Science.gov

Sample records for oxidizing enzymes elucidating

  1. Prolonged fasting identifies heat shock protein 10 as a Sirtuin 3 substrate: elucidating a new mechanism linking mitochondrial protein acetylation to fatty acid oxidation enzyme folding and function.

    PubMed

    Lu, Zhongping; Chen, Yong; Aponte, Angel M; Battaglia, Valentina; Gucek, Marjan; Sack, Michael N

    2015-01-23

    Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function. PMID:25505263

  2. Sulfite oxidizing enzymes

    PubMed Central

    Feng, Changjian; Tollin, Gordon; Enemark, John H.

    2007-01-01

    Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme

  3. Elucidation of metabolic pathways from enzyme classification data.

    PubMed

    McDonald, Andrew G; Tipton, Keith F

    2014-01-01

    The IUBMB Enzyme List is widely used by other databases as a source for avoiding ambiguity in the recognition of enzymes as catalytic entities. However, it was not designed for metabolic pathway tracing, which has become increasingly important in systems biology. A Reactions Database has been created from the material in the Enzyme List to allow reactions to be searched by substrate/product, and pathways to be traced from any selected starting/seed substrate. An extensive synonym glossary allows searches by many of the alternative names, including accepted abbreviations, by which a chemical compound may be known. This database was necessary for the development of the application Reaction Explorer ( http://www.reaction-explorer.org ), which was written in Real Studio ( http://www.realsoftware.com/realstudio/ ) to search the Reactions Database and draw metabolic pathways from reactions selected by the user. Having input the name of the starting compound (the "seed"), the user is presented with a list of all reactions containing that compound and then selects the product of interest as the next point on the ensuing graph. The pathway diagram is then generated as the process iterates. A contextual menu is provided, which allows the user: (1) to remove a compound from the graph, along with all associated links; (2) to search the reactions database again for additional reactions involving the compound; (3) to search for the compound within the Enzyme List. PMID:24218216

  4. 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.

  5. Novel uses of isotope effects to elucidate enzyme reaction mechanisms

    SciTech Connect

    Hermes, J.D.

    1983-01-01

    A new technique (Multiple Isotope Effects) for determining enzyme reaction mechanisms and intrinsic isotope effects has been developed. By determining the /sup 13/C isotope effect on V/K with both a deuterated and unlabeled substrate, and the deuterium isotope effect on V/K, it is possible to tell whether the /sup 13/C-sensitive and deuterium-sensitive steps are the same or not and, if they are different, to determine which comes first in the mechanism. If the two isotope-sensitive steps are the same, 1) deuteration increases the size of the observed /sup 13/C isotope effect, and 2) the intrinsic /sup 13/C and deuterium isotope effects, and the commitments can be calculated. When the deuterium- and /sup 13/C-sensitive steps are different, deuteration decreases the size of the observed /sup 13/C isotope effect. The data fit the equation (/sup 13/(V/K)/sub H/ - 1)/(/sup 13/(V/K)/sub D/ - 1) = /sup D/(V/K)//sup D/K/sub eq/ when the deuterium-sensitive step comes first, but fit the equation (/sup 13/(V/K)/sub H/ - /sup 13/K/sub eq/)/(/sup 13/(V/K)/sub D/ - /sup 13/K/sub eq/) = /sup D/(V/K) when the /sup 13/C-sensitive step comes first.

  6. Cellulose degradation by oxidative enzymes.

    PubMed

    Dimarogona, Maria; Topakas, Evangelos; Christakopoulos, Paul

    2012-01-01

    Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs), cellobiose dehydrogenases (CDHs) and members of carbohydrate-binding module family 33 (CBM33). PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future. PMID:24688656

  7. Cellulose degradation by oxidative enzymes

    PubMed Central

    Dimarogona, Maria; Topakas, Evangelos; Christakopoulos, Paul

    2012-01-01

    Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs), cellobiose dehydrogenases (CDHs) and members of carbohydrate-binding module family 33 (CBM33). PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future. PMID:24688656

  8. 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

  9. 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.

  10. 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.

  11. [I-cell disease: elucidation of the enzyme defect and its molecular biology significance].

    PubMed

    Leroy, J G

    1989-01-01

    "I-Cell disease" (ICD) has received its name because of the innumerable intracytoplasmatic inclusions in connective tissue cells and in vitro fibroblasts derived from patients. It is a progressive disorder already recognizable in infancy. Severe disturbance of growth, coarsening facial features and moderate to severe psychomotor handicap are the most important clinical characteristics. ICD is fatal already in childhood. Prominent among many anatomo- and physiopathologic abnormalities is the paradox of a very reduced activity of many lysosomal hydrolases in connective tissue cells on the one hand and the strongly enhanced activity of these hydrolases in body fluids and culture media on the other hand. Similar, albeit less pronounced abnormalities are observed also in patients with pseudo-Hurler polydystrophy, (PHP) a related though milder disorder. In patients with either ICZ or PHP, phosphorylation of oligomannosyl type sidechains in acid hydrolases, known to be glycoproteins, does not occur. Because the mannose-6-phosphate (M-6-P) recognition marker necessary for normal transport of nascent hydrolases to the lysosomal compartment is not formed, these enzymes are not retained in the cells. The underlying cause is a functionally deficient N-acetylglucosaminephosphotransferase in ICD and PHP patients. The pathological consequences of this defect are more pronounced in fibroblasts than in parenchymatous cells, because the former lack alternative mechanisms by which hydrolases can reach lysosomes. The number of known secondary abnormalities in ICD remain useful for confirmation of the clinical diagnosis and for prenatal diagnosis. The elucidation of the metabolic defect in ICD, a rare monogenic disorder, has increased considerably knowledge on enzyme maturation and on the physiology of intracytoplasmic organelles. PMID:2686262

  12. Crystal structure of methane oxidation enzyme determined

    SciTech Connect

    Baum, R.

    1994-01-10

    A team of chemists has determined to 2.2-[angstrom] resolution the crystal structure of the hydroxylase protein of methane monooxygenase, the enzyme system responsible for the biological oxidation of methane. The hydroxylase, at a molecular weight of 251,000 daltons, if by far the largest component of methane monooxygenase. Although the crystal structure of the hydroxylase did not reveal any startling surprises about the enzyme-many features of the hydroxylase had been inferred previously from modeling and spectroscopic studies -- obtaining it is a significant achievement. For one thing, the crystal structure unambiguously confirms aspects of the enzyme structure that been at least somewhat speculative. The three-dimensional structure of the enzyme, the chemist say, also provides important insight into biological methane oxidation, including how methane, a relatively inert gas, might diffuse to and bind near the active site of the enzyme. The structure points to particular amino acid residues that are likely to participate in catalysis, and clarifies the structure of the dinuclear iron core of the enzyme.

  13. 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. PMID:27497174

  14. Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function.

    PubMed

    Lisi, George P; Patrick Loria, J

    2016-02-01

    Conformational motions play an essential role in enzyme function, often facilitating the formation of enzyme-substrate complexes and/or product release. Although considerable debate remains regarding the role of molecular motions in the conversion of enzymatic substrates to products, numerous examples have found motions to be crucial for optimization of enzyme scaffolds, effective substrate binding, and product dissociation. Conformational fluctuations are often rate-limiting to enzyme catalysis, primarily through product release, with the chemical reaction occurring much more quickly. As a result, the direct involvement of motions at various stages along the enzyme reaction coordinate remains largely unknown and untested. In the following review, we describe the use of solution NMR techniques designed to probe various timescales of molecular motions and detail examples in which motions play a role in propagating catalytic effects from the active site and directly participate in essential aspects of enzyme function. PMID:26952190

  15. Small-molecule probes elucidate global enzyme activity in a proteomic context

    PubMed Central

    Lee, Jun-Seok; Yoo, Young-Hwa; Yoon, Chang No

    2014-01-01

    The recent dramatic improvements in high-resolution mass spectrometry (MS) have revolutionized the speed and scope of proteomic studies. Conventional MS-based proteomics methodologies allow global protein profiling based on expression levels. Although these techniques are promising, there are numerous biological activities yet to be unveiled, such as the dynamic regulation of enzyme activity. Chemical proteomics is an emerging field that extends these types proteomic profiling. In particular, activity-based protein profiling (ABPP) utilizes small-molecule probes to monitor enzyme activity directly in living intact subjects. In this mini-review, we summarize the unique roles of smallmolecule probes in proteomics studies and highlight some recent examples in which this principle has been applied. [BMB Reports 2014; 47(3): 149-157] PMID:24499666

  16. A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes

    SciTech Connect

    Yin, Si-Tao; Huang, Hao; Zhang, Yu-Hang; Zhou, Zi-Ren; Song, Ai-Xin; Hong, Fa-Shui; Hu, Hong-Yu

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer A deubiquitinating enzyme has its unique substrate specificity for deubiquitination. Black-Right-Pointing-Pointer We have established an activity assay for ubiquitin C-terminal hydrolases. Black-Right-Pointing-Pointer This assay can be applicable to other deubiquitinating enzymes. -- Abstract: Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub{sup F45W}-Xaa) and di-ubiquitin chains (Ub{sup F45W}-diUb). After removal of the intact substrate by Ni{sup 2+}-NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub{sup F45W} product. The results show that three UCHs, i.e. UCH-L1, UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs.

  17. 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.

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

    PubMed

    Sun, Ren; Wang, Liya

    2014-10-01

    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. PMID:25215937

  19. Synthesis and Structural Elucidation of Triazolylmolybdenum(VI) Oxide Hybrids and Their Behavior as Oxidation Catalysts.

    PubMed

    Lysenko, Andrey B; Senchyk, Ganna A; Domasevitch, Konstantin V; Hauser, Jürg; Fuhrmann, Daniel; Kobalz, Merten; Krautscheid, Harald; Neves, Patrícia; Valente, Anabela A; Gonçalves, Isabel S

    2015-09-01

    A large family of bifunctional 1,2,4-triazole molecular tectons (tr) has been explored for engineering molybdenum(VI) oxide hybrid solids. Specifically, tr ligands bearing auxiliary basic or acidic groups were of the type amine, pyrazole, 1H-tetrazole, and 1,2,4-triazole. The organically templated molybdenum(VI) oxide solids with the general compositions [MoO3(tr)], [Mo2O6(tr)], and [Mo2O6(tr)(H2O)2] were prepared under mild hydrothermal conditions or by refluxing in water. Their crystal structures consist of zigzag chains, ribbons, or helixes of alternating cis-{MoO4N2} or {MoO5N} polyhedra stapled by short [N-N]-tr bridges that for bitriazole ligands convert the motifs into 2D or 3D frameworks. The high thermal (235-350 °C) and chemical stability observed for the materials makes them promising for catalytic applications. The molybdenum(VI) oxide hybrids were successfully explored as versatile oxidation catalysts with tert-butyl hydroperoxide (TBHP) or aqueous H2O2 as an oxygen source, at 70 °C. Catalytic performances were influenced by the different acidic-basic properties and steric hindrances of coordinating organic ligands as well as the structural dimensionality of the hybrid. PMID:26280712

  20. 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

  1. Pathway profiling in Mycobacterium tuberculosis: elucidation of cholesterol-derived catabolite and enzymes that catalyze its metabolism.

    PubMed

    Thomas, Suzanne T; VanderVen, Brian C; Sherman, David R; Russell, David G; Sampson, Nicole S

    2011-12-23

    Mycobacterium tuberculosis, the bacterium that causes tuberculosis, imports and metabolizes host cholesterol during infection. This ability is important in the chronic phase of infection. Here we investigate the role of the intracellular growth operon (igr), which has previously been identified as having a cholesterol-sensitive phenotype in vitro and which is important for intracellular growth of the mycobacteria. We have employed isotopically labeled low density lipoproteins containing either [1,7,15,22,26-(14)C]cholesterol or [1,7,15,22,26-(13)C]cholesterol and high resolution LC/MS as tools to profile the cholesterol-derived metabolome of an igr operon-disrupted mutant (Δigr) of M. tuberculosis. A partially metabolized cholesterol species accumulated in the Δigr knock-out strain that was absent in the complemented and parental wild-type strains. Structural elucidation by multidimensional 1H and 13C NMR spectroscopy revealed the accumulated metabolite to be methyl 1β-(2'-propanoate)-3aα-H-4α-(3'-propanoic acid)-7aβ-methylhexahydro-5-indanone. Heterologously expressed and purified FadE28-FadE29, an acyl-CoA dehydrogenase encoded by the igr operon, catalyzes the dehydrogenation of 2'-propanoyl-CoA ester side chains in substrates with structures analogous to the characterized metabolite. Based on the structure of the isolated metabolite, enzyme activity, and bioinformatic annotations, we assign the primary function of the igr operon to be degradation of the 2'-propanoate side chain. Therefore, the igr operon is necessary to completely metabolize the side chain of cholesterol metabolites. PMID:22045806

  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. 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. PMID:27411370

  4. Products from enzyme-catalyzed oxidations of norcarenes.

    PubMed

    Newcomb, Martin; Lansakara-P, Dharmika S P; Kim, Hye-Yeong; Chandrasena, R Esala P; Lippard, Stephen J; Beauvais, Laurance G; Murray, Leslie J; Izzo, Viviana; Hollenberg, Paul F; Coon, Minor J

    2007-02-16

    Recent studies revealed that norcarane (bicyclo[4.1.0]heptane) is oxidized to 2-norcarene (bicyclo[4.1.0]-hept-2-ene) and 3-norcarene (bicyclo[4.1.0]hept-3-ene) by iron-containing enzymes and that secondary oxidation products from the norcarenes complicate mechanistic probe studies employing norcarane as the substrate (Newcomb, M.; Chandrasena, R. E. P.; Lansakara-P., D. S. P.; Kim, H.-Y.; Lippard, S. J.; Beauvais, L. G.; Murray, L. J.; Izzo, V.; Hollenberg, P. F.; Coon, M. J. J. Org. Chem. 2007, 72, 1121-1127). In the present work, the product profiles from the oxidations of 2-norcarene and 3-norcarene by several enzymes were determined. Most of the products were identified by GC and GC-mass spectral comparison to authentic samples produced independently; in some cases, stereochemical assignments were made or confirmed by 2D NMR analysis of the products. The enzymes studied in this work were four cytochrome P450 enzymes, CYP2B1, CYPDelta2E1, CYPDelta2E1 T303A, and CYPDelta2B4, and three diiron-containing enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. The oxidation products from the norcarenes identified in this work are 2-norcaranone, 3-norcaranone, syn- and anti-2-norcarene oxide, syn- and anti-3-norcarene oxide, syn- and anti-4-hydroxy-2-norcarene, syn- and anti-2-hydroxy-3-norcarene, 2-oxo-3-norcarene, 4-oxo-2-norcarene, and cyclohepta-3,5-dienol. Two additional, unidentified oxidation products were observed in low yields in the oxidations. In matched oxidations, 3-norcarene was a better substrate than 2-norcarene in terms of turnover by factors of 1.5-15 for the enzymes studied here. The oxidation products found in enzyme-catalyzed oxidations of the norcarenes are useful for understanding the complex product mixtures obtained in norcarane oxidations. PMID:17288367

  5. 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…

  6. 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

  7. 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

  8. 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. PMID:2119387

  9. 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. PMID:25604951

  10. Regulation of antioxidant enzymes in lung after oxidant injury.

    PubMed Central

    Quinlan, T; Spivack, S; Mossman, B T

    1994-01-01

    Studies have implicated active oxygen species (AOS) in the pathogenesis of various lung diseases. Many chemical and physical agents in the environment are potent generators of AOS, including ozone, hyperoxia, mineral dusts, paraquat, etc. These agents produce AOS by different mechanisms, but frequently the lung is the primary target of toxicity, and exposure results in damage to lung tissue to varying degrees. The lung has developed defenses to AOS-mediated damage, which include antioxidant enzymes, the superoxide dismutases [copper-zinc (CuZnSOD) and manganese-containing (MnSOD)], catalase, and glutathione peroxidase (GPX). In this review, antioxidant defenses to environmental stresses in the lung as well as in isolated pulmonary cells following exposure to a number of different oxidants, are summarized. Each oxidant appears to induce a different pattern of antioxidant enzyme response in the lung, although some common trends, i.e., induction of MnSOD following oxidants inducing inflammation or pulmonary fibrosis, in responses to oxidants occur. Responses may vary between the different cell types in the lung as a function of cell-cycle or other factors. Increases in MnSOD mRNA or immunoreactive protein in response to certain oxidants may serve as a biomarker of AOS-mediated damage in the lung. Images Figure 3. PMID:7523104

  11. DJ-1 upregulates anti-oxidant enzymes and attenuates hypoxia/re-oxygenation-induced oxidative stress by activation of the nuclear factor erythroid 2-like 2 signaling pathway.

    PubMed

    Yan, Yu-Feng; Yang, Wen-Jie; Xu, Qiang; Chen, He-Ping; Huang, Xiao-Shan; Qiu, Ling-Yu; Liao, Zhang-Ping; Huang, Qi-Ren

    2015-09-01

    DJ-1 protein, as a multifunctional intracellular protein, has an important role in transcriptional regulation and anti-oxidant stress. A recent study by our group showed that DJ-1 can regulate the expression of certain anti‑oxidant enzymes and attenuate hypoxia/re‑oxygenation (H/R)‑induced oxidative stress in the cardiomyocyte cell line H9c2; however, the detailed molecular mechanisms have remained to be elucidated. Nuclear factor erythroid 2‑like 2 (Nrf2) is an essential transcription factor that regulates the expression of several anti‑oxidant genes via binding to the anti‑oxidant response element (ARE). The present study investigated whether activation of the Nrf2 pathway is responsible for the induction of anti‑oxidative enzymes by DJ‑1 and contributes to the protective functions of DJ‑1 against H/R‑induced oxidative stress in H9c2 cells. The results demonstrated that DJ‑1‑overexpressing H9c2 cells exhibited anti‑oxidant enzymes, including manganese superoxide dismutase, catalase and glutathione peroxidase, to a greater extent and were more resistant to H/R‑induced oxidative stress compared with native cells, whereas DJ‑1 knockdown suppressed the induction of these enzymes and further augmented the oxidative stress injury. Determination of the importance of Nrf2 in DJ‑1‑mediated anti‑oxidant enzymes induction and cytoprotection against oxidative stress induced by H/R showed that overexpression of DJ‑1 promoted the dissociation of Nrf2 from its cytoplasmic inhibitor Keap1, resulting in enhanced levels of nuclear translocation, ARE‑binding and transcriptional activity of Nrf2. Of note, Nrf2 knockdown abolished the DJ‑1‑mediated induction of anti‑oxidant enzymes and cytoprotection against oxidative stress induced by H/R. In conclusion, these findings indicated that activation of the Nrf2 pathway is a critical mechanism by which DJ-1 upregulates anti-oxidative enzymes and attenuates H/R-induced oxidative stress in H9c2

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. Flume experiments elucidate relationships between stream morphology, hyporheic residence time, and nitrous oxide production

    NASA Astrophysics Data System (ADS)

    Quick, Annika; Farrell, Tiffany B.; Reeder, William Jeffrey; Feris, Kevin P.; Tonina, Daniele; Benner, Shawn G.

    2015-04-01

    The hyporheic zone is a potentially important producer of nitrous oxide, a powerful greenhouse gas. The location and magnitude of nitrous oxide generation within the hyporheic zone involves complex interactions between multiple nitrogen species, redox conditions, microbial communities, and hydraulics. To better understand nitrous oxide generation and emissions from streams, we conducted large-scale flume experiments in which we monitored pore waters along hyporheic flow paths within stream dune structures. Measurements of dissolved oxygen, ammonia, nitrate, nitrite, and dissolved nitrous oxide showed distinct spatial relationships reflecting redox changes along flow paths. Using residence times along a flow path, clear trends in oxygen conditions and nitrogen species were observed. Three dune sizes were modeled, resulting in a range of residence times, carbon reactivity levels and respiration rates. We found that the magnitude and location of nitrous oxide production in the hyporheic zone is related to nitrate loading, dune morphology, and residence time. Specifically, increasing exogenous nitrate levels in surface water to approximately 3 mg/L resulted in an increase in dissolved N2O concentrations greater than 500% (up to 10 µg/L N-N2O) in distinct zones of specific residence times. We also found, however, that dissolved N2O concentrations decreased to background levels further along the flow path due to either reduction of nitrous oxide to dinitrogen gas or degassing. The decrease in measurable N2O along a flow path strongly suggests an important relationship between dune morphology, residence time, and nitrous oxide emissions from within stream sediments. Relating streambed morphology and loading of nitrogen species allows for prediction of nitrous oxide production in the hyporheic zone of natural systems.

  14. Transient, oxidant-induced antioxidant transcript and enzyme levels correlate with greater oxidant-resistance in paraquat-resistant Conyza bonariensis.

    PubMed

    Ye, B; Gressel, J

    2000-06-01

    The elucidation of mechanisms plants use to overcome oxidative stress is facilitated where there is intra-specific genetic variability. The differential induction of higher levels of mRNAs, cytosol and chloroplast antioxidant enzyme activities, and proteins occurred after sub-lethal paraquat treatment of the oxidant-resistant biotype of Conyza bonariensis (L.) Cronq. By 6 h after sub-lethal paraquat treatment the activities of superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), dehydroascorbate reductase (EC 1.8.5), monodehydroascorbate reductase (EC 1.6.5.4), and glutathione peroxidase (EC 1.11.19) had increased, peaking at 24 h and then slowly reverting back to the basal level. Similarly, the levels of mRNAs encoding these enzymes were enhanced by 12 h and peaked at 18-24 h after sub-lethal paraquat treatment. The time courses of the transient elevation of both transcript and antioxidant enzyme levels correlated with a further transient 2.5- to 3.0-fold increase of paraquat resistance, which occurred only in the constitutively resistant biotype. The individual enzymes seem to be part of a coordinately controlled oxidant tolerance in the resistant biotype, utilizing oxidant-induced, increasingly abundant transcript levels, upon which more antioxidant enzymes were synthesized. PMID:10923703

  15. 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

  16. Elucidating molecular iridium water oxidation catalysts using metal-organic frameworks: a comprehensive structural, catalytic, spectroscopic, and kinetic study.

    PubMed

    Wang, Cheng; Wang, Jin-Liang; Lin, Wenbin

    2012-12-01

    As a new class of porous, crystalline, molecular materials, metal-organic frameworks (MOFs) have shown great promise as recyclable and reusable single-site solid catalysts. Periodic order and site isolation of the catalytic struts in MOFs facilitate the studies of their activities and reaction mechanisms. Herein we report the construction of two highly stable MOFs (1 and 2) using elongated dicarboxylate bridging ligands derived from Cp*Ir(L)Cl complexes (L = dibenzoate-substituted 2,2'-bipyridine, bpy-dc, or dibenzoate-substituted 2-phenylpyridine, ppy-dc) and Zr(6)O(4)(OH)(4)(carboxylate)(12) cuboctahedral secondary building units (SBUs) and the elucidation of water oxidation pathways of the Cp*Ir(L)Cl catalysts using these MOFs. We carried out detailed kinetic studies of Ce(4+)-driven water oxidation reactions (WORs) catalyzed by the MOFs using UV-vis spectroscopy, phosphorescent oxygen detection, and gas chromatographic analysis. These results confirmed not only water oxidation activity of the MOFs but also indicated oxidative degradation of the Cp* rings during the WOR. The (bpy-dc)Ir(H(2)O)(2)XCl (X is likely a formate or acetate group) complex resulted from the oxidative degradation process was identified as a competent catalyst responsible for the water oxidation activity of 1. Further characterization of the MOFs recovered from WORs using X-ray photoelectron, diffuse-reflectance UV-vis absorption, luminescence, and infrared spectroscopies supported the identity of (bpy-dc)Ir(H(2)O)(2)XCl as an active water oxidation catalyst. Kinetics of MOF-catalyzed WORs were monitored by Ce(4+) consumptions and fitted with a reaction-diffusion model, revealing an intricate relationship between reaction and diffusion rates. Our work underscores the opportunity in using MOFs as well-defined single-site solid catalytic systems to reveal mechanistic details that are difficult to obtain for their homogeneous counterparts. PMID:23136923

  17. Interplay of Experiment and Theory in Elucidating Mechanisms of Oxidation Reactions by a Nonheme Ru(IV)O Complex.

    PubMed

    Dhuri, Sunder N; Cho, Kyung-Bin; Lee, Yong-Min; Shin, Sun Young; Kim, Jin Hwa; Mandal, Debasish; Shaik, Sason; Nam, Wonwoo

    2015-07-01

    A comprehensive experimental and theoretical study of the reactivity patterns and reaction mechanisms in alkane hydroxylation, olefin epoxidation, cyclohexene oxidation, and sulfoxidation reactions by a mononuclear nonheme ruthenium(IV)-oxo complex, [Ru(IV)(O)(terpy)(bpm)](2+) (1), has been conducted. In alkane hydroxylation (i.e., oxygen rebound vs oxygen non-rebound mechanisms), both the experimental and theoretical results show that the substrate radical formed via a rate-determining H atom abstraction of alkanes by 1 prefers dissociation over oxygen rebound and desaturation processes. In the oxidation of olefins by 1, the observations of a kinetic isotope effect (KIE) value of 1 and styrene oxide formation lead us to conclude that an epoxidation reaction via oxygen atom transfer (OAT) from the Ru(IV)O complex to the C═C double bond is the dominant pathway. Density functional theory (DFT) calculations show that the epoxidation reaction is a two-step, two-spin-state process. In contrast, the oxidation of cyclohexene by 1 affords products derived from allylic C-H bond oxidation, with a high KIE value of 38(3). The preference for H atom abstraction over C═C double bond epoxidation in the oxidation of cyclohexene by 1 is elucidated by DFT calculations, which show that the energy barrier for C-H activation is 4.5 kcal mol(-1) lower than the energy barrier for epoxidation. In the oxidation of sulfides, sulfoxidation by the electrophilic Ru-oxo group of 1 occurs via a direct OAT mechanism, and DFT calculations show that this is a two-spin-state reaction in which the transition state is the lowest in the S = 0 state. PMID:26075466

  18. 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

    2015-10-01

    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. PMID:26325666

  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. 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.

  1. 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

    2016-06-01

    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. PMID:27192445

  2. Use of isotope effects in the elucidation of aliphatic oxidation as catalyzed by cytochrome P-450

    SciTech Connect

    Jones, J.P.

    1987-01-01

    Two selectively deuterated n-octanes (octane-1-/sup 2/H/sub 3/ and octane-1, 2, 3-/sup 2/H/sub 7/) were synthesized and subjected to hydroxylation by phenobarbital-induced rat liver microsomes and purified cytochrome P-450b. The results of these experiments provide evidence which clarifies the interplay between a branched reaction pathway and the equilibration of an enzyme-substrate complex, in determining the magnitude of an observed isotope effect. An equation is derived that allows limits to be placed on the intrinsic isotope effect. The intrinsic isotope effect for the formation of 1-octanol was determined by this equation to lie between 9.5 and 9.8. The intramolecular isotope effect for octane-1-/sup 2/H/sub 3/ was separated into its primary and secondary components using the method of Hanzlik. The primary effect was found to lie between 7.3 and 7.9 while the secondary effect was found to lie between 1.09-1.14. These data are consistent with a symmetrical reaction coordinate with 15% of the observed isotope effect being due to the secondary effects. A slight deviation from the rule of the geometric means was observed. An identical analysis was performed for ortho, meta and para-xylenes that contained deuterium in one methyl group. The secondary isotope effects varied dramatically, from between 0.93 to 1.19, due to violation of the rule of the geometric mean. The observed isotope effects appeared to vary as a function of the distance between the methyl groups in the various methyl groups of xylene with ortho having the largest isotope effect, meta a median isotope effect or an isotope effect similar in magnitude to ortho, and para the smallest effect.

  3. Structural Aspects of Several Oxide Glasses as Elucidated by Multinuclear NMR.

    NASA Astrophysics Data System (ADS)

    Zhong, Jianhui

    1988-03-01

    NMR is sensitive to many interactions that the nucleus experiences with its environment. Included among these interactions are two that were heavily exploited in this thesis. They are the electric quadrupole interaction and the chemical shift interaction. These interactions yield structural information on short-range order and atom coordinations, which are very valuable and important to the understanding of properties and microstructures of glasses. Both the static or MASS NMR at high field and low field CW NMR were utilized to obtain information concerning the coordinations and local environments of several oxide glasses. For some systems, other spectroscopic methods (thermal analysis, X-ray and IR spectroscopies) were also used to assist NMR studies. In Chapter I relevant NMR theory and detection techniques used for this work are introduced. In Chapters 2-4, some structural aspects of alkali borate, borosilicate or alkali boroaluminate glasses are studied. In particular, boron coordinations in these glasses are carefully reinvestigated. The relationship between the change in boron coordinations and the changes in macroscopic characteristics of the glasses (such as electric conductivity and the mixed alkali effects) are studied. In mixed alkali glasses, the postulated alkali -pairing model provides a reasonable explanation for the structural changes. Chapter 5 is dedicated to the ^{71}Ga and ^{69 }Ga NMR studies of alkali gallate glasses. The glassforming range is explored and the gallium atom coordinations are studied. A structural model for the glass systems is suggested based on the distribution of galliums of different coordinations and structural parameters (quadrupole coupling constants, asymmetry parameters, and isotropic chemical shifts, etc.). A study of ^{31} P spectra in lead-iron-phosphate nuclear waste glasses is presented in Chapter 6. By subjecting the samples to various magnetic field strengths and different temperatures, information on the local

  4. Oxidative degradation of bisphenol a by crude enzyme prepared from potato.

    PubMed

    Xuan, Ying Ji; Endo, Yasushi; Fujimoto, Kenshiro

    2002-10-23

    When crude enzymes prepared from some vegetables and fruits were incubated with bisphenol A (2,2-bis(4-hydroxyphenyl)propane, BPA) at 37 degrees C, BPA was oxidized by crude enzymes from potato, eggplant, and lettuce. The crude enzyme prepared from potato (Solanum tuberosum) had the strongest oxidative activity for BPA. Its optimal temperature and pH were 40-45 degrees C and 8.0, respectively. More than 95% of BPA was oxidized after the incubation with potato enzyme for 60 min. BPA gave two oxidation products besides insoluble compounds during the oxidation by potato enzyme. The oxidation products were identified to be 4[1-(4-hydroxyphenyl)-1-methyl-ethyl]-benzene-1,2-diol and 4[1-(4-hydroxyphenyl)-1-methyl-ethyl]-benzene-1,3-diol. Enzymatically oxidized BPA lost the estrogen-like activity to enhance the growth of human breast cancer (MCF-7) cells. PMID:12381152

  5. 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. PMID:24287412

  6. 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.

  7. 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 ...

  8. 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.

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

    PubMed

    Lawton, Thomas J; Rosenzweig, Amy C

    2016-08-01

    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

  10. 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

  11. 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

  12. 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.

  13. Oxidation of nonionic detergents by cytochrome P450 enzymes.

    PubMed

    Hosea, N A; Guengerich, F P

    1998-05-15

    Nonionic phenolic detergents are commonly used in the purification of membrane-associated proteins. Triton N-101 was shown to be oxidized by NADPH-fortified human liver microsomes and recombinant human cytochromes P450 (P450). Oxidation was monitored using HPLC and the fluorescence properties of Triton N-101 and other alkylphenol ethoxylate detergents, which are similar to those of anisole. Human liver microsomes and recombinantly expressed reconstituted P450 3A4-oxidized Triton N-101 in a concentration-dependent manner which could be inhibited by ketoconazole, a P450 3A4-selective inhibitor. Triton N-101 inhibition of testosterone oxidation by human liver microsomes was of a mixed nature but mainly non-competitive. Electrospray ionization mass spectrometry and tandem mass spectrometry indicated that the major product formed was hydroxylated on the alkyl moiety. Human liver microsomes also oxidized other Tritons (X-100 and X-114), Emulgens 911 and 913, and Tergitol NP-10 to a similar extent. P450s 1A1, 1A2, and 2C9 also oxidized Triton N-101 but to a lesser extent than P450 3A4. We conclude that Triton N-101 and similar nonionic detergents are oxidized by P450 3A4 and some other P450s. PMID:9606971

  14. 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. PMID:26334845

  15. 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. PMID:27094408

  16. 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.

  17. 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. PMID:24211189

  18. 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

  19. 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

  20. 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. PMID:24034427

  1. Photobiomodulation Therapy Decreases Oxidative Stress in the Lung Tissue after Formaldehyde Exposure: Role of Oxidant/Antioxidant Enzymes.

    PubMed

    Silva Macedo, Rodrigo; Peres Leal, Mayara; Braga, Tarcio Teodoro; Barioni, Éric Diego; de Oliveira Duro, Stephanie; Ratto Tempestini Horliana, Anna Carolina; Câmara, Niels Olsen Saraiva; Marcourakis, Tânia; Farsky, Sandra Helena Poliselli; Lino-Dos-Santos-Franco, Adriana

    2016-01-01

    Formaldehyde is ubiquitous pollutant that induces oxidative stress in the lung. Several lung diseases have been associated with oxidative stress and their control is necessary. Photobiomodulation therapy (PBMT) has been highlighted as a promissory treatment, but its mechanisms need to be better investigated. Our objective was to evaluate the effects of PBMT on the oxidative stress generated by FA exposure. Male Wistar rats were submitted to FA exposure of 1% or vehicle (3 days) and treated or not with PBMT (1 and 5 h after each FA exposure). Rats treated only with laser were used as control. Twenty-four hours after the last FA exposure, we analyzed the effects of PBMT on the generation of nitrites and hydrogen peroxide, oxidative burst, glutathione reductase, peroxidase, S-transferase enzyme activities, the gene expression of nitric oxide, cyclooxygenase, superoxide dismutase, the catalase enzyme, and heme oxygenase-1. PBMT reduced the generation of nitrites and hydrogen peroxide and increased oxidative burst in the lung cells. A decreased level of oxidant enzymes was observed which were concomitantly related to an increased level of antioxidants. This study provides new information about the antioxidant mechanisms of PBMT in the lung and might constitute an important tool for lung disease treatment. PMID:27293324

  2. Photobiomodulation Therapy Decreases Oxidative Stress in the Lung Tissue after Formaldehyde Exposure: Role of Oxidant/Antioxidant Enzymes

    PubMed Central

    Braga, Tarcio Teodoro; Barioni, Éric Diego; de Oliveira Duro, Stephanie; Ratto Tempestini Horliana, Anna Carolina; Câmara, Niels Olsen Saraiva; Marcourakis, Tânia; Farsky, Sandra Helena Poliselli; Lino-dos-Santos-Franco, Adriana

    2016-01-01

    Formaldehyde is ubiquitous pollutant that induces oxidative stress in the lung. Several lung diseases have been associated with oxidative stress and their control is necessary. Photobiomodulation therapy (PBMT) has been highlighted as a promissory treatment, but its mechanisms need to be better investigated. Our objective was to evaluate the effects of PBMT on the oxidative stress generated by FA exposure. Male Wistar rats were submitted to FA exposure of 1% or vehicle (3 days) and treated or not with PBMT (1 and 5 h after each FA exposure). Rats treated only with laser were used as control. Twenty-four hours after the last FA exposure, we analyzed the effects of PBMT on the generation of nitrites and hydrogen peroxide, oxidative burst, glutathione reductase, peroxidase, S-transferase enzyme activities, the gene expression of nitric oxide, cyclooxygenase, superoxide dismutase, the catalase enzyme, and heme oxygenase-1. PBMT reduced the generation of nitrites and hydrogen peroxide and increased oxidative burst in the lung cells. A decreased level of oxidant enzymes was observed which were concomitantly related to an increased level of antioxidants. This study provides new information about the antioxidant mechanisms of PBMT in the lung and might constitute an important tool for lung disease treatment. PMID:27293324

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

    PubMed Central

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

    1975-01-01

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

  4. 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.

  5. 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.

  6. Flume experiments elucidate relationships between microbial genetics, nitrogen species and hydraulics in controlling nitrous oxide production in the hyporheic zone

    NASA Astrophysics Data System (ADS)

    Quick, A. M.; Farrell, T. B.; Reeder, W. J.; Feris, K. P.; Tonina, D.; Benner, S. G.

    2014-12-01

    The hyporheic zone is a potentially important producer of nitrous oxide, a powerful greenhouse gas. The location and magnitude of nitrous oxide generation within the hyporheic zone involves complex interactions between multiple nitrogen species, redox conditions, microbial communities, and hydraulics. To better understand nitrous oxide generation and emissions from streams, we conducted large-scale flume experiments in which we monitored pore waters along hyporheic flow paths within stream dune structures. Measured dissolved oxygen, ammonia, nitrate, nitrite, and dissolved nitrous oxide showed distinct spatial relationships reflecting redox changes along flow paths. Denitrifying genes (nosZ, nirS, and nirK), determined using qPCR, were spatially associated with abundances of nitrogen species. Using residence times along a flow path, clear trends in oxygen conditions, genes encoding for microbial catalysis, and nitrogen species were observed. Hotspots of targeted genes correlated with hotspots for conversion of nitrogen species, including nitrous oxide production and conversion to dinitrogen. Trends were apparent regardless of dune size, allowing for the possibility to apply observed relationships to multiple streambed morphologies. Relating streambed morphology and loading of nitrogen species allows for prediction of nitrous oxide production in the hyporheic zone.

  7. Metal enzymes in "impossible" microorganisms catalyzing the anaerobic oxidation of ammonium and methane.

    PubMed

    Reimann, Joachim; Jetten, Mike S M; Keltjens, Jan T

    2015-01-01

    Ammonium and methane are inert molecules and dedicated enzymes are required to break up the N-H and C-H bonds. Until recently, only aerobic microorganisms were known to grow by the oxidation of ammonium or methane. Apart from respiration, oxygen was specifically utilized to activate the inert substrates. The presumed obligatory need for oxygen may have resisted the search for microorganisms that are capable of the anaerobic oxidation of ammonium and of methane. However extremely slowly growing, these "impossible" organisms exist and they found other means to tackle ammonium and methane. Anaerobic ammonium-oxidizing (anammox) bacteria use the oxidative power of nitric oxide (NO) by forging this molecule to ammonium, thereby making hydrazine (N2H4). Nitrite-dependent anaerobic methane oxidizers (N-DAMO) again take advantage of NO, but now apparently disproportionating the compound into dinitrogen and dioxygen gas. This intracellularly produced dioxygen enables N-DAMO bacteria to adopt an aerobic mechanism for methane oxidation.Although our understanding is only emerging how hydrazine synthase and the NO dismutase act, it seems clear that reactions fully rely on metal-based catalyses known from other enzymes. Metal-dependent conversions not only hold for these key enzymes, but for most other reactions in the central catabolic pathways, again supported by well-studied enzymes from model organisms, but adapted to own specific needs. Remarkably, those accessory catabolic enzymes are not unique for anammox bacteria and N-DAMO. Close homologs are found in protein databases where those homologs derive from (partly) known, but in most cases unknown species that together comprise an only poorly comprehended microbial world. PMID:25707470

  8. 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

  9. Mechanistic elucidation of C-H oxidation by electron rich non-heme iron(IV)-oxo at room temperature.

    PubMed

    Rana, Sujoy; Dey, Aniruddha; Maiti, Debabrata

    2015-10-01

    Non-heme iron(IV)-oxo species form iron(III) intermediates during hydrogen atom abstraction (HAA) from the C-H bond. While synthesizing a room temperature stable, electron rich, non-heme iron(IV)-oxo compound, we obtained iron(III)-hydroxide, iron(III)-alkoxide and hydroxylated-substrate-bound iron(II) as the detectable intermediates. The present study revealed that a radical rebound pathway was operative for benzylic C-H oxidation of ethylbenzene and cumene. A dissociative pathway for cyclohexane oxidation was established based on UV-vis and radical trap experiments. Interestingly, experimental evidence including O-18 labeling and mechanistic study suggested an electron transfer mechanism to be operative during C-H oxidation of alcohols (e.g. benzyl alcohol and cyclobutanol). The present report, therefore, unveils non-heme iron(IV)-oxo promoted substrate-dependent C-H oxidation pathways which are of synthetic as well as biological significance. PMID:26277913

  10. Enzyme-catalyzed Oxidation Facilitates the Return of Fluorescence for Single-Walled Carbon Nanotubes

    PubMed Central

    Chiu, Cheuk Fai; Barth, Brian A.; Kotchey, Gregg P.; Zhao, Yong; Gogick, Kristy A.; Saidi, Wissam A.; Petoud, Stéphane; Star, Alexander

    2013-01-01

    In this work, we studied enzyme-catalyzed oxidation of single-walled carbon nanotubes (SWCNTs) produced by the high-pressure carbon monoxide (HiPco) method. While oxidation via strong acids introduced defects sites on SWCNTs and suppressed their near-infrared (NIR) fluorescence, our results indicated that the fluorescence of SWCNTs was restored upon enzymatic oxidation, which provided new evidence that the reaction catalyzed by horseradish peroxidase (HRP) in the presence of H2O2 is mainly a defect-consuming step. These results were further supported by both UV-vis-NIR and Raman spectroscopy. Therefore, employing acid oxidation followed by HRP-catalyzed enzyme oxidation, shortened (< 300 nm in length) and NIR-fluorescent SWCNTs were produced. In contrast, when treated with myeloperoxidase (MPO), H2O2, and NaCl, the oxidized HiPco SWCNTs underwent complete oxidation (i.e. degradation). The shortened, NIR-fluorescent SWCNTs resulting from HRP-catalyzed oxidation of acid cut HiPco SWCNTs may find applications in cellular NIR imaging and drug delivery systems. PMID:23672715

  11. Enzyme-catalyzed oxidation facilitates the return of fluorescence for single-walled carbon nanotubes.

    PubMed

    Chiu, Cheuk Fai; Barth, Brian A; Kotchey, Gregg P; Zhao, Yong; Gogick, Kristy A; Saidi, Wissam A; Petoud, Stéphane; Star, Alexander

    2013-09-11

    In this work, we studied enzyme-catalyzed oxidation of single-walled carbon nanotubes (SWCNTs) produced by the high-pressure carbon monoxide (HiPco) method. While oxidation via strong acids introduced defect sites on SWCNTs and suppressed their near-infrared (NIR) fluorescence, our results indicated that the fluorescence of SWCNTs was restored upon enzymatic oxidation, providing new evidence that the reaction catalyzed by horseradish peroxidase (HRP) in the presence of H2O2 is mainly a defect-consuming step. These results were further supported by both UV-vis-NIR and Raman spectroscopy. Therefore, when acid oxidation followed by HRP-catalyzed enzyme oxidation was employed, shortened (<300 nm in length) and NIR-fluorescent SWCNTs were produced. In contrast, upon treatment with myeloperoxidase, H2O2, and NaCl, the oxidized HiPco SWCNTs underwent complete oxidation (i.e., degradation). The shortened, NIR-fluorescent SWCNTs resulting from HRP-catalyzed oxidation of acid-cut HiPco SWCNTs may find applications in cellular NIR imaging and drug delivery systems. PMID:23672715

  12. 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

  13. Star block-copolymers: enzyme-inspired catalysts for oxidation of alcohols in water.

    PubMed

    Mugemana, Clément; Chen, Ba-Tian; Bukhryakov, Konstantin V; Rodionov, Valentin

    2014-07-25

    A number of fluorous amphiphilic star block-copolymers containing a tris(benzyltriazolylmethyl)amine motif have been prepared. These polymers assembled into well-defined nanostructures in water, and their mode of assembly could be controlled by changing the composition of the polymer. The polymers were used for enzyme-inspired catalysis of alcohol oxidation. PMID:24912078

  14. Computerized pathway elucidation for hydroxyl radical-induced chain reaction mechanisms in aqueous phase advanced oxidation processes.

    PubMed

    Li, Ke; Crittenden, John

    2009-04-15

    The radical reaction mechanism that is involved in advanced oxidation processes is complex. An increasing number of trace contaminants and stringent drinking water standards call for a rule-based model to provide insight to the mechanism of the processes. A model was developed to predict the pathway of contaminant degradation and byproduct formation during advanced oxidation. The model builds chemical molecules as graph objects, which enables mathematic abstraction of chemicals and preserves chemistry information. The model algorithm enumerates all possible reaction pathways according to the elementary reactions (built as reaction rules) established from experimental observation. The method can predict minor pathways that could lead to toxic byproducts so that measures can be taken to ensure drinking water treatment safety. The method can be of great assistance to water treatment engineers and chemists who appreciate the mechanism of treatment processes. PMID:19475958

  15. 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. PMID:23969010

  16. 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. PMID:25478736

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

    SciTech Connect

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

    1987-11-01

    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.

  18. Identification and characterization of the two-enzyme system catalyzing oxidation of EDTA in the EDTA-degrading bacterial strain DSM 9103.

    PubMed Central

    Witschel, M; Nagel, S; Egli, T

    1997-01-01

    In a gram-negative isolate (DSM 9103) able to grow with EDTA as the sole source of carbon, nitrogen, and energy, the first two steps of the catabolic pathway for EDTA were elucidated. They consisted of the sequential oxidative removal of two acetyl groups, resulting in the formation of glyoxylate. An enzyme complex that catalyzes the removal of two acetyl groups was purified and characterized. In the reaction, ethylenediaminetriacetate (ED3A) was formed as an intermediate and N,N'-ethylenediaminediacetate was the end product. The enzyme complex consisted of two components: component A' (cA'), most likely a monooxygenase, which catalyzes the cleavage of EDTA and ED3A while consuming oxygen and reduced flavin mononucleotide (FMN)-H2, and component B' (cB'), an NADH2:FMN oxidoreductase that provides FMNH2 for cA'. cB' could be replaced by other NADH2:FMN oxidoreductases such as component B of the nitrilotriacetate monooxygenase or the NADH2:FMN oxidoreductase from Photobacterium fischeri. The EDTA-oxidizing enzyme complex accepted EDTA as a substrate only when it was complexed with Mg2+, Zn2+, Mn2+, Co2+, or Cu2+. Moreover, the enzyme complex catalyzed the removal of acetyl groups from several other aminopolycarboxylic acids that possess three or more acetyl groups. PMID:9371437

  19. Serum Antioxidative Enzymes Levels and Oxidative Stress Products in Age-Related Cataract Patients

    PubMed Central

    Chang, Dong; Zhang, Xuefei; Rong, Shengzhong; Sha, Qian; Liu, Peipei; Han, Tao; Pan, Hongzhi

    2013-01-01

    Purpose. To investigate the activity of antioxidative enzymes and the products of oxidative stress in patients with age-related cataracts and compare the findings with those in healthy control subjects. Method. Sixty patients with age-related cataract and sixty healthy controls of matched age and gender were included in this study. Serum samples were obtained to detect the antioxidative enzymes of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and oxidation degradation products of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), conjugated diene (CD), advanced oxidation protein products (AOPP), protein carbonyl (PC), and 8-hydroxydeoxyguanosine (8-OHdG). Results. Serum SOD, GSH-Px, and CAT activities in cataract group were significantly decreased as compared to the control subjects (P < 0.05). The levels of MDA, 4-HNE, and CD in cataract patients were significantly higher than those in the control subjects (P < 0.05, P < 0.01). Cataract patients had higher levels of 8-OHdG, AOPP, and PC with respect to the comparative group of normal subjects (P < 0.01). And there was no statistical significance in concentration of antioxidative enzymes and oxidative stress products in patients with different subtype cataract. Conclusions. Oxidative stress is an important risk factor in the development of age-related cataract, and augmentation of the antioxidant defence systems may be of benefit to prevent or delay cataractogenesis. PMID:23781296

  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. Synthesis, structural elucidation, and application of a pyrazolylpyridine-molybdenum oxide composite as a heterogeneous catalyst for olefin epoxidation.

    PubMed

    Figueiredo, Sónia; Gomes, Ana C; Neves, Patrícia; Amarante, Tatiana R; Paz, Filipe A Almeida; Soares, Rosário; Lopes, André D; Valente, Anabela A; Pillinger, Martyn; Gonçalves, Isabel S

    2012-08-01

    The reaction of [MoO(2)Cl(2)(pypzEA)] (1) (pypzEA = ethyl[3-(pyridin-2-yl)-1H-pyrazol-1-yl]acetate) with water in a Teflon-lined stainless steel autoclave (100 °C) or in an open reflux system leads to the isolation of the molybdenum oxide/pyrazolylpyridine composite material [Mo(2)O(6)(HpypzA)] (2; HpypzA = [3-(pyridinium-2-yl)-1H-pyrazol-1-yl]acetate). The solid state structure of 2 was solved through single crystal and powder X-ray diffraction analyses in conjunction with information derived from FT-IR and (13)C CP MAS NMR spectroscopies and elemental analyses. In the asymmetric unit of 2, two crystallographically distinct Mo(6+) centers are bridged by a syn,syn-carboxylate group of HpypzA. The periodic repetition of these units along the a axis of the unit cell leads to the formation of a one-dimensional composite polymer, (∞)(1)[Mo(2)O(6)(HpypzA)]. The outstretched pyrazolylpyridine groups of adjacent polymers interdigitate to form a zipper-like motif, generating strong onset π-π contacts between adjacent rings of coordinated HpypzA molecules. The composite oxide 2 is a stable heterogeneous catalyst for liquid-phase olefin epoxidation. PMID:22830308

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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. PMID:23201694

  7. Determining iron oxide nanoparticle heating efficiency and elucidating local nanoparticle temperature for application in agarose gel-based tumor model.

    PubMed

    Shah, Rhythm R; Dombrowsky, Alexander R; Paulson, Abigail L; Johnson, Margaret P; Nikles, David E; Brazel, Christopher S

    2016-11-01

    Magnetic iron oxide nanoparticles (MNPs) have been developed for magnetic fluid hyperthermia (MFH) cancer therapy, where cancer cells are treated through the heat generated by application of a high frequency magnetic field. This heat has also been proposed as a mechanism to trigger release of chemotherapy agents. In each of these cases, MNPs with optimal heating performance can be used to maximize therapeutic effect while minimizing the required dosage of MNPs. In this study, the heating efficiencies (or specific absorption rate, SAR) of two types of MNPs were evaluated experimentally and then predicted from their magnetic properties. MNPs were also incorporated in the core of poly(ethylene glycol-b-caprolactone) micelles, co-localized with rhodamine B fluorescent dye attached to polycaprolactone to monitor local, nanoscale temperatures during magnetic heating. Despite a relatively high SAR produced by these MNPs, no significant temperature rise beyond that observed in the bulk solution was measured by fluorescence in the core of the magnetic micelles. MNPs were also incorporated into a macro-scale agarose gel system that mimicked a tumor targeted by MNPs and surrounded by healthy tissues. The agarose-based tumor models showed that targeted MNPs can reach hyperthermia temperatures inside a tumor with a sufficient MNP concentration, while causing minimal temperature rise in the healthy tissue surrounding the tumor. PMID:27523991

  8. 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.

