs treated with haloperidol

    PubMed Central

    GUMULEC, JAROMIR; RAUDENSKA, MARTINA; HLAVNA, MARIAN; STRACINA, TIBOR; SZTALMACHOVA, MARKETA; TANHAUSEROVA, VERONIKA; PACAL, LUKAS; RUTTKAY-NEDECKY, BRANISLAV; SOCHOR, JIRI; ZITKA, ONDREJ; BABULA, PETR; ADAM, VOJTECH; KIZEK, RENE; NOVAKOVA, MARIE; MASARIK, MICHAL

    2013-01-01

    Guinea pigs (Cavia porcellus) were treated with haloperidol (HP), and free radical (FR) and ferric reducing antioxidant power (FRAP) assays were used to determine oxidative stress levels. Furthermore, the superoxide dismutase (SOD), glutathione reductase (GR) and glutathione-S-transferase (GST) activity levels were detected and glucose levels and the reduced and oxidized glutathione (GSH/GSSG) ratio were measured in HP-treated and untreated guinea pigs. The present study demonstrated that the administration of HP causes significant oxidative stress in guinea pigs (P=0.022). In animals treated with HP, the activity of GST was significantly increased compared with a placebo (P= 0.007). The elevation of SOD and GR activity levels and increase in the levels of glutathione (GSH) in HP-treated animals were not statistically significant. In the HP-untreated animals, a significant positive correlation was observed between oxidative stress detected by the FR method and GST (r=0.88, P=0.008) and SOD (r=0.86, P= 0.01) activity levels, respectively. A significant negative correlation between the levels of plasma glucose and oxidative stress detected by the FRAP method was observed (r=−0.78, P=0.04). Notably, no significant correlations were observed in the treated animals. In the HP-treated group, two subgroups of animals were identified according to their responses to oxidative stress. The group with higher levels of plasma HP had higher enzyme activity and reactive oxygen species production compared with the group with lower plasma levels of HP. The greatest difference in activity (U/μl) between the two groups of animals was for GR. PMID:23403848

  19. Determination of oxidative stress and activities of antioxidant enzymes in guinea pigs treated with haloperidol.

    PubMed

    Gumulec, Jaromir; Raudenska, Martina; Hlavna, Marian; Stracina, Tibor; Sztalmachova, Marketa; Tanhauserova, Veronika; Pacal, Lukas; Ruttkay-Nedecky, Branislav; Sochor, Jiri; Zitka, Ondrej; Babula, Petr; Adam, Vojtech; Kizek, Rene; Novakova, Marie; Masarik, Michal

    2013-02-01

    Guinea pigs (Cavia porcellus) were treated with haloperidol (HP), and free radical (FR) and ferric reducing antioxidant power (FRAP) assays were used to determine oxidative stress levels. Furthermore, the superoxide dismutase (SOD), glutathione reductase (GR) and glutathione-S-transferase (GST) activity levels were detected and glucose levels and the reduced and oxidized glutathione (GSH/GSSG) ratio were measured in HP-treated and untreated guinea pigs. The present study demonstrated that the administration of HP causes significant oxidative stress in guinea pigs (P=0.022). In animals treated with HP, the activity of GST was significantly increased compared with a placebo (P= 0.007). The elevation of SOD and GR activity levels and increase in the levels of glutathione (GSH) in HP-treated animals were not statistically significant. In the HP-untreated animals, a significant positive correlation was observed between oxidative stress detected by the FR method and GST (r=0.88, P=0.008) and SOD (r=0.86, P= 0.01) activity levels, respectively. A significant negative correlation between the levels of plasma glucose and oxidative stress detected by the FRAP method was observed (r=-0.78, P=0.04). Notably, no significant correlations were observed in the treated animals. In the HP-treated group, two subgroups of animals were identified according to their responses to oxidative stress. The group with higher levels of plasma HP had higher enzyme activity and reactive oxygen species production compared with the group with lower plasma levels of HP. The greatest difference in activity (U/μl) between the two groups of animals was for GR.

  20. Energetics of proton release on the first oxidation step in the water-oxidizing enzyme

    PubMed Central

    Saito, Keisuke; William Rutherford, A.; Ishikita, Hiroshi

    2015-01-01

    In photosystem II (PSII), the Mn4CaO5 cluster catalyses the water splitting reaction. The crystal structure of PSII shows the presence of a hydrogen-bonded water molecule directly linked to O4. Here we show the detailed properties of the H-bonds associated with the Mn4CaO5 cluster using a quantum mechanical/molecular mechanical approach. When O4 is taken as a μ-hydroxo bridge acting as a hydrogen-bond donor to water539 (W539), the S0 redox state best describes the unusually short O4–OW539 distance (2.5 Å) seen in the crystal structure. We find that in S1, O4 easily releases the proton into a chain of eight strongly hydrogen-bonded water molecules. The corresponding hydrogen-bond network is absent for O5 in S1. The present study suggests that the O4-water chain could facilitate the initial deprotonation event in PSII. This unexpected insight is likely to be of real relevance to mechanistic models for water oxidation. PMID:26442814

  1. Electrochemical enzyme-less urea sensor based on nano-tin oxide synthesized by hydrothermal technique.

    PubMed

    Ansari, S G; Fouad, H; Shin, Hyung-Shik; Ansari, Z A

    2015-12-05

    Nano-Tin oxide was synthesized using hydrothermal method at 150 °C for 6 h and then thin films were deposited by electrophoretic method at an optimized voltage of 100 V for 5 min on electropolished aluminum substrate. Spherical particles of about 30-50 nm diameters are observed with partial agglomeration when observed under electron microscope, which are tetragonal rutile structure. XPS results showed peaks related to Sn 4d, Sn 3d, O 1s & C 1s with spin-orbit splitting of 8.4 eV for Sn 3d. Feasibility studies of enzyme less urea sensing characteristics of nano-tin oxide thin films are exhibited herein. The deposited films have been used for enzyme less urea sensing from 1 to 20 mM concentration in buffer solution. The sensors were characterized electrochemically to obtain cyclic voltammogram as a function of urea concentration and scan rate. The sensitivity is estimated as 18.9 μA/mM below 5 mM and 2.31 μA/mM above 5 mM with a limit of detection of 0.6 mM.

  2. Facile, high efficiency immobilization of lipase enzyme on magnetic iron oxide nanoparticles via a biomimetic coating

    PubMed Central

    2011-01-01

    Background Immobilization of lipase on appropriate solid supports is one way to improve their stability and activity, and can be reused for large scale applications. A sample, cost- effective and high loading capacity method is still challenging. Results A facile method of lipase immobilization was developed in this study, by the use of polydopamine coated magnetic nanoparticles (PD-MNPs). Under optimal conditions, 73.9% of the available lipase was immobilized on PD-MNPs, yielding a lipase loading capacity as high as 429 mg/g. Enzyme assays revealed that lipase immobilized on PD-MNPs displayed enhanced pH and thermal stability compared to free lipase. Furthermore, lipase immobilized on PD-MNPs was easily isolated from the reaction medium by magnetic separation and retained more than 70% of initial activity after 21 repeated cycles of enzyme reaction followed by magnetic separation. Conclusions Immobilization of enzyme onto magnetic iron oxide nanoparticles via poly-dopamine film is economical, facile and efficient. PMID:21649934

  3. Nitric Oxide Mitigates Salt Stress by Regulating Levels of Osmolytes and Antioxidant Enzymes in Chickpea

    PubMed Central

    Ahmad, Parvaiz; Abdel Latef, Arafat A.; Hashem, Abeer; Abd_Allah, Elsayed F.; Gucel, Salih; Tran, Lam-Son P.

    2016-01-01

    This work was designed to evaluate whether external application of nitric oxide (NO) in the form of its donor S-nitroso-N-acetylpenicillamine (SNAP) could mitigate the deleterious effects of NaCl stress on chickpea (Cicer arietinum L.) plants. SNAP (50 μM) was applied to chickpea plants grown under non-saline and saline conditions (50 and 100 mM NaCl). Salt stress inhibited growth and biomass yield, leaf relative water content (LRWC) and chlorophyll content of chickpea plants. High salinity increased electrolyte leakage, carotenoid content and the levels of osmolytes (proline, glycine betaine, soluble proteins and soluble sugars), hydrogen peroxide (H2O2) and malondialdehyde (MDA), as well as the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase in chickpea plants. Expression of the representative SOD, CAT and APX genes examined was also up-regulated in chickpea plants by salt stress. On the other hand, exogenous application of NO to salinized plants enhanced the growth parameters, LRWC, photosynthetic pigment production and levels of osmolytes, as well as the activities of examined antioxidant enzymes which is correlated with up-regulation of the examined SOD, CAT and APX genes, in comparison with plants treated with NaCl only. Furthermore, electrolyte leakage, H2O2 and MDA contents showed decline in salt-stressed plants supplemented with NO as compared with those in NaCl-treated plants alone. Thus, the exogenous application of NO protected chickpea plants against salt stress-induced oxidative damage by enhancing the biosyntheses of antioxidant enzymes, thereby improving plant growth under saline stress. Taken together, our results demonstrate that NO has capability to mitigate the adverse effects of high salinity on chickpea plants by improving LRWC, photosynthetic pigment biosyntheses, osmolyte accumulation and antioxidative defense system. PMID:27066020

  4. Green tea diet decreases PCB 126-induced oxidative stress in mice by upregulating antioxidant enzymes

    PubMed Central

    Newsome, Bradley J; Petriello, Michael C; Han, Sung Gu; Murphy, Margaret O; Eske, Katryn E; Sunkara, Manjula; Morris, Andrew J; Hennig, Bernhard

    2013-01-01

    Superfund chemicals such as polychlorinated biphenyls pose a serious human health risk due to their environmental persistence and link to multiple diseases. Selective bioactive food components such as flavonoids have been shown to ameliorate PCB toxicity, but primarily in an in vitro setting. Here, we show that mice fed a green tea-enriched diet and subsequently exposed to environmentally relevant doses of coplanar PCB exhibit decreased overall oxidative stress primarily due to the upregulation of a battery of antioxidant enzymes. C57BL/6 mice were fed a low fat diet supplemented with green tea extract (GTE) for 12 weeks and exposed to 5 μmol PCB 126/kg mouse weight (1.63 mg/kg-day) on weeks 10, 11 and 12 (total body burden: 4.9 mg/kg). F2-Isoprostane and its metabolites, established markers of in vivo oxidative stress, measured in plasma via HPLC-MS/MS exhibited five-fold decreased levels in mice supplemented with GTE and subsequently exposed to PCB compared to animals on a control diet exposed to PCB. Livers were collected and harvested for both mRNA and protein analyses, and it was determined that many genes transcriptionally controlled by AhR and Nrf2 proteins were upregulated in PCB-exposed mice fed the green tea supplemented diet. An increased induction of genes such as SOD1, GSR, NQO1 and GST, key antioxidant enzymes, in these mice (green tea plus PCB) may explain the observed decrease in overall oxidative stress. A diet supplemented with green tea allows for an efficient antioxidant response in the presence of PCB 126 which supports the emerging paradigm that healthful nutrition may be able to bolster and buffer a physiological system against the toxicities of environmental pollutants. PMID:24378064

  5. Impaired metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes.

    PubMed

    Baraibar, Martin; Hyzewicz, Janek; Rogowska-Wrzesinska, Adelina; Bulteau, Anne-Laure; Prip-Buus, Carina; Butler-Browne, Gillian; Friguet, Bertrand

    2014-10-01

    Accumulation of damaged macromolecules, including irreversibly oxidized proteins, is a hallmark of cellular and organismal ageing. Failure of protein homesotasis is a major contributor to the age-related accumulation of damaged proteins. In skeletal muscle, tissue maintenance and regeneration is assured by resident adult stem cells known as satellite cells. During senescence their replication and differentiation is compromised contributing to sarcopenia. In this study we have addressed the impact of oxidatively modified proteins in the impaired metabolism of senescent human satellite cells. By using a targeted proteomics analysis we have found that proteins involved in protein quality control and glycolytic enzymes are the main targets of oxidation (carbonylation) and modification with advanced glycation/lipid peroxidation end products during replicative senescence of satellite cells. Inactivation of the proteasome in aged cells appeared as a key contributor to the accumulation of such damaged proteins. Untargeted metabolomic profiling and functional analyses indicated glucose metabolism impairment in senescent cells, although mitochondrial respiration remained unaffected. A metabolic shift leading to increased mobilization of non-carbohydrate substrates as branched chain amino acids or long chain fatty acids was observed in senescent cells. In addition, phospho-and glycerolipids metabolism was altered. Increased levels of acyl-carnitines indicated augmented turnover of storage and membrane lipids for energy production. Such changes reflect alterations in membrane composition and dysregulation of sphingolipids signaling during senescence. This study establishes a new concept connecting oxidative protein modifications with the altered cellular metabolism associated with the senescent phenotype. In addition, these findings highlight the molecular mechanisms implicated in satellite cells dysfunction during ageing, paving the road for future therapeutic interventions

  6. Effects of Microgravity On Oxidative and Antioxidant Enzymes In Mouse Hindlimb Muscle

    NASA Technical Reports Server (NTRS)

    Girten, B.; Hoopes, R.; Steele, M.; Morony, S.; Bateman, T. A.; Sun, S. (Technical Monitor)

    2002-01-01

    Gastrocnemius muscle of mice were analyzed in order to examine the effects of 12 days of microgravity on the oxidative enzyme climate synthase (CS) and the antioxidant enzyme superoxide dismutase (SOD). The female C57BL/6J mice utilized for this study were part of the Commercial Biomedical Testing Module (CBTM) payload that flew aboard STS-108. Mice were housed in Animal Enclosure Modules (AEMs) provided by NASA Ames. The flight (FLT) group and the ground control (CON) group each had 12 mice per group. The AEMs that held the CON group operated on a 48-hour delay from the FLT group and were located inside the Orbital Environmental Simulator (OES) at Kennedy Space Center. The temperature, CO2 and relative humidity inside the OES was regulated based on downlinked information from the shuttle middeck. Student T tests were used to compare groups and a p < 0.05 was used to determine statistical significance. Results indicated that CS levels for the FLT group were significantly lower than the CON group while the SOD levels were significantly higher. The CS FLT mean was 19% lower and the SOD FLT mean was 17% higher than the respective CON group means. Although these findings are among the first muscle enzyme values reported for mice from a shuttle mission, these results are similar to some results previously reported for rats exposed to microgravity or hindlimb suspension. The changes seen during the CBTM payload are reflective of the deconditioning that takes place with disuse of the hindlimbs and indicate that muscle enzyme changes induced by disuse deconditioning are similar in both rodent species.

  7. Effects of Microgravity On Oxidative and Antioxidant Enzymes In Mouse Hindlimb Muscle

    NASA Technical Reports Server (NTRS)

    Girten, B.; Hoopes, R.; Steele, M.; Morony, S.; Bateman, T. A.; Sun, S. (Technical Monitor)

    2002-01-01

    Gastrocnemius muscle of mice were analyzed in order to examine the effects of 12 days of microgravity on the oxidative enzyme climate synthase (CS) and the antioxidant enzyme superoxide dismutase (SOD). The female C57BL/6J mice utilized for this study were part of the Commercial Biomedical Testing Module (CBTM) payload that flew aboard STS-108. Mice were housed in Animal Enclosure Modules (AEMs) provided by NASA Ames. The flight (FLT) group and the ground control (CON) group each had 12 mice per group. The AEMs that held the CON group operated on a 48-hour delay from the FLT group and were located inside the Orbital Environmental Simulator (OES) at Kennedy Space Center. The temperature, CO2 and relative humidity inside the OES was regulated based on downlinked information from the shuttle middeck. Student T tests were used to compare groups and a p < 0.05 was used to determine statistical significance. Results indicated that CS levels for the FLT group were significantly lower than the CON group while the SOD levels were significantly higher. The CS FLT mean was 19% lower and the SOD FLT mean was 17% higher than the respective CON group means. Although these findings are among the first muscle enzyme values reported for mice from a shuttle mission, these results are similar to some results previously reported for rats exposed to microgravity or hindlimb suspension. The changes seen during the CBTM payload are reflective of the deconditioning that takes place with disuse of the hindlimbs and indicate that muscle enzyme changes induced by disuse deconditioning are similar in both rodent species.

  8. Human erythrocyte delta-aminolevulinate dehydratase inhibition by monosaccharides is not mediated by oxidation of enzyme sulfhydryl groups.

    PubMed

    Gabriel, D; Pivetta, L; Folmer, V; Soares, J C M; Augusti, G R; Nogueira, C W; Zeni, G; Rocha, J B T

    2005-08-01

    The heme pathway enzyme delta-aminolevulinate dehydratase is a good marker for oxidative stress and metal intoxication. This sulfhydryl enzyme is inhibited in such oxidative pathologies as lead, mercury and aluminum intoxication, exposure to selenium organic species and diabetes. Oxidative stress is a complicating factor in diabetes, inducing non-enzymatic glucose-mediated reactions that change protein structures and impair enzyme functions. We have studied the effects of high glucose, fructose and ribose concentrations on delta-ALA-D activity in vitro. These reducing sugars inhibited delta-ALA-D with efficacies in the order fructose=ribose>glucose. The possible mechanism of glucose inhibition was investigated using lysine, DTT, and t-butylamine. Oxidation of the enzyme's critical sulfhydryl groups was not involved because DTT had no effect. We concluded that high concentrations of reducing sugars or their autoxidation products inhibit delta-ALA-D by a mechanism not related to thiol oxidation. Also, we are not able to demonstrate that the formation of a Schiff base with the critical lysine residue of the enzyme is involved in the inhibition of delta-ALA-D by hexoses.

  9. The structural analysis of the pro-oxidant copper-binding site of denatured apo-H43R SOD1 and the elucidation of the origin of the acquisition of the pro-oxidant activity.

    PubMed

    Fujimaki, Nobuhiro; Miura, Takashi; Nakabayashi, Takakazu

    2016-02-14

    The pathogenesis of amyotrophic lateral sclerosis (ALS) is associated with mutations of Cu,Zn-superoxide dismutase (SOD1), which is a representative antioxidant enzyme. A previous study showed that the denatured apo-form of an ALS-linked mutant of human SOD1, His43 → Arg (H43R), obtains pro-oxidant activity as the reverse behavior of the native antioxidant activity by rebinding Cu(2+), which is considered to be closely related to the development of ALS. The Cu(2+)-binding site in denatured apo-H43R can be regarded as the center of the pro-oxidant activity, causing cellular oxidative stress. In the present study, the structure of the Cu(2+)-binding site of denatured apo-H43R was investigated to clarify the mechanism of the acquisition of the pro-oxidant activity. His residues constructing the Cu(2+)-binding site in denatured apo-H43R were experimentally assigned by absorption and fluorescence-based assays of SOD1 mutants, in which each of the seven His residues in H43R SOD1 is replaced with Ala. It was found that His120 is not involved with the Cu(2+)-binding site after denaturation, although the other His residues constructing the metal-binding site remain constant after denaturation. The disappearance of His120 from the Cu(2+)-binding site is therefore considered to be one of the important factors in obtaining the pro-oxidant activity. The mechanism of the acquisition of the pro-oxidant activity is discussed based on the results obtained.

  10. Antioxidant enzyme expression, lipid peroxidation, and protein oxidation in human myometrium with parturition.

    PubMed

    Khan, Raheela N; Matharoo-Ball, B; Shaw, R W

    2010-01-01

    Oxygen levels fluctuate considerably during human labor leading to hypoxia and reoxygenation of the uteroplacental unit and in some cases may compromise the progression of labor. Our aim was to assess the possible contribution of oxidative stress to the onset of labor. Thiobarbituric acid was used as a marker of lipid peroxidation along with Western blotting using anti-dinitrophenylhydrazine (DNPH) to assess protein carbonylation in myometrial samples obtained before and after the onset of term and preterm labor. Levels of key antioxidative enzymes were also compared. Higher levels of lipid peroxidation were observed in myometrial samples obtained during term or preterm labor. Reduced levels of glutathione peroxidase (GSHPx) were also encountered in these 2 groups. Conversely, protein carbonyl content was higher in laboring term and preterm myometrial samples. Levels of catalase (CAT) and superoxide dismutase (SOD) were unaltered across all 4 groups. Lipids in the laboring myometrium are susceptible to oxidative injury possibly due to diminished protection as a result of lower GSHPx activity. The reason for enhanced protein carbonylation suggests differential mechanisms governing protein turnover in the pregnant compared with the parturient uterus. Localized, oxidant damage of human myometrium may be a causal factor in difficult deliveries.

  11. Intracellular oxidant activity, antioxidant enzyme defense system, and cell senescence in fibroblasts with trisomy 21.

    PubMed

    Rodríguez-Sureda, Víctor; Vilches, Ángel; Sánchez, Olga; Audí, Laura; Domínguez, Carmen

    2015-01-01

    Down's syndrome (DS) is characterized by a complex phenotype associated with chronic oxidative stress and mitochondrial dysfunction. Overexpression of genes on chromosome-21 is thought to underlie the pathogenesis of the major phenotypic features of DS, such as premature aging. Using cultured fibroblasts with trisomy 21 (T21F), this study aimed to ascertain whether an imbalance exists in activities, mRNA, and protein expression of the antioxidant enzymes SOD1, SOD2, glutathione-peroxidase, and catalase during the cell replication process in vitro. T21F had high SOD1 expression and activity which led to an interenzymatic imbalance in the antioxidant defense system, accentuated with replicative senescence. Intracellular ROS production and oxidized protein levels were significantly higher in T21F compared with control cells; furthermore, a significant decline in intracellular ATP content was detected in T21F. Cell senescence was found to appear prematurely in DS cells as shown by SA-β-Gal assay and p21 assessment, though not apoptosis, as neither p53 nor the proapoptotic proteins cytochrome c and caspase 9 were altered in T21F. These novel findings would point to a deleterious role of oxidatively modified molecules in early cell senescence of T21F, thereby linking replicative and stress-induced senescence in cultured cells to premature aging in DS.

  12. Intracellular Oxidant Activity, Antioxidant Enzyme Defense System, and Cell Senescence in Fibroblasts with Trisomy 21

    PubMed Central

    Rodríguez-Sureda, Víctor; Vilches, Ángel; Sánchez, Olga; Audí, Laura; Domínguez, Carmen

    2015-01-01

    Down's syndrome (DS) is characterized by a complex phenotype associated with chronic oxidative stress and mitochondrial dysfunction. Overexpression of genes on chromosome-21 is thought to underlie the pathogenesis of the major phenotypic features of DS, such as premature aging. Using cultured fibroblasts with trisomy 21 (T21F), this study aimed to ascertain whether an imbalance exists in activities, mRNA, and protein expression of the antioxidant enzymes SOD1, SOD2, glutathione-peroxidase, and catalase during the cell replication process in vitro. T21F had high SOD1 expression and activity which led to an interenzymatic imbalance in the antioxidant defense system, accentuated with replicative senescence. Intracellular ROS production and oxidized protein levels were significantly higher in T21F compared with control cells; furthermore, a significant decline in intracellular ATP content was detected in T21F. Cell senescence was found to appear prematurely in DS cells as shown by SA-β-Gal assay and p21 assessment, though not apoptosis, as neither p53 nor the proapoptotic proteins cytochrome c and caspase 9 were altered in T21F. These novel findings would point to a deleterious role of oxidatively modified molecules in early cell senescence of T21F, thereby linking replicative and stress-induced senescence in cultured cells to premature aging in DS. PMID:25852816

  13. Enzyme-catalysed synthesis and reactions of benzene oxide/oxepine derivatives of methyl benzoates.

    PubMed

    Boyd, Derek R; Sharma, Narain D; Harrison, John S; Malone, John F; McRoberts, W Colin; Hamilton, John T G; Harper, David B

    2008-04-07

    A series of twelve benzoate esters was metabolised, by species of the Phellinus genus of wood-rotting fungi, to yield the corresponding benzyl alcohol derivatives and eight salicylates. The isolation of a stable oxepine metabolite, from methyl benzoate, allied to evidence of the migration and retention of a carbomethoxy group (the NIH Shift), during enzyme-catalysed ortho-hydroxylation of alkyl benzoates to form salicylates, is consistent with a mechanism involving an initial arene epoxidation step. This mechanism was confirmed by the isolation of a remarkably stable, optically active, substituted benzene oxide metabolite of methyl 2-(trifluoromethyl)benzoate, which slowly converted into the racemic form. The arene oxide was found to undergo a cycloaddition reaction with 4-phenyl-1,2,4-triazoline-3,5-dione to yield a crystalline cycloadduct whose structure and racemic nature was established by X-ray crystallography. The metabolite was also found to undergo some novel benzene oxide reactions, including epoxidation to give an anti-diepoxide, base-catalysed hydrolysis to form a trans-dihydrodiol and acid-catalysed aromatisation to yield a salicylate derivative via the NIH Shift of a carbomethoxy group.

  14. Serum prolidase activity, oxidative stress, and antioxidant enzyme levels in patients with renal cell carcinoma.

    PubMed

    Pirinççi, Necip; Kaba, Mehmet; Geçit, İlhan; Güneş, Mustafa; Yüksel, Mehmet Bilgehan; Tanık, Serhat; Arslan, Ayşe; Demir, Halit

    2016-02-01

    Prolidase is a member of the matrix metalloproteinase family. It plays a vital role in collagen turnover, matrix remodeling, and cell growth. Reactive oxygen species (ROS) have been implicated in the pathogenesis of various diseases, including cancers. Oxidative stress can cause tumor angiogenesis and may be carcinogenic. However, the relationship between antioxidant capacity and various cancers has been researched in several clinical trials. In our study, we aimed to identify serum prolidase activity, oxidative stress, and antioxidant enzyme levels in patients with renal tumors and to evaluate their relationships with each other. A total of 37 male patients with renal cell cancer and with a mean age of 56.28 ± 3.1 were included in the study. The control group comprising 36 male patients (mean age 56.31 ± 2.9) was randomly selected among the volunteers. Serum samples for measurement of superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione-S-transferase (GST), malondialdehyde (MDA), glutathione (GSH), and prolidase levels were kept at -20°C until they were used. Serum prolidase activity and MDA levels were significantly higher in renal cancer patients than in controls (all, p < 0.05), while SOD, GSHPx, and GST levels were significantly lower (p < 0.05). Our results indicate that increased prolidase seems to be related to increased oxidative stress along with decreased antioxidant levels in renal cancer. © The Author(s) 2013.

  15. The Protein Oxidation Repair Enzyme Methionine Sulfoxide Reductase A Modulates Aβ Aggregation and Toxicity In Vivo

    PubMed Central

    Minniti, Alicia N.; Arrazola, Macarena S.; Bravo-Zehnder, Marcela; Ramos, Francisca; Inestrosa, Nibaldo C.

    2015-01-01

    Abstract Aims: To examine the role of the enzyme methionine sulfoxide reductase A-1 (MSRA-1) in amyloid-β peptide (Aβ)-peptide aggregation and toxicity in vivo, using a Caenorhabditis elegans model of the human amyloidogenic disease inclusion body myositis. Results: MSRA-1 specifically reduces oxidized methionines in proteins. Therefore, a deletion of the msra-1 gene was introduced into transgenic C. elegans worms that express the Aβ-peptide in muscle cells to prevent the reduction of oxidized methionines in proteins. In a constitutive transgenic Aβ strain that lacks MSRA-1, the number of amyloid aggregates decreases while the number of oligomeric Aβ species increases. These results correlate with enhanced synaptic dysfunction and mislocalization of the nicotinic acetylcholine receptor ACR-16 at the neuromuscular junction (NMJ). Innovation: This approach aims at modulating the oxidation of Aβ in vivo indirectly by dismantling the methionine sulfoxide repair system. The evidence presented here shows that the absence of MSRA-1 influences Aβ aggregation and aggravates locomotor behavior and NMJ dysfunction. The results suggest that therapies which boost the activity of the Msr system could have a beneficial effect in managing amyloidogenic pathologies. Conclusion: The absence of MSRA-1 modulates Aβ-peptide aggregation and increments its deleterious effects in vivo. Antioxid. Redox Signal. 22, 48–62. PMID:24988428

  16. Attenuation of angiotensin converting enzyme inhibitor induced cough by iron supplementation: role of nitric oxide.

    PubMed

    Bhalla, Payal; Singh, Narinder Pal; Ravi, Krishnan

    2011-12-01

    The present study examined whether (1) the cough associated with angiotensin converting enzyme inhibitor therapy is attenuated by oral intake of iron and anti-oxidants, and (2) nitric oxide (NO) has any role in this attenuation. Of the 100 patients under investigation, cough occurred in 28 of them with preponderance in females. All the 28 patients were followed up for six weeks: the first two weeks were the observation period and the remaining four weeks the experimentation period. After the observation period, 11 patients received a single oral dose of ferrous sulphate (200 mg), eight received vitamin E (200 mg, o.d.) and vitamin C (150 mg, o.d.) and nine were given placebo during the experimentation period. Cough scoring, serum NO and malondialdehyde (MDA) levels were determined during both the periods. While there were significant decreases in cough scores, NO and MDA levels between these two periods in the iron group, cough scores and MDA level decreased significantly in the anti-oxidant group. None of these parameters changed in the control group. NO level was found to be increased significantly in patients who developed cough (n = 28) compared with those who did not cough (n = 72). These results suggest that iron supplementation suppresses cough in patients on ACE-I therapy through its effect on NO generation.

  17. The key nickel enzyme of methanogenesis catalyses the anaerobic oxidation of methane.

    PubMed

    Scheller, Silvan; Goenrich, Meike; Boecher, Reinhard; Thauer, Rudolf K; Jaun, Bernhard

    2010-06-03

    Large amounts (estimates range from 70 Tg per year to 300 Tg per year) of the potent greenhouse gas methane are oxidized to carbon dioxide in marine sediments by communities of methanotrophic archaea and sulphate-reducing bacteria, and thus are prevented from escaping into the atmosphere. Indirect evidence indicates that the anaerobic oxidation of methane might proceed as the reverse of archaeal methanogenesis from carbon dioxide with the nickel-containing methyl-coenzyme M reductase (MCR) as the methane-activating enzyme. However, experiments showing that MCR can catalyse the endergonic back reaction have been lacking. Here we report that purified MCR from Methanothermobacter marburgensis converts methane into methyl-coenzyme M under equilibrium conditions with apparent V(max) (maximum rate) and K(m) (Michaelis constant) values consistent with the observed in vivo kinetics of the anaerobic oxidation of methane with sulphate. This result supports the hypothesis of 'reverse methanogenesis' and is paramount to understanding the still-unknown mechanism of the last step of methanogenesis. The ability of MCR to cleave the particularly strong C-H bond of methane without the involvement of highly reactive oxygen-derived intermediates is directly relevant to catalytic C-H activation, currently an area of great interest in chemistry.

  18. Minerals Masquerading As Enzymes: Abiotic Oxidation Of Soil Organic Matter In An Iron-Rich Humid Tropical Forest Soil

    NASA Astrophysics Data System (ADS)

    Hall, S. J.; Silver, W. L.

    2010-12-01

    Oxidative reactions play an important role in decomposing soil organic matter fractions that resist hydrolytic degradation, and fundamentally affect the cycling of recalcitrant soil carbon across ecosystems. Microbial extracellular oxidative enzymes (e.g. lignin peroxidases and laccases) have been assumed to provide a dominant role in catalyzing soil organic matter oxidation, while other potential oxidative mechanisms remain poorly explored. Here, we show that abiotic reactions mediated by the oxidation of ferrous iron (Fe(II)) could explain high potential oxidation rates in humid tropical forest soils, which often contain high concentrations of Fe(II) and experience rapid redox fluctuations between anaerobic and aerobic conditions. These abiotic reactions could provide an additional mechanism to explain high rates of decomposition in these ecosystems, despite frequent oxygen deficits. We sampled humid tropical forest soils in Puerto Rico, USA from various topographic positions, ranging from well-drained ridges to riparian valleys that experience broad fluctuations in redox potential. We measured oxidative activity by adding the model humic compound L-DOPA to soil slurries, followed by colorimetric measurements of the supernatant solution over time. Dilute hydrogen peroxide was added to a subset of slurries to measure peroxidative activity. We found that oxidative and peroxidative activity correlated positively with soil Fe(II) concentrations, counter to prevailing theory that low redox potential should suppress oxidative enzymes. Boiling or autoclaving sub-samples of soil slurries to denature any enzymes present typically increased peroxidative activity and did not eliminate oxidative activity, further suggesting the importance of an abiotic mechanism. We found substantial differences in the oxidation products of the L-DOPA substrate generated by our soil slurries in comparison with oxidation products generated by a purified enzyme (mushroom tyrosinase

  19. Correlation between Antioxidant Enzyme Activity, Free Iron Content and Lipid Oxidation in Four Lines of Korean Native Chicken Meat

    PubMed Central

    Kim, Hye-Kyung; Cho, Chang-Yeon; Lee, Cheol-Koo

    2016-01-01

    This study was conducted to observe the association between antioxidant enzyme activity, free iron content and lipid oxidation of Korean native chicken (KNC) meat during refrigerated storage. Four lines of KNC (Yeonsan ogye, Hyunin black, Hoengseong yakdak and Hwangbong) were raised under similar conditions. A total of 16 roosters were randomly sampled and slaughtered at the age of 12 mon. The breast and thigh meats were stored aerobically for 10 d at 4℃. Although thigh meat had higher antioxidant enzyme activity, it was more susceptible to lipid oxidation and released more iron during storage than breast meat. Aerobic refrigerated storage for 10 d significantly decreased the activity of antioxidant enzymes and increased the amount of free iron and malondialdehyde. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were negatively correlated with lipid oxidation, whereas that of catalase was not. The amount of free iron was positively associated with lipid oxidation. We concluded that chicken line did not affect strongly on antioxidant enzyme activity and lipid oxidation in breast meat of KNC. However, the thigh meat of Hwangbong and Hyunin black had higher SOD and GSH-Px activity, respectively, and lower malondialdehyde contents than that of other chickens. SOD, GSH-Px and free iron play significant roles in meat lipid oxidation during refrigerated storage. PMID:27499663

  20. Metabolism of (-)-cis- and (-)-trans-rose oxide by cytochrome P450 enzymes in human liver microsomes.

    PubMed

    Nakahashi, Hiroshi; Yamamura, Yuuki; Usami, Atsushi; Rangsunvigit, Pramoch; Malakul, Pomthong; Miyazawa, Mitsuo

    2015-12-01

    The in vitro metabolism of (-)-cis- and (-)-trans-rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9-oxidized metabolite were determined using gas chromatography-mass spectrometry (GC-MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (-)-cis- and (-)-trans-rose oxide. CYP1A2 also efficiently oxidized (-)-cis-rose oxide at the 9-position but not (-)-trans-rose oxide. α-Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti-CYP2B6 antibody inhibited (-)-cis-rose oxide 9-hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (-)-trans-rose oxide was suppressed by thioTEPA and anti-CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide formations correlated with (S)-mephenytoin N-demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (-)-cis- and (-)-trans-rose oxide in human liver microsomes.

  1. Novel insights into the fungal oxidation of monoaromatic and biarylic environmental pollutants by characterization of two new ring cleavage enzymes.

    PubMed

    Schlüter, Rabea; Lippmann, Ramona; Hammer, Elke; Gesell Salazar, Manuela; Schauer, Frieder

    2013-06-01

    The phenol-degrading yeast Trichosporon mucoides can oxidize and detoxify biarylic environmental pollutants such as dibenzofuran, diphenyl ether and biphenyl by ring cleavage. The degradation pathways are well investigated, but the enzymes involved are not. The high similarity of hydroxylated biphenyl derivatives and phenol raised the question if the enzymes of the phenol degradation are involved in ring cleavage or whether specific enzymes are necessary. Purification of enzymes from T. mucoides with catechol cleavage activity demonstrated the existence of three different enzymes: a classical catechol-1,2-dioxygenase (CDO), not able to cleave the aromatic ring system of 3,4-dihydroxybiphenyl, and two novel enzymes with a high affinity towards 3,4-dihydroxybiphenyl. The comparison of the biochemical characteristics and mass spectrometric sequence data of these three enzymes demonstrated that they have different substrate specificities. CDO catalyzes the ortho-cleavage of dihydroxylated monoaromatic compounds, while the two novel enzymes carry out a similar reaction on biphenyl derivatives. The ring fission of 3,4-dihydroxybiphenyl by the purified enzymes results in the formation of (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid. These results suggest that the ring cleavage enzymes catalyzing phenol degradation are not involved in the ring cleavage of biarylic compounds by this yeast, although some intermediates of the phenol metabolism may function as inducers.

  2. A dual enzyme functionalized nanostructured thulium oxide based interface for biomedical application.

    PubMed

    Singh, Jay; Roychoudhury, Appan; Srivastava, Manish; Solanki, Pratima R; Lee, Dong Won; Lee, Seung Hee; Malhotra, B D

    2014-01-21

    In this paper, we present results of the studies related to fabrication of a rare earth metal oxide based efficient biosensor using an interface based on hydrothermally prepared nanostructured thulium oxide (n-Tm2O3). A colloidal solution of prepared nanorods has been electrophoretically deposited (EPD) onto an indium-tin-oxide (ITO) glass substrate. The n-Tm2O3 nanorods are found to provide improved sensing characteristics to the electrode interface in terms of electroactive surface area, diffusion coefficient, charge transfer rate constant and electron transfer kinetics. The structural and morphological studies of n-Tm2O3 nanorods have been carried out by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopic techniques. This interfacial platform has been used for fabrication of a total cholesterol biosensor by immobilizing cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto a Tm2O3 nanostructured surface. The results of response studies of the fabricated ChEt-ChOx/n-Tm2O3/ITO bioelectrode show a broad linear range of 8-400 mg dL(-1), detection limit of 19.78 mg (dL cm(-2))(-1), and high sensitivity of 0.9245 μA (mg per dL cm(-2))(-1) with a response time of 40 s. Further, this bioelectrode has been utilized for estimation of total cholesterol with negligible interference (3%) from analytes present in human serum samples. The utilization of this n-Tm2O3 modified electrode for enzyme-based biosensor analysis offers an efficient strategy and a novel interface for application of the rare earth metal oxide materials in the field of electrochemical sensors and bioelectronic devices.

  3. Response of oxidative enzyme activities to nitrogen deposition affects soil concentrations of dissolved organic carbon

    USGS Publications Warehouse

    Waldrop, M.P.; Zak, D.R.

    2006-01-01

    Recent evidence suggests that atmospheric nitrate (NO3- ) deposition can alter soil carbon (C) storage by directly affecting the activity of lignin-degrading soil fungi. In a laboratory experiment, we studied the direct influence of increasing soil NO 3- concentration on microbial C cycling in three different ecosystems: black oak-white oak (BOWO), sugar maple-red oak (SMRO), and sugar maple-basswood (SMBW). These ecosystems span a broad range of litter biochemistry and recalcitrance; the BOWO ecosystem contains the highest litter lignin content, SMRO had intermediate lignin content, and SMBW leaf litter has the lowest lignin content. We hypothesized that increasing soil solution NO 3- would reduce lignolytic activity in the BOWO ecosystem, due to a high abundance of white-rot fungi and lignin-rich leaf litter. Due to the low lignin content of litter in the SMBW, we further reasoned that the NO3- repression of lignolytic activity would be less dramatic due to a lower relative abundance of white-rot basidiomycetes; the response in the SMRO ecosystem should be intermediate. We increased soil solution NO3- concentrations in a 73-day laboratory incubation and measured microbial respiration and soil solution dissolved organic carbon (DOC) and phenolics concentrations. At the end of the incubation, we measured the activity of ??-glucosidase, N-acetyl-glucosaminidase, phenol oxidase, and peroxidase, which are extracellular enzymes involved with cellulose and lignin degradation. We quantified the fungal biomass, and we also used fungal ribosomal intergenic spacer analysis (RISA) to gain insight into fungal community composition. In the BOWO ecosystem, increasing NO 3- significantly decreased oxidative enzyme activities (-30% to -54%) and increased DOC (+32% upper limit) and phenolic (+77% upper limit) concentrations. In the SMRO ecosystem, we observed a significant decrease in phenol oxidase activity (-73% lower limit) and an increase in soluble phenolic concentrations

  4. A dual enzyme functionalized nanostructured thulium oxide based interface for biomedical application

    NASA Astrophysics Data System (ADS)

    Singh, Jay; Roychoudhury, Appan; Srivastava, Manish; Solanki, Pratima R.; Lee, Dong Won; Lee, Seung Hee; Malhotra, B. D.

    2013-12-01

    In this paper, we present results of the studies related to fabrication of a rare earth metal oxide based efficient biosensor using an interface based on hydrothermally prepared nanostructured thulium oxide (n-Tm2O3). A colloidal solution of prepared nanorods has been electrophoretically deposited (EPD) onto an indium-tin-oxide (ITO) glass substrate. The n-Tm2O3 nanorods are found to provide improved sensing characteristics to the electrode interface in terms of electroactive surface area, diffusion coefficient, charge transfer rate constant and electron transfer kinetics. The structural and morphological studies of n-Tm2O3 nanorods have been carried out by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopic techniques. This interfacial platform has been used for fabrication of a total cholesterol biosensor by immobilizing cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) onto a Tm2O3 nanostructured surface. The results of response studies of the fabricated ChEt-ChOx/n-Tm2O3/ITO bioelectrode show a broad linear range of 8-400 mg dL-1, detection limit of 19.78 mg (dL cm-2)-1, and high sensitivity of 0.9245 μA (mg per dL cm-2)-1 with a response time of 40 s. Further, this bioelectrode has been utilized for estimation of total cholesterol with negligible interference (3%) from analytes present in human serum samples. The utilization of this n-Tm2O3 modified electrode for enzyme-based biosensor analysis offers an efficient strategy and a novel interface for application of the rare earth metal oxide materials in the field of electrochemical sensors and bioelectronic devices.In this paper, we present results of the studies related to fabrication of a rare earth metal oxide based efficient biosensor using an interface based on hydrothermally prepared nanostructured thulium oxide (n-Tm2O3). A colloidal solution of prepared

  5. Oxidative stress enzyme and histopathological lesions in Colossoma macropomum (pisces, ariidae) for environmental impact assessment

    NASA Astrophysics Data System (ADS)

    Andrade, Ticianne de Sousa de Oliveira Mota; Sousa, Debora Batista Pinheiro; Dantas, Janaina Gomes; Castro, Jonatas da Silva; Neta, Raimunda Nonata Fortes Carvalho

    2015-12-01

    This study used oxidative stress enzyme (Glutathione S-Transferase and Catalase), histopathological lesions (Branchial lesions) and biometric data in the freshwater fish tambaqui, Colossoma macropomum, to assess environmental impacts in an Environmental Protection Area at São Luis, Brazil. Fish were sampled from two locations (A1 = contaminated area and A2 = reference site) within the protected area on four occasions. The activity of catalase (CAT) and glutathione S-transferase (GST) in C. macropomum was compared with biometric data and histopathological lesions. Results have shown that biometric data decreased significantly in fish (p<0.05) at the contaminated site. The activity of CAT was higher in fish specifically caught in A1. A significant difference was observed in the GST activity in the liver of C. macropomum when comparing fish from the contaminated site and those from the reference site (p<0.05).

  6. Anti-oxidative enzyme changes associated with chickpea calli tolerant to Ascochyta rabiei culture filtrate.

    PubMed

    Kumar, Prabhat; Sangwan, M S; Mehta, Naresh; Kumar, Santosh

    2014-05-01

    Ascochyta blight caused by Ascochyta rabiei (Pass.) Labrousse is a major biotic constraint in production of chickpea. In the present investigation, all chickpea genotypes [E100Y (m), Gaurav, Pb-7 and L550] induced 100% callus on standard medium with greenish colour and fragile structure. These calli were used for in vitro screening against pathogen, A. rabiei culture filtrate at 0, 5, 10, 15 and 20% concentrations. Survival rate of calli in all chickpea's genotypes were reduced significantly at higher concentration (15%) of culture filtrate. The culture filtrate concentration of 20 % was lethal for calli of all chickpea's genotypes. Hence, biochemical changes viz. total soluble proteins and activities of anti-oxidative enzymes (polyphenol oxidase, peroxidase and catalase) were estimated at 15% and below concentration of culture filtrate. Tolerant calli of resistant genotype, E100 Y (m) revealed significantly higher total soluble proteins (10.04 mg g⁻¹ f.wt. of callus) and activity of anti-oxidative enzymes, polyphenol oxidase (9.0 unit absorbance change min⁻¹ mg⁻¹ protein) and peroxidase (19.09 unit absorbance change min⁻¹ mg⁻¹ protein) and lower catalase (18.65 μ moles of H2O2 utilized min⁻¹ mg⁻¹ protein) at higher (15%) concentration of culture filtrate followed by moderately resistant (Gaurav), and susceptible genotypes (Pb-7 and L550). Thus, higher polyphenol oxidase and peroxidase and lower catalase activity in chickpea's genotypes against culture filtrate of A. rabiei could be used as parameters for screening resistant genotypes to pathogen, A. rabiei.

  7. Unravelling the Interactions between Hydrolytic and Oxidative Enzymes in Degradation of Lignocellulosic Biomass by Sporothrix carnis under Various Fermentation Conditions

    PubMed Central

    Ogunyewo, Olusola A.; Olajuyigbe, Folasade M.

    2016-01-01

    The mechanism underlying the action of lignocellulolytic enzymes in biodegradation of lignocellulosic biomass remains unclear; hence, it is crucial to investigate enzymatic interactions involved in the process. In this study, degradation of corn cob by Sporothrix carnis and involvement of lignocellulolytic enzymes in biodegradation were investigated over 240 h cultivation period. About 60% degradation of corn cob was achieved by S. carnis at the end of fermentation. The yields of hydrolytic enzymes, cellulase and xylanase, were higher than oxidative enzymes, laccase and peroxidase, over 144 h fermentation period. Maximum yields of cellulase (854.4 U/mg) and xylanase (789.6 U/mg) were at 96 and 144 h, respectively. Laccase and peroxidase were produced cooperatively with maximum yields of 489.06 U/mg and 585.39 U/mg at 144 h. Drastic decline in production of cellulase at 144 h (242.01 U/mg) and xylanase at 192 h (192.2 U/mg) indicates that they play initial roles in biodegradation of lignocellulosic biomass while laccase and peroxidase play later roles. Optimal degradation of corn cob (76.6%) and production of hydrolytic and oxidative enzymes were achieved with 2.5% inoculum at pH 6.0. Results suggest synergy in interactions between the hydrolytic and oxidative enzymes which can be optimized for improved biodegradation. PMID:26881077

  8. Effects of nanomaterials on luciferase with significant protection and increased enzyme activity observed for zinc oxide nanomaterials.

    PubMed

    Barber, S; Abdelhakiem, M; Ghosh, K; Mitchell, L; Spidle, R; Jacobs, B; Washington, L; Li, J; Wanekaya, A; Glaspell, G; DeLong, R K

    2011-12-01

    This principle goal of this research was to examine the effects of various nanomaterials on the activity and behavior of the firefly enzyme luciferase. Nanomaterials have been found to stabilize, and in some instances, shown to increase the activity of enzymes. In this study gold, manganese oxide (MnO), and zinc oxide (ZnO) nanomaterials were utilized in order to test their effects on enzyme activity. Luciferase was used because its activity is easy to analyze, as it typically produces a large amount of bioluminescence easily detected by a Microtiter plate reader. Following incubation with the various nanomaterials, luciferase was subjected to degradation by several protein denaturing agents, such as heat, SDS, urea, ethanol, protease, hydrogen peroxide, and pH changes. Results indicated that luciferase activity is indeed affected when combined with nanomaterials, accompanied by both increases and decreases in enzyme activity depending on the type of nanomaterial and denaturing agent used. In most of the experiments, when incubated with ZnO nanomaterials, luciferase depicted significant increases in activity and bioluminescence. Additional experiments, in which human A375 cells were treated with luciferase-nanomaterial mixtures, also depicted increased enzyme activity and bioluminescence for luciferase incubated with ZnO nanomaterials. Ultimately, our findings indicated that when luciferase was subjected to multiple types of denaturation, zinc oxide nanomaterials dramatically preserved and increased enzyme activity and bioluminescence.

