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Sample records for polyamine oxidase activity

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

    PubMed

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

    2016-09-01

    Retinal vascular injury is a major cause of vision impairment in ischemic retinopathies. Insults such as hyperoxia, oxidative stress and inflammation contribute to this pathology. Previously, we showed that hyperoxia-induced retinal neurodegeneration is associated with increased polyamine oxidation. Here, we are studying the involvement of polyamine oxidases in hyperoxia-induced injury and death of retinal vascular endothelial cells. New-born C57BL6/J mice were exposed to hyperoxia (70% O2) from postnatal day (P) 7 to 12 and were treated with the polyamine oxidase inhibitor MDL 72527 or vehicle starting at P6. Mice were sacrificed after different durations of hyperoxia and their retinas were analyzed to determine the effects on vascular injury, microglial cell activation, and inflammatory cytokine profiling. The results of this analysis showed that MDL 72527 treatment significantly reduced hyperoxia-induced retinal vascular injury and enhanced vascular sprouting as compared with the vehicle controls. These protective effects were correlated with significant decreases in microglial activation as well as levels of inflammatory cytokines and chemokines. In order to model the effects of polyamine oxidation in causing microglial activation in vitro, studies were performed using rat brain microvascular endothelial cells treated with conditioned-medium from rat retinal microglia stimulated with hydrogen peroxide. Conditioned-medium from activated microglial cultures induced cell stress signals and cell death in microvascular endothelial cells. These studies demonstrate the involvement of polyamine oxidases in hyperoxia-induced retinal vascular injury and retinal inflammation in ischemic retinopathy, through mechanisms involving cross-talk between endothelial cells and resident retinal microglia. PMID:27239699

  2. Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity1[C][W

    PubMed Central

    Kim, Dong Wook; Watanabe, Kanako; Murayama, Chihiro; Izawa, Sho; Niitsu, Masaru; Michael, Anthony J.; Berberich, Thomas; Kusano, Tomonobu

    2014-01-01

    The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development. PMID:24906355

  3. Inhibition of polyamine oxidase activity affects tumor development during the maize-Ustilago maydis interaction.

    PubMed

    Jasso-Robles, Francisco Ignacio; Jiménez-Bremont, Juan Francisco; Becerra-Flora, Alicia; Juárez-Montiel, Margarita; Gonzalez, María Elisa; Pieckenstain, Fernando Luis; García de la Cruz, Ramón Fernando; Rodríguez-Kessler, Margarita

    2016-05-01

    Ustilago maydis is a biotrophic plant pathogenic fungus that leads to tumor development in the aerial tissues of its host, Zea mays. These tumors are the result of cell hypertrophy and hyperplasia, and are accompanied by the reprograming of primary and secondary metabolism of infected plants. Up to now, little is known regarding key plant actors and their role in tumor development during the interaction with U. maydis. Polyamines are small aliphatic amines that regulate plant growth, development and stress responses. In a previous study, we found substantial increases of polyamine levels in tumors. In the present work, we describe the maize polyamine oxidase (PAO) gene family, its contribution to hydrogen peroxide (H2O2) production and its possible role in tumor development induced by U. maydis. Histochemical analysis revealed that chlorotic lesions and maize tumors induced by U. maydis accumulate H2O2 to significant levels. Maize plants inoculated with U. maydis and treated with the PAO inhibitor 1,8-diaminooctane exhibit a notable reduction of H2O2 accumulation in infected tissues and a significant drop in PAO activity. This treatment also reduced disease symptoms in infected plants. Finally, among six maize PAO genes only the ZmPAO1, which encodes an extracellular enzyme, is up-regulated in tumors. Our data suggest that H2O2 produced through PA catabolism by ZmPAO1 plays an important role in tumor development during the maize-U. maydis interaction. PMID:26926794

  4. Antiviral activity of oxidized polyamines.

    PubMed

    Bachrach, U

    2007-08-01

    Polyamines, oxidized by serum amine oxidase, yield aminoaldehydes and hydrogen peroxide. Acrolein may be formed from the aminoaldehydes by a spontaneous beta-elimination process. These oxidation products "oxidized polyamines" inhibit bacterial growth and exhibit anticancer activity. The antimicrobial activity of oxidized polyamines is not limited to bacteria; and the inactivation of bacterial viruses, plant viruses and animal viruses, was also reported. Bacteriophages of the T-odd series are permeable and were inactivated by oxidized polyamines. The inactive phages absorb to their bacterial host and injected their DNA, which formed a stable inactive complex with the aminoaldehydes. Aminoaldehydes, synthesized chemically, also inactivated viruses. The growth of the plant viruses: Tobacco mosaic virus, Potato virus X and Alfalfa mosaic virus was also inhibited by oxidized polyamines. The animal viruses, which were inactivated by oxidized polyamines included Myxoviruses (influenza and Newcastle disease viruses), West Nile, vaccinia and Sindbis viruses. These findings may have practical implications. PMID:17429570

  5. POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis POLYAMINE OXIDASE 5

    PubMed Central

    Liu, Taibo; Wook Kim, Dong; Niitsu, Masaru; Berberich, Thomas; Kusano, Tomonobu

    2014-01-01

    POLYAMINE OXIDASE 1 (OsPAO1), from rice (Oryza sativa), and POLYAMINE OXIDASE 5 (AtPAO5), from Arabidopsis (Arabidopsis thaliana), are enzymes sharing high identity at the amino acid level and with similar characteristics, such as polyamine specificity and pH preference; furthermore, both proteins localize to the cytosol. A loss-of-function Arabidopsis mutant, Atpao5–2, was hypersensitive to low doses of exogenous thermospermine but this phenotype could be rescued by introduction of the wild-type AtPAO5 gene. Introduction of OsPAO1, under the control of a constitutive promoter, into Atpao5–2 mutants also restored normal thermospermine sensitivity, allowing growth in the presence of low levels of thermospermine, along with a concomitant decrease in thermospermine content in plants. By contrast, introduction of OsPAO3, which encodes a peroxisome-localized polyamine oxidase, into Atpao5–2 plants could not rescue any of the mutant phenotypes in the presence of thermospermine. These results suggest that OsPAO1 is the functional ortholog of AtPAO5. PMID:25763711

  6. POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis POLYAMINE OXIDASE 5.

    PubMed

    Liu, Taibo; Wook Kim, Dong; Niitsu, Masaru; Berberich, Thomas; Kusano, Tomonobu

    2014-07-25

    POLYAMINE OXIDASE 1 (OsPAO1), from rice (Oryza sativa), and POLYAMINE OXIDASE 5 (AtPAO5), from Arabidopsis (Arabidopsis thaliana), are enzymes sharing high identity at the amino acid level and with similar characteristics, such as polyamine specificity and pH preference; furthermore, both proteins localize to the cytosol. A loss-of-function Arabidopsis mutant, Atpao5-2, was hypersensitive to low doses of exogenous thermospermine but this phenotype could be rescued by introduction of the wild-type AtPAO5 gene. Introduction of OsPAO1, under the control of a constitutive promoter, into Atpao5-2 mutants also restored normal thermospermine sensitivity, allowing growth in the presence of low levels of thermospermine, along with a concomitant decrease in thermospermine content in plants. By contrast, introduction of OsPAO3, which encodes a peroxisome-localized polyamine oxidase, into Atpao5-2 plants could not rescue any of the mutant phenotypes in the presence of thermospermine. These results suggest that OsPAO1 is the functional ortholog of AtPAO5. PMID:25061821

  7. Reducing Cytoplasmic Polyamine Oxidase Activity in Arabidopsis Increases Salt and Drought Tolerance by Reducing Reactive Oxygen Species Production and Increasing Defense Gene Expression

    PubMed Central

    Sagor, G. H. M.; Zhang, Siyuan; Kojima, Seiji; Simm, Stefan; Berberich, Thomas; Kusano, Tomonobu

    2016-01-01

    The link between polyamine oxidases (PAOs), which function in polyamine catabolism, and stress responses remains elusive. Here, we address this issue using Arabidopsis pao mutants in which the expression of the five PAO genes is knocked-out or knocked-down. As the five single pao mutants and wild type (WT) showed similar response to salt stress, we tried to generate the mutants that have either the cytoplasmic PAO pathway (pao1 pao5) or the peroxisomal PAO pathway (pao2 pao3 pao4) silenced. However, the latter triple mutant was not obtained. Thus, in this study, we used two double mutants, pao1 pao5 and pao2 pao4. Of interest, pao1 pao5 mutant was NaCl- and drought-tolerant, whereas pao2 pao4 showed similar sensitivity to those stresses as WT. To reveal the underlying mechanism of salt tolerance, further analyses were performed. Na uptake of the mutant (pao1 pao5) decreased to 75% of WT. PAO activity of the mutant was reduced to 62% of WT. The content of reactive oxygen species (ROS) such as hydrogen peroxide, a reaction product of PAO action, and superoxide anion in the mutant became 81 and 72% of the levels in WT upon salt treatment. The mutant contained 2.8-fold higher thermospermine compared to WT. Moreover, the mutant induced the genes of salt overly sensitive-, abscisic acid (ABA)-dependent- and ABA-independent- pathways more strongly than WT upon salt treatment. The results suggest that the Arabidopsis plant silencing cytoplasmic PAOs shows salinity tolerance by reducing ROS production and strongly inducing subsets of stress-responsive genes under stress conditions. PMID:26973665

  8. Reducing Cytoplasmic Polyamine Oxidase Activity in Arabidopsis Increases Salt and Drought Tolerance by Reducing Reactive Oxygen Species Production and Increasing Defense Gene Expression.

    PubMed

    Sagor, G H M; Zhang, Siyuan; Kojima, Seiji; Simm, Stefan; Berberich, Thomas; Kusano, Tomonobu

    2016-01-01

    The link between polyamine oxidases (PAOs), which function in polyamine catabolism, and stress responses remains elusive. Here, we address this issue using Arabidopsis pao mutants in which the expression of the five PAO genes is knocked-out or knocked-down. As the five single pao mutants and wild type (WT) showed similar response to salt stress, we tried to generate the mutants that have either the cytoplasmic PAO pathway (pao1 pao5) or the peroxisomal PAO pathway (pao2 pao3 pao4) silenced. However, the latter triple mutant was not obtained. Thus, in this study, we used two double mutants, pao1 pao5 and pao2 pao4. Of interest, pao1 pao5 mutant was NaCl- and drought-tolerant, whereas pao2 pao4 showed similar sensitivity to those stresses as WT. To reveal the underlying mechanism of salt tolerance, further analyses were performed. Na uptake of the mutant (pao1 pao5) decreased to 75% of WT. PAO activity of the mutant was reduced to 62% of WT. The content of reactive oxygen species (ROS) such as hydrogen peroxide, a reaction product of PAO action, and superoxide anion in the mutant became 81 and 72% of the levels in WT upon salt treatment. The mutant contained 2.8-fold higher thermospermine compared to WT. Moreover, the mutant induced the genes of salt overly sensitive-, abscisic acid (ABA)-dependent- and ABA-independent- pathways more strongly than WT upon salt treatment. The results suggest that the Arabidopsis plant silencing cytoplasmic PAOs shows salinity tolerance by reducing ROS production and strongly inducing subsets of stress-responsive genes under stress conditions. PMID:26973665

  9. Electron-microscopic cytochemical localization of diamine and polyamine oxidases in pea and maize tissues

    NASA Technical Reports Server (NTRS)

    Slocum, R. D.; Furey MJ, 3. d.

    1991-01-01

    An electron-microscopic cytochemical method was used to localize diamine oxidase (DAO) in pea and polyamine oxidase (PAO) in maize (Zea mays L.). The method, based on the precipitation of amine-oxidase-generated H2O2 by CeCl3, was shown to be specific for DAO and PAO and permitted their localization in plant tissues with a high degree of resolution. Both enzymes are localized exclusively in the cell wall. Both DAO- and PAO-activity staining is most intense in the middle lamellar region of the wall and in cells exhibiting highly lignified walls. The oxidases could provide H2O2 for peroxidase-mediated cross-linking reactions in the cell wall and may, in this capacity, play a role in the regulation of plant growth.

  10. Inhibition of pig liver and Zea mays L. polyamine oxidase: a comparative study.

    PubMed

    Federico, R; Leone, L; Botta, M; Binda, C; Angelini, R; Venturini, G; Ascenzi, P

    2001-01-01

    Polyamine oxidase (PAO) is involved in polyamine metabolism and production of hydrogen peroxide in animal and plants, thus representing a key system in development and programmed cell death. In the present study, the inhibitory effect of amiloride, p-aminobenzamidine, clonidine, 4',6-diamidino-2-phenyl-indole (DAPI), gabexate mesylate, guazatine, and N,N'-bis(2,3-butadienyl)-1,4-butane-diamine (MDL72527) on the catalytic activity of pig liver and Zea mays L. PAO, Lens culinaris L. and Pisum sativum L. and swine kidney copper amine oxidase, bovine trypsin, as well as neuronal constitutive nitric oxide synthase (NOS-I) was investigated. Moreover, agmatine and N(3) -prenylagmatine (G3) were observed to inhibit pig liver and Zea mays L. PAO, bovine trypsin, and NOS-I action, but were substrates for Lens culinaris L., Pisum sativum L. and swine kidney copper amine oxidase. Guazatine and G3 inhibited selectively Zea mays L. PAO with K(i) values of 7.5 x 10(-9) M and 1.5 x 10(-8) M, respectively (at pH 6.5 and 25.0 degrees C). As a whole, the data reported here represent examples of enzyme cross-inhibition, and appear to be relevant in view of the use of cationic L-arginine-and imidazole-based compounds as drugs. PMID:11342283

  11. Potential anticancer application of polyamine oxidation products formed by amine oxidase: a new therapeutic approach.

    PubMed

    Agostinelli, E; Tempera, G; Viceconte, N; Saccoccio, S; Battaglia, V; Grancara, S; Toninello, A; Stevanato, R

    2010-02-01

    The polyamines spermine, spermidine and putrescine are ubiquitous cell components. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper-containing amine oxidases. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. In tumors, polyamines and amine oxidases are increased as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H(2)O(2) and aldehydes produced by the reaction. This study demonstrated that multidrug-resistant (MDR) cancer cells (colon adenocarcinoma and melanoma) are significantly more sensitive than the corresponding wild-type (WT) ones to H(2)O(2) and aldehydes, the products of BSAO-catalyzed oxidation of spermine. Transmission electron microscopy (TEM) observations showed major ultrastructural alterations of the mitochondria. These were more pronounced in MDR than in WT cells. Increasing the incubation temperature from 37 to 42 degrees Celsius enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumor growth, particularly well if the enzyme has been conjugated to a biocompatible hydrogel polymers. Since both wild-type and MDR cancer cells after pre-treatment with MDL 72527, a lysosomotropic compound, are sensitized to subsequent exposure to BSAO/spermine, it is conceivable that combined treatment with a lysosomotropic compound and BSAO/spermine would be effective against tumor cells. It is of interest to search for such novel compounds, which might be promising for application in a therapeutic setting. PMID:20012114

  12. Copper-Containing Amine Oxidases and FAD-Dependent Polyamine Oxidases Are Key Players in Plant Tissue Differentiation and Organ Development

    PubMed Central

    Tavladoraki, Paraskevi; Cona, Alessandra; Angelini, Riccardo

    2016-01-01

    Plant polyamines are catabolized by two classes of amine oxidases, the copper amine oxidases (CuAOs) and the flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). These enzymes differ to each other in substrate specificity, catalytic mechanism and subcellular localization. CuAOs and PAOs contribute to several physiological processes both through the control of polyamine homeostasis and as sources of biologically-active reaction products. CuAOs and PAOs have been found at high level in the cell-wall of several species belonging to Fabaceae and Poaceae families, respectively, especially in tissues fated to undertake extensive wall loosening/stiffening events and/or in cells undergoing programmed cell death (PCD). Apoplastic CuAOs and PAOs have been shown to play a key role as a source of H2O2 in light- or developmentally-regulated differentiation events, thus influencing cell-wall architecture and maturation as well as PCD. Moreover, growing evidence suggests a key role of intracellular CuAOs and PAOs in several facets of plant development. Here, we discuss recent advances in understanding the contribution of different CuAOs/PAOs, as well as their cross-talk with different intracellular and apoplastic metabolic pathways, in tissue differentiation and organ development. PMID:27446096

  13. Genome Wide Association Mapping for the Tolerance to the Polyamine Oxidase Inhibitor Guazatine in Arabidopsis thaliana.

    PubMed

    Atanasov, Kostadin E; Barboza-Barquero, Luis; Tiburcio, Antonio F; Alcázar, Rubén

    2016-01-01

    Guazatine is a potent inhibitor of polyamine oxidase (PAO) activity. In agriculture, guazatine is used as non-systemic contact fungicide efficient in the protection of cereals and citrus fruits against disease. The composition of guazatine is complex, mainly constituted by a mixture of synthetic guanidated polyamines (polyaminoguanidines). Here, we have studied the effects from exposure to guazatine in the weed Arabidopsis thaliana. We report that micromolar concentrations of guazatine are sufficient to inhibit growth of Arabidopsis seedlings and induce chlorosis, whereas germination is barely affected. We observed the occurrence of quantitative variation in the response to guazatine between 107 randomly chosen Arabidopsis accessions. This enabled us to undertake genome-wide association (GWA) mapping that identified a locus on chromosome one associated with guazatine tolerance. CHLOROPHYLLASE 1 (CLH1) within this locus was studied as candidate gene, together with its paralog (CLH2). The analysis of independent clh1-2, clh1-3, clh2-3, clh2-2, and double clh1-2 clh2-3 mutant alleles indicated that CLH1 and/or CLH2 loss-of-function or expression down-regulation promote guazatine tolerance in Arabidopsis. We report a natural mechanism by which Arabidopsis populations can overcome toxicity by the fungicide guazatine. PMID:27092150

  14. Genome Wide Association Mapping for the Tolerance to the Polyamine Oxidase Inhibitor Guazatine in Arabidopsis thaliana

    PubMed Central

    Atanasov, Kostadin E.; Barboza-Barquero, Luis; Tiburcio, Antonio F.; Alcázar, Rubén

    2016-01-01

    Guazatine is a potent inhibitor of polyamine oxidase (PAO) activity. In agriculture, guazatine is used as non-systemic contact fungicide efficient in the protection of cereals and citrus fruits against disease. The composition of guazatine is complex, mainly constituted by a mixture of synthetic guanidated polyamines (polyaminoguanidines). Here, we have studied the effects from exposure to guazatine in the weed Arabidopsis thaliana. We report that micromolar concentrations of guazatine are sufficient to inhibit growth of Arabidopsis seedlings and induce chlorosis, whereas germination is barely affected. We observed the occurrence of quantitative variation in the response to guazatine between 107 randomly chosen Arabidopsis accessions. This enabled us to undertake genome-wide association (GWA) mapping that identified a locus on chromosome one associated with guazatine tolerance. CHLOROPHYLLASE 1 (CLH1) within this locus was studied as candidate gene, together with its paralog (CLH2). The analysis of independent clh1-2, clh1-3, clh2-3, clh2-2, and double clh1-2 clh2-3 mutant alleles indicated that CLH1 and/or CLH2 loss-of-function or expression down-regulation promote guazatine tolerance in Arabidopsis. We report a natural mechanism by which Arabidopsis populations can overcome toxicity by the fungicide guazatine. PMID:27092150

  15. Identification and biochemical characterization of polyamine oxidases in amphioxus: Implications for emergence of vertebrate-specific spermine and acetylpolyamine oxidases.

    PubMed

    Wang, Huihui; Liu, Baobao; Li, Hongyan; Zhang, Shicui

    2016-01-10

    Polyamine oxidases (PAOs) have been identified in a wide variety of animals, as well as in fungi and plant. Generally, plant PAOs oxidize spermine (Spm), spermidine (Spd) and their acetylated derivatives, N(1)-acetylspermine (N(1)-Aspm) and N(1)-acetylspermidine (N(1)-Aspd), while yeast PAOs oxidize Spm, N(1)-Aspm and N(1)-Aspd, but not Spd. By contrast, two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of Spm and N(1)-Aspm/N(1)-Aspd, respectively. However, our knowledge on the biochemical and structural characterization of PAOs remains rather limited, and their evolutionary history is still enigmatic. In this study, two amphioxus (Branchiostoma japonicum) PAO genes, named Bjpao1 and Bjpao2, were cloned and characterized. Both Bjpao1 and Bjpao2 displayed distinct tissue-specific expression patterns. Notably, rBjPAO1 oxidized both spermine and spermidine, but not N(1)-acetylspermine, whereas rBjPAO2 oxidizes both spermidine and N(1)-acetylspermine, but not spermine. To understand structure-function relationship, the enzymatic activities of mutant BjPAOs that were generated by site-directed mutagenesis and expressed in E. coli were examined, The results indicate that the residues H64, K301 and T460 in rBjPAO1, and H69, K315 and T467 in rBjPAO2 were all involved in substrate binding and enzyme catalytic activity to some extent. Based on our results and those of others, a model depicting the divergent evolution and functional specialization of vertebrate SMO and APAO genes is proposed. PMID:26367330

  16. POLYAMINE OXIDASE2 of Arabidopsis contributes to ABA mediated plant developmental processes.

    PubMed

    Wimalasekera, Rinukshi; Schaarschmidt, Frank; Angelini, Riccardo; Cona, Alessandra; Tavladoraki, Parasklevi; Scherer, Günther F E

    2015-11-01

    Polyamines (PA) are catabolised by two groups of amine oxidases, the copper-binding amine oxidases (CuAOs) and the FAD-binding polyamine oxidases (PAOs). Previously, we have shown that CuAO1 is involved in ABA associated growth responses and ABA- and PA-mediated rapid nitric oxide (NO) production. Here we report the differential regulation of expression of POLYAMINE OXIDASE2 of Arabidopsis (AtPAO2) in interaction with ABA, nitrate and ammonium. Without ABA treatment germination, cotyledon growth and fresh weight of pao2 knockdown mutants as well as PAO2OX over-expressor plants were comparable to those of the wild type (WT) plants irrespective of the N source. In the presence of ABA, in pao2 mutants cotyledon growth and fresh weights were more sensitive to inhibition by ABA while PAO2OX over-expressor plants showed a rather similar response to WT. When NO3(-) was the only N source primary root lengths and lateral root numbers were lower in pao2 mutants both without and with exogenous ABA. PAO2OX showed enhanced primary and lateral root growth in media with NO3(-) or NH4(+). Vigorous root growth of PAO2OX and the hypersensitivity of pao2 mutants to ABA suggest a positive function of AtPAO2 in root growth. ABA-induced NO production in pao2 mutants was lower indicating a potential contributory function of AtPAO2 in NO-mediated effects on root growth. PMID:26310141

  17. Inhibition of polyamine oxidase prevented cyclin-dependent kinase inhibitor-induced apoptosis in HCT 116 colon carcinoma cells.