  9. 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

  10. 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

    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

    2015-12-01

    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

  11. Glutathione peroxidase in yeast. Presence of the enzyme and induction by oxidative conditions.

    PubMed

    Galiazzo, F; Schiesser, A; Rotilio, G

    1987-09-30

    The presence of glutathione peroxidase activity is reported for the first time for a wild type strain of Saccharomyces cerevisiae. Both forms of enzyme, i.e. that specifically active toward H2O2 alone and that decomposing also organic peroxides, were found to be present. The H2O2 specific form disappeared when cells were grown in the absence of oxygen, while the other form was much increased under the same conditions. Addition of copper to the culture greatly increased both forms. The results show that glutathione peroxidase is to be included, as an important component that is also highly responsive to oxidative environments, in the enzyme defense system of yeast against oxidative damage. PMID:3311044

  12. Cloning, expression and characterization of a gene encoding nitroalkane-oxidizing enzyme from Streptomyces ansochromogenes.

    PubMed

    Zhang, Jihui; Tan, Huarong

    2002-12-01

    A nitroalkane-oxidizing enzyme gene (naoA) was cloned from a genomic DNA library of Streptomyces ansochromogenes 7100. The deduced protein (NaoA) of this gene contains 363 amino acids and has high similarity to several nitroalkane-oxidizing enzymes from various micro-organisms. The naoA gene was subcloned into an expression vector pET23b and overexpressed in Escherichia coli BL21(DE3). The protein was then purified, and its characteristics were studied. Experimental results showed that NaoA can convert 1-nitropropane, 2-nitropropane and nitroethane into the corresponding carbonyl compounds. The optimal pH and temperature for NaoA was found to be pH 7-8 and 48-56 degrees C, respectively. The Km of NaoA for nitroethane is approximately 26.8 mm. NADH and nitro blue tetrazolium are strong inhibitors of NaoA, and thiol compounds and superoxide dismutase partially inhibit the enzyme activity. Therefore, superoxide may be an essential intermediate in the oxidation of nitroalkane by NaoA. PMID:12473127

  13. Biochemical assessment of oxidative status versus liver enzymes in patients with chronic fascioliasis.

    PubMed

    Kamel, Hanan H; Sarhan, Rania M; Saad, Ghada A

    2015-12-01

    The aim of this study was to examine the oxidative status in Egyptian patients suffering chronic fascioliasis. The relationship between serum malondialdehyde (MDA) levels, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities was investigated in relation to the level of liver enzymes; ALT and AST compared to healthy controls. Twenty patients versus ten controls were included in the study. Among cases the MDA, CAT, AST and ALT were higher than controls, while SOD and GPX higher values were present among controls. There was a highly significant difference between cases and controls as regard MDA, CAT, SOD, GPX, and AST, and a significant difference regarding ALT. The findings of increased serum lipid peroxidation and decreased antioxidant enzymes in erythrocytes of chronic fascioliasis patients indicated the presence of persistent inflammation and oxidative stress which confirms the underlying pathogenesis and reflected the stage of infection providing a baseline data for comparison between normal and infected patients guided by the level of liver enzymes in relation to oxidative status. PMID:26688624

  14. Studies on the effect of sodium arsenate on the enzymes of carbohydrate metabolism, brush border membrane, and oxidative stress in the rat kidney.

    PubMed

    Shahid, Faaiza; Rizwan, Sana; Khan, Md Wasim; Khan, Sara Anees; Naqshbandi, Ashreeb; Yusufi, Ahad Noor Khan

    2014-03-01

    Arsenic is an environmental pollutant and its contamination in drinking water poses serious world wide environmental health threats. It produces multiple adverse effects in various tissues, including the kidney. However, biochemical mechanism and renal response to its toxic insult are not completely elucidated. We hypothesized that sodium arsenate (ARS) induces oxidative stress and alters the structure and metabolic functions of kidney. Male Wistar rats were administered ARS (10 mg/kg body weight/day), intraperitoneally daily for 10 days. ARS administration increased blood urea nitrogen, serum creatinine, cholesterol, glucose, and phospholipids but decreased inorganic phosphate, indicating kidney toxicity. The activity of brush border membrane (BBM) enzymes significantly lowered in both cortex and medulla. Activity of hexokinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenases, and NADP-malic enzyme significantly increased whereas malate dehydrogenase, glucose-6-phosphatase, and fructose 1,6 bis phosphatase decreased by ARS exposure. The activity of superoxide dismutase, GSH-peroxidase, and catalase were selectively altered in renal tissues along with an increase in lipid peroxidation. The present results indicated that ARS induced oxidative stress caused severe renal damage that resulted in altered levels of carbohydrate metabolism and BBM enzymes. PMID:24562057

  15. Evidence of short-range electron transfer of a redox enzyme on graphene oxide electrodes.

    PubMed

    Martins, Marccus V A; Pereira, Andressa R; Luz, Roberto A S; Iost, Rodrigo M; Crespilho, Frank N

    2014-09-01

    Direct electron transfer (DET) between redox enzymes and electrode surfaces is of growing interest and an important strategy in the development of biofuel cells and biosensors. Among the nanomaterials utilized at electrode/enzyme interfaces to enhance the electronic communication, graphene oxide (GO) has been identified as a highly promising candidate. It is postulated that GO layers decrease the distance between the flavin cofactor (FAD/FADH2) of the glucose oxidase enzyme (GOx) and the electrode surface, though experimental evidence concerning the distance dependence of the rate constant for heterogeneous electron-transfer (k(het)) has not yet been observed. In this work, we report the experimentally observed DET of the GOx enzyme adsorbed on flexible carbon fiber (FCF) electrodes modified with GO (FCF-GO), where the k(het) between GO and electroactive GOx has been measured at a structurally well-defined interface. The curves obtained from the Marcus theory were used to obtain k(het), by using the model proposed by Chidsey. In agreement with experimental data, this model proved to be useful to systematically probe the dependence of electron transfer rates on distance, in order to provide an empirical basis to understand the origin of interfacial DET between GO and GOx. We also demonstrate that the presence of GO at the enzyme/electrode interface diminishes the activation energy by decreasing the distance between the electrode surface and FAD/FADH2. PMID:24676540

  16. Selective oxidation of enzyme extracts for improved quantification of peroxidase activity.

    PubMed

    Jiang, Shu; Penner, Michael H

    2015-05-01

    Natural components endogenous to plant material extracts often interfere with traditional peroxidase assays by reducing the oxidized product generated as a result of the peroxidase-catalyzed reaction. This leads to an underestimation of peroxidase activity when the oxidized product provides the signal for enzyme activity quantification. This article describes a relatively simple way to alleviate complications arising due to the presence of such confounding compounds. The method is based on using 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the reducing substrate. The oxidized product of the reaction is ABTS(+), the accumulation of which can be followed spectrophotometrically. It is shown here that one can selectively inactivate the endogenous compounds that confound the peroxidase assay by treating the enzyme preparation with the oxidized product itself, ABTS(+), prior to initiating the quantification assay. This approach is selective for those compounds likely to interfere with peroxidase quantification. The presented method is shown to alleviate the complications associated with lag phases typical of plant extract peroxidase assays and, thus, to more accurately reflect total peroxidase activity. The presented assay is expected to be applicable to the wide range of biological systems for which the determination of peroxidase activity is desired. PMID:25640588

  17. Upregulation of endogenous antioxidants and phase 2 enzymes by the red wine polyphenol, resveratrol in cultured aortic smooth muscle cells leads to cytoprotection against oxidative and electrophilic stress.

    PubMed

    Li, Yunbo; Cao, Zhuoxiao; Zhu, Hong

    2006-01-01

    Resveratrol (3,4',5-trihydroxystilbene), a polyphenolic compound found in mulberries, grapes and red wine has been demonstrated to be capable of protecting against oxidative cardiovascular pathophysiology. However, the underlying cellular and biochemical mechanisms remain to be elucidated. This study was undertaken to determine if resveratrol could upregulate endogenous antioxidants and phase 2 enzymes in cultured aortic smooth muscle cells (ASMCs), and if such increased cellular defenses could provide protection against oxidative and electrophilic vascular cell injury. Incubation of rat ASMCs with resveratrol at low micromolar concentrations resulted in a significant induction of a scope of cellular antioxidants and phase 2 enzymes in a concentration- and/or time-dependent fashion. These cytoprotective factors include superoxide dismutase, catalase, glutathione, glutathione reductase, glutathione peroxidase, glutathione S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NOQ1). Notably, induction of catalase, GST, and NOQ1 was most remarkable among the above resveratrol-inducible antioxidants and phase 2 enzymes. Moreover, resveratrol treatment also significantly increased the mRNA expression of catalase, GSTA1, and NQO1 in a time-dependent manner. Pretreatment of ASMCs with resveratrol afforded a remarkable protection against xanthine oxidase (XO)/xanthine- or 4-hydroxy-2-nonenal-induced cytotoxicity, as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Resveratrol pretreatment also led to a marked reduction in intracellular accumulation of reactive oxygen species in ASMCs after incubation with XO/xanthine. Taken together, this study demonstrates that a scope of key endogenous antioxidants and phase 2 enzymes in cultured ASMCs can be upregulated by resveratrol at low micromolar concentrations, and that such chemically-elevated cellular defenses rendered cells increased resistance to oxidative and electrophilic

  18. 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. PMID:26536075

  19. Trifluorosubstrates as mechanistic probes for an FMN-dependent l-2-hydroxy acid-oxidizing enzyme.

    PubMed

    Lederer, Florence; Vignaud, Caroline; North, Paul; Bodevin, Sabrina

    2016-09-01

    A controversy exists with respect to the mechanism of l-2-hydroxy acid oxidation by members of a family of FMN-dependent enzymes. A so-called carbanion mechanism was initially proposed, in which the active site histidine abstracts the substrate α-hydrogen as a proton, followed by electron transfer from the carbanion to the flavin. But an alternative mechanism was not incompatible with some results, a mechanism in which the active site histidine instead picks up the substrate hydroxyl proton and a hydride transfer occurs. Even though more recent experiments ruling out such a mechanism were published (Rao & Lederer (1999) Protein Science 7, 1531-1537), a few authors have subsequently interpreted their results with variant enzymes in terms of a hydride transfer. In the present work, we analyse the reactivity of trifluorolactate, a substrate analogue, with the flavocytochrome b2 (Fcb2) flavodehydrogenase domain, compared to its reactivity with an NAD-dependent lactate dehydrogenase (LDH), for which this compound is known to be an inhibitor (Pogolotti & Rupley (1973) Biochem. Biophys. Res. Commun, 55, 1214-1219). Indeed, electron attraction by the three fluorine atoms should make difficult the removal of the α-H as a hydride. We also analyse the reactivity of trifluoropyruvate with the FMN- and NAD-dependent enzymes. The results substantiate a different effect of the fluorine substituents on the two enzymes compared to their normal substrates. In the discussion we analyse the conclusions of recent papers advocating a hydride transfer mechanism for the family of l-2-hydroxy acid oxidizing FMN-dependent enzymes. PMID:27155230

  20. Constitutively Elevated Levels of Putrescine and Putrescine-Generating Enzymes Correlated with Oxidant Stress Resistance in Conyza bonariensis and Wheat.

    PubMed

    Ye, B.; Muller, H. H.; Zhang, J.; Gressel, J.

    1997-12-01

    Oxidant stress resistance in Conyza bonariensis and wheat (Triticum aestivum) has been correlated with high levels of antioxidant enzyme activities. Additionally, external oxidant stresses can increase a plant's levels of the enzymes of polyamine biosynthesis and polyamines, especially putrescine. We investigated the constitutive relationships between putrescine, putrescine-generating enzymes, and oxidant stress resistance in wheat and C. bonariensis. Putrescine was Constitutively elevated (2.5- to 5.7-fold) in 2-week-old-resistant wheat and C. bonariensis biotypes, which correlated with a 10- to 15-fold increase in paraquat oxidant resistance. Arginine and ornithine decarboxylase activities doubled, along with higher putrescine levels in resistant C. bonariensis. The variations in levels of putrescine and arginine and ornithine decarboxylase activities paralleled the constitutive variation of antioxidant enzymes, as well as oxidant resistance. Higher levels of both putrescine and antioxidant enzyme activities occurred during a peak of oxidant resistance at 10 weeks, when paraquat resistance in C. bonariensis plants is >50-fold greater than in the sensitive biotype. Application of 100 [mu]M putrescine can double oxidant-stress resistance in the resistant C. bonariensis. Putrescine may play an important role in contributing to the base level of oxidant resistance found at the nonpeak period. PMID:12223875

  1. Constitutively Elevated Levels of Putrescine and Putrescine-Generating Enzymes Correlated with Oxidant Stress Resistance in Conyza bonariensis and Wheat.

    PubMed Central

    Ye, B.; Muller, H. H.; Zhang, J.; Gressel, J.

    1997-01-01

    Oxidant stress resistance in Conyza bonariensis and wheat (Triticum aestivum) has been correlated with high levels of antioxidant enzyme activities. Additionally, external oxidant stresses can increase a plant's levels of the enzymes of polyamine biosynthesis and polyamines, especially putrescine. We investigated the constitutive relationships between putrescine, putrescine-generating enzymes, and oxidant stress resistance in wheat and C. bonariensis. Putrescine was Constitutively elevated (2.5- to 5.7-fold) in 2-week-old-resistant wheat and C. bonariensis biotypes, which correlated with a 10- to 15-fold increase in paraquat oxidant resistance. Arginine and ornithine decarboxylase activities doubled, along with higher putrescine levels in resistant C. bonariensis. The variations in levels of putrescine and arginine and ornithine decarboxylase activities paralleled the constitutive variation of antioxidant enzymes, as well as oxidant resistance. Higher levels of both putrescine and antioxidant enzyme activities occurred during a peak of oxidant resistance at 10 weeks, when paraquat resistance in C. bonariensis plants is >50-fold greater than in the sensitive biotype. Application of 100 [mu]M putrescine can double oxidant-stress resistance in the resistant C. bonariensis. Putrescine may play an important role in contributing to the base level of oxidant resistance found at the nonpeak period. PMID:12223875

  2. Endothelial targeting of nanocarriers loaded with antioxidant enzymes for protection against vascular oxidative stress and inflammation

    PubMed Central

    Hood, Elizabeth D.; Chorny, Michael; Greineder, Colin F.; Alferiev, Ivan; Levy, Robert J.; Muzykantov, Vladimir R.

    2015-01-01

    Endothelial-targeted delivery of antioxidant enzymes, catalase and superoxide dismutase (SOD), is promising strategy for protecting organs and tissues from inflammation and oxidative stress. Here we describe Protective Antioxidant Carriers for Endothelial Targeting (PACkET), the first carriers capable of targeted endothelial delivery of both catalase and SOD. PACkET formed through controlled precipitation loaded ~30% enzyme and protected it from proteolytic degradation, whereas attachment of PECAM monoclonal antibodies to surface of the enzyme-loaded carriers, achieved without adversely affecting their stability and functionality, provided targeting. Isotope tracing and microscopy showed that PACkET exhibited specific endothelial binding and internalization in vitro. Endothelial targeting of PACkET was validated in vivo by specific (vs IgG-control) accumulation in the pulmonary vasculature after intravenous injection achieving 33% of injected dose at 30 min. Catalase loaded PACkET protects endothelial cells from killing by H2O2 and alleviated the pulmonary edema and leukocyte infiltration in mouse model of endotoxin-induced lung injury, whereas SOD-loaded PACkET mitigated cytokine-induced endothelial pro-inflammatory activation and endotoxin-induced lung inflammation. These studies indicate that PACkET offers a modular approach for vascular targeting of therapeutic enzymes. PMID:24480537

  3. 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.

  4. Elucidation of lead-induced oxidative stress in Talinum triangulare roots by analysis of antioxidant responses and DNA damage at cellular level.

    PubMed

    Kumar, Abhay; Prasad, M N V; Mohan Murali Achary, V; Panda, Brahma B

    2013-07-01

    Hydroponic experiments were performed with Talinum triangulare (Jacq.) Willd. focusing the root cellular biochemistry with special emphasis on DNA damage, structural, and elemental analyses in Pb(NO3)2 exposed with 0, 0.25, 0.5, 0.75, 1.0, and 1.25 mM for 7 days. Lead (Pb) increased reactive oxygen species production, lipid peroxidation, protein oxidation, cell death, and DNA damage and decreased the protein content in a dose-dependent manner. Likewise, a dose-dependent induction of antioxidative enzymes superoxide dismutase and catalase by Pb was evident. Ascorbate peroxidase on the other hand responded biphasically to Pb treatments by showing induction at low (0.25 and 0.50) and repression at high (0.75-1.25 mM) concentrations. The estimation of proline content also indicated a similar biphasic trend. Scanning electron microscope and energy-dispersive X-ray spectroscopy analysis showed that 1.25 mM Pb treatment resulted in ultrastructural modifications in roots and stem tissue that was marked by the change in the elemental profile. The findings pointed to the role of oxidative stress in the underlying Pb phytotoxicity and genotoxicity in T. triangulare. PMID:23263755

  5. 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. PMID:25620714

  6. Model analysis for enhancement of enzyme-catalyzed alcohol oxidation by solvent extraction of product

    SciTech Connect

    Hidaka, Nobuyuki; Matsumoto, Toshitatsu; Morooka, Shigeharu

    1995-07-01

    Enzymatic oxidation of ethanol and butanol was conducted with alcohol oxidase in a one-phase system of water and a two-phase system of water and toluene. The conversion of the reaction in the two-phase system was higher than that in the one-phase system. The inhibition of the enzyme was relieved in the two-phase system by extracting the aldehyde product into the organic phase. The dissolved oxygen concentration in the water phase also affected the reaction rate. A kinetic model of the reaction was developed by considering both the inhibition of enzyme activity and the dissolved oxygen concentration. Parameters used in the model were evaluated experimentally. The reaction rate calculated using the model was in good agreement with the data.

  7. Novel triterpene oxidizing activity of Arabidopsis thaliana CYP716A subfamily enzymes.

    PubMed

    Yasumoto, Shuhei; Fukushima, Ery O; Seki, Hikaru; Muranaka, Toshiya

    2016-02-01

    Triterpenoids have diverse chemical structures and bioactivities. Cytochrome P450 monooxygenases play a key role in their structural diversification. In higher plants, CYP716A subfamily enzymes are triterpene oxidases. In this study, Arabidopsis thaliana CYP716A1 and CYP716A2 were characterized by heterologously expressing them in simple triterpene-producing yeast strains. In contrast to the C-28 oxidative activity of CYP716A1 shown in several CYP716A subfamily enzymes, remarkably, CYP716A2 displayed 22α-hydroxylation activity against α-amyrin that has not been previously reported, which produces the cytotoxic triterpenoid, 22α-hydroxy-α-amyrin. Our results contribute to the enrichment of the molecular toolbox that allows for the combinatorial biosynthesis of diverse triterpenoids. PMID:26801524

  8. 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. PMID:23357484

  9. 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

  10. A comparative genomic analysis of the oxidative enzymes potentially involved in lignin degradation by Agaricus bisporus.

    PubMed

    Doddapaneni, Harshavardhan; Subramanian, Venkataramanan; Fu, Bolei; Cullen, Dan

    2013-06-01

    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 putative glyoxal oxidase-encoding genes (GLXs)], 12 laccases sensu stricto and 109 cytochrome P450 monooxygenases. Comparative analyses of these enzymes in Ab with those of the white-rot fungus, Phanerochaete chrysosporium, the brown-rot fungus, Postia placenta, the coprophilic litter fungus, Coprinopsis cinerea and the ectomychorizal fungus, Laccaria bicolor, revealed enzyme diversity consistent with adaptation to substrates rich in humic substances and partially degraded plant material. For instance, relative to wood decay fungi, Ab cytochrome P450 genes were less numerous (109 gene models), distributed among distinctive families, and lacked extensive duplication and clustering. Viewed together with P450 transcript accumulation patterns in three tested growth conditions, these observations were consistent with the unique Ab lifestyle. Based on tandem gene arrangements, a certain degree of gene duplication seems to have occurred in this fungus in the copper radical oxidase (CRO) and the laccase gene families. In Ab, high transcript levels and regulation of the heme-thiolate peroxidases, two manganese peroxidases and the three GLX-like genes are likely in response to complex natural substrates, including lignocellulose and its derivatives, thereby suggesting an important role in lignin degradation. On the other hand, the expression patterns of the related CROs suggest a developmental role in this fungus. Based on these observations, a brief comparative genomic overview of the Ab oxidative enzyme machinery is presented. PMID:23583597

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. Involvement of oxidants and oxidant-generating enzyme(s) in tumour-necrosis-factor-alpha-mediated apoptosis: role for lipoxygenase pathway but not mitochondrial respiratory chain.

    PubMed

    O'Donnell, V B; Spycher, S; Azzi, A

    1995-08-15

    Cellular signalling by the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) has been suggested to involve generation of low levels of reactive oxygen species (ROS). Certain antioxidants and metal chelators can inhibit cytotoxicity and gene expression in response to TNF alpha in numerous cell types. However, neither the source nor function of TNF alpha-induced oxidant generation is known. Using specific inhibitors, we ruled out involvement of several oxidant-generating enzymes [cyclo-oxygenase (indomethacin), cytochrome P-450 (metyrapone), nitric oxide synthase (NG-methyl-L-arginine), NADPH oxidase (iodonium diphenyl), xanthine oxidase (allopurinol), ribonucleotide reductase (hydroxyurea)] in TNF alpha-mediated apoptosis of the murine fibrosarcoma line, L929. We also demonstrated no role for mitochondrial-derived radicals/respiratory chain in the lytic pathway using specific inhibitors/uncouplers (rotenone, KCN, carboxin, fluoroacetate, antimycin, malonate, carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and chloramphenicol-derived respiration-deficient cells. Significant ROS (H2O2, O2-.) generation was not observed in response to TNF alpha in L929 cells using four separate assays. Also, prevention of intracellular H2O2 removal by inhibition of catalase did not potentiate TNF alpha-mediated cell death. These data suggest that neither H2O2 nor O2-. plays a direct role in TNF alpha cytotoxicity. Finally, we suggest a central role for lipoxygenase in TNF alpha-mediated lysis. Three inhibitors of this radical-generating signalling pathway, including an arachidonate analogue (5,8,11,14-eicosatetraynoic acid), could protect cells against TNF alpha. The inhibitor nordihydroguaiaretic acid is also a radical scavenger, but it could not protect cells from ROS toxicity at concentrations that effectively prevented TNF alpha killing. Therefore protection by nordihydroguaiaretic acid cannot be due to scavenging of cytotoxic H2O or O2-.. The lipoxygenase product

  16. 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

  17. 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

    2015-12-01

    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. PMID:26381425

  18. Oxidation of aliphatic olefins by toluene dioxygenase: enzyme rates and product identification.

    PubMed Central

    Lange, C C; Wackett, L P

    1997-01-01

    Toluene dioxygenase from Pseudomonas putida F1 has been studied extensively with aromatic substrates. The present work examined the toluene dioxygenase-catalyzed oxidation of various halogenated ethenes, propenes, butenes and nonhalogenated cis-2-pentene, an isomeric mix of 2-hexenes, cis-2-heptene, and cis-2-octene as substrates for toluene dioxygenase. Enzyme specific activities were determined for the more water-soluble C2 to C5 compounds and ranged from <4 to 52 nmol per min per mg of protein. Trichloroethene was oxidized at a rate of 33 nmol per min per mg of protein. Products from enzyme reactions were identified by gas chromatography-mass spectrometry. Proton and carbon nuclear magnetic resonance spectroscopy of compounds from whole-cell incubation confirmed the identity of products. Substrates lacking a halogen substituent on sp2 carbon atoms were dioxygenated, while those with halogen and one or more unsubstituted allylic methyl groups were monooxygenated to yield allylic alcohols. 2,3-Dichloro-1-propene, containing both a halogenated double bond and a halogenated allylic methyl group, underwent monooxygenation with allylic rearrangement to yield an isomeric mixture of cis- and trans-2,3-dichloro-2-propene-1-ol. PMID:9190800

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

    PubMed

    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

  20. 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

  1. Formaldehyde induces lung inflammation by an oxidant and antioxidant enzymes mediated mechanism in the lung tissue.

    PubMed

    Lino-dos-Santos-Franco, Adriana; Correa-Costa, Matheus; Durão, Ana Carolina Cardoso dos Santos; de Oliveira, Ana Paula Ligeiro; Breithaupt-Faloppa, Ana Cristina; Bertoni, Jônatas de Almeida; Oliveira-Filho, Ricardo Martins; Câmara, Niels Olsen Saraiva; Marcourakis, Tânia; Tavares-de-Lima, Wothan

    2011-12-15

    Formaldehyde (FA) is an indoor and outdoor pollutant widely used by many industries, and its exposure is associated with inflammation and oxidative stress in the airways. Our previous studies have demonstrated the role of reactive oxygen species (ROS) in lung inflammation induced by FA inhalation but did not identify source of the ROS. In the present study, we investigate the effects of FA on the activities and gene expression of glutathione peroxidase (GPX), glutathione reductase (GR), glutathione S-transferase (GST), superoxide dismutase (SOD) 1 and 2, catalase (CAT), nitric oxide synthase (iNOS and cNOS) and cyclooxygenase (COX) 1 and 2. The hypothesized link between NADPH-oxidase, nitric oxide synthase and cyclooxygenase, the lung inflammation after FA inhalation was also investigated. For experiments, male Wistar rats were submitted to FA inhalation (1%, 90 min daily) for 3 consecutive days. The treatments with apocynin and indomethacin before the FA exposure reduced the number of neutrophils recruited into the lung. Moreover, the treatments with apocynin and indomethacin blunted the effect of FA on the generation of IL-1β, while the treatments with L-NAME and apocynin reduced the generation of IL-6 by lung explants when compared to the untreated group. FA inhalation increased the levels of NO and hydrogen peroxide by BAL cells cultured and the treatments with apocynin and l-NAME reduced these generations. FA inhalation did not modify the activities of GPX, GR, GST and CAT but reduced the activity of SOD when compared to the naïve group. Significant increases in SOD-1 and -2, CAT, iNOS, cNOS and COX-1 expression were observed in the FA group compared to the naïve group. The treatments with apocynin, indomethacin and L-NAME reduced the gene expression of antioxidant and oxidant enzymes. In conclusion, our results indicate that FA causes a disruption of the physiological balance between oxidant and antioxidant enzymes in lung tissue, most likely favoring the

  2. Cytochrome bo from Escherichia coli: reaction of the oxidized enzyme with hydrogen peroxide.

    PubMed Central

    Watmough, N J; Cheesman, M R; Greenwood, C; Thomson, A J

    1994-01-01

    Oxidized cytochrome bo reacts rapidly with micromolar concentrations of H2O2 to form a single derivative. The electronic absorption spectrum of this compound differs from that of the oxidized form of the enzyme reported by this laboratory [Watmough, Cheesman, Gennis, Greenwood and Thomson (1993) FEBS Lett. 319, 151-154]. It is characterized by a Soret maximum at 411 nm, increased absorbance at 555 nm, and reduced intensity at 624 nm. The apparent dissociation constant for this process is of the order of 4 x 10(-6) M, and the bimolecular rate constant for the formation of the new compound is (1.25-1.7) x 10(3) M-1.s-1. Electronic absorption difference spectroscopy shows this product to be identical with the compound formed from the reaction of the mixed-valence form of the enzyme with dioxygen. Investigation of this compound by room-temperature magnetic c.d. spectroscopy shows haem o to be neither high-spin nor low-spin ferric, but to have a spectrum characteristic of an oxyferryl species. There is no evidence for oxidation of the porphyrin ring. Therefore the binuclear centre of this species must consist of an oxyferryl haem (S = 1) coupled to a Cu(II) ion (S = 1/2) to form a new paramagnetic centre. The reaction was also followed by X-band e.p.r. spectroscopy, and this showed the disappearance in parallel with the formation of the oxyferryl species, of the broad g = 3.7, signal which arises from the weakly coupled binuclear centre in the oxidized enzyme. Since no new e.p.r.-detectable paramagnetic species were observed, the Cu(II) ion is presumed to be coupled to another paramagnet, possibly an organic radical. There is no evidence in the electronic absorption spectrum to indicate further reaction of cytochrome bo with H2O2 to form a second species. We argue that the circumstances of formation of this oxyferryl species are the same as those for the P form of cytochrome c oxidase, a species often regarded as containing a bound peroxide ion. The implications of

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

    2008-04-01

    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. PMID:18362966

  9. 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. PMID:25852816

  10. 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

  11. 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

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

    PubMed

    Utama, Dicky Tri; Lee, Seung Gyu; Baek, Ki Ho; Kim, Hye-Kyung; Cho, Chang-Yeon; Lee, Cheol-Koo; Lee, Sung Ki

    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

  13. 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

  14. 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. PMID:24301799

  15. 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. PMID:22408903

  16. 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

  17. 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).

  18. Morphological characteristics, oxidative stability and enzymic hydrolysis of amylose-fatty acid complexes.

    PubMed

    Marinopoulou, Anna; Papastergiadis, Efthimios; Raphaelides, Stylianos N; Kontominas, Michael G

    2016-05-01

    Complexes of amylose with fatty acids varying in carbon chain length and degree of unsaturation were prepared at 30, 50 or 70°C by dissolving amylose in 0.1N KOH and mixing with fatty acid potassium soap solution. The complexes were obtained in solid form as precipitates after neutralization. SEM microscopy revealed that the morphology of the complexes was that of ordered lamellae separated from amorphous regions whereas confocal laser scanning microscopy showed images of the topography of the guest molecules in the complex matrix. FTIR spectroscopy revealed that the absorption peak attributed to carbonyl group of free fatty acid was shifted when the fatty acid was in the form of amylose complex. Thermo-gravimetry showed that the unsaturated fatty acids were effectively protected from oxidation when they were complexed with amylose whereas enzymic hydrolysis experiments showed that the guest molecules were quantitatively released from the amylose complexes. PMID:26877002

  19. 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

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

    PubMed

    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

    2015-07-24

    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. 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

  2. The Relationship between Coenzyme Q10, Oxidative Stress, and Antioxidant Enzymes Activities and Coronary Artery Disease

    PubMed Central

    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. PMID:22645453

  3. 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. PMID:25958541

  4. Evidence for a complex of three beta-oxidation enzymes in Escherichia coli: induction and localization.

    PubMed Central

    O'Brien, W J; Frerman, F E

    1977-01-01

    The enzymes for beta-oxidation of fatty acids in inducible and constitutive strains of Escherichia coli were assayed in soluble and membrane fractions of disrupted cells by using fatty acid and acyl-coenzyme A (CoA) substrates containing either 4 or 16 carbon atoms in the acyl moieties. Cell fractionation was monitored, using succinic dehydrogenase as a membrane marker and glucose 6-phosphate dehydrogenase as a soluble marker. Acyl-CoA synthetase activity was detected exclusively in the membrane fraction, whereas acyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities that utilized both C4 and C16 acyl-CoA substrates were isolated from the soluble fraction. 3-Hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities assayed with both C4 and C16 acyl-CoA substrates co-chromatographed on gel filtration and ion-exchange columns and cosedimented in glycerol gradients. The data show that these three enzyme activities of the fad regulon can be isolated as a multienzyme complex. This complex dissociates in very dilute preparations; however, in those preparations where the three activities are separated, the fractionated species retain activity with both C4 and C16 acyl-CoA substrates. PMID:334745

  5. A graphene oxide-based enzyme-free signal amplification platform for homogeneous DNA detection.

    PubMed

    Zhang, Zhen; Liu, Yufei; Ji, Xinghu; Xiang, Xia; He, Zhike

    2014-10-01

    A graphene oxide (GO) based enzyme-free signal amplification platform for homogeneous DNA sensing is developed with simplicity and high sensitivity. In the absence of the target DNA, labeled hairpin probe 1 (H1) and probe 2 (H2) were adsorbed on the surface of GO, resulting in the fluorescence quenching of the dyes and minimizing the background fluorescence. The addition of the target DNA facilitated the formation of double-stranded DNA (dsDNA) between H1 and H2, causing the probes to separate from GO and release the target DNA through a strand displacement reaction. Meanwhile, the whole reaction started anew. This is an excellent isothermal signal amplification technique without the involvement of enzymes. By monitoring the change of the fluorescence intensity, the target DNA not only can be determined in buffer solution, but also can be detected in 1% serum solution spiked with a series of concentrations of the target DNA. In addition, the consumption amount of the probes in this method is lower than that in traditional molecular beacon methods. PMID:25058563

  6. 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. PMID:26289332

  7. 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.

  8. 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. PMID:26751827

  9. 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

    2014-12-01

    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. PMID:25384038

  10. 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. PMID:10984077

  11. Insights into enzyme point mutation effect by molecular simulation: phenylethylamine oxidation catalyzed by monoamine oxidase A.

    PubMed

    Oanca, Gabriel; Purg, Miha; Mavri, Janez; Shih, Jean C; Stare, Jernej

    2016-05-21

    The I335Y point mutation effect on the kinetics of phenylethylamine decomposition catalyzed by monoamine oxidase A was elucidated by means of molecular simulation. The established empirical valence bond methodology was used in conjunction with the free energy perturbation sampling technique and a classical force field representing the state of reactants and products. The methodology allows for the simulation of chemical reactions, in the present case the breaking of the α-C-H bond in a phenylethylamine substrate and the subsequent hydrogen transfer to the flavin cofactor, resulting in the formation of the N-H bond on flavin. The empirical parameters were calibrated against the experimental data for the simulated reaction in a wild type protein and then used for the calculation of the reaction free energy profile in the I335Y mutant. In very good agreement with the measured kinetic data, mutation increases the free energy barrier for the rate limiting step by slightly more than 1 kcal mol(-1) and consequently decreases the rate constant by about an order of magnitude. The magnitude of the computed effect slightly varies with simulation settings, but always remains in reasonable agreement with the experiment. Analysis of trajectories reveals a major change in the interaction between phenyl rings of the substrate and the neighboring Phe352 residue upon the I335Y mutation due to the increased local polarity, leading to an attenuated quadrupole interaction between the rings and destabilization of the transition state. Additionally, the increased local polarity in the mutant allows for a larger number of water molecules to be present near the active site, effectively shielding the catalytic effect of the enzyme and contributing to the increased barrier. PMID:27121693

  12. Angiotensin-converting enzyme inhibitor prevents oxidative stress, inflammation, and fibrosis in carbon tetrachloride-treated rat liver.

    PubMed

    Reza, Hasan Mahmud; Tabassum, Nabila; Sagor, Md Abu Taher; Chowdhury, Mohammed Riaz Hasan; Rahman, Mahbubur; Jain, Preeti; Alam, Md Ashraful

    2016-01-01

    Hepatic fibrosis is a common feature of chronic liver injury, and the involvement of angiotensin II in such process has been studied earlier. We hypothesized that anti-angiotensin II agents may be effective in preventing hepatic fibrosis. In this study, Long Evans female rats were used and divided into four groups such as Group-I, Control; Group-II, Control + ramipril; Group-III, CCl4; and Group-IV, CCl4 + ramipril. Group II and IV are treated with ramipril for 14 d. At the end of treatment, the livers were removed, and the level of hepatic marker enzymes (aspartate aminotransferase, Alanine aminotransferase, and alkaline phosphatase), nitric oxide, advanced protein oxidation product , catalase activity, and lipid peroxidation were determined. The degree of fibrosis was evaluated through histopathological staining with Sirius red and trichrome milligan staining. Carbon-tetrachloride (CCl4) administration in rats developed hepatic dysfunction and raised the hepatic marker enzymes activities significantly. CCl4 administration in rats also produced oxidative stress, inflammation, and fibrosis in liver. Furthermore, angiotensinogen-inhibitor ramipril normalized the hepatic enzymes activities and improved the antioxidant enzyme catalase activity. Moreover, ramipril treatment ameliorated lipid peroxidation and hepatic inflammation in CCl4-treated rats. Ramipril treatment also significantly reduced hepatic fibrosis in CCl4-administered rats. In conclusion, our investigation suggests that the antifibrotic effect of ramipril may be attributed to inhibition of angiotensin-II mediated oxidative stress and inflammation in liver CCl4-administered rats. PMID:26862777

  13. Cysteine Oxidation Reactions Catalyzed by a Mononuclear Non-heme Iron Enzyme (OvoA) in Ovothiol Biosynthesis

    PubMed Central

    2015-01-01

    OvoA in ovothiol biosynthesis is a mononuclear non-heme iron enzyme catalyzing the oxidative coupling between histidine and cysteine. It can also catalyze the oxidative coupling between hercynine and cysteine, yet with a different regio-selectivity. Due to the potential application of this reaction for industrial ergothioneine production, in this study, we systematically characterized OvoA by a combination of three different assays. Our studies revealed that OvoA can also catalyze the oxidation of cysteine to either cysteine sulfinic acid or cystine. Remarkably, these OvoA-catalyzed reactions can be systematically modulated by a slight modification of one of its substrates, histidine. PMID:24684381

  14. 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

  15. Black tea polyphenols modulate xenobiotic-metabolizing enzymes, oxidative stress and adduct formation in a rat hepatocarcinogenesis model.

    PubMed

    Murugan, Ramalingam Senthil; Uchida, Koji; Hara, Yukihiko; Nagini, Siddavaram

    2008-10-01

    The present study was designed to investigate the modulatory effects of black tea polyphenols (Polyphenon-B) on phase I and phase II xenobiotic-metabolizing enzymes and oxidative stress in a rat model of hepatocellular carcinoma (HCC). Liver tumours induced in male Sprague-Dawley rats by dietary administration of rho-dimethylaminoazobenzene (DAB) increased cytochrome P450 (total and CYP1A1, 1A2 and 2B isoforms), cytochrome b(5), cytochrome b(5) reductase, glutathione S-transferase (GST total and GST-P isoform) and gamma-glutamyltranspeptidase (GGT) with decrease in quinone reductase (QR). This was accompanied by enhanced lipid and protein oxidation and compromised antioxidant defences associated with increased expression of the oxidative stress markers 4-hydroxynonenal (4-HNE), anti-hexanoyl lysine (HEL), dibromotyrosine (DiBrY) and 8-hydroxy 2-deoxyguanosine (8-OHdG). Dietary administration of Polyphenon-B effectively suppressed DAB-induced hepatocarcinogenesis, as evidenced by reduced preneoplastic and neoplastic lesions, modulation of xenobiotic-metabolizing enzymes and amelioration of oxidative stress. Thus, it can be concluded that Polyphenon-B acts as an effective chemopreventive agent by modulating xenobiotic-metabolizing enzymes and mitigating oxidative stress in an in vivo model of hepatocarcinogenesis. PMID:18985486

  16. 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. PMID:23911531

  17. 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.

  18. Modulation of oxidative DNA damage by repair enzymes XRCC1 and hOGG1.

    PubMed

    Rihs, Hans-Peter; Marczynski, Boleslaw; Lotz, Anne; Raulf-Heimsoth, Monika; Brüning, Thomas

    2012-01-01

    The influence of DNA repair gene polymorphisms (XRCC1: Arg194Trp, Arg280His, Arg399Gln; APE1: Asp148Glu; hOGG1: Ser326Cys) on oxidative DNA damage is controversial and was investigated in 214 German workers with occupational exposure to vapors and aerosols of bitumen,compared to 87 German construction workers without exposure, who were part of the Human Bitumen Study. Genotypes were determined by real-time polymerase chain reaction (PCR), and actual smoking habits by a questionnaire and cotinine analysis. Oxidative DNA damage in white blood cells (WBC) collected pre- and postshift was measured as 8-oxodGuo adducts/10(6) dGuo by a hjigh-performance liquid chromatography electron capture detection (HPLC-ECD) method, followed by calculation of the difference between post- and preshift values (Δ8-oxodGuo/10(6) dGuo). The 214 bitumen exposed workers showed higher median Δ8-oxodGuo values than the 87 references. In the whole study group (n=301) there was a trend for increasing adduct values for XRCC1 Arg(GG)399Gln(AA) during a shift, especially in nonsmokers (n=108. Referents (n=87) displayed a similar trend for hOGG1 Ser(CC)326Cys(GG). In contrast, XRCC1 Arg(GG)280His(AA) showed a decrease of median Δ8-oxodGuo/10(6) dGuo values in workers with exposure to vapors and aerosols of bitumen (n=214), especially in smokers (n=145). XRCC1 Arg194Trp and APE1 Asp148Glu displayed no marked association with Δ8-oxodGuo levels. Data indicate that the combination of different variants in DNA damage repair enzymes may modulate the production of 8-oxoguanine adducts in WBC produced by xenobiotics during a shift. PMID:22686320

  19. Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX*

    PubMed Central

    Bradley, Justin M.; Marritt, Sophie J.; Kihlken, Margaret A.; Haynes, Kate; Hemmings, Andrew M.; Berks, Ben C.; Cheesman, Myles R.; Butt, Julea N.

    2012-01-01

    SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (Em) at pH 7.0 of approximately +210, −340, and −400 mV for the His/Met, His/Cys−, and active site His/CysS−-ligated heme, respectively. Exposing SoxAX to S2O42−, a substrate analog with Em ∼−450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (Em ∼−1140 mV), allows cyanide to displace the cysteine persulfide (CysS−) ligand to the active site heme. This provides the first evidence for the dissociation of CysS− that has been proposed as a key event in SoxAX catalysis. PMID:23060437

  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. PMID:26932500

  1. Reduction of the Mn cluster of the water-oxidizing enzyme by nitric oxide: formation of an S(-2) state.

    PubMed

    Schansker, Gert; Goussias, Charilaos; Petrouleas, Vasili; Rutherford, A William

    2002-03-01

    The manganese cluster of the oxygen-evolving enzyme of photosystem II is chemically reduced upon interaction with nitric oxide at -30 degrees C. The state formed gives rise to an S = 1/2 multiline EPR signal [Goussias, Ch., Ioannidis, N., and Petrouleas, V. (1997) Biochemistry 36, 9261] that is attributed to a Mn(II)- Mn(III) dimer [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581]. In this work, we sought to establish whether the state could be assigned to a specific, reduced S state by using flash oxymetry, chlorophyll a fluorescence, and electron paramagnetic resonance spectroscopy. With the Joliot-type O(2) electrode, the first maximum of oxygen evolution was observed on the sixth or seventh flash. Three saturating pre-flashes were required to convert the flash pattern characteristic of NO-reduced samples to that of the untreated control (i.e., O(2) evolution maximum on the third flash). Measurements of the S state-dependent level of chlorophyll fluorescence in NO-treated PSII showed a three-flash downshift compared to untreated controls. In the EPR study, the maximum S(2) multi-line EPR signal was observed after the fourth flash. The results from all three methods are consistent with the Mn cluster being in a redox state corresponding to an S(-2) state in a majority of centers after treatment with NO. We were unable to generate the Mn(II)-Mn(III) multi-line signal using hydrazine as a reductant; it appears that the valence distribution and possibly the structure of the Mn cluster in the S(-2) state are dependent on the nature of the reductant that is used. PMID:11863444

  2. 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. PMID:24815822

  3. Desaturase reactions complicate the use of norcarane as a mechanistic probe. Unraveling the mixture of twenty-plus products formed in enzyme-catalyzed oxidations of norcarane.

    PubMed

    Newcomb, Martin; Chandrasena, R Esala P; Lansakara-P, Dharmika S P; Kim, Hye-Yeong; Lippard, Stephen J; Beauvais, Laurance G; Murray, Leslie J; Izzo, Viviana; Hollenberg, Paul F; Coon, Minor J

    2007-02-16

    Norcarane, bicyclo[4.1.0]heptane, has been widely used as a mechanistic probe in studies of oxidations catalyzed by several iron-containing enzymes. We report here that, in addition to oxygenated products, norcarane is also oxidized by iron-containing enzymes in desaturase reactions that give 2-norcarene and 3-norcarene. Furthermore, secondary products from further oxidation reactions of the norcarenes are produced in yields that are comparable to those of the minor products from oxidation of the norcarane. We studied oxidations catalyzed by a representative spectrum of iron-containing enzymes including four cytochrome P450 enzymes, CYP2B1, CYPDelta2B4, CYPDelta2E1, and CYPDelta2E1 T303A, and three diiron enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. 2-Norcarene and 3-norcarene and their oxidation products were found in all reaction mixtures, accounting for up to half of the oxidation products in some cases. In total, more than 20 oxidation products were identified from the enzyme-catalyzed reactions of norcarane. The putative radical-derived product from the oxidation of norcarane, 3-hydroxymethylcyclohexene (21), and the putative cation-derived product from the oxidation of norcarane, cyclohept-3-enol (22), coelute with other oxidation products on low-polarity GC columns. The yields of product 21 found in this study are smaller than those previously reported for the same or similar enzymes in studies where the products from norcarene oxidations were ignored, and therefore, the limiting values for lifetimes of radical intermediates produced in the enzyme-catalyzed oxidation reactions are shorter than those previously reported. PMID:17288366

  4. Effect of high-intensity intermittent swimming training on fatty acid oxidation enzyme activity in rat skeletal muscle.

    PubMed

    Terada, Shin; Tabata, Izumi; Higuchi, Mitsuru

    2004-02-01

    We previously reported that high-intensity exercise training significantly increased citrate synthase (CS) activity, a marker of oxidative enzyme, in rat skeletal muscle to a level equaling that attained after low-intensity prolonged exercise training (Terada et al., J Appl Physiol 90: 2019-2024, 2001). Since mitochondrial oxidative enzymes and fatty acid oxidation (FAO) enzymes are often increased simultaneously, we assessed the effect of high-intensity intermittent swimming training on FAO enzyme activity in rat skeletal muscle. Male Sprague-Dawley rats (3 to 4 weeks old) were assigned to a 10-day period of high-intensity intermittent exercise training (HIT), low-intensity prolonged exercise training (LIT), or sedentary control conditions. In the HIT group, the rats repeated fourteen 20 s swimming sessions with a weight equivalent to 14-16% of their body weight. Between the exercise sessions, a 10 s pause was allowed. Rats in the LIT group swam 6 h/day in two 3 h sessions separated by 45 min of rest. CS activity in the triceps muscle of rats in the HIT and LIT groups was significantly higher than that in the control rats by 36 and 39%, respectively. Furthermore, 3-beta hydroxyacyl-CoA dehydrogenase (HAD) activity, an important enzyme in the FAO pathway in skeletal muscle, was higher in the two training groups than in the control rats (HIT: 100%, LIT: 88%). No significant difference in HAD activity was observed between the two training groups. In conclusion, the present investigation demonstrated that high-intensity intermittent swimming training elevated FAO enzyme activity in rat skeletal muscle to a level similar to that attained after 6 h of low-intensity prolonged swimming exercise training. PMID:15040848

  5. 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

  6. 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

    2015-01-01

    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

  7. 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. PMID:23744122

  8. 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

  9. Enzymes involved in the anaerobic oxidation of n-alkanes: from methane to long-chain paraffins

    PubMed Central

    Callaghan, Amy V.

    2013-01-01

    Anaerobic microorganisms play key roles in the biogeochemical cycling of methane and non-methane alkanes. To date, there appear to be at least three proposed mechanisms of anaerobic methane oxidation (AOM). The first pathway is mediated by consortia of archaeal anaerobic methane oxidizers and sulfate-reducing bacteria (SRB) via “reverse methanogenesis” and is catalyzed by a homolog of methyl-coenzyme M reductase. The second pathway is also mediated by anaerobic methane oxidizers and SRB, wherein the archaeal members catalyze both methane oxidation and sulfate reduction and zero-valent sulfur is a key intermediate. The third AOM mechanism is a nitrite-dependent, “intra-aerobic” pathway described for the denitrifying bacterium, ‘Candidatus Methylomirabilis oxyfera.’ It is hypothesized that AOM proceeds via reduction of nitrite to nitric oxide, followed by the conversion of two nitric oxide molecules to dinitrogen and molecular oxygen. The latter can be used to functionalize the methane via a particulate methane monooxygenase. With respect to non-methane alkanes, there also appear to be novel mechanisms of activation. The most well-described pathway is the addition of non-methane alkanes across the double bond of fumarate to form alkyl-substituted succinates via the putative glycyl radical enzyme, alkylsuccinate synthase (also known as methylalkylsuccinate synthase). Other proposed mechanisms include anaerobic hydroxylation via ethylbenzene dehydrogenase-like enzymes and an “intra-aerobic” denitrification pathway similar to that described for ‘Methylomirabilis oxyfera.’ PMID:23717304

  10. 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. PMID:25660326

  11. 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.

  12. 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. PMID:6144677

  13. Elucidation of the regio- and chemoselectivity of enzymatic allylic oxidations with Pleurotus sapidus – conversion of selected spirocyclic terpenoids and computational analysis

    PubMed Central

    Weidmann, Verena; Schaffrath, Mathias; Zorn, Holger

    2013-01-01

    Summary Allylic oxidations of olefins to enones allow the efficient synthesis of value-added products from simple olefinic precursors like terpenes or terpenoids. Biocatalytic variants have a large potential for industrial applications, particularly in the pharmaceutical and food industry. Herein we report efficient biocatalytic allylic oxidations of spirocyclic terpenoids by a lyophilisate of the edible fungus Pleurotus sapidus. This ‘’mushroom catalysis’’ is operationally simple and allows the conversion of various unsaturated spirocyclic terpenoids. A number of new spirocyclic enones have thus been obtained with good regio- and chemoselectivity and chiral separation protocols for enantiomeric mixtures have been developed. The oxidations follow a radical mechanism and the regioselectivity of the reaction is mainly determined by bond-dissociation energies of the available allylic CH-bonds and steric accessibility of the oxidation site. PMID:24204436

  14. 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. PMID:16401082

  15. Oxidative stress enzymes in Ficus religiosa L.: biochemical, histochemical and anatomical evidences.

    PubMed

    Smitha, R B; Bennans, Thomas; Mohankumar, C; Benjamin, Sailas

    2009-04-01

    In order to unveil the reasons behind the successful survival of Ficus religiosa L. grown under normal and adverse habitats (AH), i.e., on concrete roof tops were subjected to biochemical, histochemical and physiological studies with a focus on reactive oxygen species (ROS) and oxidative stress enzymes (OSE). The specific objectives were: to localize the OSE, peroxidase (POX) and catalase (CAT); to localize and quantify the main ROS, hydrogen peroxide (H(2)O(2)); to estimate the activities of POX, CAT and glycolate oxidase (GO); and to study the diurnal variations in stomatal activity by scanning electron microscopy (SEM). From the results, plants grown in AH showed 55% higher H(2)O(2) production with about 30% increase in POX activity. Of the three substrates tested for POX activity (guaiacol, ascorbate and o-dianisidine), o-dianisidine was found as the preferred substrate of F. religiosa POX with about 7-fold more activity over its counterparts. Cytosolic POX activity showed 11-fold increase over cell wall bound POX. Similarly, CAT activity in specimens from AH showed about 2-fold increase during day time. The physiological interaction between CAT and its substrate H(2)O(2) in the plant was determined by quantifying H(2)O(2) and assaying the CAT, in which CAT showed 4-fold increases in activity, especially during night. F. religiosa has higher amount of H(2)O(2) deposition during night than day time, which was in correlation high CAT activity during night, coupled with scotoactive opening of stomata as shown by the SEM images. Moreover, GO did not show much habitat-dependent variation. In toto, F. religiosa grown in AH showed elevated production of ROS and their scavenging OSE, which is the direct evidence for drought stress and also giving an insight into its evolution and ecological niche. PMID:19162506

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

    PubMed

    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

  17. 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

  18. 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). PMID:11746892

  19. 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

  20. Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

    PubMed Central

    Zhao, J; Williams, C C; Last, R L

    1998-01-01

    The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated. PMID:9501110

  1. 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…

  2. 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. PMID:25043555

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

    PubMed

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

    2016-05-01

    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

  4. 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

  5. Immobilization of Enzymes by Electrochemical and Chemical Oxidative Polymerization of L-DOPA to Fabricate Amperometric Biosensors and Biofuel Cells.