  9. Enzyme-free glucose sensor based on Au nanobouquet fabricated indium tin oxide electrode.

    PubMed

    Lee, Jin-Ho; El-Said, Waleed Ahmed; Oh, Byung-Keun; Choi, Jeong-Woo

    2014-11-01

    In this study, we demonstrated a simple, rapid and inexpensive fabrication method to develop a novel gold nanobouquet structure fabricated indium tin oxide (GNB/ITO) electrode based on electrochemical deposition of gold ions onto ITO substrate. The morphology of the fabricated electrode surface was characterized by scanning electron microscopy (SEM) to confirm the GNB formation. Enzyme-free detection of glucose using a GNB/ITO electrode was described with high sensitivity and selectivity based on cyclic voltammetry assay. The results demonstrate a linear relation within wide concentration range (500 nM to 10 mM) of glucose, with a correlation coefficient of 0.988. The interference effect of uric acid was effectively avoided for the detection of glucose (1 μM to 10 mM). Moreover, the developed sensor was applied to determine the concentration of glucose in the presence of human serum to indicate the ability of GNB/ITO electrodes in real samples. Hence, newly developed GNB/ITO electrode has potential application in enzyme-free glucose sensor with highly sensitivity and selectivity.

  10. Oxidation of naphthalene by a multicomponent enzyme system from Pseudomonas sp. strain NCIB 9816.

    PubMed Central

    Ensley, B D; Gibson, D T; Laborde, A L

    1982-01-01

    The initial reactions in the oxidation of naphthalene by Pseudomonas sp. strain NCIB 9816 involves the enzymatic incorporation of one molecule of oxygen into the aromatic nucleus to form (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The enzyme catalyzing this reaction, naphthalene dioxygenase, was resolved into three protein components, designated A, B, and C, by DEAE-cellulose chromatography. Incubation of naphthalene with components A, B, and C in the presence of NADH resulted in the formation of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The ratio of oxygen and NADH utilization to product formation was 1:1:1. NADPH also served as an electron donor for naphthalene oxygenation. However, its activity was less than 50% of that observed with NADH. Component A showed NAD(P)H-cytochrome c reductase activity which was stimulated by the addition of flavin adenine dinucleotide and flavin mononucleotide. A similar stimulation was observed when these flavin nucleotides were added to the naphthalene dioxygenase assay system. These preliminary observations indicate that naphthalene dioxygenase has properties in common with both monooxygenase and dioxygenase multicomponent enzyme systems. Images PMID:7037744

  11. Prenylcysteine oxidase 1, a pro-oxidant enzyme of low density lipoproteins.

    PubMed

    Herrera-Marcos, Luis V; Lou-Bonafonte, Jose M; Martinez-Gracia, Maria V; Arnal, Carmen; Navarro, María A; Osada, Jesus

    2018-01-01

    Elevated levels of low density lipoproteins (LDLs) cause atherosclerotic disease, and proteomic analyses have found that these lipoproteins are endowed with prenylcysteine lyase. This systematic review summarizes current understanding of this enzyme, now known as prenylcysteine oxidase 1 (PCYOX1), which hydrolyzes the thioether bond of prenylcysteines in the final step in the degradation of prenylated proteins, releasing hydrogen peroxide, cysteine and the isoprenoid aldehyde. Despite the high variability of the PCYOX1 gene, no polymorphism has yet been associated with any disease. The liver, which is responsible for vehiculization of the enzyme in lipoproteins, is one of the main organs responsible for its expression, together with the gastrointestinal tract, kidney, male reproductive tissue and muscle. Moreover, although hepatic mRNA expression is sensitive to diet and hormones, the repercussion of these changes in LDLs containing PCYOX1 has not been addressed. One consequence of its elevated activity could be an increase in hydrogen peroxide, which might help to propagate the oxidative burden of LDLs, thus making PCYOX1 a potential pharmacological target and a new biomarker in cardiovascular disease.

  12. Stage-specific distribution of oxidative radicals and antioxidant enzymes in the midgut of Leptinotarsa decemlineata.

    PubMed

    Krishnan, Natraj; Kodrík, Dalibor; Turanli, Ferit; Sehnal, Frantisek

    2007-01-01

    The titers of reactive oxygen species (ROS) represented by superoxide anion and general peroxides, and the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), are regulated in the midgut of the Colorado potato beetle (CPB) relative to the gut compartment, developmental stage, and food intake. ROS concentration is low in the potato leaves but it is very high in their digest in insect's anterior midgut. It is proposed that intensive ROS production in this gut region is linked to the processing of allelochemicals. SOD and CAT activities, low oxygen tension, and unidentified redox systems that maintain a slightly reducing milieu in the midgut lumen (pe+pH=6.95 declining to 5.36), obviously contribute to the decrease of ROS concentration along the gut length to a minimum in the wall of posterior midgut region. SOD and CAT activities are higher in the potato leaves than in the midgut tissues but the role of plant enzymes in ROS elimination within the gut lumen remains to be shown. A lower level of ROS and a higher antioxidant potential in the adult than in the larval midgut indicate stage specificity in the management of oxidative stress. The antioxidant defense is high in the diapausing adults that contain no detectable superoxide and about ten times less peroxides than the reproducing adults.

  13. The relationship between coenzyme Q10, oxidative stress, and antioxidant enzymes activities and coronary artery disease.

    PubMed

    Lee, Bor-Jen; Lin, Yi-Chin; Huang, Yi-Chia; Ko, Ya-Wen; Hsia, Simon; Lin, Ping-Ting

    2012-01-01

    A higher oxidative stress may contribute to the pathogenesis of coronary artery disease (CAD). The purpose of this study was to investigate the relationship between coenzyme Q10 concentration and lipid peroxidation, antioxidant enzymes activities and the risk of CAD. Patients who were identified by cardiac catheterization as having at least 50% stenosis of one major coronary artery were assigned to the case group (n = 51). The control group (n = 102) comprised healthy individuals with normal blood biochemical values. The plasma coenzyme Q10, malondialdehyde (MDA) and antioxidant enzymes activities (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx)) were measured. Subjects with CAD had significant lower plasma coenzyme Q10, CAT and GPx activities and higher MDA and SOD levels compared to those of the control group. The plasma coenzyme Q10 was positively correlated with CAT and GPx activities and negatively correlated with MDA and SOD. However, the correlations were not significant after adjusting for the potential confounders of CAD with the exception of SOD. A higher level of plasma coenzyme Q10 (≥ 0.52 μmol/L) was significantly associated with reducing the risk of CAD. Our results support the potential cardioprotective impact of coenzyme Q10.

  14. Antioxidant enzymes and oxidative stress in the erythrocytes of iron deficiency anemic patients supplemented with vitamins.

    PubMed

    Madhikarmi, Nirjala Laxmi; Murthy, Kora Rudraiah Siddalinga

    2014-01-01

    Iron deficiency anemia is one of the major causes of morbidity and mortality worldwide. Evidences from epidemiological and clinical studies suggest a possible correlation between antioxidant levels and the anemic disease risk. The present work is to investigate antioxidant levels and lipid peroxidation in anemic patients. A number of 30 patients (15 males and 15 females) were selected for the study. Likewise, 30 age- and gender-matched healthy volunteers (15 males and 15 females) were selected with their informed consent. Patients and healthy subjects were supplemented with vitamins C and E for 15 days. The lipid peroxidation both in plasma and erythrocyte lysates was determined by thiobarbituric acid reactive substances and lipid peroxides. The antioxidant vitamins A, C, and E and total antioxidant activity were also analyzed. The antioxidant enzyme superoxide dismutase, catalase, and glutathione peroxidase were also determined. Based on analysis, we found that the increase in lipid peroxidation was higher in the anemic subjects before vitamin supplementation, which was statistically significant at P<0.05. The antioxidant enzymes were higher in the patients before antioxidant supplementation when compared with patients after vitamin supplementation. Our data revealed higher oxidative stress before vitamin supplementation in iron deficiency anemic patients and after supplementation, lower lipid peroxidation and increased antioxidant vitamins were achieved.

  15. Antioxidant enzymes activities of Burkholderia spp. strains-oxidative responses to Ni toxicity.

    PubMed

    Dourado, M N; Franco, M R; Peters, L P; Martins, P F; Souza, L A; Piotto, F A; Azevedo, R A

    2015-12-01

    Increased agriculture production associated with intense application of herbicides, pesticides, and fungicides leads to soil contamination worldwide. Nickel (Ni), due to its high mobility in soils and groundwater, constitutes one of the greatest problems in terms of environmental pollution. Metals, including Ni, in high concentrations are toxic to cells by imposing a condition of oxidative stress due to the induction of reactive oxygen species (ROS), which damage lipids, proteins, and DNA. This study aimed to characterize the Ni antioxidant response of two tolerant Burkholderia strains (one isolated from noncontaminated soil, SNMS32, and the other from contaminated soil, SCMS54), by measuring superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione S-transferase (GST) activities. Ni accumulation and bacterial growth in the presence of the metal were also analyzed. The results showed that both strains exhibited different trends of Ni accumulation and distinct antioxidant enzymes responses. The strain from contaminated soil (SCMS54) exhibited a higher Ni biosorption and exhibited an increase in SOD and GST activities after 5 and 12 h of Ni exposure. The analysis of SOD, CAT, and GR by nondenaturing PAGE revealed the appearance of an extra isoenzyme in strain SCMS54 for each enzyme. The results suggest that the strain SCMS54 isolated from contaminated soil present more plasticity with potential to be used in soil and water bioremediation.

  16. The oxidative dealkylation of insecticidal phosphoric acid triesters by mammalian liver enzymes

    PubMed Central

    Donninger, C.; Hutson, D. H.; Pickering, B. A.

    1972-01-01

    1. The dealkylation of the insecticidal phosphoric acid triester, 2-chloro-1-(2,4-dichlorophenyl)vinyl diethyl phosphate, proceeds in mammalian liver slices via an oxidative mechanism and not by hydrolysis. 2. The enzyme that catalyses the reaction is located in the microsomal fraction of liver homogenate and is dependent for activity on molecular oxygen and NADPH. 3. There are large species differences between rat, mouse, rabbit and dog in the activity of the enzymes, the relative rates of dealkylation being 1, 8, 24 and 88 respectively in liver slices. 4. Dimethyl and di-isopropyl phosphate triesters are also dealkylated by rabbit liver microsomal preparations. 5. The mechanism of dealkylation involves hydroxylation at the α-carbon atom of an alkyl group, which is removed as the corresponding aldehyde, and is thus analogous to that of similar reactions catalysed by the microsomal mixed-function oxidases. 6. The relevance of these findings in the toxicology of phosphoric acid triesters is discussed. PMID:5075276

  17. MITOCHONDRIAL LOCALIZATION OF OXIDATIVE ENZYMES: STAINING RESULTS WITH TWO TETRAZOLIUM SALTS

    PubMed Central

    Novikoff, Alex B.; Shin, Woo-Yung; Drucker, Joan

    1961-01-01

    A comparison is made of the staining results obtained with Nitro-BT and MTT-Co++ as acceptors when DPNH is the substrate in frozen sections of cold formol-calcium-fixed rat kidney (normal and following ligation of the blood vessels) and human liver containing lipofuscin granules. The kidney results are evaluated in terms of mitochondrial morphology seen after classical mitochondrial stains and in electron micrographs. It is concluded that, except for formazan deposition on lipid-aqueous interfaces, Nitro-BT staining indicates the intracellular localization of oxidative enzymes, at least at the level of light microscopy. In contrast, the use of MTT-Co++ is not reliable for such intracellular localizations. The deposition of the formazan of MTT-Co++ is determined in large part by physicochemical factors other than enzyme localization. Despite marked abnormalities of the mitochondria in cells of the ligated kidney, MTT-Co++ formazan is generally deposited in the same dotlike fashion as in cells of normal kidney. PMID:13729759

  18. Antioxidant Enzymes and Oxidative Stress in the Erythrocytes of Iron Deficiency Anemic Patients Supplemented with Vitamins

    PubMed Central

    Laxmi Madhikarmi, Nirjala; Rudraiah Siddalinga Murthy, Kora

    2014-01-01

    Background: Iron deficiency anemia is one of the major causes of morbidity and mortality worldwide. Evidences from epidemiological and clinical studies suggest a possible correlation between antioxidant levels and the anemic disease risk. The present work is to investigate antioxidant levels and lipid peroxidation in anemic patients. Methods: A number of 30 patients (15 males and 15 females) were selected for the study. Likewise, 30 age- and gender-matched healthy volunteers (15 males and 15 females) were selected with their informed consent. Patients and healthy subjects were supplemented with vitamins C and E for 15 days. The lipid peroxidation both in plasma and erythrocyte lysates was determined by thiobarbituric acid reactive substances and lipid peroxides. The antioxidant vitamins A, C, and E and total antioxidant activity were also analyzed. The antioxidant enzyme superoxide dismutase, catalase, and glutathione peroxidase were also determined. Results: Based on analysis, we found that the increase in lipid peroxidation was higher in the anemic subjects before vitamin supplementation, which was statistically significant at P<0.05. The antioxidant enzymes were higher in the patients before antioxidant supplementation when compared with patients after vitamin supplementation. Conclusion: Our data revealed higher oxidative stress before vitamin supplementation in iron deficiency anemic patients and after supplementation, lower lipid peroxidation and increased antioxidant vitamins were achieved. PMID:24518548

  19. A highly sensitive electrochemical biosensor for catechol using conducting polymer reduced graphene oxide-metal oxide enzyme modified electrode.

    PubMed

    Sethuraman, V; Muthuraja, P; Anandha Raj, J; Manisankar, P

    2016-10-15

    The fabrication, characterization and analytical performances were investigated for a catechol biosensor, based on the PEDOT-rGO-Fe2O3-PPO composite modified glassy carbon (GC) electrode. The graphene oxide (GO) doped conducting polymer poly (3,4-ethylenedioxythiophene) (PEDOT) was prepared through electrochemical polymerization by potential cycling. Reduction of PEDOT-GO was carried out by amperometric method. Fe2O3 nanoparticles were synthesized in ethanol by hydrothermal method. The mixture of Fe2O3, PPO and glutaraldehyde was casted on the PEDOT-rGO electrode. The surface morphology of the modified electrodes was studied by FE-SEM and AFM. Cyclic voltammetric studies of catechol on the enzyme modified electrode revealed higher reduction peak current. Determination of catechol was carried out successfully by Differential Pulse Voltammetry (DPV) technique. The fabricated biosensor investigated shows a maximum current response at pH 6.5. The catechol biosensor exhibited wide sensing linear range from 4×10(-8) to 6.20×10(-5)M, lower detection limit of 7×10(-9)M, current maxima (Imax) of 92.55µA and Michaelis-Menten (Km) constant of 30.48µM. The activation energy (Ea) of enzyme electrode is 35.93KJmol(-1) at 50°C. There is no interference from d-glucose and l-glutamic acid, ascorbic acid and o-nitrophenol. The PEDOT-rGO-Fe2O3-PPO biosensor was stable for at least 75 days when stored in a buffer at about 4°C.

  20. Oxidation of Monolignols by Members of the Berberine Bridge Enzyme Family Suggests a Role in Plant Cell Wall Metabolism*

    PubMed Central

    Daniel, Bastian; Pavkov-Keller, Tea; Steiner, Barbara; Dordic, Andela; Gutmann, Alexander; Nidetzky, Bernd; Sensen, Christoph W.; van der Graaff, Eric; Wallner, Silvia; Gruber, Karl; Macheroux, Peter

    2015-01-01

    Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family. PMID:26037923

  1. The activities of antioxidant enzymes in response to oxidative stresses and hormones in paraquat-tolerant Rehmannia glutinosa plants.

    PubMed

    Choi, Dong Geun; Yoo, Nam Hee; Yu, Chang Yeon; de Los Reyes, Benildo; Yun, Song Joong

    2004-09-30

    All members of R. glutinosa show the unique characteristic of intrinsic tolerance to paraquat (PQ). Antioxidant enzymes have been proposed to be the primary mechanism of PQ resistance in several plant species. Therefore, the antioxidant enzyme systems of R. glutinosa were evaluated by comparatively analyzing cellular antioxidant enzyme levels, and their responses of oxidative stresses and hormones. The levels of ascorbate peroxidase (APX), glutathione reductase (GR), non-specific peroxidase (POX), and superoxide dismutase (SOD) were 7.3-, 4.9-, 2.7- and 1.6-fold higher in PQ-tolerant R. glutinosa than in PQ-susceptible soybeans. However, the activity of catalase (CAT) was about 12-fold higher in the soybeans. The activities of antioxidant enzymes reduced after PQ treatment in the two species, with the exception of POX and SOD in R. glutinosa, which increased by about 40 %. Interestingly, the activities of APX, SOD and POX in R. glutinosa, relative to those in soybeans, were further increased by 49, 67 and 93 % after PQ treatment. The considerably higher intrinsic levels, and increases in the relative activities of antioxidant enzymes in R. glutinosa under oxidative stress support the possible role of these enzymes in the PQ tolerance of R. glutinosa. However, the relatively lower levels of SOD versus PQ tolerance, and the mixed responses of antioxidant enzymes to stresses and hormones, suggest a possible alternative mechanism(s) for PQ tolerance in R. glutinosa.

  2. A specific affinity reagent to distinguish aldehyde dehydrogenases and oxidases. Enzymes catalyzing aldehyde oxidation in an adult moth

    SciTech Connect

    Tasayco, M.L.; Prestwich, G.D. )

    1990-02-25

    Aldehyde dehydrogenase (ALDH) and oxidase (AO) enzymes from the tissue extracts of male and female tobacco budworm moth (Heliothis virescens) were identified after electrophoretic protein separation. AO activity was visualized using formazan- or horseradish peroxidase-mediated staining coupled to the AO-catalyzed oxidation of benzaldehyde. A set of six soluble AO enzymes with isoelectric points from pI 4.6 to 5.3 were detected primarily in the antennal extracts. Partially purified antennal AO enzymes also oxidized both (Z)-9-tetradecenal and (Z)-11-hexadecenal, the two major pheromone components of this moth. ALDH activity was detected using a tritium-labeled affinity reagent based on a known irreversible inhibitor of this enzyme. This labeled vinyl ketone, (3H)(Z)-1,11-hexadecadien-3-one, was synthesized and used to covalently modify the soluble ALDH enzymes from tissue extracts. Molecular subunits of potential ALDH enzymes were visualized in the fluorescence autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the antenna, head, and leg tissues. Covalent modification of these protein subunits decreased specifically in the presence of excess pheromone aldehyde or benzaldehyde. Labeled vinyl ketones are thus novel tools for the identification of molecular subunits of ALDH enzymes.

  3. Coupling osmium complexes to epoxy-functionalised polymers to provide mediated enzyme electrodes for glucose oxidation.

    PubMed

    Ó Conghaile, Peter; Pöller, Sascha; MacAodha, Domhnall; Schuhmann, Wolfgang; Leech, Dónal

    2013-05-15

    Newly synthesised osmium complex-modified redox polymers were tested for potential application as mediators in glucose oxidising enzyme electrodes for application to biosensors or biofuel cells. Coupling of osmium complexes containing amine functional groups to epoxy-functionalised polymers of variable composition provides a range of redox polymers with variation possible in redox potential and physicochemical properties. Properties of the redox polymers as mediators for glucose oxidation were investigated by co-immobilisation onto graphite with glucose oxidase or FAD-dependent glucose dehydrogenase using a range of crosslinkers and in the presence and absence of multiwalled carbon nanotubes. Electrodes prepared by immobilising [P20-Os(2,2'-bipyridine)2(4-aminomethylpyridine)Cl].PF6, carbon nanotubes and glucose oxidase exhibit glucose oxidation current densities as high as 560μAcm(-2) for PBS containing 100mM glucose at 0.45V vs. Ag/AgCl. Films prepared by crosslinking [P20-Os(4,4'-dimethoxy-2,2'-bipyridine)2(4-aminomethylpyridine)Cl].PF6, an FAD-dependent glucose dehydrogenase, and carbon nanotubes achieve current densities of 215μAcm(-2) in 5mM glucose at 0.2V vs. Ag/AgCl, showing some promise for application to glucose oxidising biosensors or biofuel cells.

  4. Zinc oxide nanoparticles cause inhibition of microbial denitrification by affecting transcriptional regulation and enzyme activity.

    PubMed

    Zheng, Xiong; Su, Yinglong; Chen, Yinguang; Wan, Rui; Liu, Kun; Li, Mu; Yin, Daqiang

    2014-12-02

    Over the past few decades, human activities have accelerated the rates and extents of water eutrophication and global warming through increasing delivery of biologically available nitrogen such as nitrate and large emissions of anthropogenic greenhouse gases. In particular, nitrous oxide (N2O) is one of the most important greenhouse gases, because it has a 300-fold higher global warming potential than carbon dioxide. Microbial denitrification is a major pathway responsible for nitrate removal, and also a dominant source of N2O emissions from terrestrial or aquatic environments. However, whether the release of zinc oxide nanoparticles (ZnO NPs) into the environment affects microbial denitrification is largely unknown. Here we show that the presence of ZnO NPs lead to great increases in nitrate delivery (9.8-fold higher) and N2O emissions (350- and 174-fold higher in the gas and liquid phases, respectively). Our data further reveal that ZnO NPs significantly change the transcriptional regulations of glycolysis and polyhydroxybutyrate synthesis, which causes the decrease in reducing powers available for the reduction of nitrate and N2O. Moreover, ZnO NPs substantially inhibit the gene expressions and catalytic activities of key denitrifying enzymes. These negative effects of ZnO NPs on microbial denitrification finally cause lower nitrate removal and higher N2O emissions, which is likely to exacerbate water eutrophication and global warming.

  5. Insulin, catecholamines, glucose and antioxidant enzymes in oxidative damage during different loads in healthy humans.

    PubMed

    Koska, J; Blazícek, P; Marko, M; Grna, J D; Kvetnanský, R; Vigas, M

    2000-01-01

    Exercise, insulin-induced hypoglycemia and oral glucose loads (50 g and 100 g) were used to compare the production of malondialdehyde and the activity of antioxidant enzymes in healthy subjects. Twenty male volunteers participated in the study. Exercise consisted of three consecutive work loads on a bicycle ergometer of graded intensity (1.5, 2.0, and 2.5 W/kg, 6 min each). Hypoglycemia was induced by insulin (Actrapid MC Novo, 0.1 IU/kg, i.v.). Oral administration of 50 g and 100 g of glucose was given to elevate plasma glucose. The activity of superoxide dismutase (SOD) was determined in red blood cells, whereas glutathione peroxidase (GSH-Px) activity was measured in whole blood. The concentration of malondialdehyde (MDA) was determined by HPLC, catecholamines were assessed radioenzymatically and glucose was measured by the glucose-oxidase method. Exercise increased MDA concentrations, GSH-Px and SOD activities as well as plasma noradrenaline and adrenaline levels. Insulin hypoglycemia increased plasma adrenaline levels, but the concentrations of MDA and the activities of GSH-Px and SOD were decreased. Hyperglycemia increased plasma MDA concentrations, but the activities of GSH-Px and SOD were significantly higher after a larger dose of glucose only. Plasma catecholamines were unchanged. These results indicate that the transient increase of plasma catecholamine and insulin concentrations did not induce oxidative damage, while glucose already in the low dose was an important triggering factor for oxidative stress.

  6. Dynamic changes of anti-oxidative enzymes of 10 wheat genotypes at soil water deficits.

    PubMed

    Shao, Hong Bo; Liang, Zong Suo; Shao, Ming An; Sun, Qun

    2005-05-25

    Drought is a world-spread problem seriously influencing crop production and quality, the loss of which is the total for other natural disasters, with increasing global climate change making the situation more serious. Wheat is the staple food for more than 35% of world population, so wheat anti-drought physiology study is of importance to wheat production and biological breeding for the sake of coping with abiotic and biotic conditions. Much research is involved in this hot topic, but the pace of progress is not so large because of drought resistance being a multiple-gene-control quantitative character and wheat genome being larger (16,000 Mb). On the other hand, stress adaptive mechanisms are quite different, with stress degree, different growth and developmental stages, time course, materials and experimental plots, thus increasing the complexity of the issue in question. Additionally, a little study is related to the whole life circle of wheat, which cannot provide a comprehensive understanding of its anti-drought machinery. We selected 10 kinds of wheat genotypes as materials, which have potential to be applied in practice, and measured change of relative physiological indices through wheat whole growing developmental circle (i.e. seedling, tillage and maturing). Here, we reported the dynamic anti-oxidative results of whole stage (i.e. seedling, tillage and maturing) in terms of activities of POD, SOD, CAT of 10 wheat genotypes as follows: (1) 10 wheat genotypes can be grouped into three kinds (A, B and C, respectively) according to their changing trend of the measured indices; (2) A group performed better resistance drought under the condition of treatment level 1, whose activities of anti-oxidative enzymes (POD, SOD, CAT) were higher; (3) B group exhibited stronger anti-drought under treatment level 2, whose activities of anti-oxidative enzymes were higher; (4) C group expressed anti-drought to some extent under treatment level 3, whose activities of anti-oxidative

  7. [1-butanol synthesis by Escherichia coli cells through butyryl-CoA formation by heterologous enzymes of clostridia and native enzymes of fatty acid beta-oxidation].

    PubMed

    Gulevich, A Iu; Skorokhodova, A Iu; Morzhakova, A A; Antonova, S V; Sukhozhenko, A V; Shakulov, R S; Debabov, V G

    2012-01-01

    Anaerobic biosynthesis of 1-butanol from glucose is investigated in recombinant Escherichia coli strains which form butyryl-CoA using the heterologous enzyme complex of clostridia or as a result of a reversal in the action of native enzymes of the fatty acid beta-oxidation pathway. It was revealed that when the basic pathways of acetic and lactic acid formation are inactivated due to deletions in the ackA, pta, poxB, and ldhA genes, the efficiency of butyryl-CoA biosynthesis and its reduced product, i.e., 1-butanol, by two types of recombinant stains is comparable. The limiting factor for 1-butanol production by the obtained strains is the low substrate specificity of the basic CoA-dependent alcohol/aldehyde AdhE dehydrogenase from E. coli to butyryl-CoA. It was concluded that, in order to construct an efficient 1-butanol producer based on a model strain synthesizing butyryl-CoA as a result of a reversal in fatty acid beta-oxidation enzymes, it is necessary to provide intensive formation of acetyl-CoA and enhanced activity of alternative alcohol and aldehyde dehydrogenases in the cells of a strain.

  8. The clinical research for Ganoderan's effect on preventing and treating cerebral arteriosclerosis through inhibiting NADPH oxidizing enzyme expression.

    PubMed

    Feng, Zhang Li; Fang, Teng Jun; Qian, Yuan Xue; Rong, Wang Huan

    2014-07-01

    A lot of researches have verified that produced excessive reactive oxygen is one of the hazard factors causing atherosclerosis. NADPH oxidase is the main protease of vascular cell's producing reactive oxygen, the expression of its relevant subunits is closely correlated with the occurring and development process of atherosclerosis. Oxidizing reaction could damage organism tissue cells, ganoderan has very significant effect on the anti-oxidizing function of cell. The pharmaceutical research of ganoderan has significant meaning in curing diabetes mellitus, preventing and controlling arteriosclerosis. This paper is mainly to discuss the effect of anoderan's inhibiting NADPH oxidizing enzyme expression on preventing and treating cerebral arteriosclerosis and its action mechanism.

  9. Inactivation of enzymes and oxidative modification of proteins by stimulated neutrophils.

    PubMed

    Oliver, C N

    1987-02-15

    Differentiated, stimulated HL-60 cells and freshly isolated, stimulated neutrophils inactivate glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) either inside or outside of Escherichia coli. Stimulated neutrophils also inactivate at least four endogenous enzymes which are inactivated by mixed-function oxidation (MFO) systems in vitro (L. Fucci, C.N. Oliver, M.J. Coon, and E.R. Stadtman (1983) Proc. Natl. Acad. Sci. USA 80, 1521-1525). The inactivation of glutamine synthetase by stimulated neutrophils exhibits characteristics similar to those previously described using both enzymic and nonenzymic MFO systems (R.L. Levine, C.N. Oliver, R.M. Fulks, and E.R. Stadtman (1981) Proc. Natl. Acad. Sci. USA 78, 2120-2124). Although the reaction occurs in the absence of Fe(III), it is stimulated by added Fe (III). Inactivation required molecular oxygen and is partially inhibited by Mn(II), catalase, superoxide dismutase, and metal chelators, ethylenediaminetetraacetic acid and o-phenanthroline. Both the kinetics and the extent of glutamine synthetase inactivation differ when neutrophils are stimulated with phorbol esters compared with formylated peptides. Glutamine synthetase inactivation catalyzed by MFO systems is accompanied by the formation of protein carbonyl derivatives which form stable hydrazones when treated with 2,4-dinitrophenylhydrazine. Multiple carbonyl derivatives are formed in the soluble protein fraction of stimulated neutrophils and these derivatives collectively exhibit an absorbance spectrum similar to that of glutamine synthetase inactivated by liver microsomal cytochrome P-450 MFO system (K. Nakamura, C.N. Oliver, and E.R. Stadtman (1985) Arch. Biochem. Biophys. 240, 319-329).

  10. Electrochemically reduced graphene and iridium oxide nanoparticles for inhibition-based angiotensin-converting enzyme inhibitor detection.

    PubMed

    Kurbanoglu, Sevinc; Rivas, Lourdes; Ozkan, Sibel A; Merkoçi, Arben

    2017-02-15

    In this work, a novel biosensor based on electrochemically reduced graphene oxide and iridium oxide nanoparticles for the detection of angiotensin-converting enzyme inhibitor drug, captopril, is presented. For the preparation of the biosensor, tyrosinase is immobilized onto screen printed electrode by using 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-Hydroxysuccinimide coupling reagents, in electrochemically reduced graphene oxide and iridium oxide nanoparticles matrix. Biosensor response is characterized towards catechol, in terms of graphene oxide concentration, number of cycles to reduce graphene oxide, volume of iridium oxide nanoparticles and tyrosinase solution. The designed biosensor is used to inhibit tyrosinase activity by Captopril, which is generally used to treat congestive heart failure. It is an angiotensin-converting enzyme inhibitor that operates via chelating copper at the active site of tyrosinase and thioquinone formation. The captopril detections using both inhibition ways are very sensitive with low limits of detection: 0.019µM and 0.008µM for chelating copper at the active site of tyrosinase and thioquinone formation, respectively. The proposed methods have been successfully applied in captopril determination in spiked human serum and pharmaceutical dosage forms with acceptable recovery values. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Markers of oxidative stress and erythrocyte antioxidant enzyme activity in older men and women with differing physical activity.

    PubMed

    Rowiński, Rafał; Kozakiewicz, Mariusz; Kędziora-Kornatowska, Kornelia; Hübner-Woźniak, Elżbieta; Kędziora, Józef

    2013-11-01

    The aim of the present study was to examine the relationship between markers of oxidative stress and erythrocyte antioxidant enzyme activity and physical activity in older men and women. The present study included 481 participants (233 men and 248 women) in the age group 65-69 years (127 men and 125 women) and in the age group 90 years and over (106 men and 123 women). The classification of respondents by physical activity was based on answers to the question if, in the past 12 months, they engaged in any pastimes which require physical activity. The systemic oxidative stress status was assessed by measuring plasma iso-PGF2α and protein carbonyl concentration as well as erythrocyte antioxidant enzymes activity, i.e., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). The concentration of plasma iso-PGF2α and protein carbonyls (CP) was lower in groups of younger men and women compared to the respective older groups. In all examined groups, physical activity resulted in decrease of these oxidative stress markers and simultaneously caused adaptive increase in the erythrocyte SOD activity. Additionally, in active younger men CAT, GPx, and GR activities were higher than in sedentary ones. In conclusion, oxidative stress increase is age-related, but physical activity can reduce oxidative stress markers and induce adaptive increase in the erythrocyte antioxidant enzyme activity, especially SOD, even in old and very old men and women.

  12. Anaerobic Toluene Catabolism of Thauera aromatica: the bbs Operon Codes for Enzymes of β Oxidation of the Intermediate Benzylsuccinate

    PubMed Central

    Leuthner, Birgitta; Heider, Johann

    2000-01-01

    The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent β-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in β oxidation of benzylsuccinate are unknown. We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting of bbs (beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the β-oxidation pathway of benzylsuccinate. Two of the genes, bbsE and bbsF, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown Thauera aromatica. The bbsG gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in Escherichia coli and detection of enzyme activity. The further enzymes of the pathway are probably encoded by bbsH (enoyl-CoA hydratase), bbsCD (3-hydroxyacyl-CoA dehydrogenase), and bbsB (3-oxoacyl-CoA thiolase). The operon contains two additional genes, bbsA and bbsI, for which no obvious function could be derived. The bbs operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the bbsA gene. This represents the first known promoter site in Thauera spp. PMID:10629170

  13. Oxidations of N-(3-indoleethyl) cyclic aliphatic amines by horseradish peroxidase: the indole ring binds to the enzyme and mediates electron-transfer amine oxidation.

    PubMed

    Ling, Ke-Qing; Li, Wen-Shan; Sayre, Lawrence M

    2008-01-23

    Although oxidations of aromatic amines by horseradish peroxidase (HRP) are well-known, typical aliphatic amines are not substrates of HRP. In this study, the reactions of N-benzyl and N-methyl cyclic amines with HRP were found to be slow, but reactions of N-(3-indoleethyl) cyclic amines were 2-3 orders of magnitude faster. Analyses of pH-rate profiles revealed a dominant contribution to reaction by the amine-free base forms, the only species found to bind to the enzyme. A metabolic study on a family of congeneric N-(3-indoleethyl) cyclic amines indicated competition between amine and indole oxidation pathways. Amine oxidation dominated for the seven- and eight-membered azacycles, where ring size supports the change in hybridization from sp3 to sp2 that occurs upon one-electron amine nitrogen oxidation, whereas only indole oxidation was observed for the six-membered ring congener. Optical difference spectroscopic binding data and computational docking simulations suggest that all the arylalkylamine substrates bind to the enzyme through their aromatic termini with similar binding modes and binding affinities. Kinetic saturation was observed for a particularly soluble substrate, consistent with an obligatory role of an enzyme-substrate complexation preceding electron transfer. The significant rate enhancements seen for the indoleethylamine substrates suggest the ability of the bound indole ring to mediate what amounts to medium long-range electron-transfer oxidation of the tertiary amine center by the HRP oxidants. This is the first systematic investigation to document aliphatic amine oxidation by HRP at rates consistent with normal metabolic turnover, and the demonstration that this is facilitated by an auxiliary electron-rich aromatic ring.

  14. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  15. Endosulfan induces oxidative stress and changes on detoxication enzymes in the aquatic macrophyte Myriophyllum quitense.

    PubMed

    Menone, Mirta L; Pesce, Silvia F; Díaz, María P; Moreno, Víctor J; Wunderlin, Daniel A

    2008-03-01

    Endosulfan (1) is a chlorinated insecticide still in use in both developed and emerging countries. Although its toxicity on animals has been studied in the last years, scarce information is available on its effects on plants. In this study, we exposed the aquatic macrophyte Myriophyllum quitense to environmentally relevant concentrations of endosulfan (microg/L) (1) for a short time, simulating exposures that might occur after either accidental spills or toxic run-off from agricultural areas. The main goal was to evaluate changes in both detoxication and antioxidant enzymatic systems of this plant upon exposure to endosulfan (1). Thus, we measured the activities of catalase (CAT), soluble and membrane associated glutathione-S-transferases (s- and m-GSTs) and glutathione reductase (GR), as well as the hydrogen peroxide (H2O2) content. Results showed that endosulfan (1) exerts oxidative stress on M. quitense, which was evidenced by the increase of CAT activity and the H2O2 content in exposed plants. At 5 microg/L endosulfan (1), we found a generalized induction of activities of tested enzymes, indicating that this xenobiotic activates the protection system of this plant, increasing its capacity to scavenge reactive oxygen species. On the other hand, we did not find significant changes at 0.02 microg/L endosulfan (1), which is the maximal concentration allowed for freshwater. We conclude that runoff events, which can produce significant amounts of endosulfan (1) in aquatic environments during short time, can result in oxidative stress on M. quitense, and probably on similar macrophytes.

  16. Alcohol oxidizing enzymes and ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells

    PubMed Central

    Bhopale, Kamlesh K.; Falzon, Miriam; Ansari, G. A. S.

    2016-01-01

    Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disease causing significant morbidity and mortality. Due to lack of a suitable animal model, the underlying mechanism of ACP is poorly understood. Chronic alcohol abuse inhibits alcohol dehydrogenase (ADH) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) in the pancreas frequently damaged during chronic ethanol abuse. Earlier, we reported a concentration-dependent formation of FAEEs and cytotoxicity in ethanol-treated rat pancreatic tumor (AR42J) cells, which express high FAEE synthase activity as compared to ADH and cytochrome P450 2E1. Therefore, the present study was undertaken to investigate the role of various ethanol oxidizing enzymes in ethanol-induced pancreatic acinar cell injury. Confluent AR42J cells were pre-treated with inhibitors of ADH class I and II [4-methylpyrazole (MP)] or class I, II, and III [1,10-phenanthroline (PT)], cytochrome P450 2E1 (trans-1,2-dichloroethylene) or catalase (sodium azide) followed by incubation with 800 mg% ethanol at 37°C for 6 h. Ethanol metabolism, cell viability, cytotoxicity (apoptosis and necrosis), cell proliferation status, and formation of FAEEs in AR42J cells were measured. The cell viability and cell proliferation rate were significantly reduced in cells pretreated with 1,10-PT + ethanol followed by those with 4-MP + ethanol. In situ formation of FAEEs was twofold greater in cells incubated with l,10-PT + ethanol and ~1.5-fold in those treated with 4-MP + ethanol vs. respective controls. However, cells treated with inhibitors of cytochrome P450 2E1 or catalase in combination of ethanol showed no significant changes either for FAEE formation, cell death or proliferation rate. Therefore, an impaired ADH class I—III catalyzed oxidation of ethanol appears to be a key contributing factor in ethanol-induced pancreatic injury via formation of nonoxidative metabolites of ethanol. PMID:24281792

  17. Alcohol oxidizing enzymes and ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells.

    PubMed

    Bhopale, Kamlesh K; Falzon, Miriam; Ansari, G A S; Kaphalia, Bhupendra S

    2014-04-01

    Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disease causing significant morbidity and mortality. Due to lack of a suitable animal model, the underlying mechanism of ACP is poorly understood. Chronic alcohol abuse inhibits alcohol dehydrogenase (ADH) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) in the pancreas frequently damaged during chronic ethanol abuse. Earlier, we reported a concentration-dependent formation of FAEEs and cytotoxicity in ethanol-treated rat pancreatic tumor (AR42J) cells, which express high FAEE synthase activity as compared to ADH and cytochrome P450 2E1. Therefore, the present study was undertaken to investigate the role of various ethanol oxidizing enzymes in ethanol-induced pancreatic acinar cell injury. Confluent AR42J cells were pre-treated with inhibitors of ADH class I and II [4-methylpyrazole (MP)] or class I, II, and III [1,10-phenanthroline (PT)], cytochrome P450 2E1 (trans-1,2-dichloroethylene) or catalase (sodium azide) followed by incubation with 800 mg% ethanol at 37°C for 6 h. Ethanol metabolism, cell viability, cytotoxicity (apoptosis and necrosis), cell proliferation status, and formation of FAEEs in AR42J cells were measured. The cell viability and cell proliferation rate were significantly reduced in cells pretreated with 1,10-PT + ethanol followed by those with 4-MP + ethanol. In situ formation of FAEEs was twofold greater in cells incubated with 1,10-PT + ethanol and ∼1.5-fold in those treated with 4-MP + ethanol vs. respective controls. However, cells treated with inhibitors of cytochrome P450 2E1 or catalase in combination of ethanol showed no significant changes either for FAEE formation, cell death or proliferation rate. Therefore, an impaired ADH class I-III catalyzed oxidation of ethanol appears to be a key contributing factor in ethanol-induced pancreatic injury via formation of nonoxidative metabolites of ethanol.

  18. Iron oxide filled magnetic carbon nanotube-enzyme conjugates for recycling of amyloglucosidase: toward useful applications in biofuel production process.

    PubMed

    Goh, Wei Jiang; Makam, Venkata S; Hu, Jun; Kang, Lifeng; Zheng, Minrui; Yoong, Sia Lee; Udalagama, Chammika N B; Pastorin, Giorgia

    2012-12-11

    Biofuels are fast advancing as a new research area to provide alternative sources of sustainable and clean energy. Recent advances in nanotechnology have sought to improve the efficiency of biofuel production, enhancing energy security. In this study, we have incorporated iron oxide nanoparticles into single-walled carbon nanotubes (SWCNTs) to produce magnetic single-walled carbon nanotubes (mSWCNTs). Our objective is to bridge both nanotechnology and biofuel production by immobilizing the enzyme, Amyloglucosidase (AMG), onto mSWCNTs using physical adsorption and covalent immobilization, with the aim of recycling the immobilized enzyme, toward useful applications in biofuel production processes. We have demonstrated that the enzyme retains a certain percentage of its catalytic efficiency (up to 40%) in starch prototype biomass hydrolysis when used repeatedly (up to ten cycles) after immobilization on mSWCNTs, since the nanotubes can be easily separated from the reaction mixture using a simple magnet. The enzyme loading, activity, and structural changes after immobilization onto mSWCNTs were also studied. In addition, we have demonstrated that the immobilized enzyme retains its activity when stored at 4 °C for at least one month. These results, combined with the unique intrinsic properties of the nanotubes, pave the way for greater efficiency in carbon nanotube-enzyme bioreactors and reduced capital costs in industrial enzyme systems.

  19. Fast solubilization of recalcitrant cellulosic biomass by the basidiomycete fungus Laetisaria arvalis involves successive secretion of oxidative and hydrolytic enzymes.

    PubMed

    Navarro, David; Rosso, Marie-Noëlle; Haon, Mireille; Olivé, Caroline; Bonnin, Estelle; Lesage-Meessen, Laurence; Chevret, Didier; Coutinho, Pedro M; Henrissat, Bernard; Berrin, Jean-Guy

    2014-01-01

    Enzymatic breakdown of lignocellulosic biomass is a known bottleneck for the production of high-value molecules and biofuels from renewable sources. Filamentous fungi are the predominant natural source of enzymes acting on lignocellulose. We describe the extraordinary cellulose-deconstructing capacity of the basidiomycete Laetisaria arvalis, a soil-inhabiting fungus. The L. arvalis strain displayed the capacity to grow on wheat straw as the sole carbon source and to fully digest cellulose filter paper. The cellulolytic activity exhibited in the secretomes of L. arvalis was up to 7.5 times higher than that of a reference Trichoderma reesei industrial strain, resulting in a significant improvement of the glucose release from steam-exploded wheat straw. Global transcriptome and secretome analyses revealed that L. arvalis produces a unique repertoire of carbohydrate-active enzymes in the fungal taxa, including a complete set of enzymes acting on cellulose. Temporal analyses of secretomes indicated that the unusual degradation efficiency of L. arvalis relies on its early response to the carbon source, and on the finely tuned sequential secretion of several lytic polysaccharide monooxygenases and hydrolytic enzymes targeting cellulose. The present study illustrates the adaptation of a litter-rot fungus to the rapid breakdown of recalcitrant plant biomass. The cellulolytic capabilities of this basidiomycete fungus result from the rapid, selective and successive secretion of oxidative and hydrolytic enzymes. These enzymes expressed at critical times during biomass degradation may inspire the design of improved enzyme cocktails for the conversion of plant cell wall resources into fermentable sugars.

  20. Mechanistic investigation of trimethylamine-N-oxide reduction catalysed by biomimetic molybdenum enzyme models.

    PubMed

    Fortino, M; Marino, T; Russo, N; Sicilia, E

    2016-03-28

    In this paper, we report a theoretical investigation of the reduction reaction mechanism of Me3NO using molybdenum containing systems that are functional and structural analogues of trimethylamine N-oxide reductase mononuclear molybdenum enzyme. The reactivity of the monooxomolybdenum(IV) benzenedithiolato complex and its derivatives to carbamoyl (t-BuNHCO) and acylamino (t-BuCONH) substituents on the benzene rings in both cis and trans arrangements was explored. The calculated energy profiles describing the steps of two mechanisms of attack considered viable (named cis- and trans-attack) by the Me3NO substrate at cis and trans positions with respect to the oxo ligand show that the attack on cis is energetically more favourable than the attack on trans. Along the pathway for the cis-attack the first step of the reaction, that is rate-determining for all the studied compounds, is the approach of the substrate to the Mo centre in cis to the oxo ligand that causes a distortion of the initial square-pyramidal geometry of the complex. The reaction steps involved in the trans position attack were also explored. Calculations confirm that, as previously suggested, the introduction of ligands able to form intramolecular NH···S hydrogen bonds accelerates the reduction of the Me3NO substrate and contributes to the tuning of the reactivity of molybdoenzyme models.

  1. Oxidative stress enzyme status and frequency of micronuclei in heroin addicts in Turkey.

    PubMed

    Soykut, Buğra; Eken, Ayşe; Erdem, Onur; Akay, Cemal; Aydın, Ahmet; Çetin, Mustafa Kemal; Dilbaz, Nesrin

    2013-11-01

    Heroin is among the most widely used and dangerous addictive opiate. The World Health Organization (WHO) estimated that more than 15 million people are under the influence of opiate addiction. The aim of this study was to investigate copper zinc-superoxide dismutase (Cu,Zn-SOD), catalase (CAT) and selenium-dependent glutathione peroxidase (Se-GPx) antioxidant enzyme activities, malondialdehyde (MDA) levels and the frequency of micronuclei (MN) in addicts using heroin, the most commonly abused opiate in Turkey. Addicts were defined as individuals diagnosed according to "Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV)" criteria by the "Alcohol and Substance Abuse Treatment and Education Centre-Ankara (AMATEM)". The control group had no addiction. In comparisons between the groups, a significant decrease in Cu,Zn-SOD activity and increases in MDA levels and MN frequency were observed in addicts. It can be concluded that opiates may cause oxidative stress and that antioxidant supplementation, in addition to pharmacological and psychiatric approaches, can reduce the toxicological effects of these opiates.

  2. A Thermostable Bilirubin-Oxidizing Enzyme from Activated Sludge Isolated by a Metagenomic Approach

    PubMed Central

    Kimura, Nobutada; Kamagata, Yoichi

    2016-01-01

    A gene coding for a multicopper oxidase (BopA) was identified through the screening of a metagenomic library constructed from wastewater treatment activated sludge. The recombinant BopA protein produced in Escherichia coli exhibited oxidation activity toward 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of copper, suggesting that BopA is laccase. A bioinformatic analysis of the bopA gene sequence indicated that it has a phylogenetically bacterial origin, possibly derived from a bacterium within the phylum Deinococcus-Thermus. Purified BopA exhibited maximum activity at pH 7.5 with bilirubin as its substrate and was found to be active over a markedly broad pH range from 6 to 11. It also showed notable thermostability; its activity remained intact even after a heat treatment at 90°C for 60 min. This enzyme is a thermostable-bilirubin oxidase that exhibits markedly higher thermostability than that previously reported for laccases. PMID:27885197

  3. Hydrogen peroxide-scavenging enzymes impart tolerance to high temperature induced oxidative stress in sugarcane.

    PubMed

    Srivastava, Sangeeta; Pathak, Ashwini Dutt; Gupta, Prashant Shekhar; Shrivastava, Ashok Kumar; Srivastava, Arun Kumar

    2012-05-01

    Seventy-one genotypes of sugarcane from diverse agro-climatic zones of India viz. peninsular, northwest, north-central and eastern zones, were screened for their tolerance to high temperature stress based on the damage to leaf biomass i.e. necrosis of leaf-tips and margins, and rolling of leaves. Nine selected genotypes showing variable response to heat injury were tested for activity pattern of isoforms of two H2O2-scavenging enzymes; ascorbate peroxidase (APX) and catalase (CAT), under high temperature induced oxidative stress. Changes in the activity of APX and CAT isozymes in leaves corresponded to the level of tolerance of genotypes towards heat injury which was substantiated by the highly negative correlation coefficients of heat injury levels of leaves vs. integrated density of APX and CAT isozyme bands. This indicated that the criteria of higher expression of CATs' andAPXs', the two major reactive oxygen species scavenging proteins in leaves may be used to screen large seedling populations and germplasm for high temperature tolerance.