    PubMed

    Gürkan, Ajda Coker; Arisan, Elif Damla; Obakan, Pinar; Palavan-Ünsal, Narçin

    2013-12-01

    Roscovitine and purvalanol are novel cyclin-dependent kinase (CDK) inhibitors that prevent cell proliferation and induce apoptotic cell death in various cancer cell lines. Although a number of studies have demonstrated the potential apoptotic role of roscovitine, there is limited data about the therapeutic efficiency of purvalanol on cancer cells. The natural polyamines (PAs) putrescine, spermidine, and spermine have essential roles in the regulation of cell differentiation, growth, and proliferation, and increased levels of these compounds have been associated with cancer progression. Recently, depletion of intracellular PA levels because of modulation of PA catabolic enzymes was shown to be an indicator of the efficacy of chemotherapeutic agents. In this study, our aim was to investigate the potential role of PA catabolic enzymes in CDK inhibitor-induced apoptosis in HCT 116 colon carcinoma cells. Exposure of cells to roscovitine or purvalanol decreased cell viability in a dose- and time-dependent manner. The selected concentrations of roscovitine and purvalanol inhibited cell viability by 50 % compared with control cells and induced apoptosis by activating the mitochondria-mediated pathway in a caspase-dependent manner. However, the apoptotic effect of purvalanol was stronger than that of roscovitine in HCT 116 cells. In addition, we found that CDK inhibitors decreased PA levels and significantly upregulated expression of key PA catabolic enzymes such as polyamine oxidase (PAO) and spermine oxidase (SMO). MDL-72,527, a specific inhibitor of PAO and SMO, decreased apoptotic potential of CDK inhibitors on HCT 116 cells. Moreover, transient silencing of PAO was also reduced prevented CDK inhibitor-induced apoptosis in HCT 116 cells. We conclude that the PA catabolic pathway, especially PAO, is a critical target for understanding the molecular mechanism of CDK inhibitor-induced apoptosis. PMID:23892915

  18. Involvement of Polyamine Oxidase-Produced Hydrogen Peroxide during Coleorhiza-Limited Germination of Rice Seeds

    PubMed Central

    Chen, Bing-Xian; Li, Wen-Yan; Gao, Yin-Tao; Chen, Zhong-Jian; Zhang, Wei-Na; Liu, Qin-Jian; Chen, Zhuang; Liu, Jun

    2016-01-01

    Seed germination is a complicated biological process that requires regulated enzymatic and non-enzymatic reactions. The action of polyamine oxidase (PAO) produces hydrogen peroxide (H2O2), which promotes dicot seed germination. However, whether and, if so, how PAOs regulate monocot seed germination via H2O2 production is unclear. Herein, we report that the coleorhiza is the main physical barrier to radicle protrusion during germination of rice seed (a monocot seed) and that it does so in a manner similar to that of dicot seed micropylar endosperm. We found that H2O2 specifically and steadily accumulated in the coleorhizae and radicles of germinating rice seeds and was accompanied by increased PAO activity as the germination percentage increased. These physiological indexes were strongly decreased in number by guazatine, a PAO inhibitor. We also identified 11 PAO homologs (OsPAO1–11) in the rice genome, which could be classified into four subfamilies (I, IIa, IIb, and III). The OsPAO genes in subfamilies I, IIa, and IIb (OsPAO1–7) encode PAOs, whereas those in subfamily III (OsPAO8–11) encode histone lysine-specific demethylases. In silico-characterized expression profiles of OsPAO1–7 and those determined by qPCR revealed that OsPAO5 is markedly upregulated in imbibed seeds compared with dry seeds and that its transcript accumulated to a higher level in embryos than in the endosperm. Moreover, its transcriptional abundance increased gradually during seed germination in water and was inhibited by 5 mM guazatine. Taken together, these results suggest that PAO-generated H2O2 is involved in coleorhiza-limited rice seed germination and that OsPAO5 expression accounts for most PAO expression and activity during rice seed germination. These findings should facilitate further study of PAOs and provide valuable information for functional validation of these proteins during seed germination of monocot cereals. PMID:27570530

  19. Involvement of Polyamine Oxidase-Produced Hydrogen Peroxide during Coleorhiza-Limited Germination of Rice Seeds.

    PubMed

    Chen, Bing-Xian; Li, Wen-Yan; Gao, Yin-Tao; Chen, Zhong-Jian; Zhang, Wei-Na; Liu, Qin-Jian; Chen, Zhuang; Liu, Jun

    2016-01-01

    Seed germination is a complicated biological process that requires regulated enzymatic and non-enzymatic reactions. The action of polyamine oxidase (PAO) produces hydrogen peroxide (H2O2), which promotes dicot seed germination. However, whether and, if so, how PAOs regulate monocot seed germination via H2O2 production is unclear. Herein, we report that the coleorhiza is the main physical barrier to radicle protrusion during germination of rice seed (a monocot seed) and that it does so in a manner similar to that of dicot seed micropylar endosperm. We found that H2O2 specifically and steadily accumulated in the coleorhizae and radicles of germinating rice seeds and was accompanied by increased PAO activity as the germination percentage increased. These physiological indexes were strongly decreased in number by guazatine, a PAO inhibitor. We also identified 11 PAO homologs (OsPAO1-11) in the rice genome, which could be classified into four subfamilies (I, IIa, IIb, and III). The OsPAO genes in subfamilies I, IIa, and IIb (OsPAO1-7) encode PAOs, whereas those in subfamily III (OsPAO8-11) encode histone lysine-specific demethylases. In silico-characterized expression profiles of OsPAO1-7 and those determined by qPCR revealed that OsPAO5 is markedly upregulated in imbibed seeds compared with dry seeds and that its transcript accumulated to a higher level in embryos than in the endosperm. Moreover, its transcriptional abundance increased gradually during seed germination in water and was inhibited by 5 mM guazatine. Taken together, these results suggest that PAO-generated H2O2 is involved in coleorhiza-limited rice seed germination and that OsPAO5 expression accounts for most PAO expression and activity during rice seed germination. These findings should facilitate further study of PAOs and provide valuable information for functional validation of these proteins during seed germination of monocot cereals. PMID:27570530

  20. Mechanistic and Structural Analyses of the Role of His67 in the Yeast Polyamine Oxidase Fms1

    SciTech Connect

    Adachi, Mariya S.; Taylor, Alexander B.; Hart, P. John; Fitzpatrick, Paul F.

    2012-07-25

    The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N1-acetylspermine to spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. Within the active site of Fms1, His67 is positioned to form hydrogen bonds with the polyamine substrate. This residue is also conserved in other polyamine oxidases. The catalytic properties of H67Q, H67N, and H67A Fms1 have been characterized to evaluate the role of this residue in catalysis. With both spermine and N1-acetylspermine as the amine substrate, the value of the first-order rate constant for flavin reduction decreases 2-3 orders of magnitude, with the H67Q mutation having the smallest effect and H67N the largest. The k{sub cat}/K{sub O2} value changes very little upon mutation with N{sup 1}-acetylspermine as the amine substrate and decreases only an order of magnitude with spermine. The k{sub cat}/K{sub M}-pH profiles with N{sup 1}-acetylspermine are bell-shaped for all the mutants; the similarity to the profile of the wild-type enzyme rules out His67 as being responsible for either of the pK{sub a} values. The pH profiles for the rate constant for flavin reduction for all the mutant enzymes similarly show the same pK{sub a} as wild-type Fms1, about {approx}7.4; this pK{sub a} is assigned to the substrate N4. The k{sub cat}/K{sub O2}-pH profiles for wild-type Fms1 and the H67A enzyme both show a pK{sub a} of about {approx}6.9; this suggests His67 is not responsible for this pH behavior. With the H67Q, H67N, and H67A enzymes the k{sub cat} value decreases when a single residue is protonated, as is the case with the wild-type enzyme. The structure of H67Q Fms1 has been determined at a resolution of 2.4 {angstrom}. The structure shows that the mutation disrupts a hydrogen bond network in the active site, suggesting that His67 is important both for direct interactions with the substrate and to maintain the overall active site structure.

  1. Polyamine homoeostasis.

    PubMed

    Persson, Lo

    2009-01-01

    The polyamines are essential for a variety of functions in the mammalian cell. Although their specific effects have not been fully elucidated, it is clear that the cellular polyamines have to be kept within certain levels for normal cell function. Polyamine homoeostasis in mammalian cells is achieved by a complex network of regulatory mechanisms affecting synthesis and degradation, as well as membrane transport of polyamines. The two key enzymes in the polyamine biosynthetic pathway, ODC (ornithine decarboxylase) and AdoMetDC (S-adenosylmethionine decarboxylase), are strongly regulated by feedback mechanisms at several levels, including transcriptional, translational and post-translational. Some of these mechanisms have been shown to be truly unique and include upstream reading frames and ribosomal frameshifting, as well as ubiquitin-independent proteasomal degradation. SSAT (spermidine/spermine N1-acetyltransferase), which is a crucial enzyme for degradation and efflux of polyamines, is also highly regulated by polyamines. A cellular excess of polyamines rapidly induces SSAT, resulting in increased degradation/efflux of the polyamines. The polyamines appear to induce both transcription and translation of the SSAT mRNA. However, the major part of the polyamine-induced increase in SSAT is caused by a marked stabilization of the enzyme against degradation by the 26S proteasome. In addition, active transport of extracellular polyamines into the cell contributes to cellular polyamine homoeostasis. Depletion of cellular polyamines rapidly induces an increased uptake of exogenous polyamines, whereas an excess of polyamines down-regulates the polyamine transporter(s). However, the protein(s) involved in polyamine transport and the exact mechanisms by which the polyamines regulate the transporter(s) are not yet known. PMID:20095967

  2. Genome-wide identification and expression analysis of the polyamine oxidase gene family in sweet orange (Citrus sinensis).

    PubMed

    Wang, Wei; Liu, Ji-Hong

    2015-01-25

    Polyamine oxidases (PAOs) are FAD-dependent enzymes associated with polyamine catabolism. In plants, increasing evidences support that PAO genes play essential roles in abiotic and biotic stresses response. In this study, six putative PAO genes (CsPAO1-CsPAO6) were unraveled in sweet orange (Citrus sinensis) using the released citrus genome sequences. A total of 203 putative cis-regulatory elements involved in hormone and stress response were predicted in 1.5-kb promoter regions at the upstream of CsPAOs. The CsPAOs can be divided into four major groups, with similar organizations with their counterparts of Arabidopsis thaliana. Transcripts of CsPAOs were detected in leaf, stem, cotyledon, and root, with the highest levels detected in the roots. The CsPAOs displayed various responses to exogenous treatments with polyamines and ABA and were differentially altered by abiotic stresses, including cold, salt, and mannitol. Overexpression of CsPAO3 in tobacco demonstrated that spermidine and spermine were decreased in the transgenic line, while putrescine was significantly enhanced, implying a potential role of this gene in polyamine back conversion. These data provide valuable knowledge for understanding the roles of the PAO genes in the future. PMID:25445392

  3. Ternary Hybrid γ-Fe2 O3 /Cr(VI) /Amine Oxidase Nanostructure for Electrochemical Sensing: Application for Polyamine Detection in Tumor Tissue.

    PubMed

    Bonaiuto, Emanuela; Magro, Massimiliano; Baratella, Davide; Jakubec, Petr; Sconcerle, Elisabetta; Terzo, Milo; Miotto, Giovanni; Macone, Alberto; Agostinelli, Enzo; Fasolato, Silvano; Venerando, Rina; Salviulo, Gabriella; Malina, Ondrej; Zboril, Radek; Vianello, Fabio

    2016-05-10

    Dichromate binds to surface-active maghemite nanoparticles (SAMNs) to form a stable core-shell nanostructures (SAMN@Cr(VI) ). The hybrid was characterized by Mössbauer spectroscopy, high-angle annular dark-field imaging, electron energy-loss spectroscopy, and electrochemical techniques, which revealed a strong interaction of dichromate with the nanoparticle surface. Electrochemical characterization showed lower charge-transfer resistance, better electrochemical performance, and more reversible electrochemical behavior with respect to naked SAMNs. Moreover, SAMN@Cr(VI) is an excellent electrocatalyst for hydrogen peroxide reduction. Furthermore, an enzyme, namely, bovine serum amine oxidase (BSAO: EC 1.4.3.6), was immobilized on SAMN@Cr(VI) by self-assembly to give a ternary hybrid nanostructured catalyst for polyamine oxidation (SAMN@Cr(VI) -BSAO). SAMN@Cr(VI) -BSAO was applied for the development of a reagentless, fast, inexpensive, and interference-free polyamine biosensor, which was successfully exploited for the discrimination of tumorous tissue from healthy tissue in human crude liver extracts. PMID:27060887

  4. Chemically induced oxidative stress increases polyamine levels by activating the transcription of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase in human hepatoma HUH7 cells.

    PubMed

    Smirnova, Olga A; Isaguliants, Maria G; Hyvonen, Mervi T; Keinanen, Tuomo A; Tunitskaya, Vera L; Vepsalainen, Jouko; Alhonen, Leena; Kochetkov, Sergey N; Ivanov, Alexander V

    2012-09-01

    Biogenic polyamines spermine and spermidine participate in numerous cellular processes including transcription, RNA processing and translation. Specifically, they counteract oxidative stress, an alteration of cell redox balance involved in generation and progression of various pathological states including cancer. Here, we investigated how chemically induced oxidative stress affects polyamine metabolism, specifically the expression and activities of enzymes catalyzing polyamine synthesis (ornithine decarboxylase; ODC) and degradation (spermidine/spermine-N(1)-acetyltransferase; SSAT), in human hepatoma cells. Oxidative stress induced the up-regulation of ODC and SSAT gene transcription mediated by Nrf2, and in case of SSAT, also by NF-κB transcription factors. Activation of transcription led to the elevated intracellular activities of both enzymes. The balance in antagonistic activities of ODC and SSAT in the stressed hepatoma cells was shifted towards polyamine biosynthesis, which resulted in increased intracellular levels of putrescine, spermidine, and spermine. Accumulation of putrescine is indicating for accelerated degradation of polyamines by SSAT - acetylpolyamine oxidase (APAO) pathway generating toxic products that promote carcinogenesis, whereas accelerated polyamine synthesis via activation of ODC is favorable for proliferation of cells including those sub-lethally damaged by oxidative stress. PMID:22579641

  5. Wounding induces changes in tuber polyamine content, polyamine metabolic gene expression, and enzyme activity during closing layer formation and initiation of wound periderm formation.

    PubMed

    Lulai, Edward C; Neubauer, Jonathan D; Olson, Linda L; Suttle, Jeffrey C

    2015-03-15

    Tuber wound-healing processes are complex, and the associated regulation and modulation of these processes are poorly understood. Polyamines (PA) are involved in modulating a variety of responses to biotic and abiotic plant stresses and have been suggested to be involved in tuber wound responses. However, the time course of wound-induced changes in tuber PA content, activity of key biosynthetic enzymes and associated gene expression has not been determined and coordinated with major wound-healing processes. The objective of this study was to determine these wound-induced changes and their coordination with wound-healing processes. Wounding induced increases in putrescine (Put) and spermidine (Spd), but had only minor effects on spermine (Spm) content during the 168 h time course which encompassed the initiation and completion of the closing layer formation, and the initiation of cell division and wound periderm formation. As determinants of the first committed step in PA biosynthesis, arginine and ornithine decarboxylase (ADC and ODC, respectively) activities were below levels of detectability in resting tubers and expression of genes encoding these two enzymes was low. Within 6h of wounding, increases in the in vitro activities of ADC and ODC and expression of their cognate genes were observed. Expression of a gene encoding S-adenosylmethionine decarboxylase, required for Spd and Spm biosynthesis, was also increased 6h after wounding and remained elevated throughout the time course. Expression of a polyamine catabolic gene, encoding polyamine oxidase, was down-regulated after wounding. Results indicated a rapid wound-induced increase in PA biosynthesis during closing layer formation and the time of nuclei entry and exit from S-phase. PA content remained elevated as wound-induced cells became meristematic and initiated formation of the wound periderm suggesting sustained involvement in wound-healing. PMID:25577734

  6. Activation of hyaluronidase by 8-methoxypsoralen-polyamine photoproducts.

    PubMed

    Shigehara, N; Kakegawa, H; Miyataka, H; Matsumoto, H; Isobe, A; Satoh, T

    1991-12-01

    Ultraviolet radiation of 8-methoxypsoralen (8-MOP) in the presence of various polyamines resulted in stable photoproducts that were very soluble in water and showed hyaluronidase-activating properties. Among them, the photoproducts obtained from the reaction systems of 8-MOP-spermine and 8-MOP-spermidine markedly activated hyaluronidase. The enzyme activity was not affected by 8-MOP alone and the photoproduct of 8-MOP (8-MOP-P). From these facts, it was suggested that the photoproducts with hyaluronidase-activating properties might play an important role in the onset of 8-MOP-induced photosensitivity. PMID:1823146

  7. Ornithine decarboxylase regulates the activity and localization of rhoA via polyamination.

    PubMed

    Mäkitie, Laura T; Kanerva, Kristiina; Andersson, Leif C

    2009-04-01

    Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine synthesis. Polyamines and ODC are connected to cell proliferation and transformation. Resting cells display a low ODC activity while normal, proliferating cells display fluctuations in ODC activity that coincide with changes in the actin cytoskeleton during the cell cycle. Cancerous cells display constitutively elevated ODC activity. Overexpression of ODC in NIH 3T3 fibroblasts induces a transformed phenotype. The cytoskeletal rearrangements during cytokinesis and cell transformation are intimately coupled to the ODC activity but the molecular mechanisms have remained elusive. In this study we investigated how ODC and polyamines influence the organization of the cytoskeleton. Given that the small G-proteins of the rho family are key modulators of the actin cytoskeleton, we investigated the molecular interactions of rhoA with ODC and polyamines. Our results show that transglutaminase-catalyzed polyamination of rhoA regulates its activity. The polyamination status of rhoA crucially influences the progress of the cell cycle as well as the rate of transformation of rat fibroblasts infected with temperature-sensitive v-src. We also show that ODC influences the intracellular distribution of rhoA. These findings provide novel insights into the mechanisms by which ODC and polyamines regulate the dynamics of the cytoskeleton during cell proliferation and transformation. PMID:19331812

  8. The polyamine analogue N1,N11-diethylnorspermine can induce chondrocyte apoptosis independently of its ability to alter metabolism and levels of natural polyamines.

    PubMed

    Stanic', Ivana; Facchini, Annalisa; Borzì, Rosa Maria; Stefanelli, Claudio; Flamigni, Flavio

    2009-04-01

    We have been investigating the effects of natural polyamines and polyamine analogues on the survival and apoptosis of chondrocytes, which are cells critical for cartilage integrity. Treatment of human C-28/I2 chondrocytes with N(1),N(11)-diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, rapidly induced spermidine/spermine N(1)-acetyltransferase (SSAT) and spermine oxidase (SMO), key enzymes of polyamine catabolism and down-regulated ornithine decarboxylase, the first enzyme of polyamine biosynthesis, thus depleting all main polyamines within 24 h. The treatment with DENSPM did not provoke cell death and caspase activation when given alone for 24 h, but caused a caspase-3 and -9 dependent apoptosis in chondrocytes further exposed to cycloheximide (CHX). In other cellular models, enhanced polyamine catabolism or polyamine depletion has been implicated as mechanisms involved in DENSPM-related apoptosis. However, the simultaneous addition of DENSPM and CHX rapidly increased caspase activity in C-28/I2 cells in the absence of SSAT and SMO induction or significant reduction of polyamine levels. Moreover, caspase activation induced by DENSPM plus CHX was not prevented by a N(1)-acetylpolyamine oxidase (PAO)/SMO inhibitor, and depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not reproduce DENSPM effects in the presence of CHX. DENSPM/CHX-induced apoptosis was associated with changes in the amount or activation of signalling kinases, Akt and MAPKs, and increased uptake of DENSPM. In conclusion, the results suggest that DENSPM can favour apoptosis in chondrocytes independently of its effects on polyamine metabolism and levels. PMID:19097065

  9. Activation of Polyphenol Oxidase of Chloroplasts 1

    PubMed Central

    Tolbert, N. E.

    1973-01-01

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

  10. Global Metabolic Profiling of Arabidopsis Polyamine Oxidase 4 (AtPAO4) Loss-of-Function Mutants Exhibiting Delayed Dark-Induced Senescence

    PubMed Central

    Sequera-Mutiozabal, Miren I.; Erban, Alexander; Kopka, Joachim; Atanasov, Kostadin E.; Bastida, Jaume; Fotopoulos, Vasileios; Alcázar, Rubén; Tiburcio, Antonio F.

    2016-01-01

    Early and more recent studies have suggested that some polyamines (PAs), and particularly spermine (Spm), exhibit anti-senescence properties in plants. In this work, we have investigated the role of Arabidopsis Polyamine Oxidase 4 (PAO4), encoding a PA back-conversion oxidase, during dark-induced senescence. Two independent PAO4 (pao4-1 and pao4-2) loss-of-function mutants have been found that accumulate 10-fold higher Spm, and this associated with delayed entry into senescence under dark conditions. Mechanisms underlying pao4 delayed senescence have been studied using global metabolic profiling by GC-TOF/MS. pao4 mutants exhibit constitutively higher levels of important metabolites involved in redox regulation, central metabolism and signaling that support a priming status against oxidative stress. During senescence, interactions between PAs and oxidative, sugar and nitrogen metabolism have been detected that additively contribute to delayed entry into senescence. Our results indicate the occurrence of metabolic interactions between PAs, particularly Spm, with cell oxidative balance and transport/biosynthesis of amino acids as a strategy to cope with oxidative damage produced during senescence. PMID:26925084

  11. Toxicity of polyamines and their metabolic products.

    PubMed

    Pegg, Anthony E

    2013-12-16

    Polyamines are ubiquitous and essential components of mammalian cells. They have multiple functions including critical roles in nucleic acid and protein synthesis, gene expression, protein function, protection from oxidative damage, the regulation of ion channels, and maintenance of the structure of cellular macromolecules. It is essential to maintain a correct level of polyamines, and this amount is tightly regulated at the levels of transport, synthesis, and degradation. Catabolic pathways generate reactive aldehydes including acrolein and hydrogen peroxide via a number of oxidases. These metabolites, particularly those from spermine, can cause significant toxicity with damage to proteins, DNA, and other cellular components. Their production can be increased as a result of infection or cell damage that releases free polyamines and activates the oxidative catabolic pathways. Since polyamines also have an important physiological role in protection from oxidative damage, the reduction in polyamine content may exacerbate the toxic potential of these agents. Increases in polyamine catabolism have been implicated in the development of diseases including stroke, other neurological diseases, renal failure, liver disease, and cancer. These results provide new opportunities for the early diagnosis, prevention, and treatment of disease. PMID:24224555

  12. Targets of polyamine dysregulation in major depression and suicide: Activity-dependent feedback, excitability, and neurotransmission.