    PubMed

    Dai, Mengzhen; Sun, Lingen; Chao, Long; Tan, Yueming; Fu, Yingchun; Chen, Chao; Xie, Qingji

    2015-05-27

    Electrochemical/chemical oxidative synthesis and biosensing/biofuel cell applications of poly(L-DOPA) (PD) are studied versus polydopamine (PDA) as a recent hotspot biomaterial. The enzyme electrode developed by coelectrodeposition of PD and glucose oxidase (GOx), uricase, or tyrosinase shows biosensing performance superior to that of the corresponding PDA-based enzyme electrode. The chemical oxidative polymerization of L-DOPA (PDC) by NaAuCl4 in GOx-containing neutral aqueous solution is used to immobilize GOx and gold nanoparticles (AuNPs). The thus-prepared chitosan (CS)/GOx-PDC-AuNPs/Au(plate)/Au electrode working in the first-generation biosensing mode responds linearly to glucose concentration with a sensitivity of 152 μA mM(-1) cm(-2), which is larger than those of the CS/GOx-PDAC-AuNPs/Au(plate)/Au electrode, the CS/GOx-poly(3-anilineboronic acid) (PABA)-AuNPs/Au(plate)/Au electrode, and the most reported GOx-based enzyme electrodes. This PDC-based enzyme electrode also works well in the second-generation biosensing mode and as an excellent bioanode in biofuel cell construction, probably because PD as an amino acid polymer has the higher biocompatibility and the more favorable affinity to the enzyme than PDA. The PD material of great convenience in synthesis, outstanding biocompatibility for preparing high-performance bionanocomposites, and strong capability of multifunctional coatings on many surfaces may find wide applications in diversified fields including biotechnology and surface-coating. PMID:25938891

  6. CLINICAL CONSEQUENCES OF UREA CYCLE ENZYME DEFICIENCIES AND POTENTIAL LINKS TO ARGININE AND NITRIC OXIDE METABOLISM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. ...

  7. 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

  8. Novel reduced graphene oxide-glycol chitosan nanohybrid for the assembly of an amperometric enzyme biosensor for phenols.

    PubMed

    Boujakhrout, Abderrahmane; Jimenez-Falcao, Sandra; Martínez-Ruiz, Paloma; Sánchez, Alfredo; Díez, Paula; Pingarrón, José M; Villalonga, Reynaldo

    2016-06-20

    A novel water-soluble graphene derivative was prepared from graphene oxide via a two-step modification approach. Graphene oxide was first functionalised with reactive epoxy groups by covalent modification with (3-glycidyloxypropyl)trimethoxysilane and further cross-linked with glycol chitosan. This graphene derivative was characterized using different microscopy and physicochemical methods and employed as a coating material for a glassy carbon electrode. The nanostructured surface was used as a support for the covalent immobilization of the enzyme laccase through cross-linking with glutaraldehyde. The enzyme electrode was tested for the amperometric detection of different phenolic compounds, which displayed excellent analytical behaviour toward catechol with a linear range of response from 200 nM to 15 μM, sensitivity of 93 mA M(-1) cm(-2), and low detection limit of 76 nM. The enzyme biosensor showed high stability when stored at 4 °C under dry conditions and was successfully employed to quantify the total phenolic compounds in commercial herbal tea samples. PMID:27186597

  9. 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

  10. Hepatic drug-oxidizing enzyme systems and urinary D-glucaric acid excretion in patients with congestive heart failure.

    PubMed Central

    Tokola, O; Pelkonen, O; Karki, N T; Luoma, P

    1975-01-01

    Drug-oxidizing enzyme systems in liver biopsy samples and the urinary excretion of D-glucaric acid were studied in two different groups of patients with cardiac insufficiency. 2. In one group of six patients, the activities of drug-metabolizing enzymes had decreased considerably as compared with the control values, but in four liver samples from patients treated with oral hypoglycaemic agents for their diabetes, activities were higher than in control samples from ten patients. 3. In the other group of seven patients, the urinary excretion of D-glucaric acid (isolated by ion-exchange chromatography) was 60% lower than in the control group of nine humans, whereas in four patients taking antiepileptic agents excretion rate was higher than control values. 4. Because the age distribution was markedly different between cardiac insufficiency and control groups, it is difficult to conclude, if the impairment of drug metabolism was a consequence of the old age or of the disease process. However, drug-oxidizing enzyme systems seem to be inducible also in old age. 5. The results support further the opinion that the urinary excretion of D-glucaric acid may be one useful index in assessing an individual's capacity to metabolize foreign compounds especially in the patients with lowered drug metabolizing capacity. PMID:786355

  11. 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.

  12. Elucidation of the mechanism for sulfur oxidation on Pt and Pt3Co electrocatalysts using in situ X-ray absorption spectroscopy

    SciTech Connect

    Ramaker, D.; Gatewood, D; Garsany, Y; Korovina, A; Swider-Lyons, K

    2009-01-01

    Adsorbed sulfur is a poison to the Pt catalysts used in proton exchange membrane fuel cells, but it can be removed by potential cycling. This process is studied for S{sub x}-poisoned nanoscale Pt- and Pt{sub 3}Co- on Vulcan carbon (Pt/VC and Pt{sub 3}Co/VC) in perchloric acid electrolyte using the {Delta}{mu} adsorbate isolation technique for in situ X-ray absorption spectroscopy. The {Delta}{mu} technique is modified to better distinguish the {Delta}{mu} signatures for H, O, and Sx on Pt. The resulting {Delta}{mu} analysis suggests that SO{sub 2} on nanoscale Pt is oxidized to bisulfate or sulfate species in two regions, near 1.05 V on the cluster edges of the Pt nanoparticle, and at higher potentials from the Pt(111) faces where oxygen is less strongly bound. The bisulfate or sulfate species desorb from the Pt surface at high potentials due to O(OH) adsorption/replacement and at low potentials due to loss of the Coulomb attraction between the bisulfate anion and the Pt. A similar oxidation process occurs for S{sub x}-poisoned Pt{sub 3}Co/VC, but at lower potentials because a ligand effect coming from Co shifts the oxidization potential of adsorbed SO{sub 2} to lower potentials while pushing OH adsorption to higher potentials. The spectroscopic results give insights into cyclic voltammetry data and are consistent with electrochemical cycling procedures for removing the sulfur.

  13. 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. PMID:26004323

  14. 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.

  15. Light-driven artificial enzymes for selective oxidation of guanosine triphosphate using water-soluble POSS network polymers.

    PubMed

    Jeon, Jong-Hwan; Tanaka, Kazuo; Chujo, Yoshiki

    2014-09-01

    The light-driven artificial enzymes were constructed to realize unnatural reactions concerning bio-significant molecules. In this manuscript, the guanosine triphosphate (GTP)-selective oxidation is reported using the network polymers composed of polyhedral oligomeric silsesquioxane (POSS). We synthesized the water-soluble POSS network polymer containing the naphthyridine ligands to capture GTP inside the networks and the ruthenium complexes to oxidize the captured GTP under light irradiation. Initially, the binding affinities of the guanosine nucleosides to the naphthyridine ligand inside the POSS network polymer were evaluated from the emission quenching experiments. Accordingly, it was observed that the naphthyridine ligand can form the stable complex only with GTP (K(a) = 5.5 × 10(6) M(-1)). These results indicate that only GTP can be captured by the network polymer. Next, the photo-catalytic activity of the ruthenium complex-modified POSS network polymer was investigated. Finally, it was revealed that the network polymer can decompose GTP efficiently under light irradiation. This is the first example, to the best of our knowledge, to offer not only the GTP-selective host polymers but also the light-driven artificial enzyme for GTP oxidation. PMID:25026217

  16. The Anopheles gambiae Oxidation Resistance 1 (OXR1) Gene Regulates Expression of Enzymes That Detoxify Reactive Oxygen Species

    PubMed Central

    Jaramillo-Gutierrez, Giovanna; Molina-Cruz, Alvaro; Kumar, Sanjeev; Barillas-Mury, Carolina

    2010-01-01

    Background OXR1 is an ancient gene, present in all eukaryotes examined so far that confers protection from oxidative stress by an unknown mechanism. The most highly conserved region of the gene is the carboxyl-terminal TLDc domain, which has been shown to be sufficient to prevent oxidative damage. Methodology/Principal Findings OXR1 has a complex genomic structure in the mosquito A. gambiae, and we confirm that multiple splice forms are expressed in adult females. Our studies revealed that OXR1 regulates the basal levels of catalase (CAT) and glutathione peroxidase (Gpx) expression, two enzymes involved in detoxification of hydrogen peroxide, giving new insight into the mechanism of action of OXR1. Gene silencing experiments indicate that the Jun Kinase (JNK) gene acts upstream of OXR1 and also regulates expression of CAT and GPx. Both OXR1 and JNK genes are required for adult female mosquitoes to survive chronic oxidative stress. OXR1 silencing decreases P. berghei oocyst formation. Unexpectedly, JNK silencing has the opposite effect and enhances Plasmodium infection in the mosquito, suggesting that JNK may also mediate some, yet to be defined, antiparasitic response. Conclusion The JNK pathway regulates OXR1 expression and OXR1, in turn, regulates expression of enzymes that detoxify reactive oxygen species (ROS) in Anopheles gambiae. OXR1 silencing decreases Plasmodium infection in the mosquito, while JNK silencing has the opposite effect and enhances infection. PMID:20567517

  17. Respiratory Burst Enzymes, Pro-Oxidants and Antioxidants Status in Bangladeshi Population with β-Thalassemia Major

    PubMed Central

    Hossain, Md. Faruk; Ismail, Md.; Tanu, Arifur Rahman; Shekhar, Hossain Uddin

    2015-01-01

    Background: Oxidative stress is intimately associated with many diseases, including β-thalassemia. Aim: The study was to estimate the status of respiratory burst enzymes, pro-oxidants, and antioxidants in β-thalassemia major patients in Bangladesh and to compare with apparently healthy individuals. Materials and Methods: A total of 49 subjects were recruited which included 25 patients (age range 5 to 40 years) with β-thalassemia major and 24 controls (age and sex matched). Superoxide dismutase (SOD) and catalase (CAT) represented respiratory burst enzymes; malondialdehyde (MDA), lipid hydroperoxide (LHP), and xanthine oxidase (XO) were measured as pro-oxidants; and glutathione S transferase (GST), vitamin C (Vit.C), and glutathione (GSH) were the measured antioxidants. Results: The activity of SOD was significantly (P < 0.001) increased by about 79% and the activity of CAT was significantly (P < 0.001) decreased by more than 34% in the blood of β-thalassemia major patients compared to the control group. The content of pro-oxidants such as MDA, LHP, and XO was significantly (P < 0.001) higher in patients by about 228%, 241.3% and 148.1% respectively compared to control group. The level of GSH and Vit.C were significantly (P = 0.000) decreased in patients by about 59% and 81% versus the healthy group, respectively; and GST activity was significantly (P < 0.001) declined by 44.25% in patients group. Conclusion: β-thalassemia major patients demonstrate raised oxidative stress compared to healthy subjects. PMID:26199921

  18. Effect of Cell Phone Use on Salivary Total Protein, Enzymes and Oxidative Stress Markers in Young Adults: A Pilot Study

    PubMed Central

    Joy, Jasmi; Sunitha, Venkatesh; Rai, Manoj P.; Rao, Suresh; Nambranathayil, Shafeeque; Baliga, Manjeshwar Shrinath

    2015-01-01

    Introduction: The present study aimed to assess the levels of salivary enzymes, protein and oxidant-antioxidant system in young college-going cell phone users. Materials and Methods: The cell users (students) were categorized in to two groups – less mobile users and high mobile users, based on the duration and frequency of cell use. Unstimulated whole saliva samples of the volunteers were analysed for amylase, lactate dehydrogenase (LDH), malondialdehdye (MDA) and glutathione (GSH). Results: High mobile users had significantly higher levels of amylase (p = 0.001), LDH (p = 0.002) and MDA (p = 0.002) in saliva, when compared to less mobile users. The marginal decrease in salivary total proteins, GSH and flow rate were statistically not significant (p >0.05). Conclusion: Significant changes in salivary enzymes and MDA suggest adverse effect of high use of cell phones on cell health. PMID:25859446

  19. Carnosic acid biosynthesis elucidated by a synthetic biology platform.

    PubMed

    Ignea, Codruta; Athanasakoglou, Anastasia; Ioannou, Efstathia; Georgantea, Panagiota; Trikka, Fotini A; Loupassaki, Sofia; Roussis, Vassilios; Makris, Antonios M; Kampranis, Sotirios C

    2016-03-29

    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

  20. Abasic and oxidized abasic site reactivity in DNA: enzyme inhibition, cross-linking, and nucleosome catalyzed reactions.

    PubMed

    Greenberg, Marc M

    2014-02-18

    Abasic lesions are a family of DNA modifications that lack Watson-Crick bases. The parent member of this family, the apurinic/apyrimidinic lesion (AP), occurs as an intermediate during DNA repair, following nucleobase alkylation, and from random hydrolysis of native nucleotides. In a given day, each cell produces between 10000 and 50000 AP lesions. A variety of oxidants including γ-radiolysis produce oxidized abasic sites, such as C4-AP, from the deoxyribose backbone. A number of potent, cytotoxic antitumor agents, such as bleomycin and the enediynes (e.g., calicheamicin, esperamicin, and neocarzinostatin) also lead to oxidized abasic sites in DNA. The absence of Watson-Crick bases prevents DNA polymerases from properly determining which nucleotide to incorporate opposite abasic lesions. Consequently, several studies have revealed that (oxidized) abasic sites are highly mutagenic. Abasic lesions are also chemically unstable, are prone to strand scission, and possess electrophilic carbonyl groups. However, researchers have only uncovered the consequences of the inherent reactivity of these electrophiles within the past decade. The development of solid phase synthesis methods for oligonucleotides that both place abasic sites in defined positions and circumvent their inherent alkaline lability has facilitated this research. Chemically synthesized oligonucleotides containing abasic lesions provide substrates that have allowed researchers to discover a range of interesting chemical properties of potential biological importance. For instance, abasic lesions form DNA-DNA interstrand cross-links, a particularly important family of DNA damage because they block replication and transcription absolutely. In addition, bacterial repair enzymes can convert an interstrand cross-link derived from C4-AP into a double-strand break, the most deleterious form of DNA damage. Oxidized abasic lesions can also inhibit DNA repair enzymes that remove damaged nucleotides. DNA polymerase

  1. 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

  2. Production of Oxidative and Hydrolytic Enzymes by Coprinus cinereus (Schaeff.) Gray from Sisal Wastes Supplemented with Cow Dung Manure.

    PubMed

    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

  3. Elucidating the two and three-body fragmentation channels on isotopically labeled nitrous oxide by a two-color asymmetric laser field

    NASA Astrophysics Data System (ADS)

    Kotsina, N.; Kaziannis, S.; Kosmidis, C.

    2016-05-01

    We report the interaction of isotopically labeled nitrous oxide (15N14N16O) time-of-flight mass spectroscopy. The phase dependence of two-body fragmentation channels for linear and circular polarization reveals that the nitrogen-nitrogen bond fission channel is in accordance with the 'selective ionization of oriented molecules' mechanism, while the nitrogen-oxygen bond rupture involves an electron recollision mechanism. For the three-body-fragmentation channel resulting in 14N3+ ions it is demonstrated that it may proceed via a concerted and/or a sequential manner. The phase dependence of the sequential channel is indicative of the following dissociation route: 15N14N16O → 15N14N + 16O → 15N + 14N + 16O.

  4. X-ray absorption spectroscopy elucidates the impact of structural disorder on electron mobility in amorphous zinc-tin-oxide thin films

    SciTech Connect

    Siah, Sin Cheng E-mail: buonassisi@mit.edu; Lee, Yun Seog; Buonassisi, Tonio E-mail: buonassisi@mit.edu; Lee, Sang Woon; Gordon, Roy G.; Heo, Jaeyeong; Shibata, Tomohiro; Segre, Carlo U.

    2014-06-16

    We investigate the correlation between the atomic structures of amorphous zinc-tin-oxide (a-ZTO) thin films grown by atomic layer deposition (ALD) and their electronic transport properties. We perform synchrotron-based X-ray absorption spectroscopy at the K-edges of Zn and Sn with varying [Zn]/[Sn] compositions in a-ZTO thin films. In extended X-ray absorption fine structure (EXAFS) measurements, signal attenuation from higher-order shells confirms the amorphous structure of a-ZTO thin films. Both quantitative EXAFS modeling and X-ray absorption near edge spectroscopy (XANES) reveal that structural disorder around Zn atoms increases with increasing [Sn]. Field- and Hall-effect mobilities are observed to decrease with increasing structural disorder around Zn atoms, suggesting that the degradation in electron mobility may be correlated with structural changes.

  5. Oxidative Modification of Rat Sulfotransferase 1A1 Activity in Hepatic Tissue Slices Correlates with Effects on the Purified Enzyme

    PubMed Central

    Dammanahalli, Jagadeesha K.

    2012-01-01

    Mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of xenobiotics as well as numerous endogenous molecules. The major aryl (phenol) SULT in rat liver, rSULT1A1, has been used extensively as a model enzyme for understanding the catalytic function of SULTs. Previous studies showed that purified rSULT1A1 displays significant catalytic changes in the presence of GSSG and other oxidants. In the present study, the effects of diamide [1,1′-azobis(N,N-dimethylformamide)] and tert-butyl hydroperoxide (TBHP) on the activity of rSULT1A1 in rat hepatic slices were compared with the effects of these oxidants on a homogeneous preparation of the enzyme. Precision-cut hepatic slices were incubated with 10 μM 7-hydroxycoumarin (7-HC) in the presence of varied concentrations of either diamide or TBHP. Analysis of the 7-hydroxycoumarin sulfate released into the incubation medium indicated that both oxidants significantly increased the sulfation of 7-HC, and this occurred at optimal concentrations of 5 and 10 μM, respectively. Cellular GSH and GSSG levels in the hepatic slices were not significantly altered from control values at these concentrations of diamide and TBHP. Exposure of homogeneous rSULT1A1 to diamide or TBHP also increased the rate of sulfation of 7-HC, although the optimal concentrations of diamide and TBHP were lower (50- and 100-fold, respectively) than those required for effects with the hepatic slices. These results indicate that both diamide and TBHP may modify the rSULT1A1 in intact cells in a manner similar to that observed with the homogeneous purified enzyme. PMID:22041107

  6. Inducible nitric oxide synthase (NOS2) expressed in septic patients is nitrated on selected tyrosine residues: implications for enzymic activity.

    PubMed Central

    Lanone, Sophie; Manivet, Philippe; Callebert, Jacques; Launay, Jean-Marie; Payen, Didier; Aubier, Michel; Boczkowski, Jorge; Mebazaa, Alexandre

    2002-01-01

    Tyrosine nitration is a post-translational protein modification with potentially significant biological implications. In the present study we demonstrate, for the first time, that tyrosine residues of human inducible nitric oxide synthase (NOS2) can be nitrated by peroxynitrite in vitro, leading to a decreased activity. Moreover, we show that NOS2 expressed in a skeletal muscle from septic patients is nitrated on selective tyrosine residues belonging to a canonic sequence. This phenomenon could be an endogenous mechanism of in vivo modulation of NOS2 enzymic activity. PMID:12097137

  7. Enzyme-substrate complementarity governs access to a cationic reaction manifold in the P450(BM3)-catalysed oxidation of cyclopropyl fatty acids.

    PubMed

    Cryle, Max J; Hayes, Patricia Y; De Voss, James J

    2012-12-01

    The products of cytochrome P450(BM3)-catalysed oxidation of cyclopropyl-containing dodecanoic acids are consistent with the presence of a cationic reaction intermediate, which results in efficient dehydrogenation of the rearranged probes by the enzyme. These results highlight the importance of enzyme-substrate complementarity, with a cationic intermediate occurring only when the probes used begin to diverge from ideal substrates for this enzyme. This also aids in reconciling literature reports supporting the presence of cationic intermediates with certain cytochrome P450 enzyme/substrate pairs. PMID:23109039

  8. 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. PMID:23871020

  9. Increased fructose absorption and oxidation after oral supplementation with sucraid enzyme: Implications for fructose malabsorption

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Revealing oxidative damage to enzymes of carbohydrate metabolism in yeast: An integration of 2D DIGE, quantitative proteomics, and bioinformatics.

    PubMed

    Boone, Cory H T; Grove, Ryan A; Adamcova, Dana; Braga, Camila P; Adamec, Jiri

    2016-07-01

    Clinical usage of lidocaine, a pro-oxidant has been linked with severe, mostly neurological complications. The mechanism(s) causing these complications is independent of the blockade of voltage-gated sodium channels. The budding yeast Saccharomyces cerevisiae lacks voltage-gated sodium channels, thus provides an ideal system to investigate lidocaine-induced protein and pathway alterations. Whole-proteome alterations leading to these complications have not been identified. To address this, S. cerevisiae was grown to stationary phase and exposed to an LC50 dose of lidocaine. The differential proteomes of lidocaine treatment and control were resolved 6 h post exposure using 2D DIGE. Amine reactive dyes and carbonyl reactive dyes were used to assess protein abundance and protein oxidation, respectively. Quantitative analysis of these dyes (⩾ 1.5-fold alteration, p ⩽ 0.05) revealed a total of 33 proteoforms identified by MS differing in abundance and/or oxidation upon lidocaine exposure. Network analysis showed enrichment of apoptotic proteins and cell wall maintenance proteins, while the abundance of proteins central to carbohydrate metabolism, such as triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, and redox proteins superoxide dismutase and peroxiredoxin were significantly decreased. Enzymes of carbohydrate metabolism, such as phosphoglycerate kinase and enolase, the TCA cycle enzyme aconitase, and multiple ATP synthase subunits were found to be oxidatively modified. Also, the activity of aconitase was found to be decreased. Overall, these data suggest that toxic doses of lidocaine induce significant disruption of glycolytic pathways, energy production, and redox balance, potentially leading to cell malfunction and death. PMID:27193513

  11. 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. PMID:16644199

  12. Elucidating hydrogen oxidation/evolution kinetics in base and acid by enhanced activities at the optimized Pt shell thickness on the Ru core

    DOE PAGESBeta

    Elbert, Katherine; Hu, Jue; Ma, Zhong; Zhang, Yu; Chen, Guangyu; An, Wei; Liu, Ping; Isaacs, Hugh S.; Adzic, Radoslav R.; Wang, Jia X.

    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

  13. Elucidating hydrogen oxidation/evolution kinetics in base and acid by enhanced activities at the optimized Pt shell thickness on the Ru core

    SciTech Connect

    Elbert, Katherine; Hu, Jue; Ma, Zhong; Zhang, Yu; Chen, Guangyu; An, Wei; Liu, Ping; Isaacs, Hugh S.; Adzic, Radoslav R.; Wang, Jia X.

    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 to 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+).

  14. 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.

  15. An Adenosine Triphosphate-Phosphate Exchange Catalyzed by a Soluble Enzyme Couple Inhibited by Uncouplers of Oxidative Phosphorylation

    PubMed Central

    Allison, William S.; Benitez, Lita V.

    1972-01-01

    The sulfenic acid form of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), which is an acyl phosphatase, will catalyze an acetyl phosphate-Pi exchange reaction. This exchange reaction is reversibly inhibited by the uncouplers of oxidative phosphorylation, 2,4-dinitrophenol, m-Cl carbonylcyanide-phenylhydrazone, pentachlorophenol, and 5-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanalide, and is irreversibly inhibited by cyanide and dicumarol. An ATP-Pi exchange reaction similar to that catalyzed by mitochondria can be simulated by a system composed of oxidized glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase (EC 2.7.1.28), 3-phosphoglycerate, ATP, 32Pi, and appropriate cofactors. The ATP-Pi exchange is inhibited by uncouplers of oxidative phosphorylation. Higher concentrations of uncouplers will also inhibit the ATPase reaction catalyzed by the coupled enzyme system. The exchange reactions catalyzed by the sulfenic acid form of glyceraldehyde-3-phosphate are consistent with a sulfenyl carboxylate intermediate. On the basis of these observations, a reaction scheme has been postulated for covalent coupling in oxidative phosphorylation that includes a sulfenyl carboxylate as a nonphosphorylated, high energy intermediate and an acyl phosphate as a phosphorylated, high energy intermediate. PMID:4507619

  16. Temperature stress, anti-oxidative enzyme activity and virus acquisition in Bemisia tabaci (Hemiptera: Aleyrodidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. 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.

  18. 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

    2011-03-01

    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. PMID:21268605

  19. 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.

  20. Protective Role of Cys-178 against the Inactivation and Oligomerization of Human Insulin-degrading Enzyme by Oxidation and Nitrosylation*

    PubMed Central

    Ralat, Luis A.; Ren, Min; Schilling, Alexander B.; Tang, Wei-Jen

    2009-01-01

    Insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, hydrolyzes several physiologically relevant peptides, including insulin and amyloid-β (Aβ). Human IDE has 13 cysteines and is inhibited by hydrogen peroxide and S-nitrosoglutathione (GSNO), donors of reactive oxygen and nitrogen species, respectively. Here, we report that the oxidative burst of BV-2 microglial cells leads to oxidation or nitrosylation of secreted IDE, leading to the reduced activity. Hydrogen peroxide and GSNO treatment of IDE reduces the Vmax for Aβ degradation, increases IDE oligomerization, and decreases IDE thermostability. Additionally, this inhibitory response of IDE is substrate-dependent, biphasic for Aβ degradation but monophasic for a shorter bradykinin-mimetic substrate. Our mutational analysis of IDE and peptide mass fingerprinting of GSNO-treated IDE using Fourier transform-ion cyclotron resonance mass spectrometer reveal a surprising interplay of Cys-178 with Cys-110 and Cys-819 for catalytic activity and with Cys-789 and Cys-966 for oligomerization. Cys-110 is near the zinc-binding catalytic center and is normally buried. The oxidation and nitrosylation of Cys-819 allow Cys-110 to be oxidized or nitrosylated, leading to complete inactivation of IDE. Cys-789 is spatially adjacent to Cys-966, and their nitrosylation and oxidation together trigger the oligomerization and inhibition of IDE. Interestingly, the Cys-178 modification buffers the inhibition caused by Cys-819 modification and prevents the oxidation or nitrosylation of Cys-110. The Cys-178 modification can also prevent the oligomerization-mediated inhibition. Thus, IDE can be intricately regulated by reactive oxygen or nitrogen species. The structure of IDE reveals the molecular basis for the long distance interactions of these cysteines and how they regulate IDE function. PMID:19808678

  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. 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

  3. Oxidised low density lipoprotein causes human macrophage cell death through oxidant generation and inhibition of key catabolic enzymes.

    PubMed

    Katouah, Hanadi; Chen, Alpha; Othman, Izani; Gieseg, Steven P

    2015-10-01

    Oxidised low density lipoprotein (oxLDL) is thought to be a significant contributor to the death of macrophage cells observed in advanced atherosclerotic plaques. Using human-derived U937 cells we have examined the effect of cytotoxic oxLDL on oxidative stress and cellular catabolism. Within 3h of the addition of oxLDL, there was a rapid, concentration dependent rise in cellular reactive oxygen species followed by the loss of cellular GSH, and the enzyme activity of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aconitase. The loss of these catabolic enzymes was accompanied by the loss of cellular ATP and lower lactate generation. Addition of the macrophage antioxidant 7,8-dihydroneopterin inhibited the ROS generation, glutathione loss and catabolic inactivation. NOX was shown to be activated by oxLDL addition while apocynin inhibited the loss of GSH and cell viability. The data suggests that oxLDL triggers an excess of ROS production through NOX activation, and catabolic failure through thiol oxidation resulting in cell death. PMID:26255116

  4. Ultrasensitive detection of uranyl by graphene oxide-based background reduction and RCDzyme-based enzyme strand recycling signal amplification.

    PubMed

    Li, Ming-Hui; Wang, Yong-Sheng; Cao, Jin-Xiu; Chen, Si-Han; Tang, Xian; Wang, Xiao-Feng; Zhu, Yu-Feng; Huang, Yan-Qin

    2015-10-15

    We proposed a novel strategy which combines graphene oxide-based background reduction with RCDzyme-based enzyme strand recycling amplification for ultrahigh sensitive detection of uranyl. The RCDzyme is designed to contain a guanine (G)-rich sequence that replaces the partial sequence in an uranyl-specific DNAzyme. This multifunctional probe can act as the target recognition element, DNAzyme and the primer of signal amplification. The presence of UO2(2+) can induce the cleavage of the substrate strands in RCDzyme. Then, each released enzyme strand can hybridize with another substrate strands to trigger many cycles of the cleavage by binding uranyl, leading to the formation of more G-quadruplexes by split guanine-rich oligonucleotide fragments. The resulting G-quadruplexes could bind to N-methyl-mesoporphyrin IX (NMM), causing an amplified detection signal for the target uranyl. Next, graphene oxide-based background reduction strategy was further employed for adsorbing free ssDNA and NMM, thereby providing a proximalis zero-background signal. The combination of RCDzyme signal amplification and proximalis zero-background signal remarkably improves the sensitivity of this method, achieving a dynamic range of two orders of magnitude and giving a detection limit down to 86 pM, which is much lower than those of related literature reports. These achievements might be helpful in the design of highly sensitive analytical platform for wide applications in environmental and biomedical fields. PMID:26000462

  5. Graphene oxide in cetyltrimethylammonium bromide (CTAB) reverse micelle: a befitting soft nanocomposite for improving efficiency of surface-active enzymes.

    PubMed

    Das, Krishnendu; Maiti, Subhabrata; Ghosh, Moumita; Mandal, Deep; Das, Prasanta Kumar

    2013-04-01

    Herein, we report the successful inclusion of 2D allotrope of carbon, graphene oxide (GO) in cetyltrimethylammonium bromide (CTAB)/isooctane/n-hexanol/water reverse micelle without compromising the stability of water-in-oil (w/o) microemulsion. This newly developed self-assembled nanocomposites act as proficient host for surface-active enzymes, lipase, horseradish peroxidase (HRP), and soybean peroxidase (SBP). Lipase activity within GO-doped CTAB reverse micelles remarkably improved by 3.8-fold compared to that was observed in only CTAB reverse micelle (second-order rate constant, k2=433±7 cm3 g(-1) s(-1)). In case of GO-doped CTAB reverse micelle, the observed enzyme activity (k2=1653±11 cm3 g(-1) s(-1)) is till date the highest ever activity of lipase in CTAB w/o microemulsions. In case of HRP and SBP, the catalytic efficiency maximally increased up to 2.6-fold and 2.3-fold, respectively. Electrostatic attraction between cationic head group of CTAB and anionic surface of GO as well as intrinsic amphiphilic character of GO possibly resulted in the confinement of this 2D nanosheet at the interface of reverse micelles. Integration of GO at the interface augmented the interfacial space in vicinity of surface-active enzyme. This enlarged interface might have accommodated higher amount of substrate and lipase with flexibility in its conformation resulting in marked improvement in the enzyme activity. Interfacial localization of GO was established by fluorescence spectroscopy. In addition, change in secondary structure of lipase in presence of 2D carbon allotrope was substantiated by circular dichroism spectroscopy. PMID:23374433

  6. 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. PMID:24985886

  7. Ocimum sanctum Linn. (Holy Basil) ethanolic leaf extract protects against 7,12-dimethylbenz(a)anthracene-induced genotoxicity, oxidative stress, and imbalance in xenobiotic-metabolizing enzymes.

    PubMed

    Manikandan, P; Murugan, R Senthil; Abbas, H; Abraham, S K; Nagini, S

    2007-09-01

    The present study was designed to evaluate the protective effects of ethanolic Ocimum sanctum leaf extract against 7,12-dimethylbenz[a]anthracene (DMBA)-induced genotoxicity, oxidative stress, and imbalance in xenobiotic-metabolizing enzymes. Four different concentrations of ethanolic O. sanctum leaf extract (100, 200, 300, and 400 mg/kg of body weight) were administered to Wistar rats by intragastric intubation for five consecutive days followed by intraperitoneal injection of DMBA (35 mg/kg of body weight) 90 minutes after the final dose of the extract. Administration of DMBA increased bone marrow micronuclei, phase I enzymes, lipid peroxidation, and protein carbonyl formation. This was accompanied by a significant decrease in the activities of phase II detoxification enzymes and antioxidants in the liver, erythrocytes, and bone marrow. Pretreatment with ethanolic O. sanctum leaf extract at a concentration of 300 mg/kg of body weight significantly reduced micronuclei formation and phase I enzymes as well as lipid and protein oxidation with enhanced antioxidant and phase II enzyme activities. The results of the present study suggest that ethanolic O. sanctum leaf extract inhibits DMBA-induced genotoxicity and oxidative stress by modulating xenobiotic-metabolizing enzymes, reducing the extent of lipid and protein oxidation and up-regulating antioxidant defenses. PMID:17887944

  8. 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. PMID:24378064

  9. 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. PMID:26221723

  10. 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

  11. 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…

  12. 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

  13. Effect of the Antihypertensive Drug Enalapril on Oxidative Stress Markers and Antioxidant Enzymes in Kidney of Spontaneously Hypertensive Rat

    PubMed Central

    Chandran, G.; Sirajudeen, K. N. S.; Swamy, M.; Samarendra, Mutum S.

    2014-01-01

    Oxidative stress has been suggested to play a role in hypertension and hypertension induced organ damage. This study examined the effect of enalapril, an antihypertensive drug, on oxidative stress markers and antioxidant enzymes in kidney of spontaneously hypertensive rat (SHR) and Nω -nitro-L-arginine methyl ester (L-NAME) administered SHR. Male rats were divided into four groups (SHR, SHR+enalapril, SHR+L-NAME, and SHR+enalapril+L-NAME). Enalapril (30 mg kg−1 day−1) was administered from week 4 to week 28 and L-NAME (25 mg kg−1 day−1) was administered from week 16 to week 28 in drinking water. Systolic blood pressure (SBP) was measured during the experimental period. At the end of experimental periods, rats were sacrificed; urine, blood, and kidneys were collected for the assessment of creatinine clearance, total protein, total antioxidant status (TAS), thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), and catalase (CAT), as well as histopathological examination. Enalapril treatment significantly enhanced the renal TAS level (P < 0.001) and SOD activity (P < 0.001), reduced the TBARS levels (P < 0.001), and also prevented the renal dysfunction and histopathological changes. The results indicate that, besides its hypotensive and renoprotective effects, enalapril treatment also diminishes oxidative stress in the kidneys of both the SHR and SHR+L-NAME groups. PMID:25254079

  14. 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. PMID:24338896

  15. Immobilization of leachable toxic soil pollutants by using oxidative enzymes. [Geotrichum candidum

    SciTech Connect

    Shannon, M.J.R.; Bartha, R.

    1988-07-01

    Screening of leachable toxic chemicals in a horseradish peroxidase-H/sub 2/O/sub 2/ immobilization system established that immobilization was promising for most phenolic pollutants but not for benzoic acid, 2,6-dinitrocresol, or dibutyl phthalate. The treatment did not mobilize inherently nonmobile pollutants such as anilines and benzo(a)pyrene. In a separate study, an extracellular laccase in the culture filtrate of Geotrichum candidum was selected from five fungal enzymes evaluated as a cost-effective substitute for horseradish peroxidase. This enzyme was used in demonstrating the immobilization and subsequent fate of /sup 14/C-labeled 4-methylphenol and 2,4-dichlorophenol in soil columns. When applied to Lakewood sand, 98.1% of 4-methylpheno was leached through with distilled water. Two days after immobilization treatment with the G. candidum culture filtrate, only 9.1% of the added 4-methylphenol was leached with the same volume of water. Of the more refractory test pollutant 2,4-dichlorophenol, 91.6% had leached at time zero and 48.5% had leached 1 day after the immobilization treatment. However, 2 weeks after immobilization, only 12.0% of the 2,4-dichlorophenol was leached compared with 61.7% from the control column that received no immobilization treatment. No remobilization of the bound pollutants was detected during 3- and 4-week incubation periods.

  16. Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification.

    PubMed

    Jones, Christopher M; Stres, Blaz; Rosenquist, Magnus; Hallin, Sara

    2008-09-01

    Denitrification is a facultative respiratory pathway in which nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) are successively reduced to nitrogen gas (N(2)), effectively closing the nitrogen cycle. The ability to denitrify is widely dispersed among prokaryotes, and this polyphyletic distribution has raised the possibility of horizontal gene transfer (HGT) having a substantial role in the evolution of denitrification. Comparisons of 16S rRNA and denitrification gene phylogenies in recent studies support this possibility; however, these results remain speculative as they are based on visual comparisons of phylogenies from partial sequences. We reanalyzed publicly available nirS, nirK, norB, and nosZ partial sequences using Bayesian and maximum likelihood phylogenetic inference. Concomitant analysis of denitrification genes with 16S rRNA sequences from the same organisms showed substantial differences between the trees, which were supported by examining the posterior probability of monophyletic constraints at different taxonomic levels. Although these differences suggest HGT of denitrification genes, the presence of structural variants for nirK, norB, and nosZ makes it difficult to determine HGT from other evolutionary events. Additional analysis using phylogenetic networks and likelihood ratio tests of phylogenies based on full-length sequences retrieved from genomes also revealed significant differences in tree topologies among denitrification and 16S rRNA gene phylogenies, with the exception of the nosZ gene phylogeny within the data set of the nirK-harboring genomes. However, inspection of codon usage and G + C content plots from complete genomes gave no evidence for recent HGT. Instead, the close proximity of denitrification gene copies in the genomes of several denitrifying bacteria suggests duplication. Although HGT cannot be ruled out as a factor in the evolution of denitrification genes, our analysis suggests that other phenomena, such gene

  17. Oxidation and structural perturbation of redox-sensitive enzymes in injured skeletal muscle.

    PubMed

    Pierce, Anson P; de Waal, Eric; McManus, Linda M; Shireman, Paula K; Chaudhuri, Asish R

    2007-12-15

    Molecular events that control skeletal muscle injury and regeneration are poorly understood. However, inflammation associated with oxidative stress is considered a key player in modulating this process. To understand the consequences of oxidative stress associated with muscle injury, inflammation, and regeneration, hind-limb muscles of C57Bl/6J mice were studied after injection of cardiotoxin (CT). Within 1 day post-CT injection, polymorphonuclear neutrophilic leukocyte accumulation was extensive. Compared to baseline, tissue myeloperoxidase (MPO) activity was elevated eight- and fivefold at 1 and 7 days post-CT, respectively. Ubiquitinylated protein was elevated 1 day postinjury and returned to baseline by 21 days. Cysteine residues of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were irreversibly oxidized within 1 day post-CT injection and were associated with protein conformational changes that fully recovered after 21 days. Importantly, protein structural alterations occurred in conjunction with significant decreases in CK activity at 1, 3, and 7 days post-CT injury. Interestingly, elevations in tissue MPO activity paralleled the time course of conformational changes in CK and GAPDH. In combination, these results demonstrate that muscle proteins in vivo are structurally and functionally altered via the generation of reactive oxygen species produced during inflammatory events after muscle injury and preceding muscle regeneration. PMID:18037124

  18. 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

  19. Modified enzyme-linked immunosorbent assay strategy using graphene oxide sheets and gold nanoparticles functionalized with different antibody types.

    PubMed

    Lin, Hongjun; Liu, Yingfu; Huo, Jingrui; Zhang, Aihong; Pan, Yiting; Bai, Haihong; Jiao, Zhang; Fang, Tian; Wang, Xin; Cai, Yun; Wang, Qingming; Zhang, Yangjun; Qian, Xiaohong

    2013-07-01

    Gold nanoparticles (GNPs) and graphene oxide (GO) sheets are excellent nano carriers in many analytical methods. In this study, a modified enzyme-linked immunosorbent assay (ELISA) strategy was developed using antibody-functionalized GO sheets and GNPs. This modification significantly reduced the limit of detection (LOD) and cost greatly of this assay. The applicability of the method was demonstrated by detecting HSP70 in a human serum sample. This result suggests that the 3G-ELISA method is feasible to detect an antigen in a complex mixture, and the LOD is up to 64-fold and the cost is as low as one-tenth of the conventional ELISA method. PMID:23713797

  20. Duration of priapism is associated with increased corporal oxidative stress and antioxidant enzymes in a rat model.

    PubMed

    Kucukdurmaz, F; Kucukgergin, C; Akman, T; Salabas, E; Armagan, A; Seckin, S; Kadıoglu, A

    2016-05-01

    Ischaemic priapism is characterised by hypoxia, hypercapnia and acidosis with resultant corporal fibrosis. Studies reported decreased erectile recovery after treatment of priapism longer than 36 h. However, a recent study revealed that half of patients with 3 days of priapism achieved recovery after T-shunt, although mechanism remains unclear. We aimed to investigate the effect of priapism duration on oxidative stress and antioxidant enzymes. Twenty-four male rats were divided into four groups. Group 1 served as control. Groups 2, 3 and 4 represented 1, 2 and 4 h, respectively, of priapism induced by vacuum device and rubber band placed at base of erect penis. After 30 min of reperfusion, penectomy and blood withdrawal were performed to investigate levels of malondialdehyde (MDA), protein carbonyl (PC), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx). Corporal MDA progressively increased with priapism duration (P = 0.01). Corporal SOD significantly differed between groups 1, 2 and 4. Also, there were significant differences in corporal GPx in groups 1 and 4 (P = 0.004) and groups 2 and 4 (P = 0.01). Corporal CAT was higher in group 4, but multivariable analysis revealed insignificant differences. Plasma MDA of the experimental groups was significantly higher than that of controls. There were no differences among groups in terms of other parameters. Increased antioxidant enzymes according to duration of priapism suggest that immediate treatment to relieve oxidative stress should be initiated in prolonged cases. However, further studies should be conducted to determine resistance mechanisms of the corpora to prolonged ischaemia. PMID:26223151

  1. S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: Further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase

    PubMed Central

    Leiper, James; Murray-Rust, Judith; McDonald, Neil; Vallance, Patrick

    2002-01-01

    The enzyme dimethylarginine dimethylaminohydrolase (DDAH) hydrolyses asymmetrically methylated arginine residues that are endogenously produced inhibitors of nitric oxide synthases (NOS). We and others have proposed that DDAH activity is a key determinant of intracellular methylarginine concentrations and that factors that regulate the activity of DDAH may modulate nitric oxide (NO) production in vivo. We recently solved the crystal structure of a bacterial DDAH and identified a Cys-His-Glu catalytic triad [Murray-Rust, J., Leiper, J. M., McAlister, M., Phelan, J., Tilley, S., Santa Maria, J., Vallance, P. & McDonald, N. (2001) Nat. Struct. Biol. 8, 679–683]. The presence of a reactive cysteine residue (Cys-249) in the active site of DDAH raised the possibility that DDAH activity might be directly regulated by S-nitrosylation of this residue by NO. In the present study, we demonstrate that recombinant DDAH is reversibly inhibited after incubation with NO donors in vitro. Similarly mammalian DDAH in cytosolic extracts is also reversibly inhibited by NO donors. In cultured endothelial cells, heterologously expressed human DDAH II was S-nitrosylated after cytokine induced expression of the inducible NOS isoforms. The implication of these findings is that under certain conditions when NO generation increases, S-nitrosylation diminishes DDAH activity and this would be expected to lead to accumulation of asymmetric dimethylarginine and inhibition of NOS. This observation may help explain why expression of iNOS often leads to inhibition of activity of constitutively expressed NOS isozymes. We also identify Cys-His-Glu as a nitrosylation motif that is conserved in a family of arginine handling enzymes. PMID:12370443

  2. 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. PMID:26212988

  3. 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. PMID:25169909

  4. Enzyme mechanism-based, oxidative DNA-protein cross-links formed with DNA polymerase β in vivo.

    PubMed

    Quiñones, Jason L; Thapar, Upasna; Yu, Kefei; Fang, Qingming; Sobol, Robert William; Demple, Bruce

    2015-07-14

    Free radical attack on the C1' position of DNA deoxyribose generates the oxidized abasic (AP) site 2-deoxyribonolactone (dL). Upon encountering dL, AP lyase enzymes such as DNA polymerase β (Polβ) form dead-end, covalent intermediates in vitro during attempted DNA repair. However, the conditions that lead to the in vivo formation of such DNA-protein cross-links (DPC), and their impact on cellular functions, have remained unknown. We adapted an immuno-slot blot approach to detect oxidative Polβ-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polβ-DPC in vivo. In a dose-dependent fashion, Polβ-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polβ-DPC under 1% O2 than under 21% O2) and even more robustly with the "chemical nuclease" 1,10-copper-ortho-phenanthroline, Cu(OP)2. Mouse embryonic fibroblasts challenged with TPZ or Cu(OP)2 also incurred Polβ-DPC. Nonoxidative agents did not generate Polβ-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polβ-DPC depended on the Ape1 AP endonuclease, which generates the Polβ lyase substrate, and they required the essential lysine-72 in the Polβ lyase active site. Oxidative Polβ-DPC had an unexpectedly short half-life (∼ 30 min) in both human and mouse cells, and their removal was dependent on the proteasome. Proteasome inhibition under Cu(OP)2 treatment was significantly more cytotoxic to cells expressing wild-type Polβ than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polβ-DPC and the biological pressure to repair them. PMID:26124145

  5. Enzyme mechanism-based, oxidative DNA–protein cross-links formed with DNA polymerase β in vivo

    PubMed Central

    Quiñones, Jason L.; Thapar, Upasna; Yu, Kefei; Fang, Qingming; Sobol, Robert William; Demple, Bruce

    2015-01-01

    Free radical attack on the C1′ position of DNA deoxyribose generates the oxidized abasic (AP) site 2-deoxyribonolactone (dL). Upon encountering dL, AP lyase enzymes such as DNA polymerase β (Polβ) form dead-end, covalent intermediates in vitro during attempted DNA repair. However, the conditions that lead to the in vivo formation of such DNA–protein cross-links (DPC), and their impact on cellular functions, have remained unknown. We adapted an immuno-slot blot approach to detect oxidative Polβ-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polβ-DPC in vivo. In a dose-dependent fashion, Polβ-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polβ-DPC under 1% O2 than under 21% O2) and even more robustly with the “chemical nuclease” 1,10-copper-ortho-phenanthroline, Cu(OP)2. Mouse embryonic fibroblasts challenged with TPZ or Cu(OP)2 also incurred Polβ-DPC. Nonoxidative agents did not generate Polβ-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polβ-DPC depended on the Ape1 AP endonuclease, which generates the Polβ lyase substrate, and they required the essential lysine-72 in the Polβ lyase active site. Oxidative Polβ-DPC had an unexpectedly short half-life (∼30 min) in both human and mouse cells, and their removal was dependent on the proteasome. Proteasome inhibition under Cu(OP)2 treatment was significantly more cytotoxic to cells expressing wild-type Polβ than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polβ-DPC and the biological pressure to repair them. PMID:26124145

  6. Oxidative stability and antioxidant enzyme activities of dry-cured bacons as affected by the partial substitution of NaCl with KCl.

    PubMed

    Wu, Haizhou; Yan, Wenjing; Zhuang, Hong; Huang, Mingming; Zhao, Jianying; Zhang, Jianhao

    2016-06-15

    This study investigated the influence of partial substitution of NaCl with KCl on protein and lipid oxidation as well as antioxidant enzyme activities in dry-cured bacons during processing. The partial substitution was 0% KCl (I), 40% KCl (II), and 70% KCl (III). Compared with 0% KCl (I), the substitution of 40% NaCl with KCl did not significantly influence the protein and lipid oxidation and antioxidant enzyme activities. The bacons that were treated with 70% KCl treatment (III) showed increased lipid oxidation and antioxidant enzyme GSH-Px activity, whereas samples treated with formulas I and II showed higher protein oxidation and antioxidant enzyme catalase activity. These results demonstrate that the substitution of NaCl with KCl by more than 40% may significantly affect protein and lipid oxidation and that for the substitution of NaCl in further processed meat products with other chloride salts, salt content is very important for control of protein and lipid biochemical changes in finished products. PMID:26868571

  7. 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

    2012-09-01

    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 (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. PMID:22894914

  8. 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.

  9. 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

  10. 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

  11. Bio-inspired artificial functional photocatalyst: biomimetic enzyme-like TiO2/reduced graphene oxide nanocomposite with excellent molecular recognition ability

    NASA Astrophysics Data System (ADS)

    Li, Wentao; Pei, Xule; Deng, Fang; Luo, Xubiao; Li, Fengcong; Xiao, Yong

    2015-05-01

    An enzyme-like TiO2/reduced graphene oxide (enzyme-TiO2/rGO) nanocomposite with molecular recognition ability was fabricated by biomimicking the geometrical and chemical complementation of the enzyme and substrate. The anatase TiO2 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-TiO2/rGO nanocomposites for 4-nitrophenol (4.71 mg g-1) is about six times that of control TiO2/rGO without the enzyme-like feature (0.79 mg g-1), and the enzyme-TiO2/rGO shows a relative selectivity coefficient of 7.24. Moreover, enzyme-TiO2/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.