  4. Degradation of Perfluorooctanoic Acid and Perfluoroctane Sulfonate by Enzyme Catalyzed Oxidative Humification Reactions

    NASA Astrophysics Data System (ADS)

    Huang, Q.

    2016-12-01

    Poly- and perfluoroalkyl substances (PFASs) are alkyl based chemicals having multiple or all hydrogens replaced by fluorine atoms, and thus exhibit high thermal and chemical stability and other unusual characteristics. PFASs have been widely used in a wide variety of industrial and consumer products, and tend to be environmentally persistent. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are two representative PFASs that have drawn particular attention because of their ubiquitous presence in the environment, resistance to degradation and toxicity to animals. This study examined the decomposition of PFOA and PFOS in enzyme catalyzed oxidative humification reactions (ECOHR), a class of reactions that are ubiquitous in the environment involved in natural organic humification. Reaction rates and influential factors were examined, and high-resolution mass spectrometry was used to identify possible products. Fluorides and partially fluorinated compounds were identified as likely products from PFOA and PFOS degradation, which were possibly formed via a combination of free radical decomposition, rearrangements and coupling processes. The findings suggest that PFOA and PFOS may be transformed during humification, and ECOHR can potentially be used for the remediation of these chemicals.

  5. A Thermostable Bilirubin-Oxidizing Enzyme from Activated Sludge Isolated by a Metagenomic Approach.

    PubMed

    Kimura, Nobutada; Kamagata, Yoichi

    2016-12-23

    A gene coding for a multicopper oxidase (BopA) was identified through the screening of a metagenomic library constructed from wastewater treatment activated sludge. The recombinant BopA protein produced in Escherichia coli exhibited oxidation activity toward 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of copper, suggesting that BopA is laccase. A bioinformatic analysis of the bopA gene sequence indicated that it has a phylogenetically bacterial origin, possibly derived from a bacterium within the phylum Deinococcus-Thermus. Purified BopA exhibited maximum activity at pH 7.5 with bilirubin as its substrate and was found to be active over a markedly broad pH range from 6 to 11. It also showed notable thermostability; its activity remained intact even after a heat treatment at 90°C for 60 min. This enzyme is a thermostable-bilirubin oxidase that exhibits markedly higher thermostability than that previously reported for laccases.

  6. In vitro effects of alloxan/copper combinations on lipid peroxidation, protein oxidation and antioxidant enzymes.

    PubMed

    Alexandrova, Albena; Petrov, L; Kessiova, Mila; Kirkova, Margarita

    2007-12-01

    The in vitro effects of alloxan and the product of its reduction dialuric acid (alone or in combination with copper ions) on lipid peroxidation, carbonyl content, GSH level and antioxidant enzyme activities in rat liver and kidney have been studied. The effects of Cu2+/alloxan and Cu2+/dialuric acid were compared with those of Fe3+/alloxan and Fe3+/dialuric acid. Unlike alloxan, dialuric acid increased liver and kidney lipid peroxidation; similar effects were registered in the presence of Fe3+. In the presence of Cu2+/dialuric acid, the lipid peroxidation was strongly inhibited and vice versa--the liver protein oxidation was increased. Alloxan and dialuric acid, as well as their combinations with Fe3+ had no effect on the total GSH level. Both substances did not affect the Cu2+-induced changes in GSH level, glucose-6-phosphate dehydrogenase and gluthatione reductase activities. In contrast, Cu2+ had no effect on dialuric-acid induced changes in gluthatione peroxidase and superoxide dismutase activities. The present in vitro results, concerning the metal dependence of the effects of alloxan and dialuric acid, are a premise for in vivo study of alloxan effects in metal-loaded animals.

  7. Effect of commercially available green and black tea beverages on drug-metabolizing enzymes and oxidative stress in Wistar rats.

    PubMed

    Yao, Hsien-Tsung; Hsu, Ya-Ru; Lii, Chong-Kuei; Lin, Ai-Hsuan; Chang, Keng-Hao; Yang, Hui-Ting

    2014-08-01

    The effect of commercially available green tea (GT) and black tea (BT) drinks on drug metabolizing enzymes (DME) and oxidative stress in rats was investigated. Male Wistar rats were fed a laboratory chow diet and GT or BT drink for 5 weeks. Control rats received de-ionized water instead of the tea drinks. Rats received the GT and BT drinks treatment for 5 weeks showed a significant increase in hepatic microsomal cytochrome P450 (CYP) 1A1 and CYP1A2, and a significant decrease in CYP2C, CYP2E1 and CYP3A enzyme activities. Results of immunoblot analyses of enzyme protein contents showed the same trend with enzyme activity. Significant increase in UDP-glucuronosyltransferase activity and reduced glutathione content in liver and lungs were observed in rats treated with both tea drinks. A lower lipid peroxide level in lungs was observed in rats treated with GT drink. Electrophoretic mobility shift assay revealed that both tea drinks decreased pregnane X receptor binding to DNA and increased nuclear factor-erythroid 2 p45-related factor 2 binding to DNA. These results suggest that feeding of both tea drinks to rats modulated DME activities and reduced oxidative stress in liver and lungs. GT drink is more effective on reducing oxidative stress than BT drink.

  8. Altered expression and activities of enzymes involved in thiamine diphosphate biosynthesis in Saccharomyces cerevisiae under oxidative and osmotic stress.

    PubMed

    Kowalska, Ewa; Kujda, Marta; Wolak, Natalia; Kozik, Andrzej

    2012-08-01

    Thiamine diphosphate (TDP) serves as a cofactor for enzymes engaged in pivotal carbohydrate metabolic pathways, which are known to be modulated under stress conditions to ensure the cell survival. Recent reports have proven a protective role of thiamine (vitamin B(1)) in the response of plants to abiotic stress. This work aimed at verifying a hypothesis that also baker's yeast, which can synthesize thiamine de novo similarly to plants and bacteria, adjust thiamine metabolism to adverse environmental conditions. Our analyses on the gene expression and enzymatic activity levels generally showed an increased production of thiamine biosynthesis enzymes (THI4 and THI6/THI6), a TDP synthesizing enzyme (THI80/THI80) and a TDP-requiring enzyme, transketolase (TKL1/TKL) by yeast subjected to oxidative (1 mM hydrogen peroxide) and osmotic (1 M sorbitol) stress. However, these effects differed in magnitude, depending on yeast growth phase and presence of thiamine in growth medium. A mutant thi4Δ with increased sensitivity to oxidative stress exhibited enhanced TDP biosynthesis as compared with the wild-type strain. Similar tendencies were observed in mutants yap1Δ and hog1Δ defective in the signaling pathways of the defense against oxidative and osmotic stress, respectively, suggesting that thiamine metabolism can partly compensate damages of yeast general defense systems.

  9. Angiotensin-I Converting Enzyme (ACE) Inhibitory and Anti-Oxidant Activities of Sea Cucumber (Actinopyga lecanora) Hydrolysates.

    PubMed

    Ghanbari, Raheleh; Zarei, Mohammad; Ebrahimpour, Afshin; Abdul-Hamid, Azizah; Ismail, Amin; Saari, Nazamid

    2015-12-04

    In recent years, food protein-derived hydrolysates have received considerable attention because of their numerous health benefits. Amongst the hydrolysates, those with anti-hypertensive and anti-oxidative activities are receiving special attention as both activities can play significant roles in preventing cardiovascular diseases. The present study investigated the angiotensin-I converting enzyme (ACE) inhibitory and anti-oxidative activities of Actinopyga lecanora (A. lecanora) hydrolysates, which had been prepared by alcalase, papain, bromelain, flavourzyme, pepsin, and trypsin under their optimum conditions. The alcalase hydrolysate showed the highest ACE inhibitory activity (69.8%) after 8 h of hydrolysis while the highest anti-oxidative activities measured by 2,2-diphenyl 1-1-picrylhydrazyl radical scavenging (DPPH) (56.00%) and ferrous ion-chelating (FIC) (59.00%) methods were exhibited after 24 h and 8 h of hydrolysis, respectively. The ACE-inhibitory and anti-oxidative activities displayed dose-dependent trends, and increased with increasing protein hydrolysate concentrations. Moreover, strong positive correlations between angiotensin-I converting enzyme (ACE) inhibitory and anti-oxidative activities were also observed. This study indicates that A. lecanora hydrolysate can be exploited as a source of functional food owing to its anti-oxidant as well as anti-hypertension functions.

  10. Identification of Manganese Superoxide Dismutase from Sphingobacterium sp. T2 as a Novel Bacterial Enzyme for Lignin Oxidation.

    PubMed

    Rashid, Goran M M; Taylor, Charles R; Liu, Yangqingxue; Zhang, Xiaoyang; Rea, Dean; Fülöp, Vilmos; Bugg, Timothy D H

    2015-10-16

    The valorization of aromatic heteropolymer lignin is an important unsolved problem in the development of a biomass-based biorefinery, for which novel high-activity biocatalysts are needed. Sequencing of the genomic DNA of lignin-degrading bacterial strain Sphingobacterium sp. T2 revealed no matches to known lignin-degrading genes. Proteomic matches for two manganese superoxide dismutase proteins were found in partially purified extracellular fractions. Recombinant MnSOD1 and MnSOD2 were both found to show high activity for oxidation of Organosolv and Kraft lignin, and lignin model compounds, generating multiple oxidation products. Structure determination revealed that the products result from aryl-Cα and Cα-Cβ bond oxidative cleavage and O-demethylation. The crystal structure of MnSOD1 was determined to 1.35 Å resolution, revealing a typical MnSOD homodimer harboring a five-coordinate trigonal bipyramidal Mn(II) center ligated by three His, one Asp, and a water/hydroxide in each active site. We propose that the lignin oxidation reactivity of these enzymes is due to the production of a hydroxyl radical, a highly reactive oxidant. This is the first demonstration that MnSOD is a microbial lignin-oxidizing enzyme.

  11. Effects of nitric oxide treatment on the cell wall softening related enzymes and several hormones of papaya fruit during storage.

    PubMed

    Guo, Qin; Wu, Bin; Chen, Weixin; Zhang, Yuli; Wang, Jide; Li, Xueping

    2014-06-01

    Papaya fruits (Carica papaya L. cv 'Sui you 2') harvested with < 5% yellow surface at the blossom end were fumigated with 60 microL/L of nitric oxide for 3 h and then stored at 20 degrees C with 85% relative humility for 20 days. The effects of nitric oxide treatment on ethylene production rate, the activities of cell wall softening related enzymes including polygalacturonase, pectin methyl esterase, pectate lyase and cellulase and the levels of hormones including indole acetic acid, abscisic acid, gibberellin and zeatin riboside were examined. The results showed that papaya fruits treated with nitric oxide had a significantly lower rate of ethylene production and a lesser loss of firmness during storage. A decrease in polygalacturonase, pectin methyl esterase, pectate lyase and cellulase activities was observed in nitric oxide treated fruit. In addition, the contents of indole acetic acid, abscisic acid and zeatin riboside were reduced in nitric oxide treated fruit, but no significant reduction in the level of gibberellin was found. These results indicate that nitric oxide treatment can effectively delay the softening and ripening of papaya fruit, likely via the regulation of cell wall softening related enzymes and certain hormones.

  12. Angiotensin-I Converting Enzyme (ACE) Inhibitory and Anti-Oxidant Activities of Sea Cucumber (Actinopyga lecanora) Hydrolysates

    PubMed Central

    Ghanbari, Raheleh; Zarei, Mohammad; Ebrahimpour, Afshin; Abdul-Hamid, Azizah; Ismail, Amin; Saari, Nazamid

    2015-01-01

    In recent years, food protein-derived hydrolysates have received considerable attention because of their numerous health benefits. Amongst the hydrolysates, those with anti-hypertensive and anti-oxidative activities are receiving special attention as both activities can play significant roles in preventing cardiovascular diseases. The present study investigated the angiotensin-I converting enzyme (ACE) inhibitory and anti-oxidative activities of Actinopyga lecanora (A. lecanora) hydrolysates, which had been prepared by alcalase, papain, bromelain, flavourzyme, pepsin, and trypsin under their optimum conditions. The alcalase hydrolysate showed the highest ACE inhibitory activity (69.8%) after 8 h of hydrolysis while the highest anti-oxidative activities measured by 2,2-diphenyl 1-1-picrylhydrazyl radical scavenging (DPPH) (56.00%) and ferrous ion-chelating (FIC) (59.00%) methods were exhibited after 24 h and 8 h of hydrolysis, respectively. The ACE-inhibitory and anti-oxidative activities displayed dose-dependent trends, and increased with increasing protein hydrolysate concentrations. Moreover, strong positive correlations between angiotensin-I converting enzyme (ACE) inhibitory and anti-oxidative activities were also observed. This study indicates that A. lecanora hydrolysate can be exploited as a source of functional food owing to its anti-oxidant as well as anti-hypertension functions. PMID:26690117

  13. Association of erythrocytes antioxidant enzymes and their cofactors with markers of oxidative stress in patients with sickle cell anemia

    PubMed Central

    Al-Naama, Lamia M.; Hassan, Mea'ad K.; Mehdi, Jawad K.

    2015-01-01

    Background: Sickle cell anemia (SCA) is an inherited blood disease with known complications as a result of certain pathophysiological dysfunctions. It has been suggested that an increase in oxidative stress contributes to the incidence of these changes. Objectives: This study investigated the oxidant/antioxidant status of patients with SCA, and evaluated the effect of SCA on antioxidant enzymes and their cofactors. Methods: The study included 42 patients with SCA (in steady state), and a control group of 50 age-matched individuals without SCA. Serum malondialdehyde (MDA), copper, zinc, ferritin and iron levels, red blood cell (RBC) superoxide dismutase (SOD) and catalase levels were measured for the SCA and control groups. Results: Significantly lower levels of antioxidant enzymes (RBC SOD and catalase) and higher serum MDA levels (biomarker of oxidative stress) were found in SCA patients compared to the control group (all p < 0.001). Increased levels of serum ferritin, iron and copper and decreased zinc concentrations were also found in the SCA patients compared to the control group (all p < 0.001). In the SCA group, there were significant negative correlations between MDA levels and RBC SOD, RBC catalase, and serum zinc levels (p < 0.01), while a significant positive correlation between MDA with serum copper and iron levels (p < 0.01) was observed. Conclusion: SCA is associated with alterations in markers of oxidative stress including an increased MDA level, decreased antioxidant enzyme levels, and altered levels of enzyme cofactors (zinc, copper, and iron). This suggests that these antioxidant enzymes could be used as effective therapeutic targets for the treatment of this disease and supplementation of patients with substances with antioxidant properties may reduce the complications of this disease. PMID:26835411

  14. ION COMPOSITION ELUCIDATION (ICE)

    EPA Science Inventory



    Ion Composition Elucidation (ICE) utilizes selected ion recording with a double focusing mass spectrometer to simultaneously determine exact masses and relative isotopic abundances from mass peak profiles. These can be determined more accurately and at higher sensitivity ...

  15. Potent induction of cellular antioxidants and phase 2 enzymes by resveratrol in cardiomyocytes: protection against oxidative and electrophilic injury.

    PubMed

    Cao, Zhuoxiao; Li, Yunbo

    2004-04-05

    Resveratrol is known to be protective against oxidative cardiovascular disorders. However, the underlying mechanisms remain unclear. This study was undertaken to determine if resveratrol could increase endogenous antioxidants and phase 2 enzymes in cardiomyocytes, and if such increased cellular defenses could provide protection against oxidative and electrophilic cell injury. Incubation of cardiac H9C2 cells with low micromolar resveratrol resulted in a significant induction of a scope of cellular antioxidants and phase 2 enzymes in a concentration- and/or time-dependent fashion. To investigate the protective effects of the resveratrol-induced cellular defenses on oxidative and electrophilic cell injury, H9C2 cells were first incubated with resveratrol, and then exposed to xanthine oxidase (XO)/xanthine, 4-hydroxy-2-nonenal or doxorubicin. We observed that resveratrol pretreatment afforded a marked protection against the above agent-mediated cytotoxicity in H9C2 cells. Moreover, the resveratrol pretreatment led to a great reduction in XO/xanthine-induced intracellular accumulation of ROS. Taken together, this study demonstrates that resveratrol induces antioxidants and phase 2 enzymes in cardiomyocytes, which is accompanied by increased resistance to oxidative and electrophilic cell injury.

  16. The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)+-dependent malic enzyme.

    PubMed

    Moreadith, R W; Lehninger, A L

    1984-05-25

    Little evidence has been available on the oxidative pathways of glutamine and glutamate, the major respiratory substrates of cancer cells. Glutamate formed from glutamine by phosphate-dependent glutaminase undergoes quantitative transamination by aerobic tumor mitochondria to yield aspartate. However, when malate is also added there is a pronounced decrease in aspartate production and a large formation of citrate and alanine, in both state 3 and 4 conditions. In contrast, addition of malate to normal rat heart, liver, or kidney mitochondria oxidizing glutamate causes a marked increase in aspartate production. Further analysis showed that extramitochondrial malate is oxidized almost quantitatively to pyruvate + CO2 by NAD(P)+-linked malic enzyme, present in the mitochondria of all tumors tested, but absent in heart, liver, and kidney mitochondria. On the other hand intramitochondrial malate generated from glutamate is oxidized quantitatively to oxalacetate by mitochondrial malate dehydrogenase of tumors. Acetyl-CoA derived from extramitochondrial malate via pyruvate and oxalacetate derived from glutamate via intramitochondrial malate are quantitatively converted into citrate, which is extruded. No evidence was found that malic enzyme of tumor mitochondria converts glutamate-derived malate into pyruvate as postulated in other reports. Possible mechanisms for the integration of mitochondrial malic enzyme and malate dehydrogenase activities in tumors are discussed.

  17. Molecular analysis of the distribution and phylogeny of the soxB gene among sulfur-oxidizing bacteria - evolution of the Sox sulfur oxidation enzyme system.

    PubMed

    Meyer, Birte; Imhoff, Johannes F; Kuever, Jan

    2007-12-01

    The soxB gene encodes the SoxB component of the periplasmic thiosulfate-oxidizing Sox enzyme complex, which has been proposed to be widespread among the various phylogenetic groups of sulfur-oxidizing bacteria (SOB) that convert thiosulfate to sulfate with and without the formation of sulfur globules as intermediate. Indeed, the comprehensive genetic and genomic analyses presented in the present study identified the soxB gene in 121 phylogenetically and physiologically divergent SOB, including several species for which thiosulfate utilization has not been reported yet. In first support of the previously postulated general involvement of components of the Sox enzyme complex in the thiosulfate oxidation process of sulfur-storing SOB, the soxB gene was detected in all investigated photo- and chemotrophic species that form sulfur globules during thiosulfate oxidation (Chromatiaceae, Chlorobiaceae, Ectothiorhodospiraceae, Thiothrix, Beggiatoa, Thiobacillus, invertebrate symbionts and free-living relatives). The SoxB phylogeny reflected the major 16S rRNA gene-based phylogenetic lineages of the investigated SOB, although topological discrepancies indicated several events of lateral soxB gene transfer among the SOB, e.g. its independent acquisition by the anaerobic anoxygenic phototrophic lineages from different chemotrophic donor lineages. A putative scenario for the proteobacterial origin and evolution of the Sox enzyme system in SOB is presented considering the phylogenetic, genomic (sox gene cluster composition) and geochemical data.

  18. Characterization of oxidative phosphorylation enzymes in Euglena gracilis and its white mutant strain W(gm)ZOflL.

    PubMed

    Krnáčová, Katarína; Rýdlová, Ivana; Vinarčíková, Michaela; Krajčovič, Juraj; Vesteg, Matej; Horváth, Anton

    2015-03-12

    The enzymes involved in Euglena oxidative phosphorylation (OXPHOS) were characterized in this study. We have demonstrated that Euglena gracilis strain Z and its stable bleached non-photosynthetic mutant strain WgmZOflL both possess fully functional OXPHOS apparatus as well as pathways requiring terminal alternative oxidase(s) and alternative mitochondrial NADH-dehydrogenase(s). Light (or dark) and plastid (non)functionality seem to have little effect on oxygen consumption, the activities of the enzymes involved in OXPHOS and the action of respiration inhibitors in Euglena. This study also demonstrates biochemical properties of complex III (cytochrome c reductase) in Euglena.

  19. Combining Solvent Isotope Effects with Substrate Isotope Effects in Mechanistic Studies of Alcohol and Amine Oxidation by Enzymes*

    PubMed Central

    Fitzpatrick, Paul F.

    2014-01-01

    Oxidation of alcohols and amines is catalyzed by multiple families of flavin-and pyridine nucleotide-dependent enzymes. Measurement of solvent isotope effects provides a unique mechanistic probe of the timing of the cleavage of the OH and NH bonds, necessary information for a complete description of the catalytic mechanism. The inherent ambiguities in interpretation of solvent isotope effects can be significantly decreased if isotope effects arising from isotopically labeled substrates are measured in combination with solvent isotope effects. The application of combined solvent and substrate (mainly deuterium) isotope effects to multiple enzymes is described here to illustrate the range of mechanistic insights that such an approach can provide. PMID:25448013

  20. Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase: transition from reversible to irreversible enzyme inhibition.

    PubMed

    Chen, Weiguo; Druhan, Lawrence J; Chen, Chun-An; Hemann, Craig; Chen, Yeong-Renn; Berka, Vladimir; Tsai, Ah-Lim; Zweier, Jay L

    2010-04-13

    Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular and cardiac function. Peroxynitrite (ONOO(-)) inactivates eNOS, but questions remain regarding the mechanisms of this process. It has been reported that inactivation is due to oxidation of the eNOS zinc-thiolate cluster, rather than the cofactor tetrahydrobiopterin (BH(4)); however, this remains highly controversial. Therefore, we investigated the mechanisms of ONOO(-)-induced eNOS dysfunction and their dose dependence. Exposure of human eNOS to ONOO(-) resulted in a dose-dependent loss of activity with a marked destabilization of the eNOS dimer. HPLC analysis indicated that both free and eNOS-bound BH(4) were oxidized during exposure to ONOO(-); however, full oxidation of protein-bound biopterin required higher ONOO(-) levels. Additionally, ONOO(-) triggered changes in the UV/visible spectrum and heme content of the enzyme. Preincubation of eNOS with BH(4) decreased dimer destabilization and heme alteration. Addition of BH(4) to the ONOO(-)-destabilized eNOS dimer only partially rescued enzyme function. In contrast to ONOO(-) treatment, incubation with the zinc chelator TPEN with removal of enzyme-bound zinc did not change the eNOS activity or stability of the SDS-resistant eNOS dimer, demonstrating that the dimer stabilization induced by BH(4) does not require zinc occupancy of the zinc-thiolate cluster. While ONOO(-) treatment was observed to induce loss of Zn binding, this cannot account for the loss of enzyme activity. Therefore, ONOO(-)-induced eNOS inactivation is primarily due to oxidation of BH(4) and irreversible destruction of the heme/heme center.

  1. High Throughput Enzyme Inhibitor Screening by Functionalized Magnetic Carbonaceous Microspheres and Graphene Oxide-Based MALDI-TOF-MS

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Li, Yan; Liu, Junyan; Deng, Chunhui; Zhang, Xiangmin

    2011-12-01

    In this work, a high throughput methodology for screening enzyme inhibitors has been demonstrated by combining enzyme immobilized magnetic carbonaceous microspheres and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with grapheme oxide as matrix. First, model enzyme acetylcholinesterase (AChE) was immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic carbonaceous (MC) microspheres, displaying a high enzyme activity and stability, and also facilitating the separation of enzyme from substrate and product. The efficiency of immobilized AChE was monitored by biochemical assay, which was carried out by mixing enzyme-immobilized MC microspheres with model substrate acetylcholine (ACh), and subsequent quantitative determination of substrate ACh and product choline using graphene oxide-based MALDI-TOF-MS with no background inference. The limit of detection (LOD) for ACh was 0.25 fmol/μL, and excellent linearity (R2 = 0.9998) was maintained over the range of 0.5 and 250 fmol/μL. Choline was quantified over the range of 0.05 and 15 pmol/μL, also with excellent linearity (R2 = 0.9994) and low LOD (0.15 fmol/μL). Good accuracy and precision were obtained for all concentrations within the range of the standard curves. All together, eight compounds (four known AChE inhibitors and four control chemical compounds with no AChE inhibit effect) were tested with our promoted methodology, and the obtained results demonstrated that our high throughput screening methodology could be a great help to the routine enzyme inhibitor screening.

  2. Titania-coated gold nanorods with expanded photocatalytic response. Enzyme-like glucose oxidation under near-infrared illumination.

    PubMed

    Ortega-Liebana, M C; Hueso, J L; Arenal, R; Santamaria, J

    2017-02-02

    Gold nanorods coated with a uniform titanium dioxide nanoshell have been prepared and used as glucose-oxidase surrogates. Remarkably, this core-shell photocatalytic nanostructure has been able to induce complete oxidation of glucose at near room temperature (32-34 °C) in a wide range of pH values with the aid of a near-infrared (NIR) irradiation source. In contrast, the uncoated gold nanorods exhibit negligible photo-oxidation response under identical experimental conditions thereby proving the photoactivity of the titania shell towards glucose oxidation. The process takes place via in situ photo-generation of singlet oxygen or hydroxyl radicals as reactive oxidative species (ROS). This underlines the role played by the core nanorods as plasmonic light harvesters in the NIR range and constitutes the first example of a NIR-activated enzyme-like catalyst.

  3. Radical intermediates in the catalytic oxidation of hydrocarbons by bacterial and human cytochrome P450 enzymes.

    PubMed

    Jiang, Yongying; He, Xiang; Ortiz de Montellano, Paul R

    2006-01-17

    Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.

  4. Age-related changes in renal expression of oxidant and antioxidant enzymes and oxidative stress markers in male SHR and WKY rats.

    PubMed

    Simão, Sónia; Gomes, Pedro; Pinto, Vanda; Silva, Elisabete; Amaral, João S; Igreja, Bruno; Afonso, Joana; Serrão, Maria Paula; Pinho, Maria João; Soares-da-Silva, Patrício

    2011-06-01

    Oxidative stress has been hypothesized to play a role in aging and age-related disorders, such as hypertension. This study compared levels of oxidative stress and renal expression of oxidant and antioxidant enzymes in male normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) at different ages (3 and 12 months). In the renal cortex of 3-month old SHR increases in hydrogen peroxide (H(2)O(2)) were accompanied by augmented expression of NADPH oxidase subunit Nox4 and decreased expression of antioxidant enzymes SOD1 and SOD3. A further increase in renal H(2)O(2) production and urinary TBARS was observed in 12-month old WKY and SHR as compared with 3-month old rats. Similarly, expressions of NADPH oxidase subunit p22(phox), SOD2 and SOD3 were markedly elevated with age in both strains. When compared with age-matched WKY, catalase expression was increased in 3-month old SHR, but unchanged in 12-month old SHR. Body weight increased with aging in both rat strains, but this increase was more pronounced in WKY. In conclusion, renal oxidative stress in 12-month old SHR is an exaggeration of the process already observed in the 3-month old SHR, whereas the occurrence of obesity in 12-month old normotensive rats may partially be responsible for the age-related increase in oxidative stress. Copyright © 2011. Published by Elsevier Inc.

  5. Endothelial Nitric Oxide Synthase and Angiotensin Converting Enzyme Gene Polymorphisms in Migraine Patients

    PubMed Central

    SİPAHİ, Tammam; GÜLDİKEN, Babürhan; KABAYEL, Levent; PALABIYIK, Orkide; ÖZKAN, Hülya; KILIÇ, Tülay Okman; SÜT, Necdet; TURGUT, Nilda

    2013-01-01

    Introduction In this study, we investigated the association of migraine with the Variable Number of Tandem Repeats (VNTR), repeated as 27 base pair, gene polymorphism in intron 4 of the endothelial nitric oxide synthase (eNOS) and the insertion/deletion of angiotensin converting enzyme (ACE) gene polymorphisms. Methods One hundred and five migraine and ninety seven healthy female control subjects were enrolled in the study. The patients were subdivided as migraine with aura and without aura, and the frequency and severity of migraine headaches were recorded. The eNOS VNTR (eNOS 4 a/b) and ACE insertion/deletion gene polymorphisms (ACE I/D) were assessed by polymerase chain reactions. Result The allele and genotype frequencies of eNOS 4 a/b gene polymorphism showed no difference between the migraine and control groups. The genotypic distribution of the ACE I/D gene polymorphism in the migraine group significantly differed from that in the control group. The DD and ID genotype increased the risk of migraine as much as 2.571 (95% CI-1.138–5.811) and 4.453 (95% CI-2.006–9.883) compared to the II genotype. The same increased risk sustained for both genotypes in the migraine with aura subgroup, but only the ID genotype remained as the risk factor in the migraine without aura subgroup (OR-3.750, 95% CI-1.493–9.420). No association of gene polymorphisms with migraine frequency and severity was observed. Conclusion Our findings support the relationship between migraine and the ACE I/D gene polymorphism. However, no association was found between migraine and the eNOS 4 a/b gene polymorphism.

  6. Oxidative metabolism in guinea pig ventricular myocytes protected from proteolytic enzyme activity.

    PubMed

    Bailey, L E; Carlos, H; Amian, A; Moon, K E

    1987-07-01

    Surface structures on guinea pig ventricular myocytes were protected from proteolytic enzyme activity with 100 KIU.ml-2 aprotinin during mechanical disaggregation. Intact myocytes, approximately 7.5 X 10(6) cells.g-1 ventricular wet weight, were separated from debris and damaged cells using Cytodex I tissue culture supports. Cellular ultrastructure did not differ from that observed in intact tissue. Neither spontaneous contractions nor contracture were ever observed in these myocytes in calcium concentrations of 10 mmol.litre-1. Dinitrophenol (0.2 mmol. litre-1) uncoupled respiration in the myocytes but only after the sarcolemma had been disrupted with Triton X100. The adenosine diphosphate to oxygen ratio of mitochondria isolated from the myocytes was 2.4(0.2) and the respiratory control index 2.6(0.3). Calcium (1.8 mmol.litre-1) increased oxygen uptake in the presence of 10 mmol.litre-1 pyruvate or 11 mmol.litre-1 glucose but not 17 mmol. litre-1 succinate. Succinate dependent oxygen consumption was greater than pyruvate dependent oxygen consumption (1090.0(190.0) and 40.1(0.8) nl.min-1.mg-1 protein respectively). The Crabtree effect was present. Oxidative metabolism was normal in cells stored at 10 degrees C for seven days but deteriorated rapidly thereafter. The results indicate that myocytes disaggregated by this procedure retain many of the morphological and metabolic characteristics of intact cardiac muscle cells and are relatively homogeneous with respect to calcium tolerance and metabolic function.

  7. Enzyme-polyelectrolyte multilayer assemblies on reduced graphene oxide field-effect transistors for biosensing applications.

    PubMed

    Piccinini, Esteban; Bliem, Christina; Reiner-Rozman, Ciril; Battaglini, Fernando; Azzaroni, Omar; Knoll, Wolfgang

    2017-06-15

    We present the construction of layer-by-layer (LbL) assemblies of polyethylenimine and urease onto reduced-graphene-oxide based field-effect transistors (rGO FETs) for the detection of urea. This versatile biosensor platform simultaneously exploits the pH dependency of liquid-gated graphene-based transistors and the change in the local pH produced by the catalyzed hydrolysis of urea. The use of an interdigitated microchannel resulted in transistors displaying low noise, high pH sensitivity (20.3µA/pH) and transconductance values up to 800 µS. The modification of rGO FETs with a weak polyelectrolyte improved the pH response because of its transducing properties by electrostatic gating effects. In the presence of urea, the urease-modified rGO FETs showed a shift in the Dirac point due to the change in the local pH close to the graphene surface. Markedly, these devices operated at very low voltages (less than 500mV) and were able to monitor urea in the range of 1-1000µm, with a limit of detection (LOD) down to 1µm, fast response and good long-term stability. The urea-response of the transistors was enhanced by increasing the number of bilayers due to the increment of the enzyme surface coverage onto the channel. Moreover, quantification of the heavy metal Cu(2+)(with a LOD down to 10nM) was performed in aqueous solution by taking advantage of the urease specific inhibition.

  8. Sodium chloride enhances cadmium tolerance through reducing cadmium accumulation and increasing anti-oxidative enzyme activity in tobacco.

    PubMed

    Zhang, Bing-Lin; Shang, Sheng-Hua; Zhang, Hai-Tao; Jabeen, Zahra; Zhang, Guo-Ping

    2013-06-01

    The effect of sodium chloride (NaCl) on cadmium (Cd) uptake, translocation, and oxidative stress was investigated using 2 tobacco cultivars differing in Cd tolerance. The growth inhibition of the tobacco plants exposed to Cd toxicity was in part alleviated by moderate addition of NaCl in the culture solution. Cadmium concentration of shoots and roots in the 2 cultivars increased with increasing Cd levels in the solution and decreased with the addition of NaCl. The addition of NaCl could alleviate the oxidative stress caused by Cd toxicity, as reflected by reduced production of malondialdehyde and recovered or enhanced activities of antioxidative enzymes catalase and glutathione peroxidase. The results also showed that the enhancement of antioxidative enzyme activity by NaCl for the tobacco plants exposed to Cd stress is related to induced Ca signaling. Copyright © 2013 SETAC.

  9. Combining solvent isotope effects with substrate isotope effects in mechanistic studies of alcohol and amine oxidation by enzymes.

    PubMed

    Fitzpatrick, Paul F

    2015-11-01

    Oxidation of alcohols and amines is catalyzed by multiple families of flavin- and pyridine nucleotide-dependent enzymes. Measurement of solvent isotope effects provides a unique mechanistic probe of the timing of the cleavage of the OH and NH bonds, necessary information for a complete description of the catalytic mechanism. The inherent ambiguities in interpretation of solvent isotope effects can be significantly decreased if isotope effects arising from isotopically labeled substrates are measured in combination with solvent isotope effects. The application of combined solvent and substrate (mainly deuterium) isotope effects to multiple enzymes is described here to illustrate the range of mechanistic insights that such an approach can provide. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment.

  10. Isolation and characterization of a 4-nitrotoluene-oxidizing enzyme from activated sludge by a metagenomic approach.

    PubMed

    Kimura, Nobutada; Sakai, Kayoko; Nakamura, Kazunori

    2010-01-01

    To isolate a biocatalytic enzyme, metagenomic libraries were constructed in fosmids from samples of activated sludge used to treat coke plant wastewater. Six indigo-producing clones were isolated from approximately 40,000 metagenomic clones in the search for the oxygenase responsible. In vitro mutagenesis and whole-sequencing revealed one open reading frame to be responsible for the production of indigo in the fosmid clones. The deduced sequence of the gene product showed 60% identity with 2-naphthoate monooxygenase from Burkholderia sp. JT1500. Subclones carrying this open reading frame (icpA) retained indigo production, and indigo-producing enzymes expressed from subclones catalyzed the oxidization of 4-nitrotoluene to form 4-nitrobenzyl alcohol. These results suggested that the icp product is an enzyme involved in catalyzing 4-nitrotoluene's oxygenation.

  11. Influence of UV-A radiation on oxidative stress and antioxidant enzymes in Mythimna separata (Lepidoptera: Noctuidae).

    PubMed

    Ali, Arif; Rashid, Muhammad Adnan; Huang, Qiu Ying; Lei, Chao-Liang

    2017-03-01

    Abiotic stress factors, including ultraviolet (UV) radiation, significantly affect insect life. UV-A radiation (320-400 nm) has been widely used for insect control since it increases the production of ROS and causes oxidative cell damage. In the present study, we evaluated the effects of UV-A irradiation on an important pest in China, the ear-cutting caterpillar, Mythimna separata (Lepidoptera: Noctuidae). We exposed 3-day-old M. separata adults to UV-A radiation for different periods of time (0, 30, 60, 90, and 120 min) and evaluated the resulting total antioxidant capacity and the activity of the antioxidant enzymes superoxide dismutase, catalase, peroxidase, and glutathione-S-transferase. The total antioxidant capacity significantly increased after exposure to UV-A radiation for 60 min but decreased after 90 and 120 min of exposure, compared with the control. The antioxidant activity of glutathione-S-transferase, superoxide dismutase, catalase, and peroxidase increased after 60-min exposure, and it was decreased at the longest exposure period 120 min. The longest exposure time period relatively activates the xenobiotic detoxifying enzymes like glutathione-S-transferase, superoxide dismutase, catalase, and peroxidase enzymes. The longest duration of UV-A radiation may cooperate with pesticide detoxification mechanism in insects, making them more susceptible to insecticides. Our results demonstrated that UV irradiation causes oxidative stress, affects the activity of antioxidant enzymes, and disturbs the physiology of M. separata adults.

  12. Mechanistic aspects of CYP74 allene oxide synthases and related cytochrome P450 enzymes

    PubMed Central

    Brash, Alan R.

    2009-01-01

    The existence of CYP5, CYP8A, and the CYP74 enzymes specialized for reaction with fatty acid peroxide substrates presents opportunities for a “different look” at the catalytic cycle of the cytochrome P450s. This review considers how the properties of the peroxide-metabolizing enzymes are distinctive, and how they tie in with those of the conventional monooxygenase enzymes. Some unusual reactions of each class have parallels in the other. As new enzyme reactions and new P450 structures emerge there will be possibilities for finding their special properties and edging this knowledge into the big picture. PMID:19747698

  13. Novel Fluorescence Arginine Analogue as a Sensor for Direct Identification and Imaging of Nitric Oxide Synthase-like Enzymes in Plants

    PubMed Central

    Chang, Kang; Guo, Tongtong; Li, Pengfei; Liu, Yin; Xu, Yufang; Fang, Yuda; Qian, Xuhong

    2016-01-01

    Nitric oxide synthase like enzyme (NOS-like enzyme), which produces nitric oxide, participates in many biological processes. However it remains unidentified and highly controversial that plants do possess a NOS-like enzyme. In this paper, a novel arginine analogue NP1 was designed and developed for the direct identification and real time tracking of NOS-like enzymes in plant by fluorescence sensing. It could bind NOS-like enzyme efficiently and enter the cell successfully. In vivo fluorescence response results directly proved that NOS-like enzymes did exist in tobacco leaf and would be stimulated by pathogen infection, which also provided a useful chemical tool for the study of the function of NOS-like enzyme in plants. PMID:27586270

  14. Trace Metal Requirements for Microbial Enzymes Involved in the Production and Consumption of Methane and Nitrous Oxide

    PubMed Central

    Glass, Jennifer B.; Orphan, Victoria J.

    2011-01-01

    Fluxes of greenhouse gases to the atmosphere are heavily influenced by microbiological activity. Microbial enzymes involved in the production and consumption of greenhouse gases often contain metal cofactors. While extensive research has examined the influence of Fe bioavailability on microbial CO2 cycling, fewer studies have explored metal requirements for microbial production and consumption of the second- and third-most abundant greenhouse gases, methane (CH4), and nitrous oxide (N2O). Here we review the current state of biochemical, physiological, and environmental research on transition metal requirements for microbial CH4 and N2O cycling. Methanogenic archaea require large amounts of Fe, Ni, and Co (and some Mo/W and Zn). Low bioavailability of Fe, Ni, and Co limits methanogenesis in pure and mixed cultures and environmental studies. Anaerobic methane oxidation by anaerobic methanotrophic archaea (ANME) likely occurs via reverse methanogenesis since ANME possess most of the enzymes in the methanogenic pathway. Aerobic CH4 oxidation uses Cu or Fe for the first step depending on Cu availability, and additional Fe, Cu, and Mo for later steps. N2O production via classical anaerobic denitrification is primarily Fe-based, whereas aerobic pathways (nitrifier denitrification and archaeal ammonia oxidation) require Cu in addition to, or possibly in place of, Fe. Genes encoding the Cu-containing N2O reductase, the only known enzyme capable of microbial N2O conversion to N2, have only been found in classical denitrifiers. Accumulation of N2O due to low Cu has been observed in pure cultures and a lake ecosystem, but not in marine systems. Future research is needed on metalloenzymes involved in the production of N2O by enrichment cultures of ammonia oxidizing archaea, biological mechanisms for scavenging scarce metals, and possible links between metal bioavailability and greenhouse gas fluxes in anaerobic environments where metals may be limiting due to sulfide

  15. Elucidation of the 1,3-sulfanylalcohol oxidation mechanism: an unusual identification of the disulfide of 3-sulfanylhexanol in sauternes botrytized wines.

    PubMed

    Sarrazin, Elise; Shinkaruk, Svitlana; Pons, Monique; Thibon, Cecile; Bennetau, Bernard; Darriet, Philippe

    2010-10-13

    A four-step purification method was developed to isolate a citrus odorant detected by gas chromatography-olfactometry (GC-O), which was apparently specific to Sauternes botrytized wines. A fragmentation pattern of the odorant was obtained by multidimensional gas chromatography-mass spectrometry-olfactometry (MDGC-MS-O). The exact mass measurement was used to determine its elemental formula as C(6)H(12)OS. On the basis of these data, the unusual structure of 3-propyl-1,2-oxathiolane was synthesized and characterized for the first time. This confirmed its identification. Its occurrence in Sauternes wine extracts was demonstrated to result from the thermal oxidative degradation of 3-sulfanylhexanol disulfide (3,3'-disulfanediyldihexan-1-ol) in the GC injector. This disulfide was synthesized and then firmly identified for the first time in Sauternes wine. Although the presence of 3-sulfanylhexanol oxidation products had previously been reported in natural extracts (but not wine), the full oxidation pathway from 3-sulfanylhexanol to 3-propyl-γ-sultine via 3,3'-disulfanediyldihexan-1-ol was clearly established for the first time. Because the disulfide has mainly been detected in Sauternes botrytized wines, this finding suggested a singular reactivity of 3-sulfanylhexanol in botrytized wines, thus opening up a wide range of new opportunities in wine chemistry.

  16. Measuring Intracellular Enzyme Concentrations: Assessing the Effect of Oxidative Stress on the Amount of Glyoxalase I

    ERIC Educational Resources Information Center

    Miranda, Hugo Vicente; Ferreira, Antonio E. N.; Quintas, Alexandre; Cordeiro, Carlos; Freire, Ana Ponces

    2008-01-01

    Enzymology is one of the fundamental areas of biochemistry and involves the study of the structure, kinetics, and regulation of enzyme activity. Research in this area is often conducted with purified enzymes and extrapolated to "in vivo" conditions. The specificity constant, k[subscript S], is the ratio between k[subscript cat] (the catalytic…

  17. Ultrasound-assisted extraction and characterization of hydrolytic and oxidative enzymes produced by solid state fermentation.

    PubMed

    Szabo, Orsolya Erzsebet; Csiszar, Emilia; Toth, Karolina; Szakacs, George; Koczka, Bela

    2015-01-01

    Ligninolytic and hydrolytic enzymes were produced with six selected fungi on flax substrate by solid state fermentation (SSF). The extracellular enzyme production of the organisms in two SSF media was evaluated by measuring the soluble protein concentration and the filter paper, endoxylanase, 1,4-β-d-glucosidase, 1,4-β-d-endoglucanase, polygalacturonase, lignin peroxidase, manganese peroxidase and laccase activities of the clear culture solutions produced by conventional extraction from the SSF materials. The SSF material of the best enzyme producer (Trichoderma virens TUB F-498) was further investigated to enhance the enzyme recovery by low frequency ultrasound treatment. Performance of both the original and ultrasound macerated crude enzyme mixtures was evaluated in degradation of the colored lignin-containing and waxy materials of raw linen fabric. Results proved that sonication (at 40%, 60% and 80% amplitudes, for 60min) did not result in reduction in the filter paper, lignin peroxidase and laccase activities of the crude enzyme solution, but has a significant positive effect on the efficiency of enzyme extraction from the SSF material. Depending on the parameters of sonication, the enzyme activities in the extracts obtained can be increased up to 129-413% of the original activities measured in the control extracts recovered by a common magnetic stirrer. Sonication also has an effect on both the enzymatic removal of the lignin-containing color materials and hydrophobic surface layer from the raw linen. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Measuring Intracellular Enzyme Concentrations: Assessing the Effect of Oxidative Stress on the Amount of Glyoxalase I

    ERIC Educational Resources Information Center

    Miranda, Hugo Vicente; Ferreira, Antonio E. N.; Quintas, Alexandre; Cordeiro, Carlos; Freire, Ana Ponces

    2008-01-01

    Enzymology is one of the fundamental areas of biochemistry and involves the study of the structure, kinetics, and regulation of enzyme activity. Research in this area is often conducted with purified enzymes and extrapolated to "in vivo" conditions. The specificity constant, k[subscript S], is the ratio between k[subscript cat] (the catalytic…

  19. Structural elucidation of monoterpene oxidation products by ion trap fragmentation using on-line atmospheric pressure chemical ionisation mass spectrometry in the negative ion mode.

    PubMed

    Warscheid, B; Hoffmann, T

    2001-01-01

    Based on ion trap mass spectrometry, an on-line method is described which provides valuable information on the molecular composition of structurally complex organic aerosols. The investigated aerosols were generated from the gas-phase ozonolysis of various C(10)H(16)-terpenes (alpha-pinene, beta-pinene, 3-carene, sabinene, limonene), and directly introduced into the ion source of the mass spectrometer. Negative ion chemical ionisation at atmospheric pressure (APCI(-)) enabled the detection of multifunctional carboxylic acid products by combining inherent sensitivity and molecular weight information. Sequential low-energy collision-induced product ion fragmentation experiments (MS(n)) were performed in order to elucidate characteristic decomposition pathways of the compounds. Dicarboxylic acids, oxocarboxylic acids and hydroxyketocarboxylic acid products could be clearly distinguished by multistage on-line MS. Furthermore, sabinonic acid and two C(9)-ether compounds were tentatively identified for the first time by applying on-line APCI(-)-MS(n). Copyright 2001 John Wiley & Sons, Ltd.

  20. Edaravone Mitigates Hexavalent Chromium-Induced Oxidative Stress and Depletion of Antioxidant Enzymes while Estrogen Restores Antioxidant Enzymes in the Rat Ovary in F1 Offspring1

    PubMed Central

    Stanley, Jone A.; Sivakumar, Kirthiram K.; Arosh, Joe A.; Burghardt, Robert C.; Banu, Sakhila K.

    2014-01-01

    ABSTRACT Environmental contamination of drinking water with chromium (Cr) has been increasing in more than 30 cities in the United States. Previous studies from our group have shown that Cr affects reproductive functions in female Sprague Dawley rats. Although it is impossible to completely remove Cr from the drinking water, it is imperative to develop effective intervention strategies to inhibit Cr-induced deleterious health effects. Edaravone (EDA), a potential inhibitor of free radicals, has been clinically used to treat cancer and cardiac ischemia. This study evaluated the efficacy of EDA against Cr-induced ovarian toxicity. Results showed that maternal exposure to CrVI in rats increased follicular atresia, decreased steroidogenesis, and delayed puberty in F1 offspring. CrVI increased oxidative stress and decreased antioxidant (AOX) enzyme levels in the ovary. CrVI increased follicle atresia by increased expression of cleaved caspase 3, and decreased expression of Bcl2 and Bcl2l1 in the ovary. EDA mitigated or inhibited the effects of CrVI on follicle atresia, pubertal onset, steroid hormone levels, and AOX enzyme activity, as well as the expression of Bcl2 and Bcl2l1 in the ovary. In a second study, CrVI treatment was withdrawn, and F1 rats were injected with estradiol (E2) (10 μg in PBS/ethanol per 100 g body weight) for a period of 2 wk to evaluate whether E2 treatment will restore Cr-induced depletion of AOX enzymes. E2 restored CrVI-induced depletion of glutathione peroxidase 1, catalase, thioredoxin 2, and peroxiredoxin 3 in the ovary. This is the first study to demonstrate the protective effects of EDA against any toxicant in the ovary. PMID:24804965

  1. Edaravone mitigates hexavalent chromium-induced oxidative stress and depletion of antioxidant enzymes while estrogen restores antioxidant enzymes in the rat ovary in F1 offspring.