    PubMed

    Limon, Agenor; Mamdani, Firoza; Hjelm, Brooke E; Vawter, Marquis P; Sequeira, Adolfo

    2016-07-01

    Major depressive disorder (MDD) is a leading cause of disability worldwide characterized by altered neuronal activity in brain regions involved in the control of stress and emotion. Although multiple lines of evidence suggest that altered stress-coping mechanisms underlie the etiology of MDD, the homeostatic control of neuronal excitability in MDD at the molecular level is not well established. In this review, we examine past and current evidence implicating dysregulation of the polyamine system as a central factor in the homeostatic response to stress and the etiology of MDD. We discuss the cellular effects of abnormal metabolism of polyamines in the context of their role in sensing and modulation of neuronal, electrical, and synaptic activity. Finally, we discuss evidence supporting an allostatic model of depression based on a chronic elevation in polyamine levels resulting in self-sustained stress response mechanisms maintained by maladaptive homeostatic mechanisms. PMID:27108532

  13. Effect of Rosuvastatin on Arginase Enzyme Activity and Polyamine Production in Experimental Breast Cancer

    PubMed Central

    Erbaş, Hakan; Bal, Oğuz; Çakır, Erol

    2015-01-01

    Background: Breast cancer is the most common malignant tumour of women around the world. As a key enzyme of the urea cycle, arginase leads to the formation of urea and ornithine from L-arginine. In the patients with several different cancers, arginase has been found to be higher and reported to be a useful biological marker. Aims: The aim of this study was to investigate the effect of rosuvastatin on serum and cancer tissue arginase enzyme activity, and ornithine and polyamine (putrescine, spermidine, spermine) levels. Study Design: Animal experiment. Methods: In this study, 50 male Balb/c mice were used. Erchlich acid tumour cells were injected into the subcutaneous part of their left foot. The mice were divided into five groups: healthy control group, healthy treatment, tumour control, treatment 1 and treatment 2. Then, 1 mg/kg and 20 mg/kg doses of rosuvastatin were given intraperitoneally. Serum and tissue arginase enzyme activities and tissue ornithine levels were determined spectrophotometrically. HPLC measurement of polyamines were applied. Results: Increased serum arginase activity and polyamine levels were significantly decreased with rosuvastatin treatment. In the tumour tissue, arginase activity and ornithine levels were significantly decreased in treatment groups compared to the tumour group. Tissue polyamine levels also decreased with rosuvastatin treatment. Conclusion: We suggest that rosuvastatin may have some protective effects on breast cancer development as it inhibits arginase enzyme activity and ornithine levels, precursors of polyamines, and also polyamine levels. This protective effect may be through the induction of nitric oxide (NO) production via nitric oxide synthase (NOS). As a promising anticancer agent, the net effects of rosuvastatin in this mechanism should be supported with more advanced studies and new parameters. PMID:25759778

  14. Antioxidant and Antimelanogenic activities of polyamine conjugates from corn bran and related hydroxycinnamic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antioxidant activity of three major polyamine conjugates, N,N'-dicoumaroyl- putrescine (DCP), N-p-coumaroyl-N'-feruloylputrescine (CFP) and N,N'-diferuloyl- putrescine (DFP) isolated from corn bran, and their related hydroxycinnamic acids, p-coumaric acid (CA) and ferulic acid (FA), were evaluat...

  15. Polyamines in cancer.

    PubMed

    Paz, Edwin A; Garcia-Huidobro, Jenaro; Ignatenko, Natalia A

    2011-01-01

    Polyamines are organic cations shown to control gene expression at the transcriptional, posttranscriptional, and translational levels. Multiple cellular oncogenic pathways are involved in regulation of transcription and translation of polyamine-metabolizing enzymes. As a consequence of genetic alterations, expression levels and activities of polyamine-metabolizing enzymes change rapidly during tumorigenesis resulting in high levels of polyamines in many human epithelial tumors. This review summarizes the mechanisms of polyamine regulation by canonical tumor suppressor genes and oncogenes, as well as the role of eukaryotic initiation factor 5A (EIF5A) in cancer. The importance of research utilizing pharmaceutical inhibitors and cancer chemopreventive strategies targeting the polyamine pathway is also discussed. PMID:21874756

  16. Altered growth and polyamine catabolism following exposure of the chocolate spot pathogen Botrytis fabae to the essential oil of Ocimum basilicum.

    PubMed

    Oxenham, Senga K; Svoboda, Katja P; Walters, Dale R

    2005-01-01

    Biomass of the fungal pathogen Botrytis fabae in liquid culture amended with two chemotypes of the essential oil of basil, Ocimum basilicum, was reduced significantly at concentrations of 50 ppm or less. The methyl chavicol chemotype oil increased the activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), but polyamine concentrations were not significantly altered. In contrast, the linalol chemotype oil decreased AdoMetDC activity in B. fabae, although again polyamine concentrations were not altered significantly. However activities of the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO) were increased significantly in B. fabae grown in the presence of the essential oil of the two chemotypes. It is suggested that the elevated activities of DAO and PAO may be responsible, in part, for the antifungal effects of the basil oil, possibly via the generation of hydrogen peroxide and the subsequent triggering of programmed cell death. PMID:16392245

  17. Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells.

    PubMed

    Ishii, Ikumi; Ikeguchi, Yoshihiko; Mano, Hiroshi; Wada, Masahiro; Pegg, Anthony E; Shirahata, Akira

    2012-02-01

    Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N(1)-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis. PMID:21809076

  18. Polyamines control of cation transport across plant membranes: implications for ion homeostasis and abiotic stress signaling

    PubMed Central

    Pottosin, Igor; Shabala, Sergey

    2014-01-01

    Polyamines are unique polycationic metabolites, controlling a variety of vital functions in plants, including growth and stress responses. Over the last two decades a bulk of data was accumulated providing explicit evidence that polyamines play an essential role in regulating plant membrane transport. The most straightforward example is a blockage of the two major vacuolar cation channels, namely slow (SV) and fast (FV) activating ones, by the micromolar concentrations of polyamines. This effect is direct and fully reversible, with a potency descending in a sequence Spm4+ > Spd3+ > Put2+. On the contrary, effects of polyamines on the plasma membrane (PM) cation and K+-selective channels are hardly dependent on polyamine species, display a relatively low affinity, and are likely to be indirect. Polyamines also affect vacuolar and PM H+ pumps and Ca2+ pump of the PM. On the other hand, catabolization of polyamines generates H2O2 and other reactive oxygen species (ROS), including hydroxyl radicals. Export of polyamines to the apoplast and their oxidation there by available amine oxidases results in the induction of a novel ion conductance and confers Ca2+ influx across the PM. This mechanism, initially established for plant responses to pathogen attack (including a hypersensitive response), has been recently shown to mediate plant responses to a variety of abiotic stresses. In this review we summarize the effects of polyamines and their catabolites on cation transport in plants and discuss the implications of these effects for ion homeostasis, signaling, and plant adaptive responses to environment. PMID:24795739

  19. Polyamines cause plasma membrane depolarization, activate Ca2+-, and modulate H+-ATPase pump activity in pea roots.

    PubMed

    Pottosin, Igor; Velarde-Buendía, Ana María; Bose, Jayakumar; Fuglsang, Anja T; Shabala, Sergey

    2014-06-01

    Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress. PMID:24723394

  20. Biosynthesis of polyamines and polyamine-containing molecules.

    PubMed

    Michael, Anthony J

    2016-08-01

    Polyamines are evolutionarily ancient polycations derived from amino acids and are pervasive in all domains of life. They are essential for cell growth and proliferation in eukaryotes and are essential, important or dispensable for growth in bacteria. Polyamines present a useful scaffold to attach other moieties to, and are often incorporated into specialized metabolism. Life has evolved multiple pathways to synthesize polyamines, and structural variants of polyamines have evolved in bacteria, archaea and eukaryotes. Among the complex biosynthetic diversity, patterns of evolutionary reiteration can be distinguished, revealing evolutionary recycling of particular protein folds and enzyme chassis. The same enzyme activities have evolved from multiple protein folds, suggesting an inevitability of evolution of polyamine biosynthesis. This review discusses the different biosynthetic strategies used in life to produce diamines, triamines, tetra-amines and branched and long-chain polyamines. It also discusses the enzymes that incorporate polyamines into specialized metabolites and attempts to place polyamine biosynthesis in an evolutionary context. PMID:27470594

  1. Antimutagenic activity of oxidase enzymes

    SciTech Connect

    Agabeili, R.A.

    1986-11-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity.

  2. Catalytically active bovine serum amine oxidase bound to fluorescent and magnetically drivable nanoparticles

    PubMed Central

    Sinigaglia, Giulietta; Magro, Massimiliano; Miotto, Giovanni; Cardillo, Sara; Agostinelli, Enzo; Zboril, Radek; Bidollari, Eris; Vianello, Fabio

    2012-01-01

    Novel superparamagnetic surface-active maghemite nanoparticles (SAMNs) characterized by a diameter of 10 ± 2 nm were modified with bovine serum amine oxidase, which used rhodamine B isothiocyanate (RITC) adduct as a fluorescent spacer-arm. A fluorescent and magnetically drivable adduct comprised of bovine serum copper-containing amine oxidase (SAMN–RITC–BSAO) that immobilized on the surface of specifically functionalized magnetic nanoparticles was developed. The multifunctional nanomaterial was characterized using transmission electron microscopy, infrared spectroscopy, mass spectrometry, and activity measurements. The results of this study demonstrated that bare magnetic nanoparticles form stable colloidal suspensions in aqueous solutions. The maximum binding capacity of bovine serum amine oxidase was approximately 6.4 mg g−1 nanoparticles. The immobilization procedure reduced the catalytic activity of the native enzyme to 30% ± 10% and the Michaelis constant was increased by a factor of 2. We suggest that the SAMN–RITC–BSAO complex, characterized by a specific activity of 0.81 IU g−1, could be used in the presence of polyamines to create a fluorescent magnetically drivable H2O2 and aldehydes-producing system. Selective tumor cell destruction is suggested as a potential future application of this system. PMID:22619559

  3. The mechanism of opiorphin-induced experimental priapism in rats involves activation of the polyamine synthetic pathway.

    PubMed

    Kanika, Nirmala Devi; Tar, Moses; Tong, Yuehong; Kuppam, Dwaraka Srinivasa Rao; Melman, Arnold; Davies, Kelvin Paul

    2009-10-01

    Intracorporal injection of plasmids encoding opiorphins into retired breeder rats can result in animals developing a priapic-like condition. Microarray analysis demonstrated that following intracorporal gene transfer of plasmids expressing opiorphins the most significantly upregulated gene in corporal tissue was the ornithine decarboxylase gene (ODC). Quantitative RT-PCR confirmed the upregulation of ODC, as well as other genes involved in polyamine synthesis, such as arginase-I and -II, polyamine oxidase, spermidine synthase, spermidine acetyltransferase (SAT), and S-adenosylmethionine decarboxylase. Western blot analysis demonstrated upregulation of arginase-I and -II, ODC, and SAT at the protein level. Levels of the polyamine putrescine were upregulated in animals treated with opiorphin-expressing plasmids compared with controls. A direct role for the upregulation of polyamine synthesis in the development of the priapic-like condition was supported by the observation that the ODC inhibitor 1,3-diaminopropane, when added to the drinking water of animals treated with plasmids expressing opiorphins, prevented experimental priapism. We also demonstrate that in sickle cell mice, another model of priapism, there is increased expression of the mouse opiorphin homologue in corporal tissue compared with the background strain at a life stage prior to evidence of priapism. At a life stage when there is onset of priapism, there is increased expression of the enzymes involved in polyamine synthesis (ODC and arginase-I and -II). Our results suggest that the upregulation of enzymes involved in the polyamine synthetic pathway may play a role in the development of experimental priapism and represent a target for the prevention of priapism. PMID:19657052

  4. Conjugation with polyamines enhances the antibacterial and anticancer activity of chloramphenicol

    PubMed Central

    Kostopoulou, Ourania N.; Kouvela, Ekaterini C.; Magoulas, George E.; Garnelis, Thomas; Panagoulias, Ioannis; Rodi, Maria; Papadopoulos, Georgios; Mouzaki, Athanasia; Dinos, George P.; Papaioannou, Dionissios; Kalpaxis, Dimitrios L.

    2014-01-01

    Chloramphenicol (CAM) is a broad-spectrum antibiotic, limited to occasional only use in developed countries because of its potential toxicity. To explore the influence of polyamines on the uptake and activity of CAM into cells, a series of polyamine–CAM conjugates were synthesized. Both polyamine architecture and the position of CAM-scaffold substitution were crucial in augmenting the antibacterial and anticancer potency of the synthesized conjugates. Compounds 4 and 5, prepared by replacement of dichloro-acetyl group of CAM with succinic acid attached to N4 and N1 positions of N8,N8-dibenzylspermidine, respectively, exhibited higher activity than CAM in inhibiting the puromycin reaction in a bacterial cell-free system. Kinetic and footprinting analysis revealed that whereas the CAM-scaffold preserved its role in competing with the binding of aminoacyl-tRNA 3′-terminus to ribosomal A-site, the polyamine-tail could interfere with the rotatory motion of aminoacyl-tRNA 3′-terminus toward the P-site. Compared to CAM, compounds 4 and 5 exhibited comparable or improved antibacterial activity, particularly against CAM-resistant strains. Compound 4 also possessed enhanced toxicity against human cancer cells, and lower toxicity against healthy human cells. Thus, the designed conjugates proved to be suitable tools in investigating the ribosomal catalytic center plasticity and some of them exhibited greater efficacy than CAM itself. PMID:24939899

  5. Activation of antibacterial autophagy by NADPH oxidases

    PubMed Central

    Huang, Ju; Canadien, Veronica; Lam, Grace Y.; Steinberg, Benjamin E.; Dinauer, Mary C.; Magalhaes, Marco A. O.; Glogauer, Michael; Grinstein, Sergio; Brumell, John H.

    2009-01-01

    Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes, promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR ligands. Engagement of either TLRs or the Fcγ receptors (FcγRs) during phagocytosis induced recruitment of the autophagy protein LC3 to phagosomes with similar kinetics. Both receptors are known to activate the NOX2 NADPH oxidase, which plays a central role in microbial killing by phagocytes through the generation of reactive oxygen species (ROS). We found that NOX2-generated ROS are necessary for LC3 recruitment to phagosomes. Antibacterial autophagy in human epithelial cells, which do not express NOX2, was also dependent on ROS generation. These data reveal a coupling of oxidative and nonoxidative killing activities of the NOX2 NADPH oxidase in phagocytes through autophagy. Furthermore, our results suggest a general role for members of the NOX family in regulating autophagy. PMID:19339495

  6. Discovery of novel polyamine analogs with anti-protozoal activity by computer guided drug repositioning.

    PubMed

    Alberca, Lucas N; Sbaraglini, María L; Balcazar, Darío; Fraccaroli, Laura; Carrillo, Carolina; Medeiros, Andrea; Benitez, Diego; Comini, Marcelo; Talevi, Alan

    2016-04-01

    Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds. PMID:26891837

  7. Polyamine depletion enhances the roscovitine-induced apoptosis through the activation of mitochondria in HCT116 colon carcinoma cells.

    PubMed

    Arısan, Elif Damla; Coker, Ajda; Palavan-Ünsal, Narçin

    2012-02-01

    Small molecule inhibitors of cyclin-dependent kinases (CDKs) show high therapeutic potential in various cancer types which are characterized by the accumulation of transformed cells due to impaired apoptotic machinery. Roscovitine, a CDK inhibitor showed to be a potent apoptotic inducer in several cancer cells. Polyamines, putrescine, spermidine and spermine, are biogenic amines involved in many cellular processes, including apoptosis. In this study, we explored the potential role of polyamines in roscovitine-induced apoptosis in HCT116 colon cancer cells. Roscovitine induced apoptosis by activating mitochondrial pathway caspases and modulating the expression of Bcl-2 family members. Depletion of polyamines by treatment with difluoromethylornithine (DFMO) increased roscovitine-induced apoptosis. Transient silencing of ornithine decarboxylase, polyamine biosynthesis enzyme and special target of DFMO also increased roscovitine-induced apoptosis in HCT116 cells. Interestingly, additional putrescine treatment was found pro-apoptotic due to the presence of non-functional ornithine decarboxylase (ODC). Finally, roscovitine altered polyamine catabolic pathway and led to decrease in putrescine and spermidine levels. Therefore, the metabolic regulation of polyamines may dictate the power of roscovitine induced apoptotic responses in HCT116 colon cancer cells. PMID:21809075

  8. Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

    PubMed Central

    Hernández-Romero, Diana; Solano, Francisco; Sanchez-Amat, Antonio

    2005-01-01

    Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds. PMID:16269713

  9. Effect of contraceptive steroids on monoamine oxidase activity

    PubMed Central

    Southgate, Jennifer; Collins, G. G. S.; Pryse-Davies, J.; Sandler, M.

    1969-01-01

    Cyclical variations in monoamine oxidase activity during the human menstrual cycle, specific to the endometrium and modified in women undergoing contraceptive steroid treatment, may reflect changes in hormonal environment. Treatment of rats with individual constituents of the contraceptive pill causes analogous changes: oestrogens inhibit and progestogens potentiate uterine monoamine oxidase activity. ImagesFig. 2Fig. 3

  10. Silencing of the polyamine catabolic key enzyme SSAT prevents CDK inhibitor-induced apoptosis in Caco-2 colon cancer cells.

    PubMed

    Çoker, A; Arısan, E D; Palavan-Ünsal, N

    2012-04-01

    Roscovitine and purvalanol are purine derivative cyclin-dependent kinase (CDK) inhibitors that induce apoptosis in various types of cancer cells. However, their impact on the apoptotic cell death mechanism requires further elucidation. Natural polyamines putrescine, spermidine and spermine play essential roles in the regulation of cell growth and proliferation. Increased levels of polyamines in cells are considered to be involved in cancer progression. Intracellular polyamine levels are under the control of several catabolic enzymes, such as spermidine/spermine-N-acetyl transferase (SSAT), acetylpolyamine oxidase (APAO) and spermine oxidase (SMO), which could be altered by several therapeutic drugs. However, the possible role of polyamines in drug-induced apoptosis has yet to be clarified. In the present study, our aim was to determine the modulation of the polyamine catabolic pathway related to CDK inhibitor-induced apoptosis in Caco-2 cells. We found that roscovitine and purvalanol (each 20 µM) induced apoptosis by activating caspase-9 and -3, and inhibiting the mitochondrial membrane potential in Caco-2 cells. CDK inhibitors decreased the intracellular putrescine and spermine levels without affecting spermidine levels. Although both roscovitine and purvalanol induced SSAT expression, they did not exert a significant effect on the APAO expression profile. SSAT transient silencing prevented roscovitine-induced apoptosis compared to parental cells. Thus, we concluded that roscovitine and purvalanol significantly induce apoptosis in Caco-2 cells by modulating the polyamine catabolism, and that SSAT could be an important target in evaluating the potential role of polyamines in apoptotic cell death. PMID:22294330

  11. Polyamine Oxidase, a Hydrogen Peroxide-Producing Enzyme, Is Up-Regulated by Light and Down-Regulated by Auxin in the Outer Tissues of the Maize Mesocotyl1

    PubMed Central

    Cona, Alessandra; Cenci, Francesco; Cervelli, Manuela; Federico, Rodolfo; Mariottini, Paolo; Moreno, Sandra; Angelini, Riccardo

    2003-01-01

    Exogenously supplied auxin (1-naphthaleneacetic acid) inhibited light-induced activity increase of polyamine oxidase (PAO), a hydrogen peroxide-producing enzyme, in the outer tissues of maize (Zea mays) mesocotyl. The same phenomenon operates at PAO protein and mRNA accumulation levels. The wall-bound to extractable PAO activity ratio was unaffected by auxin treatment, either in the dark or after light exposure. Ethylene treatment did not affect PAO activity, thus excluding an effect of auxin via increased ethylene biosynthesis. The auxin polar transport inhibitors N1-naphthylphthalamic acid or 2,3,5-triiodobenzoic acid caused a further increase of PAO expression in outer tissues after light treatment. The small increase of PAO expression, normally occurring in the mesocotyl epidermis during plant development in the dark, was also inhibited by auxin, although to a lesser extent with respect to light-exposed tissue, and was stimulated by N1-naphthylphthalamic acid or 2,3,5-triiodobenzoic acid, thus suggesting a complex regulation of PAO expression. Immunogold ultrastructural analysis in epidermal cells revealed the association of PAO with the secretory pathway and the cell walls. The presence of the enzyme in the cell walls of this tissue greatly increased in response to light treatment. Consistent with auxin effects on light-induced PAO expression, the hormone treatment inhibited the increase in immunogold staining both intraprotoplasmically and in the cell wall. These results suggest that both light and auxin finely tune PAO expression during the light-induced differentiation of the cell wall in the maize mesocotyl epidermal tissues. PMID:12586904

  12. Purvalanol A is a strong apoptotic inducer via activating polyamine catabolic pathway in MCF-7 estrogen receptor positive breast cancer cells.

    PubMed

    Obakan, Pınar; Arısan, Elif Damla; Özfiliz, Pelin; Çoker-Gürkan, Ajda; Palavan-Ünsal, Narçin

    2014-01-01

    Purvalanol A is a specific CDK inhibitor which triggers apoptosis by causing cell cycle arrest in cancer cells. Although it has strong apoptotic potential, the mechanistic action of Purvalanol A on significant cell signaling targets has not been clarified yet. Polyamines are crucial metabolic regulators affected by CDK inhibition because of their role in cell cycle progress as well. In addition, malignant cells possess impaired polyamine homeostasis with high level of intracellular polyamines. Especially induction of polyamine catabolic enzymes spermidine/spermine N1-acetyltransferase (SSAT), polyamine oxidase (PAO) and spermine oxidase (SMO) induced toxic by-products in correlation with the induction of apoptosis in cancer cells. In this study, we showed that Purvalanol A induced apoptosis in caspase- dependent manner in MCF-7 ER(+) cells, while MDA-MB-231 (ER-) cells were less sensitive against drug. In addition Bcl-2 is a critical target for Purvalanol A, since Bcl-2 overexpressed cells are more resistant to Purvalanol A-mediated apoptosis. Furthermore, exposure of MCF-7 cells to Purvalanol A triggered SSAT and PAO upregulation and the presence of PAO/SMO inhibitor, MDL 72,527 prevented Purvalanol A-induced apoptosis. PMID:24190492

  13. Immobilization of Pichia pastoris cells containing alcohol oxidase activity

    PubMed Central

    Maleknia, S; Ahmadi, H; Norouzian, D

    2011-01-01

    Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

  14. Polyamines and nonmelanoma skin cancer

    SciTech Connect

    Gilmour, Susan K.