  12. Role of Oxidative Stress in HIV-1-Associated Neurocognitive Disorder and Protection by Gene Delivery of Antioxidant Enzymes

    PubMed Central

    Louboutin, Jean-Pierre; Strayer, David

    2014-01-01

    HIV encephalopathy covers a range of HIV-1-related brain dysfunction. In the Central Nervous System (CNS), it is largely impervious to Highly Active AntiRetroviral Therapy (HAART). As survival with chronic HIV-1 infection improves, the number of people harboring the virus in their CNS increases. Neurodegenerative and neuroinflammatory changes may continue despite the use of HAART. Neurons themselves are rarely infected by HIV-1, but HIV-1 infects resident microglia, periventricular macrophages, leading to increased production of cytokines and to release of HIV-1 proteins, the most likely neurotoxins, among which are the envelope glycoprotein gp120 and HIV-1 trans-acting protein Tat. Gp120 and Tat induce oxidative stress in the brain, leading to neuronal apoptosis/death. We review here the role of oxidative stress in animal models of HIV-1 Associated Neurocognitive Disorder (HAND) and in patients with HAND. Different therapeutic approaches, including clinical trials, have been used to mitigate oxidative stress in HAND. We used SV40 vectors for gene delivery of antioxidant enzymes, Cu/Zn superoxide dismutase (SOD1), or glutathione peroxidase (GPx1) into the rat caudate putamen (CP). Intracerebral injection of SV (SOD1) or SV (GPx1) protects neurons from apoptosis caused by subsequent inoculation of gp120 and Tat at the same location. Vector administration into the lateral ventricle or cisterna magna protects from intra-CP gp120-induced neurotoxicity comparably to intra-CP vector administration. These models should provide a better understanding of the pathogenesis of HIV-1 in the brain as well as offer new therapeutic avenues. PMID:26785240

  13. 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. PMID:18948223

  14. 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

    2016-04-01

    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. PMID:26899469

  15. Use of response surface methodology to study the combined effects of UV-C and thermal process on vegetable oxidative enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. 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

  17. 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. PMID:25085218

  18. 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.

  19. 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

  20. Altered activities of kininase II, an angiotensin converting enzyme, prekallikrein, and nitric oxide in Kuwaiti patients with type 2 diabetes.

    PubMed

    Sharma, J N; Al-Shoumer, Kas; Matar, K M; Madathil, N V; Al-Moalem, Amk

    2015-06-01

    The current investigation was conducted to examine kininase II or angiotensin converting enzyme (ACE), plasma prekallikrein (PK), and nitric oxide (NO) concentrations in healthy Kuwaiti subjects and newly diagnosed Kuwaiti type 2 diabetic patients before and after treatment for 6 weeks with metformin hydrochloride 500 mg twice daily after meal. With the consent of volunteers, blood and urine samples were collected after an overnight fasting. Samples were collected from the diabetic patients before and after treatment for 6 weeks. Enzyme linked immunosorbent assay (ELISA) was carried out on the aliquoted samples to measure the concentration of kininase II. NO was detected via colorimetry. Plasma Kininase II or ACE levels were significantly (P <0.01) increased by 18% in untreated diabetics when compared with healthy volunteers. However, after treatment there was a significant decrease of 20% in their ACE levels. Plasma prekallikrein levels were raised significantly (P <0.01) by 28% in diabetic patients in contrast with the control subjects and the levels were significantly reduced (P <0.0001) by 44% after treatment with metformin hydrochloride. NO levels were found to be significantly decreased in plasma by 56% and in urine by 62% in untreated diabetic patients as compared with the healthy subjects. However, when the treated diabetic patients were compared with untreated diabetics, there was an increase of 50% in plasma and 37% in urine samples. The high levels of kininase II, prekallikrein, and reduced NO may be partly responsible for the induction of renal, cardiac, and hypertensive complications associated with type 2 diabetes. Reduced NO level is an indication of endothelial dysfunction resulting in increased blood pressure. Oral anti-diabetic treatment is associated with protective effects through the reduction of kininase II (ACE), prekallikrein, and elevation of NO levels. PMID:25964383

  1. 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.

  2. Dietary lipid quality and mitochondrial membrane composition in trout: responses of membrane enzymes and oxidative capacities.

    PubMed

    Martin, N; Bureau, D P; Marty, Y; Kraffe, E; Guderley, H

    2013-04-01

    To examine whether membrane fatty acid (FA) composition has a greater impact upon specific components of oxidative phosphorylation or on overall properties of muscle mitochondria, rainbow trout (Oncorhynchus mykiss) were fed two diets differing only in FA composition. Diet 1 was enriched in 18:1n-9 and 18:2n-6 while Diet 2 was enriched in 22:6n-3. The FA composition of mitochondrial phospholipids was strongly affected by diet. 22:6n-3 levels were twice as high (49%) in mitochondrial phospholipids of fish fed Diet 2 than in those fed Diet 1. 18:2n-6 content of the phospholipids also followed the diets, whereas 18:1n-9 changed little. All n-6 FA, most notably 22:5n-6, were significantly higher in fish fed Diet 1. Nonetheless, total saturated FA, total monounsaturated FA and total polyunsaturated FA in mitochondrial phospholipids varied little. Despite a marked impact of diet on specific FA levels in mitochondrial phospholipids, only non-phosphorylating (state 4) rates were higher in fish fed Diet 2. Phosphorylating rates (state 3), oxygen consumption due to flux through the electron transport chain complexes as well as the corresponding spectrophotometric activities did not differ with diet. Body mass affected state 4 rates and cytochrome c oxidase and F 0 F 1 ATPase activities while complex I showed a diet-specific effect of body mass. Only the minor FA that were affected by body mass were correlated with functional properties. The regulated incorporation of dietary FA into phospholipids seems to allow fish to maintain critical membrane functions even when the lipid quality of their diets varies considerably, as is likely in their natural environment. PMID:23052948

  3. 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

  4. 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

  5. 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. PMID:27126515

  6. Amelioration of Ozone-Induced Oxidative Damage in Wheat Plants Grown under High Carbon Dioxide (Role of Antioxidant Enzymes).

    PubMed Central

    Rao, M. V.; Hale, B. A.; Ormrod, D. P.

    1995-01-01

    O3-induced changes 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 CO2. High CO2 enhanced shoot biomass of wheat plants, whereas O3 exposure decreased shoot biomass. The shoot biomass was relatively unaffected in plants grown under a combination of high CO2 and O3. O3 exposure under ambient CO2 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 O3 under high CO2. O3 exposure initially enhanced the specific activities of superoxide dismutase, peroxidase, glutathione reductase, and ascorbate peroxidase irrespective of growth in ambient or high CO2. However, the specific activities decreased in plants with prolonged exposure to O3 under ambient CO2 but not in plants exposed to O3 under high CO2. Native gels revealed preferential changes in the isoform composition of superoxide dismutase, peroxidases, and ascorbate peroxidase of plants grown under a combination of high CO2 and O3. Furthermore, growth under high CO2 and O3 led to the synthesis of one new isoform of glutathione reductase. This could explain why plants grown under a combination of high CO2 and O3 are capable of resisting O3-induced damage to growth and proteins compared to plants exposed to O3 under ambient CO2. PMID:12228603

  7. 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. PMID:26554526

  8. Tight gas sands research program: Field operations and analysis. Degradation of hydroxypropyl guar fracturing fluids by enzyme, oxidative, and catalyzed oxidative breakers. Part 1. Linear hydroxypropyl guar solutions. Topical report, February 1991-December 1991

    SciTech Connect

    Craig, D.; Holditch, S.A.

    1993-12-01

    The research was designed to accomplish the following objectives: Determine the effects of pH, temperature, and polymer concentration on HPG solution degradation by enzyme, oxidative (ammonium persulfate), and catalyzed oxidative breakers (ammonium persulfate with triethanolamine); Identify the appropriate breaker system for HPG solutions in the temperature range of 120 deg F - 140 deg F; and Determine the temperature range for HPG solutions where chemical breakers are not necessary to degrade the fluid.

  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. An enzyme-aided amplification strategy for sensitive detection of DNA utilizing graphene oxide (GO) as a fluorescence quencher.

    PubMed

    Zhang, Jing; Tao, Mangjuan; Jin, Yan

    2014-07-01

    A facile, sensitive and rapid method has been developed for detection of disease-related DNA based on lambda exonuclease-aided signal amplification by utilizing graphene oxide (GO) as a fluorescence quencher. The fluorescence of the carboxyfluorescein-labeled DNA probe (F-DNA) was sharply quenched due to the electron or energy transfer between the fluorescence dye and GO. While in the presence of target DNA, the formation of a DNA hybrid released F-DNA from the surface of GO, leading to a fluorescence recovery. Then, the fluorescence enhancement was further amplified by using lambda exonuclease (λexo) to liberate target DNA for cyclic hybridization. Fluorescence polarization and gel electrophoresis further verified the reliability of the principle. Disease-related DNA can be sensitively detected based on the enzyme-aided amplification strategy. More importantly, single-base mismatched DNA can be effectively discriminated from complementary target DNA and random DNA. Therefore, it offered a universal, simple, sensitive and specific method for detection of disease-related genes. PMID:24840773

  11. Nicking enzyme and graphene oxide-based dual signal amplification for ultrasensitive aptamer-based fluorescence polarization assays.

    PubMed

    Huang, Yong; Liu, Xiaoqian; Zhang, Liangliang; Hu, Kun; Zhao, Shulin; Fang, Baizong; Chen, Zhen-Feng; Liang, Hong

    2015-01-15

    In this work, two different configurations for novel amplified fluorescence polarization (FP) aptasensors based on nicking enzyme signal amplification (NESA) and graphene oxide (GO) enhancement have been developed for ultrasensitive and selective detection of biomolecules in homogeneous solution. One approach involves the aptamer-target binding induced the stable hybridization between an aptamer probe and a fluorophore-labeled DNA probe linked to GO, and forms a nicking site-containing duplex DNA region due to the enhancement of base stacking. The second analytical method involves the target induced the assembly of two aptamer subunits into an aptamer-target complex, and then hybridizes with a fluorophore-labeled DNA probe linked to GO, forming a nicking site-containing duplex DNA region. The formation of the duplex DNA region in both methods triggers the NESA process, resulting in the release of many short DNA fragments carrying the fluorophore from GO, generating a significant decrease of the FP value that provides the readout signal for the amplified sensing process. By using the NESA coupled GO enhancement path, the sensitivity of the developed aptasensors can be significantly improved by four orders of magnitude over traditional aptamer-based homogeneous assays. Moreover, these aptasensors also exhibit high specificity for target molecules, which are capable of detecting target molecule in biological samples. Considering these qualities, the proposed FP aptasensors based NESA and GO enhancement can be expected to provide an ultrasensitive platform for amplified analysis of target molecules. PMID:25087158

  12. The Multifunctional β-Oxidation Enzyme Is Required for Full Symptom Development by the Biotrophic Maize Pathogen Ustilago maydis▿

    PubMed Central

    Klose, Jana; Kronstad, James W.

    2006-01-01

    The transition from yeast-like to filamentous growth in the biotrophic fungal phytopathogen Ustilago maydis is a crucial event for pathogenesis. Previously, we showed that fatty acids induce filamentation in U. maydis and that the resulting hyphal cells resemble the infectious filaments observed in planta. To explore the potential metabolic role of lipids in the morphological transition and in pathogenic development in host tissue, we deleted the mfe2 gene encoding the multifunctional enzyme that catalyzes the second and third reactions in β-oxidation of fatty acids in peroxisomes. The growth of the strains defective in mfe2 was attenuated on long-chain fatty acids and abolished on very-long-chain fatty acids. The mfe2 gene was not generally required for the production of filaments during mating in vitro, but loss of the gene blocked extensive proliferation of fungal filaments in planta. Consistent with this observation, mfe2 mutants exhibited significantly reduced virulence in that only 27% of infected seedlings produced tumors compared to 88% tumor production upon infection by wild-type strains. Similarly, a defect in virulence was observed in developing ears upon infection of mature maize plants. Specifically, the absence of the mfe2 gene delayed the development of teliospores within mature tumor tissue. Overall, these results indicate that the ability to utilize host lipids contributes to the pathogenic development of U. maydis. PMID:16998075

  13. Amplified inhibition of the electrochemical signal of ferrocene by enzyme-functionalized graphene oxide nanoprobe for ultrasensitive immunoassay.

    PubMed

    Lai, Guosong; Cheng, Hui; Xin, Dinghong; Zhang, Haili; Yu, Aimin

    2016-01-01

    A nanoprobe-induced signal inhibition mechanism was designed for ultrasensitive electrochemical immunoassay at a chitosan-ferrocene (CS-Fc) based immunosensor. The nanoprobe was prepared by covalently loading signal antibody and high-content horseradish peroxidase (HRP) on the graphene oxide (GO) nanocarrier. The immunosensor was prepared through the stepwise assembly of gold nanoparticles (Au NPs) and capture antibody at a CS-Fc modified electrode. After sandwich immunoreaction, the GO-HRP nanoprobes were quantitatively captured onto the immunosensor surface and thus induced the production of a layer of insoluble film through the enzymatically catalytic reaction of the HRP labels. Both the dielectric immunocomplex formed on the immunosensor surface and the enzymatic precipitate with low electroconductivity led to the electrochemical signal decease of the Fc indicator, which was greatly amplified by the multi-enzyme signal amplification of the nanoprobe. Based on this amplified signal inhibition mechanism, a new ultrasensitive electrochemical immunoassay method was developed. Using carcinoembryonic antigen as a model analyte, this method showed a wide linear range over 5 orders of magnitude with a detection limit down to 0.54 pg/mL. Besides, the immunosensor showed good specificity, acceptable reproducibility and stability as well as satisfactory reliability for the serum sample analysis. PMID:26703270

  14. 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

  15. 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.

  16. Increased tolerance to oxidative stress in transgenic tobacco expressing a wheat oxalate oxidase gene via induction of antioxidant enzymes is mediated by H2O2.

    PubMed

    Wan, Xiaoqing; Tan, Jiali; Lu, Shaoyun; Lin, Chuyu; Hu, Yihong; Guo, Zhenfei

    2009-05-01

    Hydrogen peroxide (H(2)O(2)) plays a key role in the regulation of plant responses to various environmental stresses and modulates the expression of related genes including those encoding antioxidant enzymes. A wheat oxalate oxidase (OxO) gene was transformed and expressed in tobacco for production of H(2)O(2). The transgenic plants exhibited enhanced OxO activities and H(2)O(2) concentrations, which was blocked by inhibitors of OxO. The transgenic plants showed increased tolerance to methyl viologen (MV) or high light-induced oxidative stress in both short-time and long-time tests by measuring their maximal photochemical efficiency of PSII (F(v)/F(m)), ion leakage and malondialdehyde. Higher activities and transcripts of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) were observed in the transgenic plants compared to their wild-type controls under normal growth conditions. Pretreatments with inhibitors of OxO and scavenger of H(2)O(2) blocked the increase of tolerance to MV-induced or high light-induced oxidative stress, as well as the induction of antioxidant enzyme activities. Pretreatments with H(2)O(2) increased tolerance to oxidative stresses and antioxidant enzyme activities. It is suggested that H(2)O(2) produced by OxO in the transgenic tobacco plants triggers the signaling pathways to upregulate expressions of antioxidant enzyme genes, which in turn results in the increase of tolerance to MV-induced and high light-induced oxidative stresses. PMID:19508366

  17. Curcumin Induces Nrf2 Nuclear Translocation and Prevents Glomerular Hypertension, Hyperfiltration, Oxidant Stress, and the Decrease in Antioxidant Enzymes in 5/6 Nephrectomized Rats

    PubMed Central

    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. PMID:22919438

  18. Mammalian mitochondrial beta-oxidation.

    PubMed Central

    Eaton, S; Bartlett, K; Pourfarzam, M

    1996-01-01

    The enzymic stages of mammalian mitochondrial beta-oxidation were elucidated some 30-40 years ago. However, the discovery of a membrane-associated multifunctional enzyme of beta-oxidation, a membrane-associated acyl-CoA dehydrogenase and characterization of the carnitine palmitoyl transferase system at the protein and at the genetic level has demonstrated that the enzymes of the system itself are incompletely understood. Deficiencies of many of the enzymes have been recognized as important causes of disease. In addition, the study of these disorders has led to a greater understanding of the molecular mechanism of beta-oxidation and the import, processing and assembly of the beta-oxidation enzymes within the mitochondrion. The tissue-specific regulation, intramitochondrial control and supramolecular organization of the pathway is becoming better understood as sensitive analytical and molecular techniques are applied. This review aims to cover enzymological and organizational aspects of mitochondrial beta-oxidation together with the biochemical aspects of inherited disorders of beta-oxidation and the intrinsic control of beta-oxidation. PMID:8973539

  19. Zinc oxide-montmorillonite hybrid influences diarrhea, intestinal mucosal integrity, and digestive enzyme activity in weaned pigs.

    PubMed

    Hu, Caihong; Song, Juan; You, Zhaotong; Luan, Zhaoshuang; Li, Weifen

    2012-11-01

    One hundred-eighty piglets (Duroc × Landrace × Yorkshire), with an average initial weight of 7.4 kg weaned at 27 ± 1 days of age, were used to evaluate the effects of dietary zinc oxide-montmorillonite hybrid (ZnO-MMT) on growth performance, diarrhea, intestinal mucosal integrity, and digestive enzyme activity. All pigs were allotted to five treatments and fed with the basal diets supplemented with 0, 250, 500, and 750 mg/kg of Zn as ZnO-MMT or 2,000 mg/kg of Zn as ZnO. The results showed that supplementation with 500 or 750 mg/kg of Zn from ZnO-MMT and 2,000 mg/kg of Zn from ZnO improved average daily gain, enhanced average daily feed intake, decreased fecal scores at 4, 8, and 14 days postweaning, reduced intestinal permeability which was evident from the reduced lactulose recovery and urinary lactulose/mannitol ratio, and improved the activities of protease, amylase, lipase, trypsin, and chymotrypsin both in pancreas and small intestinal contents of pigs as compared with the control. Supplemental 250 mg/kg of Zn from ZnO-MMT also decreased fecal scores at 8 and 14 days postweaning, decreased urinary lactulose/mannitol ratio, and improved chymotrypsin activity in pancreas and small intestinal contents as well as protease activity in small intestinal contents compared with control. Moreover, the above indexes of weanling pigs fed with 500 or 750 mg/kg of Zn as ZnO-MMT did not differ from those fed with 2,000 mg/kg of Zn as ZnO. The results demonstrated that supplementation with 500 or 750 mg/kg of Zn from ZnO-MMT was as efficacious as 2,000 mg/kg of Zn from ZnO in improving growth performance, alleviating postweaning diarrhea, and enhancing intestinal mucosal integrity and the digestive enzyme activities in pancreas and small intestinal contents of pigs. The results that feeding lower concentrations of ZnO-MMT to weanling pigs maintained performance will be beneficial for the environment and for sustaining swine production. PMID:22539019

  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. PMID:27049122

  1. Cu-ZSM-5: A biomimetic inorganic model for methane oxidation

    PubMed Central

    Vanelderen, Pieter; Hadt, Ryan G.; Smeets, Pieter J.; Solomon, Edward I.; Schoonheydt, Robert A.; Sels, Bert F.

    2012-01-01

    The present work highlights recent advances in elucidating the methane oxidation mechanism of inorganic Cu-ZSM-5 biomimic and in identifying the reactive intermediates that are involved. Such molecular understanding is important in view of upgrading abundantly available methane, but also to comprehend the working mechanism of genuine Cu-containing oxidation enzymes. PMID:23487537

  2. Properties of substances inhibiting aggregation of oxidized GAPDH: Data on the interaction with the enzyme and the impact on its intracellular content.

    PubMed

    Lazarev, Vladimir F; Nikotina, Alina D; Semenyuk, Pavel I; Evstafyeva, Diana B; Mikhaylova, Elena R; Muronetz, Vladimir I; Shevtsov, Maxim A; Tolkacheva, Anastasia V; Dobrodumov, Anatoly V; Shavarda, Alexey L; Guzhova, Irina V; Margulis, Boris A

    2016-06-01

    This data is related to our paper "Small molecules preventing GAPDH aggregation are therapeutically applicable in cell and rat models of oxidative stress" (Lazarev et al. [1]) where we explore therapeutic properties of small molecules preventing GAPDH aggregation in cell and rat models of oxidative stress. The present article demonstrates a few of additional properties of the chemicals shown to block GAPDH aggregation such as calculated site for targeting the enzyme, effects on GAPDH glycolytic activity, influence on GAPDH intracellular level and anti-aggregate activity of pure polyglutamine exemplifying a denatured protein. PMID:27054152

  3. Phenolic Extract from Moringa oleifera Leaves Inhibits Key Enzymes Linked to Erectile Dysfunction and Oxidative Stress in Rats' Penile Tissues.

    PubMed

    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 Fe(2+)-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 Fe(2+)-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 Fe(2+), 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 Fe(2+)-induced MDA production, and radical (OH (∗) , NO (∗) ) scavenging and Fe(2+)-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

  4. Tailoring Enzyme-Like Activities of Gold Nanoclusters by Polymeric Tertiary Amines for Protecting Neurons Against Oxidative Stress.

    PubMed

    Liu, Ching-Ping; Wu, Te-Haw; Lin, Yu-Lung; Liu, Chia-Yeh; Wang, Sabrina; Lin, Shu-Yi

    2016-08-01

    The cytotoxicity of nanozymes has drawn much attention recently because their peroxidase-like activity can decompose hydrogen peroxide (H2 O2 ) to produce highly toxic hydroxyl radicals (•OH) under acidic conditions. Although catalytic activities of nanozymes are highly associated with their surface properties, little is known about the mechanism underlying the surface coating-mediated enzyme-like activities. Herein, it is reported for the first time that amine-terminated PAMAM dendrimer-entrapped gold nanoclusters (AuNCs-NH2 ) unexpectedly lose their peroxidase-like activity while still retaining their catalase-like activity in physiological conditions. Surprisingly, the methylated form of AuNCs-NH2 (i.e., MAuNCs-N(+) R3 , where R = H or CH3 ) results in a dramatic recovery of the intrinsic peroxidase-like activity while blocking most primary and tertiary amines (1°- and 3°-amines) of dendrimers to form quaternary ammonium ions (4°-amines). However, the hidden peroxidase-like activity is also found in hydroxyl-terminated dendrimer-encapsulated AuNCs (AuNCs-OH, inside backbone with 3°-amines), indicating that 3°-amines are dominant in mediating the peroxidase-like activity. The possible mechanism is further confirmed that the enrichment of polymeric 3°-amines on the surface of dendrimer-encapsulated AuNCs provides sufficient suppression of the critical mediator •OH for the peroxidase-like activity. Finally, it is demonstrated that AuNCs-NH2 with diminished cytotoxicity have great potential for use in primary neuronal protection against oxidative damage. PMID:27346719

  5. Characterization of the chitinolytic machinery of Enterococcus faecalis V583 and high-resolution structure of its oxidative CBM33 enzyme.

    PubMed

    Vaaje-Kolstad, Gustav; Bøhle, Liv Anette; Gåseidnes, Sigrid; Dalhus, Bjørn; Bjørås, Magnar; Mathiesen, Geir; Eijsink, Vincent G H

    2012-02-17

    Little information exists for the ability of enterococci to utilize chitin as a carbon source. We show that Enterococcus faecalis V583 can grow on chitin, and we describe two proteins, a family 18 chitinase (ef0361; EfChi18A) and a family 33 CBM (carbohydrate binding module) (ef0362; EfCBM33A) that catalyze chitin conversion in vitro. Various types of enzyme activity assays showed that EfChi18A has functional properties characteristic of an endochitinase. EfCBM33A belongs to a recently discovered family of enzymes that cleave glycosidic bonds via an oxidative mechanism and that act synergistically with classical hydrolytic enzymes (i.e., chitinases). The structure and function of this protein were probed in detail. An ultra-high-resolution crystal structure of EfCBM33A revealed details of a conserved binding surface that is optimized to interact with chitin and contains the catalytic center. Chromatography and mass spectrometry analyses of product formation showed that EfCBM33A cleaves chitin via the oxidative mechanism previously described for CBP21 from Serratia marcescens. Metal-depletion studies showed that EfCBM33A is a copper enzyme. In the presence of an external electron donor, EfCBM33A boosted the activity of EfChi18A, and combining the two enzymes led to rapid and complete conversion of β-chitin to chitobiose. This study provides insight into the structure and function of the CBM33 family of enzymes, which, together with their fungal counterpart called GH61, currently receive considerable attention in the biomass processing field. PMID:22210154

  6. DNA-mediated supercharged fluorescent protein/graphene oxide interaction for label-free fluorescence assay of base excision repair enzyme activity.

    PubMed

    Wang, Zhen; Li, Yong; Li, Lijun; Li, Daiqi; Huang, Yan; Nie, Zhou; Yao, Shouzhuo

    2015-09-01

    The interaction between supercharged green fluorescent protein (ScGFP) and graphene oxide (GO) as well as the resulting quenching effect of GO on ScGFP were investigated. Based on this unique quenching effect and the DNA-mediated ScGFP/GO interaction, a label-free fluorescence method has been established for homogeneously assaying the activity and inhibition of base excision repair enzyme. PMID:26208330

  7. The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose.

    PubMed

    Westereng, Bjørge; Ishida, Takuya; Vaaje-Kolstad, Gustav; Wu, Miao; Eijsink, Vincent G H; Igarashi, Kiyohiko; Samejima, Masahiro; Ståhlberg, Jerry; Horn, Svein J; Sandgren, Mats

    2011-01-01

    Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency. PMID:22132148

  8. Investigating the effect of gallium curcumin and gallium diacetylcurcumin complexes on the structure, function and oxidative stability of the peroxidase enzyme and their anticancer and antibacterial activities.

    PubMed

    Jahangoshaei, Parisa; Hassani, Leila; Mohammadi, Fakhrossadat; Hamidi, Akram; Mohammadi, Khosro

    2015-10-01

    Curcumin has a wide spectrum of biological and pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, antimicrobial and anticancer activities. Complexation of curcumin with metals has gained attention in recent years for improvement of its stability. In this study, the effect of gallium curcumin and gallium diacetylcurcumin on the structure, function and oxidative stability of horseradish peroxidase (HRP) enzyme were evaluated by spectroscopic techniques. In addition to the enzymatic investigation, the cytotoxic effect of the complexes was assessed on bladder, MCF-7 breast cancer and LNCaP prostate carcinoma cell lines by MTT assay. Furthermore, antibacterial activity of the complexes against S. aureus and E. coli was explored by dilution test method. The results showed that the complexes improve activity of HRP and also increase its tolerance against the oxidative condition. After addition of the complexes, affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism, intrinsic and synchronous fluorescence spectra showed that the enzyme structure around the catalytic heme group becomes less compact and also the distance between the heme group and tryptophan residues increases due to binding of the complexes to HRP. On the whole, it can be concluded that the change in the enzyme structure upon binding to the gallium curcumin and gallium diacetylcurcumin complexes results in an increase in the antioxidant efficiency and activity of the peroxidise enzyme. The result of anticancer and antibacterial activities suggested that the complexes exhibit the potential for cancer treatment, but they have no significant antibacterial activity. PMID:26369539

  9. A structural basis for the regulation of an H-NOX-associated cyclic-di-GMP synthase/phosphodiesterase enzyme by nitric oxide-bound H-NOX.

    PubMed

    Lahiri, Tanaya; Luan, Bowu; Raleigh, Daniel P; Boon, Elizabeth M

    2014-04-01

    Biofilms are surface-attached communities of bacteria enclosed in a polysaccharide matrix. Bacteria in a biofilm are extremely resistant to antibiotics. Several recent reports have linked the signaling molecule nitric oxide (NO) with biofilm dispersal. We have previously reported that an H-NOX (heme-nitric oxide/oxygen binding) protein in the biofilm-dwelling bacterium Shewanella woodyi mediates NO-induced biofilm dispersal. In S. woodyi, H-NOX (SwH-NOX) is cocistronic with a gene encoding a dual-functioning diguanylate cyclase/phosphodiesterase enzyme, designated here as HaCE (H-NOX-associated cyclic-di-GMP processing enzyme). Enzymes such as these are responsible for regulating the intracellular concentrations of cyclic-di-GMP, a secondary signaling molecule essential to biofilm formation in bacteria. We have demonstrated that NO-bound SwH-NOX regulates both enzymatic activities of SwHaCE, resulting in decreased cellular cyclic-di-GMP levels and disruption of biofilm formation. Thus, H-NOX/HaCE represents a potential drug target for regulating biofilm formation. In this work, the SwH-NOX surface residues critical for the formation of a protein complex with SwHaCE are identified using nuclear magnetic resonance, fluorescence quenching, and cosedimentation. Enzyme assays confirm this protein-protein interface and its importance for H-NOX/HaCE function. PMID:24628400

  10. A Structural Basis for the Regulation of an H-NOX-Associated Cyclic-di-GMP Synthase/Phosphodiesterase Enzyme by Nitric Oxide-Bound H-NOX

    PubMed Central

    2015-01-01

    Biofilms are surface-attached communities of bacteria enclosed in a polysaccharide matrix. Bacteria in a biofilm are extremely resistant to antibiotics. Several recent reports have linked the signaling molecule nitric oxide (NO) with biofilm dispersal. We have previously reported that an H-NOX (heme-nitric oxide/oxygen binding) protein in the biofilm-dwelling bacterium Shewanella woodyi mediates NO-induced biofilm dispersal. In S. woodyi, H-NOX (SwH-NOX) is cocistronic with a gene encoding a dual-functioning diguanylate cyclase/phosphodiesterase enzyme, designated here as HaCE (H-NOX-associated cyclic-di-GMP processing enzyme). Enzymes such as these are responsible for regulating the intracellular concentrations of cyclic-di-GMP, a secondary signaling molecule essential to biofilm formation in bacteria. We have demonstrated that NO-bound SwH-NOX regulates both enzymatic activities of SwHaCE, resulting in decreased cellular cyclic-di-GMP levels and disruption of biofilm formation. Thus, H-NOX/HaCE represents a potential drug target for regulating biofilm formation. In this work, the SwH-NOX surface residues critical for the formation of a protein complex with SwHaCE are identified using nuclear magnetic resonance, fluorescence quenching, and cosedimentation. Enzyme assays confirm this protein–protein interface and its importance for H-NOX/HaCE function. PMID:24628400

  11. The Putative Endoglucanase PcGH61D from Phanerochaete chrysosporium Is a Metal-Dependent Oxidative Enzyme that Cleaves Cellulose

    PubMed Central

    Westereng, Bjørge; Ishida, Takuya; Vaaje-Kolstad, Gustav; Wu, Miao; Eijsink, Vincent G. H.; Igarashi, Kiyohiko; Samejima, Masahiro; Ståhlberg, Jerry; Horn, Svein J.; Sandgren, Mats

    2011-01-01

    Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency. PMID:22132148

  12. Amelioration Effect of Zinc and Iron Supplementation on Selected Oxidative Stress Enzymes in Liver and Kidney of Cadmium-Treated Male Albino Rat

    PubMed Central

    Jamakala, Obaiah; Rani, Usha A.

    2015-01-01

    Cadmium (Cd) is a highly toxic, nonessential heavy metal with many industrial uses that can contribute to a well-defined spectrum of diseases in animals as well as in humans. The present study examines the effect of zinc (Zn) and iron (Fe) supplementation on oxidative stress enzymes in Cd-treated rats. Wistar strain male albino rats were treated with cadmium chloride (CdCl2) at a dose of 1/10th LD50/48 h, that is, 22.5 mg/kg body weight for 7, 15, and 30 days (d) time intervals. The 15d Cd-treated rats were divided into three groups. The first group received Zn (12 mg/kg), second group Fe (40 mg/kg) alone, and third group supplemented with both Zn and Fe and observed for 7, 15, and 30d. After the specific time intervals, rats were decapitated and oxidative stress enzymes like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were assayed in liver and kidney. Simultaneously lipid peroxidation (LPO) levels were also measured. A significant elevation in LPO levels with decreased activity levels of CAT, SOD, GPx, and GST were observed during Cd intoxication. With Zn and/or Fe supplementation, a significant reversal in the oxidative stress enzymes was observed. Our study reveals that combination of Zn and Fe supplementation is effective in detoxifying the Cd body burden from the test tissues. PMID:26862254

  13. Amelioration Effect of Zinc and Iron Supplementation on Selected Oxidative Stress Enzymes in Liver and Kidney of Cadmium-Treated Male Albino Rat.

    PubMed

    Jamakala, Obaiah; Rani, Usha A

    2015-01-01

    Cadmium (Cd) is a highly toxic, nonessential heavy metal with many industrial uses that can contribute to a well-defined spectrum of diseases in animals as well as in humans. The present study examines the effect of zinc (Zn) and iron (Fe) supplementation on oxidative stress enzymes in Cd-treated rats. Wistar strain male albino rats were treated with cadmium chloride (CdCl2) at a dose of 1/10(th) LD50/48 h, that is, 22.5 mg/kg body weight for 7, 15, and 30 days (d) time intervals. The 15d Cd-treated rats were divided into three groups. The first group received Zn (12 mg/kg), second group Fe (40 mg/kg) alone, and third group supplemented with both Zn and Fe and observed for 7, 15, and 30d. After the specific time intervals, rats were decapitated and oxidative stress enzymes like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were assayed in liver and kidney. Simultaneously lipid peroxidation (LPO) levels were also measured. A significant elevation in LPO levels with decreased activity levels of CAT, SOD, GPx, and GST were observed during Cd intoxication. With Zn and/or Fe supplementation, a significant reversal in the oxidative stress enzymes was observed. Our study reveals that combination of Zn and Fe supplementation is effective in detoxifying the Cd body burden from the test tissues. PMID:26862254

  14. Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme*

    PubMed Central

    Coelho, Fernando R.; Iqbal, Asif; Linares, Edlaine; Silva, Daniel F.; Lima, Filipe S.; Cuccovia, Iolanda M.; Augusto, Ohara

    2014-01-01

    The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp32 residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp32 residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1WT and hSOD1G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp32 residue in the process. The results showed that Trp32 residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp32 residue (bovine SOD1 and hSOD1W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp32 residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1. PMID:25237191

  15. Coordination modes of tyrosinate-ligated catalase-type heme enzymes: magnetic circular dichroism studies of Plexaura homomalla allene oxide synthase, Mycobacterium avium ssp. paratuberculosis protein-2744c, and bovine liver catalase in their ferric and ferrous states.

    PubMed

    Bandara, D M Indika; Sono, Masanori; Bruce, Grant S; Brash, Alan R; Dawson, John H

    2011-12-01

    Bovine liver catalase (BLC), catalase-related allene oxide synthase (cAOS) from Plexaura homomalla, and a recently isolated protein from the cattle pathogen Mycobacterium avium ssp. paratuberculosis (MAP-2744c (MAP)) are all tyrosinate-ligated heme enzymes whose crystal structures have been reported. cAOS and MAP have low (<20%) sequence similarity to, and significantly different catalytic functions from, BLC. cAOS transforms 8R-hydroperoxy-eicosatetraenoic acid to an allene epoxide, whereas the MAP protein is a putative organic peroxide-dependent peroxidase. To elucidate factors influencing the functions of these and related heme proteins, we have investigated the heme iron coordination properties of these tyrosinate-ligated heme enzymes in their ferric and ferrous states using magnetic circular dichroism and UV-visible absorption spectroscopy. The MAP protein shows remarkable spectral similarities to cAOS and BLC in its native Fe(III) state, but clear differences from ferric proximal heme ligand His93Tyr Mb (myoglobin) mutant, which may be attributed to the presence of an Arg(+)-N(ω)-H···¯O-Tyr (proximal heme axial ligand) hydrogen bond in the first three heme proteins. Furthermore, the spectra of Fe(III)-CN¯, Fe(III)-NO, Fe(II)-NO (except for five-coordinate MAP), Fe(II)-CO, and Fe(II)-O(2) states of cAOS and MAP, but not H93Y Mb, are also similar to the corresponding six-coordinate complexes of BLC, suggesting that a tyrosinate (Tyr-O¯) is the heme axial ligand trans to the bound ligands in these complexes. The Arg(+)-N(ω)-H to ¯O-Tyr hydrogen bond would be expected to modulate the donor properties of the proximal tyrosinate oxyanion and, combined with the subtle differences in the catalytic site structures, affect the activities of cAOS, MAP and BLC. PMID:22104301

  16. Effect of the French Oak Wood Extract Robuvit on Markers of Oxidative Stress and Activity of Antioxidant Enzymes in Healthy Volunteers: A Pilot Study

    PubMed Central

    Orszaghova, Zuzana; Laubertova, Lucia; Sabaka, Peter; Rohdewald, Peter; Durackova, Zdenka; Muchova, Jana

    2014-01-01

    We examined in vitro antioxidant capacity of polyphenolic extract obtained from the wood of oak Quercus robur (QR), Robuvit, using TEAC (Trolox equivalent antioxidant capacity) method and the effect of its intake on markers of oxidative stress, activity of antioxidant enzymes, and total antioxidant capacity in plasma of 20 healthy volunteers. Markers of oxidative damage to proteins, DNA, and lipids and activities of Cu/Zn-superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined in the erythrocytes. We have found an in vitro antioxidant capacity of Robuvit of 6.37 micromole Trolox equivalent/mg of Robuvit. One month intake of Robuvit in daily dose of 300 mg has significantly decreased the serum level of advanced oxidation protein products (AOPP) and lipid peroxides (LP). Significantly increased activities of SOD and CAT as well as total antioxidant capacity of plasma after one month intake of Robuvit have been shown. In conclusion, we have demonstrated for the first time that the intake of Robuvit is associated with decrease of markers of oxidative stress and increase of activity of antioxidant enzymes and total antioxidant capacity of plasma in vivo. PMID:25254080

  17. Association between maternal micronutrient status, oxidative stress, and common genetic variants in antioxidant enzymes at 15 weeks׳ gestation in nulliparous women who subsequently develop preeclampsia

    PubMed Central

    Mistry, Hiten D.; Gill, Carolyn A.; Kurlak, Lesia O.; Seed, Paul T.; Hesketh, John E.; Méplan, Catherine; Schomburg, Lutz; Chappell, Lucy C.; Morgan, Linda; Poston, Lucilla

    2015-01-01

    Preeclampsia is a pregnancy-specific condition affecting 2–7% of women and a leading cause of perinatal and maternal morbidity and mortality. Deficiencies of specific micronutrient antioxidant activities associated with copper, selenium, zinc, and manganese have previously been linked to preeclampsia at the time of disease. Our aims were to investigate whether maternal plasma micronutrient concentrations and related antioxidant enzyme activities are altered before preeclampsia onset and to examine the dependence on genetic variations in these antioxidant enzymes. Predisease plasma samples (15±1 weeks׳ gestation) were obtained from women enrolled in the international Screening for Pregnancy Endpoints (SCOPE) study who subsequently developed preeclampsia (n=244) and from age- and BMI-matched normotensive controls (n=472). Micronutrient concentrations were measured by inductively coupled plasma mass spectrometry; associated antioxidant enzyme activities, selenoprotein-P, ceruloplasmin concentration and activity, antioxidant capacity, and markers of oxidative stress were measured by colorimetric assays. Sixty-four tag–single-nucleotide polymorphisms (SNPs) within genes encoding the antioxidant enzymes and selenoprotein-P were genotyped using allele-specific competitive PCR. Plasma copper and ceruloplasmin concentrations were modestly but significantly elevated in women who subsequently developed preeclampsia (both P<0.001) compared to controls (median (IQR), copper, 1957.4 (1787, 2177.5) vs 1850.0 (1663.5, 2051.5) µg/L; ceruloplasmin, 2.5 (1.4, 3.2) vs 2.2 (1.2, 3.0) µg/ml). There were no differences in other micronutrients or enzymes between groups. No relationship was observed between genotype for SNPs and antioxidant enzyme activity. This analysis of a prospective cohort study reports maternal micronutrient concentrations in combination with associated antioxidant enzymes and SNPs in their encoding genes in women at 15 weeks׳ gestation that subsequently

  18. Rational enzyme redesign

    SciTech Connect

    Ornstein, R.L.

    1994-05-01

    Protein engineering is first a means of elucidating structure-function relations in an enzyme, and second, a means of changing a protein to make it serve a different, but generally related, purpose. In principle, one may change the functional characteristics of an enzyme by altering its substrate specificity, kinetics, optimum range of activity, and chemical mechanism. Obviously one cannot make all possible combinations of amino acid changes for even the smallest enzyme, so the essential question is which changes to make. The intent of rational protein/enzyme redesign is to alter a protein/enzyme in a timely and premeditated fashion. This article provides an outline of the process of rational enzyme redesign.

  19. Applications of B3LYP Method for Enzyme Reactions: O2 Reduction by Cytochrome c Oxidase and Ubiquinol Oxidation by Cytochrome bc1 Complex

    NASA Astrophysics Data System (ADS)

    Yoshioka, Yasunori

    2007-11-01

    Mechanisms of dioxygen reduction by fully reduced cytochrome c oxidase and ubiquinol oxidation by cytochrome bc1 complex were examined by B3LYP method with the broken-symmetry. In the dioxygen reduction, it was found that protons to form FeOOH and FeOOH2 are provided through the K-channel, and at the same time an electron transfer occurs to FeOO from the reduced Cu(I) through the prophyrin ring. The mechanism is proposed for first H2O formation. In the ubiquinol oxidation, it was found that the ubiquinol can be docked in both His181 of Rieske iron-sulfur protein and Glu272 of cytochrome b protein. Two protons and one electron transfer simultaneously from ubiquinol to His181 and Glu272 to yield one-electron reduced [2Fe-2S] cluster from the oxidized state. The concerted proton and electron transfers were found in both enzymes.

  20. Strictosidine-related enzymes involved in the alkaloid biosynthesis of Uncaria tomentosa root cultures grown under oxidative stress.

    PubMed

    Vera-Reyes, Ileana; Huerta-Heredia, Ariana A; Ponce-Noyola, Teresa; Flores-Sanchez, Isvett Josefina; Esparza-García, Fernando; Cerda-García-Rojas, Carlos M; Trejo-Tapia, Gabriela; Ramos-Valdivia, Ana C

    2013-01-01

    The activity and gene expression of strictosidine-related enzymes in Uncaria tomentosa root cultures exposed to oxidative stress were studied. Elicitation with 0.2 mM hydrogen peroxide (H2 O2 ) or a combination of 0.8 mM buthionine sulfoximine and 0.2 mM jasmonic acid (BSO-JA) increased peroxidase activities by twofold at Day 8 and glutathione reductase by 1.4-fold at Day 5 in H2 O2 elicited cultures respect to the control. Production of monoterpenoid oxindole alkaloids (MOA), 3α-dihydrocadambine, and dolichantoside was stimulated after H2 O2 elicitation, reaching levels of 886.4 ± 23.6, 847.7 ± 25.4, and 87.5 ± 7.2 µg/g DW, at Day 8 which were 1.7-, 2.1-, and 2.3-fold higher relative to control. BSO-JA elicited cultures produced about twice alkaloids than H2 O2 -treated cultures, following a biphasic pattern with maxima at 0.5 and 8 days. Alkaloid production was preceded by increase in strictosidine synthase (STR) and strictosidine glucosidase (SGD) activities. After elicitation with H2 O2 or BSO-JA, the STR activity (pKat/mg protein) increased by 1.9-fold (93.8 ± 17.8 at 24 h) or 2.5-fold (102.4 ± 2.2 at 6 h) and the SGD activity (pKat/mg protein) by 2.8-fold (245.2 ± 14.4 at 6 h) or 4.2-fold (421.2 ± 1.8 at 18 h) relative to control. STR and SGD transcripts were upregulated after elicitation. H2 O2 -treated roots showed higher levels of STR at 48-192 h and SGD at 24-48 h, while BSO-JA treatments showed STR increased at 12 h and SGD at 24 h. Also, LC/ESI-MS confirmed the biosynthesis of dolichantoside from N-ω-methyltryptamine and secologanin by U. tomentosa protein extracts. PMID:23606578

  1. Epigallocatechin-3-gallate modulates anti-oxidant defense enzyme expression in murine submandibular and pancreatic exocrine gland cells and human HSG cells.

    PubMed

    Dickinson, Douglas; DeRossi, Scott; Yu, Hongfang; Thomas, Cristina; Kragor, Chris; Paquin, Becky; Hahn, Emily; Ohno, Seiji; Yamamoto, Tetsuya; Hsu, Stephen

    2014-05-01

    Sjogren's syndrome (SS) and type-1 diabetes are prevalent autoimmune diseases in the USA. We reported previously that epigallocatechin-3-gallate (EGCG) prevented and delayed the onset of autoimmune disease in non-obese diabetic (NOD) mice, a model for both SS and type-1 diabetes. EGCG also normalized the levels of proteins related to DNA repair and anti-oxidant activity in NOD.B10.Sn-H2 mice, a model for primary SS, prior to disease onset. The current study examined the effect of EGCG on the expression of anti-oxidant enzymes in the submandibular salivary gland and the pancreas of NOD mice and cultured human salivary gland acinar cells. NOD mice consuming 0.2% EGCG daily dissolved in water showed higher protein levels of peroxiredoxin 6 (PRDX6), a major anti-oxidant defense protein, and catalase, while the untreated NOD mice exhibited significantly lowered levels of PRDX6. Similarly, pancreas samples from water-fed NOD mice were depleted in PRDX6 and superoxide dismutase, while EGCG-fed mice showed high levels of these anti-oxidant enzymes. In cultured HSG cells EGCG increased PRDX6 levels significantly, and this was inhibited by p38 and JNK inhibitors, suggesting that the EGCG-mediated increase in protective anti-oxidant capacity is regulated in part through mitogen-activated protein kinase pathway signaling. This mechanism may explain the higher levels of PRDX6 found in EGCG-fed NOD mice. These preclinical observations warrant future preclinical and clinical studies to determine whether EGCG or green tea polyphenols could be used in novel preventive and therapeutic approaches against autoimmune diseases and salivary dysfunction involving oxidative stress. PMID:24444391

  2. Bioavailability of catechins from guaraná (Paullinia cupana) and its effect on antioxidant enzymes and other oxidative stress markers in healthy human subjects.

    PubMed

    Yonekura, Lina; Martins, Carolina Aguiar; Sampaio, Geni Rodrigues; Monteiro, Marcela Piedade; César, Luiz Antônio Machado; Mioto, Bruno Mahler; Mori, Clara Satsuki; Mendes, Thaíse Maria Nogueira; Ribeiro, Marcelo Lima; Arçari, Demetrius Paiva; Torres, Elizabeth Aparecida Ferraz da Silva

    2016-07-13

    We assessed the effects of guaraná (Paullinia cupana) consumption on plasma catechins, erythrocyte antioxidant enzyme activity (superoxide dismutase, catalase and glutathione peroxidase) and biomarkers of oxidative stress (ex vivo LDL oxidation, plasma total antioxidant status and ORAC, and lymphocyte single cell gel electrophoresis) in healthy overweight subjects. Twelve participants completed a 15-day run-in period followed by a 15-day intervention with a daily intake of 3 g guaraná seed powder containing 90 mg (+)-catechin and 60 mg (-)-epicatechin. Blood samples were taken on the first and last day of the intervention period, fasting and 1 h post-dose. The administration of guaraná increased plasma ORAC, while reducing ex vivo LDL oxidation (only in the first study day) and hydrogen peroxide-induced DNA damage in lymphocytes, at 1 h post-dose. Plasma catechin (0.38 ± 0.12 and 0.44 ± 0.18 nmol mL(-1)), epicatechin (0.59 ± 0.18 and 0.64 ± 0.25 nmol mL(-1)) and their methylated metabolites were observed at 1 h post-dose but were almost negligible after overnight fasting. The activities of catalase (in both study days) and glutathione peroxidase (in the last intervention day) increased at 1 h post-dose. Furthermore, the activity of both enzymes remained higher than the basal levels in overnight-fasting individuals on the last intervention day, suggesting a prolonged effect of guaraná that continues even after plasma catechin clearance. In conclusion, guaraná catechins are bioavailable and contribute to reduce the oxidative stress of clinically healthy individuals, by direct antioxidant action of the absorbed phytochemicals and up-regulation of antioxidant/detoxifying enzymes. PMID:27302304

  3. 4-Hydroxy-2-nonenal upregulates endogenous antioxidants and phase 2 enzymes in rat H9c2 myocardiac cells: protection against overt oxidative and electrophilic injury.

    PubMed

    Zhu, Hong; Zhang, Li; Xi, Xiaoqing; Zweier, Jay L; Li, Yunbo

    2006-08-01

    This study was undertaken to determine if 4-hydroxy-2-nonenal (HNE) could upregulate antioxidants and phase 2 enzymes in rat H9c2 myocardiac cells, and if the upregulated defenses led to cytoprotection against oxidative and electrophilic injury. Incubation of H9c2 cells with HNE at noncytotoxic concentrations resulted in significant induction of cellular catalase, glutathione (GSH), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase 1 (NQO1), as determined by enzyme activity and/or protein expression. HNE treatment caused increased mRNA expression of catalase, gamma-glutamylcysteine ligase, GST-A1, and NQO1. Pretreatment of H9c2 cells with HNE led to significant protection against cytotoxicity induced by reactive oxygen and nitrogen species. HNE-pretreated cells also exhibited increased resistance to injury elicited by subsequent cytotoxic concentrations of HNE. Taken together, this study demonstrates that several antioxidants and phase 2 enzymes in H9c2 cells are upregulated by HNE and that the increased defenses afford protection against overt oxidative and electrophilic cardiac cell injury. PMID:17015266

  4. Chemoprevention of rat mammary carcinogenesis by black tea polyphenols: modulation of xenobiotic-metabolizing enzymes, oxidative stress, cell proliferation, apoptosis, and angiogenesis.

    PubMed

    Kumaraguruparan, R; Seshagiri, P B; Hara, Y; Nagini, S

    2007-09-01

    Chemoprevention of dietary constituents has emerged as a cost-effective approach to control the incidence of breast cancer. The present study was therefore designed to evaluate the chemopreventive efficacy of black tea polyphenols (Polyphenon-B) during the preinitiation phase of 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary carcinogenesis using xenobiotic-metabolizing enzymes, cellular redox status, cell proliferation, apoptosis, and angiogenesis as biomarkers of chemoprevention. Intragastric administration of DMBA induced adenocarcinomas that showed enhanced activities of phase I carcinogen activation and phase II detoxification enzymes with increased lipid and protein oxidation and decrease in antioxidant status. This was associated with increased cell proliferation, angiogenesis, and evasion of apoptosis as revealed by upregulation of proliferating cell nuclear antigen (PCNA), Bcl-2, and vascular endothelial growth factor (VEGF), and downregulation of Bax, caspase 3, and poly(ADP-ribose) polymerase (PARP). Dietary administration of Polyphenon-B effectively suppressed the incidence of mammary tumors as evidenced by modulation of xenobiotic-metabolizing enzymes and oxidant-antioxidant status, inhibition of cell proliferation and angiogenesis, and induction of apoptosis. The present study provides evidence that Polyphenon-B exerts multifunctional inhibitory effects on DMBA-induced mammary carcinogenesis and suggests that it can be developed as a potential chemopreventive agent. PMID:17415784

  5. The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa)

    PubMed Central

    Gómez-Manzo, Saúl; Escamilla, José E.; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M. H.; Sosa-Torres, Martha Elena

    2015-01-01

    Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2–C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde. PMID:25574602

  6. The oxidative fermentation of ethanol in Gluconacetobacter diazotrophicus is a two-step pathway catalyzed by a single enzyme: alcohol-aldehyde Dehydrogenase (ADHa).