    PubMed

    Stanley, Jone A; Sivakumar, Kirthiram K; Arosh, Joe A; Burghardt, Robert C; Banu, Sakhila K

    2014-07-01

    Environmental contamination of drinking water with chromium (Cr) has been increasing in more than 30 cities in the United States. Previous studies from our group have shown that Cr affects reproductive functions in female Sprague Dawley rats. Although it is impossible to completely remove Cr from the drinking water, it is imperative to develop effective intervention strategies to inhibit Cr-induced deleterious health effects. Edaravone (EDA), a potential inhibitor of free radicals, has been clinically used to treat cancer and cardiac ischemia. This study evaluated the efficacy of EDA against Cr-induced ovarian toxicity. Results showed that maternal exposure to CrVI in rats increased follicular atresia, decreased steroidogenesis, and delayed puberty in F1 offspring. CrVI increased oxidative stress and decreased antioxidant (AOX) enzyme levels in the ovary. CrVI increased follicle atresia by increased expression of cleaved caspase 3, and decreased expression of Bcl2 and Bcl2l1 in the ovary. EDA mitigated or inhibited the effects of CrVI on follicle atresia, pubertal onset, steroid hormone levels, and AOX enzyme activity, as well as the expression of Bcl2 and Bcl2l1 in the ovary. In a second study, CrVI treatment was withdrawn, and F1 rats were injected with estradiol (E₂) (10 μg in PBS/ethanol per 100 g body weight) for a period of 2 wk to evaluate whether E₂ treatment will restore Cr-induced depletion of AOX enzymes. E₂ restored CrVI-induced depletion of glutathione peroxidase 1, catalase, thioredoxin 2, and peroxiredoxin 3 in the ovary. This is the first study to demonstrate the protective effects of EDA against any toxicant in the ovary.

  2. Characteristics of the nitric oxide synthase-catalyzed conversion of arginine to N-hydroxyarginine, the first oxygenation step in the enzymic synthesis of nitric oxide.

    PubMed

    Campos, K L; Giovanelli, J; Kaufman, S

    1995-01-27

    The nitric oxide synthase-catalyzed conversion of L-arginine to L-citrulline and nitric oxide is known to be the sum of two partial reactions: oxygenation of arginine to N-hydroxyarginine, followed by oxygenation of N-hydroxyarginine to citrulline and nitric oxide. Whereas the conversion of N-hydroxyarginine to citrulline and nitric oxide has been the subject of a number of studies, the oxygenation of arginine to N-hydroxyarginine has received little attention. Here we show that substrate amounts of rat cerebellar nitric oxide synthase, in the absence of added NADPH, catalyze the conversion of arginine to N-hydroxyarginine as the dominant product. The product appears not to be tightly bound to the enzyme. A maximum of 0.16 mol of N-hydroxyarginine/mol of nitric oxide synthase subunit was formed. The reaction requires oxygen and the addition of Ca2+/calmodulin and is stimulated 3-fold by tetrahydrobiopterin. Upon addition of NADPH, citrulline is formed exclusively. Conversion of N-hydroxyarginine to citrulline, like the first partial reaction, requires Ca2+/calmodulin and is stimulated by tetrahydrobiopterin but differs from the first partial reaction in being completely dependent upon addition of NADPH. These results indicate that brain nitric oxide synthase contains an endogenous reductant that can support oxygenation of arginine but not of N-hydroxyarginine. The reductant is not NADPH, since the amount of nitric oxide synthase-bound NADPH is appreciably less than the amount required for N-hydroxyarginine synthesis. Possible candidates for this role are discussed in relation to proposed mechanisms of action of nitric oxide synthase.

  3. Increased Oxidative Stress and Imbalance in Antioxidant Enzymes in the Brains of Alloxan-Induced Diabetic Rats

    PubMed Central

    Ceretta, Luciane B.; Réus, Gislaine Z.; Abelaira, Helena M.; Ribeiro, Karine F.; Zappellini, Giovanni; Felisbino, Francine F.; Steckert, Amanda V.; Dal-Pizzol, Felipe; Quevedo, João

    2012-01-01

    Diabetes Mellitus (DM) is associated with pathological changes in the central nervous system (SNC) as well as alterations in oxidative stress. Thus, the main objective of this study was to evaluate the effects of the animal model of diabetes induced by alloxan on memory and oxidative stress. Diabetes was induced in Wistar rats by using a single injection of alloxan (150 mg/kg), and fifteen days after induction, the rats memory was evaluated through the use of the object recognition task. The oxidative stress parameters and the activity of antioxidant enzymes, superoxide dismutase (SOD), and catalase (CAT) were measured in the rat brain. The results showed that diabetic rats did not have alterations in their recognition memory. However, the results did show that diabetic rats had increases in the levels of superoxide in the prefrontal cortex, and in thiobarbituric acid reactive species (TBARS) production in the prefrontal cortex and in the amygdala in submitochondrial particles. Also, there was an increase in protein oxidation in the hippocampus and striatum, and in TBARS oxidation in the striatum and amygdala. The SOD activity was decreased in diabetic rats in the striatum and amygdala. However, the CAT activity was increased in the hippocampus taken from diabetic rats. In conclusion, our findings illustrate that the animal model of diabetes induced by alloxan did not cause alterations in the animals' recognition memory, but it produced oxidants and an imbalance between SOD and CAT activities, which could contribute to the pathophysiology of diabetes. PMID:22645603

  4. Pyrosequencing Reveals Soil Enzyme Activities and Bacterial Communities Impacted by Graphene and Its Oxides.

    PubMed

    Rong, Yan; Wang, Yi; Guan, Yina; Ma, Jiangtao; Cai, Zhiqiang; Yang, Guanghua; Zhao, Xiyue

    2017-10-10

    Graphene (GN) and graphene oxides (GOs) are novel carbon nanomaterial; they have been attracting much attention because of their excellent properties and are widely applied in many areas, including energy, electronics, biomedicine, environmental science, etc. With industrial production and consumption of GN/GO, they will inevitably enter the soil and water environments. GN/GO may directly cause certain harm to microorganisms and lead to ecological and environmental risks. GOs are GN derivatives with abundant oxygen-containing functional groups in their graphitic backbone. The structure and chemistry of GN show obvious differences compared to those of GO, which lead to the different environmental behaviors. In this study, four different types of soil (S1-S4) were employed to investigate the effect of GN and GO on soil enzymatic activity, microbial population, and bacterial community through pyrosequencing of 16S rRNA gene amplicons. The results showed that soil enzyme activity (invertase, protease, catalase, and urease) and microbial population (bacteria, actinomycetes, and fungi) changed after GN/GO release into soils. Soil microbial community species are more rich, and the diversity also increases after GO/GN application. The phylum of Proteobacteria increased at 90 days after treatment (DAT) after GN/GO application. The phylum of Chloroflexi occurred after GN application at 90 DAT in S1 soil and reached 4.6%. Proteobacteria was the most abundant phylum in S2, S3, and S4 soils; it ranged from 43.6 to 71.4% in S2 soil, from 45.6 to 73.7% in S3 soil, and from 38.1 to 56.7% in S4 soil. The most abundant genera were Bacillus (37.5-47.0%) and Lactococcus (28.0-39.0%) in S1 soil, Lysobacter and Flavobacterium in S2 soil, Pedobacter in S3 soil, and Massilia in S4 soil. The effect of GN and GO on the soil microbial community is time-dependent, and there are no significant differences between the samples at 10 and 90 DAT.

  5. Different enzymes are involved in anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation in Thiobacillus denitrificans

    NASA Astrophysics Data System (ADS)

    Zhou, P.; Beller, H. R.

    2011-12-01

    Thiobacillus denitrificans is a widespread, obligate chemolithoautotrophic bacterium that is capable of anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation. Both of these processes can mediate the mobility of uranium in contaminated aquifers and thereby influence the long-term efficacy of in situ reductive immobilization of uranium at DOE sites. T. denitrificans has been found at uranium-contaminated sites, including a contaminated aquifer at Oak Ridge National Laboratory. We previously reported that two membrane-associated, diheme, c-type cytochromes (a c4 cytochrome, Tbd_0187, and a c5 cytochrome, Tbd_0146) were involved in nitrate-dependent U(IV) oxidation in T. denitrificans. To date, these are the only genes identified to be involved in this process. In this poster, we report on work with T. denitrificans focused on determining whether the enzymes that were demonstrated to be involved in anaerobic, nitrate-dependent U(IV) oxidation are also involved in nitrate-dependent Fe(II) oxidation. Using a genetic system in T. denitrificans that enables us to create insertion mutants and complement them in trans, we constructed a series of insertion mutants. These included strains with mutations in the genes known to be associated with U(IV) oxidation (Tbd_0146 and Tbd_0187) as well as other genes encoding membrane-associated c-type cytochromes (a group of proteins that we hypothesize to be catalyzing Fe(II) oxidation). Anaerobic cell suspension assays were carried out to determine whether any of these mutants were defective in nitrate-dependent Fe(II) oxidation. We observed that the Tbd_0146 and Tbd_0187 mutants were not defective in nitrate-dependent Fe(II) oxidation, nor were any of the other c-type cytochrome mutants tested (including a Tbd_0146-Tbd_0187 double mutant). The finding that different enzymes are associated with nitrate-dependent Fe(II) and U(IV) oxidation has led us to pursue genome-wide studies in T. denitrificans to determine the genes associated

  6. 2-Benzoxazolinone (BOA) induced oxidative stress, lipid peroxidation and changes in some antioxidant enzyme activities in mung bean (Phaseolus aureus).

    PubMed

    Batish, D R; Singh, H P; Setia, N; Kaur, S; Kohli, R K

    2006-01-01

    2-Benzoxazolinone (BOA), a well-known allelochemical with strong phytotoxicity, is a potential herbicidal candidate. The aim of the present study was to determine whether phytotoxicity of BOA is due to induction of oxidative stress caused by generation of reactive oxygen species (ROS) and the changes in levels of antioxidant enzymes induced in response to BOA. Effect of BOA was studied on electrolyte leakage, lipid peroxidation (LP), hydrogen peroxide (H(2)O(2)) generation, proline (PRO) accumulation, and activities of antioxidant enzymes-superoxide dismutase (SOD, 1.15.1.1), ascorbate peroxidase (APX, 1.11.1.11), guaiacol peroxidase (GPX, 1.11.1.7), catalase (CAT, 1.11.1.6) and glutathione reductase (GR, 1.6.4.2) in Phaseolus aureus (mung bean). BOA significantly enhanced malondialdehyde (MDA) content, a product of LP, in both leaves and roots of mung bean. The amount of H(2)O(2), a product of oxidative stress, and endogenous PRO increased many-fold in response to BOA. Accumulation of PRO, MDA and H(2)O(2) indicates the cellular damage in the target tissue caused by ROS generated by BOA. In response to BOA, there was a significant increase in the activities of scavenging enzymes SOD, APX, GPX, CAT, and GR in root and leaf tissue of mung bean. At 5 mM BOA, GR activity in roots showed a nearly 22-fold increase over that in control. The present study concludes that BOA induces oxidative stress in mung bean through generation of ROS and upregulation of activities of various scavenging enzymes.

  7. Antioxidant Enzyme Activity, Iron Content and Lipid Oxidation of Raw and Cooked Meat of Korean Native Chickens and Other Poultry

    PubMed Central

    Muhlisin; Utama, Dicky Tri; Lee, Jae Ho; Choi, Ji Hye; Lee, Sung Ki

    2016-01-01

    This study was conducted to observe antioxidant enzyme activity, iron content and lipid oxidation of Korean native chickens and other poultry. The breast and thigh meat of three Korean native chicken breeds including Woorimatdak, Hyunin black and Yeonsan ogye, and three commercial poultry breeds including the broiler, White Leghorn and Pekin duck (Anasplatyrhyncos domesticus) were studied. The analyses of the antioxidant enzymes activity, iron content and lipid oxidation were performed in raw and cooked samples. The activity of catalase (CAT) in the thigh meat was higher than that of the breast meat of three Korean native chickens and the broiler, respectively. The activity of glutathione peroxidase (GPx) in the uncooked thigh meat of three Korean native chickens was higher than that of the breasts. The breast meat of Woorimatdak and Pekin duck had higher superoxide dismutase (SOD) activity than the others, while only the thigh meat of Pekin duck had the highest activity. Cooking inactivated CAT and decreased the activity of GPx and SOD. The thigh meat of Woorimatdak, White Leghorn, Yeonsan ogye and Hyunin black contained more total iron than the breast meat of those breeds. The heme-iron lost during cooking ranged from 3.2% to 14.8%. It is noted that the thigh meat had higher thiobarbituric acid reactive substances values than the breast in all chicken breeds. Though Woorimatdak showed higher antioxidant enzyme activity and lower released-iron percentage among Korean native chickens, no differences were found on lipid oxidation. We confirm that the dark meat of poultry exhibited higher antioxidant enzyme activity and contained more iron than the white meat. PMID:26954148

  8. Induction of phase 2 enzymes by serum oxidized polyamines through activation of Nrf2: effect of the polyamine metabolite acrolein.

    PubMed

    Kwak, Mi-Kyoung; Kensler, Thomas W; Casero, Robert A

    2003-06-06

    The naturally occurring polycationic polyamines including putrescine, spermidine, and spermine play an important role in cell growth, differentiation, and gene expression. However, circulating polyamines are potential substrates for several oxidizing enzymes including copper-containing serum amine oxidase. These enzymes are capable of oxidizing serum polyamines to several toxic metabolites including aldehydes and H(2)O(2). In this study, we investigated the effects of polyamines as inducers of phase 2 enzymes and other genes that promote cell survival in a cell culture system in the presence of bovine serum. Spermidine and spermine (50 microM) increased NAD(P)H quinone oxidoreductase (NQO1) activity up to 3-fold in murine keratinocyte PE cells. Transcript levels for glutathione S-transferase (GST) A1, GST M1, NQO1, gamma-glutamylcysteine ligase regulatory subunit, and UDP-glucuronyltransferase 1A6 were significantly increased by spermidine and this effect was mediated through the antioxidant response element (ARE). The ARE from the mouse GST A1 promoter was activated about 9-fold by spermine and 5-fold by spermidine treatment, but could be inhibited by the amine oxidase inhibitor, aminoguanidine, suggesting that acrolein or hydrogen peroxide generated from polyamines by serum amine oxidase may be mediators for phase 2 enzyme induction. Elevations of ARE-luciferase expression and NQO1 enzyme activity by spermidine were not affected by catalase, while both were completely repressed by aldehyde dehydrogenase treatment. Direct addition of acrolein to PE cells induced multiple phase 2 genes and elevated nuclear levels of Nrf2, a transcription factor that binds to the ARE. Expression of mutant Nrf2 repressed the activation of the ARE-luciferase reporter by polyamines and acrolein. These results indicate that spermidine and spermine increase the expression of phase 2 genes in cells grown in culture through activation of the Nrf2-ARE pathway by generating the sulfhydryl

  9. Transformation products of pharmaceuticals in surface waters and wastewater formed during photolysis and advanced oxidation processes - degradation, elucidation of byproducts and assessment of their biological potency.

    PubMed

    Fatta-Kassinos, D; Vasquez, M I; Kümmerer, K

    2011-10-01

    The significance of transformation products of pharmaceuticals resulting from the parent compounds during natural and technical photolytic processes and advanced oxidation processes has only recently started to attract the interest of the scientific community. Even though relevant studies have now started to produce important knowledge, still many gaps exist that hinder the in-depth and broad understanding of the extent of the potential problems stemming from the presence of such compounds in the environment and the applicability of such techniques for wastewater and potable water treatment. The great diversity of pharmaceutical compounds, the variety of processes and conditions applied by the various research groups active in the field, and the endless list of potential biological endpoints that could potentially be explored, coupled with the limitations related to the analytical capabilities presently available, are some of the crucial parameters that characterize this challenging research direction. This review paper tries to highlight some of the most relevant studies performed so far and to summarize the parameters that prevent scientists from reaching comprehensive conclusions in relation to the formation, fate, and effects of transformation products of pharmaceutical compounds during photo-driven and advanced oxidation processes.

  10. Serum antioxidant enzymes activities and oxidative stress levels in patients with acute ischemic stroke: influence on neurological status and outcome.

    PubMed

    Milanlioglu, Aysel; Aslan, Mehmet; Ozkol, Halil; Çilingir, Vedat; Nuri Aydın, Mehmet; Karadas, Sevdegül

    2016-03-01

    Oxidative stress is well believed to play a role in the pathogenesis of acute ischemic stroke. Reports on antioxidant enzyme activities in patients with stroke are conflicting. Therefore, the aim of this study was to investigate serum antioxidant enzyme activities and oxidative stress levels in patients with acute ischemic stroke within 1st, 5th, and 21st day after stroke onset and also the relationship between these results and the clinical status of patients. The current study comprised 45 patients with acute ischemic stroke and 30 healthy controls. Serum malondialdehyde (MDA) levels, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase activities were measured spectrophotometrically. Serum MDA levels were significantly higher in acute ischemic stroke patients within 24 h after stroke onset than controls (p < 0.05), whereas serum catalase activity was significantly lower (p < 0.05). There were no significant differences in GSH-Px and SOD activities. Serum catalase and SOD activities were significantly lower in fifth day than those of controls (both, p < 0.05) but GSH-Px activity and MDA levels did not change (p > 0.05). Serum SOD activity was significantly lower in 21st day compared to SOD activity of controls (p < 0.05) but MDA levels, GSH-Px, and CAT activities did not change significantly. Our study demonstrated that acute ischemic stroke patients have increased oxidative stress and decreased antioxidant enzymes activities. These findings indicated that an imbalance of oxidant and antioxidant status might play a role in the pathogenesis of acute ischemic stroke.

  11. The Impact of Non-Enzymatic Reactions and Enzyme Promiscuity on Cellular Metabolism during (Oxidative) Stress Conditions.

    PubMed

    Piedrafita, Gabriel; Keller, Markus A; Ralser, Markus

    2015-09-10

    Cellular metabolism assembles in a structurally highly conserved, but functionally dynamic system, known as the metabolic network. This network involves highly active, enzyme-catalyzed metabolic pathways that provide the building blocks for cell growth. In parallel, however, chemical reactivity of metabolites and unspecific enzyme function give rise to a number of side products that are not part of canonical metabolic pathways. It is increasingly acknowledged that these molecules are important for the evolution of metabolism, affect metabolic efficiency, and that they play a potential role in human disease-age-related disorders and cancer in particular. In this review we discuss the impact of oxidative and other cellular stressors on the formation of metabolic side products, which originate as a consequence of: (i) chemical reactivity or modification of regular metabolites; (ii) through modifications in substrate specificity of damaged enzymes; and (iii) through altered metabolic flux that protects cells in stress conditions. In particular, oxidative and heat stress conditions are causative of metabolite and enzymatic damage and thus promote the non-canonical metabolic activity of the cells through an increased repertoire of side products. On the basis of selected examples, we discuss the consequences of non-canonical metabolic reactivity on evolution, function and repair of the metabolic network.

  12. The Impact of Non-Enzymatic Reactions and Enzyme Promiscuity on Cellular Metabolism during (Oxidative) Stress Conditions

    PubMed Central

    Piedrafita, Gabriel; Keller, Markus A; Ralser, Markus

    2015-01-01

    Cellular metabolism assembles in a structurally highly conserved, but functionally dynamic system, known as the metabolic network. This network involves highly active, enzyme-catalyzed metabolic pathways that provide the building blocks for cell growth. In parallel, however, chemical reactivity of metabolites and unspecific enzyme function give rise to a number of side products that are not part of canonical metabolic pathways. It is increasingly acknowledged that these molecules are important for the evolution of metabolism, affect metabolic efficiency, and that they play a potential role in human disease—age-related disorders and cancer in particular. In this review we discuss the impact of oxidative and other cellular stressors on the formation of metabolic side products, which originate as a consequence of: (i) chemical reactivity or modification of regular metabolites; (ii) through modifications in substrate specificity of damaged enzymes; and (iii) through altered metabolic flux that protects cells in stress conditions. In particular, oxidative and heat stress conditions are causative of metabolite and enzymatic damage and thus promote the non-canonical metabolic activity of the cells through an increased repertoire of side products. On the basis of selected examples, we discuss the consequences of non-canonical metabolic reactivity on evolution, function and repair of the metabolic network. PMID:26378592

  13. Myogenin Induces a Shift of Enzyme Activity from Glycolytic to Oxidative Metabolism in Muscles of Transgenic Mice

    PubMed Central

    Hughes, Simon M.; Chi, Maggie M.-Y.; Lowry, Oliver H.; Gundersen, Kristian

    1999-01-01

    Physical training regulates muscle metabolic and contractile properties by altering gene expression. Electrical activity evoked in muscle fiber membrane during physical activity is crucial for such regulation, but the subsequent intracellular pathway is virtually unmapped. Here we investigate the ability of myogenin, a muscle-specific transcription factor strongly regulated by electrical activity, to alter muscle phenotype. Myogenin was overexpressed in transgenic mice using regulatory elements that confer strong expression confined to differentiated post-mitotic fast muscle fibers. In fast muscles from such mice, the activity levels of oxidative mitochondrial enzymes were elevated two- to threefold, whereas levels of glycolytic enzymes were reduced to levels 0.3–0.6 times those found in wild-type mice. Histochemical analysis shows widespread increases in mitochondrial components and glycogen accumulation. The changes in enzyme content were accompanied by a reduction in fiber size, such that many fibers acquired a size typical of oxidative fibers. No change in fiber type-specific myosin heavy chain isoform expression was observed. Changes in metabolic properties without changes in myosins are observed after moderate endurance training in mammals, including humans. Our data suggest that myogenin regulated by electrical activity may mediate effects of physical training on metabolic capacity in muscle. PMID:10225962

  14. Enhancement of the activity of enzyme immobilized on polydopamine-coated iron oxide nanoparticles by rational orientation of formate dehydrogenase.

    PubMed

    Gao, Xin; Ni, Kefeng; Zhao, Chengcheng; Ren, Yuhong; Wei, Dongzhi

    2014-10-20

    Immobilization of enzymes onto nanoparticles and retention of their structure and activity, which may be related to the orientation of enzymes on nanoparticles, remain a challenge. Here, we developed a novel enzyme-orientation strategy to enhance the activity of formate dehydrogenase immobilized on polydopamine-coated iron oxide nanoparticles via site-directed mutation. Seven mutants were constructed based on homology modeling of formate dehydrogenase and immobilized on polydopamine-coated iron oxide nanoparticles to investigate the influence of these mutations on immobilization. The immobilized mutant C242A/C275V/C363V/K389C demonstrated the highest immobilization yield and retained 90% of its initial activity, which was about 3-fold higher than that of wild-type formate dehydrogenase. Moreover, co-immobilization of formate dehydrogenase and leucine dehydrogenase was performed for the synthesis of l-tert-leucine. The catalytic efficiency of the co-immobilized mutant C242A/C275V/C363V/K389C and leucine dehydrogenase increased by more than 4-fold compared to that of co-immobilized wild-type formate dehydrogenase and leucine dehydrogenase. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Hepatic fatty acid oxidation enzyme activities are stimulated in rats fed the brown seaweed, Undaria pinnatifida (wakame).

    PubMed

    Murata, M; Ishihara, K; Saito, H

    1999-01-01

    The activities of hepatic enzymes involved in fatty acid synthesis and oxidation were compared in rats fed diets containing different proportions of dried powder of the brown seaweed, Undaria pinnatifida (wakame). Rats were fed diets containing 0, 0.5, 1.0, 2. 0, 5.0 and 10 g/100 g of dried wakame powder. Experimental diets were adjusted to provide consistent amounts of most nutrients, but mineral concentrations were not standardized. After the 21-d feeding period, serum and liver triacylglycerol levels in rats fed diets in which wakame constituted at least 2% were significantly lower than those in rats fed the control diet. The activity of glucose-6-phosphate dehydrogenase was significantly lower in rats fed the 5 and 10% wakame diets than in rats fed the control diet. In contrast, 10% wakame diet increased activities of enzymes involved in the beta-oxidation pathway including hepatic carnitine palmitoyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase, enoyl-CoA hydratase and 2,4-dienoyl-CoA reductase. Some differences were detected in rats fed 5% wakame as well. These results suggest that alterations of the activities of enzymes involved in fatty acid metabolism in the liver are responsible for the serum triacylglycerol-lowering effect of dietary wakame. Thus, wakame may be useful as a food to prevent hyperlipidemia.

  16. Spectroscopic identification of tri-n-octylphosphine oxide (TOPO) impurities and elucidation of their roles in cadmium selenide quantum-wire growth.

    PubMed

    Wang, Fudong; Tang, Rui; Kao, Jeff L-F; Dingman, Sean D; Buhro, William E

    2009-04-08

    Tri-n-octylphosphine oxide (TOPO) is the most commonly used solvent for the synthesis of colloidal nanocrystals. Here we show that the use of different batches of commercially obtained TOPO solvent introduces significant variability into the outcomes of CdSe quantum-wire syntheses. This irreproducibility is attributed to varying amounts of phosphorus-containing impurities in the different TOPO batches. We employ (31)P NMR to identify 10 of the common TOPO impurities. Their beneficial, harmful, or negligible effects on quantum-wire growth are determined. The impurity di-n-octylphosphinic acid (DOPA) is found to be the important beneficial TOPO impurity for the reproducible growth of high-quality CdSe quantum wires. DOPA is shown to beneficially modify precursor reactivity through ligand substitution. The other significant TOPO impurities are ranked according to their abilities to similarly influence precursor reactivity. The results are likely of general relevance to most nanocrystal syntheses conducted in TOPO.

  17. Effects of Francisella tularensis, Salmonella typhimurium and Streptococcus pneumoniae Infections on Oxidative, Glycolytic and Lysosomal Enzyme Activity in Red and White Skeletal Muscle in the Rat,

    DTIC Science & Technology

    1981-03-30

    Oxidative, Glycolytic and Lysosomal. 6. PERFORMING ORG. REPORT NUMBER Enzyme Activity in Red and W~hite Skeletal Muscle _______________ 7AU THOR(.) 8...block number) S Muscle Enzymes Salmonella Infections Francisella Bacteria Tularemia Pneumococcus Streptococcus 20 A5 RAcr (contLau. os reverse side it nec...34*esa nd Idenftif y by block nubr Since opinions differ as to whether the oxidative and glycolytic capabilities of skeletal muscle are altered in

  18. Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance

    SciTech Connect

    Beckman, J.S.; Minor, R.L. Jr.; White, C.W.; Repine, J.E.; Rosen, G.M.; Freeman, B.A.

    1988-05-15

    Covalent conjugation of superoxide dismutase and catalase with polyethylene glycol (PEG) increases the circulatory half-lives of these enzymes from <10 min to 40 h, reduced immunogenicity, and decreases sensitivity to proteolysis. Because PEG has surface active properties and can induce cell fusion, the authors hypothesized that PEG conjugation could enhance cell binding and association of normally membrane-impermeable enzymes. Incubation of cultured porcine aortic endothelial cells with /sup 125/I-PEG-catalase or /sup 125/I-PEG-superoxide dismutase produced a linear, concentration-dependent increase in cellular enzyme activity and radioactivity. Fluorescently labeled PEG-superoxide dismutase incubated with endothelial cells showed a vesicular localization. Mechanical injury to cell monolayers, which is known to stimulate endocytosis, further increased the uptake of fluorescent PEG-superoxide dismutase. Addition of PEG and PEG-conjugated enzymes perturbed the spin-label binding environment, indicative of producing an increase in plasma membrane fluidity. Thus, PEG conjugation to superoxide dismutase and catalase enhances cell association of these enzymes in a manner which increases cellular enzyme activities and provides prolonged protection from partially reduced oxygen species.

  19. Oxidative release of nitrite from 2-nitrotoluene by a three-component enzyme system from Pseudomonas sp. strain JS42.

    PubMed

    An, D; Gibson, D T; Spain, J C

    1994-12-01

    Pseudomonas sp. strain JS42 utilizes 2-nitrotoluene (2NT) as the sole source of carbon and energy for growth. Intact cells catalyze the oxidation of 2NT to 3-methylcatechol and nitrite in a reaction that requires molecular oxygen. Cell extracts oxidized 2NT to 3-methylcatechol and nitrite in the presence of NAD(P)H and ferrous iron. Ion-exchange chromatography yielded three protein fractions (A, B, and C) which were all required for the oxidation of 2NT to 3-methylcatechol and nitrite. Component B (reductase2NT) catalyzed a NAD(P)H-dependent reduction of cytochrome c. Solutions of component A (ISP2NT) were brown and showed absorption maxima at 458 and 324 nm. Two major bands with M(r)s 52,500 and 28,000 were observed when ISP2NT was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Component C could be replaced by ferredoxin NAP from the Pseudomonas putida NCIB 9816-4 naphthalene dioxygenase system and was given the designation ferredoxin2NT. Experiments with 18O2 showed that both oxygen atoms were added to the aromatic ring of 2NT to yield 3-methylcatechol. The enzyme is a new multicomponent enzyme system which we have designated 2NT 2,3-dioxygenase.

  20. Oxidative release of nitrite from 2-nitrotoluene by a three-component enzyme system from Pseudomonas sp. strain JS42.

    PubMed Central

    An, D; Gibson, D T; Spain, J C

    1994-01-01

    Pseudomonas sp. strain JS42 utilizes 2-nitrotoluene (2NT) as the sole source of carbon and energy for growth. Intact cells catalyze the oxidation of 2NT to 3-methylcatechol and nitrite in a reaction that requires molecular oxygen. Cell extracts oxidized 2NT to 3-methylcatechol and nitrite in the presence of NAD(P)H and ferrous iron. Ion-exchange chromatography yielded three protein fractions (A, B, and C) which were all required for the oxidation of 2NT to 3-methylcatechol and nitrite. Component B (reductase2NT) catalyzed a NAD(P)H-dependent reduction of cytochrome c. Solutions of component A (ISP2NT) were brown and showed absorption maxima at 458 and 324 nm. Two major bands with M(r)s 52,500 and 28,000 were observed when ISP2NT was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Component C could be replaced by ferredoxin NAP from the Pseudomonas putida NCIB 9816-4 naphthalene dioxygenase system and was given the designation ferredoxin2NT. Experiments with 18O2 showed that both oxygen atoms were added to the aromatic ring of 2NT to yield 3-methylcatechol. The enzyme is a new multicomponent enzyme system which we have designated 2NT 2,3-dioxygenase. Images PMID:8002568

  1. Repeated application of composted tannery sludge affects differently soil microbial biomass, enzymes activity, and ammonia-oxidizing organisms.

    PubMed

    Araújo, Ademir Sérgio Ferreira; Lima, Luciano Moura; Santos, Vilma Maria; Schmidt, Radomir

    2016-10-01

    Repeated application of composted tannery sludge (CTS) changes the soil chemical properties and, consequently, can affect the soil microbial properties. The aim of this study was to evaluate the responses of soil microbial biomass and ammonia-oxidizing organisms to repeated application of CTS. CTS was applied repeatedly during 6 years, and, at the sixth year, the soil microbial biomass, enzymes activity, and ammonia-oxidizing organisms were determined in the soil. The treatments consisted of 0 (without CTS application), 2.5, 5, 10, and 20 t ha(-1) of CTS (dry basis). Soil pH, EC, SOC, total N, and Cr concentration increased with the increase in CTS rate. Soil microbial biomass did not change significantly with the amendment of 2.5 Mg ha(-1), while it decreased at the higher rates. Total and specific enzymes activity responded differently after CTS application. The abundance of bacteria did not change with the 2.5-Mg ha(-1) CTS treatment and decreased after this rate, while the abundance of archaea increased significantly with the 2.5-Mg ha(-1) CTS treatment. Repeated application of different CTS rates for 6 years had different effects on the soil microbial biomass and ammonia-oxidizing organisms as a response to changes in soil chemical properties.

  2. Enzyme-functionalized vascular grafts catalyze in-situ release of nitric oxide from exogenous NO prodrug.

    PubMed

    Wang, Zhihong; Lu, Yaxin; Qin, Kang; Wu, Yifan; Tian, Yingping; Wang, Jianing; Zhang, Jimin; Hou, Jingli; Cui, Yun; Wang, Kai; Shen, Jie; Xu, Qingbo; Kong, Deling; Zhao, Qiang

    2015-07-28

    Nitric oxide (NO) is an important signaling molecule in cardiovascular system, and the sustained release of NO by endothelial cells plays a vital role in maintaining patency and homeostasis. In contrast, lack of endogenous NO in artificial blood vessel is believed to be the main cause of thrombus formation. In this study, enzyme prodrug therapy (EPT) technique was employed to construct a functional vascular graft by immobilization of galactosidase on the graft surface. The enzyme-functionalized grafts exhibited excellent catalytic property in decomposition of the exogenously administrated NO prodrug. Localized and on-demand release of NO was demonstrated by in vitro release assay and fluorescent probe tracing in an ex vivo model. The immobilized enzyme retained catalytic property even after subcutaneous implantation of the grafts for one month. The functional vascular grafts were implanted into the rat abdominal aorta with a 1-month monitoring period. Results showed effective inhibition of thrombus formation in vivo and enhancement of vascular tissue regeneration and remodeling on the grafts. Thus, we create an enzyme-functionalized vascular graft that can catalyze prodrug to release NO locally and sustainably, indicating that this approach may be useful to develop new cell-free vascular grafts for treatment of vascular diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Nitric oxide inhibits specific enzymes in the Krebs cycle and the respiratory chain of rat hepatocyte mitochondria

    SciTech Connect

    Stadler, J.; Billiar, T.R.; Curran, R.D.; Kim, R.; Simmons, R.L. )

    1990-02-26

    Nitric oxide (NO) is a highly-reactive molecule produced from L-arginine as recently described. In macrophages and tumor cells, NO inhibits specific mitochondrial enzymes presumably by attacking their intrinsic 4Fe-4S centers. The susceptible enzymes include aconitase of the Krebs cycle and oxidoreductase (complex II) of the electron transport chain. The authors have recently demonstrated that hepatocytes (HC) produce NO in large amounts in response to endotoxin and inflammatory cytokines. To determine whether HC suffer a similar enzyme inhibition, the authors exposed rat HC to increasing concentrations of NO solutions for 5 minutes. The activity of aconitase, complex 1, complex 2, and complex 4 (cytochrome oxidase) was determined by measuring O{sub 2} consumption after addition of enzyme-specific substrates. An NO concentration-dependent inhibition of aconitase, complex 1, and complex 2 was measured. After exposure to 0.6 mM solution, the activity of aconitase was blocked to non-measurable values while complex 1 was reduced to 11 + 8%, and complex 2 to 36 + 2% of the activity of control HC. Complex 4 of the respiratory chain remained intact at 100 + 8%. These data indicate that HC, like other cell types, are susceptible to inhibition of important steps of energy production by NO. As NO is produced in response to septic stimuli, this mechanism may play a role in the metabolic dysfunction of HC in sepsis.

  4. Self-assembled monolayers of 1-alkenes on oxidized platinum surfaces as platforms for immobilized enzymes for biosensing

    NASA Astrophysics Data System (ADS)

    Alonso, Jose Maria; Bielen, Abraham A. M.; Olthuis, Wouter; Kengen, Servé W. M.; Zuilhof, Han; Franssen, Maurice C. R.

    2016-10-01

    Alkene-based self-assembled monolayers grafted on oxidized Pt surfaces were used as a scaffold to covalently immobilize oxidase enzymes, with the aim to develop an amperometric biosensor platform. NH2-terminated organic layers were functionalized with either aldehyde (CHO) or N-hydroxysuccinimide (NHS) ester-derived groups, to provide anchoring points for enzyme immobilization. The functionalized Pt surfaces were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (CA), infrared reflection absorption spectroscopy (IRRAS) and atomic force microscopy (AFM). Glucose oxidase (GOX) was covalently attached to the functionalized Pt electrodes, either with or without additional glutaraldehyde crosslinking. The responses of the acquired sensors to glucose concentrations ranging from 0.5 to 100 mM were monitored by chronoamperometry. Furthermore, lactate oxidase (LOX) and human hydroxyacid oxidase (HAOX) were successfully immobilized onto the PtOx surface platform. The performance of the resulting lactate sensors was investigated for lactate concentrations ranging from 0.05 to 20 mM. The successful attachment of active enzymes (GOX, LOX and HAOX) on Pt electrodes demonstrates that covalently functionalized PtOx surfaces provide a universal platform for the development of oxidase enzyme-based sensors.

  5. A comparative genomic analysis of the oxidative enzymes potentially involved in lignin degradation by Agaricus bisporus

    Treesearch

    Harshavardhan Doddapaneni; Venkataramanan Subramanian; Bolei Fu; Dan Cullen

    2013-01-01

    The oxidative enzymatic machinery for degradation of organic substrates in Agaricus bisporus (Ab) is at the core of the carbon recycling mechanisms in this fungus. To date, 156 genes have been tentatively identified as part of this oxidative enzymatic machinery, which includes 26 peroxidase encoding genes, nine copper radical oxidase [including three...

  6. Functional network analysis of obese and lean Göttingen minipigs elucidates changes in oxidative and inflammatory networks in obese pigs.

    PubMed

    Boonen, Harrie C M; Moesgaard, Sophia G; Birck, Malene M; Christoffersen, Berit O; Cirera, Susanna; Heegaard, Peter M H; Højbøge, Tina Rødgaard; Jensen, Lars J; Mortensen, Alan; Olsen, Lisbeth Høier; Sheykhzade, Majid; Tang, Jiaowei; Lykkesfeldt, Jens

    2014-12-01

    The Göttingen minipig model of obesity is used in pre-clinical research to predict clinical outcome of new treatments for metabolic diseases. However, treatment effects often remain unnoticed when using single parameter statistical comparisons due to the small numbers of animals giving rise to large variation and insufficient statistical power. The purpose of this study was to perform a correlation matrix analysis of multiple multi-scale parameters describing co-segregation of traits in order to identify differences between lean and obese minipigs. More than 40 parameters, ranging from physical, cardiovascular, inflammatory and metabolic markers were measured in lean and obese animals. Correlation matrix analysis was performed using permutation test and bootstrapping at different levels of significance. Single parameter comparisons yielded significant differences between lean and obese animals mainly for known physical traits. On the other hand, functional network analysis revealed new co-segregations, particularly in the domain of inflammatory and oxidative stress markers in the obese animals that were not present in the lean. Functional networks of lean or obese minipigs could be utilised to assess drug effects and predict changes in parameters with a certain degree of precision, on the basis of the networks confidence intervals. Comparison of functional networks in minipigs with those of human clinical data may be used to identify common parameters or co-segregations related to obesity between animal models and man.

  7. Phagocytosis and nitric oxide production by peritoneal adherent cells in response to Candida albicans in aging: a collaboration to elucidate the pathogenesis of denture stomatitis.

    PubMed

    Gardizani, Taiane Priscila; Pinke, Karen Henriette; Lima, Heliton Gustavo de; Lara, Vanessa Soares

    2017-01-01

    The aim of this study was to investigate the influence of aging on the internalization and the production of nitric oxide (NO) by peritoneal adherent cells (PAC), in response to Candida albicans (C. albicans). PAC obtained from young and aged mice were challenged with dead or viable C. albicans by using predetermined proportions (cells:yeast) for 30 and 120 minutes. Phagocytosis was analyzed by acridine orange dye, and NO production by the Griess reaction. C. albicans phagocytosis by PAC from aged mice was similar to that of young mice, although the cells from older mice cells present more internalized fungi compared with matched control. In addition, a tendency towards impaired NO production by peritoneal mononuclear phagocytes from aged mice was observed. PAC from aged mice may capture and store many fungi, which in turn may mean that these cells are effectively unable to eliminate fungi, probably due to impaired NO production. Therefore, considering the important role of C. albicans overgrowth in the pathogenesis of DS and the aspects observed in this study, aging may favor the onset and severity of local candidosis such as DS and its systemic forms.

  8. Production of Oxidative and Hydrolytic Enzymes by Coprinus cinereus (Schaeff.) Gray from Sisal Wastes Supplemented with Cow Dung Manure

    PubMed Central

    Raymond, Prosper; Mshandete, Anthony Manoni; Kajumulo Kivaisi, Amelia

    2015-01-01

    The activity of oxidative and hydrolytic enzymes of the edible and medicinal white rot fungi Coprinus cinereus (Schaeff.) Gray mushroom was observed during mycelia growth and fruiting body development in solid substrate fermentation using sisal waste fractions amended with cow dung manure as supplement. Laccase had the highest titre value among the five detected enzymes. Its activity was higher during mycelia growth compared to fruiting phase, with 10% supplemented substrate formulation unmixed sisal leaf decortication residues [abbreviated SL : SB (100 : 0)] displaying the highest activity of 39.45 ± 12.05 Ug−1. Lignin peroxidase (LiP) exhibited a characteristic wave-like pattern with the highest peaks found either during full mycelia colonization or soon after first flush harvest; the highest activity of 1.93 ± 0.62 Ug−1 was observed on unsupplemented SL : SB (100 : 0) substrate formulation during mycelia colonization. For hydrolytic enzymes, the highest carboxymethyl cellulase (CMCase) activity of 2.03 ± 0.70 Ug−1 was observed on 20% supplemented SL : SB (0 : 100) after first flush; that of pectinase (1.90 ± 0.32 Ug−1) was revealed after third flush on 10% supplemented SL : SB (0 : 100) substrate formulation while 10% supplemented SL : SB (25 : 75) exhibited the highest xylanase activity (1.23 ± 0.12 Ug−1) after first flush. These findings show that the activities of both oxidative and hydrolytic enzymes were regulated in line with developmental phase of growth of Coprinus cinereus. PMID:26664748

  9. Alpha-Lipoic Acid Supplementation Protects Enzymes from Damage by Nitrosative and Oxidative Stress

    PubMed Central

    Hiller, Sylvia; DeKroon, Robert; Hamlett, Eric D.; Xu, Longquan; Osorio, Cristina; Robinette, Jennifer; Winnik, Witold; Simington, Stephen; Maeda, Nobuyo; Alzate, Oscar; Yi, Xianwen

    2017-01-01

    Background S-nitrosylation of mitochondrial enzymes involved in energy transfer under nitrosative stress may result in ATP deficiency. We investigated whether α-lipoic acid, a powerful antioxidant, could alleviate nitrosative stress by regulating S-nitrosylation, which could result in retaining the mitochondrial enzyme activity. Methods In this study, we have identified the S-nitrosylated forms of subunit 1 of dihydrolipoyllysine succinyltransferase (complex III), and subunit 2 of the α-ketoglutarate dehydrogenase complex by implementing a fluorescence-based differential quantitative proteomics method. Results We found that the activities of these two mitochondrial enzymes were partially but reversibly inhibited by S-nitrosylation in cultured endothelial cells, and that their activities were partially restored by supplementation of α-lipoic acid. We show that protein S-nitrosylation affects the activity of mitochondrial enzymes that are central to energy supply, and that α-lipoic acid protects mitochondrial enzymes by altering S-nitrosylation levels. Conclusions Inhibiting protein S-nitrosylation with α-lipoic acid seems to be a protective mechanism against nitrosative stress. General significance Identification and characterization of these new protein targets should contribute to expanding the therapeutic power of α-lipoic acid and to a better understanding of the underlying antioxidant mechanisms. PMID:26344063

  10. Phagocytosis and nitric oxide production by peritoneal adherent cells in response to Candida albicans in aging: a collaboration to elucidate the pathogenesis of denture stomatitis

    PubMed Central

    GARDIZANI, Taiane Priscila; PINKE, Karen Henriette; de LIMA, Heliton Gustavo; LARA, Vanessa Soares

    2017-01-01

    Abstract Elderly denture wearers are commonly affected by Candida-associated denture stomatitis (DS), an inflammatory process of the oral mucosa strongly associated with Candida spp and other microorganisms, as well as local and systemic factors. The impaired immune response against pathogens is among the inherent host factors that have been also associated with the pathogenesis of DS. Mononuclear phagocytes respond to the pathogens through phagocytosis followed by the production of several substances inside the phagosomes, among them are the reactive nitrogen species (RNS). A failure in these mechanisms may contribute to the DS development. Objective The aim of this study was to investigate the influence of aging on the internalization and the production of nitric oxide (NO) by peritoneal adherent cells (PAC), in response to Candida albicans (C. albicans). Material and methods PAC obtained from young and aged mice were challenged with dead or viable C. albicans by using predetermined proportions (cells:yeast) for 30 and 120 minutes. Phagocytosis was analyzed by acridine orange dye, and NO production by the Griess reaction. Results C. albicans phagocytosis by PAC from aged mice was similar to that of young mice, although the cells from older mice cells present more internalized fungi compared with matched control. In addition, a tendency towards impaired NO production by peritoneal mononuclear phagocytes from aged mice was observed. Conclusions PAC from aged mice may capture and store many fungi, which in turn may mean that these cells are effectively unable to eliminate fungi, probably due to impaired NO production. Therefore, considering the important role of C. albicans overgrowth in the pathogenesis of DS and the aspects observed in this study, aging may favor the onset and severity of local candidosis such as DS and its systemic forms. PMID:28678945

  11. Elucidating hydrogen oxidation/evolution kinetics in base and acid by enhanced activities at the optimized Pt shell thickness on the Ru core

    DOE PAGES

    Elbert, Katherine; Hu, Jue; Ma, Zhong; ...

    2015-10-05

    Hydrogen oxidation and evolution on Pt in acid are facile processes, while in alkaline electrolytes, they are 2 orders of magnitude slower. Thus, developing catalysts that are more active than Pt for these two reactions is important for advancing the performance of anion exchange membrane fuel cells and water electrolyzers. Herein, we detail a 4-fold enhancement of Pt mass activity that we achieved using single-crystalline Ru@Pt core–shell nanoparticles with two-monolayer-thick Pt shells, which doubles the activity on Pt–Ru alloy nanocatalysts. For Pt specific activity, the two- and one-monolayer-thick Pt shells exhibited enhancement factors of 3.1 and 2.3, respectively, compared tomore » the Pt nanocatalysts in base, differing considerably from the values of 1 and 0.4, respectively, in acid. To explain such behavior and the orders of magnitude difference in activity on going from acid to base, we performed kinetic analyses of polarization curves over a wide range of potential from –250 to 250 mV using the dual-pathway kinetic equation. From acid to base, the activation free energies increase the most for the Volmer reaction, resulting in a switch of the rate-determining step from the Tafel to the Volmer reaction, and a shift to a weaker optimal hydrogen binding energy. Furthermore, the much higher activation barrier for the Volmer reaction in base than in acid is ascribed to one or both of the two catalyst-insensitive factors: slower transport of OH– than H+ in water and a stronger O–H bond in water molecules (HO–H) than in hydrated protons (H2O–H+).« less

  12. Carnosic acid biosynthesis elucidated by a synthetic biology platform

    PubMed Central

    Ignea, Codruta; Athanasakoglou, Anastasia; Ioannou, Efstathia; Georgantea, Panagiota; Trikka, Fotini A.; Loupassaki, Sofia; Roussis, Vassilios; Makris, Antonios M.

    2016-01-01

    Synthetic biology approaches achieving the reconstruction of specific plant natural product biosynthetic pathways in dedicated microbial “chassis” have provided access to important industrial compounds (e.g., artemisinin, resveratrol, vanillin). However, the potential of such production systems to facilitate elucidation of plant biosynthetic pathways has been underexplored. Here we report on the application of a modular terpene production platform in the characterization of the biosynthetic pathway leading to the potent antioxidant carnosic acid and related diterpenes in Salvia pomifera and Rosmarinus officinalis. Four cytochrome P450 enzymes are identified (CYP76AH24, CYP71BE52, CYP76AK6, and CYP76AK8), the combined activities of which account for all of the oxidation events leading to the biosynthesis of the major diterpenes produced in these plants. This approach develops yeast as an efficient tool to harness the biotechnological potential of the numerous sequencing datasets that are increasingly becoming available through transcriptomic or genomic studies. PMID:26976595

  13. Synthesis, structure elucidation and redox properties of 99Tc complexes of lacunary Wells Dawson polyoxometalates: insights into molecular 99Tc - metal oxide interactions

    SciTech Connect

    McGregor, Donna; Burton-Pye, Benjamin P.; Howell, Robertha C.; Mbomekalle, Israel M.; Lukens Jr, Wayne W.; Bian, Fang; Mausolf, Edward; Poineau, Frederic; Czerwinski, Kenneth R; Francesconi, Lynn C.