    2007-11-01

    Elevated levels of polyamines have long been associated with skin tumorigenesis. Tightly regulated metabolism of polyamines is critical for cell survival and normal skin homeostasis, and these controls are dysregulated in skin tumorigenesis. A key enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC) is upregulated in skin tumors compared to normal skin. Use of transgenic mouse models has demonstrated that polyamines play an essential role in the early promotional phase of skin tumorigenesis. The formation of skin tumors in these transgenic mice is dependent upon polyamine biosynthesis, especially putrescine, since treatment with inhibitors of ODC activity blocks the formation of skin tumors and causes the rapid regression of existing tumors. Although the mechanism by which polyamines promote skin tumorigenesis are not well understood, elevated levels of polyamines have been shown to stimulate epidermal proliferation, alter keratinocyte differentiation status, increase neovascularization, and increase synthesis of extracellular matrix proteins in a manner similar to that seen in wound healing. It is becoming increasingly apparent that elevated polyamine levels activate not only epidermal cells but also underlying stromal cells in the skin to promote the development and progression of skin tumors. The inhibition of polyamine biosynthesis has potential to be an effective chemoprevention strategy for nonmelanoma skin cancer.

  15. MicroRNAs, polyamines, and the activities antioxidant enzymes are associated with in vitro rooting in white pine (Pinus strobus L.).

    PubMed

    Fei, Yunjun; Xiao, Bo; Yang, Man; Ding, Qiong; Tang, Wei

    2016-01-01

    Molecular mechanism of in vitro rooting in conifer is not fully understood. After establishment of a regeneration procedure in eastern white pine (Pinus strobus L.) using mature embryos as explants to induce shoot formation on medium containing 3 μM IAA, 6 μM BA and 6 μM TDZ and induce root formation on medium containing 0.001-0.05 μM IAA, 0.001-0.05 μM IBA, 0.001-0.05 μM TDZ, we have investigated the changes of polyamine content and the activities of antioxidant enzymes during in vitro rooting in P. strobus. Our results demonstrated that putrescine (Put), spermidine (Spd), and spermine (Spm) did not increase in P. strobus during the first week of rooting on medium supplemented with 0.01 μM indole-3-acetic acid (IAA), whereas the levels of Put, Spd, and Spm increased during the 1st-3rd week of culture on medium with IAA, and then decreased on medium with IAA. No such a change in Put, Spd, and Spm was observed on medium without IAA. Measurement of antioxidant enzyme activity demonstrated that the activities of polyphenol oxidase, catalase, and peroxidase slightly increased in the first week of culture and reached to the highest peak in the 3rd-5th week of culture. Quantitative RT-PCR results indicated that miR160 was increased on the 7th day, miR162, miR397, and miR408 was increased from the 21th to 35th day, miR857 was increased on the 35th day, and miR827 was increased on the 49th day. These results demonstrated that enhanced polyamine biosynthesis, antioxidant enzyme activity, and microRNAs are correlated with the root induction and formation in P. strobus. PMID:27069836

  16. Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis.

    PubMed

    Wiel, C; Augert, A; Vincent, D F; Gitenay, D; Vindrieux, D; Le Calvé, B; Arfi, V; Lallet-Daher, H; Reynaud, C; Treilleux, I; Bartholin, L; Lelievre, E; Bernard, D

    2013-01-01

    Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability. PMID:24113189

  17. Monoamine oxidase inhibitory activities of heterocyclic chalcones.

    PubMed

    Minders, Corné; Petzer, Jacobus P; Petzer, Anél; Lourens, Anna C U

    2015-11-15

    Studies have shown that natural and synthetic chalcones (1,3-diphenyl-2-propen-1-ones) possess monoamine oxidase (MAO) inhibition activities. Of particular importance to the present study is a report that a series of furanochalcones acts as MAO-B selective inhibitors. Since the effect of heterocyclic substitution, other than furan (and more recently thiophene, piperidine and quinoline) on the MAO inhibitory properties of the chalcone scaffold remains unexplored, the aim of this study was to synthesise and evaluate further heterocyclic chalcone analogues as inhibitors of the human MAOs. For this purpose, heterocyclic chalcone analogues that incorporate pyrrole, 5-methylthiophene, 5-chlorothiophene and 6-methoxypyridine substitution were examined. Seven of the nine synthesised compounds exhibited IC50 values <1 μM for the inhibition of MAO-B, with all compounds exhibiting higher affinities for MAO-B compared to the MAO-A isoform. The most potent MAO-B inhibitor (4h) displays an IC50 value of 0.067 μM while the most potent MAO-A inhibitor (4e) exhibits an IC50 value of 3.81 μM. It was further established that selected heterocyclic chalcones are reversible and competitive MAO inhibitors. 4h, however, may exhibit tight-binding to MAO-B, a property linked to its thiophene moiety. We conclude that high potency chalcones such as 4h represent suitable leads for the development of MAO-B inhibitors for the treatment of Parkinson's disease and possibly other neurodegenerative disorders. PMID:26432037

  18. Polyamine aza-cyclic compounds demonstrate anti-proliferative activity in vitro but fail to control tumour growth in vivo.

    PubMed

    Wong, Pui Ee; Tetley, Laurence; Dufès, Christine; Chooi, Kar Wai; Bolton, Katherine; Schätzlein, Andreas G; Uchegbu, Ijeoma F

    2010-11-01

    Cationic polyamines such as the poly(propylenimine) dendrimers (DAB16) are anti-tumour agents (Dufes et al., 2005, Cancer Res 65:8079-8084). Their mechanism of action is poorly understood, but the lack of in vivo toxicity suggests cancer specificity. To explore this polyamine pharmacophore we cross-linked the aza-cyclic compound, hexacyclen, with 1,4-dibromobutane or 1,8-dibromooctane to yield the polyamines [poly(butylhexacyclen)--CL4] or [poly(octylhexacyclen)--CL8] respectively, both free of primary amines. We characterised the compounds and their respective nanoparticles and examined their interaction with the putative targets of the cationic polyamines: the cell membrane and DNA. Like DAB 16, CL4 and CL8 bind plasmid DNA and all three compounds interrupted the cell cycle of A431 epidermoid carcinoma cells in the S-phase. Additionally all three compounds disrupted erythrocyte membranes, with CL8 and DAB 16 being more active, in this respect, than CL4. CL4 (IC(50) =775.1 µg mL(-1)) and CL8 (IC(50) =8.4 µg mL(-1)), in a similar manner to DAB 16, were anti-proliferative against A431 cells; although unlike DAB 16, CL4 and CL8 were not tumouricidal against A431 xenografts in mice, indicating that primary amines may play an important role in the in vivo activity of DAB 16. PMID:20845462

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

    PubMed Central

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

    2015-01-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surpring because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism. PMID:25701385

  20. Polyamine Block of Inwardly Rectifying Potassium Channels

    PubMed Central

    Kurata, Harley T.; Cheng, Wayland W.L.; Nichols, Colin G.

    2011-01-01

    Polyamine blockade of inwardly rectifying potassium (Kir) channels underlies their steep voltage-dependence observed in native cells. The structural determinants of polyamine blockade and the structure-activity profile of endogenous polyamines requires specialized methodology for characterizing polyamine interactions with Kir channels. Recent identification and growing interest in the structure and function of prokaryotic Kir channels (KirBacs) has driven the development of new techniques for measuring ion channel activity. Several methods for measuring polyamine interactions with prokaryotic and eukaryotic Kir channels are discussed. PMID:21318869

  1. Towards squalamine mimics: synthesis and antibacterial activities of head-to-tail dimeric sterol-polyamine conjugates.

    PubMed

    Chen, Wen-Hua; Wennersten, Christine; Moellering, Robert C; Regen, Steven L

    2013-03-01

    Four dimeric sterol-polyamine conjugates have been synthesized from the homo- and hetero-connection of monomeric sterol-polyamine analogs in a head-to-tail manner. These dimeric conjugates show strong antibacterial activity against a broad spectrum of Gram-positive bacteria, whereas their corresponding activities against Gram-negative bacteria are relatively moderate. Though no significant difference was observed in the activities of these conjugates, cholic acid-containing dimeric conjugates generally exhibit higher activities than the corresponding deoxycholic acid-derived analogs. This is in contrast to the finding that a monomeric deoxycholic acid-spermine conjugate was more active than the corresponding cholic acid-derived analog. PMID:23495155

  2. Polyamines in plant physiology

    NASA Technical Reports Server (NTRS)

    Galston, A. W.; Sawhney, R. K.

    1990-01-01

    The diamine putrescine, the triamine spermidine, and the tetramine spermine are ubiquitous in plant cells, while other polyamines are of more limited occurrence. Their chemistry and pathways of biosynthesis and metabolism are well characterized. They occur in the free form as cations, but are often conjugated to small molecules like phenolic acids and also to various macromolecules. Their titer varies from approximately micromolar to more than millimolar, and depends greatly on environmental conditions, especially stress. In cereals, the activity of one of the major polyamine biosynthetic enzymes, arginine decarboxylase, is rapidly and dramatically increased by almost every studied external stress, leading to 50-fold or greater increases in putrescine titer within a few hours. The physiological significance of this increase is not yet clear, although most recent work suggests an adaptive, protective role. Polyamines produced through the action of ornithine decarboxylase, by contrast, seem essential for DNA replication and cell division. The application of exogenous polyamines produces effects on patterns of senescence and morphogenesis, suggesting but not proving a regulatory role for polyamines in these processes. The evidence for such a regulatory role is growing.

  3. Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus.

    PubMed

    Sakamoto, J; Koga, E; Mizuta, T; Sato, C; Noguchi, S; Sone, N

    1999-04-21

    Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. We previously identified and purified an alternative oxidase, cytochrome bd-type quinol oxidase, from a mutant of Bacillus stearothermophilus defective in the caa3-type oxidase activity (J. Sakamoto et al., FEMS Microbiol. Lett. 143 (1996) 151-158). Compared with proteobacterial counterparts, B. stearothermophilus cytochrome bd showed lower molecular weights of the two subunits, shorter wavelength of alpha-band absorption maximum due to heme D, and lower quinol oxidase activity. Preincubation with menaquinone-2 enhanced the enzyme activity up to 40 times, suggesting that, besides the catalytic site, there is another quinone-binding site which largely affects the enzyme activity. In order to clarify the molecular basis of the differences of cytochromes bd between B. stearothermophilus and proteobacteria, the genes encoding for the B. stearothermophilus bd was cloned based on its partial peptide sequences. The gene for subunit I (cbdA) encodes 448 amino acid residues with a molecular weight of 50195 Da, which is 14 and 17% shorter than those of Escherichia coli and Azotobacter vinelandii, respectively, and CbdA lacks the C-terminal half of the long hydrophilic loop between the putative transmembrane segments V and VI (Q loop), which has been suggested to include the substrate quinone-binding site for the E. coli enzyme. The gene for subunit II (cbdB) encodes 342 residues with a molecular weight of 38992 Da. Homology search indicated that the B. stearothermophilus cbdAB has the highest sequence similarity to ythAB in B. subtilis genome rather than to cydAB, the first set of cytochrome bd genes identified in the genome. Sequence comparison of cytochromes bd and their homologs from various organisms demonstrates that the proteins can be classified into two subfamilies, a proteobacterial type including E. coli bd and a

  4. CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity

    PubMed Central

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

    2013-01-01

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

  5. Polyphenol oxidase activity in co-ensiled temperate grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO) and its o-diphenol substrates have been shown to effectively decrease proteolytic activity during the ensiling of forages such as red clover. Orchardgrass and smooth bromegrass both contain high levels of PPO activity, but lack appropriate levels of o-diphenols to adequately...

  6. Methods to evaluate alterations in polyamine metabolism caused by Helicobacter pylori infection.

    PubMed

    Gobert, Alain P; Chaturvedi, Rupesh; Wilson, Keith T

    2011-01-01

    Helicobacter pylori is a Gram-negative bacteria that infects the human stomach of half of the world's -population. Colonization is followed by infiltration of the gastric mucosa by lymphocytes and myeloid cells. These cells are activated by various bacterial factors, causing them to produce immune/inflammatory mediators, including reactive nitrogen species and polyamines that contribute to cellular damage and the pathogenesis of H. pylori-associated gastric cancer. In vitro experiments have revealed that H. pylori induces macrophage polyamine production by upregulation of the arginase 2/ornithine decarboxylase (ODC) metabolic pathway and enhances hydrogen peroxide synthesis through the activity of spermidine oxidase (SMO). In this chapter, we present a survey of the methods used to analyze the induction and the role of the enzymes related to polyamine metabolism, i.e., arginase, ODC, and SMO in H. pylori-infected macrophages. PMID:21318889

  7. First Evidence for Polyamine Conjugation Mediated by an Enzymic Activity in Plants 1

    PubMed Central

    Serafini-Fracassini, Donatella; Del Duca, Stefano; D'Orazi, Dario

    1988-01-01

    An enzyme activity, found for the first time in plants, mainly located in the 22,000g supernatant of the crude extract of sprout apices of Helianthus tuberosus L. cv OB1 tubers, is able in vitro to covalently bind polyamines to endogenous substrates of different molecular weights. The major assay parameters, such as pH, dithiothreitol, and extract concentrations were optimized. The time course of the reaction, the dependence on putrescine concentration, its competition with histamine, the capacity to bind spermidine and spermine better than putrescine, the stability of the reaction product and analysis of the latter by sodium dodecyl sulfate polyacrylamide gel electrophoresis and thin-layer chromatography suggest that putrescine is linked to endogenous substrates by means of an enzyme reaction that shows some similarities with transglutaminase activities detected in animals. However, the activities of the crude extract and of a fraction eluted from a Sephadex G25 column were not affected by CaCl2 concentrations lower or equal to 5 millimolar; 4 or 10 millimolar EGTA caused a very small reduction; higher concentrations of CaCl2 and 15 millimolar or more of EDTA were inhibitory. N,N′-dimethylcasein was not recognized as a substrate. These data indicate that this activity also displays some characteristics which are different from those of animal transglutaminases. Images Fig. 1 Fig. 6 PMID:16666220

  8. Lysyl oxidase binds transforming growth factor-beta and regulates its signaling via amine oxidase activity.

    PubMed

    Atsawasuwan, Phimon; Mochida, Yoshiyuki; Katafuchi, Michitsuna; Kaku, Masaru; Fong, Keith S K; Csiszar, Katalin; Yamauchi, Mitsuo

    2008-12-01

    Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-beta1 (TGF-beta1), a potent growth factor abundant in bone, the effect of LOX on TGF-beta1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-beta1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-beta1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-beta1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling. PMID:18835815

  9. Drugs related to monoamine oxidase activity.

    PubMed

    Fišar, Zdeněk

    2016-08-01

    Progress in understanding the role of monoamine neurotransmission in pathophysiology of neuropsychiatric disorders was made after the discovery of the mechanisms of action of psychoactive drugs, including monoamine oxidase (MAO) inhibitors. The increase in monoamine neurotransmitter availability, decrease in hydrogen peroxide production, and neuroprotective effects evoked by MAO inhibitors represent an important approach in the development of new drugs for the treatment of mental disorders and neurodegenerative diseases. New drugs are synthesized by acting as multitarget-directed ligands, with MAO, acetylcholinesterase, and iron chelation as targets. Basic information is summarized in this paper about the drug-induced regulation of monoaminergic systems in the brain, with a focus on MAO inhibition. Desirable effects of MAO inhibition include increased availability of monoamine neurotransmitters, decreased oxidative stress, decreased formation of neurotoxins, induction of pro-survival genes and antiapoptotic factors, and improved mitochondrial functions. PMID:26944656

  10. NADPH OXIDASE: STRUCTURE AND ACTIVATION MECHANISMS (REVIEW). NOTE I.

    PubMed

    Filip-Ciubotaru, Florina; Manciuc, Carmen; Stoleriu, Gabriela; Foia, Liliana

    2016-01-01

    NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase), with its generically termed NOX isoforms, is the major source of ROS (reactive oxigen species) in biological systems. ROS are small oxygen-derived molecules with an important role in various biological processes (physiological or pathological). If under physiological conditions some processes are beneficial and necessary for life, under pathophysiological conditions they are noxious, harmful. NADPH oxidases are present in phagocytes and in a wide variety of nonphagocytic cells. The enzyme generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling them to molecular oxygen to produce superoxide anion, a reactive free-radical. Structurally, NADPH oxidase is a multicomponent enzyme which includes two integral membrane proteins, glycoprotein gp9 1 Phox and adaptor protein p22(phox), which together form the heterodimeric flavocytochrome b558 that constitutes the core of the enzyme. During the resting state, the multidomain regulatory subunits p40P(phox), p47(phox), p67(Phox) are located in the cytosol organized as a complex. The activation of phagocytic NADPH oxidase occurs through a complex series of protein interactions. PMID:27125069

  11. Phosphoproteins and the activation of the neutrophil respiratory burst oxidase

    SciTech Connect

    Okamura, N.; Curnutte, J.T.; Babior, B.M.

    1987-05-01

    The respiratory burst oxidase is a neutrophil enzyme that converts oxygen to O/sub 2//sup -/. It is dormant in resting cells but is activated when the cells are exposed to phorbol myristate acetate (PMA). PMA also induces the incorporation of /sup 32/P into certain neutrophil proteins. To determine whether phosphorylation of these proteins is related to oxidase activation, protein phosphorylation was studied in patients with chronic granulomatous disease (GCD), a group of inherited conditions in which oxidase activity is missing. In normals, neutrophil activation by PMA is associated with the phosphorylation inter alia of 48K proteins at pI 7.3 and 7.8. There is also inconstant phosphorylation of a 48K protein at pI 6.8. In 4 patients with X-linked chronic granulomatous disease (CGD), phosphorylation of pp48/6.8 and pp48/7.3 was absent, while in autosomal recessive CGD, phosphorylation of all 3 of these proteins was absent in 3 patients and significantly diminished in a fourth. These results suggest that the phosphorylation of these proteins is related to the activation of the respiratory burst oxidase. By peptide mapping, these 3 proteins appear to consist of a single peptide species whose pI variability may be due to post-translational modification. The only phosphoamino acid found in pp48/7.3 was phosphoserine.

  12. Activity of glucose oxidase functionalized onto magnetic nanoparticles

    PubMed Central

    Kouassi, Gilles K; Irudayaraj, Joseph; McCarty, Gregory

    2005-01-01

    Background Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties. Methods Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy. Results The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94–100% while thiophene acetylation was 66–72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticles Conclusion Binding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological applications PMID:15762994

  13. IRON REGULATES XANTHINE OXIDASE ACTIVITY IN THE LUNG

    EPA Science Inventory

    The iron chelator deferoxamine has been reported to inhibit both xanthine oxidase (XO) and xanthine dehydrogenase activity, but the relationship of this effect to the availability of iron in the cellular and tissue environment remains unexplored. XO and total xanthine oxidoreduct...

  14. Polyamine analog TBP inhibits proliferation of human K562 chronic myelogenous leukemia cells by induced apoptosis

    PubMed Central

    WANG, QING; WANG, YAN-LIN; WANG, KAI; YANG, JIAN-LIN; CAO, CHUN-YU

    2015-01-01

    The aim of the present study was to investigate the effects of the novel polyamine analog tetrabutyl propanediamine (TBP) on the growth of K562 chronic myelogenous leukemia (CML) cells and the underlying mechanism of these effects. MTT was used for the analysis of cell proliferation and flow cytometry was performed to analyze cell cycle distribution. DNA fragmentation analysis and Annexin V/propidium iodide double staining were used to identify apoptotic cells. The activity of the key enzymes in polyamine catabolism was detected using chemiluminescence. TBP can induce apoptosis and significantly inhibit K562 cell proliferation in a time- and dose-dependent manner. TBP treatment significantly induced the enzyme activity of spermine oxidase and acetylpolyamine oxidase in K562 cells, and also enhanced the inhibitory effect of the antitumor drug doxorubicin on K562 cell proliferation. As a novel polyamine analog, TBP significantly inhibited proliferation and induced apoptosis in K562 cells by upregulating the activity of the key enzymes in the polyamine catabolic pathways. TBP also increased the sensitivity of the K562 cells to the antitumor drug doxorubicin. These data indicate an important potential value of TBP for clinical therapy of human CML. PMID:25435975

  15. Wounding induces changes in tuber polyamine content, polyamine metabolic gene expression, and enzyme activity during closing layer formation and initiation of wound periderm formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tuber wound-healing processes are complex, and the associated regulation and modulation of these processes are poorly understood. Polyamines (PA) have been shown to be involved in modulating a variety of responses to biotic and abiotic plant stresses and have been suggested to be involved in tuber ...

  16. Actions of arginine polyamine on voltage and ligand-activated whole cell currents recorded from cultured neurones.

    PubMed Central

    Scott, R. H.; Sweeney, M. I.; Kobrinsky, E. M.; Pearson, H. A.; Timms, G. H.; Pullar, I. A.; Wedley, S.; Dolphin, A. C.

    1992-01-01

    1. Toxins from invertebrates have proved useful tools for investigation of the properties of ion channels. In this study we describe the actions of arginine polyamine which is believed to be a close analogue of FTX, a polyamine isolated from the American funnel web spider, Agelenopsis aperta. 2. Voltage-activated Ca2+ currents and Ca(2+)-dependent Cl- currents recorded from rat cultured dorsal root ganglion neurones were reversibly inhibited by arginine polyamine (AP; 0.001 to 100 microM). Low voltage-activated T-type Ca2+ currents were significantly more sensitive to AP than high voltage-activated Ca2+ currents. The IC50 values for the actions of AP on low and high voltage-activated Ca2+ currents were 10 nM and 3 microM respectively. AP was equally effective in inhibiting high voltage-activated currents carried by Ba2+, Sr2+ or Ca2+. However, AP-induced inhibition of Ca2+ currents was attenuated by increasing the extracellular Ca2+ concentration from 2 mM to 10 mM. 3. The actions of AP on a Ca(2+)-independent K+ current were more complex, 1 microM AP enhanced this current but 10 microM AP had a dual action, initially enhancing but then inhibiting the K+ current. 4. gamma-Aminobutyric acid-activated Cl- currents were also reversibly inhibited by 1 to 10 microM AP. In contrast N-methyl-D-aspartate currents recorded from rat cultured cerebellar neurones were greatly enhanced by 10 microM AP. 5. We conclude that at a concentration of 10 nM, AP is a selective inhibitor of low threshold T-type voltage-activated Ca2+ currents.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1380382

  17. Phenol oxidase activity in secondary transformed peat-moorsh soils

    NASA Astrophysics Data System (ADS)

    Styła, K.; Szajdak, L.