    PubMed

    Gómez-Manzo, Saúl; Escamilla, José E; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M H; Sosa-Torres, Martha Elena

    2015-01-01

    Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde. PMID:25574602

  7. Detoxifying Enzymes at the Cross-Roads of Inflammation, Oxidative Stress, and Drug Hypersensitivity: Role of Glutathione Transferase P1-1 and Aldose Reductase

    PubMed Central

    Sánchez-Gómez, Francisco J.; Díez-Dacal, Beatriz; García-Martín, Elena; Agúndez, José A. G.; Pajares, María A.; Pérez-Sala, Dolores

    2016-01-01

    Phase I and II enzymes are involved in the metabolism of endogenous reactive compounds as well as xenobiotics, including toxicants and drugs. Genotyping studies have established several drug metabolizing enzymes as markers for risk of drug hypersensitivity. However, other candidates are emerging that are involved in drug metabolism but also in the generation of danger or costimulatory signals. Enzymes such as aldo-keto reductases (AKR) and glutathione transferases (GST) metabolize prostaglandins and reactive aldehydes with proinflammatory activity, as well as drugs and/or their reactive metabolites. In addition, their metabolic activity can have important consequences for the cellular redox status, and impacts the inflammatory response as well as the balance of inflammatory mediators, which can modulate epigenetic factors and cooperate or interfere with drug-adduct formation. These enzymes are, in turn, targets for covalent modification and regulation by oxidative stress, inflammatory mediators, and drugs. Therefore, they constitute a platform for a complex set of interactions involving drug metabolism, protein haptenation, modulation of the inflammatory response, and/or generation of danger signals with implications in drug hypersensitivity reactions. Moreover, increasing evidence supports their involvement in allergic processes. Here, we will focus on GSTP1-1 and aldose reductase (AKR1B1) and provide a perspective for their involvement in drug hypersensitivity. PMID:27540362

  8. Detoxifying Enzymes at the Cross-Roads of Inflammation, Oxidative Stress, and Drug Hypersensitivity: Role of Glutathione Transferase P1-1 and Aldose Reductase.

    PubMed

    Sánchez-Gómez, Francisco J; Díez-Dacal, Beatriz; García-Martín, Elena; Agúndez, José A G; Pajares, María A; Pérez-Sala, Dolores

    2016-01-01

    Phase I and II enzymes are involved in the metabolism of endogenous reactive compounds as well as xenobiotics, including toxicants and drugs. Genotyping studies have established several drug metabolizing enzymes as markers for risk of drug hypersensitivity. However, other candidates are emerging that are involved in drug metabolism but also in the generation of danger or costimulatory signals. Enzymes such as aldo-keto reductases (AKR) and glutathione transferases (GST) metabolize prostaglandins and reactive aldehydes with proinflammatory activity, as well as drugs and/or their reactive metabolites. In addition, their metabolic activity can have important consequences for the cellular redox status, and impacts the inflammatory response as well as the balance of inflammatory mediators, which can modulate epigenetic factors and cooperate or interfere with drug-adduct formation. These enzymes are, in turn, targets for covalent modification and regulation by oxidative stress, inflammatory mediators, and drugs. Therefore, they constitute a platform for a complex set of interactions involving drug metabolism, protein haptenation, modulation of the inflammatory response, and/or generation of danger signals with implications in drug hypersensitivity reactions. Moreover, increasing evidence supports their involvement in allergic processes. Here, we will focus on GSTP1-1 and aldose reductase (AKR1B1) and provide a perspective for their involvement in drug hypersensitivity. PMID:27540362

  9. Responses of soil hydrolytic enzymes, ammonia-oxidizing bacteria and archaea to nitrogen applications in a temperate grassland in Inner Mongolia.

    PubMed

    Zhang, Xinyu; Tang, Yuqian; Shi, Yao; He, Nianpeng; Wen, Xuefa; Yu, Qiang; Zheng, Chunyu; Sun, Xiaomin; Qiu, Weiwen

    2016-01-01

    We used a seven-year urea gradient applied field experiment to investigate the effects of nitrogen (N) applications on soil N hydrolytic enzyme activity and ammonia-oxidizing microbial abundance in a typical steppe ecosystem in Inner Mongolia. The results showed that N additions inhibited the soil N-related hydrolytic enzyme activities, especially in 392 kg N ha(-1 )yr(-1) treatment. As N additions increased, the amoA gene copy ratios of ammonia-oxidizing archaea (AOA) to ammonia-oxidizing bacteria (AOB) decreased from 1.13 to 0.65. Pearson correlation analysis showed that the AOA gene copies were negatively related with NH4(+)-N content. However, the AOB gene copies were positively correlated with NO3(-)-N content. Moderate N application rates (56-224 kg N ha(-1 )yr(-1)) accompanied by P additions are beneficial to maintaining the abundance of AOB, as opposed to the inhibition of highest N application rate (392 kg N ha(-1 )yr(-1)) on the abundance of AOB. This study suggests that the abundance of AOB and AOA would not decrease unless N applications exceed 224 kg N ha(-1 )yr(-1) in temperate grasslands in Inner Mongolia. PMID:27596731

  10. Responses of soil hydrolytic enzymes, ammonia-oxidizing bacteria and archaea to nitrogen applications in a temperate grassland in Inner Mongolia

    PubMed Central

    Zhang, Xinyu; Tang, Yuqian; Shi, Yao; He, Nianpeng; Wen, Xuefa; Yu, Qiang; Zheng, Chunyu; Sun, Xiaomin; Qiu, Weiwen

    2016-01-01

    We used a seven-year urea gradient applied field experiment to investigate the effects of nitrogen (N) applications on soil N hydrolytic enzyme activity and ammonia-oxidizing microbial abundance in a typical steppe ecosystem in Inner Mongolia. The results showed that N additions inhibited the soil N-related hydrolytic enzyme activities, especially in 392 kg N ha−1 yr−1 treatment. As N additions increased, the amoA gene copy ratios of ammonia-oxidizing archaea (AOA) to ammonia-oxidizing bacteria (AOB) decreased from 1.13 to 0.65. Pearson correlation analysis showed that the AOA gene copies were negatively related with NH4+-N content. However, the AOB gene copies were positively correlated with NO3−-N content. Moderate N application rates (56–224 kg N ha−1 yr−1) accompanied by P additions are beneficial to maintaining the abundance of AOB, as opposed to the inhibition of highest N application rate (392 kg N ha−1 yr−1) on the abundance of AOB. This study suggests that the abundance of AOB and AOA would not decrease unless N applications exceed 224 kg N ha−1 yr−1 in temperate grasslands in Inner Mongolia. PMID:27596731

  11. Nitro-Oleic Acid Reduces J774A.1 Macrophage Oxidative Status and Triglyceride Mass: Involvement of Paraoxonase2 and Triglyceride Metabolizing Enzymes.

    PubMed

    Rosenblat, Mira; Rom, Oren; Volkova, Nina; Aviram, Michael

    2016-08-01

    Nitro-fatty acids possess anti-atherogenic properties, but their effects on macrophage oxidative status and lipid metabolism that play important roles in atherosclerosis development are unclear. This study compared the effects of nitro-oleic acid (OLA-NO2) with those of native oleic acid (OLA) on intracellular reactive oxygen species (ROS) generation, anti-oxidants and metabolism of triglycerides and cholesterol in J774A.1 macrophages. Upon incubating the cells with physiological concentrations of OLA-NO2 (0-1 µM) or with equivalent levels of OLA, ROS levels measured by 2, 7-dichlorofluorescein diacetate, decreased dose-dependently, but the anti-oxidative effects of OLA-NO2 were significantly augmented. Copper ion addition increased ROS generation in OLA treated macrophages without affecting OLA-NO2 treated cells. These effects could be attributed to elevated glutathione levels and to increased activity and expression of paraoxonase2 that were observed in OLA-NO2 vs OLA treated cells. Beneficial effects on triglyceride metabolism were noted in OLA-NO2 vs OLA treated macrophages in which cellular triglycerides were reduced due to attenuated biosynthesis and accelerated hydrolysis of triglycerides. Accordingly, OLA-NO2 treated cells demonstrated down-regulation of diacylglycerol acyltransferase1, the key enzyme in triglyceride biosynthesis, and increased expression of hormone-sensitive lipase and adipose triglyceride lipase that regulate triglyceride hydrolysis. Finally, OLA-NO2 vs OLA treatment resulted in modest but significant beneficial effects on macrophage cholesterol metabolism, reducing cholesterol biosynthesis rate and low density lipoprotein influx into the cells, while increasing high density lipoprotein-mediated cholesterol efflux from the macrophages. Collectively, compared with OLA, OLA-NO2 modestly but significantly reduces macrophage oxidative status and cellular triglyceride content via modulation of cellular anti-oxidants and triglyceride

  12. Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency

    PubMed Central

    Schiff, Manuel; Haberberger, Birgit; Xia, Chuanwu; Mohsen, Al-Walid; Goetzman, Eric S.; Wang, Yudong; Uppala, Radha; Zhang, Yuxun; Karunanidhi, Anuradha; Prabhu, Dolly; Alharbi, Hana; Prochownik, Edward V.; Haack, Tobias; Häberle, Johannes; Munnich, Arnold; Rötig, Agnes; Taylor, Robert W.; Nicholls, Robert D.; Kim, Jung-Ja; Prokisch, Holger; Vockley, Jerry

    2015-01-01

    Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunctions. PMID:25721401

  13. The Radical S-Adenosyl-l-methionine Enzyme MftC Catalyzes an Oxidative Decarboxylation of the C-Terminus of the MftA Peptide.

    PubMed

    Bruender, Nathan A; Bandarian, Vahe

    2016-05-24

    Ribosomally synthesized post-translationally modified peptides (RiPPs) are encoded in the genomes of a wide variety of microorganisms, in the proximity of open reading frames that encode enzymes that conduct extensive modifications, many of which are novel. Recently, members of the radical S-adenosyl-l-methionine (SAM) superfamily have been identified in these biosynthetic clusters. Herein, we demonstrate the putative radical SAM enzyme, MftC, oxidatively decarboxylates the C-terminus of the MftA peptide in the presence of the accessory protein MftB. The reaction catalyzed by MftC expands the repertoire of peptide-based radical SAM chemistry beyond the intramolecular cross-links. PMID:27158836

  14. Effect of temperature on oxidative stress parameters and enzyme activity in tissues of Cape River crab (Potamanautes perlatus) following exposure to silver nanoparticles (AgNP).

    PubMed

    Walters, Chavon R; Cheng, Paul; Pool, Edmund; Somerset, Vernon

    2016-01-01

    Biomarkers of oxidative stress have been widely used in environmental assessments to evaluate the effects of exposure of aquatic organisms to contaminants from various anthropogenic sources. Silver nanoparticles (AgNP), the most produced NP worldwide and used in several consumer products, are known to produce oxidative stress in aquatic organisms. Similarly, temperature is also known to affect reactive oxygen species (ROS) by influencing the inputs of contaminants into the environment, as well as altering behavior, fate, and transport. Aquatic ecosystems are affected by both anthropogenic releases of contaminants and increased temperature. To test this hypothesis, the influence of AgNP and temperature in the response to multiple biomarkers of oxidative stress was studied in the gills and hepatopancreas of the Cape River crab Potamonautes perlatus. Responses were assessed through activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and the nonenzymatic antioxidant glutathione S-transferase (GST). The response of the oxidative stress biomarkers analyzed was always higher in hepatopancreas than in gills. Elevated temperatures (28°C) induced oxidative stress by increasing SOD, CAT, and GST activities, particularly at 100 µg/ml AgNP. These data indicate that AgNP-mediated toxicity to P. perlatus is modulated by elevated temperatures, but this relationship is not linear. Co-effects of AgNP and temperature are reported for the first time in P. perlatus. PMID:26730549

  15. Cytotoxicity of zinc oxide nanoparticles on antioxidant enzyme activities and mRNA expression in the cocultured C2C12 and 3T3-L1 cells.

    PubMed

    Pandurangan, Muthuraman; Veerappan, Muthuviveganandavel; Kim, Doo Hwan

    2015-02-01

    The present study was aimed to investigate the dose-dependent effect of zinc oxide (ZnO) nanoparticles on antioxidant enzyme activities and messenger RNA (mRNA) expression in the cocultured C2C12 and 3T3-L1 cells. Coculturing experiments are 3D and more reliable compared to mono-culture (2D) experiment. Even though, there are several studies on ZnO nanoparticle-mediated cytotoxicity, but there are no studies on the effect of ZnO nanoparticle on antioxidant enzyme activities and mRNA expression in the cocultured C2C12 and 3T3-L1 cells. A cytotoxicity assay was carried out to determine the effect of ZnO nanoparticles on the C2C12 and 3T3-L1 cell viability. At higher concentration of ZnO nanoparticles, C2C12 and 3T3-L1 cells almost die. ZnO nanoparticles increased reactive oxygen species (ROS) and lipid peroxidation and reduced glutathione (GSH) levels in a dose-dependent manner in the C2C12 and 3T3-L1 cells. In addition, ZnO nanoparticles increased antioxidant enzyme activities and their mRNA expression in the C2C12 and 3T3-L1 cells. In conclusion, the present study showed that ZnO nanoparticles increased oxidative stress, antioxidant enzyme activities, and their mRNA expression in the cocultured C2C12 and 3T3-L1 cells. PMID:25380643

  16. Evaluation of 3-hydroxybutyrate as an enzyme-protective agent against heating and oxidative damage and its potential role in stress response of poly(3-hydroxybutyrate) accumulating cells.

    PubMed

    Obruca, Stanislav; Sedlacek, Petr; Mravec, Filip; Samek, Ota; Marova, Ivana

    2016-02-01

    Poly(3-hydroxybutyrate) (PHB) is a common carbon- and energy-storage compound simultaneously produced and degraded into its monomer 3-hydroxybutyrate (3HB) by numerous bacteria and Archae in a metabolic pathway called the PHB cycle. We investigated 3HB as a chemical chaperone capable of protecting model enzymes, namely lipase and lysozyme, from adverse effects of high temperature and oxidation. Heat-mediated denaturation of lipase in the presence or absence of 3HB was monitored by dynamic light scattering (DLS) revealing a significant protective effect of 3HB which increased as its concentration rose. Furthermore, when compared at the same molar concentration, 3HB showed a greater protective effect than the well-known chemical chaperones trehalose and hydroxyectoine. The higher protective effect of 3HB was also confirmed when employing differential scanning calorimetry (DSC) and lysozyme as a model enzyme. Furthermore, 3HB was capable of protecting lipase not only against thermal-mediated denaturation but also against oxidative damage by Cu(2+) and H2O2; its protection was higher than that of trehalose and comparable to that of hydroxyectoine. Taking into account that the PHB-producing strain Cupriavidus necator H16 reveals a 16.5-fold higher intracellular concentration than the PHB non-producing mutant C. necator PHB(-4), it might be expected that the functional PHB cycle might be responsible for maintaining a higher intracellular level of 3HB which, aside from other positive aspects of functional PHB metabolism, enhances stress resistance of bacterial strains capable of simultaneous PHB synthesis and mobilization. In addition, 3HB can be used in various applications and formulations as an efficient enzyme-stabilizing and enzyme-protecting additive. PMID:26590589

  17. Improving the direct electron transfer in monolithic bioelectrodes prepared by immobilization of FDH enzyme on carbon-coated anodic aluminum oxide films

    NASA Astrophysics Data System (ADS)

    Castro-Muñiz, Alberto; Hoshikawa, Yasuto; Komiyama, Hiroshi; Nakayama, Wataru; Itoh, Tetsuji; Kyotani, Takashi

    2016-02-01

    The present work reports the preparation of binderless carbon-coated porous films and the study of their performance as monolithic bioanodes. The films were prepared by coating anodic aluminum oxide (AAO) films with a thin layer of nitrogen-doped carbon by chemical vapor deposition. The films have cylindrical straight pores with controllable diameter and length. These monolithic films were used directly as bioelectrodes by loading the films with D-fructose dehydrogenase (FDH), an oxidoreductase enzyme that catalyzes the oxidation of D-fructose to 5-keto-D-fructose. The immobilization of the enzymes was carried out by physical adsorption in liquid phase and with an electrostatic attraction method. The latter method takes advantage of the fact that FDH is negatively charged during the catalytic oxidation of fructose. Thus the immobilization was performed under the application of a positive voltage to the CAAO film in a FDH-fructose solution in McIlvaine buffer (pH 5) at 25 ºC. As a result, the FDH modified electrodes with the latter method show much better electrochemical response than that with the conventional physical adsorption method. Due to the singular porous structure of the monolithic films, which consists of an array of straight and parallel nanochannels, it is possible to rule out the effect of the diffusion of the D-fructose into the pores. Thus the improvement in the performance upon using the electrostatic attraction method can be ascribed not only to a higher uptake, but also to a more appropriate molecule orientation of the enzyme units on the surface of the electrodes.

  18. Characterization of enzymes in the oxidation of 1,2-propanediol to D: -(-)-lactic acid by Gluconobacter oxydans DSM 2003.

    PubMed

    Wei, Liujing; Yang, Xuepeng; Gao, Keliang; Lin, Jinping; Yang, Shengli; Hua, Qiang; Wei, Dongzhi

    2010-09-01

    Although Gluconobacter oxydans can convert 1,2-propanediol to D: -(-)-lactic acid, the enzyme(s) responsible for the conversion has remain unknown. In this study, the membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter oxydans DSM 2003 was purified and confirmed to be essential for the process of D: -(-)-lactic acid production by gene knockout and complementation studies. A 25 percent decrease in D: -(-)-lactic acid production was found for the aldehyde dehydrogenase (ALDH) deficient strain of G. oxydans DSM 2003, indicating that this enzyme is involved in the reaction but not necessary. It is the first report that reveals the function of ADH and ALDH in the biooxidation of 1,2-propanediol to D: -(-)-lactic acid by G. oxydans DSM 2003. PMID:20300886

  19. Gallic acid modulates cerebral oxidative stress conditions and activities of enzyme-dependent signaling systems in streptozotocin-treated rats.

    PubMed

    Kade, I J; Rocha, J B T

    2013-04-01

    Redox imbalances and altered signaling processes in the brain are characteristic features of diabetic complications. Hence, the present study therefore sought to evaluate the effect of gallic acid (GA) on disturbed redox systems and activity of neurotransmission signaling dependent enzymes such as sodium pump, purinergic enzymes and acetylcholinesterase in diabetic animal models. We observed that GA markedly improves the antioxidant status of diabetic animals. Furthermore, the diminution of the activity of Na(+)/K(+)-ATPase and increased activities of acetylcholinesterase and the purinergic enzymes associated with diabetes progression were reversed to normalcy with the administration of GA in diabetic animals. Hence, we conclude that GA is a potential candidate in the management of neuronal dysfunction that often accompanied complications associated with diabetic hyperglycemia. PMID:23381106

  20. Tea Catechins Protect Goat Skeletal Muscle against H₂O₂-Induced Oxidative Stress by Modulating Expression of Phase 2 Antioxidant Enzymes.

    PubMed

    Zhong, Rong-Zhen; Fang, Yi; Qin, Gui-Xin; Li, Hao-Yang; Zhou, Dao-Wei

    2015-09-16

    To study the mechanisms of tea catechins (TCs) in goat muscles against oxidative stress, skeletal muscle cells (SMCs) induced by H2O2 or not were incubated with TCs or 3H-1,2-dithiole-3-thione (D3T) and were defined as H2O2, H2O2D3T, H2O2TC, D3T, and TC treatments, respectively. Results showed that, similar to effects of D3T, TCs regulated mRNA and protein expression of antioxidant enzymes by suppressing Keap1 protein expression in SMCs from 1.58 ± 0.12 to 0.71 ± 0.21 and 1.03 ± 0.11 in H2O2TC and TC groups, respectively; however, effects differed in oxidative condition of cells and among enzymes. In stressed cells, TCs increased catalase and glutathione S-transferases (GST) activities (P < 0.001), whereas both enzymes' activities decreased (P < 0.001) to 2.97 ± 0.37 U/mg protein or 42.1 ± 1.85 mU/mg protein, respectively, in unstressed SMCs. Subsequently, an in vivo experiment in goats fed grain supplemented with TCs or D3T following infusion with H2O2 was conducted to further verify mechanisms of TC action. As seen in vitro, TCs reduced Keap1 protein expression (P < 0.001) from 2.11 ± 0.37 to 1.34 ± 0.13 and 1.43 ± 0.23 in H2O2TC and TC groups, respectively, in muscle. However, dietary TCs increased plasma CuZn superoxide dismutase and GST activities (P < 0.001) regardless of oxidative stress. Moreover, feeding TCs to goats under both conditions increased meat color and tenderness (P ≤ 0.001). In conclusion, TCs protected goat muscles against oxidative stress and subsequently improved meat quality by modulating phase 2 antioxidant enzymes and Keap1 expression. PMID:26118494

  1. Green tea extract protects against nonalcoholic steatohepatitis in ob/ob mice by decreasing oxidative and nitrative stress responses induced by proinflammatory enzymes.

    PubMed

    Chung, Min-Yu; Park, Hea Jin; Manautou, Jose E; Koo, Sung I; Bruno, Richard S

    2012-04-01

    Oxidative and nitrative stress responses resulting from inflammation exacerbate liver injury associated with nonalcoholic steatohepatitis (NASH) by inducing lipid peroxidation and protein nitration. The objective of this study was to investigate whether the anti-inflammatory properties of green tea extract (GTE) would protect against NASH by suppressing oxidative and nitrative damage mediated by proinflammatory enzymes. Obese mice (ob/ob) and their 5-week-old C57BL6 lean littermates were fed 0%, 0.5% or 1% GTE for 6 weeks (n=12-13 mice/group). In obese mice, hepatic lipid accumulation, inflammatory infiltrates and serum alanine aminotransferase activity were markedly increased, whereas these markers of hepatic steatosis, inflammation and injury were significantly reduced among obese mice fed GTE. GTE also normalized hepatic 4-hydroxynonenal and 3-nitro-tyrosine (N-Tyr) concentrations to those observed in lean controls. These oxidative and nitrative damage markers were correlated with alanine aminotransferase (P<.05; r=0.410-0.471). Improvements in oxidative and nitrative damage by GTE were also associated with lower hepatic nicotinamide adenine dinucleotide phosphate oxidase activity. Likewise, GTE reduced protein expression levels of hepatic myeloperoxidase and inducible nitric oxide synthase and decreased the concentrations of nitric oxide metabolites. Correlative relationships between nicotinamide adenine dinucleotide phosphate oxidase and hepatic 4-hydroxynonenal (r=0.364) as well as nitric oxide metabolites and N-Tyr (r=0.598) suggest that GTE mitigates lipid peroxidation and protein nitration by suppressing the generation of reactive oxygen and nitrogen species. Further study is warranted to determine whether GTE can be recommended as an effective dietary strategy to reduce the risk of obesity-triggered NASH. PMID:21543212

  2. Effects of cerium oxide supplementation to laying hen diets on performance, egg quality, some antioxidant enzymes in serum and lipid oxidation in egg yolk.

    PubMed

    Bölükbaşı, S C; Al-Sagan, A A; Ürüşan, H; Erhan, M K; Durmuş, O; Kurt, N

    2016-08-01

    This study was conducted to determine the effects of dietary cerium oxide levels (0, 100, 200, 300 or 400 mg/kg) on the laying performance, egg quality, some blood serum parameters and egg lipid peroxidation of laying hen. In total, one hundred and twenty 22-week-old brown Lohman LSL laying hens were randomly assigned to five groups equally (n = 24). Each treatment was replicated six times. Dietary supplementation of cerium oxide had no significant effect on feed intake and egg weight. The addition of cerium oxide to the laying hens' feed improved feed conversion ratio and increased (p < 0.05) egg production. Quality criteria of egg for except shell breaking strength were not affected by supplementing cerium oxide. In particular, supplementation of 200 and 300 mg/kg cerium oxide to the laying hens feed led to a significant (p < 0. 01) increase in egg shell breaking strength. Calcium and phosphorus concentration of serum increased significantly (p < 0.05) with supplementation of 100 mg/kg cerium oxide to laying hen diets. It was also observed that serum superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentration decreased significantly with supplementation of cerium oxide in diets. Inclusion of cerium oxide resulted in a significant reduction in thiobarbituric acid reactive substance (TBARS) values in egg yolk in this study. It can be concluded that the addition of cerium oxide had positive effects on egg production, feed conversion ratio and egg shelf life. Based on the results of this study, it could be advised to supplement laying hens feed with cerium oxide as feed additives. PMID:26847677

  3. Mannitol alleviates chromium toxicity in wheat plants in relation to growth, yield, stimulation of anti-oxidative enzymes, oxidative stress and Cr uptake in sand and soil media.

    PubMed

    Adrees, Muhammad; Ali, Shafaqat; Iqbal, Muhammad; Aslam Bharwana, Saima; Siddiqi, Zeenat; Farid, Mujahid; Ali, Qasim; Saeed, Rashid; Rizwan, Muhammad

    2015-12-01

    Chromium (Cr) is one of the most phytotoxic metals in the agricultural soils and its concentration is continuously increasing mainly through anthropogenic activities. Little is known on the role of mannitol (M) on plant growth and physiology under metal stress. The aim of this study was to investigate the mechanism of growth amelioration and antioxidant enzyme activities in Cr-stressed wheat (Triticum aestivum L. cv. Lasani 2008) by exogenously applied mannitol. For this, wheat seedlings were sown in pots containing soil or sand and subjected to increasing Cr concentration (0, 0.25 and 0.5mM) in the form of of K2Cr2O7 with and without foliar application of 100mM mannitol. Plants were harvested after four months and data regarding growth characteristics, biomass, photosynthetic pigments, and antioxidant enzymes were recorded. Mannitol application increased plant biomass, photosynthetic pigments and antioxidant enzymes while decreased Cr uptake and accumulation in plants as compared to Cr treatments alone. In this study, we observed that M applied exogenously to Cr-stressed wheat plants, which normally cannot synthesize M, improved their Cr tolerance by increasing growth, photosynthetic pigments and enhancing activities of antioxidant enzymes and by decreasing Cr uptake and translocation in wheat plants. From this study, it can be concluded that M could be used to grow crops on marginally contaminated soils for which separate remediation techniques are time consuming and not cost effective. PMID:26164268

  4. Phylogenetic Relationships and Functional Genes: Distribution of a Gene (mnxG) Encoding a Putative Manganese-Oxidizing Enzyme in Bacillus Species▿

    PubMed Central

    Mayhew, Lisa E.; Swanner, Elizabeth D.; Martin, Andy P.; Templeton, Alexis S.

    2008-01-01

    Several Bacillus and Paenibacillus species were isolated from Fe and Mn oxide minerals precipitating at a deep subsurface oxic-anoxic interface at Henderson Molybdenum Mine, Empire, CO. The isolates were investigated for their Mn(II)-oxidizing potential and interrogated for possession of the mnxG gene, a gene that codes for a putative Mn(II)-oxidizing enzyme in Bacillus species. Seven of eight Bacillus species were capable of Mn(II) oxidation; however, the mnxG gene was detected in only one isolate. Using sequences of known Bacillus species both with and without amplifiable mnxG genes and Henderson Mine isolates, the 16S rRNA and mnxG gene phylogenies were compared to determine if 16S rRNA sequences could be used to predict the presence or absence of an amplifiable mnxG gene within the genomes of the isolates. We discovered a strong correspondence between 16S rRNA sequence similarity and the presence/absence of an amplifiable mnxG gene in the isolates. The data revealed a complex phylogenetic distribution of the mnxG gene in which vertical inheritance and gene loss influence the distribution of the gene among the Bacillus species included in this study. Comparisons of 16S rRNA and functional gene phylogenies can be used as a tool to aid in unraveling the history and dispersal of the mnxG gene within the Bacillus clade. PMID:18849460

  5. Protective effect of marine mangrove Rhizophora apiculata on acetic acid induced experimental colitis by regulating anti-oxidant enzymes, inflammatory mediators and nuclear factor-kappa B subunits.

    PubMed

    V, Vinod Prabhu; C, Guruvayoorappan

    2014-01-01

    Ulcerative colitis is a disease that causes inflammation and ulcer in the lining of the large intestine. In this study we investigate the effect of Rhizophora apiculata (R. apiculata) on acetic acid induced colitis in mouse model. Experimental animals were randomized into four groups: normal untreated, colitis control, R. apiculata treated group and sulfasalazine treated group. R. apiculata significantly (p<0.01) decreased macroscopic score and wet weight of damaged colon compared to colitis control. This effect was confirmed biochemically by significant (p<0.01) reduction of colitis associated increase in myeloperoxidase activity. R. apiculata significantly (p<0.05) increased anti-oxidant enzymes such as superoxide dismutase (SOD) and glutathione (GSH) levels compared to colitis control. R. apiculata significantly (p<0.01) reduced lipid peroxides (LPO), nitric oxide (NO) and inflammatory mediators such as myeloperoxidase (MPO), lactate dehydrogenase (LDH), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α) expressions compared to colitis control. R. apiculata treatment significantly (p<0.01) inhibits the translocation of NF-kB p65 and p50 subunits. Taken together these findings suggest that R. apiculata prevents acetic acid induced colitis in experimental mouse model and may serve as an excellent anti-oxidant and anti-inflammatory agent that could potentially be useful as a (natural) therapy for inflammatory bowel disease (IBD). PMID:24269623

  6. Evaluation of Various Packaging Systems on the Activity of Antioxidant Enzyme, and Oxidation and Color Stabilities in Sliced Hanwoo (Korean Cattle) Beef Loin during Chill Storage

    PubMed Central

    Kang, Sun Moon; Kang, Geunho; Seong, Pil-Nam; Park, Beomyoung; Cho, Soohyun

    2014-01-01

    The effects of various packaging systems, vacuum packaging (VACP), medium oxygen-modified atmosphere packaging (50% O2/20% CO2/30% N2, MOMAP), MOMAP combined with vacuum skin packaging (VSP-MOMAP), high oxygen-MAP (80% O2/20% CO2/0% N2, HOMAP), and HOMAP combined with VSP (VSP-HOMAP), on the activity of antioxidant enzyme, and oxidation and color stabilities in sliced Hanwoo (Korean cattle) beef loin were investigated at 4°C for 14 d. Higher (p<0.05) superoxide dismutase activity and total reducing ability were maintained in VSP-MOMAP beef than in HOMAP beef. Lipid oxidation (2-thiobarbituric acid reactive substances, TBARS) was significantly (p<0.05) retarded in MOMAP, VSP-MOMAP, and VSP-HOMAP beef compared with HOMAP beef. Production of nonheme iron content was lower (p<0.05) in VSP-MOMAP beef than in HOMAP beef. Red color (a*) was kept higher (p<0.05) in VSP-MOMAP beef compared with MOMAP, HOMAP, and VSP-HOMAP beef. However, VACP beef was found to have the most positive effects on the antioxidant activity, oxidation and red color stabilities among the various packaged beef. These findings suggested that VSP-MOMAP was second to VACP in improving oxidation and color stabilities in sliced beef loin during chill storage. PMID:25178378

  7. Distributive Processing by the Iron(II)/α-Ketoglutarate-Dependent Catalytic Domains of the TET Enzymes Is Consistent with Epigenetic Roles for Oxidized 5-Methylcytosine Bases.

    PubMed

    Tamanaha, Esta; Guan, Shengxi; Marks, Katherine; Saleh, Lana

    2016-08-01

    The ten-eleven translocation (TET) proteins catalyze oxidation of 5-methylcytosine ((5m)C) residues in nucleic acids to 5-hydroxymethylcytosine ((5hm)C), 5-formylcytosine ((5f)C), and 5-carboxycytosine ((5ca)C). These nucleotide bases have been implicated as intermediates on the path to active demethylation, but recent reports have suggested that they might have specific regulatory roles in their own right. In this study, we present kinetic evidence showing that the catalytic domains (CDs) of TET2 and TET1 from mouse and their homologue from Naegleria gruberi, the full-length protein NgTET1, are distributive in both chemical and physical senses, as they carry out successive oxidations of a single (5m)C and multiple (5m)C residues along a polymethylated DNA substrate. We present data showing that the enzyme neither retains (5hm)C/(5f)C intermediates of preceding oxidations nor slides along a DNA substrate (without releasing it) to process an adjacent (5m)C residue. These findings contradict a recent report by Crawford et al. ( J. Am. Chem. Soc. 2016 , 138 , 730 ) claiming that oxidation of (5m)C by CD of mouse TET2 is chemically processive (iterative). We further elaborate that this distributive mechanism is maintained for TETs in two evolutionarily distant homologues and posit that this mode of function allows the introduction of (5m)C forms as epigenetic markers along the DNA. PMID:27362828

  8. Dietary antioxidant supplementation enhances lipid and protein oxidative stability of chicken broiler meat through promotion of antioxidant enzyme activity1

    PubMed Central

    Delles, Rebecca M.; Xiong, Youling L.; True, Alma D.; Ao, Touying; Dawson, Karl A.

    2014-01-01

    Recent nutrigenomic studies have shown that animal nutrition can have a major influence on tissue gene expression. Dietary antioxidant supplements can enhance the quality of meat through modification of tissue metabolic processes. This study investigated the influence of dietary antioxidants and quality of oil on the oxidative and enzymatic properties of chicken broiler breast meat stored in an oxygen-enriched package (HiOx: 80% O2/20% CO2) in comparison with air-permeable polyvinylchloride (PVC) or skin packaging systems during retail display at 2 to 4°C for up to 21 d. Broilers were fed either a diet with a low-oxidized (peroxide value 23 mEq of O2/kg) or high-oxidized (peroxide value 121 mEq of O2/kg) oil, supplemented with or without an algae-based Se yeast and organic mineral antioxidant pack for 42 d. Lipid and protein oxidation and tissue enzymatic activity were analyzed. In all packaging systems, lipid oxidation (TBA reactive substances) was inhibited by up to 32.5% (P < 0.05) with an antioxidant-supplemented diet when compared with diets without antioxidants, particularly in the HiOx and PVC systems. Protein sulfhydryls were significantly protected by antioxidant diets (e.g., by 14.6 and 17.8% for low-and high-oxidized dietary groups, respectively, in PVC d 7 samples). Glutathione peroxidase, catalase, and superoxide dismutase activities were significantly higher (P < 0.05) in antioxidant-supplemented diets compared with the basal diet, regardless of oil quality. Also, serum carbonyls were lower in broilers fed a low-oxidized antioxidant-supplemented treatment. The results demonstrate that dietary antioxidants can minimize the oxidative instability of proteins and lipids, and the protection may be linked to improved cellular antioxidant enzymatic activity. PMID:24879706

  9. Dietary antioxidant supplementation enhances lipid and protein oxidative stability of chicken broiler meat through promotion of antioxidant enzyme activity.

    PubMed

    Delles, Rebecca M; Xiong, Youling L; True, Alma D; Ao, Touying; Dawson, Karl A

    2014-06-01

    Recent nutrigenomic studies have shown that animal nutrition can have a major influence on tissue gene expression. Dietary antioxidant supplements can enhance the quality of meat through modification of tissue metabolic processes. This study investigated the influence of dietary antioxidants and quality of oil on the oxidative and enzymatic properties of chicken broiler breast meat stored in an oxygen-enriched package (HiOx: 80% O2/20% CO2) in comparison with air-permeable polyvinylchloride (PVC) or skin packaging systems during retail display at 2 to 4°C for up to 21 d. Broilers were fed either a diet with a low-oxidized (peroxide value 23 mEq of O2/kg) or high-oxidized (peroxide value 121 mEq of O2/kg) oil, supplemented with or without an algae-based Se yeast and organic mineral antioxidant pack for 42 d. Lipid and protein oxidation and tissue enzymatic activity were analyzed. In all packaging systems, lipid oxidation (TBA reactive substances) was inhibited by up to 32.5% (P < 0.05) with an antioxidant-supplemented diet when compared with diets without antioxidants, particularly in the HiOx and PVC systems. Protein sulfhydryls were significantly protected by antioxidant diets (e.g., by 14.6 and 17.8% for low-and high-oxidized dietary groups, respectively, in PVC d 7 samples). Glutathione peroxidase, catalase, and superoxide dismutase activities were significantly higher (P < 0.05) in antioxidant-supplemented diets compared with the basal diet, regardless of oil quality. Also, serum carbonyls were lower in broilers fed a low-oxidized antioxidant-supplemented treatment. The results demonstrate that dietary antioxidants can minimize the oxidative instability of proteins and lipids, and the protection may be linked to improved cellular antioxidant enzymatic activity. PMID:24879706

  10. Vitamin E and C supplementation reduces oxidative stress, improves antioxidant enzymes and positive muscle work in chronically loaded muscles of aged rats

    PubMed Central

    Ryan, Michael J.; Dudash, Holly J.; Docherty, Megan; Geronilla, Kenneth B.; Baker, Brent A.; Haff, G. Gregory; Cutlip, Robert G.; Alway, Stephen E.

    2010-01-01

    Aging is associated with increased oxidative stress. Muscle levels of oxidative stress are further elevated with exercise. The purpose of this study was to determine if dietary antioxidant supplementation would improve muscle function and cellular markers of oxidative stress in response to chronic repetitive loading in aging. The dorsiflexors of the left limb of aged and young adult Fischer 344 Brown x Norway rats were loaded 3 times weekly for 4.5 weeks using 80 maximal stretch-shortening contractions per session. The contralateral limb served as the intra-animal control. The rats were randomly assigned to a diet supplemented with Vitamin E and Vitamin C or normal non-supplemented rat chow. Biomarkers of oxidative stress were measured in the tibialis anterior muscle. Repetitive loading exercise increased maximal isometric force, negative and positive work in the dorsiflexors of young adult rats. Only positive work increased in the aged animals that were supplemented with Vitamin E and C. Markers of oxidative stress (H2O2, total GSH, GSH/GSSG ratio, malondialdehyde and 8-OHdG) increased in the tibialis anterior muscles from aged and young adult animals with repetitive loading, but Vitamin E and C supplements attenuated this increase. MnSOD activity increased with supplementation in the young adult animals. CuZnSOD and catalase activity increased with supplementation in young adult and aged animals and GPx activity increased with exercise in the non-supplemented young adult and aged animals. The increased levels of endogenous antioxidant enzymes after Vitamin E and C supplementation appear to be regulated by post-transcriptional modifications that are affected differently by age, exercise, and supplementation. These data suggest that antioxidant supplementation improves indices of oxidative stress associated with repetitive loading exercise and aging and improve the positive work output of muscles in aged rodents. PMID:20705127

  11. Metabolism of monoterpenes: oxidation of isopiperitenol to isopiperitenone, and subsequent isomerization to piperitenone by soluble enzyme preparations from peppermint (Mentha piperita) leaves

    SciTech Connect

    Kjonaas, R.B.; Venkatachalam, K.V.; Croteau, R.

    1985-04-01

    Soluble enzyme extracts from peppermint leaves, when treated with polystyrene resin to remove endogenous monoterpenes and assayed with unlabeled substrates coupled with capillary gas-liquid chromatographic/mass spectrometric detection methods, were shown to oxidize isopiperitenol to isopiperitenone, and to isomerize isopiperitenone to piperitenone. The enzymes responsible for the monoterpenol dehydrogenation and the subsequent allylic isomerization were separated and partially purified by chromatography on Sephadex G-150, and were shown to have molecular weights of approximately 66,000 and 54,000, respectively. The general properties of the NAD-dependent dehydrogenase were examined, and specificity studies indicated that a double bond adjacent to the carbinol carbon was a required structural feature of the monoterpenol substrate. General properties of the isomerase were also determined, and it was demonstrated that the double bond migration catalyzed by this enzyme involved an intramolecular 1,3-hydrogen transfer. These enzymatic transformations represent two key steps in the metabolic pathway for the conversion of the initially formed cyclic olefin, (+/-)-limonene, to (-)-menthol and related monoterpenes characteristic of peppermint. Some stereochemical features of these reactions, and of the overall biogenetic scheme, are described. 39 references, 5 figures.

  12. Chemoprevention of rat mammary carcinogenesis by Azadirachta indica leaf fractions: modulation of hormone status, xenobiotic-metabolizing enzymes, oxidative stress, cell proliferation and apoptosis.

    PubMed

    Vinothini, G; Manikandan, P; Anandan, R; Nagini, S

    2009-08-01

    We evaluated the chemopreventive potential of the ethyl acetate fraction (EAF) and methanolic fraction (MF) of Azadirachta indica (neem) leaf on 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinogenesis. Estradiol and estrogen receptor status, xenobiotic-metabolizing enzyme activities, redox status, DNA and protein modifications, and the expression of cell proliferation, and apoptosis related proteins in the mammary gland and liver were used as biomarkers of chemoprevention. Administration of both EAF and MF at a dose of 10mg/kg bw effectively suppressed tumour incidence. Chemoprevention by neem leaf fractions was associated with modulation of hormone and receptor status, xenobiotic-metabolising enzymes, and lipid and protein oxidation, with upregulation of antioxidants, inhibition of oxidative DNA damage, protein modification, and cell proliferation, and induction of apoptosis. However EAF rich in constituent phytochemicals was more effective than MF in modulating multiple molecular targets. These results provide evidence for the chemopreventive efficacy of neem leaf fractions in the rat mammary tumour model. PMID:19427891

  13. Recyclable enzyme mimic of cubic Fe3O4 nanoparticles loaded on graphene oxide-dispersed carbon nanotubes with enhanced peroxidase-like catalysis and electrocatalysis

    SciTech Connect

    Wang, Hua; Li, Shuai; Si, Yanmei; Sun, Zhongzhao; Li, Shuying; Lin, Yuehe

    2014-01-01

    Fe3O4 nanoparticles as nanocatalysts may present peroxidase-like catalysis activities and high electrocatalysis if loaded on conductive carbon nanotube (CNT) supports; however, their catalysis performances in an aqueous system might still be challenged by the poor aqueous dispersion of hydrophobic carbon supports and/or low stability of loaded iron catalysts. In this work, amphiphilic graphene oxide nanosheets were employed as “surfactant” to disperse CNTs to create stable graphene oxide-dispersed CNT (GCNT) supports in water for covalently loading cubic Fe3O4 nanoparticles with improved distribution and binding efficiency. Compared with original Fe3O4 nanos and CNT-loaded Fe3O4 nanocomplex, the prepared GCNT–Fe3O4 nanocomposite could achieve higher aqueous stability and, especially, much stronger peroxidase-like catalysis and electrocatalysis to H2O2, presumably resulting from the synergetic effects of two conductive carbon supports and cubic Fe3O4 nanocatalysts effectively loaded. Colorimetric and direct electrochemical detections of H2O2 and glucose using the GCNT–Fe3O4 nanocomposite were conducted with high detection sensitivities, demonstrating the feasibility of practical sensing applications. Such a magnetically recyclable “enzyme mimic” may circumvent some disadvantages of natural protein enzymes and common inorganic catalysts, featuring the multi-functions of high peroxidase-like catalysis, strong electrocatalysis, magnetic separation/recyclability, environmental stability, and direct H2O2 electrochemistry.

  14. In vitro metabolism of α7 neuronal nicotinic receptor agonist AZD0328 and enzyme identification for its N-oxide metabolite.

    PubMed

    Zhou, Diansong; Zhang, Minli; Ye, Xiaomei; Gu, Chungang; Piser, Timothy M; Lanoue, Bernard A; Schock, Sara A; Cheng, Yi-Fang; Grimm, Scott W

    2011-03-01

    1. AZD0328 was pharmacologically characterized as a α7 neuronal nicotinic receptor agonist intended for treatment of Alzheimer's disease. In vitro AZD0328 cross species metabolite profile and enzyme identification for its N-oxide metabolite were evaluated in this study. 2. AZD0328 was very stable in the human hepatocyte incubation, whereas extensively metabolized in rat, dog and guinea pig hepatocyte incubations. The N-oxidation metabolite (M6) was the only metabolite detected in human hepatocyte incubations, and it also appeared to be the major in vitro metabolic pathway in a number of preclinical species. In addition, N-glucuronide metabolite of AZD0328 was observed in human liver microsomes. 3. Other metabolic pathways in the preclinical species include hydroxylation in azabicyclo octane or furopyridine part of the molecule. Pyridine N-methylation of AZD0328 (M2) was identified as a dog specific metabolite, not observed in human or other preclinical species. 4. Multiple enzymes including CYP2D6, CYP3A4/5, FMO1 and FMO3 catalyzed AZD0328 metabolism. The potential for AZD0328 to be inhibited clinically by co-administered drugs or genetic polymorphism is relative low. PMID:21226652

  15. Tamarind seed coat extract restores reactive oxygen species through attenuation of glutathione level and antioxidant enzyme expression in human skin fibroblasts in response to oxidative stress

    PubMed Central

    Nakchat, Oranuch; Nalinratana, Nonthaneth; Meksuriyen, Duangdeun; Pongsamart, Sunanta

    2014-01-01

    Objective To investigate the role and mechanism of tamarind seed coat extract (TSCE) on normal human skin fibroblast CCD-1064Sk cells under normal and oxidative stress conditions induced by hydrogen peroxide (H2O2). Methods Tamarind seed coats were extracted with boiling water and then partitioned with ethyl acetate before the cell analysis. Effect of TSCE on intracellular reactive oxygen species (ROS), glutathione (GSH) level, antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activity including antioxidant protein expression was investigated. Results TSCE significantly attenuated intracellular ROS in the absence and presence of H2O2 by increasing GSH level. In the absence of H2O2, TSCE significantly enhanced SOD and catalase activity but did not affected on GPx. Meanwhile, TSCE significantly increased the protein expression of SOD and GPx in H2O2-treated cells. Conclusions TSCE exhibited antioxidant activities by scavenging ROS, attenuating GSH level that could protect human skin fibroblast cells from oxidative stress. Our results highlight the antioxidant mechanism of tamarind seed coat through an antioxidant enzyme system, the extract potentially benefits for health food and cosmeceutical application of tamarind seed coat. PMID:25182723

  16. Fungal cellulose degradation by oxidative enzymes: from dysfunctional GH61 family to powerful lytic polysaccharide monooxygenase family.

    PubMed

    Morgenstern, Ingo; Powlowski, Justin; Tsang, Adrian

    2014-11-01

    Our understanding of fungal cellulose degradation has shifted dramatically in the past few years with the characterization of a new class of secreted enzymes, the lytic polysaccharide monooxygenases (LPMO). After a period of intense research covering structural, biochemical, theoretical and evolutionary aspects, we have a picture of them as wedge-like copper-dependent metalloenzymes that on reduction generate a radical copper-oxyl species, which cleaves mainly crystalline cellulose. The main biological function lies in the synergism of fungal LPMOs with canonical hydrolytic cellulases in achieving efficient cellulose degradation. Their important role in cellulose degradation is highlighted by the wide distribution and often numerous occurrences in the genomes of almost all plant cell-wall degrading fungi. In this review, we provide an overview of the latest achievements in LPMO research and consider the open questions and challenges that undoubtedly will continue to stimulate interest in this new and exciting group of enzymes. PMID:25217478

  17. Fungal cellulose degradation by oxidative enzymes: from dysfunctional GH61 family to powerful lytic polysaccharide monooxygenase family

    PubMed Central

    Powlowski, Justin; Tsang, Adrian

    2014-01-01

    Our understanding of fungal cellulose degradation has shifted dramatically in the past few years with the characterization of a new class of secreted enzymes, the lytic polysaccharide monooxygenases (LPMO). After a period of intense research covering structural, biochemical, theoretical and evolutionary aspects, we have a picture of them as wedge-like copper-dependent metalloenzymes that on reduction generate a radical copper-oxyl species, which cleaves mainly crystalline cellulose. The main biological function lies in the synergism of fungal LPMOs with canonical hydrolytic cellulases in achieving efficient cellulose degradation. Their important role in cellulose degradation is highlighted by the wide distribution and often numerous occurrences in the genomes of almost all plant cell-wall degrading fungi. In this review, we provide an overview of the latest achievements in LPMO research and consider the open questions and challenges that undoubtedly will continue to stimulate interest in this new and exciting group of enzymes. PMID:25217478

  18. Nitric oxide (no), citrulline - no cycle enzymes, glutamine synthetase and oxidative stress in anoxia (hypobaric hypoxia) and reperfusion in rat brain.

    PubMed

    Swamy, M; Salleh, Mohd Jamsani Mat; Sirajudeen, K N S; Yusof, Wan Roslina Wan; Chandran, G

    2010-01-01

    Nitric oxide is postulated to be involved in the pathophysiology of neurological disorders due to hypoxia/ anoxia in brain due to increased release of glutamate and activation of N-methyl-D-aspartate receptors. Reactive oxygen species have been implicated in pathophysiology of many neurological disorders and in brain function. To understand their role in anoxia (hypobaric hypoxia) and reperfusion (reoxygenation), the nitric oxide synthase, argininosuccinate synthetase, argininosuccinate lyase, glutamine synthetase and arginase activities along with the concentration of nitrate /nitrite, thiobarbituric acid reactive substances and total antioxidant status were estimated in cerebral cortex, cerebellum and brain stem of rats subjected to anoxia and reperfusion. The results of this study clearly demonstrated the increased production of nitric oxide by increased activity of nitric oxide synthase. The increased activities of argininosuccinate synthetase and argininosuccinate lyase suggest the increased and effective recycling of citrulline to arginine in anoxia, making nitric oxide production more effective and contributing to its toxic effects. The decreased activity of glutamine synthetase may favor the prolonged availability of glutamic acid causing excitotoxicity leading to neuronal damage in anoxia. The increased formation of thiobarbituric acid reactive substances and decreased total antioxidant status indicate the presence of oxidative stress in anoxia and reperfusion. The increased arginase and sustained decrease of GS activity in reperfusion group likely to be protective. PMID:20567615

  19. Molecular Phylogeny and Intricate Evolutionary History of the Three Isofunctional Enzymes Involved in the Oxidation of Protoporphyrinogen IX

    PubMed Central

    Kobayashi, Koichi; Masuda, Tatsuru; Tajima, Naoyuki; Wada, Hajime; Sato, Naoki

    2014-01-01

    Tetrapyrroles such as heme and chlorophyll are essential for biological processes, including oxygenation, respiration, and photosynthesis. In the tetrapyrrole biosynthesis pathway, protoporphyrinogen IX oxidase (Protox) catalyzes the formation of protoporphyrin IX, the last common intermediate for the biosynthesis of heme and chlorophyll. Three nonhomologous isofunctional enzymes, HemG, HemJ, and HemY, for Protox have been identified. To reveal the distribution and evolution of the three Protox enzymes, we identified homologs of each along with other heme biosynthetic enzymes by whole-genome clustering across three domains of life. Most organisms possess only one of the three Protox types, with some exceptions. Detailed phylogenetic analysis revealed that HemG is mostly limited to γ-Proteobacteria whereas HemJ may have originated within α-Proteobacteria and transferred to other Proteobacteria and Cyanobacteria. In contrast, HemY is ubiquitous in prokaryotes and is the only Protox in eukaryotes, so this type may be the ancestral Protox. Land plants have a unique HemY homolog that is also shared by Chloroflexus species, in addition to the main HemY homolog originating from Cyanobacteria. Meanwhile, organisms missing any Protox can be classified into two groups; those lacking most heme synthetic genes, which necessarily depend on external heme supply, and those lacking only genes involved in the conversion of uroporphyrinogen III into heme, which would use a precorrin2-dependent alternative pathway. However, hemN encoding coproporphyrinogen IX oxidase was frequently found in organisms lacking Protox enzyme, which suggests a unique role of this gene other than in heme biosynthesis. PMID:25108393

  20. Novel functions of the α-ketoglutarate dehydrogenase complex may mediate diverse oxidant-induced changes in mitochondrial enzymes associated with Alzheimer’s disease

    PubMed Central

    Shi, Qingli; Xu, Hui; Kleinman, Wayne A.; Gibson, Gary E.