    2011-01-10

    The isotope 99Tc (beta max: 250 keV, half-life: 2 x 105 year) is an abundant product of uranium-235 fission in nuclear reactors and is present throughout the radioactive waste stored in underground tanks at Hanford and Savannah River. Understanding and controlling the extensive redox chemistry of 99Tc is important to identify tunable strategies to separate 99Tc from spent fuel and from waste tanks and once separated, to identify and develop an appropriately stable waste-form for 99Tc. Polyoxometalates (POMs), nanometer sized models for metal oxide solid-state materials, are used in this study to provide a molecular level understanding of the speciation and redox chemistry of incorporated 99Tc. In this study, 99Tc complexes of the (alpha 2-P2W17O61)10- and (alpha 1-P2W17O61)10- isomers were prepared. Ethylene glycol was used as a"transfer ligand" to minimize the formation of TcO2 cdot xH2O. The solution structures, formulations, and purity of TcVO(alpha 1/alpha 2-P2W17O61)7- were determined by multinuclear NMR. X-ray Absorption Spectroscopy of the complexes are in agreement with the formulation and structures determined from 31P and 183W NMR. Preliminary electrochemistry results are consistent with the EXAFS results, showing a facile reduction of the TcVO(alpha 1-P2W17O61)7- species compared to the TcVO(alpha 2-P2W17O61)7- analog. The alpha1- defect is unique in that a basic oxygen atom is positioned toward the alpha1- site and the TcVO center appears to form a dative metal-metal bond with a framework W site. These attributes may lead to the assistance of protonation events that facilitate reduction. Electrochemistry comparison shows that the ReV analogs are about 200 mV more difficult to reduce in accordance with periodic trends.

  14. Synthesis, Structure Elucidation, and Redox Properties of [superscript 99]Tc Complexes of Lacunary Wells-Dawson Polyoxometalates: Insights into Molecular [superscript 99]Tc-Metal Oxide Interactions

    SciTech Connect

    McGregor, Donna; Burton-Pye, Benjamin P.; Howell, Robertha C.; Mbomekalle, Israel M.; Lukens, Jr., Wayne W.; Bian, Fang; Mausolf, Edward; Poineau, Frederic; Czerwinski, Kenneth R.; Francesconi, Lynn C.

    2011-11-17

    The isotope {sup 99}Tc ({beta}{sub max}, 293.7; half-life, 2.1 x 10{sup 5} years) is an abundant product of uranium-235 fission in nuclear reactors and is present throughout the radioactive waste stored in underground tanks at the Hanford and Savannah River sites. Understanding and controlling the extensive redox chemistry of {sup 99}Tc is important in identifying tunable strategies to separate {sup 99}Tc from spent fuel and from waste tanks and, once separated, to identify and develop an appropriately stable waste form for {sup 99}Tc. Polyoxometalates (POMs), nanometer-sized models for metal oxide solid-state materials, are used in this study to provide a molecular level understanding of the speciation and redox chemistry of incorporated {sup 99}Tc. In this study, {sup 99}Tc complexes of the ({alpha}{sub 2}-P{sub 2}W{sub 17}O{sub 61}){sup 10-} and ({alpha}{sub 1}-P{sub 2}W{sub 17}O{sub 61}){sup 10-} isomers were prepared. Ethylene glycol was used as a 'transfer ligand' to minimize the formation of TcO{sub 2} {center_dot} xH{sub 2}O. The solution structures, formulations, and purity of TcVO({alpha}{sub 1}/{alpha}{sub 2}-P{sub 2}W{sub 17}O{sub 61}){sup 7-} were determined by multinuclear NMR. X-ray absorption spectroscopy of the complexes is in agreement with the formulation and structures determined from {sup 31}P and {sup 183}W NMR. Preliminary electrochemistry results are consistent with the EXAFS results, showing a facile reduction of the TcVO({alpha}{sub 1}-P{sub 2}W{sub 17}O{sub 61}){sup 7-} species compared to the TcVO({alpha}{sub 2}-P{sub 2}W{sub 17}O{sub 61}){sup 7-} analog. The {alpha}{sub 1} defect is unique in that a basic oxygen atom is positioned toward the {alpha}{sub 1} site, and the Tc{sup V}O center appears to form a dative metal-metal bond with a framework W site. These attributes may lead to the assistance of protonation events that facilitate reduction. Electrochemistry comparison shows that the ReV analogs are about 200 mV more difficult to

  15. Synthesis, structure elucidation, and redox properties of 99Tc complexes of lacunary Wells-Dawson polyoxometalates: insights into molecular 99Tc-metal oxide interactions.

    PubMed

    McGregor, Donna; Burton-Pye, Benjamin P; Howell, Robertha C; Mbomekalle, Israel M; Lukens, Wayne W; Bian, Fang; Mausolf, Edward; Poineau, Frederic; Czerwinski, Kenneth R; Francesconi, Lynn C

    2011-03-07

    The isotope (99)Tc (β(max), 293.7; half-life, 2.1 × 10(5) years) is an abundant product of uranium-235 fission in nuclear reactors and is present throughout the radioactive waste stored in underground tanks at the Hanford and Savannah River sites. Understanding and controlling the extensive redox chemistry of (99)Tc is important in identifying tunable strategies to separate (99)Tc from spent fuel and from waste tanks and, once separated, to identify and develop an appropriately stable waste form for (99)Tc. Polyoxometalates (POMs), nanometer-sized models for metal oxide solid-state materials, are used in this study to provide a molecular level understanding of the speciation and redox chemistry of incorporated (99)Tc. In this study, (99)Tc complexes of the (α(2)-P(2)W(17)O(61))(10-) and (α(1)-P(2)W(17)O(61))(10-) isomers were prepared. Ethylene glycol was used as a "transfer ligand" to minimize the formation of TcO(2)·xH(2)O. The solution structures, formulations, and purity of Tc(V)O(α(1)/α(2)-P(2)W(17)O(61))(7-) were determined by multinuclear NMR. X-ray absorption spectroscopy of the complexes is in agreement with the formulation and structures determined from (31)P and (183)W NMR. Preliminary electrochemistry results are consistent with the EXAFS results, showing a facile reduction of the Tc(V)O(α(1)-P(2)W(17)O(61))(7-) species compared to the Tc(V)O(α(2)-P(2)W(17)O(61))(7-) analog. The α(1) defect is unique in that a basic oxygen atom is positioned toward the α(1) site, and the Tc(V)O center appears to form a dative metal-metal bond with a framework W site. These attributes may lead to the assistance of protonation events that facilitate reduction. Electrochemistry comparison shows that the Re(V) analogs are about 200 mV more difficult to reduce in accordance with periodic trends. © 2011 American Chemical Society

  16. Time course study of oxidative and nitrosative stress and antioxidant enzymes in K2Cr2O7-induced nephrotoxicity

    PubMed Central

    Pedraza-Chaverrí, José; Barrera, Diana; Medina-Campos, Omar N; Carvajal, Raymundo C; Hernández-Pando, Rogelio; Macías-Ruvalcaba, Norma A; Maldonado, Perla D; Salcedo, Marcos I; Tapia, Edilia; Saldívar, Liliana; Castilla, María E; Ibarra-Rubio, María E

    2005-01-01

    Background Potassium dichromate (K2Cr2O7)-induced nephrotoxicity is associated with oxidative and nitrosative stress. In this study we investigated the relation between the time course of the oxidative and nitrosative stress with kidney damage and alterations in the following antioxidant enzymes: Cu, Zn superoxide dismutase (Cu, Zn-SOD), Mn-SOD, glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT). Methods Nephrotoxicity was induced in rats by a single injection of K2Cr2O7. Groups of animals were sacrificed on days 1,2,3,4,6,8,10, and 12. Nephrotoxicity was evaluated by histological studies and by measuring creatinine clearance, serum creatinine, blood urea nitrogen (BUN), and urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) and total protein. Oxidative and nitrosative stress were measured by immunohistochemical localization of protein carbonyls and 3-nitrotyrosine, respectively. Cu, Zn-SOD, Mn-SOD, and CAT were studied by immunohistochemical localization. The activity of total SOD, CAT, GPx, and GR was also measured as well as serum and kidney content of chromium and urinary excretion of NO2 -/NO3-. Data were compared by two-way analysis of variance followed by a post hoc test. Results Serum and kidney chromium content increased reaching the highest value on day 1. Nephrotoxicity was made evident by the decrease in creatinine clearance (days 1–4) and by the increase in serum creatinine (days 1–4), BUN (days 1–6), urinary excretion of NAG (days 1–4), and total protein (day 1–6) and by the structural damage to the proximal tubules (days 1–6). Oxidative and nitrosative stress were clearly evident on days 1–8. Urinary excretion of NO2-/NO3- decreased on days 2–6. Mn-SOD and Cu, Zn-SOD, estimated by immunohistochemistry, and total SOD activity remained unchanged. Activity of GPx decreased on days 3–12 and those of GR and CAT on days 2–10. Similar findings were observed by immunohistochemistry of CAT. Conclusion These

  17. Interaction of vitamin E and exercise training on oxidative stress and antioxidant enzyme activities in rat skeletal muscles.

    PubMed

    Chang, Chen-Kang; Huang, Hui-Yu; Tseng, Hung-Fu; Hsuuw, Yan-Der; Tso, Tim K

    2007-01-01

    It has been shown that free radicals are increased during intensive exercise. We hypothesized that vitamin E (vit E) deficiency, which will increase oxidative stress, would augment the training-induced adaptation of antioxidant enzymes. This study investigated the interaction effect of vit E and exercise training on oxidative stress markers and activities of antioxidant enzymes in red quadriceps and white gastrocnemius of rats in a 2x2 design. Thirty-two male rats were divided into trained vit E-adequate, trained vit E-deficient, untrained vit E-adequate, and untrained vit E-deficient groups. The two trained groups swam 6 h/day, 6 days/week for 8 weeks. The two vit E-deficient groups consumed vit E-free diet for 8 weeks. Vitamin E-training interaction effect was significant on thiobarbituric acid reactive substances (TBARSs), glutathione peroxidase (GPX), and superoxide dismutase (SOD) in both muscles. The trained vit E-deficient group showed the highest TBARS and GPX activity and the lowest SOD activity in both muscles. A significant vit E effect on glutathione reductase and catalase was present in both muscles. Glutathione reductase and catalase activities were significantly lower in the two vit E-adequate groups combined than in the two vit E-deficient groups combined in both muscles. This study shows that vit E status and exercise training have interactive effect on oxidative stress and GPX and SOD activities in rat skeletal muscles. Vitamin E deprivation augmented the exercise-induced elevation in GPX activity while inhibiting exercise-induced SOD activity, possibly through elevated oxidative stress.

  18. Temperature stress, anti-oxidative enzyme activity and virus acquisition in Bemisia tabaci (Hemiptera: Aleyrodidae)

    USDA-ARS?s Scientific Manuscript database

    In most eukaryotic systems, antioxidants provide protection when cells are exposed to stressful environmental conditions. Antioxidants, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase, function in a stepwise series with SOD initially preventing oxidative damage by conve...

  19. Enzymes of respiratory iron oxidation. Progress report, March 1990--June 1992

    SciTech Connect

    Blake, R. II

    1992-12-31

    This report describes experimental progress in characterizing and identifying redox proteins in a number of iron-oxidizing bacteria. Sections of the paper are entitled (1) In Situ electrolysis was explored to achieve enhanced yields of iron-oxidizing bacteria, (2)Structure/function studies were performed on redox-active biomolecules from Thiobacillus ferrooxidans, (3) Novel redox-active biomolecules were demonstrated in other iron autotrophs, and (4) New probes of metalloprotein electron-transfer reactions were synthesized and characterized.

  20. Increased fructose absorption and oxidation after oral supplementation with sucraid enzyme: Implications for fructose malabsorption

    USDA-ARS?s Scientific Manuscript database

    The value of Sucraid enzyme supplements for correction of sucrase deficiency in CSID is well documented. Sucraid is an invertase derived from brewer’s yeast which has been characterized as hydrolyzing sucrose to glucose and fructose. However, sacrosidase may have another role: conversion of fructose...

  1. Metal Oxide Nanosensors Using Polymeric Membranes, Enzymes and Antibody Receptors as Ion and Molecular Recognition Elements

    PubMed Central

    Willander, Magnus; Khun, Kimleang; Ibupoto, Zafar Hussain

    2014-01-01

    The concept of recognition and biofunctionality has attracted increasing interest in the fields of chemistry and material sciences. Advances in the field of nanotechnology for the synthesis of desired metal oxide nanostructures have provided a solid platform for the integration of nanoelectronic devices. These nanoelectronics-based devices have the ability to recognize molecular species of living organisms, and they have created the possibility for advanced chemical sensing functionalities with low limits of detection in the nanomolar range. In this review, various metal oxides, such as ZnO-, CuO-, and NiO-based nanosensors, are described using different methods (receptors) of functionalization for molecular and ion recognition. These functionalized metal oxide surfaces with a specific receptor involve either a complex formation between the receptor and the analyte or an electrostatic interaction during the chemical sensing of analytes. Metal oxide nanostructures are considered revolutionary nanomaterials that have a specific surface for the immobilization of biomolecules with much needed orientation, good conformation and enhanced biological activity which further improve the sensing properties of nanosensors. Metal oxide nanostructures are associated with certain unique optical, electrical and molecular characteristics in addition to unique functionalities and surface charge features which shows attractive platforms for interfacing biorecognition elements with effective transducing properties for signal amplification. There is a great opportunity in the near future for metal oxide nanostructure-based miniaturization and the development of engineering sensor devices. PMID:24841244

  2. Antioxidant enzyme activities are affected by salt content and temperature and influence muscle lipid oxidation during dry-salted bacon processing.

    PubMed

    Jin, Guofeng; He, Lichao; Yu, Xiang; Zhang, Jianhao; Ma, Meihu

    2013-12-01

    Fresh pork bacon belly was used as material and manufactured into dry-salted bacon through salting and drying-ripening. During processing both oxidative stability and antioxidant enzyme stability were evaluated by assessing peroxide value (PV), thiobarbituric acid reactive substances (TBARS) and activities of catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and their correlations were also analysed. The results showed that all antioxidant enzyme activities decreased (p<0.05) until the end of process; GSH-Px was the most unstable one followed by catalase. Antioxidant enzyme activities were negatively correlated with TBARS (p<0.05), but the correlations were decreased with increasing process temperature. Salt showed inhibitory effect on all antioxidant enzyme activities and was concentration dependent. These results indicated that when process temperature and salt content were low at the same time during dry-salted bacon processing, antioxidant enzymes could effectively control lipid oxidation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Epoxidation of the methamphetamine pyrolysis product, trans-phenylpropene, to trans-phenylpropylene oxide by CYP enzymes and stereoselective glutathione adduct formation

    SciTech Connect

    Sanga, Madhu; Younis, Islam R.; Tirumalai, Padma S.; Bland, Tina M.; Banaszewska, Monica; Konat, Gregory W.; Tracy, Timothy S.; Gannett, Peter M.; Callery, Patrick S. . E-mail: pcallery@hsc.wvu.edu

    2006-03-01

    Pyrolytic products of smoked methamphetamine hydrochloride are well established. Among the various degradation products formed, trans-phenylpropene (trans-{beta}-methylstyrene) is structurally similar to styrene analogues known to be bioactivated by CYP enzymes. In human liver microsomes, trans-phenylpropene was converted to the epoxide trans-phenylpropylene oxide (trans-2-methyl-3-phenyloxirane) and cinnamyl alcohol. Incubation of trans-phenylpropene with microsomes in the presence of enzyme-specific P450 enzyme inhibitors indicated the involvement of CYP2E1, CYP1A2, and CYP3A4 enzymes. Both (R,R)-phenylpropylene oxide and (S,S)-phenylpropylene oxide were formed in human liver microsomal preparations. Enantiomers of trans-phenylpropylene oxide were stereoselectively and regioselectively conjugated in a Phase II drug metabolism reaction catalyzed by human liver cytosolic enzymes consisting of conjugation with glutathione. The structure of the phenylpropylene oxide-glutathione adduct is consistent with nucleophilic ring-opening by attack at the benzylic carbon. Exposure of cultured C6 glial cells to (S,S)-phenylpropylene oxide produced a cytotoxic response in a concentration-dependent manner based on cell degeneration and death.

  4. Epoxidation of the methamphetamine pyrolysis product, trans-phenylpropene, to trans-phenylpropylene oxide by CYP enzymes and stereoselective glutathione adduct formation.

    PubMed

    Sanga, Madhu; Younis, Islam R; Tirumalai, Padma S; Bland, Tina M; Banaszewska, Monica; Konat, Gregory W; Tracy, Timothy S; Gannett, Peter M; Callery, Patrick S

    2006-03-01

    Pyrolytic products of smoked methamphetamine hydrochloride are well established. Among the various degradation products formed, trans-phenylpropene (trans-beta-methylstyrene) is structurally similar to styrene analogues known to be bioactivated by CYP enzymes. In human liver microsomes, trans-phenylpropene was converted to the epoxide trans-phenylpropylene oxide (trans-2-methyl-3-phenyloxirane) and cinnamyl alcohol. Incubation of trans-phenylpropene with microsomes in the presence of enzyme-specific P450 enzyme inhibitors indicated the involvement of CYP2E1, CYP1A2, and CYP3A4 enzymes. Both (R,R)-phenylpropylene oxide and (S,S)-phenylpropylene oxide were formed in human liver microsomal preparations. Enantiomers of trans-phenylpropylene oxide were stereoselectively and regioselectively conjugated in a Phase II drug metabolism reaction catalyzed by human liver cytosolic enzymes consisting of conjugation with glutathione. The structure of the phenylpropylene oxide-glutathione adduct is consistent with nucleophilic ring-opening by attack at the benzylic carbon. Exposure of cultured C6 glial cells to (S,S)-phenylpropylene oxide produced a cytotoxic response in a concentration-dependent manner based on cell degeneration and death.

  5. Role of anti-oxidant enzymes superoxide dismutase and catalase in the development of cataract : study of serum levels in patients with senile and diabetic cataracts.

    PubMed

    Maurya, O P S; Mohanty, Lipa; Bhaduri, Gautam; Chandra, Abhishek

    2006-07-01

    Cataract is a universal phenomenon in old age and the most common cause of blindness worldover. Oxidative stress is believed to be a key factor in the gradual loss of transparency of lens. Free radicals formed result in derangement and opacification of lens fibres. Superoxide dismutase, catalase and glutathione peroxidase are major anti-oxidant enzymes, which protect against free radicals. The purpose of this study is to evaluate the role of anti-oxidant enzymes - superoxide dismutase and catalase in the development of cataract in senile and diabetic persons. Serum levels of major anti-oxidant enzymes-superoxide dismutase and catalase were estimated in 20 patients with diabetic cataract, and matched with another 20 patients of senile cataract taken as control. Estimation of superoxide dismutase was done by improved spectrophotometric assay based on epinephrine auto-oxidation at 480nm, while catalase estimation was done by the method of Hugo Aebi. The mean serum levels of superoxide dismutase and catalase were lower as age increased. These enzymes were also significantly lower in diabetic cataracts (9.13 and 16.42 units/ml) as compared to senile cataracts (25.30 and 57.27units/ml). Oxidative stress is one of the major factors contributing to cataract formation. In diabetics where there is increased oxidative stress, the serum levels of the major anti-oxidant enzymes decrease, which lead to early cataract formation. It may be concluded that oxidative stress is an important factor in the development of diabetic cataracts and anti-oxidants may have a role in decreasing the incidence of cataract.

  6. A highly active and oxidation-resistant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chemical modification.

    PubMed

    Grøn, H; Bech, L M; Branner, S; Breddam, K

    1990-12-27

    The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1-10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222. It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.

  7. Use of a chemiluminescent detector for quantitation of nitric oxide produced in assays of denitrifying enzymes. [Achromobacter cycloblastes; Pseudomonas perfectomarini

    SciTech Connect

    Pai, G.; Payne, W.J.; Le Gall, J.

    1987-05-01

    The authors developed a closed-flow system that continuously sweeps away gases evolved in enzyme assay mixtures into a commercially available oxides of nitrogen analyzer for the quantitation of any nitric oxide (NO) present in these gases. The system is particularly useful for the study of both the stoichiometry and kinetics of NO production from nitrite by cell-free extracts of denitrifying bacteria and by the purified denitrifying nitrite reductases. In addition to its much greater sensitivity when compared to standard gas chromatographic techniques, the method offers some unique advantages since it allows measurements of initial reaction velocities, without the problems of product inhibition, which often limit kinetic studies of denitrifying enzymes. The system also allows the immediate quantitation of any NO produced and so would be useful in detecting the transient presence of this very reactive free radical, if it is produced as a free intermediate in whole cell suspensions of denitrifying bacteria respiring nitrate. The apparatus has been calibrated using a purified copper nitrite reductase from Achromobacter cycloclastes and cell-free extracts from Pseudomonas Perfectomarini, and its utility in studies of the kinetics and stoichiometry of NO production from nitrite confirmed by comparison with results obtained using manometric and gas chromatographic techniques.

  8. Galantamine administration reduces reactive astrogliosis and upregulates the anti-oxidant enzyme catalase in rats submitted to neonatal hypoxia ischemia.

    PubMed

    Odorcyk, F K; Nicola, F; Duran-Carabali, L E; Figueiró, F; Kolling, J; Vizuete, A; Konrath, E L; Gonçalves, C A; Wyse, A T S; Netto, C A

    2017-11-01

    Neonatal hypoxia ischemia (HI) plays a role in the etiology of several neurological pathologies and causes severe sequelae. Acetylcholine is a neurotransmitter in the central nervous system and cholinesterase inhibitors have demonstrated a positive action over HI induced deficits. In order to evaluate the effects of pre and post-hypoxia administrations of galantamine, a cholinesterase inhibitor, in a model of perinatal HI, Wistar rats in the post-natal day 7 (PND7) were subjected to a combination of unilateral occlusion of the right carotid artery with the exposure to a 1h hypoxia. Intraperitoneal injections of galantamine were administered in two different protocols: one pre and other post-hypoxia. The analysis of brain structures volume at PND45 showed that pre-hypoxia galantamine treatment prevented tissue injury to the ipsilesional hippocampus. Also, immunofluorescence showed HI-induced increase in the number of astrocytes that was prevented by pre-hypoxia treatment. Biochemical analysis was performed in the ipsilesional hippocampus at PND8 and revealed that pre-hypoxia galantamine treatment: 1) prevented the neuronal loss induced by HI; 2) reduced the HI-induced hypertrophy of astrocytes; and 3) caused an increase in the activity of the anti-oxidant enzyme catalase. Overall, treatment with galantamine was able to prevent the brain damage, increase the survival of neurons, reduce astrocytic reaction and increase the activity of the anti-oxidant enzyme catalase in rats submitted to neonatal hypoxia ischemia. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

  9. Crystal Structure of Reduced and of Oxidized Peroxiredoxin IV Enzyme Reveals a Stable Oxidized Decamer and a Non-disulfide-bonded Intermediate in the Catalytic Cycle*

    PubMed Central

    Cao, Zhenbo; Tavender, Timothy J.; Roszak, Aleksander W.; Cogdell, Richard J.; Bulleid, Neil J.

    2011-01-01

    Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue. PMID:21994946

  10. Salivary aldehyde dehydrogenase--reversible oxidation of the enzyme and its inhibition by caffeine, investigated using fluorimetric method.

    PubMed

    Wierzchowski, Jacek; Pietrzak, Monika; Szelag, Malgorzata; Wroczyński, Piotr

    2008-05-01

    We have applied fluorimetric method to monitor aldehyde dehydrogenase (ALDH*) activity in human saliva samples to study inactivation, reactivation and inhibition of the enzyme. Saliva samples were collected to buffer stock solution, containing various thiols, and assayed in the presence of the fluorogenic substrate 6-dimethylamino-2-naphthaldehyde and NAD(+). Fluorescence of the produced 6-dimethylamino-2-naphthalene carboxylate was used to measure the reaction rate. Kinetic parameters for the highly fluorogenic substrate, 6-dimethylamino-2-naphthaldehyde were measured, with apparent K(m) of 7.9 microM at pH 7.3. The apparent K(m) for NAD(+) was 1.2 microM. The observed ALDH activity is unstable in the absence of thiols, but can be stabilized by 1mM glutathione, and inactivated enzyme can be re-activated within 10 min by treatment of 0.5 mM DTT. Two-assay procedure was applied to measure degree of inactivation of ALDH in saliva samples. It was found that degree of ALDH inactivation in fresh samples, stabilized by glutathione, is between 0% and 90%, with average value ca. 40%. Caffeine and theophylline were shown to be moderate inhibitors of salivary ALDH. Oxidation of the salivary ALDH in fresh saliva may be reliably measured using fluorimetric two-assay procedure. Preliminary statistics indicate that in most individuals this enzyme is partially inactive. Inhibition of the salivary ALDH by caffeine may have consequences for nutrition safety.

  11. Fabrication of carbon-felt-based multi-enzyme immobilized anodes to oxidize sucrose for biofuel cells.

    PubMed

    Handa, Yutaka; Yamagiwa, Kiyofumi; Ikeda, Yutaro; Yanagisawa, Yuichi; Watanabe, Shinya; Yabuuchi, Naoaki; Komaba, Shinichi

    2014-07-21

    A carbon-felt-based multi-enzyme immobilized bioanode for biofuel cells has been successfully developed. The combination of four enzymes, namely, invertase, mutarotase, glucose oxidase, and fructose dehydrogenase, makes it possible to use sucrose--a disaccharide--as fuel for the bioanode. The new electrode exhibits a high oxidation current density of about 12 mA cm(-2) (0.25 V vs. Ag/AgCl) in a McIlvaine buffer solution containing sucrose at pH 6.0 in the half-cell configuration. A sucrose/O(2) biofuel cell composed of the bioanode and an opposite biocathode, including bilirubin oxidase as the enzymatic electrocatalyst, was fabricated, and the new device demonstrated a maximum power density of 2.90 mW cm(-2) with an open-circuit voltage of 0.69 V in the McIlvaine buffer solution. The biofuel cell fabricated using our multi-enzyme anode operates in commercially available beverages that contain only sugar, even without glucose.

  12. Enzymatic fuel cells based on electrodeposited graphite oxide/cobalt hydroxide/chitosan composite-enzyme electrode.

    PubMed

    Uk Lee, Hee; Young Yoo, Hah; Lkhagvasuren, Tseveg; Seok Song, Yoon; Park, Chulhwan; Kim, Jungbae; Wook Kim, Seung

    2013-04-15

    Enzymatic fuel cells (EFCs) use redox enzymes with high electron transfer rates that lead to high power density from bioavailable substrates. However, EFCs are limited by the difficult electrical wiring of the enzymes to the electrode. Therefore, deposition of Co(OH)₂ onto graphite oxide (GO) was improved for efficient wiring of the enzymes. The GO/Co(OH)₂/chitosan composites were electrodeposited for immobilization of glucose oxidase (GOD) or laccase on an Au electrode, respectively. The electrical properties of the bioelectrode according to cyclic voltammetry were improved using GO/Co(OH)₂/chitosan composites. The anode and cathode system was composed of GOD and laccase as biocatalysts and glucose/oxygen as substrates under ambient conditions (pH 7.0 and 25 °C). The EFC using GO/Co(OH)₂/chitosan composites with a mediator delivered a high power density of up to 517±3.3 μW/cm² at 0.46 V and open circuit voltage of 0.60 V. These results provide a promising direction for further development and application of EFCs.

  13. Enzyme-free electrochemical immunosensor based on methylene blue and the electro-oxidation of hydrazine on Pt nanoparticles.

    PubMed

    Dutta, Gorachand; Nagarajan, Sureshbabu; Lapidus, Lisa J; Lillehoj, Peter B

    2017-06-15

    Enzyme-free electrochemical sensors enable rapid, high sensitivity measurements without the limitations associated with enzyme reporters. However, the performance of non-enzymatic electrochemical sensors tends to suffer from slow electrode kinetics and poor signal stability. We report a new enzyme-free electrochemical immunosensor based on a unique competitive detection scheme using methylene blue (MB), hydrazine and platinum nanoparticles (Pt NPs). This scheme is coupled with a robust immunosandwich format employing a MB-labelled detection antibody as a non-enzymatic reporter. In the presence of the target antigen, surface-immobilized MB consumes interfacial hydrazine thereby diminishing the electro-oxidation of hydrazine on Pt NPs. Thus, the concentration of the antigen is directly proportional to the reduction in the electrochemical signal. For proof-of-concept, this sensor was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), an important malaria biomarker, in unadulterated human saliva samples. Chronocoulometric measurements showed that this platform exhibits pM-range sensitivity, high specificity and good reproducibility, making it well suited for many biosensing applications including noninvasive diagnostic testing.

  14. Organization of the Escherichia coli aerobic enzyme complexes of oxidative phosphorylation in dynamic domains within the cytoplasmic membrane

    PubMed Central

    Erhardt, Heiko; Dempwolff, Felix; Pfreundschuh, Moritz; Riehle, Marc; Schäfer, Caspar; Pohl, Thomas; Graumann, Peter; Friedrich, Thorsten

    2014-01-01

    The Escherichia coli cytoplasmic membrane contains the enzyme complexes of oxidative phosphorylation (OXPHOS). Not much is known about their supramolecular organization and their dynamics within the membrane in this model organism. In mitochondria and other bacteria, it was demonstrated by nondenaturing electrophoretic methods and electron microscopy that the OXPHOS complexes are organized in so-called supercomplexes, stable assemblies with a defined number of the individual enzyme complexes. To investigate the organization of the E. coli enzyme complexes of aerobic OXPHOS in vivo, we established fluorescent protein fusions of the NADH:ubiquinone oxidoreductase, the succinate:ubiquinone oxidoreductase, the cytochrome bd-I, and the cytochrome bo3 terminal oxidases, and the FoF1 ATP-synthase. The fusions were integrated in the chromosome to prevent artifacts caused by protein overproduction. Biochemical analysis revealed that all modified complexes were fully assembled, active, and stable. The distribution of the OXPHOS complexes in living cells was determined using total internal reflection fluorescence microscopy. The dynamics within the membrane were detected by fluorescence recovery after photobleaching. All aerobic OXPHOS complexes showed an uneven distribution in large mobile patches within the E. coli cytoplasmic membrane. It is discussed whether the individual OXPHOS complexes are organized as clustered individual complexes, here called “segrazones.” PMID:24729508

  15. sup 15 N and sup 13 C NMR studies of ligands bound to the 280,000-dalton protein porphobilinogen synthase elucidate the structures of enzyme-bound product and a Schiff base intermediate

    SciTech Connect

    Jaffe, E.K.; Rajagopalan, J.S. ); Markham, G.D. )

    1990-09-11

    Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). Despite the 280,000-dalton size of PBGS, much can be learned about the reaction mechanism through {sup 13}C and {sup 15}N NMR. The authors knowledge, these studies represent the largest protein complex for which individual nuclei have been characterized by {sup 13}C or {sup 15}N NMR. Here they extend their {sup 13}C NMR studies to PBGS complexes with (3,3-{sup 2}H{sub 2},3-{sup 13}C)ALA and report {sup 15}N NMR studies of ({sup 15}N)ALA bound to PBGS. As in their previous {sup 13}C NMR studies, observation of enzyme-bound {sup 15}N-labeled species was facilitated by deuteration at nitrogens that are attached to slowly exchanging hydrogens. For holo-PBGS at neutral pH, the NMR spectra reflect the structure of the enzyme-bound product porphobilinogen (PBG), whose chemical shifts are uniformly consistent with deprotonation of the amino group whose solution pK{sub a} is 11. Despite this local environment, the protons of the amino group are in rapid exchange with solvent. For methyl methanethiosulfonate (MMTS) modified PBGS, the NMR spectra reflect the chemistry of an enzyme-bound Schiff base intermediate that is formed between C{sub 4} of ALA and an active-site lysine. The {sup 13}C chemical shift of (3,3-{sup 2}H{sub 2},3-{sup 13}C)ALA confirms that the Schiff base is an imine of E stereochemistry. By comparison to model imines formed between ({sup 15}N)ALA and hydrazine or hydroxylamine, the {sup 15}N chemical shift of the enzyme-bound Schiff base suggests that the free amino group is an environment resembling partial deprotonation. Deprotonation of the amino group would facilitate formation of a Schiff base between the amino group of the enzyme-bound Schiff base and C{sub 4} of the second ALA substrate. This is the first evidence supporting carbon-nitrogen bond formation as the initial site of interaction between the two substrate molecules.

  16. Hypersalinity and hydrogen peroxide upregulation of gene expression of antioxidant enzymes in Ulva fasciata against oxidative stress.

    PubMed

    Sung, Ming-Shiuan; Hsu, Yi-Ting; Hsu, Yuan-Ting; Wu, Tzure-Meng; Lee, Tse-Min

    2009-01-01

    The modulation of manganese superoxide dismutase (MnSOD), FeSOD, ascorbate peroxidase (APX), glutathione reductase (GR), and catalase (CAT) gene expression and activities and antioxidants in Ulva fasciata against hypersalinity (90 per thousand)-induced oxidative stress was studied. Increases in H(2)O(2) contents but no changes in lipid peroxidation and protein carbonyl group contents suggest oxidative damage did not occur in 90 per thousand condition. Antioxidants were consumed for reactive oxygen species (ROS) scavenging indicated by decreased ascorbate and glutathione contents by 90 per thousand. Antioxidant enzymes were differently expressed by 90 per thousand for ROS removal. MnSOD activity and transcript increased 1 h after 90 per thousand treatment with a peak at hour 3, while FeSOD activity increased fast to the plateau after 1 h and its transcript increased after 3 h. APX activity increased 1 h after 90 per thousand but its transcript rose till 3 h, and GR activity increased after 1 h with a peak at hour 3 but its transcript increased till 3 h. CAT activity and transcript increased after 12 h. Enzyme activity is transcriptionally regulated by 90 per thousand except a fast increase in FeSOD, APX, and GR activities during 1 h. APX is responsible for early H(2)O(2) decomposition while CAT scavenges H(2)O(2) in the later period. The inhibition of 90 per thousand induced increase of H(2)O(2) content and FeSOD activity and transcript by treatment of a H(2)O(2) scavenger, dimethylthiourea, and the increase of FeSOD transcript of 30 per thousand grown thalli by H(2)O(2) treatment suggest that H(2)O(2) mediates the upregulation of FeSOD by hypersalinity while other enzymes is modulated by factors other than H(2)O(2).

  17. A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

    PubMed

    Acharya, A S; Taniuchi, H

    1976-11-25

    The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure

  18. Oxidation of biphenyl by a multicomponent enzyme system from Pseudomonas sp. strain LB400.

    PubMed Central

    Haddock, J D; Nadim, L M; Gibson, D T

    1993-01-01

    Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. This organism also cooxidizes several chlorinated biphenyl congeners. Biphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of biphenyl to cis-2,3-dihydroxy-2,3-dihydrobiphenyl. Incorporation of both atoms of molecular oxygen into the substrate was shown with 18O2. The nonlinear relationship between enzyme activity and protein concentration suggested that the enzyme is composed of multiple protein components. Ion-exchange chromatography of the cell extract gave three protein fractions that were required together to restore enzymatic activity. Similarities with other multicomponent aromatic hydrocarbon dioxygenases indicated that biphenyl dioxygenase may consist of a flavoprotein and iron-sulfur proteins that constitute a short electron transport chain involved in catalyzing the incorporation of both atoms of molecular oxygen into the aromatic ring. Images PMID:8419290

  19. Oxidation of methyl groups by grass grubs and vertebrate liver enzymes.

    PubMed

    Hook, G E; Smith, J N

    1967-02-01

    1. Oxidation rates of p-nitrotoluene, p-acetamidotoluene and p-toluidine by intact grass grubs and vertebrate liver preparations were measured. 2. The effect of p-substitution in increasing the rate of conversion of the methyl into a carboxy group was in the order acetamido> nitro[unk] amino in mice and grass grubs. 3. Rates of oxidation of the N-methyl group in some alkylaryl N-methylcarbamates was measured and the effect of ring substituents in increasing the rate was in the order hydrogen> o-methyl or o-isopropyl> p-methyl or p-isopropyl> m-methyl or m-isopropyl. 4. Rates of oxidation of the N-methyl groups were similar to those of the p-substituted toluenes.

  20. Allometric scaling in centrarchid fish: origins of intra- and inter-specific variation in oxidative and glycolytic enzyme levels in muscle.

    PubMed

    Davies, Rhiannon; Moyes, Christopher D

    2007-11-01

    The influence of body size on metabolic rate, muscle enzyme activities and the underlying patterns of mRNA for these enzymes were explored in an effort to explain the genetic basis of allometric variation in metabolic enzymes. We studied two pairs of sister species of centrarchid fish: black bass (largemouth bass Micropterus salmoides and smallmouth bass Micropterus dolomieui) and sunfish (pumpkinseed Lepomis gibbosus and bluegill Lepomis macrochirus). Our goal was to assess the regulatory basis of both intraspecific and interspecific variation relative to body size, as well as to gain insights into the evolutionary constraints within lineages. Whole animal routine metabolic rate showed scaling coefficients not significantly different from 1, ranging from (+0.87 to +0.96). However, there were significant effects of body size on the specific activities of oxidative and glycolytic enzymes. Mass-specific activity of the oxidative enzyme citrate synthase (CS) scaled negatively with body size in each species, with scaling coefficients ranging from -0.15 to -0.19, whereas the glycolytic enzyme pyruvate kinase (PK) showed positive scaling, with scaling coefficients ranging from +0.08 to +0.23. The ratio of mass-specific enzyme activity in PK to CS increased with body size, whereas the ratio of mRNA transcripts of PK to CS was unaffected, suggesting the enzyme relationships were not due simply to transcriptional regulation of both genes. The mass-dependent differences in PK activities were best explained by transcriptional regulation of the muscle PK gene; PK mRNA was a good predictor of PK specific enzyme activity within species and between species. Conversely, CS mRNA did not correlate with CS specific enzyme activities, suggesting post-transcriptional mechanisms may explain the observed inter-specific and intraspecific differences in oxidative enzymes.

  1. Kinetic and solvent deuterium isotope effects in the oxidation of putrescine catalysed by enzyme diamine oxidase.

    PubMed

    Pałka, Katarzyna; Szymańska, Jolanta; Kańska, Marianna

    2013-01-01

    In this study, the kinetic isotope effects and solvent isotope effects in the reaction of the deamination of [(1R)-(2)H ] putrescine--catalysed by enzyme diamine oxidase (EC 1.4.3.6)--were determined using a non-competitive spectroscopic method. Putrescine, stereospecifically labelled with deuterium, was obtained by enzymatic decarboxylation of l-ornithine that was carried out in a fully deuteriated incubation medium.

  2. Direct electron transfer with enzymes on nanofiliform titanium oxide films with electron-transport ability.

    PubMed

    Lee, So-Yoon; Matsuno, Ryosuke; Ishihara, Kazuhiko; Takai, Madoka

    2013-03-15

    Direct electron transfer (DET) from biomolecules to electrode is a process without electron-mediators, thus superior selectivity and sensitivity is expected in order to monitor electron transfer between electrode and biomolecules without any mediator interference. However, DET is difficult because a redox center which is an electron active center of proteins such as enzymes is buried deep. So, a unique electrode nanostructure to reach the redox center is a critical factor. Here we have systematically investigated terms for DET using various nanofiliformed electrode morphologies and enzyme concentrations. It is pointed out that the reaction site is below 100 nm, the ration amounts of adsorbed enzyme per surface area are below 1.0 are contributed to the DET. As a great application, we have developed a biosensor monitoring the hydrogen peroxide (H(2)O(2)) detecting capability from peroxidase directly. For the fabricated HRP/nTOF/Ti-electrodes observed the catalytic current value was linear according to the increase in the concentration of H(2)O(2) up to 100 μM, which indicates a good potential for an H(2)O(2) biosensor. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Green tea diet decreases PCB 126-induced oxidative stress in mice by up-regulating antioxidant enzymes.

    PubMed

    Newsome, Bradley J; Petriello, Michael C; Han, Sung Gu; Murphy, Margaret O; Eske, Katryn E; Sunkara, Manjula; Morris, Andrew J; Hennig, Bernhard

    2014-02-01

    Superfund chemicals such as polychlorinated biphenyls pose a serious human health risk due to their environmental persistence and link to multiple diseases. Selective bioactive food components such as flavonoids have been shown to ameliorate PCB toxicity, but primarily in an in vitro setting. Here, we show that mice fed a green tea-enriched diet and subsequently exposed to environmentally relevant doses of coplanar PCB exhibit decreased overall oxidative stress primarily due to the up-regulation of a battery of antioxidant enzymes. C57BL/6 mice were fed a low-fat diet supplemented with green tea extract (GTE) for 12 weeks and exposed to 5 μmol PCB 126/kg mouse weight (1.63 mg/kg-day) on weeks 10, 11 and 12 (total body burden: 4.9 mg/kg). F2-isoprostane and its metabolites, established markers of in vivo oxidative stress, measured in plasma via HPLC-MS/MS exhibited fivefold decreased levels in mice supplemented with GTE and subsequently exposed to PCB compared to animals on a control diet exposed to PCB. Livers were collected and harvested for both messenger RNA and protein analyses, and it was determined that many genes transcriptionally controlled by aryl hydrocarbon receptor and nuclear factor (erythroid-derived 2)-like 2 proteins were up-regulated in PCB-exposed mice fed the green tea-supplemented diet. An increased induction of genes such as SOD1, GSR, NQO1 and GST, key antioxidant enzymes, in these mice (green tea plus PCB) may explain the observed decrease in overall oxidative stress. A diet supplemented with green tea allows for an efficient antioxidant response in the presence of PCB 126, which supports the emerging paradigm that healthful nutrition may be able to bolster and buffer a physiological system against the toxicities of environmental pollutants. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Role of Myeloperoxidase Oxidants in the Modulation of Cellular Lysosomal Enzyme Function: A Contributing Factor to Macrophage Dysfunction in Atherosclerosis?

    PubMed Central

    Ismael, Fahd O.; Barrett, Tessa J.; Sheipouri, Diba; Brown, Bronwyn E.; Davies, Michael J.; Hawkins, Clare L.

    2016-01-01

    Low-density lipoprotein (LDL) is the major source of lipid within atherosclerotic lesions. Myeloperoxidase (MPO) is present in lesions and forms the reactive oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). These oxidants modify LDL and have been strongly linked with the development of atherosclerosis. In this study, we examined the effect of HOCl, HOSCN and LDL pre-treated with these oxidants on the function of lysosomal enzymes responsible for protein catabolism and lipid hydrolysis in murine macrophage-like J774A.1 cells. In each case, the cells were exposed to HOCl or HOSCN or LDL pre-treated with these oxidants. Lysosomal cathepsin (B, L and D) and acid lipase activities were quantified, with cathepsin and LAMP-1 protein levels determined by Western blotting. Exposure of J774A.1 cells to HOCl or HOSCN resulted in a significant decrease in the activity of the Cys-dependent cathepsins B and L, but not the Asp-dependent cathepsin D. Cathepsins B and L were also inhibited in macrophages exposed to HOSCN-modified, and to a lesser extent, HOCl-modified LDL. No change was seen in cathepsin D activity or the expression of the cathepsin proteins or lysosomal marker protein LAMP-1. The activity of lysosomal acid lipase was also decreased on treatment of macrophages with each modified LDL. Taken together, these results suggest that HOCl, HOSCN and LDL modified by these oxidants could contribute to lysosomal dysfunction and thus perturb the cellular processing of LDL, which could be important during the development of atherosclerosis. PMID:27997605

  5. Selenium ameliorates arsenic induced oxidative stress through modulation of antioxidant enzymes and thiols in rice (Oryza sativa L.).

    PubMed

    Kumar, Amit; Singh, Rana Pratap; Singh, Pradyumna Kumar; Awasthi, Surabhi; Chakrabarty, Debasis; Trivedi, Prabodh Kumar; Tripathi, Rudra Deo

    2014-09-01

    Arsenic (As) contamination of rice is a major problem for South-East Asia. In the present study, the effect of selenium (Se) on rice (Oryza sativa L.) plants exposed to As was studied in hydroponic culture. Arsenic accumulation, plant growth, thiolic ligands and antioxidative enzyme activities were assayed after single (As and Se) and simultaneous supplementations (As + Se). The results indicated that the presence of Se (25 µM) decreased As accumulation by threefold in roots and twofold in shoots as compared to single As (25 µM) exposed plants. Arsenic induced oxidative stress in roots and shoots was significantly ameliorated by Se supplementation. The observed positive response was found associated with the increased activities of ascorbate peroxidase (APX; EC 1.11.1.11), catalase (CAT; EC 1.11.1.6) and glutathione peroxidase (GPx; EC 1.11.1.9) and induced levels of non-protein thiols (NPTs), glutathione (GSH) and phytochelatins (PCs) in As + Se exposed plants as compared to single As treatment. Selenium supplementation modulated the thiol metabolism enzymes viz., γ-glutamylcysteine synthetase (γ-ECS; EC 6.3.2.2), glutathione-S-transferase (GST; EC 2.5.1.18) and phytochelatin synthase (PCS; EC 2.3.2.15). Gene expression analysis of several metalloid responsive genes (LOX, SOD and MATE) showed upregulation during As stress, however, significant downregulation during As + Se exposure as compared to single As treatment. Gene expressions of enzymes of antioxidant and GSH and PC biosynthetic systems, such as APX, CAT, GPx, γ-ECS and PCS were found to be significantly positively correlated with their enzyme activities. The findings suggested that Se supplementation could be an effective strategy to reduce As accumulation and toxicity in rice plants.

  6. Effect of fat feeding on pro-oxidant and anti-oxidant enzyme systems in rat intestine: possible role in the turnover of enterocytes.

    PubMed

    Turan, Aasma; Gill, Ravinder; Dudeja, Pradeep K; Mohan, Harsh; Mahmood, Akhtar

    2009-06-01

    Immature epithelial cells generated in the crypt base undergo differentiation while progressing to the villus tip, where the cells upon apoptosis are detached from the underlying muscular tissue. We previously reported that lipid peroxidation might be involved in the turnover of enterocytes across the crypt-villus axis in rat intestine (Dig Dis Sci 52:1840-1844, 2007). To examine whether long-term feeding of fat with different fatty-acid composition influences this process, in the present study we investigated the effect of feeding fish oil (n - 3) and corn oil (n - 6) polyunsaturated fatty acids on lipid per-oxidation and anti-oxidant systems in different epithelial cell fractions isolated in rat intestine. Feeding fish oil or corn oil markedly enhanced lipid per-oxidation levels of enterocytes throughout villus height compared with control, but there was no difference in the distribution profile of pro- and anti-oxidant enzyme systems and lipid per-oxidation across the crypt-villus axis under these conditions. Analysis of lipid peroxidation levels in different cell fractions revealed that the thiobarbituric acid reactive substance were 9- to 11-fold higher at the villus tip compared with at the crypt base. The activities of glutathione reductase and glutathione-S-transferase were 2- to 5-fold higher in villus tip compared to the crypt region. However, the activities of superoxide dismutase and catalase were 6- to 8-fold high at the crypt base compared with at villus tip cells. Immunocytolocalization of superoxide dismutase showed high staining in crypt base compared with that in villus, tip cells. These findings further suggest that generation of reactive oxygen species in enterocytes across the crypt-villus axis may be involved in turnover of enterocytes across the crypt-villus unit in rat intestine.

  7. An Improved Method for Studying the Enzyme-Catalyzed Oxidation of Glucose Using Luminescent Probes

    ERIC Educational Resources Information Center

    Bare, William D.; Pham, Chi V.; Cuber, Matthew; Demas, J. N.