    2009-04-01

    The chemical composition of peat depends on the geobotanical conditions of its formation and on the depth of sampling. The evolution of hydrogenic peat soils is closely related to the genesis of peat and to the changes in water conditions. Due to a number of factors including oscillation of ground water level, different redox potential, changes of aerobic conditions, different plant communities, and root exudes, and products of the degradation of plant remains, peat-moorsh soils may undergo a process of secondary transformation conditions (Sokolowska et al. 2005; Szajdak et al. 2007). Phenol oxidase is one of the few enzymes able to degrade recalcitrant phenolic materials as lignin (Freeman et al. 2004). Phenol oxidase enzymes catalyze polyphenol oxidation in the presence of oxygen (O2) by removing phenolic hydrogen or hydrogenes to from radicals or quinines. These products undergo nucleophilic addition reactions in the presence or absence of free - NH2 group with the eventual production of humic acid-like polymers. The presence of phenol oxidase in soil environments is important in the formation of humic substances a desirable process because the carbon is stored in a stable form (Matocha et al. 2004). The investigations were carried out on the transect of peatland 4.5 km long, located in the Agroecological Landscape Park host D. Chlapowski in Turew (40 km South-West of Poznań, West Polish Lowland). The sites of investigation were located along Wyskoć ditch. The following material was taken from four chosen sites marked as Zbechy, Bridge, Shelterbelt and Hirudo in two layers: cartel (0-50cm) and cattle (50-100cm). The object of this study was to characterize the biochemical properties by the determination of the phenol oxidize activity in two layers of the four different peat-moors soils used as meadow. The phenol oxidase activity was determined spectrophotometrically by measuring quinone formation at λmax=525 nm with catechol as substrate by method of Perucci

  18. Physiological polyamines: simple primordial stress molecules

    PubMed Central

    Rhee, H J; Kim, Eui-Jin; Lee, J K

    2007-01-01

    Abstract Physiological polyamines are ubiquitous polycations with pleiotropic biochemical activities, including regulation of gene expression, cell proliferation and modulation of cell signalling. Reports that the polyamines with cytoprotective activities were induced by diverse stresses raised the hypothesis that physiological polyamines may play a role in inducing stress response. In a wide range of organisms, physiological polyamines were not only induced by diverse stresses, such as reactive oxygen species (ROS), heat, ultraviolet (UV) and psychiatric stress but were able to confer beneficial effects for survival. Recent biochemical and genetic evidences show that polyamines can function as an ROS scavenger, acid tolerance factor and chemical chaperone, and positive regulators for expression of stress response genes which may explain their protective functions against diverse stresses. Taken together, these data suggest that physiological polyamines can function as primordial stress molecules in bacteria, plants and mammals, and may play an essential role in regulation of pathogen-host interactions. PMID:17760833

  19. Studies on polyamine and ornithine metabolism in rat colon: effects of two synergistically. Acting inducers of ornithine decarboxylase activity

    SciTech Connect

    Stanley, B.A.

    1987-01-01

    Ornithine decarboxylase (ODC) activity in rat colon mucosa was determined by the release of /sup 14/CO/sub 2/ from radiolabeled ornithine in the presence (total enzyme) or absence (holoenzyme) of added pyridoxal-5'-phosphate (PLP). Total leucine incorporation into acid-precipitable protein over 30 minute was calculated by dividing the /sup 3/H-leucine in protein by the specific activity of the intracellular leucine. Amino acids, polyamines, and PLP-semicarbazide were quantified by high pressure liquid chromatography. Ornithine aminotransaminase activity (OAT) was measured as the quantity of pyrolline (5-carboxy) produced from alpha-ketoglutarate and ornithine. After 10 weeks on a high or no vitamin B/sub 6/ diet, no change in basal ODC activity was seen; however, sodium deoxycholate instillation in vitamin B/sub 6/ deficient rats led to a large increase in total but not holo-ODC activity. In rats fed normal chow diet, no increases in mucosal PLP levels were seen after either treatment. Increases in general protein synthesis rate could not account for the peaks in ODC activity after either stimulus. Putrescine increases were proportional to peaks of ODC activity after either stimulus, while spermine levels remained depressed for 18 hours after starvation/refeeding. Ornithine levels were increased after either stimulus, and this increase was linked to decreases in OAT activity, indicating short-term coordination of overall ornithine metabolism to favor polyamine biosynthesis.

  20. The use of novel C-methylated spermidine derivatives to investigate the regulation of polyamine metabolism.

    PubMed

    Hyvönen, Mervi T; Keinänen, Tuomo A; Khomutov, Maxim; Simonian, Alina; Weisell, Janne; Kochetkov, Sergey N; Vepsäläinen, Jouko; Alhonen, Leena; Khomutov, Alex R

    2011-07-14

    The polyamines are organic polycations present at millimolar concentrations in eukaryotic cells where they participate in the regulation of vital cellular functions including proliferation and differentiation. Biological evaluation of rationally designed polyamine analogs is one of the cornerstones of polyamine research. Here we have synthesized and characterized novel C-methylated spermidine analogs, that is, 2-methylspermidine, 3-methylspermidine, and 8-methylspermidine. 3-Methylspermidine was found to be metabolically stable in DU145 cells, while 8-methylspermidine was a substrate for spermidine/spermine N(1)-acetyltransferase (SSAT) and 2-methylspermidine was a substrate for both SSAT and acetylpolyamine oxidase. All the analogs induced the splicing of the productive mRNA splice variant of SSAT, overcame growth arrest induced by 72-h treatment with ornithine decarboxylase (ODC) inhibitor α-difluoromethylornithine, and were transported via the polyamine transporter. Surprisingly, 2-methylspermidine was a weak downregulator of ODC activity in DU145 cells. Our data demonstrates that it is possible to radically alter the biochemical properties of a polyamine analog by changing the position of the methyl group. PMID:21639123

  1. Contribution of polyamines metabolism and GABA shunt to chilling tolerance induced by nitric oxide in cold-stored banana fruit.

    PubMed

    Wang, Yansheng; Luo, Zisheng; Mao, Linchun; Ying, Tiejin

    2016-04-15

    Effect of exogenous nitric oxide (NO) on polyamines (PAs) catabolism, γ-aminobutyric acid (GABA) shunt, proline accumulation and chilling injury of banana fruit under cold storage was investigated. Banana fruit treated with NO sustained lower chilling injury index than the control. Notably elevated nitric oxide synthetase activity and endogenous NO level were observed in NO-treated banana fruit. PAs contents in treated fruit were significantly higher than control fruit, due to the elevated activities of arginine decarboxylase and ornithine decarboxylase. NO treatment increased the activities of diamine oxidase, polyamine oxidase and glutamate decarboxylase, while reduced GABA transaminase activity to lower levels compared with control fruit, which resulted the accumulation of GABA. Besides, NO treatment upregulated proline content and significantly enhanced the ornithine aminotransferase activity. These results indicated that the chilling tolerance induced by NO treatment might be ascribed to the enhanced catabolism of PAs, GABA and proline. PMID:26616957

  2. Potential xanthine oxidase inhibitory activity of endophytic Lasiodiplodia pseudotheobromae.

    PubMed

    Kapoor, Neha; Saxena, Sanjai

    2014-07-01

    Xanthine oxidase is considered as a potential target for treatment of hyperuricemia. Hyperuricemia is predisposing factor for gout, chronic heart failure, atherosclerosis, tissue injury, and ischemia. To date, only two inhibitors of xanthine oxidase viz. allopurinol and febuxostat have been clinically approved for used as drugs. In the process of searching for new xanthine oxidase inhibitors, we screened culture filtrates of 42 endophytic fungi using in vitro qualitative and quantitative XO inhibitory assays. The qualitative assay exhibited potential XO inhibition by culture filtrates of four isolates viz. #1048 AMSTITYEL, #2CCSTITD, #6AMLWLS, and #96 CMSTITNEY. The XO inhibitory activity was present only in the chloroform extract of the culture filtrates. Chloroform extract of culture filtrate #1048 AMSTITYEL exhibited the highest inhibition of XO with an IC50 value of 0.61 μg ml(-1) which was better than allopurinol exhibiting an IC50 of 0.937 μg ml(-1) while febuxostat exhibited a much lower IC50 of 0.076 μg ml(-1). Further, molecular phylogenetic tools and morphological studies were used to identify #1048 AMSTITYEL as Lasiodiplodia pseudotheobromae. This is the first report of an endophytic Lasiodiplodia pseudotheobromae from Aegle marmelos exhibiting potential XO Inhibitory activity. PMID:24801403

  3. External action of di- and polyamines on maxi calcium-activated potassium channels: an electrophysiological and molecular modeling study.

    PubMed Central

    Weiger, T M; Langer, T; Hermann, A

    1998-01-01

    In this study we compared polyamines to various diamines, and we modeled flexibility as well as hydrophobicity properties of these molecules to examine possible structural differences that could explain their external effects on the channels. The natural polyamines (putrescine, cadaverine, spermidine, spermine) and diamines increasing in CH2 chain length from C2 to C12 were used to probe maxi calcium-activated potassium (BK) channels in GH3 pituitary tumor cells when applied extracellularly. In single-channel recordings we found polyamines as well as diamines up to 1,10-diaminodecane to be ineffective in altering channel current amplitudes or kinetics. In contrast, 1,12-diamino dodecane (1,12-DD) was found to be a reversible blocker, with a blocking site at an electrical distance (z delta) of 0.72 within the channel. It reduced single-channel current amplitude, mean channel open time, and channel open probability. In computer simulations structural data, such as flexibility, hydration, and log D values, were calculated. 1,12-DD showed the largest flexibility of all diamines (minimum N-N distance 9.9 A) combined with a marked hydrophobicity due to a 4-5 A hydrophobic intersegment between hydrophilic ends in the molecule, as confirmed by GRID water probe maps and a log D value of -1.82 at pH 7.2. We propose that the amount of hydration of the molecule, more than its flexibility, constitutes an essential parameter for its ability to act as a channel blocker. PMID:9533685

  4. Renalase Prevents AKI Independent of Amine Oxidase Activity

    PubMed Central

    Wang, Ling; Velazquez, Heino; Moeckel, Gilbert; Chang, John; Ham, Ahrom; Lee, H. Thomas; Safirstein, Robert

    2014-01-01

    AKI is characterized by increased catecholamine levels and hypertension. Renalase, a secretory flavoprotein that oxidizes catecholamines, attenuates ischemic injury and the associated increase in catecholamine levels in mice. However, whether the amine oxidase activity of renalase is involved in preventing ischemic injury is debated. In this study, recombinant renalase protected human proximal tubular (HK-2) cells against cisplatin- and hydrogen peroxide–induced necrosis. Similarly, genetic depletion of renalase in mice (renalase knockout) exacerbated kidney injury in animals subjected to cisplatin-induced AKI. Interestingly, compared with the intact renalase protein, a 20–amino acid peptide (RP-220), which is conserved in all known renalase isoforms, but lacks detectable oxidase activity, was equally effective at protecting HK-2 cells against toxic injury and preventing ischemic injury in wild-type mice. Furthermore, in vitro treatment with RP-220 or recombinant renalase rapidly activated Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinases and downregulated c-Jun N-terminal kinase. In summary, renalase promotes cell survival and protects against renal injury in mice through the activation of intracellular signaling cascades, independent of its ability to metabolize catecholamines, and we have identified the region of renalase required for these effects. Renalase and related peptides show potential as therapeutic agents for the prevention and treatment of AKI. PMID:24511138

  5. Polyamine acetylation modulates polyamine metabolic flux, a prelude to broader metabolic consequences.

    PubMed

    Kramer, Debora L; Diegelman, Paula; Jell, Jason; Vujcic, Slavoljub; Merali, Salim; Porter, Carl W

    2008-02-15

    Recent studies suggest that overexpression of the polyamine-acetylating enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) significantly increases metabolic flux through the polyamine pathway. The concept derives from the observation that SSAT-induced acetylation of polyamines gives rise to a compensatory increase in biosynthesis and presumably to increased flow through the pathway. Despite the strength of this deduction, the existence of heightened polyamine flux has not yet been experimentally demonstrated. Here, we use the artificial polyamine precursor 4-fluoro-ornithine to measure polyamine flux by tracking fluorine unit permeation of polyamine pools in human prostate carcinoma LNCaP cells. Conditional overexpression of SSAT was accompanied by a massive increase in intracellular and extracellular acetylated spermidine and by a 6-20-fold increase in biosynthetic enzyme activities. In the presence of 300 microM 4-fluoro-ornithine, SSAT overexpression led to the sequential appearance of fluorinated putrescine, spermidine, acetylated spermidine, and spermine. As fluorinated polyamines increased, endogenous polyamines decreased, so that the total polyamine pool size remained relatively constant. At 24 h, 56% of the spermine pool in the induced SSAT cells was fluorine-labeled compared with only 12% in uninduced cells. Thus, SSAT induction increased metabolic flux by approximately 5-fold. Flux could be interrupted by inhibition of polyamine biosynthesis but not by inhibition of polyamine oxidation. Overall, the findings are consistent with a paradigm whereby flux is initiated by SSAT acetylation of spermine and particularly spermidine followed by a marked increase in key biosynthetic enzymes. The latter sustains the flux cycle by providing a constant supply of polyamines for subsequent acetylation by SSAT. The broader metabolic implications of this futile metabolic cycling are discussed in detail. PMID:18089555

  6. Effect of processing on polyamine content and bioactive peptides released after in vitro gastrointestinal digestion of infant formulas.

    PubMed

    Gómez-Gallego, C; Recio, I; Gómez-Gómez, V; Ortuño, I; Bernal, M J; Ros, G; Periago, M J

    2016-02-01

    This study examined the influence of processing on polyamines and peptide release after the digestion of a commercial infant formula designed for children during the first months of life. Polyamine oxidase activity was not suppressed during the manufacturing process, which implicates that polyamine concentrations were reduced over time and during infant formula self-life. In gel electrophoresis, in vitro gastrointestinal digestion of samples with reduced amount of enzymes and time of digestion shows an increase in protein digestibility, reflected in the increase in nonprotein nitrogen after digestion and the disappearance of β-lactoglobulin and α-lactalbumin bands in gel electrophoresis. Depending on the sample, between 22 and 87 peptides were identified after gastrointestinal digestion. A peptide from β-casein f(98-105) with the sequence VKEAMAPK and antioxidant activity appeared in all of the samples. Other peptides with antioxidant, immunomodulatory, and antimicrobial activities were frequently found, which could have an effect on infant health. The present study confirms that the infant formula manufacturing process determines the polyamine content and peptidic profile after digestion of the infant formula. Because compositional dissimilarity between human milk and infant formula in polyamines and proteins could be responsible for some of the differences in health reported between breast-fed and formula-fed children, these changes must be taken into consideration because they may have a great effect on infant nutrition and development. PMID:26686732

  7. Catalase-peroxidases (KatG) exhibit NADH oxidase activity.

    PubMed

    Singh, Rahul; Wiseman, Ben; Deemagarn, Taweewat; Donald, Lynda J; Duckworth, Harry W; Carpena, Xavi; Fita, Ignacio; Loewen, Peter C

    2004-10-01

    Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG. PMID:15280362

  8. Peroxidase and peroxidase-oxidase activities of isolated human myeloperoxidases.

    PubMed Central

    Svensson, B E; Domeij, K; Lindvall, S; Rydell, G

    1987-01-01

    Isolated neutrophils from healthy donors were used for the isolation of four highly purified forms of myeloperoxidase as determined by spectral (A430/A280 ratio 0.80-0.87) and enzyme-activity measurements. Although the myeloperoxidases exhibited different elution profiles on cation-exchange chromatography, gel filtration indicated similar relative molecular masses. When these forms were assayed for peroxidase and peroxidase-oxidase activities with several substrates, they all exhibited virtually the same specific activities. These results suggest that possible functional differences between the enzymes may be related to differences in their sites of action rather than to differences in enzyme activity. Myeloperoxidase from a patient with chronic myeloid leukaemia also revealed a similar heterogeneity on cation-exchange chromatography. However, this myeloperoxidase contained in addition one form with a lower and one form with a higher relative molecular mass, as indicated by gel-filtration chromatography. PMID:3036098

  9. Cell-free NADPH oxidase activation assays: "in vitro veritas".

    PubMed

    Pick, Edgar

    2014-01-01

    The superoxide (O2 (∙-))-generating NADPH oxidase complex of phagocytes comprises a membrane-imbedded heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of Nox2 and p22 (phox) ) and four cytosolic regulatory proteins, p47 (phox) , p67 (phox) , p40 (phox) , and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2 (∙-) generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by specific signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome. A consequent conformational change in Nox2 initiates the electron "flow" along a redox gradient, from NADPH to oxygen, leading to the one-electron reduction of molecular oxygen to O2 (∙-). Methodological difficulties in the dissection of this complex mechanism led to the design "cell-free" systems (also known as "broken cells" or in vitro systems). In these, membrane receptor stimulation and all or part of the signal transduction sequence are missing, the accent being placed on the actual process of "NADPH oxidase assembly," thus on the formation of the complex between cytochrome b 558 and the cytosolic components and the resulting O2 (∙-) generation. Cell-free assays consist of a mixture of the individual components of the NADPH oxidase complex, derived from resting phagocytes or in the form of purified recombinant proteins, exposed in vitro to an activating agent (distinct from and unrelated to whole cell stimulants), in the presence of NADPH and oxygen. Activation is commonly quantified by measuring the primary product of the reaction, O2 (∙-), trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of the linear rate of O2 (∙-) production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount

  10. Binding of cations of group IA and IIA to bovine serum amine oxidase: effect on the activity.

    PubMed Central

    Di Paolo, Maria Luisa; Scarpa, Marina; Corazza, Alessandra; Stevanato, Roberto; Rigo, Adelio

    2002-01-01

    In this paper, we report on the presence of cation binding areas on bovine serum amine oxidase, where metal ions of the groups IA and IIA, such as Na(+), K(+), Cs(+), Mg(2+), and Ca(2+), bind with various affinities. We found a cation-binding area that influences the enzyme activity if occupied, so that the catalytic reaction may be altered by some physiologically relevant cations, such as Ca(2+) and K(+). This binding area appears to be localized inside the enzyme active site, because some of these cations act as competitive inhibitors when highly charged amines, such as spermine and spermidine, are used as substrates. In particular, dissociation constant values (K(d)) of 23 and 27 mM were measured for Cs(+) and Ca(2+), respectively, using, as substrate, spermine, a polyamine of plasma. An additional cation-binding area, where metal ions such as Cs(+) (K(d) congruent with 0.1 mM) and Na(+) (K(d) congruent with 54 mM) bind without affecting the enzyme activity, was found by NMR. PMID:12324440

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

    PubMed Central

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

    2015-01-01

    The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression. PMID:26672986

  12. Inheritance of polyphenol oxidase activity in wheat breeding lines derived from matings of low polyphenol oxidase parents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO) in grain plays a major role in time-dependent discoloration of wheat (Triticum aestivum L.) products, especially fresh noodles. Breeding wheat cultivars with low or nil PPO activity can reduce the undesirable product darkening. The low PPO line PI 117635 was crossed to two...

  13. Triethylenetetramine modulates polyamine and energy metabolism and inhibits cancer cell proliferation.

    PubMed

    Hyvönen, Mervi T; Ucal, Sebahat; Pasanen, Markku; Peräniemi, Sirpa; Weisell, Janne; Khomutov, Maxim; Khomutov, Alex R; Vepsäläinen, Jouko; Alhonen, Leena; Keinänen, Tuomo A

    2016-05-15

    Polyamine metabolism is an attractive anticancer drug target, since polyamines are absolutely required for cellular proliferation, and increased levels of polyamines and their biosynthetic enzyme ornithine decarboxylase (ODC) are associated with cancer. Triethylenetetramine (TETA) is a charge-deficient isosteric analogue of the polyamine spermidine (Spd) and a Cu(II)-chelating compound used for the treatment of Wilson's disease, and it has been implicated as a potential anticancer therapeutic drug. In the present study, we studied the effects of TETA in comparison with two other Cu(II)-chelators, D-penicillamine (PA) and tetrathiomolybdate (TTM), on polyamine metabolism in DU145 prostate carcinoma, MCF-7 breast carcinoma and JEG-3 choriocarcinoma cells. TETA induced antizyme, down-regulated ODC and inhibited [(14)C] Spd uptake. Moreover, it completely prevented α-difluoromethylornithine (DFMO)-induced increase in [(14)C] Spd uptake, and inhibited [(14)C] putrescine (Put) uptake and ODC activity in vivo Seven-day treatment of DU145 cells with TETA caused growth cessation by reducing intracellular polyamine levels and suppressing the formation of hypusinated eukaryotic translation initiation factor 5A (eIF5A). TETA or its N-acetylated metabolites also inhibited spermine (Spm), diamine and semicarbazide-sensitive amine oxidases and decreased the level of intracellular reactive oxygen species. Moreover, TETA inhibited the utilization of Put as energy source via the tricarboxylic acid (TCA) cycle, as indicated by decreased production of (14)CO2 from [(14)C] Put. These results indicate that TETA attacks multiple proven anticancer drug targets not attributed to copper chelation, which warrants further studies to reveal its potential in cancer chemoprevention and cure. PMID:27001865

  14. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

    PubMed

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-03-29

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo. PMID:26976571

  15. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

    PubMed Central

    Molitor, Christian; Mauracher, Stephan Gerhard

    2016-01-01

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme’s interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate–enzyme complexes were performed, and a key residue was identified that influences the plant PPO’s acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their—so far unknown—natural substrates in vivo. PMID:26976571

  16. CDK inhibitors induce mitochondria-mediated apoptosis through the activation of polyamine catabolic pathway in LNCaP, DU145 and PC3 prostate cancer cells.