    2011-01-01

    Measures in autopsied brains from Alzheimer’s Disease (AD) patients reveal a decrease in the activity of α-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity. The present experiments tested whether both changes could be caused by the common oxidant H2O2 and to probe the mechanism underlying these changes. Since the response to H2O2 is modified by the level of the E2k subunit of KGDHC, the interaction of MDH and KGDHC was studied in cells with varying levels of E2k. In cells with only 23% of normal E2k protein levels, one hour treatment with H2O2 decreased KGDHC and increased MDH activity as well as the mRNA level for both cytosolic and mitochondrial MDH. The increase in MDH did not occur in cells with 100% or 46% of normal E2k. Longer treatments with H2O2 inhibited the activity of both enzymes. Glutathione is a major regulator of cellular redox state and can modify enzyme activities. H2O2 converts reduced glutathione (GSH) to oxidized glutathione (GSSG), which reacts with protein thiols. Treatment of purified KGDHC with GSSG leads to glutathionylation of all three KGDHC subunits. Thus, cellular glutathione level was manipulated by two means to determine the effect on KGDHC and MDH activities. Both buthionine sulfoximine (BSO), which inhibits glutathione synthesis without altering redox state, and H2O2 diminished glutathione to a similar level after 24 hrs. However, H2O2, but not BSO, reduced KGDHC and MDH activities, and the reduction was greater in the E2k-23 line. These findings suggest that the E2k may mediate diverse responses of KGDHC and MDH to oxidants. In addition, the differential response of activities to BSO and H2O2 together with the in vitro interaction of KGDHC with GSSG suggests that glutathionylation is one possible mechanism underlying oxidative stress-induced inhibition of the TCA cycle enzymes. PMID:18206986

  1. Neutralizing a Surface Charge on the FMN Subdomain Increases the Activity of Neuronal Nitric-oxide Synthase by Enhancing the Oxygen Reactivity of the Enzyme Heme-Nitric Oxide Complex*

    PubMed Central

    Haque, Mohammad Mahfuzul; Fadlalla, Mohammed; Wang, Zhi-Qiang; Ray, Sougata Sinha; Panda, Koustubh; Stuehr, Dennis J.

    2009-01-01

    Nitric-oxide synthases (NOSs) are calmodulin-dependent flavoheme enzymes that oxidize l-Arg to nitric oxide (NO) and l-citrulline. Their catalytic behaviors are complex and are determined by their rates of heme reduction (kr), ferric heme-NO dissociation (kd), and ferrous heme-NO oxidation (kox). We found that point mutation (E762N) of a conserved residue on the enzyme's FMN subdomain caused the NO synthesis activity to double compared with wild type nNOS. However, in the absence of l-Arg, NADPH oxidation rates suggested that electron flux through the heme was slower in E762N nNOS, and this correlated with the mutant having a 60% slower kr. During NO synthesis, little heme-NO complex accumulated in the mutant, compared with ∼50–70% of the wild-type nNOS accumulating as this complex. This suggested that the E762N nNOS is hyperactive because it minimizes buildup of an inactive ferrous heme-NO complex during NO synthesis. Indeed, we found that kox was 2 times faster in the E762N mutant than in wild-type nNOS. The mutational effect on kox was independent of calmodulin. Computer simulation and experimental measures both indicated that the slower kr and faster kox of E762N nNOS combine to lower its apparent Km,O2 for NO synthesis by at least 5-fold, which in turn increases its V/Km value and enables it to be hyperactive in steady-state NO synthesis. Our work underscores how sensitive nNOS activity is to changes in the kox and reveals a novel means for the FMN module or protein-protein interactions to alter nNOS activity. PMID:19473991

  2. Astaxanthin protects ARPE-19 cells from oxidative stress via upregulation of Nrf2-regulated phase II enzymes through activation of PI3K/Akt

    PubMed Central

    Li, Zhongrui; Dong, Xin; Liu, Hongling; Chen, Xi; Shi, Huanqi; Fan, Yan; Hou, Dingshan

    2013-01-01

    Purpose Oxidative stress on retinal pigment epithelial (RPE) cells is thought to play a crucial role in the development and progression of age-related macular degeneration. Astaxanthin (AST) is a carotenoid that shows significant antioxidant properties. This study was designed to investigate the protective effect of AST on ARPE-19 cells against oxidative stress and the possible underlying mechanism. Methods ARPE-19 cells exposed to different doses of H2O2 were incubated with various concentrations of AST and cell viability subsequently detected with the (4-[3-[4-iodophenyl]-2–4(4-nitrophenyl)-2H-5- tetrazolio-1,3-benzene disulfonate]; WST-1) assay. The apoptosis rate and intracellular levels of reactive oxygen species (ROS) were measured with flow cytometry. NAD(P)H quinine oxidoreductase 1 (NQO1), hemeoxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and glutamate-cysteine ligase catalytic subunit (GCLC) expression were examined with real-time PCR and western blotting. The nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein and the expression levels of cleaved caspase-3 and protein kinase B proteins were evaluated with western blotting. Results AST clearly reduced H2O2-induced cell viability loss, cell apoptosis, and intracellular generation of ROS. Furthermore, treatment with AST activated the Nrf2-ARE pathway by inducing Nrf2 nuclear localization. Consequently, Phase II enzymes NQO1, HO-1, GCLM, and GCLC mRNA and proteins were increased. AST inhibited expression of H2O2-induced cleaved caspase-3 protein. Activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway was involved in the protective effect of AST on the ARPE-19 cells. Conclusions AST protected ARPE-19 cells against H2O2-induced oxidative stress via Nrf2-mediated upregulation of the expression of Phase II enzymes involving the PI3K/Akt pathway. PMID:23901249

  3. Mammary gene expression and activity of antioxidant enzymes and oxidative indicators in the blood, milk, mammary tissue and ruminal fluid of dairy cows fed flax meal.

    PubMed

    Schogor, Ana Luiza Bachmann; Palin, Marie-France; Santos, Geraldo Tadeu dos; Benchaar, Chaouki; Lacasse, Pierre; Petit, Hélène V

    2013-11-01

    The effects of flax meal (FM) on the activity of antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)) in the blood, mammary tissue and ruminal fluid, and oxidative stress indicators (thiobarbituric acid-reactive substances(TBARS) and 1,1-diphenyl-2-picrylhydrazyl-scavenging activity) in the milk, plasma and ruminal fluid of dairy cows were determined.The mRNA abundance of the antioxidant enzymes and oxidative stress-related genes was assessed in mammary tissue. A total of eight Holstein cows were used in a double 4 x 4 Latin square design. There were four treatments in the diet: control with no FM(CON) or 5% FM (5FM), 10% FM (10FM) and 15% FM (15FM). There was an interaction between treatment and time for plasma GPx and CAT activities. Cows supplemented with FM had a linear reduction in TBARS at 2 h after feeding, and there was no treatment effect at 0, 4 and 6 h after feeding. TBARS production decreased in the milk of cows fed the 5FM and 10FM diets. There was a linear increase in nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) mRNA abundance in mammary tissue with FM supplementation.A linear trend for increased mRNA abundance of the CAT gene was observed with higher concentrations of FM. The mRNA abundance of CAT, GPx1, GPx3, SOD1, SOD2, SOD3 and nuclear factor of k light polypeptide gene enhancer in B-cells (NFKB) genes was not affected by the treatment. These findings suggest that FM supplementation can improve the oxidative status of Holstein cows as suggested by decreased TBARS production in ruminal fluid 2 h post-feeding and increased NFE2L2/nuclear factor-E2-related factor 2 (Nrf2) mRNA abundance in mammary tissue. PMID:23578516

  4. Identification of enzymes and regulatory proteins in Escherichia coli that are oxidized under nitrogen, carbon, or phosphate starvation

    PubMed Central

    Noda, Yasuko; Berlett, Barbara S.; Stadtman, Earl R.; Aponte, Angel; Morgan, Meghan; Shen, Rong-Fong

    2007-01-01

    Using proteomic technologies, we identified 62 proteins that are oxidized to carbonyl derivatives during growth of Escherichia coli under nitrogen starvation (NS), carbon starvation (CS), and phosphate starvation (PS) conditions. The carbonylated proteins were converted to 2,4-dinitrophenylhydrazone derivatives and these were identified using Western blotting and mass spectrometry by searching E. coli proteins in the Swiss-Prot and/or NCBI databases. Fourteen of the oxidized proteins were formed under both NS and CS conditions, and only three proteins were specifically oxidized under PS conditions. Interestingly, the carbonyl content of proteins in crude extracts of cells harvested after 48 h of stationary growth under NS and CS was significantly lower than that observed at mid-log and end-log phases of growth. In contrast, the carbonyl content of proteins in extracts of cells grown under PS conditions was fairly constant during comparable periods of growth. PMID:18003895

  5. Oxidative and nitrative stress and pro-inflammatory cytokines in Mucopolysaccharidosis type II patients: effect of long-term enzyme replacement therapy and relation with glycosaminoglycan accumulation.

    PubMed

    Jacques, Carlos Eduardo Diaz; Donida, Bruna; Mescka, Caroline P; Rodrigues, Daiane G B; Marchetti, Desirèe P; Bitencourt, Fernanda H; Burin, Maira G; de Souza, Carolina F M; Giugliani, Roberto; Vargas, Carmen Regla

    2016-09-01

    Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by a deficient activity of iduronate-2-sulfatase, leading to abnormal accumulation of glycosaminoglycans (GAG). The main treatment for MPS II is enzyme replacement therapy (ERT). Previous studies described potential benefits of six months of ERT against oxidative stress in patients. Thus, the aim of this study was to investigate oxidative, nitrative and inflammatory biomarkers in MPS II patients submitted to long term ERT. It were analyzed urine and blood samples from patients on ERT (mean time: 5.2years) and healthy controls. Patients presented increased levels of lipid peroxidation, assessed by urinary 15-F2t-isoprostane and plasmatic thiobarbituric acid-reactive substances. Concerning to protein damage, urinary di-tyrosine (di-Tyr) was increased in patients; however, sulfhydryl and carbonyl groups in plasma were not altered. It were also verified increased levels of urinary nitrate+nitrite and plasmatic nitric oxide (NO) in MPS II patients. Pro-inflammatory cytokines IL-1β and TNF-α were increased in treated patients. GAG levels were correlated to di-Tyr and nitrate+nitrite. Furthermore, IL-1β was positively correlated with TNF-α and NO. Contrastingly, we did not observed alterations in erythrocyte superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities, in reduced glutathione content and in the plasmatic antioxidant capacity. Although some parameters were still altered in MPS II patients, these results may suggest a protective role of long-term ERT against oxidative stress, especially upon oxidative damage to protein and enzymatic and non-enzymatic defenses. Moreover, the redox imbalance observed in treated patients seems to be GAG- and pro-inflammatory cytokine-related. PMID:27251652

  6. Elucidation of the Teixobactin Pharmacophore.

    PubMed

    Yang, Hyunjun; Chen, Kevin H; Nowick, James S

    2016-07-15

    This paper elucidates the teixobactin pharmacophore by comparing the arginine analogue of teixobactin Arg10-teixobactin to seven homologues with varying structure and stereochemistry. The roles of the guanidinium group at position 10, the stereochemistry of the macrolactone ring, and the "tail" comprising residues 1-5 are investigated. The guanidinium group is not necessary for activity; Lys10-teixobactin is more active than Arg10-teixobactin against Gram-positive bacteria in minimum inhibitory concentration (MIC) assays. The relative stereochemistry of the macrolactone ring is important. Diastereomer l-Thr8,Arg10-teixobactin is inactive, and diastereomer d-allo-Ile11,Arg10-teixobactin is less active. The macrolactone ring is critical; seco-Arg10-teixobactin is inactive. The absolute stereochemistry is not important; the enantiomer ent-Arg10-teixobactin is comparable in activity. The hydrophobic N-terminal tail is important. Truncation of residues 1-5 results in loss of activity, and replacement of residues 1-5 with a dodecanoyl group partially restores activity. These findings pave the way for developing simpler homologues of teixobactin with enhanced pharmacological properties. PMID:27232661

  7. Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

    PubMed

    Lavrukhin, O V; Lloyd, R S

    2000-12-12

    Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates. PMID:11106507

  8. Enzymological and Structural Studies of the Mechanism of Promiscuous Substrate Recognition by the Oxidative DNA Repair Enzyme AlkB

    SciTech Connect

    Yu, B.; Hunt, J

    2009-01-01

    Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N6-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher kcat and Km for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this 'kcat/Km compensation,' which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O2 substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O2 binding before nucleotide substrate binding.

  9. Spectroscopic Analyses of the Biofuels-Critical Phytochemical Coniferyl Alcohol and Its Enzyme-Catalyzed Oxidation Products

    SciTech Connect

    Achyuthan, Komandoor; Adams, Paul; Simmons, Blake; Singh, Anup

    2011-07-13

    Lignin composition (monolignol types of coniferyl, sinapyl or p-coumaryl alcohol) is causally related to biomass recalcitrance. We describe multiwavelength (220, 228, 240, 250, 260, 290, 295, 300, 310 or 320 nm) absorption spectroscopy of coniferyl alcohol and its laccase- or peroxidase-catalyzed products during real time kinetic, pseudo-kinetic and endpoint analyses, in optical turn on or turn off modes, under acidic or basic conditions. Reactions in microwell plates and 100 mu L volumes demonstrated assay miniaturization and high throughput screening capabilities. Bathochromic and hypsochromic shifts along with hyperchromicity or hypochromicity accompanied enzymatic oxidations by laccase or peroxidase. The limits of detection and quantitation of coniferyl alcohol averaged 2.4 and 7.1 mu M respectively, with linear trend lines over 3 to 4 orders of magnitude. Coniferyl alcohol oxidation was evident within 10 minutes or with 0.01 mu g/mL laccase and 2 minutes or 0.001 mu g/mL peroxidase. Detection limit improved to 1.0 mu M coniferyl alcohol with Km of 978.7 +/- 150.7 mu M when examined at 260 nm following 30 minutes oxidation with 1.0 mu g/mL laccase. Our assays utilized the intrinsic spectroscopic properties of coniferyl alcohol or its oxidation products for enabling detection, without requiring chemical synthesis or modification of the substrate or product(s). These studies facilitate lignin compositional analyses and augment pretreatment strategies for reducing biomass recalcitrance.

  10. Hepatic ischemia-reperfusion syndrome after partial liver resection (LR): hepatic venous oxygen saturation, enzyme pattern, reduced and oxidized glutathione, procalcitonin and interleukin-6.

    PubMed

    Kretzschmar, Michael; Krüger, Antie; Schirrmeister, Wulf

    2003-06-01

    The hepatic ischemia-reperfusion syndrome was investigated in 28 patients undergoing elective partial liver resection with intraoperative occlusion of hepatic inflow (Pringle maneuver) using the technique of liver vein catheterization. Hepatic venous oxygen saturation (ShvO2) was monitored continuously up to 24 hours after surgery. Aspartate aminotransferase, glutamate dehydrogenase, gamma-glutamyl transpeptidase, pseudocholinesterase, alpha-glutathione S-transferase, reduced and oxidized glutathione, procalcitonine, and interleukin-6 were serially measured both before and after Pringle maneuver during the resection and postoperatively in arterial and/or hepatic venous blood. ShvO2 measurement demonstrated that peri- and postoperative management was suitable to maintain an optimal hepatic oxygen supply. As expected, we were able to demonstrate a typical enzyme pattern of postischemic liver injury. There was a distinct decrease of reduced glutathione levels both in arterial and hepatic venous plasma after LR accompanied by a strong increase in oxidized glutathione concentration during the phase of reperfusion. We observed increases in procalcitonin and interleukin-6 levels both in arterial and hepatic venous blood after declamping. Our data support the view that liver resection in man under conditions of inflow occlusion resulted in ischemic lesion of the liver (loss of glutathione synthesizing capacity with disturbance of protection against oxidative stress) and an additional impairment during reperfusion (liberation of reactive oxygen species, local and systemic inflammation reaction with cytokine production). Additionally, we found some evidence for the assumption that the liver has an export function for reduced glutathione into plasma in man. PMID:12877355

  11. Investigation of Propolis’ Effect on Thiobarbituric Acid Reactive Substances and Anti-Oxidant Enzyme Levels of Hippocampus in Diabetic Rats Induced by Streptozotocin

    PubMed Central

    Köksal, Burcu; Emre, Memet Hanifi; Polat, Alaadin

    2015-01-01

    BACKGROUND: Propolis is an organic resinous viscous substance collected from flower bud and plant sprig by bees. Propolis has a potential treatment agent for oxidative damage caused by diabetes in hippocampus due to its flavonoid and phenolic content. AIM: In this study effect of propolis on thiobarbituric acid reactive substances and anti-oxidative enzyme levels of hippocampus in diabetic rats induced by streptozotocin was investigated. MATERIALS AND METHODS: The study involved measuring levels of SOD, CAT, GSH-Px and TBARs in hippocampus tissue of STZ-induced diabetic rats (Adult Male Sprague Dawley rats) after applying propolis for one month. The subjects of the study were composed of 51 rats randomly assigned to four groups (Control, STZ, P+STZ and STZ+P). For analysis of data, Kruskal Wallis Test was utilized. RESULTS: The findings of the study showed that there were no significant difference in the levels of TBARS, SOD, CAT and GSH-Px of hippocampus across the groups. CONCLUSION: Propolis application in four-week duration does not have effect on TBARS, SOD, CAT and GSH-Px levels of hippocampus of diabetic rats. These findings mean that more time for observing oxidative harms on hippocampus is needed. PMID:27275196

  12. Virgin Olive Oil Enriched with Its Own Phenols or Complemented with Thyme Phenols Improves DNA Protection against Oxidation and Antioxidant Enzyme Activity in Hyperlipidemic Subjects.

    PubMed

    Romeu, Marta; Rubió, Laura; Sánchez-Martos, Vanessa; Castañer, Olga; de la Torre, Rafael; Valls, Rosa M; Ras, Rosa; Pedret, Anna; Catalán, Úrsula; López de las Hazas, María del Carmen; Motilva, María J; Fitó, Montserrat; Solà, Rosa; Giralt, Montserrat

    2016-03-01

    The effects of virgin olive oil (VOO) enriched with its own phenolic compounds (PC) and/or thyme PC on the protection against oxidative DNA damage and antioxidant endogenous enzymatic system (AEES) were estimated in 33 hyperlipidemic subjects after the consumption of VOO, VOO enriched with its own PC (FVOO), or VOO complemented with thyme PC (FVOOT). Compared to pre-intervention, 8-hydroxy-2'-deoxyguanosine (a marker for DNA damage) decreased in the FVOO intervention and to a greater extent in the FVOOT with a parallel significant increase in olive and thyme phenolic metabolites. Superoxide dismutase (AEES enzyme) significantly increased in the FVOO intervention and to a greater extent in the FVOOT with a parallel significant increase in thyme phenolic metabolites. When all three oils were compared, FVOOT appeared to have the greatest effect in protecting against oxidative DNA damage and improving AEES. The sustained intake of a FVOOT improves DNA protection against oxidation and AEES probably due to a greater bioavailability of thyme PC in hyperlipidemic subjects. PMID:26889783

  13. Enzyme markers

    MedlinePlus

    ... or defects passed down through families (inherited) can affect how enzymes work. Some enzymes are affected by several genes. Test results are usually reported as a percentage of normal enzyme activity.

  14. Coordinated Changes in Antioxidative Enzymes Protect the Photosynthetic Machinery from Salinity Induced Oxidative Damage and Confer Salt Tolerance in an Extreme Halophyte Salvadora persica L.

    PubMed

    Rangani, Jaykumar; Parida, Asish K; Panda, Ashok; Kumari, Asha

    2016-01-01

    Salinity-induced modulations in growth, photosynthetic pigments, relative water content (RWC), lipid peroxidation, photosynthesis, photosystem II efficiency, and changes in activity of various antioxidative enzymes were studied in the halophyte Salvadora persica treated with various levels of salinity (0, 250, 500, 750, and 1000 mM NaCl) to obtain an insight into the salt tolerance ability of this halophyte. Both fresh and dry biomass as well as leaf area (LA) declined at all levels of salinity whereas salinity caused an increase in leaf succulence. A gradual increase was observed in the Na(+) content of leaf with increasing salt concentration up to 750 mM NaCl, but at higher salt concentration (1000 mM NaCl), the Na(+) content surprisingly dropped down to the level of 250 mM NaCl. The chlorophyll and carotenoid contents of the leaf remained unaffected by salinity. The photosynthetic rate (PN), stomatal conductance (gs), the transpiration rate (E), quantum yield of PSII (ΦPSII), photochemical quenching (qP), and electron transport rate remained unchanged at low salinity (250 to 500 mM NaCl) whereas, significant reduction in these parameters were observed at high salinity (750 to 1000 mM NaCl). The RWC% and water use efficiency (WUE) of leaf remained unaffected by salinity. The salinity had no effect on maximum quantum efficiency of PS II (Fv/Fm) which indicates that PS II is not perturbed by salinity-induced oxidative damage. Analysis of the isoforms of antioxidative enzymes revealed that the leaves of S. persica have two isoforms each of Mn-SOD and Fe-SOD and one isoform of Cu-Zn SOD, three isoforms of POX, two isoforms of APX and one isoform of CAT. There was differential responses in activity and expression of different isoforms of various antioxidative enzymes. The malondialdehyde (MDA) content (a product of lipid peroxidation) of leaf remained unchanged in S. persica treated with various levels of salinity. Our results suggest that the absence of pigment

  15. Coordinated Changes in Antioxidative Enzymes Protect the Photosynthetic Machinery from Salinity Induced Oxidative Damage and Confer Salt Tolerance in an Extreme Halophyte Salvadora persica L.

    PubMed Central

    Rangani, Jaykumar; Parida, Asish K.; Panda, Ashok; Kumari, Asha

    2016-01-01

    Salinity-induced modulations in growth, photosynthetic pigments, relative water content (RWC), lipid peroxidation, photosynthesis, photosystem II efficiency, and changes in activity of various antioxidative enzymes were studied in the halophyte Salvadora persica treated with various levels of salinity (0, 250, 500, 750, and 1000 mM NaCl) to obtain an insight into the salt tolerance ability of this halophyte. Both fresh and dry biomass as well as leaf area (LA) declined at all levels of salinity whereas salinity caused an increase in leaf succulence. A gradual increase was observed in the Na+ content of leaf with increasing salt concentration up to 750 mM NaCl, but at higher salt concentration (1000 mM NaCl), the Na+ content surprisingly dropped down to the level of 250 mM NaCl. The chlorophyll and carotenoid contents of the leaf remained unaffected by salinity. The photosynthetic rate (PN), stomatal conductance (gs), the transpiration rate (E), quantum yield of PSII (ΦPSII), photochemical quenching (qP), and electron transport rate remained unchanged at low salinity (250 to 500 mM NaCl) whereas, significant reduction in these parameters were observed at high salinity (750 to 1000 mM NaCl). The RWC% and water use efficiency (WUE) of leaf remained unaffected by salinity. The salinity had no effect on maximum quantum efficiency of PS II (Fv/Fm) which indicates that PS II is not perturbed by salinity-induced oxidative damage. Analysis of the isoforms of antioxidative enzymes revealed that the leaves of S. persica have two isoforms each of Mn-SOD and Fe-SOD and one isoform of Cu-Zn SOD, three isoforms of POX, two isoforms of APX and one isoform of CAT. There was differential responses in activity and expression of different isoforms of various antioxidative enzymes. The malondialdehyde (MDA) content (a product of lipid peroxidation) of leaf remained unchanged in S. persica treated with various levels of salinity. Our results suggest that the absence of pigment

  16. The in vivo infusion of hydrogen peroxide induces oxidative stress and differentially affects the activities of small intestinal carbohydrate digestive enzymes in the neonatal pig.

    PubMed

    Lackeyram, D; Mine, Y; Widowski, T; Archbold, T; Fan, M Z

    2012-12-01

    Chronic fatigue syndrome (CFS) is characterized by persistent and relapsing fatigue that involves oxidative stress in its pathogenesis. We tested the hypothesis that a decrease in key carbohydrate-digesting enzyme activity in the gut is one of the major biological mechanisms of developing CFS in liquid formula-fed neonatal pigs with in vivo infusion of H(2)O(2). Piglets at 7 to 10 d of age were fitted with an intraperitoneal catheter, allowed a 3-d post surgical recovery, and infused with either H(2)O(2) at 5 mmol/kg BW (PER; n = 8) or the same volume of saline (CON; n = 8) in six 20-ml doses daily for a period of 10 d. During this period, animal behavior was monitored, blood samples collected, and jejunal enzyme activity kinetic experiments for lactase, sucrase, maltase, and maltase-glucoamylase were conducted. Plasma concentration of reduced glutathione remained similar (P > 0.05) to the pre-infusion level over the study duration in the CON group whereas this was 65% lower (P < 0.05) than the pre-infusion level in the PER group. Piglets experiencing oxidative stress had an overall lower (P < 0.05) physical mobility and the maximal jejunal specific activities [μmol/(mg protein · min)] for lactase (PER, 6.54 ± 0.68 vs. CON, 12.65 ± 0.69) and maltase (PER, 57.39 ± 1.02 vs. CON, 75.60 ± 1.04), respectively. However, differences were not observed (P > 0.05) in the maximal specific activities [μmol/(mg protein · min)] of sucrase (PER, 10.50 ± 1.37 vs. CON, 12.40 ± 1.55) and maltase-glucoamylase (PER, 0.71 ± 0.08 vs. CON, 0.70 ± 0.07) between the 2 groups. In conclusion, infusion of a suitable dose of H(2)O(2) induced CFS in the neonatal pigs. Oxidative stress in vivo differentially affected the maximal activities of important small intestinal carbohydrate-digesting enzymes in neonatal pigs fed a dairy milk-based liquid formula. PMID:23365398

  17. Species Differences in the Oxidative Desulfurization of a Thiouracil-Based Irreversible Myeloperoxidase Inactivator by Flavin-Containing Monooxygenase Enzymes.

    PubMed

    Eng, Heather; Sharma, Raman; Wolford, Angela; Di, Li; Ruggeri, Roger B; Buckbinder, Leonard; Conn, Edward L; Dalvie, Deepak K; Kalgutkar, Amit S

    2016-08-01

    N1-Substituted-6-arylthiouracils, represented by compound 1 [6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one], are a novel class of selective irreversible inhibitors of human myeloperoxidase. The present account is a summary of our in vitro studies on the facile oxidative desulfurization in compound 1 to a cyclic ether metabolite M1 [5-(2,4-dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one] in NADPH-supplemented rats (t1/2 [half-life = mean ± S.D.] = 8.6 ± 0.4 minutes) and dog liver microsomes (t1/2 = 11.2 ± 0.4 minutes), but not in human liver microsomes (t1/2 > 120 minutes). The in vitro metabolic instability also manifested in moderate-to-high plasma clearances of the parent compound in rats and dogs with significant concentrations of M1 detected in circulation. Mild heat deactivation of liver microsomes or coincubation with the flavin-containing monooxygenase (FMO) inhibitor imipramine significantly diminished M1 formation. In contrast, oxidative metabolism of compound 1 to M1 was not inhibited by the pan cytochrome P450 inactivator 1-aminobenzotriazole. Incubations with recombinant FMO isoforms (FMO1, FMO3, and FMO5) revealed that FMO1 principally catalyzed the conversion of compound 1 to M1. FMO1 is not expressed in adult human liver, which rationalizes the species difference in oxidative desulfurization. Oxidation by FMO1 followed Michaelis-Menten kinetics with Michaelis-Menten constant, maximum rate of oxidative desulfurization, and intrinsic clearance values of 209 μM, 20.4 nmol/min/mg protein, and 82.7 μl/min/mg protein, respectively. Addition of excess glutathione essentially eliminated the conversion of compound 1 to M1 in NADPH-supplemented rat and dog liver microsomes, which suggests that the initial FMO1-mediated S-oxygenation of compound 1 yields a sulfenic acid intermediate capable of redox cycling to the parent compound in a glutathione-dependent fashion or undergoing further oxidation to a more

  18. Catalyzed enzyme electrodes

    DOEpatents

    Zawodzinski, Thomas A.; Wilson, Mahlon S.; Rishpon, Judith; Gottesfeld, Shimshon

    1993-01-01

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  19. Magnetically responsive enzyme powders

    NASA Astrophysics Data System (ADS)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-04-01

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (-20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties.

  20. Catalyzed enzyme electrodes

    SciTech Connect

    Zawodzinski, T.A.; Wilson, M.S.; Rishpon, J.; Gottesfeld, S.

    1992-12-31

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid, polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  1. Catalyzed enzyme electrodes

    SciTech Connect

    Zawodzinski, T.A.; Wilson, M.S.; Rishpon, J.; Gottesfeld, S.

    1993-07-13

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  2. Effect of Aloe vera gel extract on antioxidant enzymes and azoxymethane-induced oxidative stress in rats.

    PubMed

    Anilakumar, K R; Sudarshanakrishna, K R; Chandramohan, G; Ilaiyaraja, N; Khanum, Farhath; Bawa, A S

    2010-08-01

    The present work was undertaken with a view to study the effect of oral feeding of 2% Aloe vera gel extract (AGE) for 30 days on azoxymethane (AOM)-induced oxidative stress in rats. It was observed that AOM administration resulted in a significant increase in malondialdehyde and conjugated dienes, with reduction in hepatic glutathione (GSH), vitamin A and uric acid contents. AOM-induced reduction in hepatic GSH and uric acid was brought back to normal by AGE. There was a significant raise in hepatic catalase, superoxide dismutase and glucose-6-phosphate dehydrogenase (G-6-PD) activities as a result of feeding of the extract. Ingestion of the extract effected reduction in AOM-induced colonic GSH-peroxidase, G-6-PD and glutathione S-transferase and femur bone marrow micronuclei formation. Hence, it is suggested that Aloe vera gel extract possess the ability to reduce AOM- induced oxidative stress and toxicity in liver. PMID:21341543

  3. Enzyme-free Detection of Hydrogen Peroxide from Cerium Oxide Nanoparticles Immobilized on Poly(4-vinylpyridine) Self-Assembled Monolayers

    SciTech Connect

    Gaynor, James D.; Karakoti, Ajay S.; Inerbaev, Talgat; Sanghavi, Shail P.; Nachimuthu, Ponnusamy; Shutthanandan, V.; Seal, Sudipta; Thevuthasan, Suntharampillai

    2013-05-02

    A single layer of oxygen-deficient cerium oxide nanoparticles (CNPs) are immobilized on microscopic glass slide using poly(4-vinylpyridine) (PVP) self-assembled monolayers (SAMs). A specific colorimetric property of CNPs when reacted with hydrogen peroxide allows for the direct, single-step peroxide detection which can be used in medical diagnosis and explosives detection. Multiple PVP-CNP immobilized layers improve sensitivity of detection and the sensor can be regenerated for reuse.

  4. Analysis of Oxidative Stress Enzymes and Structural and Functional Proteins on Human Aortic Tissue from Different Aortopathies

    PubMed Central

    Soto, María Elena; Soria-Castro, Elizabeth; Guarner Lans, Verónica; Muruato Ontiveros, Eleazar; Iván Hernández Mejía, Benjamín; Jorge Martínez Hernandez, Humberto; Barragán García, Rodolfo; Herrera, Valentín; Pérez-Torres, Israel

    2014-01-01

    The role of oxidative stress in different aortopathies is evaluated. Thirty-two tissue samples from 18 men and 14 women were divided into: 4 control (C) subjects, 11 patients with systemic arterial hypertension (SAH), 4 with variants of Marfan's syndrome (MV), 9 with Marfan's syndrome (M), 2 with Turner's syndrome, and 2 with Takayasu's arteritis (TA). Aorta fragments were homogenized. Lipoperoxidation (LPO), copper-zinc and manganese superoxide dismutase (Mn and Cu-Zn-SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), endothelial nitric oxide synthase (eNOS), nitrates and nitrites (NO3−/NO2−), and type IV collagen, and laminin were evaluated. There was an increase in Mn- and Cu-Zn-SOD activity in SAH, MV, M, and Turner's syndrome. There was also an increase in CAT activity in M and Turner' syndrome. GPx and GST activity decreased and LPO increased in all groups. eNOS was decreased in SAH, MV, and M and NO3−/NO2− were increased in SAH and TA. Type IV collagen was decreased in Turner's syndrome and TA. Laminin γ-1 was decreased in MV and increased in M. In conclusion, similarities and differences in oxidative stress in the different aortopathies studied including pathologies with aneurysms were found with alterations in SOD, CAT, GPx, GST, and eNOS activity that modify subendothelial basement membrane proteins. PMID:25101153

  5. Analysis of oxidative stress enzymes and structural and functional proteins on human aortic tissue from different aortopathies.

    PubMed

    Soto, María Elena; Soria-Castro, Elizabeth; Lans, Verónica Guarner; Ontiveros, Eleazar Muruato; Mejía, Benjamín Iván Hernández; Hernandez, Humberto Jorge Martínez; García, Rodolfo Barragán; Herrera, Valentín; Pérez-Torres, Israel

    2014-01-01

    The role of oxidative stress in different aortopathies is evaluated. Thirty-two tissue samples from 18 men and 14 women were divided into: 4 control (C) subjects, 11 patients with systemic arterial hypertension (SAH), 4 with variants of Marfan's syndrome (MV), 9 with Marfan's syndrome (M), 2 with Turner's syndrome, and 2 with Takayasu's arteritis (TA). Aorta fragments were homogenized. Lipoperoxidation (LPO), copper-zinc and manganese superoxide dismutase (Mn and Cu-Zn-SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), endothelial nitric oxide synthase (eNOS), nitrates and nitrites (NO3(-)/NO2(-)), and type IV collagen, and laminin were evaluated. There was an increase in Mn- and Cu-Zn-SOD activity in SAH, MV, M, and Turner's syndrome. There was also an increase in CAT activity in M and Turner' syndrome. GPx and GST activity decreased and LPO increased in all groups. eNOS was decreased in SAH, MV, and M and NO3 (-)/NO2 (-) were increased in SAH and TA. Type IV collagen was decreased in Turner's syndrome and TA. Laminin γ-1 was decreased in MV and increased in M. In conclusion, similarities and differences in oxidative stress in the different aortopathies studied including pathologies with aneurysms were found with alterations in SOD, CAT, GPx, GST, and eNOS activity that modify subendothelial basement membrane proteins. PMID:25101153

  6. Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100

    PubMed Central

    Delporte, Cédric; Boudjeltia, Karim Zouaoui; Noyon, Caroline; Furtmüller, Paul G.; Nuyens, Vincent; Slomianny, Marie-Christine; Madhoun, Philippe; Desmet, Jean-Marc; Raynal, Pierre; Dufour, Damien; Koyani, Chintan N.; Reyé, Florence; Rousseau, Alexandre; Vanhaeverbeek, Michel; Ducobu, Jean; Michalski, Jean-Claude; Nève, Jean; Vanhamme, Luc; Obinger, Christian; Malle, Ernst; Van Antwerpen, Pierre

    2014-01-01

    Oxidation of LDL by the myeloperoxidase (MPO)-H2O2-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H2O2-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H2O2-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL. PMID:24534704

  7. Essential oil from lemon peels inhibit key enzymes linked to neurodegenerative conditions and pro-oxidant induced lipid peroxidation.

    PubMed

    Oboh, Ganiyu; Olasehinde, Tosin A; Ademosun, Ayokunle O

    2014-01-01

    This study sought to investigate the effects of essential oil from lemon (Citrus limoni) peels on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities in vitro. The essential oil was extracted by hydrodistillation, dried with anhydrous Na2SO4 and characterized using gas chromatography. Antioxidant properties of the oil and inhibition of pro-oxidant-induced lipid peroxidation in rats brain homogenate were also assessed. The essential oil inhibited AChE and BChE activities in a concentration-dependent manner. GC analysis revealed the presence of sabinene, limonene, α-pinene, β-pinene, neral, geranial, 1,8-cineole, linalool, borneol, α-terpineol, terpinen-4-ol, linalyl acetate and β-caryophyllene. Furthermore, the essential oil exhibited antioxidant activities as typified by ferric reducing property, Fe(2+)-chelation and radicals [DPPH, ABTS, OH, NO] scavenging abilities. The inhibition of AChE and BChE activities, inhibition of pro-oxidant induced lipid peroxidation and antioxidant activities could be possible mechanisms for the use of the essential oil in the management and prevention of oxidative stress-induced neurodegeneration. PMID:24599102

  8. Activity-Based Protein Profiling Reveals Mitochondrial Oxidative Enzyme Impairment and Restoration in Diet-Induced Obese Mice

    SciTech Connect

    Sadler, Natalie C.; Angel, Thomas E.; Lewis, Michael P.; Pederson, Leeanna M.; Chauvigne-Hines, Lacie M.; Wiedner, Susan D.; Zink, Erika M.; Smith, Richard D.; Wright, Aaron T.

    2012-10-24

    High-fat diet (HFD) induced obesity and concomitant development of insulin resistance (IR) and type 2 diabetes mellitus have been linked to mitochondrial dysfunction. However, it is not clear whether mitochondrial dysfunction is a direct effect of a HFD or if the mitochondrial function is reduced with increased HFD duration. We hypothesized that the function of mitochondrial oxidative and lipid metabolism functions in skeletal muscle mitochondria for HFD mice are similar or elevated relative to standard diet (SD) mice, thereby IR is neither cause nor consequence of mitochondrial dysfunction. We applied a chemical probe approach to identify functionally reactive ATPases and nucleotide-binding proteins in mitochondria isolated from skeletal muscle of C57Bl/6J mice fed HFD or SD chow for 2-, 8-, or 16-weeks; feeding time points known to induce IR. A total of 293 probe-labeled proteins were identified by mass spectrometry-based proteomics, of which 54 differed in abundance between HFD and SD mice. We found proteins associated with the TCA cycle, oxidative phosphorylation (OXPHOS), and lipid metabolism were altered in function when comparing SD to HFD fed mice at 2-weeks, however by 16-weeks HFD mice had TCA cycle, β-oxidation, and respiratory chain function at levels similar to or higher than SD mice.

  9. Alterations in antioxidant enzyme activities and oxidative damage in alcoholic rat tissues: protective role of Thespesia populnea.

    PubMed

    Pandanaboina, Sahitya Chetan; Kondeti, Shanmugam Ramudu; Rajbanshi, Sangeetha Lakshmi; Kunala, Pavan Nagavenkata; Pandanaboina, Saritha; Pandanaboina, Murali Mohan; Wudayagiri, Rajendra

    2012-05-01

    Recent advances in our understanding of the pathogenesis of alcohol-induced hepato-renal injury and the development of new approaches to its treatment have been reported in various works. This study involves alcohol-induced oxidative stress linked to the metabolism of ethanol involving both mitochondrial and peroxisomal fractions of liver and kidney. Alcohol treatment resulted in the depletion of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), Glutathione-S-Transferase (GST) activities, and reduced glutathione (GSH) content, higher level of malondialdehyde (MDA) and lower levels of protein carbonyls (PC) causing malfunction of hepatic and renal tissues, when compared to control rats. Thespesia populnea (TP) leaf extracts, administered to chronic alcohol ingested rats, were envisaged to possess significant antioxidant defence properties and help in the recovery of tissues from alcohol-induced oxidative damage. The results showed that degenerative changes in hepatic and renal cells of alcoholic groups were minimized by the administration of TP leaf extracts as also revealed by histopathological examination. The current findings indicate that treatment with TP extracts reduces alcohol-induced oxidative stress, thereby protecting the hepatic and renal tissue from alcohol-induced damage. PMID:26434274

  10. Genistein modulates oxidative stress in breast cancer cell lines according to ERα/ERβ ratio: effects on mitochondrial functionality, sirtuins, uncoupling protein 2 and antioxidant enzymes.

    PubMed

    Nadal-Serrano, Mercedes; Pons, Daniel Gabriel; Sastre-Serra, Jorge; Blanquer-Rosselló, M del Mar; Roca, Pilar; Oliver, Jordi

    2013-09-01

    Genistein is a biologically active isoflavone with estrogenic activity and can be found in a variety of soy products. This natural compound displays a wide array of biological activities, but it is best known for its ability to inhibit cancer progression, especially for hormone-related ones such as breast cancer. Genistein has been shown to bind both the estrogen receptor alpha (ERα) and the estrogen receptor beta (ERβ), although it has a higher affinity for the ERβ. The ERα/ERβ ratio is a prognostic marker for breast tumors, and ERβ expression could indicate the presence of tumors more benign in state, whereas ERα indicates malignant tumors. The objective of the present study was to investigate the effects of genistein on oxidative stress and mitochondrial functionality through its interaction with the estrogen receptor in breast cancer cell lines with different ERα/ERβ ratios. The lower ERα/ERβ ratio T47D cell line showed lower oxidative stress and greater mitochondrial functionality, along with an up-regulation of uncoupling protein 2 and sirtuins. On the other hand, genistein-treated MCF-7 cell line, with the highest ERα/ERβ ratio, reported no changes for the control situation. On the whole, our results show different genistein effects depending on ERα/ERβ ratio for oxidative stress regulation, mitochondrial functionality, and modulation of UCPs, antioxidant enzymes and sirtuins in breast cancer cell lines. Effects of genistein on oxidative stress and mitochondria could be due at least in part, to a higher ERβ presence, but could also be due to up-regulation of ERβ caused by the genistein treatment. PMID:23871935

  11. Supplementation with l-arginine stabilizes plasma arginine and nitric oxide metabolites, suppresses elevated liver enzymes and peroxidation in sickle cell anaemia.

    PubMed

    Jaja, S I; Ogungbemi, S O; Kehinde, M O; Anigbogu, C N

    2016-06-01

    The effect of l-arginine on liver function in SCD has received little or no attention. The effect of a chronic, oral, low-dose supplementation with l-arginine (1gm/day for 6 weeks) on some liver enzymes, lipid peroxidation and nitric oxide metabolites was studied in 20 normal (non-sickle cell anaemia; NSCA) subjects and 20 sickle cell anaemia (SCA) subjects. Ten milliliters of blood was withdrawn from an ante-cubital vein for the estimation of plasma arginine concentration ([R]), alanine aminotransaminase (ALT), aspartate aminotransaminase (AST) and alkaline phosphatase (ALP), plasma total bilirubin concentration [TB], malondialdehyde concentration [MDA] and nitric oxide metabolites concentration [NOx]. Before supplementation, ALT, AST, ALP (p<0.05 respectively) and TB (p<0.001) were higher in SCA subjects than in NSCA subjects. [R] and [NOx] were higher in NSCA subjects (p<0.001 and p<0.05 respectively). Supplementation caused greater percent increases in [R], and [NOX] in SCA than in NSCA subjects (p<0.001 in each case). l-Arginine caused greater percent reductions in ALT and AST in SCA subjects but greater percent reduction in ALP in NSCA subjects (p<0.001 in each case). Changes in [MDA] and [TB] in the two groups were similar. Study shows that chronic, oral, low-dose supplementation with l-arginine improved liver function, oxidative stress, plasma arginine concentration and nitric oxide metabolites levels in NSCA and SCA subjects. Responses in SCA subjects to l-arginine were more sensitive than in NSCA subjects. PMID:27156372

  12. Effect of slaughter age on the antioxidant enzyme activity, color, and oxidative stability of Korean Hanwoo (Bos taurus coreanae) cow beef.

    PubMed

    Cho, Soohyun; Kang, Geunho; Seong, Pil-Nam; Park, Beomyoung; Kang, Sun Moon

    2015-10-01

    This study investigated the effect of slaughter age on the antioxidant enzyme activity, lipid and protein oxidation, and color stability in striploins (M. longissimus lumborum) from Korean Hanwoo (Bos taurus coreanae) cows of different age groups (1.9 to 3.7, 4.0 to 4.8, 5.0 to 5.7, 6.0 to 6.9, and 7.5 to 11.5yr). Myoglobin content and the activities of catalase, superoxide dismutase, and glutathione peroxidase were significantly (P<0.05) increased in older cow beef. During refrigerated storage, 2-thiobarbituric acid reactive substances and protein carbonyls were significantly (P<0.05) increased in the meat from the older cows. The beef from older cows was darker and had lower color stability. These findings suggest that slaughter age has a negative effect on the color and lipid stability of Hanwoo cow beef. PMID:26025115

  13. Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

    PubMed

    Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. PMID:22770942

  14. Nitric oxide (NO), citrulline-NO cycle enzymes, glutamine synthetase, and oxidative status in kainic acid-mediated excitotoxicity in rat brain.

    PubMed

    Swamy, Mummedy; Sirajudeen, Kuttulebbai N S; Chandran, Govindasamy

    2009-01-01

    Neuronal excitation, involving the excitatory glutamate receptors, is recognized as an important underlying mechanism in neurodegenerative disorders. To understand their role in excitotoxicity, the nitric oxide synthase (NOS), argininosuccinate synthetase (AS), argininosuccinate lyase (AL), glutamine synthetase (GS), and arginase activities, along with the concentration of nitrate/nitrite, thiobarbituric acid-reactive substances (TBARS), and total antioxidant status (TAS), were estimated in the cerebral cortex, cerebellum, and brain stem of rats subjected to kainic acid-mediated excitotoxicity. The results of this study clearly demonstrated the increased production of NO by increased activity of NOS. The increased activities of AS and AL suggest the increased and effective recycling of citrulline to arginine in excitotoxicity, making NO production more effective and contributing to its toxic effects. The decreased activity of GS may favor the prolonged availability of glutamic acid, causing excitotoxicity, leading to neuronal damage. The increased formation of TBARS and decreased TAS indicate the presence of oxidative stress in excitotoxicity. PMID:19793024

  15. Effect of different methods of hypoxic exercise training on free radical oxidation and antioxidant enzyme activity in the rat brain.

    PubMed

    Li, Jie; Wang, Yuxia

    2013-11-01

    The effects of different modes of hypoxic exercise training on free radical production and antioxidant enzyme activity in the brain of rats were investigated in this study. A total of 40 healthy 2-month-old male Wister rats were randomly assigned to 5 groups according to different training modes. Endurance training sessions were performed for 5 weeks under different normoxic (atmospheric pressure ~632 mmHg, altitude ~1,500 m) and hypoxic conditions (atmospheric pressure ~493 mmHg, altitude ~3,500 m) at the same relative intensity. The superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activity and the malondialdehyde (MDA) content of the brain were evaluated by spectrophotometric analysis. Compared to the low-training low (LL) group, the SOD activity was significantly increased by 68.73, 54.28 and 304.02% in the high-training high (HH), high-training low (HL) and high-exercise high-training low (HHL) groups, respectively. However, no obvious change was observed for the low-training high (LH) group. In comparison to the LL group, the GSH-Px activity was found to be significantly higher in the HH, HL, LH and HHL groups. Similarly, in comparison to the LL group, the CAT activity exhibited a significant increase in the HH, HL, LH and HHL groups. Compared to the LL group, the MDA content was significantly increased in the HH, HL and HHL groups, although no significant difference was detected for the LH group. Following exhaustive exercise, the antioxidant enzyme activities in the rat brains were immediately improved in all the hypoxia modes. Moreover, the free radical production was increased after all the modes of hypoxic exercise training, with the LH mode being the only exception. PMID:24649054

  16. Effects of small peptides, probiotics, prebiotics, and synbiotics on growth performance, digestive enzymes, and oxidative stress in orange-spotted grouper, Epinephelus coioides, juveniles reared in artificial seawater

    NASA Astrophysics Data System (ADS)

    Wang, Tao; Cheng, Yongzhou; Chen, Xiaoyan; Liu, Zhaopu; Long, Xiaohua

    2016-04-01

    Aquaculture production efficiency may increase by using feed additives. This study investigated the effects of diff erent dietary additives [w/w: 2% small peptides, 0.01% probiotics (Bacillus licheniformis) and 0.2% prebiotics (inulin)] on growth performance, digestive enzyme activities, and oxidative stress in juvenile Epinephelus coioides reared in artificial seawater of two salt concentrations (13.5 vs. 28.5). Weight gain rate was significantly higher in fish fed the diet supplemented with small peptides, B. licheniformis, inulin, or synbiotics than that in fish fed the basal diet; the greatest weight gain rate was found in fish fed the small peptide treatment [56.0% higher than basal diet]. Higher feed efficiency was detected in fish fed the diet supplemented with small peptides than that of fish in the other dietary treatments. Total protease activity in the stomach and intestines was highest in fish fed the small peptide-treated diet, whereas lipase activity was highest in those fed synbiotics (combination of Bacillus licheniformis and inulin) than that in fish fed the other treatments. Antioxidant enzyme (total superoxide dismutase and catalase) activities and hepatic malondialdehyde content were higher in fish receiving the dietary supplements and maintained in artificial seawater containing 13.5 salinity compared with those in the control (28.5). Hepatic catalase activity in grouper fed the diets with small peptides or synbiotics decreased significantly compared with that in control fish. Overall, the three types of additives improved growth rate of juvenile grouper and digestive enzymes activities to varying degrees but did not effectively improve antioxidant capacity under low-salinity stress conditions.