    2007-01-01

    A new method is presented for measuring the rate of the oxidation of glucose in the presence of glucose oxidase. The improved method employs luminescence measurements to directly determine the concentration of oxygen in real time, thus obviating complicated reaction schemes employed in previous methods. Our method has been used to determine…

  8. An Improved Method for Studying the Enzyme-Catalyzed Oxidation of Glucose Using Luminescent Probes

    ERIC Educational Resources Information Center

    Bare, William D.; Pham, Chi V.; Cuber, Matthew; Demas, J. N.

    2007-01-01

    A new method is presented for measuring the rate of the oxidation of glucose in the presence of glucose oxidase. The improved method employs luminescence measurements to directly determine the concentration of oxygen in real time, thus obviating complicated reaction schemes employed in previous methods. Our method has been used to determine…

  9. Oxidation of Acenaphthene and Acenaphthylene by Human Cytochrome P450 Enzymes

    PubMed Central

    Shimada, Tsutomu; Takenaka, Shigeo; Murayama, Norie; Yamazaki, Hiroshi; Kim, Joo-Hwan; Kim, Donghak; Yoshimoto, Francis K.; Guengerich, F. Peter; Komori, Masayuki

    2016-01-01

    Acenaphthene and acenaphthylene, two known environmental polycyclic aromatic hydrocarbon (PAH) pollutants, were incubated at 50 µM concentrations in a standard reaction mixture with human P450s 2A6, 2A13, 1B1, 1A2, 2C9, and 3A4 and the oxidation products were determined using HPLC and LC-MS. HPLC analysis showed that P450 2A6 converted acenaphthene and acenaphthylene to several mono- and di-oxygenated products. LC-MS analysis of acenaphthene oxidation by P450s indicated the formation of 1-acenaphthenol as a major product, with turnover rates of 6.7, 4.5, and 3.6 nmol product formed/min/nmol P450 for P450 2A6, 2A13, and 1B1, respectively. Acenaphthylene oxidation by P450 2A6 showed the formation of 1,2-epoxyacenaphthene as a major product (4.4 nmol epoxide formed/min/nmol P450) and also several mono- and di-oxygenated products. P450 2A13, 1B1, 1A2, 2C9, and 3A4 formed 1,2-epoxyacenaphthene at rates of 0.18, 5.3 2.4, 0.16, and 3.8 nmol/min nmol P450, respectively. 1-Acenaphthenol, which induced Type I binding spectra with P450 2A13, was further oxidized by P450 2A13 but not P450 2A6. 1,2-Epoxyacenaphthene induced Type I binding spectra with P450 2A6 and 2A13 (Ks 1.8 and 0.16 µM, respectively) and was also oxidized to several oxidation products by these P450s. Molecular docking analysis suggested different orientations of acenaphthene, acenaphthylene, 1-acenaphthenol, and 1,2-epoxyacenaphthene in their interactions with P450 2A6 and 2A13. Neither these four PAHs induced umu gene expression in a Salmonella typhimurium NM tester strain. These results suggest, for the first time, that acenaphthene and acenaphthylene are oxidized by human P450s 2A6 and 2A13 and other P450s to form several mono- and di-oxygenated products. The results are of use in considering the biological and toxicological significance of these environmental PAHs in humans. PMID:25642975

  10. Oxidative stress and inflammation in mucopolysaccharidosis type IVA patients treated with enzyme replacement therapy.

    PubMed

    Donida, Bruna; Marchetti, Desirèe P; Biancini, Giovana B; Deon, Marion; Manini, Paula R; da Rosa, Helen T; Moura, Dinara J; Saffi, Jenifer; Bender, Fernanda; Burin, Maira G; Coitinho, Adriana S; Giugliani, Roberto; Vargas, Carmen Regla

    2015-05-01

    Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism due to the deficient activity of N-acetylgalactosamine-6-sulfate sulfatase that leads to accumulation of the keratan sulfate and chondroitin 6-sulfate in body fluids and in lysosomes. The pathophysiology of this lysosomal storage disorder is not completely understood. The aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokine and GAG levels in MPS IVA patients. We analyzed urine and blood samples from patients under ERT (n=17) and healthy age-matched controls (n=10-15). Patients presented a reduction of antioxidant defense levels, assessed by a decrease in glutathione content and by an increase in superoxide dismutase activity in erythrocytes. Concerning lipid and protein damage, it was verified increased urine isoprostanes and di-tyrosine levels and decreased plasma sulfhydryl groups in MPS IVA patients compared to controls. MPS IVA patients showed higher DNA damage than control group and this damage had an oxidative origin in both pyrimidine and purine bases. Interleukin 6 was increased in patients and presented an inverse correlation with GSH levels, showing a possible link between inflammation and oxidative stress in MPS IVA disease. The data presented suggest that pro-inflammatory and pro-oxidant states occur in MPS IVA patients even under ERT. Taking these results into account, supplementation of antioxidants in combination with ERT can be a tentative therapeutic approach with the purpose of improving the patient's quality of life. To the best of our knowledge, this is the first study relating MPS IVA patients with oxidative stress. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Lifestyle predictors of oxidant and antioxidant enzyme activities and total antioxidant capacity in healthy women: a cross-sectional study.

    PubMed

    Mahasneh, Amjad A; Zhang, Yali; Zhao, Hua; Ambrosone, Christine B; Hong, Chi-Chen

    2016-12-01

    The aim of this study was to identify demographic and modifiable lifestyle factors that may be related to endogenous oxidant and antioxidant activity measured in blood specimens from putatively healthy women recruited at the Roswell Park Cancer Institute (Buffalo, NY, USA). Total glutathione (TGSH), catalase (CAT), CuZn-superoxide dismutase (CuZn-SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and myeloperoxidase (MPO) activity, and total antioxidant capacity (TAC) were measured in 124 healthy women, and associations with epidemiological factors were tested using general linear models. There were significant differences in oxidant and antioxidant enzyme activities according to lifestyle factors, after adjusting for duration of blood storage and season of blood draw. Compared to women who consumed ≤2.8 servings of fruits and vegetables daily, those consuming >5.3 servings had on average 31 % lower MPO activity (p-trend = 0.02), as a marker of oxidative stress, 16 % higher antioxidant GPx activity (p-trend = 0.08), and 9 % higher TAC (p-trend = 0.05). Obese women (body mass index, BMI ≥ 30) in contrast showed 17 % lower antioxidant GPx activity, 44 % higher MPO activity (p-trend = 0.03), and 10 % higher TAC (p-trend = 0.03) compared to women with normal BMI < 25. Smoking was associated with higher TGSH activity (p-trend = 0.01) and lower TAC (p-trend = 0.05). Higher TAC levels were most strongly associated with increasing age (standardized β = 0.40, p < 0.0001), BMI (standardized β = 0.17, p = 0.03), and GPx activity (standardized β = 0.23, p = 0.005), and inversely associated with CuZn-SOD activity (standardized β = -0.14, p = 0.07). Physical activity levels, multivitamin use, and alcohol intake were not associated with TAC. Our data indicate that endogenous oxidant and antioxidant enzyme activities are associated with lifestyle factors and, therefore, may be potentially modifiable, with implications

  12. Zebrafish high-throughput screening to study the impact of dissolvable metal oxide nanoparticles on the hatching enzyme, ZHE1.

    PubMed

    Lin, Sijie; Zhao, Yan; Ji, Zhaoxia; Ear, Jason; Chang, Chong Hyun; Zhang, Haiyuan; Low-Kam, Cecile; Yamada, Kristin; Meng, Huan; Wang, Xiang; Liu, Rong; Pokhrel, Suman; Mädler, Lutz; Damoiseaux, Robert; Xia, Tian; Godwin, Hilary A; Lin, Shuo; Nel, André E

    2013-05-27

    The zebrafish is emerging as a model organism for the safety assessment and hazard ranking of engineered nanomaterials. In this Communication, the implementation of a roboticized high-throughput screening (HTS) platform with automated image analysis is demonstrated to assess the impact of dissolvable oxide nanoparticles on embryo hatching. It is further demonstrated that this hatching interference is mechanistically linked to an effect on the metalloprotease, ZHE 1, which is responsible for degradation of the chorionic membrane. The data indicate that 4 of 24 metal oxide nanoparticles (CuO, ZnO, Cr2 O3 , and NiO) could interfere with embryo hatching by a chelator-sensitive mechanism that involves ligation of critical histidines in the ZHE1 center by the shed metal ions. A recombinant ZHE1 enzymatic assay is established to demonstrate that the dialysates from the same materials responsible for hatching interference also inhibit ZHE1 activity in a dose-dependent fashion. A peptide-based BLAST search identifies several additional aquatic species that express enzymes with homologous histidine-based catalytic centers, suggesting that the ZHE1 mechanistic paradigm could be used to predict the toxicity of a large number of oxide nanoparticles that pose a hazard to aquatic species.

  13. Effect of Tai Chi exercise on DNA damage, antioxidant enzymes, and oxidative stress in middle-age adults.

    PubMed

    Goon, J A; Aini, A H Noor; Musalmah, M; Anum, M Y Yasmin; Nazaimoon, W M Wan; Ngah, W Z Wan

    2009-01-01

    The biochemical mechanisms involving oxidative stress to explain the relationship between exercise and healthy aging are still unclear. Tai Chi participants and matched sedentary volunteers age 45 and above were enrolled. Glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) activities; levels of DNA damage using the comet assay; and malondialdehyde (MDA) and advanced glycation end products (AGE) were determined at 0, 6, and 12 months. Tai Chi subjects had decreased normal and increased mildly damaged DNA with elevated GPx activity after 6 months (n=25). Plasma MDA and AGE concentrations decreased significantly after 12 months (n=15) accompanied by increased SOD activity. This may be attributed to the hormesis effect, whereby mild induction of oxidative stress at the first 6 months of exercise resulted in stimulation of antioxidant defenses. These parameters were unchanged in the sedentary subjects in the first 6 months (n=27) except for elevated SOD activity. After 12 months, the sedentary subjects (n=17) had decreased normal DNA and increased severely damaged DNA with unaltered MDA and AGE levels while SOD and GPx activities were significantly elevated. Regular Tai Chi exercise stimulated endogenous antioxidant enzymes and reduced oxidative damage markers.

  14. Effects of dietary sodium selenite and selenium yeast on antioxidant enzyme activities and oxidative stability of chicken breast meat.

    PubMed

    Ahmad, Hussain; Tian, Jinke; Wang, Jianjun; Khan, Muhammad Ammar; Wang, Yuanxiao; Zhang, Lili; Wang, Tian

    2012-07-25

    The effects of sodium selenite (SS) and selenium yeast (SY) alone and in combination (MS) on the selenium (Se) content, antioxidant enzyme activities (AEA), total antioxidant capacity (TAC), and oxidative stability of chicken breast meat were investigated. The results showed that the highest (p < 0.05) glutathione peroxidase (GSH-Px) activity was found in the SS-supplemented chicken breast meat; however, SY and MS treatments significantly increased (p < 0.05) the Se content and the activities of catalase (CAT), total superoxide dismutase (T-SOD), and TAC, but decreased (p < 0.05) the malondialdehyde (MDA) content at 42 days of age. Twelve days of storage at 4 °C decreased (p < 0.05) the activity of the GSH-Px, but CAT, T-SOD, and TAC remained stable. SY decreased the lipid oxidation more effectively in chicken breast meat. It was concluded that SY and MS are more effective than SS in increasing the AEA, TAC, and oxidative stability of chicken breast meat.

  15. Oxidative stress and antioxidant enzymes activities in the African catfish, Clarias gariepinus, experimentally challenged with Escherichia coli and Vibrio fischeri.

    PubMed

    Adeyemi, Joseph A

    2014-04-01

    The impacts of bacterial infection on cultivated fish species, African catfish, were investigated using oxidative stress biomarkers [lipid peroxidation (LPO) and protein carbonylation] and the activities of important antioxidant/detoxifying enzymes [catalase and glutathione S-transferase (GST)]. Fish were inoculated via oral gavage with one of the following treatments: 1 × 10(5) CFU/ml of Escherichia coli (EC1), 2 × 10(5) CFU/ml of E. coli (EC2), 1 × 10(5) CFU/ml of Vibrio fischeri (V1), 2 × 10(5) CFU/ml of V. fischeri (V2), gavaged with distilled water and not gavaged. Fish were maintained in the laboratory for 7 days after the bacterial inoculation, and the levels of LPO, protein carbonylation, GST, and catalase activities were determined in the muscle, gills, and liver of fish. Fish inoculated with bacteria (either E. coli or V. fischeri) had a significant higher levels of tissue LPO, protein carbonylation, and GST activities in a tissue-specific pattern (liver > muscle > gills). This appears to be related with the levels of bacterial inoculation, with effects more pronounced in fish inoculated with either EC2 or V2. The catalase activity did not differ significantly between the inoculated and fish that were not inoculated. The results of this study indicate that bacterial inoculation could result in oxidative stress in fish, and liver has a higher rate of oxidative stress per mg tissue compared to the gills and the muscle.

  16. A highly sensitive enzyme-amplified immunosensor based on a nanoporous niobium oxide (Nb2O5) electrode.

    PubMed

    Lee, Chang-Soo; Kwon, Dohyoung; Yoo, Jeng Eun; Lee, Byung Gun; Choi, Jinsub; Chung, Bong Hyun

    2010-01-01

    We report on the development of an enzyme-amplified sandwich-type immunosensor based on a thin gold film sputtered on an anodic nanoporous niobium oxide (Au@Nb(2)O(5)) electrode. The electrocatalytic activity of enzymatically amplified electroactive species and a stable electrode consisting of Au@Nb(2)O(5) were used to obtain a powerful signal amplification of the electrochemical immunobiosensor. The method using this electrochemical biosensor based on an Au@Nb(2)O(5) electrode provides a much better performance than those based on conventional bulk gold or niobium oxide electrodes. Our novel approach does not require any time-consuming cleaning steps to yield reproducible electrochemical signals. In addition, the strong adhesion of gold films on the niobium oxide electrodes offers a very stable substrate during electrochemical biosensing. Cyclic voltammetry measurements indicate that non-specific binding of proteins to the modified Au@Nb(2)O(5) surface is sufficiently low to be ignored in the case of our novel system. Finally, we demonstrated the ability of the biosensor based on an Au@Nb(2)O(5) offering the enhanced performance with a high resolution and sensitivity. Therefore, it is expected that the biosensor based on an Au@Nb(2)O(5) has great potential for highly efficient biological devices.

  17. Biomimetic oxidation studies. 9. Mechanistic aspects of the oxidation of alcohols with functional,active site methane monooxygenase enzyme models in aqueous solution

    SciTech Connect

    Rabion, A. ||; Chen, S.; Wang, J.; Buchanan, R.M.; Seris, J.L.; Fish, R.H. |

    1995-12-13

    The syntheses of biomimetic complexes that mimic the major structural features of the hydroxylase component of methane monooxygenase enzyme (MMO) and, more importantly, that provide similar alkane functionalization activity, in the presence of an oxidant, have been of great interest to the discipline of bioinorganic chemistry. In this communication, we will demonstrate the feasibility of conducting biomimetic oxidation studies in H{sub 2}O with soluble substrates, i.e., alcohols (cyclohexanol, benzyl alcohol), using H{sub 2}O-stable MMO mimics at pH 4.2, and the oxidant, tert-butyl hydroperoxide (TBHP). Both the Mitusunobu procedure and the mesylate displacement reaction proceeded with complete inversion of the stereo-center and provided optically pure penultimate intermediate (>99.9% ee). The synthesis was completed by reduction of the nitro group under standard conditions to deliver LY300164 in 87%. In summary, we have developed an efficient and environmentally benign synthesis of the 5H-2,3-benzodiazepine LY300164 that provides the optically pure compound in 51% overall yield. Intramolecular hydrazone alkylation led to a remarkably facile and selective formation of the benzodiazepine. Furthermore, the application of resins to whole-cell-based biotransformations should find general utility for similar reactions that are complicated by component inhibition and product isolation. 11 refs., 1 fig.

  18. Oxidative Stress and Digestive Enzyme Activity of Flatfish Larvae in a Changing Ocean.

    PubMed

    Pimentel, Marta S; Faleiro, Filipa; Diniz, Mário; Machado, Jorge; Pousão-Ferreira, Pedro; Peck, Myron A; Pörtner, Hans O; Rosa, Rui

    2015-01-01

    Until now, it is not known how the antioxidant and digestive enzymatic machinery of fish early life stages will change with the combined effects of future ocean acidification and warming. Here we show that high pCO2 (~1600 μatm) significantly decreased metabolic rates (up to 27.4 %) of flatfish larvae, Solea senegalensis, at both present (18 °C) and warmer temperatures (+4 °C). Moreover, both warming and hypercapnia increased the heat shock response and the activity of antioxidant enzymes, namely catalase (CAT) and glutathione S-transferase (GST), mainly in post-metamorphic larvae (30 dph). The lack of changes in the activity of CAT and GST of pre-metamorphic larvae (10 dph) seems to indicate that earlier stages lack a fully-developed antioxidant defense system. Nevertheless, the heat shock and antioxidant responses of post-metamorphic larvae were not enough to avoid the peroxidative damage, which was greatly increased under future environmental conditions. Digestive enzymatic activity of S. senegalensis larvae was also affected by future predictions. Hypercapnic conditions led to a decrease in the activity of digestive enzymes, both pancreatic (up to 26.1 % for trypsin and 74.5 % for amylase) and intestinal enzymes (up to 36.1 % for alkaline phosphatase) in post-metamorphic larvae. Moreover, the impact of ocean acidification and warming on some of these physiological and biochemical variables (namely, lower OCR and higher HSP and MDA levels) were translated into larvae performance, being significantly correlated with decreased larval growth and survival or increased incidence of skeletal deformities. The increased vulnerability of flatfish early life stages under future ocean conditions is expected to potentially determine recruitment and population dynamics in marine ecosystems.

  19. Oxidative Stress and Digestive Enzyme Activity of Flatfish Larvae in a Changing Ocean

    PubMed Central

    Pimentel, Marta S.; Faleiro, Filipa; Diniz, Mário; Machado, Jorge; Pousão-Ferreira, Pedro; Peck, Myron A.; Pörtner, Hans O.; Rosa, Rui

    2015-01-01

    Until now, it is not known how the antioxidant and digestive enzymatic machinery of fish early life stages will change with the combined effects of future ocean acidification and warming. Here we show that high pCO2 (~1600 μatm) significantly decreased metabolic rates (up to 27.4 %) of flatfish larvae, Solea senegalensis, at both present (18 °C) and warmer temperatures (+4 °C). Moreover, both warming and hypercapnia increased the heat shock response and the activity of antioxidant enzymes, namely catalase (CAT) and glutathione S-transferase (GST), mainly in post-metamorphic larvae (30 dph). The lack of changes in the activity of CAT and GST of pre-metamorphic larvae (10 dph) seems to indicate that earlier stages lack a fully-developed antioxidant defense system. Nevertheless, the heat shock and antioxidant responses of post-metamorphic larvae were not enough to avoid the peroxidative damage, which was greatly increased under future environmental conditions. Digestive enzymatic activity of S. senegalensis larvae was also affected by future predictions. Hypercapnic conditions led to a decrease in the activity of digestive enzymes, both pancreatic (up to 26.1 % for trypsin and 74.5 % for amylase) and intestinal enzymes (up to 36.1 % for alkaline phosphatase) in post-metamorphic larvae. Moreover, the impact of ocean acidification and warming on some of these physiological and biochemical variables (namely, lower OCR and higher HSP and MDA levels) were translated into larvae performance, being significantly correlated with decreased larval growth and survival or increased incidence of skeletal deformities. The increased vulnerability of flatfish early life stages under future ocean conditions is expected to potentially determine recruitment and population dynamics in marine ecosystems. PMID:26221723

  20. Structures of reduced and ligand-bound nitric oxide reductase provide insights into functional differences in respiratory enzymes.

    PubMed

    Sato, Nozomi; Ishii, Shoko; Sugimoto, Hiroshi; Hino, Tomoya; Fukumori, Yoshihiro; Sako, Yoshihiko; Shiro, Yoshitsugu; Tosha, Takehiko

    2014-07-01

    Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N2O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non-heme Fe center. We report herein on the structures of the reduced and ligand-bound forms of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3-2.7 Å, to elucidate structure-function relationships in NOR, and compare them to those of cytochrome c oxidase (CCO) that is evolutionarily related to NOR. Comprehensive crystallographic refinement of the CO-bound form of cNOR suggested that a total of four atoms can be accommodated at the binuclear center. Consistent with this, binding of bulky acetaldoxime (CH3-CH=N-OH) to the binuclear center of cNOR was confirmed by the structural analysis. Active site reduction and ligand binding in cNOR induced only ∼0.5 Å increase in the heme/non-heme Fe distance, but no significant structural change in the protein. The highly localized structural change is consistent with the lack of proton-pumping activity in cNOR, because redox-coupled conformational changes are thought to be crucial for proton pumping in CCO. It also permits the rapid decomposition of cytotoxic NO in denitrification. In addition, the shorter heme/non-heme Fe distance even in the bulky ligand-bound form of cNOR (∼4.5 Å) than the heme/Cu distance in CCO (∼5 Å) suggests the ability of NOR to maintain two NO molecules within a short distance in the confined space of the active site, thereby facilitating N-N coupling to produce a hyponitrite intermediate for the generation of N2O.

  1. The role of calcium and nitric oxide during liver enzyme release induced by increased physical forces as evidenced in partially hepatectomized rats.

    PubMed

    Díaz-Juárez, Julieta; Hernández-Muñoz, Rolando

    2011-03-01

    Although increased plasma enzyme activities could be diagnostic for tissue damage, the mechanisms controlling cellular enzyme release remain poorly understood. We found a selective and drastic elevation of serum enzyme activities accompanying rat liver regeneration after partial hepatectomy (PH), apparently controlled by a mechanism dependent on flow-bearing physical forces. In fact, this study assesses a putative role of calcium mobilization and nitric oxide (NO) production underlying rat liver enzyme release. The role of increased shear stress (by enhancing viscosity during perfusion) and the participation of cell calcium and NO were tested in isolated livers subjected to increasing flow rate. After PH, there was a drastic elevation of serum activities for liver enzyme markers, clearly predominating those of mitochondrial localization. Liver enzyme release largely depended on extracellular calcium entry, probably mediated by stretch-sensitive calcium channels, as well as by increasing NO production. However, these effects were differentially observed when comparing liver enzymes from cytoplasmic or mitochondrial compartments. Moreover, a possible role for cell-mediated mechanotransduction in liver enzyme release was suggested by increasing shear stress (high viscosity), which also selectively affected the release of the enzymes tested. Therefore, we show, for the first time, that flow-induced shear stress can control the amount of hepatic enzymes released into the bloodstream, which is largely regulated through modifications in cell calcium mobilization and production of liver NO, events markedly elevated in the proliferating rat liver. Copyright © 2011 American Association for the Study of Liver Diseases.

  2. Prolonged constriction of sciatic nerve affecting oxidative stressors & antioxidant enzymes in rat.

    PubMed

    Varija, Devarapali; Kumar, K P; Reddy, K P; Reddy, V K

    2009-05-01

    This study was carried out to determine the effects of reactive oxygen species in the balance between the pro-oxidant and antioxidant levels in experimental peripheral constriction injury induced by silver wire looping of sciatic nerve of rats. Rats were divided into experimental group 1 (silver wire ligated) and group 2 (control, sham operated). Functional and behavioural activities were assessed by a modified Basso Beattie Bresnahan (BBB) locomotory rating scale. Mechanical pain intensity was measured with Randall and Selitto apparatus. Foot positioning, toe spread, paw withdrawal threshold and paw withdrawal latency were carried out on days 1, 3, 7, 14, 21 and 28 in rats with chronic pain. Oxidative stress markers such as malondialdehyde (MDA) and advanced oxidation protein products (AOPP) were measured along with antioxidants such as glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) on day 30 after constriction in sciatic nerve, spinal cord, dorsal root ganglion, dorsal root and ventral root. Significant (P<0.05) increase in MDA, AOPP, SOD and GPx and decrease in the GSH and catalase activities in sciatic nerve, spinal cord, dorsal root ganglion, dorsal root and ventral root were observed in experimental group rats compared to control group. There was no recovery in foot positioning and toe spread. Reduced paw withdrawal threshold and paw withdrawal latency was observed in ligated rats compared to control rats. Foot positioning, toe spread, paw withdrawal threshold and paw withdrawal latency with no recovery until day 30 confirmed locomotory deficits, hyperalgesia and neuronal impairment. Oxidative stress evidenced by increased MDA, AOPP and decreased GSH and catalase support the generation of reactive oxygen species in constriction model. The present experimental model for chronic pain induced by silver wire spirally coiled around sciatic nerve may be useful for future studies.

  3. Association of serum paraoxonase enzyme activity and oxidative stress markers with dyslipidemia in obese adolescents

    PubMed Central

    Zaki, Moushira Erfan; El-Bassyouni, Hala; Kamal, Sanaa; El-Gammal, Mona; Youness, Eman

    2014-01-01

    Objectives: The aim of the present study was to investigate the serum paraoxonase 1 (PON1) concentration and oxidative stress markers and assess its relations with the biochemical parameters in obese adolescents. Materials and Methods: One hundred and fifty obese adolescents (range 16-18 years) and 150 healthy age- and sex-matched controls were enrolled in the study. The data were extracted from a project entitled “Obesity among Youth: Lifestyle and Genetic Factors” funded by the Science and Technology Development Fund, Egypt. Serum paraoxonase 1 (PON1), nitric oxide (NO), and malonaldehyde were measured. Anthropometry, fasting glucose, insulin concentrations, total cholesterol, high density lipoprotein–cholesterol, low density lipoprotein–cholesterol, triglycerides, systolic and diastolic blood pressure (BP) were measured. Insulin resistance was determined by Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). Diagnostic accuracy of oxidative markers to identify dyslipidemia was calculated with ROC analysis. Results: The study showed that PON1 activity was significantly lower in obese adolescents than controls. Obese adolescents had significant lower NO level and significant increased MA values as compared to controls. PON1 was negatively correlated with MAD and body mass index in obese subjects. Obese adolescents showed dyslipidemia and increased blood pressure and HOMA-IR values. PON1 had high area under the curve in ROC analysis for identifying dyslipidemia in obese subjects. Conclusions: Our results indicate that obese subjects have increased oxidative stress and decreased PON1 activity. The lower paraoxonase level might contribute to the greater risk of dyslipidemia, insulin resistance, high blood pressure that are considered as important components in the pathogenesis of the metabolic syndrome in obese adolescents. PMID:24944928

  4. Crosslinked redox polymer enzyme electrodes containing carbon nanotubes for high and stable glucose oxidation current.

    PubMed

    MacAodha, Domhnall; Ferrer, Maria Luisa; Conghaile, Peter Ó; Kavanagh, Paul; Leech, Dónal

    2012-11-14

    Co-immobilisation approaches for preparation of glucose-oxidising films of [Os(2,2'-bipyridine)(2)(poly-vinylimidazole)(10)Cl] and glucose oxidase on glassy carbon electrodes are compared. Electrodes prepared by crosslinking using glutaraldehyde vapour, without and with a NaBH(4) reduction, provide higher glucose oxidation current than those prepared using a well-established diepoxide method. Addition of multi walled carbon nanotubes to the film deposition solutions produces an enhanced glucose oxidation current density of 5 mA cm(-2) at 0.35 V vs. Ag/AgCl, whilst improving the operational stability of the current signal. Carbon nanotube, glutaraldehyde vapour crosslinked, films on electrodes, reduced by NaBH(4), retain 77% of initial catalytic current over 24 hours of continuous amperometric testing in a 37 °C, 50 mM phosphate buffer solution containing 150 mM NaCl and 100 mM glucose. Potential application of this approach to implantable enzymatic biofuel cells is demonstrated by production of glucose oxidation currents, under pseudo-physiological conditions, using mediating films with lower redox potentials.

  5. Quercetin changes purinergic enzyme activities and oxidative profile in platelets of rats with hypothyroidism.

    PubMed

    Baldissarelli, Jucimara; Santi, Adriana; Schmatz, Roberta; Zanini, Daniela; Cardoso, Andréia M; Abadalla, Fátima H; Thomé, Gustavo R; Murussi, Camila; Polachini, Carla R N; Delenogare, Diéssica P; Loro, Vania L; Morsch, Vera M; Schetinger, Maria R C

    2016-12-01

    Diseases related to thyroid hormones have been extensively studied because affect a large number of individuals, and these hormones participate in the regulation of the whole organism homeostasis. However, little is known about the involvement of purinergic signaling related to oxidative stress in hypothyroidism and possible therapeutic adjuncts for treatment of this disorder. Thus, the present study investigates the effects of quercetin on NTPDase, 5'-nucleotidase and adenosine deaminase activities, platelet aggregation and oxidative profile in platelets of rats with methimazole (MMI)-induced hypothyroidism. Methimazole at a concentration of 20mg/100mL was administered for 90days. From the second month the animals received quercetin 10 or 25mg/kg for 60days. Results showed that: Ecto-5'-nucleotidase activity decreased in methimazole/water group and the treatment with quercetin 25mg/kg decreased NTPDase, 5'-nucleotidase and adenosine deaminase activities. Moreover, platelet aggregation increased in methimazole/water group. Lipid peroxidation increased while superoxide dismutase and catalase activities decreased, but, interestingly, the treatment with quercetin reversed these changes. These results demonstrated that quercetin modulates adenine nucleotide hydrolysis decreasing the ADP formation and adenosine deamination. At the same time quercetin improves the oxidative profile, as well as reduces platelet aggregation, which together with the modulation in the nucleotides levels can contribute to the prevention of platelet disorders.

  6. Thiamine biosensor based on oxidative trapping of enzyme-substrate intermediate.

    PubMed

    Halma, Matilte; Doumèche, Bastien; Hecquet, Laurence; Prévot, Vanessa; Mousty, Christine; Charmantray, Franck

    2017-01-15

    In the present work, we describe a new thiamine amperometric biosensor based on thiamine pyrophosphate (ThDP)-dependent transketolase (TK)-catalyzed reaction, followed by the oxidative trapping of TK intermediate α,β-dihydroxyethylthiamine diphosphate (DHEThDP) within the enzymatic active site. For the biosensor design purpose, TK from Escherichia coli (TKec) was immobilized in Mg2Al-NO3 Layered Double Hydroxides (LDH) and the electrochemical detection was achieved with the TKec/LDH modified glassy carbon electrode (GCE). The transduction process was based on the ability of Fe(CN)6(3-) to oxidize DHEThDP to glycolic acid along with ThDP regeneration. The released Fe(CN)6(4-) was re-oxidized at +0.5V vs Ag-AgCl and the reaction was followed by chronoamperometry. The TKec/LDH/GCE biosensor was optimized using the best TK donor substrates, namely l-erythrulose and d-fructose-6-phosphate. ThDP was assayed with great sensitivity (3831mAM(-1)cm(-2)) over 20-400nM linear range.

  7. Lipid peroxidation, proteins modifications, anti-oxidant enzymes activities and selenium deficiency in the plasma of hashitoxicosis patients

    PubMed Central

    Mseddi, Malek; Ben Mansour, Riadh; Mnif, Fatma; Gargouri, Bochra; Abid, Mohamed; Guermazi, Fadhel; Attia, Hamadi; Lassoued, Saloua

    2015-01-01

    Objectives: The aim of this study was to explore the oxidative stress profile in hashitoxicosis (HTX) and to compare it with that of healthy subjects. Patients and methods: Spectrophotometric methods were used to evaluate the oxidative stress markers. The selenium level was investigated by atomic absorption. Results: High levels of thiobarbituric acid reactive species (TBARS) and conjugated dienes were found in HTX patients (p = 0.034 and p = 0.043, respectively) compared with healthy controls. For antioxidant enzymes, superoxide dismutase (SOD) and catalase activities increased, whereas that of glutathione peroxidase (GPx) decreased (p = 0.000, p = 0.014, p = 0.000, respectively) compared with controls. A reduction in the level of selenium (p = 0.029) and thiol groups (p = 0.008) were shown in patients; however, levels of carbonyl group and malondialdehyde (MDA) protein adducts decreased (p = 0.000) compared with controls. Positive correlation was shown between levels of free thyroxine (FT4) and TBARS (r = 0.711, p = 0.048) and between FT4 level and SOD activity (r = 0.713, p = 0.047). Conversely, GPx activity presented a negative correlation with FT4 and free triiodothyronine (FT3) levels (r = –0.934, p = 0.001; r = –0.993, p = 0.000, respectively). In addition, GPx activity showed positive correlation with selenium level (r = 0.981, p = 0.019) and the FT3 level correlated negatively with the level of thiol groups (r = –0.892, p = 0.017). Conclusions: This study shows the presence of an oxidative stress and selenium deficiency in HTX patients and suggests that the hyperthyroid state is strongly implicated in the establishment of this disturbed oxidative profile. PMID:26445640

  8. Saikosaponin-D attenuates heat stress-induced oxidative damage in LLC-PK1 cells by increasing the expression of anti-oxidant enzymes and HSP72.

    PubMed

    Zhang, Bao-Zhen; Guo, Xiao-Tong; Chen, Jian-Wei; Zhao, Yuan; Cong, Xia; Jiang, Zhong-Ling; Cao, Rong-Feng; Cui, Kai; Gao, Shan-Song; Tian, Wen-Ru

    2014-01-01

    Heat stress stimulates the production of reactive oxygen species (ROS), which cause oxidative damage in the kidney. This study clarifies the mechanism by which saikosaponin-d (SSd), which is extracted from the roots of Bupleurum falcatum L, protects heat-stressed pig kidney proximal tubular (LLC-PK1) cells against oxidative damage. SSd alone is not cytotoxic at concentrations of 1 or 3 μg/mL as demonstrated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To assess the effects of SSd on heat stress-induced cellular damage, LLC-PK1 cells were pretreated with various concentrations of SSd, heat stressed at 42°C for 1 h, and then returned to 37°C for 9 h. DNA ladder and MTT assays demonstrated that SSd helped to prevent heat stress-induced cellular damage when compared to untreated cells. Additionally, pretreatment with SSd increased the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) but decreased the concentration of malondialdehyde (MDA) in a dose-dependent manner when compared to controls. Furthermore, real-time PCR and Western blot analysis demonstrated that SSd significantly increased the expression of copper and zinc superoxide dismutase (SOD-1), CAT, GPx-1 and heat shock protein 72 (HSP72) at both the mRNA and protein levels. In conclusion, these results are the first to demonstrate that SSd ameliorates heat stress-induced oxidative damage by modulating the activity of anti-oxidant enzymes and HSP72 in LLC-PK1 cells.

  9. The Conversion of Nitrite to Nitrogen Oxide(s) by the Constitutive NAD(P)H-Nitrate Reductase Enzyme from Soybean 1

    PubMed Central

    Dean, John V.; Harper, James E.

    1988-01-01

    A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase [NAD(P)H-NR, pH 6.5; EC 1.6.6.2] activity from the nitric oxide and nitrogen dioxide (NO(x)) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets. Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.5]) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatography-anion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO(x) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent Km for nitrite of 0.49 millimolar, and an apparent Km for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO(x) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH2) and reduced methyl viologen (MV) can serve as electron donors for NO(x) evolution activity. The NADPH-, FMNH2-, and reduced MV-NO(x) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymer-curibenzoate, whereas, the FMNH2 and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO(x). NADPH eluted both NR and NO(x) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate- or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean. PMID:16666314

  10. Leucine accelerates blood ethanol oxidation by enhancing the activity of ethanol metabolic enzymes in the livers of SHRSP rats.

    PubMed

    Murakami, Hitoshi; Ito, Michiko; Furukawa, Yuji; Komai, Michio

    2012-12-01

    Chronic ethanol consumption induces liver diseases, such as alcoholic hepatitis and cirrhosis. The enhancement of alcohol oxidation is important in the prevention of these liver diseases. Chronic supplementation with branched chain amino acids (BCAAs) prevents liver cirrhosis. Therefore, BCAAs may be associated with enhanced ethanol oxidation. To evaluate this hypothesis, we investigated the effect of the administration of individual BCAAs on ethanol oxidation and changes in alcohol-metabolizing enzyme activities following acute alcohol intake in rats. Blood ethanol concentrations and the activities of alcohol-metabolizing enzymes, such as alcohol dehydrogenase (ADH) and low and high Km aldehyde dehydrogenase (ALDH), were measured in the liver following acute ethanol administration in rats; the ethanol was administered 30 min after the treatment with amino acids [such as leucine (Leu), isoleucine (Ile), valine (Val) or alanine (Ala)]. Leu significantly decreased the blood ethanol concentration 1 h after ethanol administration compared to the water-treated control (C) [C 0.46 ± 0.09, Leu 0.18 ± 0.04, Ile 0.27 ± 0.09, Val 0.46 ± 0.1, Ala 0.43 ± 0.06, mean ± SEM (g/l), P < 0.05]. In addition, leucine significantly stimulated ADH activity 30 min after ethanol intake [C 0.042 ± 0.014, Leu 0.090 ± 0.016, Ile 0.042 ± 0.008, Val 0.022 ± 0.010, Ala 0.070 ± 0.016, mean ± SEM (unit/mg protein), P < 0.05] and low Km ALDH activity 15 min after ethanol intake [C 0.51 ± 0.63, Leu 3.72 ± 0.66, Ile 1.26 ± 0.89, Val: ND, Ala 1.86 ± 1.57, mean ± SEM (unit/mg protein), P < 0.05]. However, leucine and its metabolite α-keto-isocaproic acid did not enhance ethanol clearance in isolated rat hepatocytes. These results indicate that leucine accelerates ethanol oxidation by indirectly enhancing ADH and low Km ALDH activities in the liver.

  11. Enzyme monolayer- and bilayer-modified tin oxide electrodes for the determination of hydrogen peroxide and glucose

    SciTech Connect

    Tatsuma, Tetsu; Okawa, Yusuke; Watanabe, Tadashi )

    1989-11-01

    An enzyme-based amperometric biosensor for hydrogen peroxide was developed, employing a horseradish peroxidase (HRP) monolayer covalently attached to a tin oxide electrode and a dissolved electron mediator. The sensor can determine hydrogen peroxide at levels down to 10{sup {minus}8} M and can be applied to a flow system. Stability of the electrode, kinetics of the surface process, and the efficiencies of different mediators were studied. As an extension, glucose oxidase (GOx) was chemically bound to the HRP-modified electrode. A GOx/HRP bilayer-modified electrode thus obtained exhibits much better performance in glucose detection limit, sensitivity, and response speed than previously reported GOx monolayer-modified electrodes.

  12. Oxidative metabolism of flunarizine and cinnarizine by microsomes from B-lymphoblastoid cell lines expressing human cytochrome P450 enzymes.

    PubMed

    Kariya, S; Isozaki, S; Uchino, K; Suzuki, T; Narimatsu, S

    1996-11-01

    The oxidative metabolism of cinnarizine [(E)-1-(diphenylmethyl)-4-(3-phenyl-2-propyl)piperazine, CZ] and flunarizine [(E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propyl)piperazine, FZ] was examined in microsomes from lymphoblastoid cells that expressed human cytochrome P450 (CYP) enzymes. Among 10 kinds of CYP enzymes examined, only CYP2D6 catalyzed p-hydroxylation of the cinnamyl phenyl ring of CZ (C-2 formation) and FZ (F-2 formation), and only CYP2B6 exhibited activity for p-hydroxylation (C-4 formation) of the diphenylmethyl group of CZ at a substrate concentration of 50 microM. On the other hand, CYP2C9 together with CYP1A1, -1A2 and/or -2A6 mediated N-desalkylation at the 1- and 4-positions of the piperazine ring of the two drugs that formed C-1 and C-3 from CZ and F-1 and F-3 from FZ, respectively, whereas CYP2C8, -2C19, -2E1 or -3A4 did not show detectable activity for these reactions under the conditions used. We then examined kinetics for the oxidative metabolism of CZ and FZ using CYP2B6 and -2D6 that have considerable activities. CYP2D6 with Km values of 2 to 4 microM had intrinsic clearance values (Vmax/Km) of 0.31 and 0.14 ml/min/nmol CYP for C-2 and F-2 formation, respectively, while CYP2B6 with a Km value of 17 microM exhibited the clearance value of 0.10 ml/min/nmol CYP for C-4 formation. These results suggest that CYP2D6 mainly mediates p-hydroxylation of the cinnamyl phenyl rings of CZ and FZ, and CYP2B6 mediates that of the diphenylmethyl group of CZ.

  13. Role of DNA repair enzymes in the cellular resistance to oxidative stress.

    PubMed

    Laval, J

    1996-01-01

    Oxidative stress occurs in cells when the equilibrium between prooxidant and antioxidant species is broken in favor of the prooxidant state. It is due to reactive oxygen species (ROS) generated either by the cellular metabolism such as phagocytosis, mitochondrial respiration, xenobiotic detoxification, or by exogenous factors such as ionizing radiation or chemical compounds performing red-ox reactions. Some ROS are extremely reactive and interact with all the macromolecules including lipids, nucleic acids and proteins. Cells have numerous defence systems to counteract the deleterious effects of ROS. Proteins and small molecules specifically eliminate ROS when they are formed. There are three species of superoxyde dismutases which transform the superoxyde anion O2- in hydrogen peroxyde H2O2 which in turn will be destroyed by peroxysomal catalase or by various peroxydases. There are numerous small molecules in the cell such as glutathion, alpha-tocopherol, vitamines A and C, melanine, etc. which are antioxydant molecules. ROS escaping destruction generate various lesions in DNA such as base modifications, degradation products of deoxyribose, chain breaks. These various lesions have been characterized and it is possible to quantitate them in the DNA of cells which have been irradiated or treated by free radical generating systems. The biological properties of the bases modified by ROS have been established. For example C8-hydroxyguanine (8-oxoG) is promutagenic since, if present in DNA during replication, it leads to incorporation of dAMP residues, leading to transversion mutation (GC-->TA). Purines whose imidazole ring is opened (Fapy residues) are stops for the DNA polymerase during DNA replication and are therefore potentially lethal lesions for the cell. Oxidized pyrimidines have comparable coding properties. Efficient DNA repair mechanisms remove these oxidized bases. In Escherichia coli cells, endonuclease III (NTH protein) and endonuclease VIII (NEI protein

  14. Oxidative dechlorination of halogenated phenols catalyzed by two distinct enzymes: Horseradish peroxidase and dehaloperoxidase.

    PubMed

    Szatkowski, Lukasz; Thompson, Matthew K; Kaminski, Rafal; Franzen, Stefan; Dybala-Defratyka, Agnieszka

    2011-01-01

    The mechanism of the dehalogenation step catalyzed by dehaloperoxidase (DHP) from Amphitrite ornata, an unusual heme-containing protein with a globin fold and peroxidase activity, has remarkable similarity with that of the classical heme peroxidase, horseradish peroxidase (HRP). Based on quantum mechanical/molecular mechanical (QM/MM) modeling and experimentally determined chlorine kinetic isotope effects, we have concluded that two sequential one electron oxidations of the halogenated phenol substrate leads to a cationic intermediate that strongly resembles a Meisenheimer intermediate - a commonly formed reactive complex during nucleophilic aromatic substitution reactions especially in the case of arenes carrying electron withdrawing groups. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. The catabolism of 2,4-xylenol and p-cresol share the enzymes for the oxidation of para-methyl group in Pseudomonas putida NCIMB 9866.

    PubMed

    Chen, Yan-Fei; Chao, Hongjun; Zhou, Ning-Yi

    2014-02-01

    Pseudomonas putida NCIMB 9866 utilizes p-cresol or 2,4-xylenol as a sole carbon and energy source. Enzymes catalyzing the oxidation of the para-methyl group of p-cresol have been studied in detail. However, those responsible for the oxidation of the para-methyl group in 2,4-xylenol catabolism are still not reported. In this study, real-time quantitative PCR analysis indicated pchC- and pchF-encoded p-cresol methylhydroxylase (PCMH) and pchA-encoded p-hydroxybenzaldehyde dehydrogenase (PHBDD) in p-cresol catabolism were also likely involved in the catabolism of 2,4-xylenol. Enzyme activity assays and intermediate identification indicated that the PCMH and PHBDD catalyzed the oxidations of 2,4-xylenol to 4-hydroxy-3-methylbenzaldehyde and 4-hydroxy-3-methylbenzaldehyde to 4-hydroxy-3-methylbenzoic acid, respectively. Furthermore, the PCMH-encoding gene pchF was found to be necessary for the catabolism of 2,4-xylenol, whereas the PHBDD-encoding gene pchA was not essential for the catabolism by gene knockout and complementation. Analyses of the maximum specific growth rate (μ m) and specific activity of the gene-knockout strain to different intermediates revealed the presence of other enzyme(s) with PHBDD activity in strain 9866. However, PHBDD played a major role in the catabolism of 2,4-xylenol in contrast to the other enzyme(s).

  16. Density functional theory (DFT) and combined quantum mechanical/molecular mechanics (QM/MM) studies on the oxygen activation step in nitric oxide synthase enzymes.

    PubMed

    de Visser, Sam P

    2009-04-01

    In this review paper, we will give an overview of recent theoretical studies on the catalytic cycle(s) of NOS (nitric oxide synthase) enzymes and in particular on the later stages of these cycles where experimental work is difficult due to the short lifetime of intermediates. NOS enzymes are vital for human health and are involved in the biosynthesis of toxic nitric oxide. Despite many experimental efforts in the field, the catalytic cycle of this important enzyme is still surrounded by many unknowns and controversies. Our theoretical studies were focused on the grey zones of the catalytic cycle, where intermediates are short-lived and experimental detection is impossible. Thus combined QM/MM (quantum mechanics/molecular mechanics) as well as DFT (density functional theory) studies on NOS enzymes and active site models have established a novel mechanism of oxygen activation and the conversion of L-arginine into N(omega)-hydroxo-arginine. Although NOS enzymes show many structural similarities to cytochrome P450 enzymes, it has long been anticipated that therefore they should have a similar catalytic cycle where molecular oxygen binds to a haem centre and is converted into an Fe(IV)-oxo haem(+*) active species (Compound I). Compound I, however, is elusive in the cytochrome P450s as well as in NOS enzymes, but indirect experimental evidence on cytochrome P450 systems combined with theoretical modelling have shown it to be the oxidant responsible for hydroxylation reactions in cytochrome P450 enzymes. By contrast, in the first catalytic cycle of NOS it has been shown that Compound I is first reduced to Compound II before the hydroxylation of arginine. Furthermore, substrate arginine in NOS enzymes appears to have a dual function, namely first as a proton donor in the catalytic cycle to convert the ferric-superoxo into a ferric-hydroperoxo complex and secondly as the substrate that is hydroxylated in the process leading to N(omega)-hydroxo-arginine.

  17. Characterization of YqjM, an Old Yellow Enzyme homolog from Bacillus subtilis involved in the oxidative stress response.

    PubMed

    Fitzpatrick, Teresa B; Amrhein, Nikolaus; Macheroux, Peter

    2003-05-30

    In this paper, we demonstrate that a protein from Bacillus subtilis (YqjM) shares many characteristic biochemical properties with the homologous yeast Old Yellow Enzyme (OYE); the enzyme binds FMN tightly but noncovalently, preferentially uses NADPH as a source of reducing equivalents, and forms charge transfer complexes with phenolic compounds such as p-hydroxybenzaldehyde. Like yeast OYE and other members of the family, YqjM catalyzes the reduction of the double bond of an array of alpha,beta-unsaturated aldehydes and ketones including nitroester and nitroaromatic compounds. Although yeast OYE was the first member of this family to be discovered in 1933 and was the first flavoenzyme ever to be isolated, the physiological role of the family still remains obscure. The finding that alpha,beta-unsaturated compounds are substrates provoked speculation that the OYE family might be involved in reductive degradation of xenobiotics or lipid peroxidation products. Here, for the first time, we demonstrate on the protein level that whereas YqjM shows a basal level of expression in B. subtilis, the addition of the toxic xenobiotic, trinitrotoluene, leads to a rapid induction of the protein in vivo denoting a role in detoxification. Moreover, we show that YqjM is rapidly induced in response to oxidative stress as exerted by hydrogen peroxide, demonstrating a potential physiological role for this enigmatic class of proteins.