    PubMed

    Arisan, Elif Damla; Obakan, Pinar; Coker-Gurkan, Ajda; Calcabrini, Annarica; Agostinelli, Enzo; Unsal, Narcin Palavan

    2014-01-01

    Androgen signaling is critical in prostate cancer development and progression. The co-existence of hormone responsive and irresponsive cells due to functional androgen receptor (AR) in prostate gland is the major obstacle in prostate cancer therapy models. Targeting aberrant cell cycle by novel cell cycle blocking agents is a promising strategy to treat various types of malignancies. Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors able to activate apoptotic cell death by inducing cell cycle arrest at G1/S and G2/M phases in cancer cells. Polyamines are unique cationic amine derivatives involved in the regulation of cell proliferation. Although the elevated intracellular level of polyamines (putrescine, spermidine and spermine) is typical for prostate gland, abnormal regulation of polyamine metabolism might result in rapid cell proliferation and, thus in prostate cancer progression. Therefore, treatment with drug-induced depletion of intracellular polyamine levels through the activated polyamine catabolism is critical to achieve successful strategies for prostate cancer. In this study we aimed to investigate the apoptotic efficiency of CDK inhibitors in three prostate cancer cell lines (LNCaP, DU145 and PC3), showing different AR expression profile. We found that both purvalanol and roscovitine were able to induce apoptosis at moderate cytotoxic concentrations by decreasing mitochondria membrane potential. The apoptotic effect of both CDK inhibitors was due to activation of caspases by modulating Bcl-2 family members. The efficiency of drugs was quite similar on the three prostate cell lines used in this study. However, DU145 cells were found the least sensitive against CDK inhibitors while purvalanol was more potent than roscovitine. Similarly to classical chemotherapeutic agents, both drugs could up-regulate polyamine catabolic enzymes (SSAT, SMO and PAO) in cell type dependent manner. Transient silencing of SSAT and/or inhibition of PAO

  17. Polyamines in soybeans.

    PubMed

    Wang, L C

    1972-07-01

    Putrescine, spermidine, and spermine were three main polyamines isolated from soybeans and partially characterized. Occurrence of polyamines in soybeans was established by separating trichloroacetic acid extracts of soybeans by cationic exchange column chromatography, identification with thin layer chromatography, paper electrophoresis, mass spectral analysis, reactions with ninhydrin and Dragendorff reagents, and spectrophotometric characteristics. Soybeans contained a minimum of 29.0 micrograms of polyamines per gram of full-fat flour. The alcohol-soluble fraction of soybeans contained polyamines also. Resting seeds contained spermidine in higher concentration than either putrescine or spermine. Spermine appeared to be present in lowest concentration. Preliminary experiments suggested that some polyamines were possibly in bound forms. PMID:16658112

  18. Changes in polyamines, auxins and peroxidase activity during in vitro rooting of Fraxinus angustifolia shoots: an auxin-independent rooting model.

    PubMed

    Tonon, G; Kevers, C; Gaspar, T

    2001-07-01

    Among shoots of Fraxinus angustifolia Vahl raised in vitro, 76% rooted after culture on root induction medium for 5 days in darkness followed by culture on root expression medium for 15 days in light. The addition of 20.7 microM indole-butyric acid (IBA) to the root induction medium did not significantly increase the rooting percentage (88%). Putrescine, spermidine, cyclohexylamine (CHA) and aminoguanidine (AG) enhanced rooting up to 100% (98.66% for AG), when applied during root induction in the absence of IBA, otherwise these compounds inhibited rooting, as did spermine and difluoromethylornithine (DFMO) + difluoromethylarginine (DFMA). The root induction phase was characterized by a temporary increase in endogenous free indole-acetic acid (IAA) and putrescine concentrations during root induction, whereas the root expression phase was characterized by increased peroxidase activity and low concentrations of polyamines. These changes were specifically associated with the rooting process and did not depend on the presence of exogenous IBA, because application of exogenous IBA enhanced the amount of IAA in the cuttings but did not affect rooting or the pattern of changes in polyamines and peroxidase. The effects of CHA, AG and DFMO + DFMA on endogenous concentrations of auxins and polyamines highlight the close relationship between the effects of IAA and putrescine in root induction and suggest that polyamine catabolism has an important role in root formation and elongation. PMID:11446994

  19. Xanthine oxidase inhibitory activity of Vietnamese medicinal plants.

    PubMed

    Nguyen, Mai Thanh Thi; Awale, Suresh; Tezuka, Yasuhiro; Tran, Quan Le; Watanabe, Hiroshi; Kadota, Shigetoshi

    2004-09-01

    Among 288 extracts, prepared from 96 medicinal plants used in Vietnamese traditional medicine to treat gout and related symptoms, 188 demonstrated xanthine oxidase (XO) inhibitory activity at 100 microg/ml, with 46 having greater than 50% inhibition. At 50 microg/ml, 168 of the extracts were active, with 21 possessing more than 50% inhibition. At 25 microg/ml, 146 extracts exhibited inhibitory activity, with 8 showing over 50% inhibition, while 126 extracts presented activity at 10 microg/ml, with 2 having greater than 50% inhibition. The MeOH extracts of Artemisia vulgaris, Caesalpinia sappan (collected at the Seven-Mountain area), Blumea balsamifera (collected in Lam Dong province), Chrysanthemum sinense and MeOH-H(2)O extract of Tetracera scandens (Khanh Hoa province) exhibited strong XO inhibitory activity with IC(50) values less than 20 microg/ml. The most active extract was the MeOH extract of the flower of C. sinense with an IC(50) value of 5.1 microg/ml. Activity-guided fractionation of the MeOH extract led to the isolation of caffeic acid (1), luteolin (2), eriodictyol (3), and 1,5-di-O-caffeoylquinic acid (4). All these compounds showed significant XO inhibitory activity in a concentration-dependent manner, and the activity of 2 was more potent (IC(50) 1.3 microM) than the clinically used drug, allopurinol (IC(50) 2.5 microM). PMID:15340229

  20. Self-Immolative Polycations as Gene Delivery Vectors and Prodrugs Targeting Polyamine Metabolism in Cancer

    PubMed Central

    2015-01-01

    Polycations are explored as carriers to deliver therapeutic nucleic acids. Polycations are conventionally pharmacological inert with the sole function of delivering therapeutic cargo. This study reports synthesis of a self-immolative polycation (DSS-BEN) based on a polyamine analogue drug N1,N11-bisethylnorspermine (BENSpm). The polycation was designed to function dually as a gene delivery carrier and a prodrug targeting dysregulated polyamine metabolism in cancer. Using a combination of NMR and HPLC, we confirm that the self-immolative polycation undergoes intracellular degradation into the parent drug BENSpm. The released BENSpm depletes cellular levels of spermidine and spermine and upregulates polyamine catabolic enzymes spermine/spermidine N1-acetyltransferase (SSAT) and spermine oxidase (SMO). The synthesized polycations form polyplexes with DNA and facilitate efficient transfection. Taking advantage of the ability of BENSpm to sensitize cancer cells to TNFα-induced apoptosis, we show that DSS-BEN enhances the cell killing activity of TNFα gene therapy. The reported findings validate DSS-BEN as a dual-function delivery system that can deliver a therapeutic gene and improve the outcome of gene therapy as a result of the intracellular degradation of DSS-BEN to BENSpm and the subsequent beneficial effect of BENSpm on dysregulated polyamine metabolism in cancer. PMID:25153488

  1. Ataxia telangiectasia mutated influences cytochrome c oxidase activity.

    PubMed

    Patel, Akshar Y; McDonald, Todd M; Spears, Larry D; Ching, James Kain; Fisher, Jonathan S

    2011-02-25

    Cells lacking ataxia telangiectasia mutated (ATM) have impaired mitochondrial function. Furthermore, mammalian cells lacking ATM have increased levels of reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) deletions in the region encoding for cytochrome c oxidase (COX). We hypothesized that ATM specifically influences COX activity in skeletal muscle. COX activity was ∼40% lower in tibialis anterior from ATM-deficient mice than for wild-type mice (P < 0.01, n = 9/group). However, there were no ATM-related differences in activity of succinate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, or complex III. Incubation of wild-type extensor digitorum longus muscles for 1h with the ATM inhibitor KU55933 caused a ∼50% reduction (P<0.05, n = 5/group) in COX activity compared to muscles incubated with vehicle alone. Among the control muscles and muscles treated with the ATM inhibitor, COX activity was correlated (r = 0.61, P<0.05) with activity of glucose 6-phosphate dehydrogenase, a key determinant of antioxidant defense through production of NADPH. Overall, the findings suggest that ATM has a protective role for COX activity. PMID:21266166

  2. Quantitative structure-activity studies on monoamine oxidase inhibitors.

    PubMed

    Johnson, C L

    1976-05-01

    Quantitative structure-activity studies were carried out on a series of N-isopropylaryl hydrazides which inhibits monoamine oxidase (MAO). The inhibitory potencies of these compounds of MAO were found to correlate with the electron-withdrawing capacity of the aryl ring substituents as estimated by both empirical Hammet sigma constants and electronic indices from molecular orbital calculations. Based on these correlations and previously published data on other classes of MAO inhibitors, a general model for the inhibitor pharmacophore is proposed: potent MAO of an aromatic ring; electron-withdrawing groups on the aromatic ring or replacing the phenyl ring with certain types of heterocyclic rings will tend to increase the potency. PMID:1271400

  3. Activation of polyamine catabolic enzymes involved in diverse responses against epibrassinolide-induced apoptosis in LNCaP and DU145 prostate cancer cell lines.

    PubMed

    Obakan, Pinar; Arisan, Elif Damla; Calcabrini, Annarica; Agostinelli, Enzo; Bolkent, Sehnaz; Palavan-Unsal, Narçin

    2014-03-01

    Epibrassinolide (EBR) is a biologically active compound of the brassinosteroids, steroid-derived plant growth regulator family. Generally, brassinosteroids are known for their cell expansion and cell division-promoting roles. Recently, EBR was shown as a potential apoptotic inducer in various cancer cells without affecting the non-tumor cell growth. Androgen signaling controls cell proliferation through the interaction with the androgen receptor (AR) in the prostate gland. Initially, the development of prostate cancer is driven by androgens. However, in later stages, a progress to the androgen-independent stage is observed, resulting in metastatic prostate cancer. The androgen-responsive or -irresponsive cells are responsible for tumor heterogeneity, which is an obstacle to effective anti-cancer therapy. Polyamines are amine-derived organic compounds, known for their role in abnormal cell proliferation as well as during malignant transformation. Polyamine catabolism-targeting agents are being investigated against human cancers. Many chemotherapeutic agents including polyamine analogs have been demonstrated to induce polyamine catabolism that depletes polyamine levels and causes apoptosis in tumor models. In our study, we aimed to investigate the mechanism of apoptotic cell death induced by EBR, related with polyamine biosynthetic and catabolic pathways in LNCaP (AR+), DU145 (AR-) prostate cancer cell lines and PNT1a normal prostate epithelial cell line. Induction of apoptotic cell death was observed in prostate cancer cell lines after EBR treatment. In addition, EBR induced the decrease of intracellular polyamine levels, accompanied by a significant ornithine decarboxylase (ODC) down-regulation in each prostate cancer cell and also modulated ODC antizyme and antizyme inhibitor expression levels only in LNCaP cells. Catabolic enzymes SSAT and PAO expression levels were up-regulated in both cell lines; however, the specific SSAT and PAO siRNA treatments prevented the

  4. Depletion of the Polyamines Spermidine and Spermine by Overexpression of Spermidine/spermine N1-Acetyltransferase1 (SAT1) Leads to Mitochondria-Mediated Apoptosis in Mammalian Cells

    PubMed Central

    Mandal, Swati; Mandal, Ajeet; Park, Myung Hee

    2015-01-01

    The polyamines putrescine, spermidine and spermine are intimately involved in the regulation of cellular growth and viability. Transduction of HEK293T cells with an adenovirus (AdSAT1) encoding a key polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase1 (SAT1), leads to a rapid depletion of spermidine and spermine, arrest in cell growth and a decline in cell viability. Annexin V/ propidium iodide Fluorescent Activated Cell Sorter (FACS) analyses, Terminal Uridine Nucleotide End- Labeling (TUNEL) and caspase 3 assays showed a clear indication of apoptosis in AdSAT1 transduced cells (at 24–72 h), but not in cells transduced with GFP-encoding adenovirus (AdGFP). Apoptosis in the polyamine-depleted cells occurs by the mitochondrial intrinsic pathway, as evidenced by loss of mitochondrial membrane potential, increase in proapoptotic Bax, decrease in anti-apoptotic Bcl-xl, Bcl2, and Mcl-1 and release of cytochrome c from mitochondria, upon transduction with AdSAT1. Moreover, transmission electron microscopy images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including cell shrinkage, nuclear fragmentation, mitochondrial alteration, vacuolization and membrane blebbing. The apoptosis appears to result largely from depletion of the polyamines, spermidine and spermine, as polyamine analogs, α-methylspermidine and N1,N12-dimethylspermine that are not substrates for SAT1 could partially restore growth and prevent apoptosis of AdSAT1-transduced cells. Inhibition of polyamine oxidases did not restore the growth of AdSAT1-transduced cells or block apoptosis, suggesting that the growth arrest and apoptosis were not induced by oxidative stress resulting from accelerated polyamine catabolism. Taken together, these data provide strong evidence that the depletion of polyamines spermidine and spermine leads to mitochondria-mediated apoptosis. PMID:25849284

  5. Polyamine catabolism: target for antiproliferative therapies in animals and stress tolerance strategies in plants.

    PubMed

    Tavladoraki, Paraskevi; Cona, Alessandra; Federico, Rodolfo; Tempera, Giampiero; Viceconte, Nikenza; Saccoccio, Stefania; Battaglia, Valentina; Toninello, Antonio; Agostinelli, Enzo

    2012-02-01

    Metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine, has been a target for antineoplastic therapy since these naturally occurring alkyl amines were found essential for normal mammalian cell growth. Intracellular polyamine concentrations are maintained at a cell type-specific set point through the coordinated and highly regulated interplay between biosynthesis, transport, and catabolism. A correlation between regulation of cell proliferation and polyamine metabolism is described. In particular, polyamine catabolism involves copper-containing amine oxidases and FAD-dependent polyamine oxidases. Several studies showed an important role of these enzymes in several developmental and disease-related processes in both animals and plants through a control on polyamine homeostasis in response to normal cellular signals, drug treatment, environmental and/or cellular stressors. The production of toxic aldehydes and reactive oxygen species, H(2)O(2) in particular, by these oxidases using extracellular and intracellular polyamines as substrates, suggests a mechanism by which the oxidases can be exploited as antineoplastic drug targets. This minireview summarizes recent advances on the physiological roles of polyamine catabolism in animals and plants in an attempt to highlight differences and similarities that may contribute to determine in detail the underlined mechanisms involved. This information could be useful in evaluating the possibility of this metabolic pathway as a target for new antiproliferative therapies in animals and stress tolerance strategies in plants. PMID:21874532

  6. Changes in lysyl oxidase (LOX) distribution and its decreased activity in keratoconus corneas.

    PubMed

    Dudakova, Lubica; Liskova, Petra; Trojek, Tomas; Palos, Michalis; Kalasova, Sarka; Jirsova, Katerina

    2012-11-01

    Inadequate cross-linking between collagen lamellae is a characteristic feature of keratoconus corneas. The formation of covalent bonds between collagen and elastin fibrils, which maintain the biomechanical properties of the cornea, is mediated by the cuproenzyme lysyl oxidase and four lysyl oxidase-like enzymes. The aim of this study was to determine the distribution of lysyl oxidase and the total lysyl oxidase activity (lysyl oxidase and the four lysyl oxidase-like enzymes) in control and keratoconic corneas. Seven control and eight keratoconic corneas were used for the imunohistochemical detection of lysyl oxidase in corneal cryosections using two different antibodies. The total lysyl oxidase activity in the culture medium of corneal fibroblasts from six explanted keratoconic and four control corneas was measured using a fluorometric assay in the presence and absence of the lysyl oxidase inhibitor beta-aminopropionitrile and determined as the production of H(2)O(2) in nM per μg of total protein. In the control tissue, the most intense signal for lysyl oxidase was present in the corneal epithelium, in which perinuclear dots brightly projecting from more or less homogenous cytoplasmic staining may represent the lysyl oxidase propeptide. Less intense staining was present in keratocytes, the extracellular matrix and in the corneal endothelium. The epithelium of the limbus and the perilimbal conjunctiva showed intense to very intense staining. The distribution of lysyl oxidase was clearly decreased in at least five of the eight keratoconic specimens. The most marked signal reduction was observed in the stromal matrix and in keratocytes. Moreover, the signal in pathological specimens revealed a more irregular pattern, including the presence of intra- and extracellular clumps in the epithelium. Interestingly, endothelial cells showed no or very weak staining in areas just beneath negative stromal tissue. The mean activity of total lysyl oxidase in the keratoconic

  7. Polyamines: fundamental characters in chemistry and biology.

    PubMed

    Agostinelli, E; Marques, M P M; Calheiros, R; Gil, F P S C; Tempera, G; Viceconte, N; Battaglia, V; Grancara, S; Toninello, A

    2010-02-01

    Polyamines are small cationic molecules required for cellular proliferation and are detected at higher concentrations in most tumour tissues, compared to normal tissues. Agmatine (AGM), a biogenic amine, is able to arrest proliferation in cell lines by depleting intracellular polyamine levels. It enters mammalian cells via the polyamine transport system. Agmatine is able to induce oxidative stress in mitochondria at low concentrations (10 or 100 microM), while at higher concentrations (e.g. 1-2 mM) it does not affect mitochondrial respiration and is ineffective in inducing any oxidative stress. As this effect is strictly correlated with the mitochondrial permeability transition induction and the triggering of the pro-apoptotic pathway, AGM may be considered as a regulator of this type of cell death. Furthermore, polyamine transport is positively correlated with the rate of cellular proliferation. By increasing the expression of antizyme, a protein that inhibits polyamine biosynthesis and transport, AGM also exhibits a regulatory effect on cell proliferation. Methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosyl-L: -methionine decarboxylase, displaying anticancer activity, is a structural analogue of the natural polyamine spermidine. MGBG has been extensively studied, preclinically as well as clinically, and its anticancer activity has been attributed to the inhibition of polyamine biosynthesis and also to its effect on mitochondrial function. Numerous findings have suggested that MGBG might be used as a chemotherapeutic agent against cancer. PMID:20013011

  8. Inhibition of apple polyphenol oxidase activity by sodium chlorite.

    PubMed

    Lu, Shengmin; Luo, Yaguang; Feng, Hao

    2006-05-17

    Sodium chlorite (SC) was shown to have strong efficacy both as a sanitizer to reduce microbial growth on produce and as a browning inhibitor on fresh-cut apples in previous experiments. This study was undertaken to investigate the inhibitory effect of SC on polyphenol oxidase (PPO) and the associated mechanisms. The experiment showed that SC had a strong inhibition of apple PPO. The extent of inhibition was influenced by SC concentration and pH. Inhibition was most prominent at pH 4.5, at which approximately 30% of enzyme activity was lost in the presence of 10 mM SC, followed closely by that at pH 4.0 with a 26% reduction in PPO activity. The inhibition mode was determined using Dixon and Lineweaver-Burk plots, which established SC to be a mixed inhibitor of apple PPO for the oxidation of catechol. Preincubation of PPO with 8 mM SC for 8 min caused a maximum of 46% activity reduction compared to noninhibited control. However, preincubation of SC with catechol for 8 min resulted in no additional loss of PPO activity. These findings provide further evidence that the inhibition of PPO activity by SC is due to the inhibition of the enzyme itself rather than removal of the substrate. PMID:19127746

  9. Characterization of polyphenol oxidase activity in Ataulfo mango.

    PubMed

    Cheema, Summervir; Sommerhalter, Monika

    2015-03-15

    Crude extracts of Ataulfo exhibited polyphenol oxidase (PPO) activity with pyrogallol, 3-methylcatechol, catechol, gallic acid, and protocatechuic acid. The substrate dependent pH optima ranged from pH 5.4 to 6.4 with Michaelis-Menten constants between 0.84 ± 0.09 and 4.6 ± 0.7 mM measured in MES or phosphate buffers. The use of acetate buffers resulted in larger Michaelis-Menten constants, up to 14.62 ± 2.03 mM. Sodium ascorbate, glutathione, and kojic acid are promising inhibitors to prevent enzymatic browning in Ataulfo. PPO activity increased with ripeness and was always higher in the skin compared to the pulp. Sodium dodecyl sulphate (SDS) enhanced PPO activity, with pulp showing a stronger increase than skin. SDS-PAGE gels stained for catecholase activity showed multiple bands, with the most prominent bands at apparent molecular weights of 53, 112, and 144 kDa. PMID:25308684

  10. Targeting Polyamines and Inflammation for Cancer Prevention

    PubMed Central

    Babbar, Naveen; Gerner, Eugene W.

    2013-01-01

    Increased polyamine synthesis and inflammation have long been associated with intraepithelial neoplasia, which are risk factors for cancer development in humans. Targeting polyamine metabolism (by use of polyamine synthesis inhibitors or polyamine catabolism activators) and inflammation (by use of nonsteroidal anti-inflammatory drugs) has been studied for many cancers, including colon, prostate, and skin. Genetic epidemiology results indicate that a genetic variant associated with the expression of a polyamine biosynthetic gene is associated with risk of colon and prostate cancers. A clinical trial of difluoromethylornithine (DFMO), a selective inhibitor of polyamine synthesis, showed that the 1 year treatment duration reduced prostate volume and serum prostate-specific antigen doubling time in men with a family history of prostate cancer. A second, clinical trial of DFMO in combination with sulindac, a NSAID in patients with prior colon polyps found that the 3-year treatment was associated with a 70% reduction of all, and over a 90% reduction of advanced and/or multiple metachronous colon adenomas. In this chapter, we discuss that similar combination prevention strategies of targeting polyamines and inflammation can be effective in reducing risk factors associated with the development of human cancers. PMID:21253788

  11. The effects of ingested aluminium on brain cytochrome oxidase activity.

    PubMed

    Mohan, N; Alleyne, T; Adogwa, A

    2009-11-01

    Aluminium has a unique combination of physical and chemical properties which has enabled man to put this metal to very wide and varied use. However prolonged exposure to aluminium ions may lead to adverse health effects. In this study, we evaluated the effects of dietary aluminium on the protein composition and the intrinsic activity of cytochrome oxidase (COX) for brain mitochondria. New Zealand white rabbits were maintained on a diet of commercial rabbit pellets and distilled water for a period of 12 weeks. For the experimental group, AlCl3, 330 mg/kg/L was added to the drinking water. When compared to the control, mitochondria isolated from the brains of the AICl3 fed rabbits showed no change in Km but an approximate 35% decrease in both the low and high affinity Vmax values. Also, whereas the protein composition of the mitochondria from both sources appeared to be normal, isolation of highly purified COX proved to be difficult and for the AlCl3 fed rabbits, a number of the enzyme's low molecular weight subunits were absent. These results appear to confirm a relationship between long term aluminium consumption and low brain COX activity; they further suggest that an altered COX structure may be the cause of the low enzymic activity. PMID:20441059

  12. Charge deficient analogues of the natural polyamines.

    PubMed

    Weisell, Janne; Hyvönen, Mervi T; Alhonen, Leena; Vepsäläinen, Jouko; Keinänen, Tuomo A; Khomutov, Alex R

    2014-01-01

    Mitochondrial dysfunction, either inherited or acquired, is associated with several diseases in humans. Depending on the cell type and location, cells are prone to multiple types of insults that may compromise their proper function. Generally, these insults are overcome by defensive mechanisms but sometimes they lead to sustained damage, requiring the action of scavenging and repair machineries to retain the viability of the cells. As a final measure, severely damaged cells are targeted to a controlled cell death pathway in order to not to compromise the well-being of the whole tissue. The polyamines, spermine and spermidine are essential cellular constituents, participating in many vital functions such as proliferation and differentiation, immune response and scavenging of reactive oxygen species. Therefore, dysregulation of polyamine metabolism is often associated with different pathological states. Polyamine acetylating enzyme spermidine/spermine-N(1)-acetyltransferase is induced by inflammation, drugs and by several other environmental insults. Resulting accelerated polyamine acetylation with accompanying polyamine biosynthesis induction i.e. activation of polyamine futile cycle generates excessive amount of hydrogen peroxide, hampers cell energy metabolism and induces mitochondrial dysfunction and biogenesis. Therefore, the drugs inhibiting polyamine metabolism are valuable in protecting mitochondria and cell energy metabolism. Here we review the current literature focusing on the applicability of chargedeficient polyamine analogs as drugs to modulate polyamine metabolism. Alteration of pK(a) of amino group(s) in a respective analog is achieved by fluorine substitution of hydrogen atom, hydroxylamine substitution of methylamine or by reducing the numbers of carbon atoms between amine groups to two instead of three or four. PMID:23701551

  13. Brain monoamine oxidase A activity predicts trait aggression.

    PubMed

    Alia-Klein, Nelly; Goldstein, Rita Z; Kriplani, Aarti; Logan, Jean; Tomasi, Dardo; Williams, Benjamin; Telang, Frank; Shumay, Elena; Biegon, Anat; Craig, Ian W; Henn, Fritz; Wang, Gene-Jack; Volkow, Nora D; Fowler, Joanna S

    2008-05-01

    The genetic deletion of monoamine oxidase A (MAO A), an enzyme that breaks down the monoamine neurotransmitters norepinephrine, serotonin, and dopamine, produces aggressive phenotypes across species. Therefore, a common polymorphism in the MAO A gene (MAOA, Mendelian Inheritance in Men database number 309850, referred to as high or low based on transcription in non-neuronal cells) has been investigated in a number of externalizing behavioral and clinical phenotypes. These studies provide evidence linking the low MAOA genotype and violent behavior but only through interaction with severe environmental stressors during childhood. Here, we hypothesized that in healthy adult males the gene product of MAO A in the brain, rather than the gene per se, would be associated with regulating the concentration of brain amines involved in trait aggression. Brain MAO A activity was measured in vivo in healthy nonsmoking men with positron emission tomography using a radioligand specific for MAO A (clorgyline labeled with carbon 11). Trait aggression was measured with the multidimensional personality questionnaire (MPQ). Here we report for the first time that brain MAO A correlates inversely with the MPQ trait measure of aggression (but not with other personality traits) such that the lower the MAO A activity in cortical and subcortical brain regions, the higher the self-reported aggression (in both MAOA genotype groups) contributing to more than one-third of the variability. Because trait aggression is a measure used to predict antisocial behavior, these results underscore the relevance of MAO A as a neurochemical substrate of aberrant aggression. PMID:18463263

  14. Brain Monoamine Oxidase-A Activity Predicts Trait Aggression

    PubMed Central

    Alia-Klein, Nelly; Goldstein, Rita Z.; Kriplani, Aarti; Logan, Jean; Tomasi, Dardo; Williams, Benjamin; Telang, Frank; Shumay, Elena; Biegon, Anat; Craig, Ian W.; Henn, Fritz; Wang, Gene-Jack; Volkow, Nora D.; Fowler, Joanna S.