  17. Juniperus communis Linn oil decreases oxidative stress and increases antioxidant enzymes in the heart of rats administered a diet rich in cholesterol.

    PubMed

    Gumral, Nurhan; Kumbul, Duygu Doguc; Aylak, Firdevs; Saygin, Mustafa; Savik, Emin

    2015-01-01

    It has been asserted that consumption of dietary cholesterol (Chol) raises atherosclerotic cardiovascular diseases and that Chol causes an increase in free radical production. Hypercholesterolemic diet has also been reported to cause changes in the antioxidant system. In our study, different doses of Juniperus communis Linn (JCL) oil, a tree species growing in Mediterranean and Isparta regions and having aromatic characteristics, were administered to rats; and the levels of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and thiobarbituric acid reactive substances assay (TBARS) were examined in the heart tissue of rats. In this study, 35 Wistar Albino male adult rats weighing approximately 250-300 g were used. The rats were divided into five groups of seven each. The control group was administered normal pellet chow, and the Chol group was administered pellet chow including 2% Chol, while 50 JCL, 100 JCL, and 200 JCL groups were administered 50, 100, and 200 mg/kg JCL oil dissolved in 0.5% sodium carboxy methyl cellulose, respectively, in addition to the pellet chow containing 2% Chol, by gavage. After 30 days, the experiment was terminated and the antioxidant enzyme activities were examined in the heart tissue of rats. While consumption of dietary Chol decreases the activities of SOD, GSH-Px, and CAT in heart tissue of rats (not significant), administeration of 200 mg/kg JCL oil in addition to Chol led to a significant increase in the activity of antioxidant enzymes. Administering Chol led to a significant increase in TBARS level. Administering 100 and 200 mg/kg JCL oil together with Chol prevented significantly the increase in lipid peroxides. As a result of the study, JCL oil showed oxidant-antioxidant effect in the heart tissue of rats. PMID:23293127

  18. Priming of pathogenesis related-proteins and enzymes related to oxidative stress by plant growth promoting rhizobacteria on rice plants upon abiotic and biotic stress challenge.

    PubMed

    García-Cristobal, J; García-Villaraco, A; Ramos, B; Gutierrez-Mañero, J; Lucas, J A

    2015-09-01

    Two plant growth promoting rhizobacteria (PGPR) were tested to evaluate their capacity to prime rice seedlings against stress challenge (salt and Xanthomonas campestris infection). As is accepted that plants respond to biotic and abiotic stresses by generation of reactive oxygen species (ROS), enzyme activities related to oxidative stress (ascorbate peroxidase (APX, EC 1.11.1.11), guaiacol peroxidase (GPX, EC 1.11.1.7), glutathione reductase (GR, EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1)) as well as the pathogenesis-related proteins (PRs) ß-1,3-glucanase (PR2, EC 3.2.1.6) and chitinase (PR3, EC 3.2.1.14) were measured at 3 time points after stress challenge. In addition, photosynthetic parameters related with fluorescence emission of photosystem II (F0, Fv/Fm, ΦPSII and NPQ) were also measured although they were barely affected. Both strains were able to protect rice seedlings against salt stress. AMG272 reduced the salt symptoms over 47% with regard to control, and L81 over 90%. Upon pathogen challenge, 90% protection was achieved by both strains. All enzyme activities related to oxidative stress were modified by the two PGPR, especially APX and SOD upon salinity stress challenge, and APX and GR upon pathogen presence. Both bacteria induced chitinase activity 24 and 48 h after pathogen inoculation, and L81 induced ß-1,3-Glucanase activity 48 h after pathogen inoculation, evidencing the priming effect. These results indicate that these strains could be used as bio-fortifying agents in biotechnological inoculants in order to reduce the effects of different stresses, and indirectly reduce the use of agrochemicals. PMID:26439659

  19. Changes of platelet antioxidative enzymes during oxidative stress: the protective effect of polyphenol-rich extract from berries of Aronia melanocarpa and grape seeds.

    PubMed

    Kedzierska, Magdalena; Olas, Beata; Wachowicz, Barbara; Stochmal, Anna; Oleszek, Wiesław; Erler, Joachim

    2011-01-01

    Aronia melanocarpa fruits (Rosaceae) and grape seeds (seeds of Vitis vinifera, Vitaceae) are two of the richest plant sources of phenolic substances, and they have been shown to have various biological activities. The aim of the present study was to investigate and compare the action of phenolic extracts (at concentrations 5-100 µg/mL) of two different plants, berries of A. melanocarpa (chokebbery) and grape seeds, on the activities of various antioxidative enzymes, the amount of glutathione (as an important component of redox status) in control the platelets and platelets treated with H(2)O(2) (the strong physiological oxidant) in vitro. The properties of these two tested extracts were also compared with the action of a well characterized antioxidative and antiplatelet commercial monomeric polyphenol - resveratrol. The extract from berries of A. melanocarpa, like the extract from grape seeds, reduced the changes in activities of different antioxidative enzymes (glutathione peroxidase, superoxide dismutase, and catalase) in platelets treated with H(2)O(2). The action of the two tested plant extracts and H(2)O(2) evoked a significant increase of reduced glutathione in platelets compared with platelets treated with H(2)O(2) only. Comparative studies indicate that the two tested plant extracts had similar antioxidative properties, and were found to be more reactive in blood platelets than the solution of resveratrol. PMID:21299394

  20. The structure and function of Alzheimer's gamma secretase enzyme complex.

    PubMed

    Krishnaswamy, Sudarsan; Verdile, Giuseppe; Groth, David; Kanyenda, Limbikani; Martins, Ralph N

    2009-01-01

    The production and accumulation of the beta amyloid protein (Abeta) is a key event in the cascade of oxidative and inflammatory processes that characterizes Alzheimer's disease (AD). A multi-subunit enzyme complex, referred to as gamma (gamma) secretase, plays a pivotal role in the generation of Abeta from its parent molecule, the amyloid precursor protein (APP). Four core components (presenilin, nicastrin, aph-1, and pen-2) interact in a high-molecular-weight complex to perform intramembrane proteolysis on a number of membrane-bound proteins, including APP and Notch. Inhibitors and modulators of this enzyme have been assessed for their therapeutic benefit in AD. However, although these agents reduce Abeta levels, the majority have been shown to have severe side effects in pre-clinical animal studies, most likely due to the enzymes role in processing other proteins involved in normal cellular function. Current research is directed at understanding this enzyme and, in particular, at elucidating the roles that each of the core proteins plays in its function. In addition, a number of interacting proteins that are not components of gamma-secretase also appear to play important roles in modulating enzyme activity. This review will discuss the structural and functional complexity of the gamma-secretase enzyme and the effects of inhibiting its activity. PMID:19958215

  1. Characterizing metabolic inhibition using electrochemical enzyme/DNA biosensors.

    PubMed

    Hull, Dominic O; Bajrami, Besnik; Jansson, Ingela; Schenkman, John B; Rusling, James F

    2009-01-15

    Studies of metabolic enzyme inhibition are necessary in drug development and toxicity investigations as potential tools to limit or prevent appearance of deleterious metabolites formed, for example, by cytochrome (cyt) P450 enzymes. In this paper, we evaluate the use of enzyme/DNA toxicity biosensors as tools to investigate enzyme inhibition. We have examined DNA damage due to cyt P450cam metabolism of styrene using DNA/enzyme films on pyrolytic graphite (PG) electrodes monitored via Ru(bpy)(3)(2+)-mediated DNA oxidation. Styrene metabolism initiated by hydrogen peroxide was evaluated with and without the inhibitors, imidazole, imidazole-4-acetic acid, and sulconazole (in micromolar range) to monitor DNA damage inhibition. The initial rates of DNA damage decreased with increased inhibitor concentrations. Linear and nonlinear fits of Michaelis-Menten inhibition models were used to determine apparent inhibition constants (K(I)*) for the inhibitors. Elucidation of the best fitting inhibition model was achieved by comparing correlation coefficients and the sum of the square of the errors (SSE) from each inhibition model. Results confirmed the utility of the enzyme/DNA biosensor for metabolic inhibition studies. A simple competitive inhibition model best approximated the data for imidazole, imidazole-4-acetic acid and sulconazole with K(I)* of 268.2, 142.3, and 204.2 microM, respectively. PMID:19099359

  2. Characterizing Metabolic Inhibition Using Electrochemical Enzyme-DNA Biosensors

    PubMed Central

    Hull, Dominic O.; Bajrami, Besnik; Jansson, Ingela; Schenkman, John B.; Rusling, James F.

    2009-01-01

    Studies of metabolic enzyme inhibition are necessary in drug development and toxicity investigations as potential tools to limit or prevent appearance of deleterious metabolites formed, for example by cytochrome (cyt) P450 enzymes. In this paper, we evaluate the use of enzyme/DNA toxicity biosensors as tools to investigate enzyme inhibition. We have examined DNA damage due to cyt P450cam metabolism of styrene using DNA/enzyme films on pyrolytic graphite (PG) electro*des monitored via Ru(bpy)32+–mediated DNA oxidation. Styrene metabolism initiated by hydrogen peroxide was evaluated with and without the inhibitors, imidazole, imidazole-4-acetic acid and sulconazole (in micromolar range) to monitor DNA damage inhibition. The initial rates of DNA damage decreased with increased inhibitor concentrations. Linear and nonlinear fits of Michaelis-Menten inhibition models were used to determine apparent inhibition constants (KI*) for the inhibitors. Elucidation of the best fitting inhibition model was achieved by comparing correlation coefficients and the sum of the square of the errors (SSE) from each inhibition model. Results confirmed the utility of the enzyme/DNA biosensor for metabolic inhibition studies. A simple competitive inhibition model best approximated the data for imidazole, imidazole-4-acetic acid and sulconazole with KI* of 268.2, 142.3 and 204.2 µM, respectively. PMID:19099359

  3. Monitoring of dihydroxyacetone production during oxidation of glycerol by immobilized Gluconobacter oxydans cells with an enzyme biosensor.

    PubMed

    Tkác, J; Navrátil, M; Sturdík, E; Gemeiner, P

    2001-03-01

    A bi-enzymatic biosensor for monitoring of dihydroxyacetone production during oxidation of glycerol by bacterial cells of Gluconobacter oxydans is presented. Galactose oxidase oxidizes dihydroxyacetone efficiently producing hydrogen peroxide, which reacts with co-immobilized peroxidase and ferrocene pre-adsorbed on graphite electrode. This mediator-based bi-enzymatic biosensor possesses very high sensitivity (4.7 µA/mM in phosphate buffer), low detection limit (0.8 µM, signal/noise = 3), short response time (22 s, 95% of steady-state) and broad linear range (0.002-0.55 mM in phosphate buffer). The effect of pH, temperature, type of buffer, as well as different stabilizers (combinations of a polyelectrolyte and a polyol) on the sensor performance were carefully optimized and discussed. Dihydroxyacetone produced during a batch conversion of glycerol by the pectate-immobilized bacteria in an air-lift reactor was determined by the biosensor and by reference spectrophotometric method. Both methods were compared and were in a very good correlation. The main advantage of the biosensor is a very short time needed for sample analysis (less than 1 min). PMID:11240195

  4. The impact of individual cytochrome P450 enzymes on oxidative metabolism of benzo[a]pyrene in human livers

    PubMed Central

    Šulc, Miroslav; Indra, Radek; Moserová, Michaela; Schmeiser, Heinz H.; Frei, Eva; Arlt, Volker M.; White, P.

    2016-01-01

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. In this study human recombinant CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5) were expressed in Supersomes™ together with their reductases, NADPH:CYP oxidoreductase, epoxide hydrolase and cytochrome b5, to investigate BaP metabolism. Human CYPs produced up to eight BaP metabolites. Among these, BaP‐7,8‐dihydrodiol and BaP‐9‐ol, which are intermediates in BaP‐derived DNA adduct formation, were mainly formed by CYP1A1 and 1B1, and to a lesser extent by CYP2C19 and 3A4. BaP‐3‐ol, a metabolite that is a ‘detoxified’ product of BaP, was formed by most human CYPs tested, although CYP1A1 and 1B1 produced it the most efficiently. Based on the amounts of the individual BaP metabolites formed by these CYPs and their expression levels in human liver, we determined their contributions to BaP metabolite formation in this organ. Our results indicate that hepatic CYP1A1 and CYP2C19 are most important in the activation of BaP to BaP‐7,8‐dihydrodiol, whereas CYP2C19, 3A4, and 1A1 are the major enzymes contributing to the formation of BaP‐9‐ol. BaP‐3‐ol is predominantly formed by hepatic CYP3A4, while CYP1A1 and 2C19 are less active. Environ. Mol. Mutagen. 57:229–235, 2016. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc. PMID:26919089

  5. Effects of acute ammonia toxicity on nitric oxide (NO), citrulline-NO cycle enzymes, arginase and related metabolites in different regions of rat brain.

    PubMed

    Swamy, M; Zakaria, Adlin Zafrulan; Govindasamy, Chandran; Sirajudeen, K N S; Nadiger, H A

    2005-10-01

    Nitric oxide (NO) is involved in many pathophysiological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS) enzymes. Citrulline formed as a by-product of the NOS reaction, can be recycled to arginine by successive actions of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) via the citrulline-NO cycle. Hyperammonemia is known to cause poorly understood perturbations of the citrulline-NO cycle. To understand the role of citrulline-NO cycle in hyperammonemia, NOS, ASS, ASL and arginase activities, as well as nitrate/nitrite (NOx), arginine, ornithine, citrulline, glutamine, glutamate and GABA were estimated in cerebral cortex (CC), cerebellum (CB) and brain stem (BS) of rats subjected to acute ammonia toxicity. NOx concentration and NOS activity were found to increase in all the regions of brain in acute ammonia toxicity. The activities of ASS and ASL showed an increasing trend whereas the arginase was not changed. The results of this study clearly demonstrated the increased formation of NO, suggesting the involvement of NO in the pathophysiology of acute ammonia toxicity. The increased activities of ASS and ASL suggest the increased and effective recycling of citrulline to arginine in acute ammonia toxicity, making NO production more effective and contributing to its toxic effects. PMID:16009439

  6. Enzyme activities associated with oxidative stress in Metarhizium anisopliae during germination, mycelial growth, and conidiation and in response to near-UV irradiation.

    PubMed

    Miller, Charles D; Rangel, Drauzio; Braga, Gilberto U L; Flint, Stephan; Kwon, Sun-Il; Messias, Claudio L; Roberts, Donald W; Anderson, Anne J

    2004-01-01

    Metarhizium anisopliae isolates have a wide insect host range, but an impediment to their commercial use as a biocontrol agent of above-ground insects is the high susceptibility of spores to the near-UV present in solar irradiation. To understand stress responses in M. anisopliae, we initiated studies of enzymes that protect against oxidative stress in two strains selected because their spores differed in sensitivity to UV-B. Spores of the more near-UV resistant strain in M. anisopliae 324 displayed different isozyme profiles for catalase-peroxidase, glutathione reductase, and superoxide dismutase when compared with the less resistant strain 2575. A transient loss in activity of catalase-peroxidase and glutathione reductase was observed during germination of the spores, whereas the intensity of isozymes displaying superoxide dismutase did not change as the mycelium developed. Isozyme composition for catalase-peroxidases and glutathione reductase in germlings changed with growth phase. UV-B exposure from lamps reduced the activity of isozymes displaying catalase-peroxidase and glutathione reductase activities in 2575 more than in 324. The major effect of solar UV-A plus UV-B also was a reduction in catalase-peroxidases isozyme level, a finding confirmed by measurement of catalase specific activity. Impaired growth of M. anisopliae after near-UV exposure may be related to reduced abilities to handle oxidative stress. PMID:15052320

  7. Crocin Abrogates Carbon Tetrachloride-Induced Renal Toxicity in Rats via Modulation of Metabolizing Enzymes and Diminution of Oxidative Stress, Apoptosis, and Inflammatory Cytokines.

    PubMed

    Hassan, Memy H; Bahashawan, Saleh A; Abdelghany, Tamer M; Abd-Allah, Gamil M; Ghobara, Mohamed M

    2015-07-01

    This work aimed at investigating the potential modulatory effects and mechanisms of crocin against CCl4 -induced nephrotoxicity. Forty male rats were allocated for three weeks treatment with corn oil, CCl4 , crocin, or crocin plus CCl4 . Crocin effectively mitigated CCl4 -induced kidney injury as evidenced by amelioration of alterations in kidney histopathology, renal weight/100 g body weight ratio and kidney functions. Crocin modulated CCl4 -induced disturbance of kidney cytochrom-P450 subfamily 2E1 and glutathione-S-transferase. The attenuation of crocin to kidney injury was also associated with suppression of oxidative stress via reduction of lipid peroxides along with induction of renal glutathione content and enhancement of superoxide dismutase, glutathione peroxidase, and catalase activities. Crocin mitigated CCl4 -induced elevation of the renal levels of tumor necrosis factor-alpha, interleukin-6, prostaglandin E2, and active caspases-3. Collectively, crocin alleviated CCl4 -induced renal damage via modulation of kidney metabolizing enzymes, suppression of oxidative stress, inhibition of inflammatory cytokines, PGE2, and active caspase3 in kidney. PMID:25899501

  8. Apigenin-7-Glycoside Prevents LPS-Induced Acute Lung Injury via Downregulation of Oxidative Enzyme Expression and Protein Activation through Inhibition of MAPK Phosphorylation

    PubMed Central

    Li, Kun-Cheng; Ho, Yu-Ling; Hsieh, Wen-Tsong; Huang, Shyh-Shyun; Chang, Yuan-Shiun; Huang, Guan-Jhong

    2015-01-01

    Apigenin-7-glycoside (AP7Glu) with multiple biological activities is a flavonoid that is currently prescribed to treat inflammatory diseases such as upper respiratory infections. Recently, several studies have shown that its anti-inflammatory activities have been strongly linked to the inhibition of secretion of pro-inflammatory proteins, such as inducible nitric oxide synthase (iNOs) and cyclooxygenase-2 (COX-2) induced through phosphorylation nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK) pathways. Additionally, inflammation, which can decrease the activities of antioxidative enzymes (AOEs) is also observed in these studies. At the same time, flavonoids are reported to promote the activities of heme oxygenase-1 (HO-1) decreased by LPS. The purpose of this study was to assess these theories in a series of experiments on the suppressive effects of AP7Glu based on LPS-induced nitric oxide production in RAW264.7 macrophages in vitro and acute lung injury in mice in vivo. After six hours of lipopolysaccharide (LPS) stimulation, pulmonary pathological, myeloperoxidase (MPO) activity, total polymorphonuclear leukocytes (PMN) cells, cytokines in bronchoalveolar lavage fluid (BALF) and AOEs, are all affected and changed. Meanwhile, our data revealed that AP7Glu not only did significantly inhibit the LPS-enhanced inflammatory activity in lung, but also exhibited anti-inflammatory effect through the MAPK and inhibitor NF-κB (IκB) pathways. PMID:25590301

  9. Antioxidant-enzyme reaction to the oxidative stress due to alpha-cypermethrin, chlorpyriphos, and pirimicarb in tomato (Lycopersicon esculentum Mill.).

    PubMed

    Chahid, Karim; Laglaoui, Amin; Zantar, Said; Ennabili, Abdeslam

    2015-11-01

    Tomato (Lycopersicon esculentum Mill.) becomes one of the world's foremost vegetables, and its world production and consumption have increased fairly quickly. The capacity to induce oxidative stress in tomato plant, exposed to three xenobiotics such as alpha-cypermethrin, chlorpyriphos, and pirimicarb, was investigated by the evaluation of lipid peroxidation by measuring malondialdehyde (MDA) rate; also, we studied the response of tomato to this stress by assessing the response of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione-s-transferase (GST), and glutathione reductase (GR). The effect of the insecticides was observed using four concentrations (25, 50, 75, and 100%) for germinating seeds and only the recommended concentration in agriculture (100%) for growing plants. Our results show an important accumulation of MDA, demonstrating the increase of lipid peroxidation in consequence of the excessive reactive oxygen species (ROS) production due to insecticide treatment. In response to this oxidative stress in tomato seedlings and plants, the activities of antioxidant-enzyme system were generally enhanced. The electrophoretic analysis showed also the apparition of new isoenzymes as the case for CAT and POD. PMID:26178835

  10. The effect of sumatriptan on nitric oxide synthase enzyme production after iatrogenic inflammation in the brain stem of adolescent rats: A randomized, controlled, experimental study

    PubMed Central

    Demirpence, Savas; Kurul, Semra Hiz; Kiray, Müge; Tugyan, Kazim; Yilmaz, Osman; Köse, Galip

    2009-01-01

    Background: Migraine is a common disabling disorder of childhood and adolescence. Despite advances in the understanding of migraine pathophysiology, treatment remains a challenge. Objectives: The aims of this study were to investigate the production of nitric oxide synthase (NOS) enzymes in the brain stem of adolescent rats, using an experimental model of migraine, and the effect of sumatriptan pretreatment on the production of the NOS enzymes. Methods: Male adolescent (aged ~2 months) Wistar rats were used in the study. The animals were anesthetized using pentobarbital. The trigeminovascular system was stimulated by injecting a proinflammatory molecule, carrageenan, into the cis-terna magna of the anesthetized rats. The animals were divided into 3 groups of equal size: (1) the study group, in which the rats were treated with sumatriptan succinate 2 hours before intracisternal carrageenan injection; (2) the sham group, in which the rats were not administered intracisternal carrageenan injection or sumatriptan pretreatment; and (3) the control group, in which the rats were administered intracisternal carrageenan injection but were not pretreated with sumatriptan. In the control and study groups, the rats were euthanized using ether anesthesia 1 hour after intracisternal carrageenan injection. Rats in the sham group were euthanized 1 hour after intracisternal catheterization. Brain tissue was removed and endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) immunohistochemistry was performed. Results: Twenty-one rats were randomized into 3 groups of 7. The mean values of the immunolabeling intensities for eNOS, nNOS, and iNOS enzymes in the brain stem were significantly lower in the sham group compared with the control group (P = 0.001, P = 0.002, and P = 0.001, respectively). The mean values of the immunolabeling intensities of eNOS, nNOS, and iNOS in the brain stem were significantly lower in the study group compared with the control group (P = 0

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

    SciTech Connect

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

    1996-06-01

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

  12. Oxidative stress in deep scattering layers: Heat shock response and antioxidant enzymes activities of myctophid fishes thriving in oxygen minimum zones

    NASA Astrophysics Data System (ADS)

    Lopes, Ana Rita; Trübenbach, Katja; Teixeira, Tatiana; Lopes, Vanessa M.; Pires, Vanessa; Baptista, Miguel; Repolho, Tiago; Calado, Ricardo; Diniz, Mário; Rosa, Rui

    2013-12-01

    Diel vertical migrators, such as myctophid fishes, are known to encounter oxygen minimum zones (OMZ) during daytime in the Eastern Pacific Ocean and, therefore, have to cope with temperature and oxidative stress that arise while ascending to warmer, normoxic surface waters at night-time. The aim of this study was to investigate the antioxidant defense strategies and heat shock response (HSR) in two myctophid species, namely Triphoturus mexicanus and Benthosema panamense, at shallow and warm surface waters (21 kPa, 20-25 °C) and at hypoxic, cold (≤1 kPa, 10 °C) mesopelagic depths. More specifically, we quantified (i) heat shock protein concentrations (HSP70/HSC70) (ii) antioxidant enzyme activities [including superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST)], and (iii) lipid peroxidation [malondialdehyde (MDA) levels]. HSP70/HSC70 levels increased in both myctophid species at warmer, well-oxygenated surface waters probably to prevent cellular damage (oxidative stress) due to increased oxygen demand under elevated temperatures and reactive oxygen species (ROS) formation. On the other hand, CAT and GST activities were augmented under hypoxic conditions, probably as preparatory response to a burst of oxyradicals during the reoxygenation phase (while ascending). SOD activity decreased under hypoxia in B. panamense, but was kept unchanged in T. mexicanus. MDA levels in B. panamense did not change between the surface and deep-sea conditions, whereas T. mexicanus showed elevated MDA and HSP70/HSC70 concentrations at warmer surface waters. This indicated that T. mexicanus seems to be not so well tuned to temperature and oxidative stress associated to diel vertical migrations. The understanding of such physiological strategies that are linked to oxygen deprivation and reoxygenation phases may provide valuable information about how different species might respond to the impacts of environmental stressors (e.g. expanding mesopelagic hypoxia

  13. Inhibitory effect of some tropical green leafy vegetables on key enzymes linked to Alzheimer's disease and some pro-oxidant induced lipid peroxidation in rats' brain.

    PubMed

    Oboh, Ganiyu; Akinyemi, Ayodele Jacobson; Ademiluyi, Adedayo Oluwaseun; Bello, Fatai Olumide

    2014-05-01

    This study sought to investigate the inhibitory effect of some commonly consumed Nigerian green leafy vegetables (raw and blanched) on acetylcholinesterase and butyrylcholinesterase (key enzyme linked to Alzheimer's disease) activities and some pro-oxidants (FeSO4, Sodium nitroprusside and Quinolinic acid) induced lipid peroxidation in rat brain in vitro. Three commonly consumed green leafy vegetables in Nigeria [Amarantus cruentus (Arowojeja), Struchium sparganophora (Ewuro-odo) and Telfairia occidentalis (Ugwu] were blanched in hot water for 10 min, and the extracts of the raw and blanched vegetables were prepared and used for subsequent analysis. The result revealed that all the vegetables inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity as well as the pro-oxidants induced lipid peroxidation in rat brain in a dose dependent manner; however, Amarantus cruentus extract (EC50 = 97.9 μg/ml) had the highest inhibitory effect on acetylcholinesterase activity while Telfairia occidentalis extract (EC50 = 52.7 μg/ml) had the highest inhibitory effect on butyrylcholinesterase activity. However, blanching of the vegetables caused a significant (P < 0.05) decrease in the inhibitory effect of the vegetables on AChE activities while it enhanced the inhibition of the pro-oxidants induced lipid peroxidation in rat brain in vitro. Therefore, some of the possible mechanism by which green leafy vegetables exert their neuroprotective activities could be through the inhibition of acetylcholinesterase and butyrylcholinesterase activities and prevention of lipid peroxidation in the brain. However, blanching of the vegetables could reduce their ability to inhibit acetylcholinesterase and butyrylcholinesterase activity. PMID:24803694

  14. Cytochrome b5 reductase, a plasma membrane redox enzyme, protects neuronal cells against metabolic and oxidative stress through maintaining redox state and bioenergetics.

    PubMed

    Hyun, Dong-Hoon; Lee, Ga-Hyun

    2015-12-01

    The plasma membrane redox system (PMRS) containing NADH-dependent reductases is known to be involved in the maintenance of redox state and bioenergetics. Neuronal cells are very vulnerable to oxidative stress and altered energy metabolism linked to mitochondrial dysfunction. However, the role of the PMRS in these pathways is far from clear. In this study, in order to investigate how cytochrome b5 reductase (b5R), one of the PM redox enzymes, regulates cellular response under stressed conditions, human neuroblastoma cells transfected with b5R were used for viability and mitochondrial functional assays. Cells transfected with b5R exhibited significantly higher levels of the NAD(+)/NADH ratio, consistent with increased levels of b5R activity. Overexpression of b5R made cells more resistant to H2O2 (oxidative stress), 2-deoxyglucose (metabolic stress), rotenone and antimycin A (energetic stress), and lactacystin (proteotoxic stress), but did not protect cells against H2O2 and serum withdrawal. Overexpression of b5R induced higher mitochondrial functions such as ATP production rate, oxygen consumption rate, and activities of complexes I and II, without formation of further reactive oxygen species, consistent with lower levels of oxidative/nitrative damage and resistance to apoptotic cell death. In conclusion, higher NAD(+)/NADH ratio and consequent more efficient mitochondrial functions are induced by the PMRS, enabling them to maintain redox state and energy metabolism under conditions of some energetic stresses. This suggests that b5R can be a target for therapeutic intervention for aging and neurodegenerative diseases. PMID:26611738

  15. Toward structural elucidation of the gamma-secretase complex

    SciTech Connect

    Li, H.; Wolfe, M. S.; Selkoe, D. J.

    2009-03-11

    {gamma}-Secretase is an intramembrane protease complex that mediates the Notch signaling pathway and the production of amyloid {beta}-proteins. As such, this enzyme has emerged as an important target for development of novel therapeutics for Alzheimer disease and cancer. Great progress has been made in the identification and characterization of the membrane complex and its biological functions. One major challenge now is to illuminate the structure of this fascinating and important protease at atomic resolution. Here, we review recent progress on biochemical and biophysical probing of the structure of the four-component complex and discuss obstacles and potential pathways toward elucidating its detailed structure.

  16. Toward structural elucidation of the γ-secretase complex

    PubMed Central

    Li, Huilin; Wolfe, Michael S.; Selkoe, Dennis J.

    2009-01-01

    γ-Secretase is an intramembrane protease complex that mediates the Notch signaling pathway and the production of amyloid β-proteins. As such, this enzyme has emerged as an important target for development of novel therapeutics for Alzheimer disease and cancer. Great progress has been made in the identification and characterization of the membrane complex and its biological functions. One major challenge now is to illuminate the structure of this fascinating and important protease at atomic resolution. Here, we review recent progress on biochemical and biophysical probing of the structure of the four-component complex and discuss barriers and potential pathways toward elucidating its detailed structure. PMID:19278647

  17. Astroglial Contribution to Brain Energy Metabolism in Humans Revealed by 13C Nuclear Magnetic Resonance Spectroscopy: Elucidation of the Dominant Pathway for Neurotransmitter Glutamate Repletion and Measurement of Astrocytic Oxidative Metabolism

    PubMed Central

    Lebon, Vincent; Petersen, Kitt F.; Cline, Gary W.; Shen, Jun; Mason, Graeme F.; Dufour, Sylvie; Behar, Kevin L.; Shulman, Gerald I.; Rothman, Douglas L.

    2010-01-01

    Increasing evidence supports a crucial role for glial metabolism in maintaining proper synaptic function and in the etiology of neurological disease. However, the study of glial metabolism in humans has been hampered by the lack of noninvasive methods. To specifically measure the contribution of astroglia to brain energy metabolism in humans, we used a novel noninvasive nuclear magnetic resonance spectroscopic approach. We measured carbon 13 incorporation into brain glutamate and glutamine in eight volunteers during an intravenous infusion of [2-13C] acetate, which has been shown in animal models to be metabolized specifically in astroglia. Mathematical modeling of the three established pathways for neurotransmitter glutamate repletion indicates that the glutamate/glutamine neurotransmitter cycle between astroglia and neurons (0.32 ± 0.07 μmol · gm−1 · min−1) is the major pathway for neuronal glutamate repletion and that the astroglial TCA cycle flux (0.14 ± 0.06 μmol · gm−1 · min−1) accounts for ~14% of brain oxygen consumption. Up to 30% of the glutamine transferred to the neurons by the cycle may derive from replacement of oxidized glutamate by anaplerosis. The further application of this approach could potentially enlighten the role of astroglia in supporting brain glutamatergic activity and in neurological and psychiatric disease. PMID:11880482

  18. Identification of metabolites from benzo[a]pyrene oxidation by ligninolytic enzymes of Polyporus sp. S133.

    PubMed

    Hadibarata, Tony; Kristanti, Risky Ayu

    2012-11-30

    The biodegradation of benzo[a]pyrene (BaP) by using Polyporus sp. S133, a white-rot fungus isolated from oil-contaminated soil was investigated. Approximately 73% of the initial concentration of BaP was degraded within 30 d of incubation. The isolation and characterization of 3 metabolites by thin layer chromatography, column chromatography, and UV-vis spectrophotometry in combination with gas chromatography-mass spectrometry, indicated that Polyporus sp. S133 transformed BaP to BaP-1,6-quinone. This quinone was further degraded in 2 ways. First, BaP-1,6-quinone was decarboxylated and oxidized to form coumarin, which was then hydroxylated to hydroxycoumarin, and finally to hydroxyphenyl acetic acid by addition of an epoxide group. Second, Polyporus sp. S133 converted BaP-1,6-quinone into a major product, 1-hydroxy-2-naphthoic acid. During degradation, free extracellular laccase was detected with reduced activity of lignin peroxidase, manganese-dependent peroxidase and 2,3-dioxygenase, suggesting that laccase and 1,2-dioxygenase might play an important role in the transformation of PAHs compounds. PMID:22835655

  19. Trimethylamine oxide, betaine and other osmolytes in deep-sea animals: depth trends and effects on enzymes under hydrostatic pressure.

    PubMed

    Yancey, P H; Rhea, M D; Kemp, K M; Bailey, D M

    2004-06-01

    Most shallow teleosts have low organic osmolyte contents, e.g. 70 mmol/kg or less of trimethylamine oxide (TMAO). Our previous work showed that TMAO contents increase with depth in muscles of several Pacific families of teleost fishes, to about 180 mmol/kg wet wt at 2.9 km depth in grenadiers. We now report that abyssal grenadiers (Coryphaenoides armatus, Macrouridae) from the Atlantic at 4.8 km depth contain 261 mmol/kg wet wt in muscle tissue. This precisely fits a linear trend extrapolated from the earlier data. We also found that anemones show a trend of increasing contents of methylamines (TMAO, betaine) and scyllo-inositol with increasing depth. Previously we found that TMAO counteracts the inhibitory effects of hydrostatic pressure on a variety of proteins. We now report that TMAO and, to a lesser extent, betaine, are generally better stabilizers than other common osmolytes (myo-inositol, taurine and glycine), in terms of counteracting the effects of pressure on NADH Km of grenadier lactate dehydrogenase and ADP Km of anemone and rabbit pyruvate kinase. PMID:15529747

  20. Effect of Excess Iodine on Oxidative Stress Markers, Steroidogenic-Enzyme Activities, Testicular Morphology, and Functions in Adult Male Rats.

    PubMed

    Chakraborty, Arijit; Mandal, Jagadis; Mondal, Chiranjit; Sinha, Sabyasachi; Chandra, Amar K

    2016-08-01

    Improper iodine intake is a major concern in public health. Chronic intake of low iodine affects gonadal functions of man and animals; however, such effects of excess iodine in male reproduction, specially on testicular morphology, testicular steroidogenic enzyme activities, sperm morphology, sperm viability, and sperm count including male hormonal profiles in reference to iodine status and thyroid hormone profiles are yet to be explored. With this background, adult male rats of 120 ± 10 gm Bw of 90 ± 5 days were divided broadly in two groups depending on the duration of the treatment for 30 and 60 days, respectively. Both the groups consisted of control animals. Excess iodine (100EI), i.e., 100 times more than its recommended level but within its tolerable ranges, was administered through gavage regularly to the first group of experimental animals for 30 and 60 days, respectively, and excessive iodine (500EI), i.e., 500 times more than its recommended level and above tolerable range in the same way and for the same durations, was administered to the other group of experimental animals. Overall results revealed that regular consumption of iodine in excess impairs reproductive functions in adult male rats depending on the dose and duration of its exposure through different mechanisms. Excess iodine accumulates in the testis which results in generation of reactive oxygen species (ROS) as evidenced by higher lipid peroxidation level as well as an imbalance in the pro-/antioxidant status inhibiting the activity of ∆(5) 3β- hydroxysteroid dehydrogenase (HSD) and 17β-HSD resulting to reduced synthesis of testosterone that causes structural and functional changes of the testis. Secondly, persistent generation of ROS in testis as a result of prolonged excess iodine exposure affects hypothalamo-pituitary-adrenal axis that stimulates synthesis and secretion of corticosterone which inhibits LH release that downregulates testosterone synthesis causing further

  1. Diminution of Hepatic Response to 7, 12-dimethylbenz(α)anthracene by Ethyl Acetate Fraction of Acacia catechu Willd. through Modulation of Xenobiotic and Anti-Oxidative Enzymes in Rats

    PubMed Central

    Kumar, Rakesh; Kaur, Rajbir; Singh, Amrit Pal; Arora, Saroj

    2014-01-01

    Background Liver is the primary metabolizing site of body and is prone to damage by exogenous as well as endogenous intoxicants. Polycyclic aromatic hydrocarbons such as 7, 12- dimethylbenz(α)anthracene (DMBA) is an exogenous hepatotoxin, which is well known for modulating phase I, II and anti-oxidative enzymes of liver. Plants contain plethora of polyphenolic compounds which can reverse the damaging effect of various xenobiotics. The present study investigated protective role of the ethyl acetate fraction of Acacia catechu Willd. (EAF) against DMBA induced alteration in hepatic metabolizing and anti-oxidative enzymes in rats. Methodology and Principal Findings The rats were subjected to hepatic damage by treating with DMBA for 7 weeks on alternative days and treatment schedule was terminated at the end of 14 weeks. The rats were euthanized at the end of protocol and livers were homogenized. The liver homogenates were used to analyse phase I (NADPH-cytochrome P450 reducatse, NADH-cytochrome b5 reductase, cytochrome P420, cytochrome b5), phase II (glutathione-S-transferase, DT diaphorase and γ-Glutamyl transpeptidase) and antioxidative enzymes (catalase, superoxide dismutase, ascorbate peroxidase, glutathione reductase, guiacol peroxidase and lactate dehydrogenase). Furthermore, other oxidative stress parameters (thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes and reduced glutathione) and liver marker enzymes (serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase and alkaline phosphatase) were also studied. The DMBA induced significant changes in activity of hepatic enzymes that was reversed by treatment with three dose levels of EAF. Conclusion It is concluded that EAF affords hepato-protection against DMBA in rats through modulation of phase I, II and anti-oxidative enzymes. PMID:24587216

  2. Effect of selenium-saturated bovine lactoferrin (Se-bLF) on antioxidant enzyme activities in human gut epithelial cells under oxidative stress.

    PubMed

    Burrow, Hannah; Kanwar, Rupinder K; Mahidhara, Ganesh; Kanwar, Jagat R

    2011-10-01

    Cancer and many chronic inflammatory diseases are associated with increased amounts of reactive oxygen species (ROS). The potential cellular and tissue damage created by ROS has significant impact on many disease and cancer states and natural therapeutics are becoming essential in regulating altered redox states. We have shown recently that iron content is a critical determinant in the antitumour activity of bovine milk lactoferrin (bLF). We found that 100% iron-saturated bLF (Fe-bLF) acts as a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy and thus has a broad utility in the treatment of cancer. Furthermore, we also studied the effects of iron saturated bLF's ability as an antioxidant in the human epithelial colon cancer cell line HT29, giving insights into the potential of bLF in its different states. Thus, metal saturated bLF could be implemented as anti-cancer neutraceutical. In this regard, we have recently been able to prepare a selenium (Se) saturated form of bLF, being up to 98% saturated. Therefore, the objectives of this study were to determine how oxidative stress induced by hydrogen peroxide (H2O2) alters antioxidant enzyme activity within HT29 epithelial colon cancer cells, and observe changes in this activity by treatments with different antioxidants ascorbic acid (AA), Apo (iron free)-bLF and selenium (Se)-bLF. The states of all antioxidant enzymes (glutathione peroxidase (GPx), glutathione reductase (GR), glutathione- s-transferase (GsT), catalase and superoxide dismutase (SOD)) demonstrated high levels within untreated HT29 cells compared to the majority of other treatments being used, even prior to H2O2 exposure. All enzymes showed significant alterations in activity when cells were treated with antioxidants AA, Apo-bLF or Se-bLF, with and/or without H2O2 exposure. Obvious indications that the Se content of the bLF potentially interacted with the glutathione (GSH)/GPx/GR/GsT associated redox system could be

  3. The enzymes associated with denitrification

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Tomlinson, G. A.

    1988-01-01

    The enzymes involved in the reduction of nitrogenous oxides are thought to be intermediates in denitrification processes. This review examines the roles of nitrate reductase, nitrite reductases, nitric oxide reductase, mechanisms of N-N bond formation, and nitrous oxide reductases.

  4. Methyl jasmonate counteracts boron toxicity by preventing oxidative stress and regulating antioxidant enzyme activities and artemisinin biosynthesis in Artemisia annua L.

    PubMed

    Aftab, Tariq; Khan, M Masroor A; Idrees, Mohd; Naeem, M; Moinuddin; Hashmi, Nadeem

    2011-07-01

    Boron is an essential plant micronutrient, but it is phytotoxic if present in excessive amounts in soil for certain plants such as Artemisia annua L. that contains artemisinin (an important antimalarial drug) in its areal parts. Artemisinin is a sesquiterpene lactone with an endoperoxide bridge. It is quite expensive compound because the only commercial source available is A. annua and the compound present in the plant is in very low concentration. Since A. annua is a major source of the antimalarial drug and B stress is a deadly threat to its cultivation, the present research was conducted to determine whether the exogenous application of methyl jasmonate (MeJA) could combat the ill effects of excessive B present in the soil. According to the results obtained, the B toxicity induced oxidative stress and reduced the stem height as well as fresh and dry masses of the plant remarkably. The excessive amounts of soil B also lowered the net photosynthetic rate, stomatal conductance, internal CO(2) concentration and total chlorophyll content in the leaves. In contrast, the foliar application of MeJA enhanced the growth and photosynthetic efficiency both in the stressed and non-stressed plants. The excessive B levels also increased the activities of antioxidant enzymes, such as catalase, peroxidase and superoxide dismutase. Endogenous H(2)O(2) and O(2)(-) levels were also high in the stressed plants. However, the MeJA application to the stressed plants reduced the amount of lipid peroxidation and stimulated the synthesis of antioxidant enzymes, enhancing the content and yield of artemisinin as well. Thus, it was concluded that MeJA might be utilized in mitigating the B toxicity and improving the content and yield of artemisinin in A. annua plant. PMID:20957501

  5. Arginase enzymes in isolated airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin

    SciTech Connect

    Bratt, Jennifer M.; Franzi, Lisa M.; Linderholm, Angela L.; Last, Michael S.; Kenyon, Nicholas J. Last, Jerold A.

    2009-02-01

    Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, L-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma. Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses - inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration - were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, N{omega}-hydroxy-nor-L-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals. We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the

  6. Nitric Oxide Synthase Enzymes in the Airways of Mice Exposed to Ovalbumin: NOS2 Expression Is NOS3 Dependent

    PubMed Central

    Bratt, Jennifer M.; Williams, Keisha; Rabowsky, Michelle F.; Last, Michael S.; Franzi, Lisa M.; Last, Jerold A.; Kenyon, Nicholas J.

    2010-01-01

    Objectives and Design. The function of the airway nitric oxide synthase (NOS) isoforms and the lung cell types responsible for its production are not fully understood. We hypothesized that NO homeostasis in the airway is important to control inflammation, which requires upregulation, of NOS2 protein expression by an NOS3-dependent mechanism. Materials or Subjects. Mice from a C57BL/6 wild-type, NOS1−/−, NOS2−/−, and NOS3−/− genotypes were used. All mice strains were systemically sensitized and exposed to filtered air or ovalbumin (OVA) aerosol for two weeks to create a subchronic model of allergen-induced airway inflammation. Methods. We measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled NO, nitrate and nitrite concentration, and airway NOS1, NOS2, and NOS3 protein content. Results. Deletion of NOS1 or NOS3 increases NOS2 protein present in the airway epithelium and smooth muscle of air-exposed animals. Exposure to allergen significantly reduced the expression of NOS2 protein in the airway epithelium and smooth muscle of the NOS3−/− strain only. This reduction in NOS2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express NOS2. NOS1−/− animals had significantly reduced goblet cell metaplasia compared to C57Bl/6 wt, NOS2−/−, and NOS3−/− allergen-exposed mice. Conclusion. The airway epithelial and smooth muscle cells maintain a stable airway NO concentration under noninflammatory conditions. This “homeostatic” mechanism is unable to distinguish between NOS derived from the different constitutive NOS isoforms. NOS3 is essential for the expression of NOS2 under inflammatory conditions, while NOS1 expression contributes to allergen-induced goblet cell metaplasia. PMID:20953358

  7. ARGINASE ENZYMES IN ISOLATED AIRWAYS FROM NORMAL AND NITRIC OXIDE SYNTHASE 2-KNOCKOUT MICE EXPOSED TO OVALBUMIN

    PubMed Central

    Bratt, Jennifer M.; Franzi, Lisa M.; Linderholm, Angela L.; Last, Michael S.; Kenyon, Nicholas J.; Last, Jerold A.

    2009-01-01

    Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, L-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma. Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses---inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration--were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, Nω-hydroxy-nor-L-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals. We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the NOS2

  8. Arginase enzymes in isolated airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin.

    PubMed

    Bratt, Jennifer M; Franzi, Lisa M; Linderholm, Angela L; Last, Michael S; Kenyon, Nicholas J; Last, Jerold A

    2009-02-01

    Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, l-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma. Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses - inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration - were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, Nomega-hydroxy-nor-l-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals. We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the

  9. Enzyme-catalyzed degradation of carbon nanomaterials

    NASA Astrophysics Data System (ADS)

    Kotchey, Gregg P.

    Carbon nanotubes and graphene, the nanoscale sp 2 allotropes of carbon, have garnered widespread attention as a result of their remarkable electrical, mechanical, and optical properties and the promise of new technologies that harness these properties. Consequently, these carbon nanomaterials (CNMs) have been employed for diverse applications such as electronics, sensors, composite materials, energy conversion devices, and nanomedicine. The manufacture and eventual disposal of these products may result in the release of CNMs into the environment and subsequent exposure to humans, animals, and vegetation. Given the possible pro-inflammatory and toxic effects of CNMs, much attention has been focused on the distribution, toxicity, and persistence of CNMs both in living systems and the environment. This dissertation will guide the reader though recent studies aimed at elucidating fundamental insight into the persistence of CNMs such as carbon nanotubes (CNTs) and graphene derivatives (i.e., graphene oxide and reduced graphene oxide). In particular, in-testtube oxidation/degradation of CNMs catalyzed by peroxidase enzymes will be examined, and the current understanding of the mechanisms underlying these processes will be discussed. Finally, an outlook of the current field including in vitro and in vivo biodegradation experiments, which have benefits in terms of human health and environmental safety, and future directions that could have implications for nanomedical applications such as imaging and drug delivery will be presented. Armed with an understanding of how and why CNMs undergo enzyme-catalyzed oxidation/biodegradation, researchers can tailor the structure of CNMs to either promote or inhibit these processes. For example, in nanomedical applications such as drug delivery, the incorporation of carboxylate functional groups could facilitate biodegradation of the nanomaterial after delivery of the cargo. Also, the incorporation of CNMs with defect sites in consumer

  10. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  11. Arginine metabolism: nitric oxide and beyond.

    PubMed Central

    Wu, G; Morris, S M

    1998-01-01

    Arginine is one of the most versatile amino acids in animal cells, serving as a precursor for the synthesis not only of proteins but also of nitric oxide, urea, polyamines, proline, glutamate, creatine and agmatine. Of the enzymes that catalyse rate-controlling steps in arginine synthesis and catabolism, argininosuccinate synthase, the two arginase isoenzymes, the three nitric oxide synthase isoenzymes and arginine decarboxylase have been recognized in recent years as key factors in regulating newly identified aspects of arginine metabolism. In particular, changes in the activities of argininosuccinate synthase, the arginases, the inducible isoenzyme of nitric oxide synthase and also cationic amino acid transporters play major roles in determining the metabolic fates of arginine in health and disease, and recent studies have identified complex patterns of interaction among these enzymes. There is growing interest in the potential roles of the arginase isoenzymes as regulators of the synthesis of nitric oxide, polyamines, proline and glutamate. Physiological roles and relationships between the pathways of arginine synthesis and catabolism in vivo are complex and difficult to analyse, owing to compartmentalized expression of various enzymes at both organ (e.g. liver, small intestine and kidney) and subcellular (cytosol and mitochondria) levels, as well as to changes in expression during development and in response to diet, hormones and cytokines. The ongoing development of new cell lines and animal models using cDNA clones and genes for key arginine metabolic enzymes will provide new approaches more clearly elucidating the physiological roles of these enzymes. PMID:9806879

  12. Activation Energy of Extracellular Enzymes in Soils from Different Biomes

    PubMed Central

    Steinweg, J. Megan; Jagadamma, Sindhu; Frerichs, Joshua; Mayes, Melanie A.

    2013-01-01

    Enzyme dynamics are being incorporated into soil carbon cycling models and accurate representation of enzyme kinetics is an important step in predicting belowground nutrient dynamics. A scarce number of studies have measured activation energy (Ea) in soils and fewer studies have measured Ea in arctic and tropical soils, or in subsurface soils. We determined the Ea for four typical lignocellulose degrading enzymes in the A and B horizons of seven soils covering six different soil orders. We also elucidated which soil properties predicted any measurable differences in Ea. β-glucosidase, cellobiohydrolase, phenol oxidase and peroxidase activities were measured at five temperatures, 4, 21, 30, 40, and 60°C. Ea was calculated using the Arrhenius equation. β-glucosidase and cellobiohydrolase Ea values for both A and B horizons in this study were similar to previously reported values, however we could not make a direct comparison for B horizon soils because of the lack of data. There was no consistent relationship between hydrolase enzyme Ea and the environmental variables we measured. Phenol oxidase was the only enzyme that had a consistent positive relationship between Ea and pH in both horizons. The Ea in the arctic and subarctic zones for peroxidase was lower than the hydrolases and phenol oxidase values, indicating peroxidase may be a rate limited enzyme in environments under warming conditions. By including these six soil types we have increased the number of soil oxidative enzyme Ea values reported in the literature by 50%. This study is a step towards better quantifying enzyme kinetics in different climate zones. PMID:23536898

  13. A new role for an old enzyme: Nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in Arabidopsis thaliana

    PubMed Central

    Desikan, Radhika; Griffiths, Rachael; Hancock, John; Neill, Steven

    2002-01-01

    The plant hormone abscisic acid (ABA), synthesized in response to water-deficit stress, induces stomatal closure via activation of complex signaling cascades. Recent work has established that nitric oxide (NO) is a key signaling molecule mediating ABA-induced stomatal closure. However, the biosynthetic origin of NO in guard cells has not yet been resolved. Here, we provide pharmacological, physiological, and genetic evidence that NO synthesis in Arabidopsis guard cells is mediated by the enzyme nitrate reductase (NR). Guard cells of wild-type Arabidopsis generate NO in response to treatment with ABA and nitrite, a substrate for NR. Moreover, NR-mediated NO synthesis is required for ABA-induced stomatal closure. However, in the NR double mutant, nia1, nia2 that has diminished NR activity, guard cells do not synthesize NO nor do the stomata close in response to ABA or nitrite, although stomatal opening is still inhibited by ABA. Furthermore, by using the ABA-insensitive (ABI) abi1–1 and abi2–1 mutants, we show that the ABI1 and ABI2 protein phosphatases are downstream of NO in the ABA signal-transduction cascade. These data demonstrate a previously uncharacterized signaling role for NR, that of mediating ABA-induced NO synthesis in Arabidopsis guard cells. PMID:12446847

  14. Multiplexed enzyme-free electrochemical immunosensor based on ZnO nanorods modified reduced graphene oxide-paper electrode and silver deposition-induced signal amplification strategy.