  18. A plasma membrane-bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite.

    PubMed

    Stöhr, C; Strube, F; Marx, G; Ullrich, W R; Rockel, P

    2001-04-01

    Purified plasma membranes (PMs) of tobacco (Nicotiana tabacum L. cv. Samsun) roots exhibited a nitrite-reducing enzyme activity that resulted in nitric oxide (NO) formation. This enzyme activity was not detected in soluble protein fractions or in PM vesicles of leaves. At the pH optimum of pH 6.0, nitrite was reduced to NO with reduced cytochrome c as electron donor at a rate comparable to the nitrate-reducing activity of root-specific succinate-dependent PM-bound nitrate reductase (PM-NR). The hitherto unknown PM-bound nitrite: NO-reductase (NI-NOR) was insensitive to cyanide and anti-NR IgG and thereby proven to be different from PM-NR. Furthermore, PM-NR and NI-NOR were separated by gel-filtration chromatography and apparent molecular masses of 310 kDa for NI-NOR and 200 kDa for PM-NR were estimated. The PM-associated NI-NOR may reduce the apoplastic nitrite produced by PM-NR in vivo and may play a role in nitrate signalling via NO formation.

  19. Polyphenols rich fraction from Geoffroea decorticans fruits flour affects key enzymes involved in metabolic syndrome, oxidative stress and inflammatory process.

    PubMed

    Costamagna, M S; Zampini, I C; Alberto, M R; Cuello, S; Torres, S; Pérez, J; Quispe, C; Schmeda-Hirschmann, G; Isla, M I

    2016-01-01

    Geoffroea decorticans (chañar), is widely distributed throughout Northwestern Argentina. Its fruit is consumed as flour, arrope or hydroalcoholic beverage. The chañar fruits flour was obtained and 39 phenolic compounds were tentatively identified by HPLC-MS/MS(n). The compounds comprised caffeic acid glycosides, simple phenolics (protocatechuic acid and vanillic acid), a glycoside of vanillic acid, p-coumaric acid and its phenethyl ester as well as free and glycosylated flavonoids. The polyphenols enriched extract with and without gastroduodenal digestion inhibited enzymes associated with metabolic syndrome, including α-amylase, α-glucosidase, lipase and hydroxyl methyl glutaryl CoA reductase. The polyphenolic extract exhibited antioxidant activity by different mechanisms and inhibited the pro-inflammatory enzymes (ciclooxygenase, lipoxygenase and phospholipase A2). The polyphenolic extract did not showed mutagenic effect by Ames test against Salmonella typhimurium TA98 and TA100 strains. These findings add evidence that chañar fruit flour may be considered a functional food with preventive properties against diseases associated with oxidative stress, inflammatory mediators and metabolic syndrome. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Testicular toxicity in cannabis extract treated mice: association with oxidative stress and role of antioxidant enzyme systems.

    PubMed

    Mandal, Tapas K; Das, Nildari S

    2010-02-01

    Intraperitoneal injection of cannabis extract at low doses (total doses ranging from 40 mg to 60 mg per mouse) induced adverse effect on testes and oxidative stress. At low doses, there was a significant increase in lipid peroxidation and decrease in testicular lipid content, but the effects were significantly less at higher doses and at the withdrawal of cannabis treatment (recovery dose). There was a marked decrease in antioxidant enzyme profiles (superoxide dismutase, catalase and glutathione peroxidase) and glutathione content at low doses, but these effects were higher at higher dose and at withdrawal of the treatment (recovery effect). Histology revealed significant shrinkage of tubular diameter and detrimental changes in seminiferous epithelium of testis with resulting lowered serum testosterone and pituitary gonadotropins (follicular stimulating [FSH] and luteinizing hormones [LH]) levels at low doses. But at higher doses and particularly at withdrawal of the treatment, regression of various germ cell layers of testes through the revival of testosterone hormone and pituitary gonadotropins (FSH and LH) were observed, indicating that recovery effects on testes became operative possibly through the corrective measure of endogenous testicular antioxidant enzymes profiles and pituitary gonadotropins hormones feedback mechanisms.

  1. Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme.

    PubMed

    Zilly, A; Souza, C G M; Barbosa-Tessmann, I P; Peralta, R M

    2002-01-01

    The ability of a Brazilian strain of Pleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase), P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorization in vivo was tested using three dyes--Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability of P. pulmonarius to decolorize industrial dyes.

  2. Caffeine 7-N-demethylation and C-8-oxidation mediated by liver microsomal cytochrome P450 enzymes in common marmosets.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Inoue, Takashi; Suzuki, Takako; Utoh, Masahiro; Sasaki, Erika; Yamazaki, Hiroshi

    2015-10-27

    1. 3-N-Demethylation of caffeine (1,3,7-trimethylxanthine) is mediated by human cytochrome P450 1A2, whereas 7-N-demethylation and C-8-hydroxylation are reportedly catalyzed by monkey P450 2C9 and rat P450 1A2, respectively. 2. Roles of marmoset P450 enzymes in caffeine oxidation were investigated using nine marmoset liver microsomes and 14 recombinantly expressed marmoset P450 enzymes. 3. Predominant caffeine 7-N-demethylation and C-8-hydroxylation activities in marmoset liver microsomes were moderately (r = 0.78, p < 0.05) and highly (r = 0.82, p < 0.01) correlated with midazolam 1'-hydroxylation activities, respectively, while the former was not strongly affected by ketoconazole or α-naphthoflavone. 4. Caffeine C-8-hydroxylation in liver microsomes was inhibited by ketoconazole and activated by α-naphthoflavone, suggesting main involvements of P450 3As. 5. Recombinant marmoset P450 3As had high Vmax/Km values for C-8-hydroxylation, comparable to Km values for marmoset liver microsomes. Marmoset P450 1As efficiently mediated caffeine 3-N-demethylation and C-8-hydroxylation with apparently lower Km values than those of liver microsomes. 6. These results collectively suggest highly active marmoset P450 3A enzymes toward caffeine 8-hydorxylaiton and involvement of multiple P450 isoforms including P450 1A in caffeine 7-N- and 3-N-demethylations in marmoset livers. Marmoset P450s have slightly different properties to human or monkey P450s regarding caffeine metabolic pathways.

  3. Magnetically triggered clustering of biotinylated iron oxide nanoparticles in the presence of streptavidinylated enzymes.

    PubMed

    Hodenius, Michael; Hieronymus, Thomas; Zenke, Martin; Becker, Christiane; Elling, Lothar; Bornemann, Jörg; Wong, John E; Richtering, Walter; Himmelreich, Uwe; De Cuyper, Marcel

    2012-09-07

    This work deals with the production and characterization of water-compatible, iron oxide based nanoparticles covered with functional poly(ethylene glycol) (PEG)-biotin surface groups (SPIO-PEG-biotin). Synthesis of the functionalized colloids occurred by incubating the oleate coated particles used as precursor magnetic fluid with anionic liposomes containing 14 mol% of a phospholipid-PEG-biotin conjugate. The latter was prepared by coupling dimyristoylphosphatidylethanolamine (DC(14:0)PE) to activated α-biotinylamido-ω -N-hydroxy-succinimidcarbonyl-PEG (NHS-PEG-biotin). Physical characterization of the oleate and PEG-biotin iron oxide nanocolloids revealed that they appear as colloidal stable clusters with a hydrodynamic diameter of 160 nm and zeta potentials of - 39 mV (oleate coated particles) and - 14 mV (PEG-biotin covered particles), respectively, as measured by light scattering techniques. Superconducting quantum interference device (SQUID) measurements revealed specific saturation magnetizations of 62-73 emu g(-1) Fe(3)O(4) and no hysteresis was observed at 300 K. MR relaxometry at 3 T revealed very high r(2) relaxivities and moderately high r(1) values. Thus, both nanocolloids can be classified as small, superparamagnetic, negative MR contrast agents. The capacity to functionalize the particles was illustrated by binding streptavidin alkaline phosphatase (SAP). It was found, however, that these complexes become highly aggregated after capturing them on the magnetic filter device during high-gradient magnetophoresis, thereby reducing the accessibility of the SAP.

  4. Role of beta-oxidation enzymes in gamma-decalactone production by the yeast Yarrowia lipolytica.

    PubMed

    Waché, Y; Aguedo, M; Choquet, A; Gatfield, I L; Nicaud, J M; Belin, J M

    2001-12-01

    Some microorganisms can transform methyl ricinoleate into gamma-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C(18)) to the C(10) precursor of gamma-decalactone, (ii) accumulation of other lactones (3-hydroxy-gamma-decalactone and 2- and 3-decen-4-olide), and (iii) gamma-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and gamma-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume gamma-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-gamma-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, beta-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the beta-oxidation flux. We also identified mutant strains that produced 26 times more gamma-decalactone than the wild-type parents.

  5. Role of β-Oxidation Enzymes in γ-Decalactone Production by the Yeast Yarrowia lipolytica

    PubMed Central

    Waché, Yves; Aguedo, Mario; Choquet, Armelle; Gatfield, Ian L.; Nicaud, Jean-Marc; Belin, Jean-Marc

    2001-01-01

    Some microorganisms can transform methyl ricinoleate into γ-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C18) to the C10 precursor of γ-decalactone, (ii) accumulation of other lactones (3-hydroxy-γ-decalactone and 2- and 3-decen-4-olide), and (iii) γ-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and γ-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume γ-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-γ-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, β-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the β-oxidation flux. We also identified mutant strains that produced 26 times more γ-decalactone than the wild-type parents. PMID:11722925

  6. Magnetically triggered clustering of biotinylated iron oxide nanoparticles in the presence of streptavidinylated enzymes

    NASA Astrophysics Data System (ADS)

    Hodenius, Michael; Hieronymus, Thomas; Zenke, Martin; Becker, Christiane; Elling, Lothar; Bornemann, Jörg; Wong, John E.; Richtering, Walter; Himmelreich, Uwe; De Cuyper, Marcel

    2012-09-01

    This work deals with the production and characterization of water-compatible, iron oxide based nanoparticles covered with functional poly(ethylene glycol) (PEG)-biotin surface groups (SPIO-PEG-biotin). Synthesis of the functionalized colloids occurred by incubating the oleate coated particles used as precursor magnetic fluid with anionic liposomes containing 14 mol% of a phospholipid-PEG-biotin conjugate. The latter was prepared by coupling dimyristoylphosphatidylethanolamine (DC14:0PE) to activated α-biotinylamido-ω -N-hydroxy-succinimidcarbonyl-PEG (NHS-PEG-biotin). Physical characterization of the oleate and PEG-biotin iron oxide nanocolloids revealed that they appear as colloidal stable clusters with a hydrodynamic diameter of 160 nm and zeta potentials of - 39 mV (oleate coated particles) and - 14 mV (PEG-biotin covered particles), respectively, as measured by light scattering techniques. Superconducting quantum interference device (SQUID) measurements revealed specific saturation magnetizations of 62-73 emu g-1 Fe3O4 and no hysteresis was observed at 300 K. MR relaxometry at 3 T revealed very high r2 relaxivities and moderately high r1 values. Thus, both nanocolloids can be classified as small, superparamagnetic, negative MR contrast agents. The capacity to functionalize the particles was illustrated by binding streptavidin alkaline phosphatase (SAP). It was found, however, that these complexes become highly aggregated after capturing them on the magnetic filter device during high-gradient magnetophoresis, thereby reducing the accessibility of the SAP.

  7. Elucidating rifampin's inducing and inhibiting effects on glyburide pharmacokinetics and blood glucose in healthy volunteers: unmasking the differential effects of enzyme induction and transporter inhibition for a drug and its primary metabolite.

    PubMed

    Zheng, H X; Huang, Y; Frassetto, L A; Benet, L Z

    2009-01-01

    The effects of single doses of intravenous (IV) ciprofloxacin and rifampin and of multiple doses of rifampin on glyburide exposure and blood glucose levels were investigated in nine healthy volunteers. A single IV dose of rifampin significantly increased the area under the concentration-time curve (AUC) of glyburide and its metabolite. Blood glucose levels were significantly lower than those observed after dosing with glyburide alone. Multiple doses of rifampin induced an increase in liver enzyme levels, leading to a marked decrease in glyburide exposure and blood glucose levels. When IV rifampin was administered after multiple doses of rifampin, the inhibition of hepatic uptake transporters masked the induction effect; however, the relative changes in AUC for glyburide and its hydroxyl metabolite were similar to those seen under noninduced conditions. The studies reported here demonstrate how measurements of the levels of both the parent drug and its primary metabolite are useful in unmasking simultaneous drug-drug induction and inhibition effects and in characterizing enzymatic vs. transporter mechanisms.

  8. Zinc oxide nanoparticles as a substitute for zinc oxide or colistin sulfate: Effects on growth, serum enzymes, zinc deposition, intestinal morphology and epithelial barrier in weaned piglets.

    PubMed

    Wang, Chao; Zhang, Ligen; Su, Weipeng; Ying, Zhixiong; He, Jintian; Zhang, Lili; Zhong, Xiang; Wang, Tian

    2017-01-01

    The objective of this study was to evaluate effects of zinc oxide nanoparticles (nano-ZnOs) as a substitute for colistin sulfate (CS) and/or zinc oxide (ZnO) on growth performance, serum enzymes, zinc deposition, intestinal morphology and epithelial barrier in weaned piglets. A total of 216 crossbred Duroc×(Landrace×Yorkshire) piglets weaned at 23 days were randomly assigned into 3 groups, which were fed with basal diets supplemented with 20 mg/kg CS (CS group), 20mg/kg CS+3000 mg/kg ZnO (CS+ZnO group), and 1200 mg/kg nano-ZnOs (nano-ZnO group) for 14 days. Results indicated that compared to CS group, supplementation of 1200 mg/kg nano-ZnOs (about 30 nm) significantly increased final body weight and average daily gain, and 3000 mg/kg ZnO plus colistin sulfate significantly increased average daily gain and decreased diarrhea rate in weaned piglets. There was no significant difference in growth performance and diarrhea rate between nano-ZnO and CS+ZnO groups. Supplementation of nano-ZnOs did not affect serum enzymes (glutamic oxalacetic transaminase, glutamic-pyruvic transaminase, and lactate dehydrogenase), but significantly increased plasma and tissue zinc concentrations (liver, tibia), improved intestinal morphology (increased duodenal and ileal villus length, crypt depth, and villus surface), enhanced mRNA expression of ZO-1 in ileal mucosa, and significantly decreased diamine oxidase activity in plasma, total aerobic bacterial population in MLN as compared to CS group. Effects of nano-ZnOs on serum enzymes, intestinal morphology, and mRNA expressions of tight junction were similar to those of high dietary ZnO plus colistin sulfate, while nano-ZnOs significantly reduced zinc concentrations of liver, tibia, and feces, and decreased total aerobic bacterial population in MLN as compared to CS+ZnO group. These results suggested that nano-ZnOs (1200 mg/kg) might be used as a substitute for colistin sulfate and high dietary ZnO in weaned piglets.

  9. Anti-inflammatory effect of Pueraria tuberosa extracts through improvement in activity of red blood cell anti-oxidant enzymes

    PubMed Central

    Pandey, Nidhi; Yadav, Durgavati; Pandey, Vivek; Tripathi, Yamini B.

    2013-01-01

    Changing life style and over-nutrition causes low-grade inflammation (LGI), with obesity and hyper-lipidemia as basic factors. The physiological state polarizes macrophages to classical type (M1), which is pro-inflammatory and promotes ectopic fat deposition in the body. Both factors induce inflammatory cascade, where free radicals (FRs) play an important role. Thus, pharmacological and non-pharmacological interventions would be effective in the management of LGI and plant products would be used as food supplement or as a drug. Previously, a study has reported the anti-oxidant potential of methanolic extract of tubers of Pueraria tuberosa (PTME) and inhibitory role of tuberosin on lipopolysaccharides-induced expression of inducible nitric oxide synthase in macrophages in an in vitro study model. Here, the effect of PTME has been explored on carrageenan-induced inflammatory changes in rats. The activity of antioxidant enzymes in red blood cell hemolysate has been assessed. PTME was orally given to rats for 9 days and periodical changes (every 3rd day) in the activity/concentration of superoxide dismutase (SOD), catalase, reduced glutathione (GSH), lipid peroxides (LPO), and C-reactive proteins (CRP) were monitored. The PTME significantly prevented carrageenan-induced decline in GSH content, lowering of catalase and SOD activity, and rise in LPO and CRP in rats in a time-dependent, sequential manner. Thus, it could be suggested that the anti-inflammatory role of PTME is primarily mediated through its FR scavenging potential. PMID:24501527

  10. Retention of enzyme activity with a boron-doped diamond electrode in the electro-oxidative nitration of lysozyme

    PubMed Central

    Iniesta, Jesús; Esclapez-Vicente, María Deseada; Heptinstall, John; Walton, David J.; Peterson, Ian R.; Mikhailov, Victor A.; Cooper, Helen J.

    2010-01-01

    In this paper we report the successful use of a non-metallic electrode material, boron-doped diamond (BDD), for the anodic electro-oxidative modification of hen egg white lysozyme (HEWL). Platinum electrodes can give rise to loss of activity of HEWL in electrosynthetic studies, whereas activity is retained on boron-doped diamond which is proposed as an effective substitute material for this purpose. We also compare literature methods of electrode pre-treatment to determine the most effective in electrosynthesis. Our findings show a decrease in total nitroprotein yield with decreasing nitrite concentration and an increase with increasing solution pH, confirming that, at a BDD electrode, the controlling factor remains the concentration of tyrosine phenolate anion. Purification of mono- and bis-nitrated HEWL and assay of enzymic activity showed better retention of activity at BDD electrode surfaces when compared to platinum. The products from electro-oxidation of HEWL at BDD were confirmed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FT-ICR) mass spectrometry, which revealed unique mass increases of +45 and +90 Da for the mono- and bis-nitrated lysozyme, respectively, corresponding to nitration at tyrosine residues. The nitration sites were confirmed as Tyr23 and Tyr20. PMID:21760652

  11. Involvement of a plastid terminal oxidase in plastoquinone oxidation as evidenced by expression of the Arabidopsis thaliana enzyme in tobacco.

    PubMed

    Joët, Thierry; Genty, Bernard; Josse, Eve-Marie; Kuntz, Marcel; Cournac, Laurent; Peltier, Gilles

    2002-08-30

    Chlororespiration has been defined as a respiratory electron transport chain in interaction with photosynthetic electron transport involving both non-photochemical reduction and oxidation of plastoquinones. Different enzymatic activities, including a plastid-encoded NADH dehydrogenase complex, have been reported to be involved in the non-photochemical reduction of plastoquinones. However, the enzyme responsible for plasquinol oxidation has not yet been clearly identified. In order to determine whether the newly discovered plastid oxidase (PTOX) involved in carotenoid biosynthesis acts as a plastoquinol oxidase in higher plant chloroplasts, the Arabidopsis thaliana PTOX gene (At-PTOX) was expressed in tobacco under the control of a strong constitutive promoter. We showed that At-PTOX is functional in tobacco chloroplasts and strongly accelerates the non-photochemical reoxidation of plastoquinols; this effect was inhibited by propyl gallate, a known inhibitor of PTOX. During the dark to light induction phase of photosynthesis at low irradiances, At-PTOX drives significant electron flow to O(2), thus avoiding over-reduction of plastoquinones, when photo- synthetic CO(2) assimilation was not fully induced. We proposed that PTOX, by modulating the redox state of intersystem electron carriers, may participate in the regulation of cyclic electron flow around photosystem I.

  12. Astaxanthin, canthaxanthin and beta-carotene differently affect UVA-induced oxidative damage and expression of oxidative stress-responsive enzymes.

    PubMed

    Camera, Emanuela; Mastrofrancesco, Arianna; Fabbri, Claudia; Daubrawa, Felicitas; Picardo, Mauro; Sies, Helmut; Stahl, Wilhelm

    2009-03-01

    Carotenoids are used for systemic photoprotection in humans. Regarding mechanisms underlying photoprotective effects of carotenoids, here we compared the modulation of UVA-related injury by carotenoids. Human dermal fibroblasts (HDF) were exposed to moderate doses of UVA, which stimulated apoptosis, increased levels of reactive oxygen species and thiobarbituric acid reactive substances, decreased antioxidant enzymes activities, promoted membrane perturbation, and induced the expression of heme oxygenase-1 (HO-1). The carotenoids astaxanthin (AX), canthaxanthin (CX) and beta-carotene (betaC) were delivered to HDF 24 h before exposure to UVA. Astaxanthin exhibited a pronounced photoprotective effect and counteracted all of the above-mentioned UVA-induced alterations to a significant extent. beta-Carotene only partially prevented the UVA-induced decline of catalase and superoxide dismutase activities, but it increased membrane damage and stimulated HO-1 expression. Moreover, betaC dose-dependently induced caspase-3 activity following UVA exposure. In contrast, CX had no effect on oxidative damage, except for HO-1 expression, which was augmented. Uptake of AX by fibroblasts was higher than that of the other two carotenoids. The photostability of the three compounds in fibroblasts was AX > CX > betaC. The data indicate that the oxo-carotenoid AX has a superior preventive effect towards photo-oxidative changes in cell culture.

  13. Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.

    PubMed Central

    Shamir, R; Johnson, W J; Morlock-Fitzpatrick, K; Zolfaghari, R; Li, L; Mas, E; Lombardo, D; Morel, D W; Fisher, E A

    1996-01-01

    Pancreatic carboxyl ester lipase (CEL) hydrolyzes cholesteryl esters (CE), triglycerides (TG), and lysophospholipids, with CE and TG hydrolysis stimulated by cholate. Originally thought to be confined to the gastrointestinal system, CEL has been reported in the plasma of humans and other mammals, implying its potential in vivo to modify lipids associated with LDL, HDL (CE, TG), and oxidized LDL (lysophosphatidylcholine, lysoPC). We measured the concentration of CEL in human plasma as 1.2+/-0.5 ng/ml (in the range reported for lipoprotein lipase). Human LDL and HDL3 reconstituted with radiolabeled lipids were incubated with purified porcine CEL without or with cholate (10 or 100 microM, concentrations achievable in systemic or portal plasma, respectively). Using a saturating concentration of lipoprotein-associated CE (4 microM), with increasing cholate concentration there was an increase in the hydrolysis of LDL- and HDL3-CE; at 100 microM cholate, the present hydrolysis per hour was 32+/-2 and 1.6+/-0.1, respectively, indicating that CEL interaction varied with lipoprotein class. HDL3-TG hydrolysis was also observed, but was only approximately 5-10% of that for HDL3-CE at either 10 or 100 microM cholate. Oxidized LDL (OxLDL) is enriched with lysoPC, a proatherogenic compound. After a 4-h incubation with CEL, the lysoPC content of OxLDL was depleted 57%. Colocalization of CEL in the vicinity of OxLDL formation was supported by demonstrating in human aortic homogenate a cholate-stimulated cholesteryl ester hydrolytic activity inhibited by anti-human CEL IgG. We conclude that CEL has the capability to modify normal human LDL and HDL composition and structure and to reduce the atherogenicity of OxLDL by decreasing its lysoPC content. PMID:8601635

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

    PubMed

    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.

  15. Isotope Effects Associated with N2O Production by Fungal and Bacterial Nitric Oxide Reductases: Implications for Enzyme Mechanisms

    NASA Astrophysics Data System (ADS)

    Hegg, E. L.; Yang, H.; Gandhi, H.; McQuarters, A.; Lehnert, N.; Ostrom, N. E.

    2014-12-01

    Nitrous oxide (N2O) is both a powerful greenhouse gas and a key participant in ozone destruction. Microbial activity accounts for over 70% of the N2O produced annually, and the atmospheric concentration of N2O continues to rise. Because the fungal and bacterial denitrification pathways are major contributors to microbial N2O production, understanding the mechanism by which NO is reduced to N2O will contribute to both N2O source tracing and quantification. Our strategy utilizes stable isotopes to probe the enzymatic mechanism of microbial N2O production. Although the use of stable isotopes to study enzyme mechanisms is not new, our approach is distinct in that we employ both measurements of isotopic preferences of purified enzyme and DFT calculations, thereby providing a synergistic combination of experimental and computational approaches. We analyzed δ18O, δ15Nα (central N atom in N2O), and δ15Nβ (terminal N atom) of N2O produced by purified fungal cytochrome P450 nitric oxide reductase (P450nor) from Histoplasma capsulatum as well as bacterial cytochrome c dependent nitric oxide reductase (cNOR) from Paracoccus denitrificans. P450nor exhibits an inverse kinetic isotope effect for Nβ (KIE = 0.9651) but a normal isotope effect for both Nα (KIE = 1.0127) and the oxygen atom (KIE = 1.0264). These results suggest a mechanism where NO binds to the ferric heme in the P450nor active site and becomes Nβ. Analysis of the NO-binding step indicated a greater difference in zero point energy in the transition state than the ground state, resulting in the inverse KIE observed for Nβ. Following protonation and rearrangement, it is speculated that this complex forms a FeIV-NHOH- species as a key intermediate. Our data are consistent with the second NO (which becomes Nα and O in the N2O product) attacking the FeIV-NHOH- species to generate a FeIII-N2O2H2 complex that enzymatically (as opposed to abiotically) breaks down to release N2O. Conversely, our preliminary data

  16. Hierarchical change in antioxidant enzyme gene expression and activity in acute cardiac rejection: role of inducible nitric oxide synthase.

    PubMed

    Nilakantan, Vani; Zhou, Xianghua; Hilton, Gail; Roza, Allan M; Adams, Mark B; Johnson, Christopher P; Pieper, Galen M

    2005-02-01

    Reactive oxygen and nitrogen may mediate inflammation injury, but the status of the antioxidant defense system that might influence this process is unknown. In the present study, we examined the expression profile of the antioxidant enzymes, manganese superoxide dismutase (MnSOD), catalase and glutathione peroxidase (GPX) in acutely rejecting cardiac allografts and the potential role of inducible nitric oxide synthase (iNOS) in modulating antioxidant gene expression and activity. Donor hearts from Lewis (isograft) or Wistar-Furth (allograft) rats were transplanted into Lewis recipient rats. A subset of the allografts received L-N6-(1-imino-ethyl) lysine (L-NIL), a specific iNOS inhibitor, beginning the day of surgery until the day of harvesting. Catalase and glutathione peroxidase (GPX) protein levels were significantly decreased by postoperative day 4 (POD4) and postoperative day 5 (POD5), respectively, in allografts compared to isografts. While CuZn superoxide dismutase (CuZn SOD) levels were unchanged, there was a 50% decrease in MnSOD protein in allografts at postoperative day 6 (POD6). The sequential loss in antioxidant protein levels was not due to transcriptional regulation since there was no change in RNA levels for any of the genes tested. L-NIL did not alter catalase protein; however, the loss of MnSOD protein at POD6 was prevented by L-NIL. Consistent with a decrease in antioxidant protein levels, there was a sequential loss in enzyme activity for MnSOD, catalase and GPX. L-NIL however, restored MnSOD and GPX activities but not catalase activity. Treatment with CsA restored both protein and enzyme activities of GPX and MnSOD but not catalase. These results indicate that the loss in MnSOD and GPX protein and activity in allografts occurs via an iNOS-dependent mechanism whereas the decrease in catalase appears to be iNOS-independent. This suggests a differential role for iNOS in regulating post-translational modification of individual antioxidant enzymes

  17. Regio- and Stereo-Selective Oxidation of a Cardiovascular Drug, Metoprolol, Mediated by Cytochrome P450 2D and 3A Enzymes in Marmoset Livers.

    PubMed

    Uehara, Shotaro; Ishii, Sakura; Uno, Yasuhiro; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2017-08-01

    A β-blocker, metoprolol, is one of the in vivo probes for human cytochrome P450 (P450) 2D6. Investigation of nonhuman primate P450 enzymes helps to improve the accuracy of the extrapolation of pharmacokinetic data from animals into humans. Common marmosets (Callithrix jacchus) are a potential primate model for preclinical research, but the detailed roles of marmoset P450 enzymes in metoprolol oxidation remain unknown. In this study, regio- and stereo-selectivity of metoprolol oxidations by a variety of P450 enzymes in marmoset and human livers were investigated in vitro. Although liver microsomes from cynomolgus monkeys and rats preferentially mediated S-metoprolol O-demethylation and R-metoprolol α-hydroxylation, respectively, those from humans, marmosets, minipigs, and dogs preferentially mediated R-metoprolol O-demethylation, in contrast to the slow rates of R- and S-metoprolol oxidation in mouse liver microsomes. R- and S-metoprolol O-demethylation activities in marmoset livers were strongly inhibited by quinidine and ketoconazole, and were significantly correlated with bufuralol 1'-hydroxylation and midazolam 1'-hydroxylation activities and also with P450 2D and 3A4 contents, which is different from the case in human livers that did not have any correlations with P450 3A-mediated midazolam 1'-hydroxylation. Recombinant human P450 2D6 enzyme and marmoset P450 2D6/3A4 enzymes effectively catalyzed R-metoprolol O-demethylation, comparable to the activities of human and marmoset liver microsomes, respectively. These results indicated that the major roles of P450 2D enzymes for the regio- and stereo-selectivity of metoprolol oxidation were similar between human and marmoset livers, but the minor roles of P450 3A enzymes were unique to marmosets. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  18. Intracellular antioxidant enzymes are not globally upregulated during hibernation in the major oxidative tissues of the 13-lined ground squirrel Spermophilus tridecemlineatus.

    PubMed

    Page, Melissa M; Peters, Craig W; Staples, James F; Stuart, Jeffrey A

    2009-01-01

    Hibernating mammals exhibit oxidative stress resistance in brain, liver and other tissues. In many animals, cellular oxidative stress resistance is associated with enhanced expression of intracellular antioxidant enzymes. Intracellular antioxidant capacity may be upregulated during hibernation to protect against oxidative damage associated with the ischemia-reperfusion that occurs during transitions between torpor and arousal. We tested the hypothesis that the 13-lined ground squirrel (Spermophilus tridecemlineatus), upregulates intracellular antioxidant enzymes in major oxidative tissues during hibernation. The two major intracellular isoforms of superoxide dismutase (MnSOD and CuZnSOD), which catalyze the first step in superoxide detoxification, were quantified in heart, brain and liver tissue using immunodetection and an in-gel activity assay. However, no differences in SOD protein expression or activity were found between active and hibernating squirrels. Measurements of glutathione peroxidase and glutathione reductase, which catalyze hydrogen peroxide removal, were not broadly upregulated during hibernation. The activity of catalase, which catalyzes an alternative hydrogen peroxide detoxification pathway, was higher in heart and brain of torpid squirrels, but lower in liver. Taken together, these data do not support the hypothesis that hibernation is associated with enhanced oxidative stress resistance due to an upregulation of intracellular antioxidant enzymes in the major oxidative tissues.

  19. Inhibition of DNA methyltransferase or histone deacetylase protects retinal pigment epithelial cells from DNA damage induced by oxidative stress by the stimulation of antioxidant enzymes.

    PubMed

    Tokarz, Paulina; Kaarniranta, Kai; Blasiak, Janusz

    2016-04-05

    Epigenetic modifications influence DNA damage response (DDR). In this study we explored the role of DNA methylation and histone acetylation in DDR in cells challenged with acute or chronic oxidative stress. We used retinal pigment epithelial cells (ARPE-19), which natively are exposed to oxidative stress due to permanent exposure to light and high blood flow. We employed a DNA methyltransferase inhibitor - RG108 (RG), or a histone deacetylase inhibitor - valproic acid (VA). ARPE-19 cells were exposed to tert-butyl hydroperoxide, an acute oxidative stress inducer, or glucose oxidase, which slowly liberates low-doses of hydrogen peroxide in the presence of glucose, creating chronic conditions. VA and RG reduced level of intracellular reactive oxygen species and DNA damage in ARPE-19 cells in normal condition and in oxidative stress. This protective effect of VA and RG was associated with the up-regulated expression of antioxidant enzyme genes: CAT, GPx1, GPx4, SOD1 and SOD2. RG decreased the number of cells in G2/M checkpoint in response to chronic oxidative stress. Neither RG nor VA changed the DNA repair or apoptosis induced by oxidative stress. Therefore, certain epigenetic manipulations may protect ARPE-19 cells from detrimental effects of oxidative stress by modulation of antioxidative enzyme gene expression, which may be further explored in pharmacological studies on oxidative stress-related eye diseases.

  20. Oxidative stress as a risk factor for osteoporosis in elderly Mexicans as characterized by antioxidant enzymes

    PubMed Central

    Sánchez-Rodríguez, Martha A; Ruiz-Ramos, Mirna; Correa-Muñoz, Elsa; Mendoza-Núñez, Víctor Manuel

    2007-01-01

    Background Oxidative stress (OxS) has recently been linked with osteoporosis; however, we do not know the influence of OxS as an independent risk factor for this disease. Methods We conducted a case-control study in 94 subjects ≥60 years of age, 50 healthy and 44 with osteoporosis. We measured total antioxidant status, plasma lipid peroxides, antioxidant activity of superoxide dismutase and glutathione peroxidase (GPx), and calculated the SOD/GPx ratio. Bone mineral density was obtained at the peripheral DXA in calcaneus using a portable Norland Apollo Densitometer®. Osteoporosis was considered when subjects had a BMD of 2.5 standard deviations or more below the mean value for young adults. Results GPx antioxidant activity was significantly lower in the group of subjects with osteoporosis in comparison with the group of healthy subjects (p < 0.01); in addition, the SOD/GPx ratio was significantly higher in the group of individuals with osteoporosis (p < 0.05). In logistic regression analysis, we found OxS to be an independent risk factor for osteoporosis (odds ratio [OR] = 2.79; 95% confidence interval [95% CI] = 1.08–7.23; p = 0.034). Conclusion Our findings suggest that OxS is an independent risk factor for osteoporosis linked to increase of SOD/GPx ratio. PMID:18088440

  1. Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution.

    PubMed

    Azim, N; Deery, E; Warren, M J; Wolfenden, B A A; Erskine, P; Cooper, J B; Coker, A; Wood, S P; Akhtar, M

    2014-03-01

    The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form.

  2. Bio-inspired artificial functional photocatalyst: biomimetic enzyme-like TiO2/reduced graphene oxide nanocomposite with excellent molecular recognition ability.

    PubMed

    Li, Wentao; Pei, Xule; Deng, Fang; Luo, Xubiao; Li, Fengcong; Xiao, Yong

    2015-05-01

    An enzyme-like TiO(2)/reduced graphene oxide (enzyme-TiO(2)/rGO) nanocomposite with molecular recognition ability was fabricated by biomimicking the geometrical and chemical complementation of the enzyme and substrate. The anatase TiO(2) nanocrystals were densely dispersed on rGO nanosheets with close interfacial contacts. With geometrical and chemical matching of target molecules and memorized cavities, the adsorption capacity of enzyme-TiO(2)/rGO nanocomposites for 4-nitrophenol (4.71 mg g(-1)) is about six times that of control TiO(2)/rGO without the enzyme-like feature (0.79 mg g(-1)), and the enzyme-TiO(2)/rGO shows a relative selectivity coefficient of 7.24. Moreover, enzyme-TiO(2)/rGO exhibits molecular recognitive photocatalytic degradation for a particular contaminant. The results demonstrate that enzyme-substrate recognition provides a convenient and powerful basis on which to biomimic and construct efficient photocatalysts with high selectivity.

  3. Pseudo-basal levels of and distribution of anti-oxidant enzyme biomarkers in Eisenia fetida and effect of exposure to phenanthrene.

    PubMed

    Shi, Zhiming; Xu, Li; Wang, Nan; Zhang, Wei; Li, Huixin; Hu, Feng

    2013-09-01

    In the paper, the pseudo-basal levels of anti-oxidant system in different earthworm life stages (juvenile and adult) and the pseudo-basal distribution in different regions of adult earthworms (pre-clitellum, clitellum and post-clitellum) were studied using filter contact tests. The effects of phenanthrene (PHE) at different exposure levels on anti-oxidant enzymes along the earthworm body were also investigated after 24 and 48h of exposure. The pseudo-basal levels of the anti-oxidant enzymes varied during the different growth phase, and results indicated that earthworm has a low oxidative risk and SOD plays important roles during the development whereas CAT and POD are more important in maintain the low ROS level in adult earthworm. The pseudo-basal distribution of the anti-oxidant enzymes along the earthworms was heterogeneous and MDA mainly located in clitellum. POD in pre-clitellum, SOD in clitellum and CAT in post-clitellum were important to eliminate excess total ROS. Time of exposure impacted the anti-oxidant enzyme activities and their distribution patterns along earthworms, from the viewpoint of which supported that exposure time was an environment stress factors. In a short exposure time (24 h), CAT and SOD in the three regions, POD in pre-clitellum and clitellum might be good indicator to a low PHE stress level (0.0629 μg cm(-2) treatments). In a long exposure time (48 h), only SOD in clitellum is a good indicator to both low and high PHE stress (0.629 μg cm(-2) treatments). Earthworm biomembrane system inflicted no oxidative damage until the stress magnitude reached or exceeds the level of exposure in low PHE concentration condition for 48 h. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Use of response surface methodology to study the combined effects of UV-C and thermal process on vegetable oxidative enzymes

    USDA-ARS?s Scientific Manuscript database

    The effects of ultraviolet processing (UV-C) (temperature, exposure time, and wavelength) and an environmental parameter (pH) were studied on three oxidative enzymes, namely, lipoxygenase (LOX), peroxidase (POD) and polyphenoloxidase (PPO) by using a central composite design. An initial screening de...

  5. Polysaccharide-based biomaterials with on-demand nitric oxide releasing property regulated by enzyme catalysis.

    PubMed

    Zhao, Qiang; Zhang, Jimin; Song, Lijie; Ji, Qing; Yao, Yao; Cui, Yun; Shen, Jie; Wang, Peng George; Kong, Deling

    2013-11-01

    The regulatory role of nitric oxide (NO) in cell signaling has been well recognized. Clinically, NO deficiency is known to be associated with severe vascular disorders, especially in patients with long-term diabetes. Exogenous compensation of NO is a promising therapeutic strategy, although the lack of stable NO compounds often lead to unsatisfactory clinical outcomes. In the present study, we report a stable comb-shaped polymer (CS-NO) using glycosylated NO compound as pendent chains and chitosan (CS) as backbone for controlled NO release. The on-demand release of NO is achieved by controlling the decomposition process of the CS-NO polymer, which is blocked by galactose and only occurs in the presence of glycosidase, making the NO releasing kinetic closely correlate with the glycosidase concentration. In addition, due to its high stability, the CS-NO polymers can also be processed into supportive membrane or injectable hydrogel, further demonstrating its clinical potential. Indeed, we report that the NO-releasing membrane inhibited platelet adhesion, prolonged activated partial thromboplastin time (APTT) as shown in the platelet-rich-plasma (PRP) assay. We also observe enhanced human umbilical vein endothelial cell growth yet suppressed vascular smooth muscle cell proliferation on the NO-contained membrane in vitro. Furthermore, in vivo administration of CS-NO solution significantly enhanced angiogenesis in diabetic mice with hind-limb ischemia. Protective effect of CS-NO was also observed against limb necrosis. Given the physiological importance of NO, the CS-NO polymer may be considered a promising option in therapeutic development against vascular disorders and diabetic feet.

  6. The evolution of eukaryotic cells from the perspective of peroxisomes: phylogenetic analyses of peroxisomal beta-oxidation enzymes support mitochondria-first models of eukaryotic cell evolution.

    PubMed

    Bolte, Kathrin; Rensing, Stefan A; Maier, Uwe-G

    2015-02-01

    Beta-oxidation of fatty acids and detoxification of reactive oxygen species are generally accepted as being fundamental functions of peroxisomes. Additionally, these pathways might have been the driving force favoring the selection of this compartment during eukaryotic evolution. Here we performed phylogenetic analyses of enzymes involved in beta-oxidation of fatty acids in Bacteria, Eukaryota, and Archaea. These imply an alpha-proteobacterial origin for three out of four enzymes. By integrating the enzymes' history into the contrasting models on the origin of eukaryotic cells, we conclude that peroxisomes most likely evolved non-symbiotically and subsequent to the acquisition of mitochondria in an archaeal host cell. © 2015 WILEY Periodicals, Inc.

  7. Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids.

    PubMed

    Iyer, Lakshminarayan M; Tahiliani, Mamta; Rao, Anjana; Aravind, L

    2009-06-01

    Modified bases in nucleic acids present a layer of information that directs biological function over and beyond the coding capacity of the conventional bases. While a large number of modified bases have been identified, many of the enzymes generating them still remain to be discovered. Recently, members of the 2-oxoglutarate- and iron(II)-dependent dioxygenase super-family, which modify diverse substrates from small molecules to biopolymers, were predicted and subsequently confirmed to catalyze oxidative modification of bases in nucleic acids. Of these, two distinct families, namely the AlkB and the kinetoplastid base J binding proteins (JBP) catalyze in situ hydroxylation of bases in nucleic acids. Using sensitive computational analysis of sequences, structures and contextual information from genomic structure and protein domain architectures, we report five distinct families of 2-oxoglutarate- and iron(II)-dependent dioxygenase that we predict to be involved in nucleic acid modifications. Among the DNA-modifying families, we show that the dioxygenase domains of the kinetoplastid base J-binding proteins belong to a larger family that includes the Tet proteins, prototyped by the human oncogene Tet1, and proteins from basidiomycete fungi, chlorophyte algae, heterolobosean amoeboflagellates and bacteriophages. We present evidence that some of these proteins are likely to be involved in oxidative modification of the 5-methyl group of cytosine leading to the formation of 5-hydroxymethylcytosine. The Tet/JBP homologs from basidiomycete fungi such as Laccaria and Coprinopsis show large lineage-specific expansions and a tight linkage with genes encoding a novel and distinct family of predicted transposases, and a member of the Maelstrom-like HMG family. We propose that these fungal members are part of a mobile transposon. To the best of our knowledge, this is the first report of a eukaryotic transposable element that encodes its own DNA-modification enzyme with a

  8. Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids

    PubMed Central

    Iyer, Lakshminarayan M.; Tahiliani, Mamta; Rao, Anjana; Aravind, L.

    2010-01-01

    Modified bases in nucleic acids present a layer of information that directs biological function over and beyond the coding capacity of the conventional bases. While a large number of modified bases have been identified, many of the enzymes generating them still remain to be discovered. Recently, members of the 2-oxoglutarate- and iron(II)-dependent dioxygenase superfamily, which modify diverse substrates from small molecules to biopolymers, were predicted and subsequently confirmed to catalyze oxidative modification of bases in nucleic acids. Of these, two distinct families, namely the AlkB and the kinetoplastid base J binding proteins (JBP) catalyze in situ hydroxylation of bases in nucleic acids. Using sensitive computational analysis of sequences, structures and contextual information from genomic structure and protein domain architectures, we report five distinct families of 2-oxoglutarate- and iron(II)-dependent dioxygenase that we predict to be involved in nucleic acid modifications. Among the DNA-modifying families, we show that the dioxygenase domains of the kinetoplastid base J-binding proteins belong to a larger family that includes the Tet proteins, prototyped by the human oncogene Tet1, and proteins from basidiomycete fungi, chlorophyte algae, heterolobosean amoeboflagellates and bacteriophages. We present evidence that some of these proteins are likely to be involved in oxidative modification of the 5-methyl group of cytosine leading to the formation of 5-hydroxymethyl-cytosine. The Tet/JBP homologs from basidiomycete fungi such as Laccaria and Coprinopsis show large lineage-specific expansions and a tight linkage with genes encoding a novel and distinct family of predicted transposases, and a member of the Maelstrom-like HMG family. We propose that these fungal members are part of a mobile transposon. To the best of our knowledge, this is the first report of a eukaryotic transposable element that encodes its own DNA-modification enzyme with a

  9. Oxidative Stress and Genetic Variants of Xenobiotic-Metabolising Enzymes Associated with COPD Development and Severity in Serbian Adults.

    PubMed

    Malic, Zivka; Topic, Aleksandra; Francuski, Djordje; Stankovic, Marija; Nagorni-Obradovic, Ljudmila; Markovic, Bojan; Radojkovic, Dragica

    2017-02-01

    The genetic and non-genetic factors that contribute to the development of chronic obstructive pulmonary disease (COPD) are still poorly understood. We investigated the potential role of genetic variants of xenobiotic-metabolising enzymes (glutathione-S-transferase M1, GSTM1; glutathione-S-transferase T1, GSTT1; microsomal epoxide hydrolase, mEH), oxidative stress (assessed by urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxodG/creatinine), sex, ageing and smoking habits on susceptibility to development of COPD and its severity in Serbian population. The investigated population consisted of 153 healthy subjects (85 males and 68 females) and 71 patients with COPD (33 males and 38 females). Detection of GSTM1*null, GSTT1*null, mEH Tyr113His and mEH His139Arg gene variants was performed by PCR/RFLP method. Urinary 8-oxodG was determined using HPLC-MS/MS, and expressed as 8-oxodG/creatinine. We revealed that increased urinary 8-oxodG/creatinine and leucocytosis are the strongest independent predictors for COPD development. Increased level of oxidative stress increased the risk for COPD in males [odds ratio (OR), 95% confidence interval (CI): 8.42, 2.26-31.28], more than in females (OR, 95% CI: 3.60, 1.37-9.45). Additionally, independent predictors for COPD were ageing in males (OR, 95% CI: 1.29, 1.12-1.48), while in females they were at least one GSTM1 or GSTT1 gene deletion in combination (OR, 95% CI: 23.67, 2.62-213.46), and increased cumulative cigarette consumption (OR, 95% CI: 1.09, 1.01-1.16). Severity of COPD was associated with the combined effect of low mEH activity phenotype, high level of oxidative stress and heavy smoking. In conclusion, early identification of GSTM1*null or GSTT1*null genotypes in females, low mEH activity phenotype in heavy smokers and monitoring of oxidative stress level can be useful diagnostic and prognostic biomarkers.

  10. Increased 8-hydroxy-2'-deoxyguanosine in plasma and decreased mRNA expression of human 8-oxoguanine DNA glycosylase 1, anti-oxidant enzymes, mitochondrial biogenesis-related proteins and glycolytic enzymes in leucocytes in patients with systemic lupus erythematosus.

    PubMed

    Lee, H-T; Lin, C-S; Lee, C-S; Tsai, C-Y; Wei, Y-H

    2014-04-01

    We measured plasma levels of the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) and leucocyte mRNA expression levels of the genes encoding the 8-OHdG repair enzyme human 8-oxoguanine DNA glycosylase 1 (hOGG1), the anti-oxidant enzymes copper/zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase-1 (GPx-1), GPx-4, glutathione reductase (GR) and glutathione synthetase (GS), the mitochondrial biogenesis-related proteins mtDNA-encoded ND 1 polypeptide (ND1), ND6, ATPase 6, mitochondrial transcription factor A (Tfam), nuclear respiratory factor 1(NRF-1), pyruvate dehydrogenase E1 component alpha subunit (PDHA1), pyruvate dehydrogenase kinase isoenzyme 1 (PDK-1) and hypoxia inducible factor-1α (HIF-1α) and the glycolytic enzymes hexokinase-II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase A (LDHa). We analysed their relevance to oxidative damage in 85 systemic lupus erythematosus (SLE) patients, four complicated SLE patients undergoing rituximab treatment and 45 healthy individuals. SLE patients had higher plasma 8-OHdG levels (P < 0·01) but lower leucocyte expression of the genes encoding hOGG1(P < 0·01), anti-oxidant enzymes (P < 0·05), mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) than healthy individuals. The increase in plasma 8-OHdG was correlated positively with the elevation of leucocyte expression of the genes encoding hOGG1 (P < 0·05), anti-oxidant enzymes (P < 0·05), several mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) in lupus patients. The patients, whose leucocyte mtDNA harboured D310 heteroplasmy, exhibited a positive correlation between the mtDNA copy number and expression of ND1, ND6 and ATPase 6 (P < 0·05) and a negative correlation between mt

  11. Effect of sodium chloride and cadmium on the growth, oxidative stress and antioxidant enzyme activities of Zygosaccharomyces rouxii

    NASA Astrophysics Data System (ADS)

    Li, Chunsheng; Xu, Ying; Jiang, Wei; Lv, Xin; Dong, Xiaoyan

    2014-06-01

    Zygosaccharomyces rouxii is a salt-tolerant yeast species capable of removing cadmium (Cd) pollutant from aqueous solution. Presently, the physiological characteristics of Z. rouxii under the stress of sodium chloride (NaCl) and Cd are poorly understood. This study investigated the effects of NaCl and Cd on the growth, oxidative stress and antioxidant enzyme activities of Z. rouxii after stress treatment for 24 h. Results showed that NaCl or Cd alone negatively affected the growth of Z. rouxii, but the growth-inhibiting effect of Cd on Z. rouxii was reduced in the presence of NaCl. Flow cytometry assay showed that under Cd stress, NaCl significantly reduced the production of reactive oxygen species (ROS) and cell death of Z. rouxii compared with those in the absence of NaCl. The activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) of Z. rouxii were significantly enhanced by 2%-6% NaCl, which likely contributed to the high salt tolerance of Z. rouxii. The POD activity was inhibited by 20 mg L-1 Cd while the SOD and CAT activities were enhanced by 8 mg L-1 Cd and inhibited by 20 mg L-1 or 50 mg L-1 Cd. The inhibitory effect of high-level Cd on the antioxidant enzyme activities of Z. rouxii was counteracted by the combined use of NaCl, especially at 6%. This probably accounted for the decrease in Cd-induced ROS production and cell death of Z. rouxii after incubation with NaCl and Cd. Our work provided physiological clues as to the use of Z. rouxii as a biosorbent for Cd removal from seawater and liquid highly salty food.