    2008-01-01

    The genetic deletion of monoamine oxidase A (MAO A, an enzyme which breaks down the monoamine neurotransmitters norepinephrine, serotonin and dopamine) produces aggressive phenotypes across species. Therefore, a common polymorphism in the MAO A gene (MAOA, MIM 309850, referred to as high or low based on transcription in non-neuronal cells) has been investigated in a number of externalizing behavioral and clinical phenotypes. These studies provide evidence linking the low MAOA genotype and violent behavior but only through interaction with severe environmental stressors during childhood. Here, we hypothesized that in healthy adult males the gene product of MAO A in the brain, rather than the gene per se, would be associated with regulating the concentration of brain amines involved in trait aggression. Brain MAO A activity was measured in-vivo in healthy non-smoking men with positron emission tomography using a radioligand specific for MAO A (clorgyline labeled with carbon 11). Trait aggression was measured with the Multidimensional Personality Questionnaire (MPQ). Here we report for the first time that brain MAO A correlates inversely with the MPQ trait measure of aggression (but not with other personality traits) such that the lower the MAO A activity in cortical and subcortical brain regions the higher the self-reported aggression (in both MAOA genotype groups) contributing to more than a third of the variability. Since trait aggression is a measure used to predict antisocial behavior, these results underscore the relevance of MAO A as a neurochemical substrate of aberrant aggression. PMID:18463263

  15. Unfolding and refolding of active apple polyphenol oxidase.

    PubMed

    Mari, S; Marquès, L; Breton, F; Karamanos, Y; Macheix, J J

    1998-11-01

    For the first time, unfolding (6 M guanidine) and refolding of partially proteolysed purified polyphenol oxidase (PPOr) was achieved, with 88% of activity recovered. Optimal refolding conditions consisted in stepwise dialysis of guanidine treated extracts, the dialysis buffers containing 1 M (NH4)2SO4 and 100 microM CuSO4. However, CuSO4 had limited effect on the recovering of PPOr activity, whereas (NH4)2SO4 was essential. Concerning the PPO tertiary structure, denaturing conditions (combinations of boiling and reducing agent) used on SDS-PAGE have shown (i) a compact tertiary structure and (ii) the presence of disulfide bonds in PPOr, accounting for the shift between 27 and 41 kDa, and 41 and 42 kDa, respectively. Resistance to proteolytic cleavage was used to study the conformational changes induced by the denaturing treatments. Folded PPOr was resistant to further proteolysis whereas unfolded PPO was totally digested, indicating the role of tertiary structure of PPOr in the resistance to proteases. PMID:9842726

  16. Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography.

    PubMed

    Yamamoto, T; Moriwaki, Y; Takahashi, S; Tsutsumi, Z; Yamakita, J; Nasako, Y; Hiroishi, K; Higashino, K

    1996-06-01

    An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 microliters of plasma and 240 microliters of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 microM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierke's disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects. PMID:8811453

  17. An alcohol oxidase of Phanerochaete chrysosporium with a distinct glycerol oxidase activity.

    PubMed

    Linke, Diana; Lehnert, Nicole; Nimtz, Manfred; Berger, Ralf G

    2014-01-01

    An intracellular alcohol oxidase (AOX) was isolated from the white-rot basidiomycete Phanerochaete chrysosporium (Pch), grown on l-lactate induction medium, and purified to electrophoretic homogeneity. The dimeric protein consisted of two identical 75kDa subunits. The open reading frame of 1,956bp resulted in a monomer consisting of 651 amino acids. The enzyme showed a pI at 5.4, a pH optimum of 9, a temperature optimum at 50°C, possessed putative conserved domains of the GMC superfamily, a FAD binding domain, and showed up to 86% homology to alcohol oxidase sequences of Gloeophyllum trabeum and Coprinopsis cinerea. As was shown for the first time for an AOX from a basidiomycete, not only methanol, but also lower primary alcohols and glycerol were accepted as substrates. An assay based on aldehyde dehydrogenase confirmed d-glyceraldehyde as the product of the reaction. A bioprocess based on this enzyme could alleviate the problems associated with the huge side-stream of glycerol occurring during the manufacture of biodiesel, yielding the green oxidant hydrogen peroxide. PMID:24910330

  18. Polyamines function in stress tolerance: from synthesis to regulation

    PubMed Central

    Liu, Ji-Hong; Wang, Wei; Wu, Hao; Gong, Xiaoqing; Moriguchi, Takaya

    2015-01-01

    Plants are challenged by a variety of biotic or abiotic stresses, which can affect their growth and development, productivity, and geographic distribution. In order to survive adverse environmental conditions, plants have evolved various adaptive strategies, among which is the accumulation of metabolites that play protective roles. A well-established example of the metabolites that are involved in stress responses, or stress tolerance, is the low-molecular-weight aliphatic polyamines, including putrescine, spermidine, and spermine. The critical role of polyamines in stress tolerance is suggested by several lines of evidence: firstly, the transcript levels of polyamine biosynthetic genes, as well as the activities of the corresponding enzymes, are induced by stresses; secondly, elevation of endogenous polyamine levels by exogenous supply of polyamines, or overexpression of polyamine biosynthetic genes, results in enhanced stress tolerance; and thirdly, a reduction of endogenous polyamines is accompanied by compromised stress tolerance. A number of studies have demonstrated that polyamines function in stress tolerance largely by modulating the homeostasis of reactive oxygen species (ROS) due to their direct, or indirect, roles in regulating antioxidant systems or suppressing ROS production. The transcriptional regulation of polyamine synthesis by transcription factors is also reviewed here. Meanwhile, future perspectives on polyamine research are also suggested. PMID:26528300

  19. Characterization of active site residues of nitroalkane oxidase.

    PubMed

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

    2010-06-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  20. Age dependency of the metabolic conversion of polyamines into amino acids in IMR-90 human embryonic lung diploid fibroblasts

    SciTech Connect

    Chen, K.Y.; Chang, Z.

    1986-07-01

    When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000-dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000-dalton protein. The authors have investigated these two polyamine-associated biochemical events in IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL). They found that (1) the metabolic labeling of the 18,000-dalton protein was about two-fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six-fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4-fold higher in the young cells as compared to the old cells, their data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR-90 cells was about eight-fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000-dalton protein were also observed in cell strains derived from patients with premature aging disease.

  1. Inhibition of polyphenol oxidases activity by various dipeptides.

    PubMed

    Girelli, Anna M; Mattei, Enrico; Messina, Antonella; Tarola, Anna M

    2004-05-19

    In an effort to develop natural and nontoxic inhibitors on the activity of mushroom polyphenol oxidase (PPO) the effect of various glycyl-dipeptides (GlyAsp, GlyGly, GlyHis, GlyLeu, GlyLys, GlyPhe, GlyPro, GlyTyr) was investigated. The inhibition study with dihydroxyphenylalanine (DOPA) as substrate is based on separation of the enzymatic reaction components by reversed phase HPLC and the UV detection of the dopachrome formed. The results have evidenced that several of tested dipeptides inhibited PPO activity in the range of 20-40% while GlyPro and GlyLeu had no effect. The study has also permitted the characterization of the following kinetic pattern: a linear-mixed-type mechanism for GlyAsp, GlyGly, GlyLys, and GlyPhe and a hyperbolic-mixed-type for GlyTyr. It was not possible to identify the inhibition mechanism for GlyHis, although it affects PPO activity. In addition the effects of GlyAsp, GlyLys and GlyHis were evaluated for lessening the browning of fresh Golden Delicious apple and Irish White Skinned potato. The effectiveness of such inhibitors was determined by the difference between the colors observed in the dipeptide-treated sample and the controls using the color space CIE-Lab system. The % browning inhibition on potato (20-50%) was greater than of apple (20-30%) by the all tested dipeptides. Only GlyLys presented the significant value of 50%. PMID:15137808

  2. Predicting Monoamine Oxidase Inhibitory Activity through Ligand-Based Models

    PubMed Central

    Vilar, Santiago; Ferino, Giulio; Quezada, Elias; Santana, Lourdes; Friedman, Carol

    2013-01-01

    The evolution of bio- and cheminformatics associated with the development of specialized software and increasing computer power has produced a great interest in theoretical in silico methods applied in drug rational design. These techniques apply the concept that “similar molecules have similar biological properties” that has been exploited in Medicinal Chemistry for years to design new molecules with desirable pharmacological profiles. Ligand-based methods are not dependent on receptor structural data and take into account two and three-dimensional molecular properties to assess similarity of new compounds in regards to the set of molecules with the biological property under study. Depending on the complexity of the calculation, there are different types of ligand-based methods, such as QSAR (Quantitative Structure-Activity Relationship) with 2D and 3D descriptors, CoMFA (Comparative Molecular Field Analysis) or pharmacophoric approaches. This work provides a description of a series of ligand-based models applied in the prediction of the inhibitory activity of monoamine oxidase (MAO) enzymes. The controlled regulation of the enzymes’ function through the use of MAO inhibitors is used as a treatment in many psychiatric and neurological disorders, such as depression, anxiety, Alzheimer’s and Parkinson’s disease. For this reason, multiple scaffolds, such as substituted coumarins, indolylmethylamine or pyridazine derivatives were synthesized and assayed toward MAO-A and MAO-B inhibition. Our intention is to focus on the description of ligand-based models to provide new insights in the relationship between the MAO inhibitory activity and the molecular structure of the different inhibitors, and further study enzyme selectivity and possible mechanisms of action. PMID:23231398

  3. Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern 1

    PubMed Central

    Chatfield, J. Mark; Armstrong, Donald J.

    1986-01-01

    The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N6-(Δ2-isopentenyl)adenine-8-14C (i6 Ade-8-14C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i6 Ade-8-14C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded. PMID:16664650

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

    PubMed Central

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

    2012-01-01

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

  5. CsPAO4 of Citrus sinensis functions in polyamine terminal catabolism and inhibits plant growth under salt stress

    PubMed Central

    Wang, Wei; Liu, Ji-Hong

    2016-01-01

    Polyamine oxidase (PAO) is a key enzyme catalyzing polyamine catabolism leading to H2O2 production. We previously demonstrated that Citrus sinensis contains six putative PAO genes, but their functions are not well understood. In this work, we reported functional elucidation of CsPAO4 in polyamine catabolism and salt stress response. CsPAO4 was localized to the apoplast and used both spermidine (Spd) and spermine (Spm) as substrates for terminal catabolism. Transgenic plants overexpressing CsPAO4 displayed prominent increase in PAO activity, concurrent with marked decrease of Spm and Spd and elevation of H2O2. Seeds of transgenic lines displayed better germination when compared with wild type (WT) under salt stress. However, both vegetative growth and root elongation of the transgenic lines were prominently inhibited under salt stress, accompanied by higher level of H2O2 and more conspicuous programmed cell death (PCD). Exogenous supply of catalase (CAT), a H2O2 scavenger, partially recovered the vegetative growth and root elongation. In addition, spermine inhibited root growth of transgenic plants. Taken together, these data demonstrated that CsPAO4 accounts for production of H2O2 causing oxidative damages under salt stress and that down-regulation of a PAO gene involved in polyamine terminal catabolism may be an alternative approach for improving salt stress tolerance. PMID:27535697

  6. CsPAO4 of Citrus sinensis functions in polyamine terminal catabolism and inhibits plant growth under salt stress.

    PubMed

    Wang, Wei; Liu, Ji-Hong

    2016-01-01

    Polyamine oxidase (PAO) is a key enzyme catalyzing polyamine catabolism leading to H2O2 production. We previously demonstrated that Citrus sinensis contains six putative PAO genes, but their functions are not well understood. In this work, we reported functional elucidation of CsPAO4 in polyamine catabolism and salt stress response. CsPAO4 was localized to the apoplast and used both spermidine (Spd) and spermine (Spm) as substrates for terminal catabolism. Transgenic plants overexpressing CsPAO4 displayed prominent increase in PAO activity, concurrent with marked decrease of Spm and Spd and elevation of H2O2. Seeds of transgenic lines displayed better germination when compared with wild type (WT) under salt stress. However, both vegetative growth and root elongation of the transgenic lines were prominently inhibited under salt stress, accompanied by higher level of H2O2 and more conspicuous programmed cell death (PCD). Exogenous supply of catalase (CAT), a H2O2 scavenger, partially recovered the vegetative growth and root elongation. In addition, spermine inhibited root growth of transgenic plants. Taken together, these data demonstrated that CsPAO4 accounts for production of H2O2 causing oxidative damages under salt stress and that down-regulation of a PAO gene involved in polyamine terminal catabolism may be an alternative approach for improving salt stress tolerance. PMID:27535697

  7. Regulation of polyamine metabolism by translational control.

    PubMed

    Perez-Leal, Oscar; Merali, Salim

    2012-02-01

    Polyamines are low molecular weight, positively charged compounds that are ubiquitous in all living cells. They play a crucial role in many biochemical processes including regulation of transcription and translation, modulation of enzyme activities, regulation of ion channels and apoptosis. A strict balance between synthesis, catabolism and excretion tightly controls the cellular concentration of polyamines. The concentrations of rate-limiting enzymes in the polyamine synthesis and degradation pathways are regulated at different levels, including transcription, translation and degradation. Polyamines can modulate the translation of most of the enzymes required for their synthesis and catabolism through feedback mechanisms that are unique for each enzyme. Translational control is associated with cis-acting and trans-acting factors that can be influenced by the concentration of polyamines through mechanisms that are not completely understood. In this review, we present an overview of the translational control mechanisms of the proteins in the polyamine pathway, including ornithine decarboxylase (ODC), ODC antizyme, S-adenosylmethionine decarboxylase and spermidine/spermine N(1) acetyltransferase, highlighting the areas where more research is needed. A better understanding of the translational control of these enzymes would offer the possibility of a novel pharmacological intervention against cancer and other diseases. PMID:21811825

  8. Polyamines interact with hydroxyl radicals in activating Ca(2+) and K(+) transport across the root epidermal plasma membranes.

    PubMed

    Zepeda-Jazo, Isaac; Velarde-Buendía, Ana María; Enríquez-Figueroa, René; Bose, Jayakumar; Shabala, Sergey; Muñiz-Murguía, Jesús; Pottosin, Igor I

    2011-12-01

    Reactive oxygen species (ROS) are integral components of the plant adaptive responses to environment. Importantly, ROS affect the intracellular Ca(2+) dynamics by activating a range of nonselective Ca(2+)-permeable channels in plasma membrane (PM). Using patch-clamp and noninvasive microelectrode ion flux measuring techniques, we have characterized ionic currents and net K(+) and Ca(2+) fluxes induced by hydroxyl radicals (OH(•)) in pea (Pisum sativum) roots. OH(•), but not hydrogen peroxide, activated a rapid Ca(2+) efflux and a more slowly developing net Ca(2+) influx concurrent with a net K(+) efflux. In isolated protoplasts, OH(•) evoked a nonselective current, with a time course and a steady-state magnitude similar to those for a K(+) efflux in intact roots. This current displayed a low ionic selectivity and was permeable to Ca(2+). Active OH(•)-induced Ca(2+) efflux in roots was suppressed by the PM Ca(2+) pump inhibitors eosine yellow and erythrosine B. The cation channel blockers gadolinium, nifedipine, and verapamil and the anionic channel blockers 5-nitro-2(3-phenylpropylamino)-benzoate and niflumate inhibited OH(•)-induced ionic currents in root protoplasts and K(+) efflux and Ca(2+) influx in roots. Contrary to expectations, polyamines (PAs) did not inhibit the OH(•)-induced cation fluxes. The net OH(•)-induced Ca(2+) efflux was largely prolonged in the presence of spermine, and all PAs tested (spermine, spermidine, and putrescine) accelerated and augmented the OH(•)-induced net K(+) efflux from roots. The latter effect was also observed in patch-clamp experiments on root protoplasts. We conclude that PAs interact with ROS to alter intracellular Ca(2+) homeostasis by modulating both Ca(2+) influx and efflux transport systems at the root cell PM. PMID:21980172

  9. Drought-inhibited ribulose-1,5-bisphosphate carboxylase activity is mediated through increased release of ethylene and changes in the ratio of polyamines in pakchoi.

    PubMed

    Huang, Xingxue; Zhou, Guolin; Yang, Wengang; Wang, Aihua; Hu, Zhenhua; Lin, Chufa; Chen, Xin

    2014-09-15

    To study the mechanisms of drought inhibiting photosynthesis and the role of PAs and ethylene, the photosynthetic rate (Pn), the maximal photochemical efficiency of PSII (Fv/Fm), the intercellular CO2 concentration (Ci), photorespiratory rate (Pr), the amount of chlorophyll (chl), antioxidant enzyme activity, ethylene levels, RuBPC (ribulose-1,5-bisphosphate carboxylase) activity and endogenous polyamine levels of pakchoi were examined, and an inhibitor of S-adenosylmethionine decarboxylase (SAMDC) and an inhibitor of ethylene synthesis and spermidine (Spd) were used to induce the change of endogenous polyamine levels. The results show that drought induced a decrease in Pn and RuBPC activity, an increase in the intercellular CO2 concentration (Ci), but no change in the actual photochemical efficiency of PSII (ΦPSII), and chlorophyll content. In addition, drought caused an increase in the free putrescine (fPut), the ethylene levels, a decrease in the Spd and spermine (Spm) levels, and the PAs/fPut ratio in the leaves. The exogenous application of Spd and amino oxiacetic acid (AOAA, an inhibitor of ethylene synthesis) markedly reversed these drought-induced effects on polyamine, ethylene, Pn, the PAs/fPut ratio and RuBPCase activity in leaves. Methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of SAMDC resulting in the inability of activated cells to synthesize Spd and Spm, exacerbates the negative effects induced by drought. These results suggest that the decrease in Pn is at least partially attributed to the decrease of RuBPC activity under drought stress and that drought inhibits RuBPC activity by decreasing the ratio of PAs/fPut and increasing the release of ethylene. PMID:25046760

  10. Cholesterol oxidase with high catalytic activity from Pseudomonas aeruginosa: Screening, molecular genetic analysis, expression and characterization.