    PubMed

    Sun, Guoqiang; Zhang, Lina; Zhang, Yan; Yang, Hongmei; Ma, Chao; Ge, Shenguang; Yan, Mei; Yu, Jinghua; Song, Xianrang

    2015-09-15

    Herein, an origami multiplexed enzyme-free electrochemical (EC) immunodevice is developed for the first time. Typically, ZnO nanorods (ZNRs) modified reduced graphene oxide (rGO)-paper electrode is used as a sensor platform, in which rGO improves the electronic transmission rate and ZNRs provide abundant sites for capture probes binding. Furthermore, by combining the large surface area of rGO and high catalytic activity of bovine serum protein (BSA)-stabilized silver nanoparticles (Ag@BSA) toward H2O2 reduction, rGO/Ag@BSA composites can be used as an excellent signal labels. The current signal is generated from the reduction of H2O2 and further amplified by a subsequent signal labels-promoted deposition of silver. Under optimal conditions, the proposed immunoassays exhibit excellent precision, high sensitivity and a wide linear range of 0.002-120 mIU mL(-1) for human chorionic gonadotropin, 0.001-110 ng mL(-1) for prostate-specific antigen, and 0.001-100 ng mL(-1) for carcinoembryonic antigen. The results for real sample analysis demonstrate that the newly constructed immunosensor arrays provide a simple and cost-effective method for clinical applications. PMID:25884731

  15. Enzyme Informatics

    PubMed Central

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  16. Nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis.

    PubMed

    Gadda, Giovanni; Francis, Kevin

    2010-01-01

    Nitronate monooxygenase (NMO), formerly referred to as 2-nitropropane dioxygenase, is an FMN-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from Neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. Over the past 5 years, a resurgence of interest on the enzymology of NMO has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. This review article summarizes the knowledge gained from these studies on NMO, which has been emerging as a model system for the investigation of anionic flavosemiquinone intermediates in the oxidative catalysis of organic molecules, and for the effect that branching of reaction intermediates has on both the kinetic parameters and isotope effects associated with enzymatic reactions. A comparison of the catalytic mechanism of NMO with other flavin-dependent enzymes that oxidize nitroalkane and nitronates is also presented. PMID:19577534

  17. Ergovaline in tall fescue and its effect on health, milk quality, biochemical parameters, oxidative status, and drug metabolizing enzymes of lactating ewes.

    PubMed

    Zbib, N; Repussard, C; Tardieu, D; Priymenko, N; Domange, C; Guerre, P

    2014-11-01

    Ergovaline (EV) produced by symbiotic association of Epichloë coenophiala with tall fescue (Lolium arundinaceum) causes toxicoses in livestock. In this study, 16 lactating ewes (BW 76.0 ± 0.6 kg) were used to determine the effects of feeding endophyte-infected (FE+) or endophyte free (FE-) tall fescue hay on animal health and performances and to investigate the putative mechanisms of action of EV. The mean EV concentrations in FE+ and FE- diets were 497 ± 52 and <5 µg/kg DM, respectively. Decreased hay consumption and BW were observed in the FE+ group. Prolactin (PRL) concentrations decreased (P < 0.02) in the FE+ group from d 3 to 28 of the study compared to the FE- group, but no consequences were observed on milk quantity or quality. Skin temperature and the thermocirculation index were lower (P < 0.05) in the FE+ than in the FE- group from d 3 to 7, but this effect disappeared from d 14 to 28. Hematocrit, mineral and biochemical, and enzymatic analyses of plasma revealed no differences between the 2 groups. Measurement of oxidative damage and antioxidant enzyme activities revealed a decrease in the activities of plasma catalase (P < 0.05), kidney glutathione reductase and peroxidase and in kidney total glutathione and malondialdehyde contents (P < 0.02) in ewes fed FE+. Hepatic flavin monooxygenase enzyme activities decreased (P < 0.01) in ewes fed FE+, except for a marked increase in the demethylation of erythromycin. This activity is linked to cytochrome P4503A content and is known to be involved in ergot alkaloid metabolism. Glutathione S-transferase activity in the kidneys decreased (P < 0.02) in the FE+ group, whereas no difference was observed in uridine diphosphate-glucuronosyltransferase activity in the liver or kidneys. The reversibility of the effect of FE+ hay on skin temperature and the increase in erythromycin N-demethylase activity may contribute to the relative resistance of ewes to EV toxicity. PMID:25253811

  18. Postnatal exposure to chromium through mother’s milk accelerates follicular atresia in F1 offspring through increased oxidative stress and depletion of antioxidant enzymes

    PubMed Central

    Stanley, Jone A.; Sivakumar, Kirthiram K.; Nithy, Thamizh K.; Arosh, Joe A.; Hoyer, Patricia B.; Burghardt, Robert C.; Banu, Sakhila K.

    2013-01-01

    Hexavalent chromium, CrVI, is a heavy metal endocrine disruptor, known as a mutagen, teratogen, and a group A carcinogen. Environmental contamination with CrVI, including drinking water, has been increasing in more than 30 cities in the United States. CrVI is rapidly converted to CrIII intracellularly, and CrIII can cause DNA strand breaks and cancer or apoptosis through different mechanisms. Our previous study demonstrated that lactational exposure to chromium results in a delay or arrest in follicle development and a decrease in steroid hormone levels in F1 female rats, both of which are mitigated (partial inhibition) by vitamin C. The current study tested the hypothesis that lactational exposure to CrIII accelerates follicle atresia in F1 offspring by increasing reactive oxygen species (ROS) and decreasing cellular antioxidants. Results showed that lactational exposure to CrIII dose-dependently increased follicular atresia and decreased steroidogenesis in postnatal day 25, 45, and 65 rats. Vitamin C mitigated or inhibited the effects of CrIII at all doses. CrIII increased hydrogen peroxide and lipid hydroperoxide in plasma and ovary; decreased the antioxidant enzymes (AOXs) GPx1, GR, SOD, and catalase; and increased glutathione S-transferase in plasma and ovary. To understand the effects of CrVI on ROS and AOXs in granulosa (GC) and theca (TC) cell compartments in the ovary, ROS levels and mRNA expression of cytosolic and mitochondrial AOXs, such as SOD1, SOD2, catalase, GLRX1, GSTM1, GSTM2, GSTA4, GR, TXN1, TXN2, TXNRD2, and PRDX3, were studied in GCs and TCs and in a spontaneously immortalized granulosa cell line (SIGC). Overall, CrVI downregulated each of the AOXs; and vitamin C mitigated the effects of CrVI on these enzymes in GCs and SIGCs, but failed to mitigate CrVI effects on GSTM1, GSTM2, TXN1, and TXN2 in TCs. Thus, these data for the first time reveal that lactational exposure to CrIII accelerated follicular atresia and decreased steroidogenesis in F1

  19. Enzymes, Industrial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes serve key roles in numerous biotechnology processes and products that are commonly encountered in the forms of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes can display regio- and stereo-specificity, p...

  20. Understanding Enzymes.

    ERIC Educational Resources Information Center

    Sinnott, M. L.

    1979-01-01

    Describes the way enzymes operate through reaction energetics, and explains that most of the catalytic power of enzymes lies in the strong noncovalent forces responsible for initial binding of substrate, which are only manifested at the transition state of the reaction. (Author/GA)

  1. Soil Enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The functionality and resilience of natural and managed ecosystems mainly rely on the metabolic abilities of microbial communities, the main source of enzymes in soils. Enzyme mediated reactions are critical in the decomposition of organic matter, cycling of nutrients, and in the breakdown of herbic...

  2. In-silico analysis and mRNA modulation of detoxification enzymes GST delta and kappa against various biotic and abiotic oxidative stressors.

    PubMed

    Chaurasia, Mukesh Kumar; Ravichandran, Gayathri; Nizam, Faizal; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Arshad, Aziz; Harikrishnan, Ramasamy; Arockiaraj, Jesu

    2016-07-01

    This study reports the comprehensive comparative information of two different detoxification enzymes such as glutathione S-transferases (GSTs) delta and kappa from freshwater giant prawn Macrobrachium rosenbergii (designated as MrGSTD and MrGSTK) by investigating their in-silico characters and mRNA modulation against various biotic and abiotic oxidative stressors. The physico-chemical properties of these cDNA and their polypeptide structure were analyzed using various bioinformatics program. The analysis indicated the variation in size of the polypeptides, presence or absence of domains and motifs and structure. Homology and phylogenetic analysis revealed that MrGSTD shared maximum identity (83%) with crustaceans GST delta, whereas MrGSTK fell in arthropods GST kappa. It is interesting to note that MrGSTD and MrGSTK shared only 21% identity; it indicated their structural difference. Structural analysis indicated that MrGSTD to be canonical dimer like shape and MrGSTK appeared to be butterfly dimer like shape, in spite of four β-sheets being conserved in both GSTs. Tissue specific gene expression analysis showed that both MrGSTD and MrGSTK are highly expressed in immune organs such as haemocyte and hepatopancreas, respectively. To understand the role of mRNA modulation of MrGSTD and MrGSTK, the prawns were inducted with oxidative stressors such as bacteria (Vibrio harveyi), virus [white spot syndrome virus (WSSV)] and heavy metal, cadmium (Cd). The analysis revealed an interesting fact that both MrGSTD and MrGSTK showed higher (P < 0.05) up-regulation at 48 h post-challenge, except MrGSTD stressed with bacteria, where it showed up-regulation at 24 h post-challenge. Overall, the results suggested that GSTs are diverse in their structure and possibly conferring their potential involvement in immune protection in crustaceans. However, further study is necessary to focus their functional differences at proteomic level. PMID:27109581

  3. Complex of vitamins and antioxidants protects low-density lipoproteins in blood plasma from free radical oxidation and activates antioxidants enzymes in erythrocytes from patients with coronary heart disease.

    PubMed

    Konovalova, G G; Lankin, V Z; Tikhaze, A K; Nezhdanova, I B; Lisina, M O; Kukharchuk, V V

    2003-08-01

    We studied the effect of a complex containing antioxidant vitamins C and E, provitamin A, and antioxidant element selenium on the contents of primary (lipid peroxides) and secondary products (malonic dialdehyde) of free radical lipid oxidation in low-density lipoproteins isolated from the plasma of patients with coronary heart disease and hypercholesterolemia by means of preparative ultracentrifugation. Activity of key antioxidant enzymes in the blood was measured during treatment with the antioxidant preparation. Combination treatment with antioxidant vitamins and antioxidant element selenium sharply decreased the contents of primary and secondary free radical oxidation products in circulating low-density lipoproteins and increased activity of antioxidant enzymes in erythrocytes. Activities of superoxide dismutase and selenium-containing glutathione peroxidase increased 1 and 2 months after the start of therapy, respectively. PMID:14631494

  4. Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase.

    PubMed Central

    Knight, Kirsty; Scrutton, Nigel S

    2002-01-01

    The reduction by NADPH of the FAD and FMN redox centres in the isolated flavin reductase domain of calmodulin-bound rat neuronal nitric oxide synthase (nNOS) has been studied by anaerobic stopped-flow spectroscopy using absorption and fluorescence detection. We show by global analysis of time-dependent photodiode array spectra, single wavelength absorption and NADPH fluorescence studies, that at least four resolvable steps are observed in stopped-flow studies with NADPH and that flavin reduction is reversible. The first reductive step represents the rapid formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP(+). The second and third steps represent further reduction of the enzyme flavins and NADP(+) release. The fourth step is attributed to the slow accumulation of an enzyme species that is inferred not to be relevant catalytically in steady-state reactions. Stopped-flow flavin fluorescence studies indicate the presence of slow kinetic phases, the timescales of which correspond to the slow phase observed in absorption and NADPH fluorescence transients. By analogy with stopped-flow studies of cytochrome P450 reductase, we attribute these slow fluorescence and absorption changes to enzyme disproportionation and/or conformational change. Unlike for the functionally related cytochrome P450 reductase, transfer of the first hydride equivalent from NADPH to nNOS reductase does not generate the flavin di-semiquinoid state. This indicates that internal electron transfer is relatively slow and is probably gated by NADP(+) release. Release of calmodulin from the nNOS reductase does not affect the kinetics of inter-flavin electron transfer under stopped-flow conditions, although the observed rate of formation of the equilibrium between the NADPH-oxidized enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP(+) is modestly slower in calmodulin-depleted enzyme. Our studies indicate the

  5. Nitrite Reductase and Nitric-oxide Synthase Activity of the Mitochondrial Molybdopterin Enzymes mARC1 and mARC2*

    PubMed Central

    Sparacino-Watkins, Courtney E.; Tejero, Jesús; Sun, Bin; Gauthier, Marc C.; Thomas, John; Ragireddy, Venkata; Merchant, Bonnie A.; Wang, Jun; Azarov, Ivan; Basu, Partha; Gladwin, Mark T.

    2014-01-01

    Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production. PMID:24500710

  6. Effects of angiotensin converting enzyme inhibitor and angiotensin II receptor antagonist combination on nitric oxide bioavailability and atherosclerotic change in Watanabe heritable hyperlipidemic rabbits.

    PubMed

    Imanishi, Toshio; Kuroi, Akio; Ikejima, Hideyuki; Kobayashi, Katsunobu; Muragaki, Yasuteru; Mochizuki, Seiichi; Goto, Masami; Yoshida, Kiyoshi; Akasaka, Takashi

    2008-03-01

    We investigated the effects of co-administration of an angiotensin-converting enzyme inhibitor (ACEI) and angiotensin type 1 receptor blocker (ARB) on nitric oxide (NO) bioavailability in genetically hyperlipidemic rabbits with our newly developed NO sensor. Plasma NO was measured using the new NO sensor in the abdominal aorta of anesthetized Watanabe heritable hyperlipidemic (WHHL) rabbits. Acetylcholine (ACh)-stimulated (20 microg in 5 min into the aortic arch) NO production was recorded after an 8 week per os pretreatment with 1) vehicle (control), 2) the ACEI enalapril (E: 3 mg/kg/day), 3) the ARB losartan (L: 30 mg/kg/day) and 4) enalapril (1.5 mg/kg/day)+losartan (15 mg/kg/day) (E+L). Intra-aortic infusion of ACh produced an increase in plasma NO concentration, which was significantly greater with all the drug treatments than with the control. E increased ACh-induced NO significantly more than L (by 6.9 nmol/L, and 4.7 nmol/L, respectively). E+L increased ACh-induced NO by 9.5 nmol/L, significantly more than either E or L. Plasma peroxynitrite concentration was 1.2 pmol/mg protein in the control group and significantly less than in the E- and L-group. The lowest peroxynitrite concentration was observed in the E+L group (0.5 pmol/mg protein), which was significantly lower than in the E-group and the L-group. Optical coherence tomography and histology of the thoracic aorta revealed that the plaque area decreased significantly more with the combination than with the monotherapy (p<0.01). In conclusion, the combined treatment with an ACEI and an ARB may have additive protective effects on endothelial function as well as atherosclerotic change. PMID:18497479

  7. Highly scalable, uniform, and sensitive biosensors based on top-down indium oxide nanoribbons and electronic enzyme-linked immunosorbent assay.

    PubMed

    Aroonyadet, Noppadol; Wang, Xiaoli; Song, Yan; Chen, Haitian; Cote, Richard J; Thompson, Mark E; Datar, Ram H; Zhou, Chongwu

    2015-03-11

    Nanostructure field-effect transistor (FET) biosensors have shown great promise for ultra sensitive biomolecular detection. Top-down assembly of these sensors increases scalability and device uniformity but faces fabrication challenges in achieving the small dimensions needed for sensitivity. We report top-down fabricated indium oxide (In2O3) nanoribbon FET biosensors using highly scalable radio frequency (RF) sputtering to create uniform channel thicknesses ranging from 50 to 10 nm. We combine this scalable sensing platform with amplification from electronic enzyme-linked immunosorbent assay (ELISA) to achieve high sensitivity to target analytes such as streptavidin and human immunodeficiency virus type 1 (HIV-1) p24 proteins. Our approach circumvents Debye screening in ionic solutions and detects p24 protein at 20 fg/mL (about 250 viruses/mL or about 3 orders of magnitude lower than commercial ELISA) with a 35% conduction change in human serum. The In2O3 nanoribbon biosensors have 100% device yield and use a simple 2 mask photolithography process. The electrical properties of 50 In2O3 nanoribbon FETs showed good uniformity in on-state current, on/off current ratio, mobility, and threshold voltage. In addition, the sensors show excellent pH sensitivity over a broad range (pH 4 to 9) as well as over the physiological-related pH range (pH 6.8 to 8.2). With the demonstrated sensitivity, scalability, and uniformity, the In2O3 nanoribbon sensor platform makes great progress toward clinical testing, such as for early diagnosis of acquired immunodeficiency syndrome (AIDS). PMID:25636984

  8. Immunocytochemical localization of calretinin-containing neurons in the rat periaqueductal gray and colocalization with enzymes producing nitric oxide: a double, double-labeling study.

    PubMed

    Barbaresi, Paolp; Quaranta, Alexia; Amoroso, Salvatore; Mensà, Emanuela; Fabri, Mara

    2012-04-01

    The pattern of distribution and colocalization of the calcium-binding protein calretinin (Cal) and of enzymes producing nitric oxide (NO) was examined in the rat periaqueductal gray matter (PAG) using two different experimental approaches, by combining Cal immunocytochemistry with NADPH-diaphorase (NADPH-d) histochemistry and with NOS immunocytochemistry, respectively. Cal-immunopositive neurons were found throughout the rostrocaudal extension of both dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl). Double-labeled neurons were found only in PAG-dl. The first experimental approach indicated that 33-41% of the NADPH-d-positive (Nadph+) cells were immunoreactive for Cal, whereas NADPH-d activity appeared in 19-26% of the Cal-immunopositive (Cal(IP) ) neurons. Two-color immunofluorescence revealed that ∼39-43% of NOS-immunoreactive (NOS(IR) ) neurons were double-labeled with Cal and ∼23% of Cal(IP) neurons expressed NOS immunoreactivity. Measurement in semithin sections of the size of the three neuronal populations found in PAG-dl, showed that Cal(IP) neurons had a cross-sectional area of 94.7 μm², whereas Nadph+ neurons and double-labeled neurons were slightly smaller, having a cross-sectional area of 90.5 and 91.4 μm², respectively. On electron microscopy, Cal(IP) axon terminals formed either symmetric or asymmetric synapses; although the latter synapses were more numerous, both types contacted preferentially Cal(IP) dendrites. These experiments suggest that PAG-dl is characterized by a high degree of heterogeneity. PMID:22121011

  9. Elucidating the redox cycle of environmental phosphorus using ion chromatography.

    PubMed

    Pech, Herbe; Vazquez, Maria G; Van Buren, Jean; Shi, Lixin; Ivey, Michelle M; Salmassi, Tina M; Pasek, Matthew A; Foster, Krishna L

    2011-09-01

    Historically, it was assumed that reactive, inorganic phosphorus present in pristine environments was solely in the form of orthophosphate. However, this assumption contradicts theories of biogenesis and the observed metabolic behavior of select microorganisms. This paper discusses the role of ion chromatography (IC) in elucidating the oxidation-reduction cycle of environmental phosphorus. These methods employ suppressed-IC, coupled with tandem conductivity and electrospray mass spectrometry detectors to identify and quantify phosphorus oxyanions in natural water, synthetic cosmochemical, and biological samples. These techniques have been used to detect phosphite and orthophosphate in geothermal hot springs. Hypophosphite, phosphite, and orthophosphate have been detected in synthetic schreibersite corrosion samples, and termite extract supernatant. Synthetic schreibersite corrosion samples were also analyzed for two poly-phosphorus compounds, hypophosphate and pyrophosphate, and results show these samples did not contain concentrations above the 1.3 and 2.0 μM respective 3σ limit of detection. These methods are readily adaptable to a variety of matrices, and contribute to the elucidation of the oxidation-reduction cycle of phosphorus oxyanions in the environment. In contrast to most studies, these techniques have been used to show that phosphorus actively participates in redox processes in both the biological and geological world. PMID:21859529

  10. Elucidation of wear mechanisms by ferrographic analysis

    NASA Technical Reports Server (NTRS)

    Jones, W. R., Jr.

    1981-01-01

    The use of ferrographic analysis in conjunction with light and scanning electron microscopy is described for the elucidation of wear mechanisms taking place in operating equipment. Example of adhesive wear, abrasive wear, corrosive wear, rolling element fatigue, lubricant breakdown, and other wear modes are illustrated. In addition, the use of magnetic solutions to precipitate nonmagnetic debris from aqueous and nonaqueous fluids is described.

  11. Does Pictorial Elucidation Foster Recollection of Idioms?

    ERIC Educational Resources Information Center

    Boers, Frank; Piquer Piriz, Ana Maria; Stengers, Helene; Eyckmans, June

    2009-01-01

    Experimental evidence suggests that pictorial elucidation helps learners comprehend and remember the meaning of second language (L2) idioms. In this article we address the question whether it also helps retention of the form of idioms, i.e. their precise lexical composition. In a small-scale experiment, the meaning of English idioms was clarified…

  12. Combinatorial Generation of Chemical Diversity by Redox Enzymes in Chaetoviridin Biosynthesis.

    PubMed

    Sato, Michio; Winter, Jaclyn M; Kishimoto, Shinji; Noguchi, Hiroshi; Tang, Yi; Watanabe, Kenji

    2016-03-18

    Chaetoviridins constitute a large family of structurally related secondary metabolites isolated from Chaetomium fungi. To elucidate the biosynthesis pathway and understand how the chemical diversity of chaetoviridins is generated, gene deletion and in vitro characterization of the four post-PKS modifications enzymes were undertaken. CazL and CazP were identified to have substrate promiscuity that facilitates the formation of nonchlorinated analogues. In addition, enzymatic oxidation and reduction combined with spontaneous dehydration and lactonization of the intermediates further expand the chemical diversity. PMID:26959241

  13. Using Genomics for Natural Product Structure Elucidation.

    PubMed

    Tietz, Jonathan I; Mitchell, Douglas A

    2016-01-01

    Natural products (NPs) are the most historically bountiful source of chemical matter for drug development-especially for anti-infectives. With insights gleaned from genome mining, interest in natural product discovery has been reinvigorated. An essential stage in NP discovery is structural elucidation, which sheds light not only on the chemical composition of a molecule but also its novelty, properties, and derivatization potential. The history of structure elucidation is replete with techniquebased revolutions: combustion analysis, crystallography, UV, IR, MS, and NMR have each provided game-changing advances; the latest such advance is genomics. All natural products have a genetic basis, and the ability to obtain and interpret genomic information for structure elucidation is increasingly available at low cost to non-specialists. In this review, we describe the value of genomics as a structural elucidation technique, especially from the perspective of the natural product chemist approaching an unknown metabolite. Herein we first introduce the databases and programs of interest to the natural products chemist, with an emphasis on those currently most suited for general usability. We describe strategies for linking observed natural product-linked phenotypes to their corresponding gene clusters. We then discuss techniques for extracting structural information from genes, illustrated with numerous case examples. We also provide an analysis of the biases and limitations of the field with recommendations for future development. Our overview is not only aimed at biologically-oriented researchers already at ease with bioinformatic techniques, but also, in particular, at natural product, organic, and/or medicinal chemists not previously familiar with genomic techniques. PMID:26456468

  14. Food Enzymes

    ERIC Educational Resources Information Center

    McBroom, Rachel; Oliver-Hoyo, Maria T.

    2007-01-01

    Many students view biology and chemistry as two unrelated, separate sciences; how these courses are generally taught in high schools may do little to change that impression. The study of enzymes provide a great opportunity for both biology and chemistry teachers to share with students the interdisciplinary nature of science. This article describes…

  15. Zinc Enzymes.

    ERIC Educational Resources Information Center

    Bertini, I.; And Others

    1985-01-01

    Discusses the role of zinc in various enzymes concerned with hydration, hydrolysis, and redox reactions. The binding of zinc to protein residues, properties of noncatalytic zinc(II) and catalytic zinc, and the reactions catalyzed by zinc are among the topics considered. (JN)

  16. Label-free and enzyme-free platform for the construction of advanced DNA logic devices based on the assembly of graphene oxide and DNA-templated AgNCs

    NASA Astrophysics Data System (ADS)

    Fan, Daoqing; Zhu, Jinbo; Liu, Yaqing; Wang, Erkang; Dong, Shaojun

    2016-02-01

    DNA-based molecular logic computation has drawn extensive attention in bioanalysis, intelligent diagnostics of diseases and other nanotechnology areas. Herein, taking 2-to-1 and 4-to-2 encoders and a 1-to-2 decoder as model molecular logic devices, we for the first time combined the quenching ability of GO (graphene oxide) to DNA-templated AgNCs with G-quadruplex-enhanced fluorescence intensity of porphyrin dyes for the construction of label-free and enzyme-free dual-output advanced DNA molecular logic devices. Also, through the application of negative logic conversion to an XOR logic gate and combined with an INHIBIT logic gate, we also operated a label-free and enzyme-free comparator.DNA-based molecular logic computation has drawn extensive attention in bioanalysis, intelligent diagnostics of diseases and other nanotechnology areas. Herein, taking 2-to-1 and 4-to-2 encoders and a 1-to-2 decoder as model molecular logic devices, we for the first time combined the quenching ability of GO (graphene oxide) to DNA-templated AgNCs with G-quadruplex-enhanced fluorescence intensity of porphyrin dyes for the construction of label-free and enzyme-free dual-output advanced DNA molecular logic devices. Also, through the application of negative logic conversion to an XOR logic gate and combined with an INHIBIT logic gate, we also operated a label-free and enzyme-free comparator. Electronic supplementary information (ESI) available: Optimization experiments, Table S1, Fig. S1-S5 in ESI. See DOI: 10.1039/c6nr00032k

  17. Elucidating novel disease mechanisms in severe asthma.

    PubMed

    Kim, Richard Y; Rae, Brittany; Neal, Rachel; Donovan, Chantal; Pinkerton, James; Balachandran, Lohis; Starkey, Malcolm R; Knight, Darryl A; Horvat, Jay C; Hansbro, Philip M

    2016-07-01

    Corticosteroids are broadly active and potent anti-inflammatory agents that, despite the introduction of biologics, remain as the mainstay therapy for many chronic inflammatory diseases, including inflammatory bowel diseases, nephrotic syndrome, rheumatoid arthritis, chronic obstructive pulmonary disease and asthma. Significantly, there are cohorts of these patients with poor sensitivity to steroid treatment even with high doses, which can lead to many iatrogenic side effects. The dose-limiting toxicity of corticosteroids, and the lack of effective therapeutic alternatives, leads to substantial excess morbidity and healthcare expenditure. We have developed novel murine models of respiratory infection-induced, severe, steroid-resistant asthma that recapitulate the hallmark features of the human disease. These models can be used to elucidate novel disease mechanisms and identify new therapeutic targets in severe asthma. Hypothesis-driven studies can elucidate the roles of specific factors and pathways. Alternatively, 'Omics approaches can be used to rapidly generate new targets. Similar approaches can be used in other diseases. PMID:27525064

  18. Elucidating novel disease mechanisms in severe asthma

    PubMed Central

    Kim, Richard Y; Rae, Brittany; Neal, Rachel; Donovan, Chantal; Pinkerton, James; Balachandran, Lohis; Starkey, Malcolm R; Knight, Darryl A; Horvat, Jay C; Hansbro, Philip M

    2016-01-01

    Corticosteroids are broadly active and potent anti-inflammatory agents that, despite the introduction of biologics, remain as the mainstay therapy for many chronic inflammatory diseases, including inflammatory bowel diseases, nephrotic syndrome, rheumatoid arthritis, chronic obstructive pulmonary disease and asthma. Significantly, there are cohorts of these patients with poor sensitivity to steroid treatment even with high doses, which can lead to many iatrogenic side effects. The dose-limiting toxicity of corticosteroids, and the lack of effective therapeutic alternatives, leads to substantial excess morbidity and healthcare expenditure. We have developed novel murine models of respiratory infection-induced, severe, steroid-resistant asthma that recapitulate the hallmark features of the human disease. These models can be used to elucidate novel disease mechanisms and identify new therapeutic targets in severe asthma. Hypothesis-driven studies can elucidate the roles of specific factors and pathways. Alternatively, 'Omics approaches can be used to rapidly generate new targets. Similar approaches can be used in other diseases. PMID:27525064

  19. Estimating rates of denitrification enzyme activity in wetland soils and direct simultaneous quantification of nitrogen and nitrous oxide by membrane inlet mass spectrometry

    EPA Science Inventory

    Denitrification enzyme activity (DEA) was measured in short-term (4 h) anaerobic assays using Membrane Inlet Mass Spectrometry (MIMS) and electron capture gas chromatography (GC-ECD). Using MIMS, modifications of the instrument and sample handling allowed for the simultaneous me...

  20. [The effect of N-stearoylethanolamine on the activity of antioxidant enzymes, content of lipid peroxidation products and nitric oxide in the blood plasma and liver of rats with induced insulin-resistance].

    PubMed

    Onopchenko, O V; Kosiakova, H V; Horid'ko, T M; Berdyshev, A H; Mehed', O F; Hula, N M

    2013-01-01

    The influence of N-stearoylethanolamine (NSE) on the content of lipid peroxidation products, activity of antioxidant enzymes and the nitric oxide level in the liver and blood plasma of rats with insulin-resistance (IR) state was investigated. IR state was induced in rats by prolonged high-fat diet (58% of energy derived from fat) for 6 months combined with one injection of streptozotocin (15 mg/kg of body weight). The existence of IR state was estimated by results of glucoso-tolerance test and blood plasma insulin content. The level of lipid peroxides products was shown to be higher in the liver of insulin resistant animals as a result of reduced superoxide dismutase and catalase activity, however, glutathione peroxidase activity was increased. The increase of nitric-oxide content in the liver and blood plasma of high-fat diet rats compared with healthy control animals was also observed. The administration of the NSE suspension per os in a dose of 50 mg/kg during 2 weeks to the rats with induced insulin-resistance state contributed to the increase of superoxide dismutase, catalase and glutathione peroxidase activity. In consequence of antioxidant enzymes activation the intensity of POL process was decreased. The NSE administration caused normalization of nitric oxide level, restoring pro-/antioxidant balance in the liver and blood plasma of rats with IR state. In conclusion, the NSE administration to the rats with insulin-resistance state restored pro-/antioxidant balance and enhanced the content of nitric oxide, therefore, improving insulin sensitivity. PMID:24479326

  1. Nitric Oxide Regulates Mitochondrial Fatty Acid Metabolism through Reversible Protein S-Nitrosylation **

    PubMed Central

    Doulias, Paschalis-Thomas; Tenopoulou, Margarita; Greene, Jennifer L.; Raju, Karthik; Ischiropoulos, Harry

    2014-01-01

    Cysteine S-nitrosylation is a posttranslational modification by which nitric oxide regulates protein function and signaling. Studies of individual proteins have elucidated specific functional roles for S-nitrosylation, but knowledge of the extent of endogenous S-nitrosylation, the sites that are nitrosylated, and the regulatory consequences of S-nitrosylation remains limited. We used mass spectrometry-based methodologies to identify 1011 S-nitrosocysteine residues in 647 proteins in various mouse tissues. We uncovered selective S-nitrosylation of enzymes participating in glycolysis, gluconeogenesis, tricarboxylic acid cycle, and oxidative phosphorylation, indicating that this posttranslational modification may regulate metabolism and mitochondrial bioenergetics. S-nitrosylation of the liver enzyme VLCAD (very long acyl-CoA dehydrogenase) at Cys238, which was absent in mice lacking endothelial nitric oxide synthase, improved its catalytic efficiency. These data implicate protein S-nitrosylation in the regulation of β-oxidation of fatty acids in mitochondria. PMID:23281369

  2. Mcy protein, a potential antidiabetic agent: evaluation of carbohydrate metabolic enzymes and antioxidant status.

    PubMed

    Marella, Saritha; Maddirela, Dilip Rajasekhar; Kumar, E G T V; Tilak, Thandaiah Krishna; Badri, Kameswara Rao; Chippada, Apparao

    2016-05-01

    The objective of the present study is to elucidate the long-term effects of anti-hyperglycemic active principle, Mcy protein (MCP), isolated from the fruits of Momordica cymbalaria on carbohydrate metabolism and oxidative stress in experimental diabetic rats. We used streptozotocin induced diabetic rats for the current studies. Our studies showed that MCP (2.5mg/kg.b.w) treatment significantly normalized the deranged activities of critical carbohydrate metabolizing enzymes, hexokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase and fructose-1,6-bis phosphatase. In addition MCP showed inhibitory activity on α-glucosidase and aldose reductase enzymes in in vitro assays. Further MCP treatment improved the antioxidant defensive mechanism by preventing deleterious oxidative products of cellular metabolism, which initiates the lipid peroxidation and by normalizing the antioxidant enzyme (catalase, superoxide dismutase, glutathione peroxidase) activities. Additional structural studies using circular dichroism spectroscopy indicate that MCP contains majorly α-helix. Our findings suggest MCP regulates blood glucose and better manage diabetes mellitus associated complications by regulating carbohydrate metabolism and by protecting from the deleterious effects of oxidative stress. PMID:26826289

  3. Why (--)deprenyl prolongs survivals of experimental animals: increase of anti-oxidant enzymes in brain and other body tissues as well as mobilization of various humoral factors may lead to systemic anti-aging effects.

    PubMed

    Kitani, Kenichi; Minami, Chiyoko; Isobe, Ken-ichi; Maehara, Kayoko; Kanai, Setsuko; Ivy, Gwen O; Carrillo, Maria-Christina

    2002-04-30

    (--)Deprenyl, a monoamine oxidase B (MAO B) inhibitor is known to upregulate activities of anti-oxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) in brain dopaminergic regions. The drug is also the sole chemical which has been repeatedly shown to increase life spans of several animal species including rats, mice, hamsters and dogs. Further, the drug was recently found to enhance anti-oxidant enzyme activities not only in brain dopaminergic regions but also in extra-brain tissues such as the heart, kidneys, adrenal glands and the spleen. We and others have also observed mobilization of many humoral factors (interferone (INF)-gamma, tumor necrosis factor (TNF)-alpha, interleukine (IL)-1beta,2,6, trophic factors, etc.) and enhancement of natural killer (NK) cell functions by (-)deprenyl administration. An apparent extension of life spans of experimental animals reported in the past may be better explained by these new observations that (-)deprenyl upregulate SOD and CAT activities not only in the brain but also in extra-brain vital organs and involve anti-tumorigenic as well as immunomodulatory effect as well. These combined drug effects may lead to the protection of the homeostatic regulations of the neuro-immuno-endocrine axis of an organism against aging. PMID:12044958

  4. Effects of pectin pentaoligosaccharide from Hawthorn ( Crataegus pinnatifida Bunge. var. Major) on the activity and mRNA levels of enzymes involved in fatty acid oxidation in the liver of mice fed a high-fat diet.

    PubMed

    Li, Tuo-Ping; Zhu, Ru-Gang; Dong, Yin-Ping; Liu, Yong-Hui; Li, Su-Hong; Chen, Gang

    2013-08-01

    The regulatory effects of haw pectin pentaoligosaccharide (HPPS) on fatty acid oxidation-related enzyme activities and mRNA levels were investigated in the liver of high fat diet induced hyperlipidemic mice. Results showed that HPPS (150 mg/kg for 10 weeks) significantly suppresses weight gain (32.3 ± 0.26 and 21.1 ± 0.14 g for high-fat diet and HPPS groups, respectively), decreases serum triacylglycerol levels (1.64 ± 0.09 and 0.91 ± 0.02 mmol/L, respectively), and increases lipid excretion in feces (55.7 ± 0.38 and 106.4 ± 0.57 mg/g for total lipid, respectively), compared to high-fat diet as control. HPPS significantly increased the hepatic fatty acid oxidation-related enzyme activities of acyl-CoA oxidase, carnitine palmitoyltransferase I, 3-ketoacyl-CoA thiolase, and 2,4-dienoyl-CoA reductase by 53.8, 74.2, 47.1, and 24.2%, respectively. Meanwhile, the corresponding mRNAs were up-regulated by 89.6, 85.8, 82.9, and 30.9%, respectively. Moreover, HPPS was able to up-regulate the gene and protein expressions of peroxisome proliferator-activated receptor α. Results suggest that continuous HPPS ingestion may be used as dietary therapy to prevent obesity and cardiovascular diseases. PMID:23855516

  5. Primary enzyme quantitation

    DOEpatents

    Saunders, G.C.

    1982-03-04

    The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

  6. Effects of supplementation with two sources and two levels of copper on meat lipid oxidation, meat colour and superoxide dismutase and glutathione peroxidase enzyme activities in Nellore beef cattle.

    PubMed

    Correa, Lísia Bertonha; Zanetti, Marcus Antonio; Del Claro, Gustavo Ribeiro; de Paiva, Fernanda Alves; da Luz e Silva, Saulo; Netto, Arlindo Saran

    2014-10-28

    In the present study, thirty-five Nellore bulls were used to determine the effects of two levels and two sources (organic and inorganic) of Cu supplementation on the oxidative stability of lipids, measured by the thiobarbituric acid-reactive substance (TBARS) test, meat colour and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzyme activities. The following treatments were used: (1) control (C) - basal diet without supplementation of Cu (7 mg Cu/kg DM); (2) I10 - basal diet supplemented with 10 mg Cu/kg DM in the form of copper sulphate (inorganic form); (3) I40 - basal diet supplemented with 40 mg Cu/kg DM in the form of copper sulphate; (4) O10 - basal diet supplemented with 10 mg Cu/kg DM in the form of copper proteinate (organic form); (5) O40 - basal diet supplemented with 40 mg Cu/kg DM in the form of copper proteinate. Lipid oxidation was determined in meat samples exposed to display, modified atmosphere (MA) and vacuum packaging (VC) conditions and in liver samples using the TBARS test. These samples were also evaluated for meat discolouration after exposure to air. The activities of SOD and GSH-Px enzymes were determined in liver samples. In display, MA and VC conditions, the TBARS values of samples from animals supplemented with 40 mg Cu/kg DM were lower than those of samples from control animals. There was no effect of treatment on the colour variables (L*, a*, b*). There was also no significant effect of treatment on hepatic TBARS concentrations and GSH-Px activity. Supplementation with Cu at 40 mg/kg, regardless of the source, induced higher hepatic SOD activity compared with the control treatment. In conclusion, Cu supplementation improved the oxidative stability of lipids in samples exposed to display, MA and VC conditions, demonstrating the antioxidant effect of this mineral. PMID:25313573

  7. Enzyme Analysis to Determine Glucose Content

    NASA Astrophysics Data System (ADS)

    Carpenter, Charles; Ward, Robert E.

    Enzyme analysis is used for many purposes in food science and technology. Enzyme activity is used to indicate adequate processing, to assess enzyme preparations, and to measure constituents of foods that are enzyme substrates. In this experiment, the glucose content of corn syrup solids is determined using the enzymes, glucose oxidase and peroxidase. Glucose oxidase catalyzes the oxidation of glucose to form hydrogen peroxide (H2O2), which then reacts with a dye in the presence of peroxidase to give a stable colored product.

  8. Enzyme stabilization: state of the art.

    PubMed

    Gianfreda, L; Scarfi, M R

    1991-02-01

    'Enzyme stabilization' is one of the most important fields in basic and applied enzymology. In basic enzymology, it is of particular relevance to understand enzyme stabilization principles first elucidating how and why the enzymes lose their biological activity and then deriving structure-stability relationships existing in enzymatic molecules. In applied enzymology, the most significant goal is to achieve useful compounds by biocatalysis. Enzymes are good catalysts in terms of high catalytic and specific activity with ability to function under mild conditions. However, they are not always ideal catalysts for practical applications because they are generally unstable and they inactivate rapidly through several mechanisms. In order to enhance enzyme stability, many strategies have been pursued in recent years. The present article is an attempt to provide detailed information about these strategies. PMID:2008180

  9. Curcuma longa polyphenols improve insulin-mediated lipid accumulation and attenuate proinflammatory response of 3T3-L1 adipose cells during oxidative stress through regulation of key adipokines and antioxidant enzymes.

    PubMed

    Septembre-Malaterre, Axelle; Le Sage, Fanny; Hatia, Sarah; Catan, Aurélie; Janci, Laurent; Gonthier, Marie-Paule

    2016-07-01

    Plant polyphenols may exert beneficial action against obesity-related oxidative stress and inflammation which promote insulin resistance. This study evaluated the effect of polyphenols extracted from French Curcuma longa on 3T3-L1 adipose cells exposed to H2 O2 -mediated oxidative stress. We found that Curcuma longa extract exhibited high amounts of curcuminoids identified as curcumin, demethoxycurcumin, and bisdemethoxycurcumin, which exerted free radical-scavenging activities. Curcuma longa polyphenols improved insulin-mediated lipid accumulation and upregulated peroxisome proliferator-activated receptor-gamma gene expression and adiponectin secretion which decreased in H2 O2 -treated cells. Curcuminoids attenuated H2 O2 -enhanced production of pro-inflammatory molecules such as interleukin-6, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and nuclear factor κappa B. Moreover, they reduced intracellular levels of reactive oxygen species elevated by H2 O2 and modulated the expression of genes encoding superoxide dismutase and catalase antioxidant enzymes. Collectively, these findings highlight that Curcuma longa polyphenols protect adipose cells against oxidative stress and may improve obesity-related metabolic disorders. © 2016 BioFactors, 42(4):418-430, 2016. PMID:27094023

  10. Solution structure of the reduced form of human peroxiredoxin-6 elucidated using zero-length chemical cross-linking and homology modelling.

    PubMed

    Rivera-Santiago, Roland F; Harper, Sandra L; Zhou, Suiping; Sriswasdi, Sira; Feinstein, Sheldon I; Fisher, Aron B; Speicher, David W

    2015-05-15

    Peroxiredoxin-6 (PRDX6) is an unusual member of the peroxiredoxin family of antioxidant enzymes that has only one evolutionarily conserved cysteine. It reduces oxidized lipids and reactive oxygen species (ROS) by oxidation of the active-site cysteine (Cys(47)) to a sulfenic acid, but the mechanism for conversion back to a thiol is not completely understood. Moreover, it has phospholipase A2 (PLA2) activity in addition to its peroxidase activity. Interestingly, some biochemical data are inconsistent with a known high-resolution crystal structure of the catalytic intermediate of the protein, and biophysical data indicate that the protein undergoes conformational changes that affect enzyme activity. In order to further elucidate the solution structure of this important enzyme, we used chemical cross-linking coupled with high-resolution MS (CX-MS), with an emphasis on zero-length cross-links. Distance constraints from high confidence cross-links were used in homology modelling experiments to determine a solution structure of the reduced form of the protein. This structure was further evaluated using chemical cross-links produced by several homo-bifunctional amine-reactive cross-linking reagents, which helped to confirm the solution structure. The results show that several regions of the reduced version of human PRDX6 are in a substantially different conformation from that shown for the crystal structure of the peroxidase catalytic intermediate. The differences between these two structures are likely to reflect catalysis-related conformational changes. These studies also demonstrate that CX-MS using zero-length cross-linking is a powerful strategy for probing protein conformational changes that is complementary to alternative methods such as crystallographic, NMR and biophysical studies. PMID:25748205

  11. [Effect of nitric oxide synthase inhibitor L-NNA on the activities of antioxidant enzymes and lipid peroxidation in blood and tissues of rats with different resistance to hypoxia].

    PubMed

    Kurhaliuk, N M

    2001-01-01

    The main components of antioxidant enzyme system (AOS) are superoxide dismutase (SOD) and glutathione reductase (GR) catalyses the conversion of the superoxide anion. The important role in AOS belongs to catalase and glutathione peroxidase which perform H2O2 to nontoxic products. Simultaneous determination of AOS activity and malonic dialdehide (MDA) concentration (the index of lipid peroxidation in tissues and blood) characterize cells complex resistance to damage factor. The effect of L-arginine, as a precursor of nitric oxide synthesis and blocator NO-synthase (Nw-nitro-L-arginine) on AOS of rats with different resistance to hypoxia under stress condition is unknown and were subject of our investigation. Experiments were done on liver and blood tissues of white laboratory rats. The experimental animals were divided on two groups depending on hypoxia factor: high resistance (HR) and low resistance (LR). The type of resistance was determined by the time of ability to respire in barocamera with oxygen deficient equal to 12.000 meters over sea level. The animals adaptation to laboratory conditions continue during 14 days after in barocamera presence. All animals were divided dependent on experiment conditions on fourth groups. The first group: intact (HR and LR) animals parentherally injected by 1 ml of 0.9% NaCl solution. The second group was subject of stress condition. The third group: HR and LR animals injected parentherally by 1 ml L-arginine (Sigma, USA) dose (600 mg/kg body weight). The fourth one: rats injected by 1 ml Nw-nitro-L-arginine (L-NNA, Sigma, USA)--the blocator of NO-synthase. The animals were decapitated 30 min after injection and stress condition under ethereal anesthesia. Activity of antioxidant system enzymes superoxide dismutase (SOD), catalase (CAT); glutathione reductase (GR), glutathione peroxidase (GP) were measured spectrophotometrically. Also was investigated the concentration of serum antioxidant ceruloplasmin (CP). Level of lipid

  12. [Elucidating hypoglycemic mechanism of Dendrobium nobile through auxiliary elucidation system for traditional Chinese medicine mechanism].

    PubMed

    Li, Man-man; Zhang, Bai-xia; He, Shuai-bing; Zheng, Rao; Zhang, Yan-ling; Wang, Yun

    2015-10-01

    To build the Dendrobium nobile -T2DM network, and elucidate the molecular mechanism of D. nobile to type 2 diabetes (T2DM). Collect the chemical composition of D. nobile and the targets on T2DM by retrieving database and documents, build the network of D. nobile to T2DM using the entity grammar systems inference rules. The molecular mechanism of D. nobile to T2DM includes: (1) regulating lipid metabolism by lowering triglyceride; (2) reducing insulin resistance; (3) protecting islet cells; (4) promoting the glucose-dependent insulin tropic peptide (GIP) secretion; (5) inhibiting calcium channel. Under the guidance of network pharmacology, through entity grammar systems inference rules we elucidate the molecular mechanism of D. nobile to T2DM, and provide the basis for the further development of health care products based on D. nobile. PMID:26975089

  13. Arabidopsis CYP94B3 encodes jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate.

    PubMed

    Kitaoka, Naoki; Matsubara, Takuya; Sato, Michio; Takahashi, Kosaku; Wakuta, Shinji; Kawaide, Hiroshi; Matsui, Hirokazu; Nabeta, Kensuke; Matsuura, Hideyuki

    2011-10-01

    The hormonal action of jasmonate in plants is controlled by the precise balance between its biosynthesis and catabolism. It has been shown that jasmonyl-L-isoleucine (JA-Ile) is the bioactive form involved in the jasmonate-mediated signaling pathway. However, the catabolism of JA-Ile is poorly understood. Although a metabolite, 12-hydroxyJA-Ile, has been characterized, detailed functional studies of the compound and the enzyme that produces it have not been conducted. In this report, the kinetics of wound-induced accumulation of 12-hydroxyJA-Ile in plants were examined, and its involvement in the plant wound response is described. Candidate genes for the catabolic enzyme were narrowed down from 272 Arabidopsis Cyt P450 genes using Arabidopsis mutants. The candidate gene was functionally expressed in Pichia pastoris to reveal that CYP94B3 encodes JA-Ile 12-hydroxylase. Expression analyses demonstrate that expression of CYP94B3 is induced by wounding and shows specific activity toward JA-Ile. Plants grown in medium containing JA-Ile show higher sensitivity to JA-Ile in cyp94b3 mutants than in wild-type plants. These results demonstrate that CYP94B3 plays a major regulatory role in controlling the level of JA-Ile in plants. PMID:21849397

  14. Deoxynivalenol induced mouse skin tumor initiation: Elucidation of molecular mechanisms in human HaCaT keratinocytes.

    PubMed

    Mishra, Sakshi; Tewari, Prachi; Chaudhari, Bhushan P; Dwivedi, Premendra D; Pandey, Haushila P; Das, Mukul

    2016-11-01

    Among food contaminants, mycotoxins are toxic to both human and animal health. Our prior studies suggest that Deoxynivalenol (DON), a mycotoxin, behaves as a tumor promoter by inducing edema, hyperplasia, ODC activity and activation of MAPK's in mouse skin. In this study, topical application of DON, 336 and 672 nmol significantly enhanced ROS levels, DNA damage and apoptosis with concomitant downregulation of Ki-67, cyclin D, cyclin E, cyclin A and cyclin-dependent kinases (CDK4 and CDK2) thereby resulting in tumor initiation in mouse skin. Further, the elucidation of molecular mechanisms of tumor initiation by DON (0.42-3.37 nmol/ml) in HaCaT keratinocytes, revealed (i) enhanced ROS generation with cell cycle phase arrest in G0/G1 phase, (ii) increase in levels of 8-OxoG (6-24 hr) and γH2AX protein, (iii) significant enhancement in oxidative stress marker enzymes LPO, GSH, GR with concomitant decrease in antioxidant enzymes catalase, GPx, GST, SOD and mitochondrial membrane potential after DON (1.68 nmol) treatment, (iv) suppression of Nrf2 translocation to nucleus, enhanced phosphorylation with subsequent activation ERK1/2, p38 and JNK MAPK's following DON (1.68 nmol) treatment, (v) overexpression of c-jun, c-fos proteins, upregulation of Bax along with downregulation of Bcl-2 proteins, (vi) increase in cytochrome-c, caspase-9, caspase-3 and poly ADP ribose polymerase levels leads to apoptosis. Pretreatment of superoxide dismutase, mannitol and ethanol to HaCaT cells resulted in significant reduction in ROS levels and apoptosis indicating the role of superoxide and hydroxyl radicals in DON induced apoptosis as an early event and skin tumor initiation as a late event. PMID:27389473

  15. Assay Methods for H2S Biogenesis and Catabolism Enzymes

    PubMed Central

    Banerjee, Ruma; Chiku, Taurai; Kabil, Omer; Libiad, Marouane; Motl, Nicole; Yadav, Pramod K.

    2015-01-01

    H2S is produced from sulfur-containing amino acids, cysteine and homocysteine, or a catabolite, 3-mercaptopyruvate, by three known enzymes: cystathionine β-synthase, γ-cystathionase, and 3-mercaptopyruvate sulfurtransferase. Of these, the first two enzymes reside in the cytoplasm and comprise the transsulfuration pathway, while the third enzyme is found both in the cytoplasm and in the mitochondrion. The following mitocho