  12. Oxidative stress determined through the levels of antioxidant enzymes and the effect of N-acetylcysteine in aluminum phosphide poisoning

    PubMed Central

    Agarwal, Avinash; Robo, Roto; Jain, Nirdesh; Gutch, Manish; Consil, Shuchi; Kumar, Sukriti

    2014-01-01

    Introduction: The primary objective of this study was to determine the serum level of antioxidant enzymes and to correlate them with outcome in patients of aluminum phosphide (ALP) poisoning and, secondly, to evaluate the effect of N-acetylcysteine (NAC) given along with supportive treatment of ALP poisoning. Design: We conducted a cohort study in patients of ALP poisoning hospitalized at a tertiary care center of North India. The treatment group and control group were enrolled during the study period of 1 year from May 2011 to April 2012. Interventions: Oxidative stress was evaluated in each subject by estimating the serum levels of the enzymes, viz. catalase, superoxide dismutase (SOD) and glutathione reductase (GR). The treatment group comprised of patients who were given NAC in addition to supportive treatment (magnesium sulfate and vasopressors, if required), while in the control group, only supportive treatment was instituted. The primary endpoint of the study was the survival of the patients. Measurements and Results: The baseline catalase (P = 0.008) and SOD (P < 0.01) levels were higher among survivors than non-survivors. Of the total patients in the study, 31 (67.4%) expired and 15 (32.6%) survived. Among those who expired, the mean duration of survival was 2.92 ± 0.40 days in the test group and 1.82 ± 0.33 days in the control group (P = 0.043). Conclusions: This study suggests that the baseline level of catalase and SOD have reduced in ALP poisoning, but baseline GR level has not suppressed but is rather increasing with due time, and more so in the treatment group. NAC along with supportive treatment may have improved survival in ALP poisoning. PMID:25316977

  13. Mycorrhiza-induced lower oxidative burst is related with higher antioxidant enzyme activities, net H2O2 effluxes, and Ca2+ influxes in trifoliate orange roots under drought stress.

    PubMed

    Zou, Ying-Ning; Huang, Yong-Ming; Wu, Qiang-Sheng; He, Xin-Hua

    2015-02-01

    Mechanisms of arbuscular mycorrhiza (AM)-induced lower oxidative burst of host plants under drought stress (DS) are not elucidated. A noninvasive microtest technology (NMT) was used to investigate the effects of Funneliformis mosseae on net fluxes of root hydrogen peroxide (H2O2) and calcium ions (Ca2+) in 5-month-old Poncirus trifoliata, in combination with catalase (CAT) and superoxide dismutase (SOD) activities as well as tissue superoxide radical (O2•-) and H2O2 concentrations under DS and well-watered (WW) conditions. A 2-month DS (55% maximum water holding capacity of growth substrates) significantly inhibited AM fungal root colonization, while AM symbiosis significantly increased plant biomass production, irrespective of water status. F. mosseae inoculation generally increased SOD and CAT activity but decreased O2•- and H2O2 concentrations in leaves and roots under WW and DS. Compared with non-AM seedlings, roots of AM seedlings had significantly higher net H2O2 effluxes and net Ca2+ influxes, especially in the meristem zone, but lower net H2O2 efflux in the elongation zone. Net Ca2+ influxes into roots were significantly positively correlated with root net H2O2 effluxes but negatively with root H2O2 concentrations. Results from this study suggest that AM-induced lower oxidative burst is related with higher antioxidant enzyme activities, root net H2O2 effluxes, and Ca2+ influxes under WW and DS.

  14. Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution

    SciTech Connect

    Azim, N.; Deery, E.; Warren, M. J.; Wolfenden, B. A. A.; Erskine, P.; Cooper, J. B. Coker, A.; Wood, S. P.; Akhtar, M.

    2014-03-01

    The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step in the biosynthesis of tetrapyrroles in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. Two near-atomic resolution structures of PBGD from B. megaterium are reported that demonstrate the time-dependent accumulation of partially oxidized forms of the cofactor, including one that possesses a tetrahedral C atom in the terminal pyrrole ring. The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form.

  15. The reduction potential of the active site disulfides of human protein disulfide isomerase limits oxidation of the enzyme by Ero1α.

    PubMed

    Chambers, Joseph E; Tavender, Timothy J; Oka, Ojore B V; Warwood, Stacey; Knight, David; Bulleid, Neil J

    2010-09-17

    Disulfide formation in newly synthesized proteins entering the mammalian endoplasmic reticulum is catalyzed by protein disulfide isomerase (PDI), which is itself thought to be directly oxidized by Ero1α. The activity of Ero1α is tightly regulated by the formation of noncatalytic disulfides, which need to be broken to activate the enzyme. Here, we have developed a novel PDI oxidation assay, which is able to simultaneously determine the redox status of the individual active sites of PDI. We have used this assay to confirm that when PDI is incubated with Ero1α, only one of the active sites of PDI becomes directly oxidized with a slow turnover rate. In contrast, a deregulated mutant of Ero1α was able to oxidize both PDI active sites at an equivalent rate to the wild type enzyme. When the active sites of PDI were mutated to decrease their reduction potential, both were now oxidized by wild type Ero1α with a 12-fold increase in activity. These results demonstrate that the specificity of Ero1α toward the active sites of PDI requires the presence of the regulatory disulfides. In addition, the rate of PDI oxidation is limited by the reduction potential of the PDI active site disulfide. The inability of Ero1α to oxidize PDI efficiently likely reflects the requirement for PDI to act as both an oxidase and an isomerase during the formation of native disulfides in proteins entering the secretory pathway.

  16. Kinetics of conjugation and oxidation of nitrobenzyl alcohols by rat hepatic enzymes.

    PubMed

    Rickert, D E; deBethizy, J D; Glover, M R; Kedderis, G L

    1985-12-01

    Previous work has suggested that quantitative differences in the in vitro and in vivo metabolism of mononitrotoluene isomers are a result of differences in the hepatic conjugation and oxidation of the first metabolic intermediates, the mononitrobenzyl alcohols. We have determined the steady-state kinetic parameters, Vmax, Km and V/K, for the metabolism of the nitrobenzyl alcohols by rat hepatic alcohol dehydrogenase, glucuronyltransferase, and sulfotransferase. 3-Nitrobenzyl alcohol was the best substrate for cytosolic alcohol dehydrogenase (Vmax = 1.48 nmoles/min/mg protein, V/K = 3.15 X 10(-3) nmoles/min/mg protein/microM, Km = 503 microM). Vmax and Km values for 4-nitrobenzyl alcohol were similar, but V/K was about 60% of that for 3-nitrobenzyl alcohol. 2-Nitrobenzyl alcohol was not metabolized by the alcohol dehydrogenase preparation used here, but it was metabolized to 2-nitrobenzoic acid by a rat liver mitochondrial preparation. 2-Nitrobenzyl alcohol was the best substrate for microsomal glucuronyltransferase (Vmax = 3.59 nmoles/min/mg protein, V/K = 11.28 X 10(-3) nmoles/min/mg protein/microM, Km = 373 microM). The Vmax for 3-nitrobenzyl alcohol was similar, but the V/K was about half and the Km was about twice that for 2-nitrobenzyl alcohol. The Vmax for 4-nitrobenzyl alcohol was about 40% and the V/K was about half that for 2-nitrobenzyl alcohol. The best substrate for cytosolic sulfotransferase was 4-nitrobenzyl alcohol (Vmax = 1.69 nmoles/min/mg protein, V/K = 37.21 X 10(-3) nmoles/min/mg protein/microM, Km = 48 microM). The Vmax values for the other two benzyl alcohols were similar, but the V/K and Km values were about 11 and 400%, respectively, of those for 4-nitrobenzyl alcohol. These data are in qualitative agreement with results obtained when the nitrobenzyl alcohols were incubated with isolated hepatocytes, but they do not allow quantitative modeling of the data from hepatocytes.

  17. Unchanged content of oxidative enzymes in fast-twitch muscle fibers and kinetics after intensified training in trained cyclists

    PubMed Central

    Christensen, Peter M; Gunnarsson, Thomas P; Thomassen, Martin; Wilkerson, Daryl P; Nielsen, Jens Jung; Bangsbo, Jens

    2015-01-01

    The present study examined if high intensity training (HIT) could increase the expression of oxidative enzymes in fast-twitch muscle fibers causing a faster oxygen uptake () response during intense (INT), but not moderate (MOD), exercise and reduce the slow component and muscle metabolic perturbation during INT. Pulmonary kinetics was determined in eight trained male cyclists (-max: 59 ± 4 (means ± SD) mL min−1 kg−1) during MOD (205 ± 12 W ∼65% -max) and INT (286 ± 17 W ∼85% -max) exercise before and after a 7-week HIT period (30-sec sprints and 4-min intervals) with a 50% reduction in volume. Both before and after HIT the content in fast-twitch fibers of CS (P < 0.05) and COX-4 (P < 0.01) was lower, whereas PFK was higher (P < 0.001) than in slow-twitch fibers. Content of CS, COX-4, and PFK in homogenate and fast-twitch fibers was unchanged with HIT. Maximal activity (μmol g DW−1 min−1) of CS (56 ± 8 post-HIT vs. 59 ± 10 pre-HIT), HAD (27 ± 6 vs. 29 ± 3) and PFK (340 ± 69 vs. 318 ± 105) and the capillary to fiber ratio (2.30 ± 0.16 vs. 2.38 ± 0.20) was unaltered following HIT. kinetics was unchanged with HIT and the speed of the primary response did not differ between MOD and INT. Muscle creatine phosphate was lower (42 ± 15 vs. 66 ± 17 mmol kg DW−1) and muscle lactate was higher (40 ± 18 vs. 14 ± 5 mmol kg DW−1) at 6 min of INT (P < 0.05) after compared to before HIT. A period of intensified training with a volume reduction did not increase the content of oxidative enzymes in fast-twitch fibers, and did not change kinetics. PMID:26152692

  18. DFT study on the oxygen transfer mechanism in nitroethenediamine based H2-receptor antagonists using the bis-dithiolene complex as the model catalyst for N-oxide reductase enzyme.

    PubMed

    Dhaked, Devendra K; Bharatam, Prasad V

    2015-01-01

    Nitroethenediamine is an important functional unit, which is present in H2-receptor antagonists. These drugs show low bioavailability due to the bacterial degradation caused by the N-oxide reductase type of enzymes present in the human colon. Quantum chemical studies have been carried out to elucidate the mechanism of metabolic degradation of nitroethenediamine in the active site of N-oxide reductase. Three different pathways have been explored for the N-oxide bond cleavage by the model system, Mo(IV) bis-dithiolene complex [Mo(OMe)(mdt)2](-), (where mdt=1,2-dimethyl-ethene-1,2-dithiolate) using B3LYP/6-311+G(d,p) and M06/6-311+G(d,p) Density Functional Theory methods. The oxygen atom transfer from the nitrogen atom of nitroethenediamine to the Mo(IV) complex, involves simultaneous weakening of the N-oxide bond and the formation of Mo-O bond through a least motion path. During this transfer, Mo center is converted from a square pyramidal geometry to a distorted octahedral geometry, to facilitate the process of oxygen atom transfer. The energy barrier for the oxygen atom transfer from the imine tautomer has been estimated to be 25.9kcal/mol however, the overall reaction has been found to be endothermic. On the other hand, oxygen transfer reaction from the nitronic acid tautomer requires 30.5kcal/mol energy leading to a highly exothermic metabolite (M-1) directly hence, this path can be considered thermodynamically favorable for this metabolite. The alternative path involving the oxygen atom transfer from the enamine tautomer requires comparatively a higher energy barrier (32.6kcal/mol) and leads to a slightly endothermic metabolite. This study established the structural and energetic details associated with the Mo(IV) bis-dithiolene complex that catalyzes the degradation of nitroethenediamine based drug molecules. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Mechanisms of nitric oxide crosstalk with reactive oxygen species scavenging enzymes during abiotic stress tolerance in plants.

    PubMed

    Arora, Dhara; Jain, Prachi; Singh, Neha; Kaur, Harmeet; Bhatla, Satish C

    2016-01-01

    Nitric oxide (NO) acts in a concentration and redox-dependent manner to counteract oxidative stress either by directly acting as an antioxidant through scavenging reactive oxygen species (ROS), such as superoxide anions (O(2)(-)*), to form peroxynitrite (ONOO(-)) or by acting as a signaling molecule, thereby altering gene expression. NO can interact with different metal centres in proteins, such as heme-iron, zinc-sulfur clusters, iron-sulfur clusters, and copper, resulting in the formation of a stable metal-nitrosyl complex or production of varied biochemical signals, which ultimately leads to modification of protein structure/function. The thiols (ferrous iron-thiol complex and nitrosothiols) are also involved in the metabolism and mobilization of NO. Thiols bind to NO and transport it to the site of action whereas nitrosothiols release NO after intercellular diffusion and uptake into the target cells. S-nitrosoglutathione (GSNO) also has the ability to transnitrosylate proteins. It is an NO˙ reservoir and a long-distance signaling molecule. Tyrosine nitration of proteins has been suggested as a biomarker of nitrosative stress as it can lead to either activation or inhibition of target proteins. The exact molecular mechanism(s) by which exogenous and endogenously generated NO (or reactive nitrogen species) modulate the induction of various genes affecting redox homeostasis, are being extensively investigated currently by various research groups. Present review provides an in-depth analysis of the mechanisms by which NO interacts with and modulates the activity of various ROS scavenging enzymes, particularly accompanying ROS generation in plants in response to varied abiotic stress.

  20. Amelioration of ozone-induced oxidative damage in wheat plants grown under high carbon dioxide: Role of antioxidant enzymes

    SciTech Connect

    Rao, M.V.; Hale, B.A.; Ormrod, D.P.

    1995-10-01

    O{sub 3}-induced in growth, oxidative damage to protein, and specific activities of certain antioxidant enzymes were investigated in wheat plants (Triticum aestivum L. cv Roblin) grown under ambient or high CO{sub 2}. High CO{sub 2} enhanced shoot biomass. The shoot biomass was relatively unaffected in plants grown under a combination of high CO{sub 2} and O{sub 3}. O{sub 3} exposure under ambient CO{sub 2} decreased photosynthetic pigments, soluble proteins, and ribulose-1,5-bisphosphate carboxylase/oxygenase protein and enhanced oxidative damage to proteins, but these effects were not observed in plants exposed to O{sub 3} under high CO{sub 2}. O{sub 3} exposure initially enhanced the specific activities of superoxide dismutase, peroxidase, glutathione reductase, and ascorbate peroxidase irrespective of growth in ambient or high CO{sub 2}. O{sub 3} exposure initially enhanced the specific activities of superoxide dismutase, peroxidase, glutathione reductase, and ascorbate peroxidase irrespective of growth in ambient or high CO{sub 2}. However, the specific activities decreased in plants with prolonged exposure to O{sub 3} under ambient CO{sub 2} but not in plants exposed to O{sub 3} under high CO{sub 2}. Native gels revealed preferential changes in the isoform composition of superoxide dismutase, peroxidases, and ascorbate peroxidase of plants grown under a combination of high CO{sub 2} and O{sub 3}. Furthermore, growth under high CO{sub 2} and O{sub 3} led to the synthesis of one new isoform of glutathione reductase. This could explain why plants grown under a combination of high CO{sub 2} and O{sub 3} are capable of resisting O{sub 3}-induced damage to growth and proteins compared to plants exposed to O{sub 3} under ambient CO{sub 2}. 66 refs., 8 figs., 1 tab.

  1. Electron pathways in catalase and peroxidase enzymic catalysis. Metal and macrocycle oxidations of iron porphyrins and chlorins

    SciTech Connect

    Hanson, L.K.; Chang, C.K.; Davis, M.S.; Fajer, J.

    1981-02-11

    Charge iterative extended Hueckel calculations are presented for compound II, the one-electron oxidation intermediate of horseradish peroxidase (HRP), and for compounds I, the two-electron oxidation transients of HRP and catalase (CAT) observed in the catalytic cycles of the hydroperoxidase enzymes. Compound II is described in terms of a ferryl configuration (O = Fe/sup IV/), and compounds I are described as ferrylporphyrin ..pi..-cation radicals. The validity of the iron ..pi..-cation calculations is supported by favorable comparison of parallel computations for porphyrin ..pi.. cations of diamagnetic metals with new and previously reported ESR results for radicals of zinc tetrabenz-, meso-tetramethyl, (/sup 14/N and /sup 15/N) tetraphenyl-, and magnesium (/sup 1/H and /sup 2/H) octaethylporphyrins. The calculated electronic configurations and unpaired spin density profiles for the ferryl ..pi.. cations satisfactorily account for the physical properties reported for compounds I of HRP (in the native protoporphyrin IX form or reconstituted with deuteroporphyrin), chloroperoxidase, and CAT. The ground states of the ..pi.. cations, a/sub 1u/ or a/sub 2u/, are determined by peripheral substitution and axial ligation, and the axial ligand of CAT I is predicted to differ from that of HRP I. The combination of model studies and calculations suggests that /sup 2/H, /sup 13/C, and /sup 15/N NMR studies of isotopically substituted proto and deutero HRP I would confirm the electronic profiles predicted. /sup 15/N NMR in particular would clearly discriminate between a/sub 1u/ and a/sub 2u/ configurations. As an additional test of the ferryl ..pi..-cation hypothesis, calculations are presented for a proposed ferrylchlorin ..pi.. cation of Neurospora crassa catalase, which contains an iron chlorin prosthetic group. Compound I of this unusual heme is predicted to occupy an a/sub 2/ ground state with the spin distribution and optical spectra reported here for synthetic chlorin

  2. Magnaporthe oryzae Effector AVR-Pii Helps to Establish Compatibility by Inhibition of the Rice NADP-Malic Enzyme Resulting in Disruption of Oxidative Burst and Host Innate Immunity.

    PubMed

    Singh, Raksha; Dangol, Sarmina; Chen, Yafei; Choi, Jihyun; Cho, Yoon-Seong; Lee, Jea-Eun; Choi, Mi-Ok; Jwa, Nam-Soo

    2016-05-31

    Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant immune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVR-Pii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen.

  3. Magnaporthe oryzae Effector AVR-Pii Helps to Establish Compatibility by Inhibition of the Rice NADP-Malic Enzyme Resulting in Disruption of Oxidative Burst and Host Innate Immunity

    PubMed Central

    Singh, Raksha; Dangol, Sarmina; Chen, Yafei; Choi, Jihyun; Cho, Yoon-Seong; Lee, Jea-Eun; Choi, Mi-Ok; Jwa, Nam-Soo

    2016-01-01

    Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant immune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVR-Pii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen. PMID:27126515

  4. Consequence of restricted mitochondrial oxidative metabolism on photosynthetic carbon assimilation in mesophyll protoplasts: Decrease in light activation of four chloroplastic enzymes.

    PubMed

    Padmasree, K.; Raghavendra, A. S.

    2001-08-01

    The patterns of light activation of 4 chloroplastic enzymes were examined in mesophyll protoplasts of pea (Pisum sativum) in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM, inhibitor of alternative pathway). The results were compared with those of DCMU (inhibitor of photosynthetic electron transport). The light activation of NADP glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), fructose-1,6-bisphosphatase (FBPase), phosphoribulokinase (PRK) (enzymes of the Calvin cycle) and NADP malate dehydrogenase (NADP-MDH) (reflects chloroplast redox state) was more pronounced at limiting CO2 (0.1 mM NaHCO3) than that at optimal CO2 (1.0 mM NaHCO3). SHAM decreased markedly (up to 33%) the light activation of all 4 enzymes, while antimycin A or oligomycin exerted only a limited effect (<10% decrease). Antimycin A or oligomycin or SHAM had no significant effect on light activation of these 4 enzymes in isolated chloroplasts. However, DCMU caused a remarkable decrease in light activation of enzymes in both protoplasts (up to 78%) and chloroplasts (up to 69%). These results suggest that the restriction of alternative pathway of mitochondrial metabolism results in a marked decrease in the light activation of key chloroplastic enzymes in mesophyll protoplasts but not in isolated chloroplasts. Such a decrease in the light activation of enzymes could be also a secondary feedback effect because of the restriction on carbon assimilation.

  5. Hazardous effect of organophosphate compound, dichlorvos in transgenic Drosophila melanogaster (hsp70-lacZ): induction of hsp70, anti-oxidant enzymes and inhibition of acetylcholinesterase.

    PubMed

    Gupta, Subash Chandra; Siddique, Hifzur Rahman; Saxena, Daya Krishna; Chowdhuri, Debapratim Kar

    2005-08-30

    We tested a working hypothesis that stress genes and anti-oxidant enzyme machinery are induced by the organophosphate compound dichlorvos in a non-target organism. Third instar larvae of Drosophila melanogaster transgenic for hsp70 were exposed to 0.1 to 100.0 ppb dichlorvos and 5.0 mM CuSO(4) (an inducer of oxidative stress and stress genes) and hsp70, and activities of acetylcholinesterase (AchE), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) product were measured. The study was further extended to examine tissue damage, if any, under such conditions. A concentration- and time-dependent increase in hsp70 and anti-oxidant enzymes was observed in the exposed organism as compared to control. A comparison of stress gene expression with SOD, CAT activities and LPO product under similar experimental conditions revealed that induction of hsp70 precedes the anti-oxidant enzyme activities in the exposed organism. Further, concomitant with a significant inhibition of AChE activity, significant induction of hsp70 was observed following chemical exposure. Mild tissue damage was observed in the larvae exposed to 10.0 ppb dichlorvos for 48 h when hsp70 expression reaches plateau. Dichlorvos at 0.1 ppb dietary concentration did not evoke significant hsp70 expression, anti-oxidant enzymes and LPO and AchE inhibition in the exposed organism, and thereby, was found to be non-hazardous to D. melanogaster. Conversely, 1.0 ppb of the test chemical stimulated a significant induction of hsp70 and anti-oxidant enzymes and significant inhibition of AchE; hence this concentration of test chemical was hazardous to the organism. The present study suggests that (a) both stress genes and anti-oxidant enzymes are stimulated as indices of cellular defense against xenobiotic hazard in D. melanogaster with hsp70 being proposed as first-tier bio-indicator of cellular hazard, (b) 0.1 ppb of the test chemical may be regarded as No Observed Adverse Effect Level (NOAEL), and

  6. Gelatin-encapsulated iron oxide nanoparticles for platinum (IV) prodrug delivery, enzyme-stimulated release and MRI.

    PubMed

    Cheng, Ziyong; Dai, Yunlu; Kang, Xiaojiao; Li, Chunxia; Huang, Shanshan; Lian, Hongzhou; Hou, Zhiyao; Ma, Pingan; Lin, Jun

    2014-08-01

    A facile method for transferring hydrophobic iron oxide nanoparticles (IONPs) from chloroform to aqueous solution via encapsulation of FITC-modified gelatin based on the hydrophobic-hydrophobic interaction is described in this report. Due to the existence of large amount of active groups such as amine groups in gelatin, the fluorescent labeling molecules of fluorescein isothiocyanate (FITC) and platinum (IV) prodrug functionalized with carboxylic groups can be conveniently conjugated on the IONPs. The nanoparticles carrying Pt(IV) prodrug exhibit good anticancer activities when the Pt(IV) complexes are reduced to Pt(II) in the intracellular environment, while the pure Pt(IV) prodrug only presents lower cytotoxicity on cancer cells. Meanwhile, fluorescence of FITC on the surface of nanoparticles was completely quenched due to the possible Förster Resonance Energy Transfer (FRET) mechanism and showed a fluorescence recovery after gelatin release and detachment from IONPs. Therefore FITC as a fluorescence probe can be used for identification, tracking and monitoring the drug release. In addition, adding pancreatic enzyme can effectively promote the gelatin release from IONPs owing to the degradation of gelatin. Noticeable darkening in magnetic resonance image (MRI) was observed at the tumor site after in situ injection of nanoparticles, indicating the IONPs-enhanced T2-weighted imaging. Our results suggest that the gelatin encapsulated Fe3O4 nanoparticles have potential applications in multi-functional drug delivery system for disease therapy, MR imaging and fluorescence sensor. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. The effect of heavy metal-induced oxidative stress on the enzymes in white rot fungus Phanerochaete chrysosporium.

    PubMed

    Zhang, Qihua; Zeng, Guangming; Chen, Guiqiu; Yan, Min; Chen, Anwei; Du, Jianjian; Huang, Jian; Yi, Bin; Zhou, Ying; He, Xiaoxiao; He, Yan

    2015-02-01

    Prevalence of heavy metals in the living environment causes chemical stress and reactive oxygen species (ROS) formation in Phanerochaete chrysosporium (P. chrysosporium). However, the mechanisms involved in ROS defense are still under investigation. In the present study, we evaluated the effect of lead- and cadmium-induced oxidative stress on the activities of catalase (CAT), peroxidase (POD), lignin peroxidase (LiP), and manganese peroxidase (MnP). A time-dependent change in all enzyme activities was observed following exposure to 50 μM cadmium and 25 μM lead. The lowest values were recorded at 4 h after exposure. Both cadmium and lead inhibited CAT and POD. The cytochrome P450 (CYP450) levels increased under 50-100 μM cadmium or lead exposure and decreased when heavy metal concentration was under 50 μM; this suggested that ROS is not the only factor that alters the CYP450 levels. The cadmium removal rate in the sample containing 900 μM taxifolin (inhibitor of CYP450) and 100 μM cadmium was reduced to 12.34 %, 9.73 % lower than that of 100 μM cadmium-induced sample, indicating CYP450 may play an indirect but key role in the process of clearance of heavy metals. The pH of the substrate solution decreased steadily during the incubation process.

  8. Effects of hexaammine cobalt (III) chloride on oxidative stress-related parameters and drug metabolizing enzymes in mice.

    PubMed

    Singh, Amarjit; Kalla, Natwar R; Sharma, Raj P; Sharma, Rajeshwar

    2007-01-01

    Hexaammine cobalt (III) chloride has been advocated as a potential anticarcinogenic compound. There is no information on the effects of this compound on oxidative stress-related parameters in animals. In the present study the effects of administration of hexaammine cobalt (III) chloride in drinking water to balb/c male mice at doses of 25, 50, and 100 ppm for 14 weeks were examined. The tissue distribution of the compound was seen in liver, kidney, lung, intestine, blood, and spleen. The effects of the compound were monitored on levels of lipid peroxidation, GSH content, and activities of SOD, catalase, GST, and Cyt P450, along with the liver and kidney function tests. The results show that the cobalt accumulated maximally in kidney followed by liver, intestine, blood, spleen, and lung in decreasing order, in a dose-dependent manner. GSH and GST also showed increase in a dose-dependent manner while SOD and catalase showed increase with the highest dose only. Liver and kidney function tests showed no untoward change with any dose at the end of the study. The results suggest an antioxidant potentiating effect of the hexaammine cobalt (III) chloride besides nontoxicity to liver and kidney. Since the ability to induce an increase of GSH and GST along with other detoxifying enzymes by anticarcinogenic agents has been reported to correlate with the inhibition of tumorigenesis, the cobalt complex might qualify as a potential cancer chemopreventive agent.

  9. Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase

    PubMed Central

    Hebbelmann, Inga; Selinski, Jennifer; Wehmeyer, Corinna; Goss, Tatjana; Voss, Ingo; Mulo, Paula; Kangasjärvi, Saijaliisa; Aro, Eva-Mari; Oelze, Marie-Luise; Dietz, Karl-Josef; Nunes-Nesi, Adriano; Do, Phuc T.; Fernie, Alisdair R.; Talla, Sai K.; Raghavendra, Agepati S.; Linke, Vera; Scheibe, Renate

    2012-01-01

    The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C3 plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck–Halliwell–Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants. PMID:22140244

  10. Phenolic Extract from Moringa oleifera Leaves Inhibits Key Enzymes Linked to Erectile Dysfunction and Oxidative Stress in Rats' Penile Tissues

    PubMed Central

    Oboh, Ganiyu; Ademiluyi, Adedayo O.; Ademosun, Ayokunle O.; Olasehinde, Tosin A.; Oyeleye, Sunday I.; Boligon, Aline A.; Athayde, Margareth L.

    2015-01-01

    This study was designed to determine the antioxidant properties and inhibitory effects of extract from Moringa oleifera leaves on angiotensin-I-converting enzyme (ACE) and arginase activities in vitro. The extract was prepared and phenolic (total phenols and flavonoid) contents, radical (nitric oxide (NO), hydroxyl (OH)) scavenging abilities, and Fe2+-chelating ability were assessed. Characterization of the phenolic constituents was done via high performance liquid chromatography-diode array detection (HPLC-DAD) analysis. Furthermore, the effects of the extract on Fe2+-induced MDA production in rats' penile tissue homogenate as well as its action on ACE and arginase activities were also determined. The extract scavenged NO∗, OH∗, chelated Fe2+, and inhibited MDA production in a dose-dependent pattern with IC50 values of 1.36, 0.52, and 0.38 mg/mL and 194.23 µg/mL, respectively. Gallic acid, chlorogenic acid, quercetin, and kaempferol were the most abundant phenolic compounds identified in the leaf extract. The extract also inhibited ACE and arginase activities in a dose-dependent pattern and their IC50 values were 303.03 and 159.59 µg/mL, respectively. The phenolic contents, inhibition of ACE, arginase, and Fe2+-induced MDA production, and radical (OH∗, NO∗) scavenging and Fe2+-chelating abilities could be some of the possible mechanisms by which M. oleifera leaves could be used in the treatment and/or management of erectile dysfunction. PMID:26557995

  11. Prevention of cumene hydroperoxide induced oxidative stress in cultured neonatal rat myocytes by scavengers and enzyme inhibitors.

    PubMed

    Persoon-Rothert, M; Egas-Kenniphaas, J M; van der Valk-Kokshoorn, E J; Mauve, I; van der Laarse, A

    1990-10-01

    Oxidative stress induced by cumene hydroperoxide was studied in cultured neonatal rat myocytes. A progressive increase of irreversible cell injury as determined by leakage of the cytoplastic enzyme alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) from the cells was noted at concentrations ranging from 25-100 microM cumene hydroperoxide (incubation time 90 min). Cumene hydroperoxide-induced damage was reduced or prevented by several compounds: the application of Trolox C, a water-soluble vitamin E analogue, and of phospholipase A2 inhibitors chlorpromazine and (to a lesser extent) quinacrine prevented alpha-HBDH release. ICRF-159, a chelator of divalent cations, ascorbic acid, a potent antioxidant, and the cysteine protease inhibitor leupeptin did not reduce the cumene hydroperoxide-induced cytotoxicity. Detoxification of hydroperoxides by the glutathione peroxidase system results in an increased flux through the pentose phosphate shunt and loss of NADPH. Glucose inhibited the cumene hydroperoxide-induced alpha-HBDH release, probably by replenishing NADPH. These results indicate that cumene hydroperoxide, after exhaustion of the glutathione system, induces irreversible injury in cultured myocytes by a mechanism that depends to a large extent on deterioration of cellular membranes caused by lipid peroxidation and phospholipase activation.

  12. Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase.

    PubMed

    Hebbelmann, Inga; Selinski, Jennifer; Wehmeyer, Corinna; Goss, Tatjana; Voss, Ingo; Mulo, Paula; Kangasjärvi, Saijaliisa; Aro, Eva-Mari; Oelze, Marie-Luise; Dietz, Karl-Josef; Nunes-Nesi, Adriano; Do, Phuc T; Fernie, Alisdair R; Talla, Sai K; Raghavendra, Agepati S; Linke, Vera; Scheibe, Renate

    2012-02-01

    The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C(3) plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck-Halliwell-Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants.

  13. Real time monitoring of layer-by-layer polyelectrolyte deposition and bacterial enzyme detection in nanoporous anodized aluminum oxide.

    PubMed

    Krismastuti, Fransiska Sri Herwahyu; Bayat, Haider; Voelcker, Nicolas H; Schönherr, Holger

    2015-04-07

    Porous anodized aluminum oxide (pAAO) is a nanostructured material, which due to its optical properties lends itself to the design of optical biosensors where interactions in the pores of this material are transduced into interferometric reflectance shifts. In this study, a pAAO-based biosensor was developed as a biosensing platform to detect proteinase K, an enzyme which is a readily available model system for the proteinase produced by Pseudomonas aeruginosa. The pAAO pore walls are decorated by means of the layer-by-layer (LbL) deposition technique using poly(sodium-4-styrenesulfonate) and poly-l-lysine as negatively and positively charged polyelectrolytes, respectively. Interferometric reflectance spectroscopy utilized to observe the optical properties of pAAO during LbL deposition shows that the deposition of the polyelectrolyte onto the pore walls increases the net refractive index, thus red-shifting the effective optical thickness (EOT). Upon incubation with proteinase K, a conspicuous blue shift of the EOT is observed, which is attributed to the destabilization of the LbL film upon enzymatic degradation of the poly-l-lysine components. This result is confirmed by scanning electron microscopy results. Finally, as a proof-of-principle, we demonstrate the ability of the label-free pAAO-based biosensing platform to detect the presence of the proteinase K in human wound fluid, highlighting the potential for detection of bacterial infections in chronic wounds.

  14. The roles of bacterial biofilm and oxidizing enzymes in the biodegradation of plastic by the bacterium Rhodococcus ruber (C208)

    NASA Astrophysics Data System (ADS)

    Sivan, A.; Gilan, I.; Santo, M.

    2011-12-01

    novel method for isolating mutants impaired in their production of biofilms (but not in their growth performance) was developed and utilized to isolate such mutants. Indeed, combining the Crystal Violet staining with confocal microscopy we were able to show that such mutants, not only contains reduced amounts of biofilm but also alters biofilm architecture. The above characterization of wild type and mutant strains can be utilized to determine the role of biofilm on the biodegradation of polyethylene. C208 produces laccase (phenol oxidase). This is an oxidizing enzyme that requires copper for induction and activity. In the presence of copper the biodegradation of mineral oil and of polyethylene, by C208, increased by up to 25% and 100%, respectively. Treatment of polyethylene films with a cell free-extract of laccase resulted in an increase of more then 40% in the carbonyl peak (indicating oxidation) as measured by FTIR. Furthermore, during 2 weeks of incubation, with C208 laccase, the molecular weight of polyethylene was reduced by 25%. It seems that laccase alone could not account for all degrading activity and, presumably, more enzyme(s) capable of degrading olefins are yet to be discovered.

  15. Spearmint induced hypothalamic oxidative stress and testicular anti-androgenicity in male rats - altered levels of gene expression, enzymes and hormones.

    PubMed

    Kumar, Vikas; Kural, Mool Raj; Pereira, B M J; Roy, Partha

    2008-12-01

    Mentha spicata Labiatae, commonly known as spearmint, can be used for various kinds of illnesses in herbal medicines and food industries. One of the prominent functions of this plant extract is its anti-androgenic activity. The present study investigated the probable correlation between oxidative stress in hypothalamic region and anti-androgenic action of this plant's aqueous extract on rats. Decreased activities of enzymes like superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase in hypothalamus of treated rats indicated spearmint induced oxidative stress. Further RT-PCR and immunoblot analysis demonstrated the decreased expression of some of the steroidogenic enzymes, cytochrome P450scc, cytochrome P450C17, 3beta-Hydroxysteroid dehydrogenase (3beta-HSD), 17beta-Hydroxysteroid dehydrogenase (17beta-HSD) and other related proteins like, steroidogenic acute regulatory protein, androgen receptor and scavenger receptor class B-1. Further, in vitro enzyme assays demonstrated depressed activities of testicular 3beta-HSD and 17beta-HSD enzymes. Histopathology indicated a decreased sperm density in cauda epididymis and degeneration of ductus deference. Our study suggested that spearmint probably induced oxidative stress in hypothalamus resulting in decreased synthesis of LH and FSH which in turn down-regulated the production of testicular testosterone through the disruption of a number of intermediate cascades.

  16. Combustion products of 1,3-butadiene inhibit catalase activity and induce expression of oxidative DNA damage repair enzymes in human bronchial epithelial cells.

    PubMed

    Kennedy, Christopher H; Catallo, W James; Wilson, Vincent L; Mitchell, James B

    2009-10-01

    1,3-Butadiene, an important petrochemical, is commonly burned off when excess amounts need to be destroyed. This combustion process produces butadiene soot (BDS), which is composed of a complex mixture of polycyclic aromatic hydrocarbons in particulates ranging in size from <1 microm to 1 mm. An organic extract of BDS is both cytotoxic and genotoxic to normal human bronchial epithelial (NHBE) cells. Based on the oxidizing potential of BDS, we hypothesized that an organic extract of this particulate matter would (1) cause enzyme inactivation due to protein amino acid oxidation and (2) induce oxidative DNA damage in NHBE cells. Thus, our aims were to determine the effect of butadiene soot ethanol extract (BSEE) on both enzyme activity and the expression of proteins involved in the repair of oxidative DNA damage. Catalase was found to be sensitive to BDS as catalase activity was potently diminished in the presence of BSEE. Using Western analysis, both the alpha isoform of human 8-oxoguanine DNA glycosylase (alpha-hOGG1) and human apurinic/apyrimidinic endonuclease (APE-1) were shown to be significantly overexpressed as compared to untreated controls after exposure of NHBE cells to BSEE. Our results indicate that BSEE is capable of effectively inactivating the antioxidant enzyme catalase, presumably via oxidation of protein amino acids. The presence of oxidized biomolecules may partially explain the extranuclear fluorescence that is detected when NHBE cells are treated with an organic extract of BDS. Overexpression of both alpha-hOGG1 and APE-1 proteins following treatment of NHBE cells with BSEE suggests that this mixture causes oxidative DNA damage.

  17. Hydroxytyrosol protects against oxidative damage by simultaneous activation of mitochondrial biogenesis and phase II detoxifying enzyme systems in retinal pigment epithelial cells.

    PubMed

    Zhu, Lu; Liu, Zhongbo; Feng, Zhihui; Hao, Jiejie; Shen, Weili; Li, Xuesen; Sun, Lijuan; Sharman, Edward; Wang, Ying; Wertz, Karin; Weber, Peter; Shi, Xianglin; Liu, Jiankang

    2010-11-01

    Studies in this laboratory have previously shown that hydroxytyrosol, the major antioxidant polyphenol in olives, protects ARPE-19 human retinal pigment epithelial cells from oxidative damage induced by acrolein, an environmental toxin and endogenous end product of lipid oxidation, that occurs at increased levels in age-related macular degeneration lesions. A proposed mechanism for this is that protection by hydroxytyrosol against oxidative stress is conferred by the simultaneous activation of two critically important pathways, viz., induction of phase II detoxifying enzymes and stimulation of mitochondrial biogenesis. Cultured ARPE-19 cells were pretreated with hydroxytyrosol and challenged with acrolein. The protective effects of hydroxytyrosol on key factors of mitochondrial biogenesis and phase II detoxifying enzyme systems were examined. Hydroxytyrosol treatment simultaneously protected against acrolein-induced inhibition of nuclear factor-E2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor coactivator 1 alpha (PPARGC1α) in ARPE-19 cells. The activation of Nrf2 led to activation of phase II detoxifying enzymes, including γ-glutamyl-cysteinyl-ligase, NADPH (nicotinamide adenine dinucleotide phosphate)-quinone-oxidoreductase 1, heme-oxygenase-1, superoxide dismutase, peroxiredoxin and thioredoxin as well as other antioxidant enzymes, while the activation of PPARGC1α led to increased protein expression of mitochondrial transcription factor A, uncoupling protein 2 and mitochondrial complexes. These results suggest that hydroxytyrosol is a potent inducer of phase II detoxifying enzymes and an enhancer of mitochondrial biogenesis. Dietary supplementation of hydroxytyrosol may contribute to eye health by preventing the degeneration of retinal pigment epithelial cells induced by oxidative stress. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Curcumin induces Nrf2 nuclear translocation and prevents glomerular hypertension, hyperfiltration, oxidant stress, and the decrease in antioxidant enzymes in 5/6 nephrectomized rats.

    PubMed

    Tapia, Edilia; Soto, Virgilia; Ortiz-Vega, Karla Mariana; Zarco-Márquez, Guillermo; Molina-Jijón, Eduardo; Cristóbal-García, Magdalena; Santamaría, José; García-Niño, Wylly Ramsés; Correa, Francisco; Zazueta, Cecilia; Pedraza-Chaverri, José

    2012-01-01

    Renal injury resulting from renal ablation induced by 5/6 nephrectomy (5/6NX) is associated with oxidant stress, glomerular hypertension, hyperfiltration, and impaired Nrf2-Keap1 pathway. The purpose of this work was to know if the bifunctional antioxidant curcumin may induce nuclear translocation of Nrf2 and prevents 5/6NX-induced oxidant stress, renal injury, decrease in antioxidant enzymes, and glomerular hypertension and hyperfiltration. Four groups of rats were studied: (1) control, (2) 5/6NX, (3) 5/6NX +CUR, and (4) CUR (n = 8-10). Curcumin was given by gavage to NX5/6 +CUR and CUR groups (60 mg/kg/day) starting seven days before surgery. Rats were studied 30 days after NX5/6 or sham surgery. Curcumin attenuated 5/6NX-induced proteinuria, systemic and glomerular hypertension, hyperfiltration, glomerular sclerosis, interstitial fibrosis, interstitial inflammation, and increase in plasma creatinine and blood urea nitrogen. This protective effect was associated with enhanced nuclear translocation of Nrf2 and with prevention of 5/6NX-induced oxidant stress and decrease in the activity of antioxidant enzymes. It is concluded that the protective effect of curcumin against 5/6NX-induced glomerular and systemic hypertension, hyperfiltration, renal dysfunction, and renal injury was associated with the nuclear translocation of Nrf2 and the prevention of both oxidant stress and the decrease of antioxidant enzymes.

  19. Benzene Exposure Alters Expression of Enzymes Involved in Fatty Acid β-Oxidation in Male C3H/He Mice.

    PubMed

    Sun, Rongli; Cao, Meng; Zhang, Juan; Yang, Wenwen; Wei, Haiyan; Meng, Xing; Yin, Lihong; Pu, Yuepu

    2016-10-31

    Benzene is a well-known hematotoxic carcinogen that can cause leukemia and a variety of blood disorders. Our previous study indicated that benzene disturbs levels of metabolites in the fatty acid β-oxidation (FAO) pathway, which is crucial for the maintenance and function of hematopoietic and leukemic cells. The present research aims to investigate the effects of benzene on changes in the expression of key enzymes in the FAO pathway in male C3H/He mice. Results showed that benzene exposure caused reduced peripheral white blood cell (WBC), red blood cell (RBC), platelet (Pit) counts, and hemoglobin (Hgb) concentration. Investigation of the effects of benzene on the expression of FA transport- and β-oxidation-related enzymes showed that expression of proteins Cpt1a, Crat, Acaa2, Aldh1l2, Acadvl, Crot, Echs1, and Hadha was significantly increased. The ATP levels and mitochondrial membrane potential decreased in mice exposed to benzene. Meanwhile, reactive oxygen species (ROS), hydrogen peroxide (H₂O₂), and malondialdehyde (MDA) levels were significantly increased in the benzene group. Our results indicate that benzene induces increased expression of FA transport and β-oxidation enzymes, mitochondrial dysfunction, and oxidative stress, which may play a role in benzene-induced hematotoxicity.

  20. Dietary supplementation of green synthesized manganese-oxide nanoparticles and its effect on growth performance, muscle composition and digestive enzyme activities of the giant freshwater prawn Macrobrachium rosenbergii.

    PubMed

    Asaikkutti, Annamalai; Bhavan, Periyakali Saravana; Vimala, Karuppaiya; Karthik, Madhayan; Cheruparambath, Praseeja

    2016-05-01

    The green synthesized Mn3O4 nanoparticles (manganese-oxide nanoparticles) using Ananas comosus (L.) peel extract was characterized by various techniques. HR-SEM photograph showed that manganese-oxide nanoparticles (Mn-oxide NPs) were spherical in shape, with an average size of 40-50 nm. The Zeta potential revealed the surface charge of Mn-oxide NPs to be negative. Further, the Mn-oxide NPs were dietary supplemented for freshwater prawn Macrobrachium rosenbergii. The experimental basal diets were supplemented with Mn-oxide NPs at the rates of 0 (control), 3.0, 6.0, 9.0, 12, 15 and 18 mg/kg dry feed weight. The as-supplemented Mn-oxide NPs were fed in M. rosenbergii for a period of 90 days. The experimental study demonstrated that prawns fed with diet supplemented with 3-18 mg Mn-oxide NPs/kg shows enhanced (P<0.05) growth performance, including final weight and weight gain (WG). Significant differences (P<0.05) in feed conversion ratio (FCR) were observed in prawn fed with different diets. Additionally, prawns fed with 3.0-18 mg/kg Mn-oxide NPs supplemented diets achieved significant (P<0.05) improvement in growth performance, digestive enzyme activities and muscle biochemical compositions, while, the prawns fed with 16 mg/kg of Mn-oxide NPs showed enhanced performance. Prawns fed on diet supplemented with 16 mg/kg Mn-oxide NPs showed significantly (P<0.05) higher total protein level. The antioxidants enzymatic activity (SOD and CAT) metabolic enzymes status in muscle and hepatopancreas showed no significant (P>0.05) alterations in prawns fed with 3.0-18 mg/kg of Mn-oxide NPs supplemented diets. Consequently, the present work proposed that 16 mg/kg of Mn-oxide NPs could be supplemented for flexible enhanced survival, growth and production of M. rosenbergii. Therefore, the data of the present study recommend the addition of 16 mg/kg of Mn-oxide NPs diet to developed prawn growth and antioxidant defense system. Copyright © 2016 Elsevier GmbH. All rights reserved.

  1. NiCl2-down-regulated antioxidant enzyme mRNA expression causes oxidative damage in the broiler(')s kidney.

    PubMed

    Guo, Hongrui; Wu, Bangyuan; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Xun; Deng, Jie; Yin, Shuang; Li, Jian; Tang, Kun

    2014-12-01

    The kidney serves as a major organ of nickel (Ni) excretion and is a target organ for acute Ni toxicity due to Ni accumulation. There are no studies on the Ni or Ni compound-regulated antioxidant enzyme mRNA expression in animals and human beings at present. This study was conducted to investigate the pathway of nickel chloride (NiCl2)-caused renal oxidative damage by the methods of biochemistry, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. Two hundred and eighty one-day-old broilers were randomly divided into four groups and fed on a control diet and three experimental diets supplemented with 300, 600, and 900 mg/kg of NiCl2 for 42 days. Dietary NiCl2 elevated the malondialdehyde (MDA), nitric oxide (NO), 8-hydroxy-2'-deoxyguanosine (8-OHdG) contents, and reduced the ability to inhibit hydroxy radical in the NiCl2-treated groups. Also, the renal inducible nitric oxide synthase (iNOS) activity and mRNA expression levels were increased. The total antioxidant (T-AOC) and activities of antioxidant enzymes including copper zinc superoxide dismutase (CuZn-SOD), manganese superoxide dismutase (Mn