    PubMed

    Doukyu, Noriyuki; Nihei, Shyou

    2015-07-01

    An extracellular cholesterol oxidase producer, Pseudomonas aeruginosa strain PA157, was isolated by a screening method to detect 6β-hydroperoxycholest-4-en-3-one-forming cholesterol oxidase. On the basis of a putative cholesterol oxidase gene sequence in the genome sequence data of P. aeruginosa strain PAO1, the cholesterol oxidase gene from strain PA157 was cloned. The mature form of the enzyme was overexpressed in Escherichia coli cells. The overexpressed enzyme formed inclusion bodies in recombinant E. coli cells grown at 20 °C and 30 °C. A soluble and active PA157 enzyme was obtained when the recombinant cells were grown at 10 °C. The purified enzyme was stable at pH 5.5 to 10 and was most active at pH 7.5-8.0, showing optimal activity at pH 7.0 and 70 °C. The enzyme retained about 90% of its activity after incubation for 30 min at 70 °C. The enzyme oxidized 3β-hydroxysteroids such as cholesterol, β-cholestanol, and β-sitosterol at high rates. The Km value and Vmax value for the cholesterol were 92.6 μM and 15.9 μmol/min/mg of protein, respectively. The Vmax value of the enzyme was higher than those of commercially available cholesterol oxidases. This is the first report to characterize a cholesterol oxidase from P. aeruginosa. PMID:25573142

  11. Identification in Marinomonas mediterranea of a novel quinoprotein with glycine oxidase activity

    PubMed Central

    Campillo-Brocal, Jonatan Cristian; Lucas-Elio, Patricia; Sanchez-Amat, Antonio

    2013-01-01

    Abstract A novel enzyme with lysine-epsilon oxidase activity was previously described in the marine bacterium Marinomonas mediterranea. This enzyme differs from other l-amino acid oxidases in not being a flavoprotein but containing a quinone cofactor. It is encoded by an operon with two genes lodA and lodB. The first one codes for the oxidase, while the second one encodes a protein required for the expression of the former. Genome sequencing of M. mediterranea has revealed that it contains two additional operons encoding proteins with sequence similarity to LodA. In this study, it is shown that the product of one of such genes, Marme_1655, encodes a protein with glycine oxidase activity. This activity shows important differences in terms of substrate range and sensitivity to inhibitors to other glycine oxidases previously described which are flavoproteins synthesized by Bacillus. The results presented in this study indicate that the products of the genes with different degrees of similarity to lodA detected in bacterial genomes could constitute a reservoir of different oxidases. PMID:23873697

  12. Biogenic and synthetic polyamines bind cationic dendrimers.

    PubMed

    Mandeville, Jean-Sebastian; Bourassa, Phillipe; Thomas, Thekkumkattil John; Tajmir-Riahi, Heidar-Ali

    2012-01-01

    Biogenic polyamines are essential for cell growth and differentiation, while polyamine analogues exert antitumor activity in multiple experimental model systems, including breast and lung cancer. Dendrimers are widely used for drug delivery in vitro and in vivo. We report the bindings of biogenic polyamines, spermine (spm), and spermidine (spmd), and their synthetic analogues, 3,7,11,15-tetrazaheptadecane.4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane.5HCl (BE-3333) to dendrimers of different compositions, mPEG-PAMAM (G3), mPEG-PAMAM (G4) and PAMAM (G4). FTIR and UV-visible spectroscopic methods as well as molecular modeling were used to analyze polyamine binding mode, the binding constant and the effects of polyamine complexation on dendrimer stability and conformation. Structural analysis showed that polyamines bound dendrimers through both hydrophobic and hydrophilic contacts with overall binding constants of K(spm-mPEG-G3) = 7.6 × 10(4) M(-1), K(spm-mPEG-PAMAM-G4) = 4.6 × 10(4) M(-1), K(spm-PAMAM-G4) = 6.6 × 10(4) M(-1), K(spmd-mPEG-G3) = 1.0 × 10(5) M(-1), K(spmd-mPEG-PAMAM-G4) = 5.5 × 10(4) M(-1), K(spmd-PAMAM-G4) = 9.2 × 10(4) M(-1), K(BE-333-mPEG-G3) = 4.2 × 10(4) M(-1), K(Be-333-mPEG-PAMAM-G4) = 3.2 × 10(4) M(-1), K(BE-333-PAMAM-G4) = 3.6 × 10(4) M(-1), K(BE-3333-mPEG-G3) = 2.2 × 10(4) M(-1), K(Be-3333-mPEG-PAMAM-G4) = 2.4 × 10(4) M(-1), K(BE-3333-PAMAM-G4) = 2.3 × 10(4) M(-1). Biogenic polyamines showed stronger affinity toward dendrimers than those of synthetic polyamines, while weaker interaction was observed as polyamine cationic charges increased. The free binding energies calculated from docking studies were: -3.2 (spermine), -3.5 (spermidine) and -3.03 (BE-3333) kcal/mol, with the following order of binding affinity: spermidine-PAMAM-G-4>spermine-PAMMAM-G4>BE-3333-PAMAM-G4 consistent with spectroscopic data. Our results suggest that dendrimers can act as carrier vehicles for delivering antitumor polyamine analogues to target tissues

  13. Polyamine-Induced Rapid Root Abscission in Azolla pinnata.

    PubMed

    Gurung, Sushma; Cohen, Michael F; Fukuto, Jon; Yamasaki, Hideo

    2012-01-01

    Floating ferns of the genus Azolla detach their roots under stress conditions, a unique adaptive response termed rapid root abscission. We found that Azolla pinnata plants exhibited dose-dependent rapid root abscission in response to the polyamines spermidine and spermine after a substantial time lag (>20 min). The duration of the time lag decreased in response to high pH and high temperature whereas high light intensity increased the time lag and markedly lowered the rate of abscission. The oxidation products of polyamines, 1,3-diaminopropane, β-alanine and hydrogen peroxide all failed to initiate root abscission, and hydroxyethyl hydrazine, an inhibitor of polyamine oxidase, did not inhibit spermine-induced root abscission. Exposure of A. pinnata to the polyamines did not result in detectable release of NO and did not affect nitrite-dependent NO production. The finding of polyamine-induced rapid root abscission provides a facile assay for further study of the mode of action of polyamines in plant stress responses. PMID:22997568

  14. The Effect of Exogenous Spermidine Concentration on Polyamine Metabolism and Salt Tolerance in Zoysiagrass (Zoysia japonica Steud) Subjected to Short-Term Salinity Stress

    PubMed Central

    Li, Shucheng; Jin, Han; Zhang, Qiang

    2016-01-01

    Salt stress, particularly short-term salt stress, is among the most serious abiotic factors limiting plant survival and growth in China. It has been established that exogenous spermidine (Spd) stimulates plant tolerance to salt stress. The present study utilized two zoysiagrass cultivars commonly grown in China that exhibit either sensitive (cv. Z081) or tolerant (cv. Z057) adaptation capacity to salt stress. The two cultivars were subjected to 200 mM salt stress and treated with different exogenous Spd concentrations for 8 days. Polyamine [diamine putrescine (Put), tetraamine spermine (Spm), and Spd], H2O2 and malondialdehyde (MDA) contents and polyamine metabolic (ADC, ODC, SAMDC, PAO, and DAO) and antioxidant (superoxide dismutase, catalase, and peroxidase) enzyme activities were measured. The results showed that salt stress induced increases in Spd and Spm contents and ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and diamine oxidase (DAO) activities in both cultivars. Exogenous Spd application did not alter polyamine contents via regulation of polyamine-degrading enzymes, and an increase in polyamine biosynthetic enzyme levels was observed during the experiment. Increasing the concentration of exogenous Spd resulted in a tendency of the Spd and Spm contents and ODC, SAMDC, DAO, and antioxidant enzyme activities to first increase and then decrease in both cultivars. H2O2 and MDA levels significantly decreased in both cultivars treated with Spd. Additionally, in both cultivars, positive correlations between polyamine biosynthetic enzymes (ADC, SAMDC), DAO, and antioxidant enzymes (SOD, POD, CAT), but negative correlations with H2O2 and MDA levels, and the Spd + Spm content were observed with an increase in the concentration of exogenous Spd. PMID:27582752

  15. The Effect of Exogenous Spermidine Concentration on Polyamine Metabolism and Salt Tolerance in Zoysiagrass (Zoysia japonica Steud) Subjected to Short-Term Salinity Stress.

    PubMed

    Li, Shucheng; Jin, Han; Zhang, Qiang

    2016-01-01

    Salt stress, particularly short-term salt stress, is among the most serious abiotic factors limiting plant survival and growth in China. It has been established that exogenous spermidine (Spd) stimulates plant tolerance to salt stress. The present study utilized two zoysiagrass cultivars commonly grown in China that exhibit either sensitive (cv. Z081) or tolerant (cv. Z057) adaptation capacity to salt stress. The two cultivars were subjected to 200 mM salt stress and treated with different exogenous Spd concentrations for 8 days. Polyamine [diamine putrescine (Put), tetraamine spermine (Spm), and Spd], H2O2 and malondialdehyde (MDA) contents and polyamine metabolic (ADC, ODC, SAMDC, PAO, and DAO) and antioxidant (superoxide dismutase, catalase, and peroxidase) enzyme activities were measured. The results showed that salt stress induced increases in Spd and Spm contents and ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), and diamine oxidase (DAO) activities in both cultivars. Exogenous Spd application did not alter polyamine contents via regulation of polyamine-degrading enzymes, and an increase in polyamine biosynthetic enzyme levels was observed during the experiment. Increasing the concentration of exogenous Spd resulted in a tendency of the Spd and Spm contents and ODC, SAMDC, DAO, and antioxidant enzyme activities to first increase and then decrease in both cultivars. H2O2 and MDA levels significantly decreased in both cultivars treated with Spd. Additionally, in both cultivars, positive correlations between polyamine biosynthetic enzymes (ADC, SAMDC), DAO, and antioxidant enzymes (SOD, POD, CAT), but negative correlations with H2O2 and MDA levels, and the Spd + Spm content were observed with an increase in the concentration of exogenous Spd. PMID:27582752

  16. Polyamines: Predictive Biomarker for HIV-Associated Neurocognitive Disorders

    PubMed Central

    Merali, Salim; Barrero, Carlos A.; Sacktor, Ned C.; Haughey, Norman J.; Datta, Prasun K.; Langford, Dianne; Khalili, Kamel

    2014-01-01

    Objectives Spermidine/spermine-N1-acetytransferase (SSAT) is the key enzyme in the catabolism of polyamines that are involved in regulating NMDA functioning. Over expression of SSAT leads to abnormal metabolic cycling and may disrupt NMDA receptor signaling. In fact, the HIV protein Tat induces neurotoxicity involving polyamine/NMDA receptor interactions. Thus, we investigated abnormal polyamine cycling in HIV+ participants with varying degrees of HIV-associated neurocognitive disorders. Methods Acetyl-polyamine (SSAT products) levels were assessed by HPLC in CSF from 99 HIV-infected participants (no cognitive impairment (NCI, n=25), asymptomatic neurocognitive impairment (ANI, n=25), mild cognitive and motor disorders (MCMD, n=24), and HIV-associated dementia (HAD, n=25)). Polyamine levels in brain tissues from a subset of participants (uninfected (n=3), NCI (n=3), and MNCD (n=3)) were also assessed. Human primary astrocytes expressing HIV Tat were assessed for levels of the SSAT activity. Results Activation of the polyamine catabolic enzyme, SSAT increases polyamine flux in brain and CSF of HIV infected individuals with HIV-associated neurocognitive disorders. CSF levels of acetylated polyamine increase with the degree of HAND severity as indicated by significantly increased acetylpolyamine levels in HAD participants compared to NCI and ANI (p<0.0001) and between MCMD and NCI and ANI (p<0.0001). In vitro studies suggest that the HIV protein Tat may be responsible in part for astrocyte-derived acetyl polyamine release. Interpretation Our data suggest that polyamine metabolism may play a pivotal role in the neurodegeneration process among HAND patients. Changes in polyamine flux may serve as a potential predictive diagnostic biomarker for different severities of HAND. PMID:25893137

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

    PubMed

    Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

    2015-11-10

    In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. PMID:26257292

  18. Involvement of NADPH oxidases in suppression of cyclooxygenase-2 promoter-dependent transcriptional activities by sesamol

    PubMed Central

    Shimizu, Satomi; Ishigamori, Rikako; Fujii, Gen; Takahashi, Mami; Onuma, Wakana; Terasaki, Masaru; Yano, Tomohiro; Mutoh, Michihiro

    2015-01-01

    Cyclooxygenase-2 (COX-2) has been shown to play an important role in colon carcinogenesis. Moreover, one of the components of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NADPH oxidase 1 (NOX1), dominantly expressed in the colon, is implicated in the pathogenesis of colon cancer. We have reported that sesamol, one of the lignans in sesame seeds, suppressed COX-2 gene transcriptional activity in human colon cancer cells, and also suppressed intestinal polyp formation in Apc-mutant mice. In the present study, we investigated the involvement of NADPH oxidase in the inhibition of COX-2 transcriptional activity by sesamol. We found that several NADPH oxidase inhibitors, such as apocynin, showed suppressive effects on COX-2 transcriptional activity. Moreover, sesamol significantly suppressed NOX1 mRNA levels in a dose-dependent manner. In addition, we demonstrated that knockdown of NOX1 successfully suppressed COX-2 transcriptional activity. These results suggest that inhibition of NADPH oxidase, especially NOX1, may be involved in the mechanism of the suppression of COX-2 transcriptional activity by sesamol. PMID:25759517

  19. Diphenol activation of the monophenolase and diphenolase activities of field bean (Dolichos lablab) polyphenol oxidase.

    PubMed

    Gowda, Lalitha R; Paul, Beena

    2002-03-13

    This paper reports a study on the hydroxylation of ferulic acid and tyrosine by field bean (Dolichos lablab) polyphenol oxidase, a reaction that does not take place without the addition of catechol. A lag period similar to the characteristic lag of tyrosinase activity was observed, the length of which decreased with increasing catechol concentration and increased with increasing ferulic acid concentration. The activation constant K(a) of catechol for ferulic acid hydroxylation reaction was 5 mM. The kinetic parameters of field bean polyphenol oxidase toward ferulic acid and tyrosine were evaluated in the presence of catechol. 4-Methyl catechol, L-dihydroxyphenylalanine, pyrogallol, and 2,3,4-trihydroxybenzoic acid, substrates with high binding affinity to field bean polyphenol oxidase, could stimulate this hydroxylation reaction. In contrast, diphenols such as protocatechuic acid, gallic acid, chlorogenic acid, and caffeic acid, which were not substrates for the oxidation reaction, were unable to bring about this activation. It is most likely that only o-diphenols that are substrates for the diphenolase serve as cosubstrates by donating electrons at the active site for the monophenolase activity. The reaction mechanism for this activation is consistent with that proposed for tyrosinase (Sanchez-Ferrer, A.; Rodriguez-Lopez, J. N.; Garcia-Canovas, F.; Garcia-Carmona, F. Biochim. Biophys. Acta 1995, 1247, 1-11). The presence of o-diphenols, viz. catechol, L-dihydroxyphenylalanine, and 4-methyl catechol, is also necessary for the oxidation of the diphenols, caffeic acid, and catechin to their quinones by the field bean polyphenol oxidase. This oxidation reaction occurs immediately with no lag period and does not occur without the addition of diphenol. The kinetic parameters for caffeic acid (K(m) = 0.08 mM, V(max) = 32440 u/mg) in the presence of catechol and the activation constant K(a) of catechol (4.6 mM) for this reaction were enumerated. The absence of a lag

  20. Hyper-responsive Toll-like receptor 7 and 9 activation in NADPH oxidase-deficient B lymphoblasts.

    PubMed

    McLetchie, Shawna; Volpp, Bryan D; Dinauer, Mary C; Blum, Janice S

    2015-12-01

    Chronic granulomatous disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-subunit leucocyte NADPH oxidase. Myeloid-derived phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of reactive oxygen species to clear engulfed pathogens. In this study we show that oxidase also influences B-cell functions, including responses to single-stranded RNA or unmethylated DNA by endosomal Toll-like receptors (TLRs) 7 and 9. In response to TLR7/9 ligands, B-cell lines derived from patients with CGD with mutations in either the NADPH oxidase p40(phox) or p47(phox) subunits produced only low levels of reactive oxygen species. Remarkably, cytokine secretion and p38 mitogen-activated protein kinase activation by these oxidase-deficient B cells was significantly increased upon TLR7/9 activation when compared with oxidase-sufficient B cells. Increased TLR responsiveness was also detected in B cells from oxidase-deficient mice. NADPH oxidase-deficient patient-derived B cells also expressed enhanced levels of TLR7 and TLR9 mRNA and protein compared with the same cells reconstituted to restore oxidase activity. These data demonstrate that the loss of oxidase function associated with CGD can significantly impact B-cell TLR signalling in response to nucleic acids with potential repercussions for auto-reactivity in patients. PMID:26340429

  1. Cholesterol: A modulator of the phagocyte NADPH oxidase activity - A cell-free study

    PubMed Central

    Masoud, Rawand; Bizouarn, Tania; Houée-Levin, Chantal

    2014-01-01

    The NADPH oxidase Nox2, a multi-subunit enzyme complex comprising membrane and cytosolic proteins, catalyzes a very intense production of superoxide ions O2•−, which are transformed into other reactive oxygen species (ROS). In vitro, it has to be activated by addition of amphiphiles like arachidonic acid (AA). It has been shown that the membrane part of phagocyte NADPH oxidase is present in lipid rafts rich in cholesterol. Cholesterol plays a significant role in the development of cardio-vascular diseases that are always accompanied by oxidative stress. Our aim was to investigate the influence of cholesterol on the activation process of NADPH oxidase. Our results clearly show that, in a cell-free system, cholesterol is not an efficient activator of NADPH oxidase like arachidonic acid (AA), however it triggers a basal low superoxide production at concentrations similar to what found in neutrophile. A higher concentration, if present during the assembly process of the enzyme, has an inhibitory role on the production of O2•−. Added cholesterol acts on both cytosolic and membrane components, leading to imperfect assembly and decreasing the affinity of cytosolic subunits to the membrane ones. Added to the cytosolic proteins, it retains their conformations but still allows some conformational change induced by AA addition, indispensable to activation of NADPH oxidase. PMID:25462061

  2. Spinach thylakoid polyphenol oxidase isolation, activation, and properties of the native chloroplast enzyme

    SciTech Connect

    Golbeck, J.H.; Cammarata, K.V.

    1981-05-01

    Polyphenol oxidase activity (E.C. 1.14,18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. Sonication releases polyphenol oxidase from the membrane largely in the latent state. In the absence of added fatty acids, the isolated enzyme spontaneously, but slowly, activates with time. Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; however, the enzyme is substrate-inhibited by the last four at concentrations near their K/sub m/. A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  3. 1-Aminocyclopropane-1-Carboxylate Oxidase Activity Limits Ethylene Biosynthesis in Rumex palustris during Submergence

    PubMed Central

    Vriezen, Wim H.; Hulzink, Raymond; Mariani, Celestina; Voesenek, Laurentius A.C.J.

    1999-01-01

    Submergence strongly stimulates petiole elongation in Rumex palustris, and ethylene accumulation initiates and maintains this response in submerged tissues. cDNAs from R. palustris corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene (RP-ACO1) were isolated from elongating petioles and used to study the expression of the corresponding gene. An increase in RP-ACO1 messenger was observed in the petioles and lamina of elongating leaves 2 h after the start of submergence. ACC oxidase enzyme activity was measured in homogenates of R. palustris shoots, and a relevant increase was observed within 12 h under water with a maximum after 24 h. We have shown previously that the ethylene production rate of submerged shoots does not increase significantly during the first 24 h of submergence (L.A.C.J. Voesenek, M. Banga, R.H. Thier, C.M. Mudde, F.M. Harren, G.W.M. Barendse, C.W.P.M. Blom [1993] Plant Physiol 103: 783–791), suggesting that under these conditions ACC oxidase activity is inhibited in vivo. We found evidence that this inhibition is caused by a reduction of oxygen levels. We hypothesize that an increased ACC oxidase enzyme concentration counterbalances the reduced enzyme activity caused by low oxygen concentration during submergence, thus sustaining ethylene production under these conditions. Therefore, ethylene biosynthesis seems to be limited at the level of ACC oxidase activity rather than by ACC synthase in R. palustris during submergence. PMID:10482674

  4. Simulated ischaemia-reperfusion conditions increase xanthine dehydrogenase and oxidase activities in rat brain slices.

    PubMed

    Battelli, M G; Buonamici, L; Virgili, M; Abbondanza, A; Contestabile, A

    1998-01-01

    Xanthine dehydrogenase and oxidase activities increased by 87% in rat brain slices after 30 min in vitro ischaemia. A further 41% increase was induced by 30 min simulated reperfusion of ischaemic slices. No conversion from the dehydrogenase to the oxidase activity was observed. The increment of enzyme activity was not due to neosynthesis of the enzyme, since it was not affected by the addition of cycloheximide during the ischaemic incubation. The increased oxygen-dependent form of the enzyme could aggravate the ischaemic brain injury by free radicals production, in particular after reperfusion. PMID:9460697

  5. A description of an HPLC assay of coproporphyrinogen III oxidase activity in mononuclear cells.

    PubMed

    Gross, U; Gerlach, R; Kühnel, A; Seifert, V; Doss, M O

    2003-01-01

    Coproporphyrinogen III oxidase is deficient in hereditary coproporphyria. An activity assay for this enzyme in mononuclear cells, besides the preparation of the substrate, are presented. The separation conditions for the product of the test protoporphyrin IX by gradient, reversed-phase high-performance liquid chromatography are given. The normal value from mononuclear cells of healthy volunteers was 138 +/- 21 pkat/g total soluble protein (mean +/- SD). The enzyme activity of a family with hereditary coproporphyria was measured. The gene carriers exhibit a specific coproporphyrinogen III oxidase activity of 61-90 pkat/g total soluble protein. PMID:14605502

  6. Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction

    SciTech Connect

    Qiu, James A.; Wilson, Heather L.; Rajagopalan, K.V.

    2012-04-18

    Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conserved in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 {angstrom} resolution, respectively.

  7. Polyamines and plant stress - Activation of putrescine biosynthesis by osmotic shock

    NASA Technical Reports Server (NTRS)

    Flores, H. E.; Galston, A. W.

    1982-01-01

    The putrescine content of oat leaf cells and protoplasts increases up to 60-fold within 6 hours of exposure to osmotic stress (0.4 to 0.6 molar sorbitol). Barley, corn, wheat, and wild oat leaves show a similar response. Increased arginine decarboxylase activity parallels the rise in putrescine, whereas ornithine decarboxylase remains unchanged. DL-alpha-Difluoromethylarginine, a specific irreversible inhibitor of arginine decarboxylase, prevents the stress-induced rise in increase in arginine decarboxylase activity and putrescine synthesis, indicating the preferential activation of this pathway.

  8. Exogenous polyamines promote osteogenic differentiation by reciprocally regulating osteogenic and adipogenic gene expression.

    PubMed

    Lee, Mon-Juan; Chen, Yuhsin; Huang, Yuan-Pin; Hsu, Yi-Chiang; Chiang, Lan-Hsin; Chen, Tzu-Yu; Wang, Gwo-Jaw

    2013-12-01

    Polyamines are naturally occurring organic polycations that are ubiquitous in all organisms, and are essential for cell proliferation and differentiation. Although polyamines are involved in various cellular processes, their roles in stem cell differentiation are relatively unexplored. In this study, we found that exogenous polyamines, putrescine, spermidine, and spermine, promoted osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) without inducing cell death or apoptosis. Alkaline phosphatase (ALP) activity and the mRNA level of osteogenic genes, including Runx2, ALP, osteopontin, and osteocalcin, were up-regulated by exogenous polyamines. When hBMSCs were cultured at high cell density favoring adipocyte formation, exogenous polyamines resulted in down-regulation of adipogenic genes such as PPARγ, aP2, and adipsin. Extracellular matrix mineralization, a marker for osteoblast maturation, was enhanced in the presence of exogenous polyamines, while lipid accumulation, an indication of adipogenic differentiation, was attenuated. Exogenous polyamines increased the mRNA expression of polyamine-modulated factor 1 (PMF-1) and its downstream effector, spermidine/spermine N(1)-acetyltransferase (SSAT), while that of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was suppressed. These results lead to possible connections between polyamine metabolism and osteogenic differentiation pathways. To summarize, this study provides evidence for the involvement of polyamines in osteogenic differentiation of hBMSCs, and is the first to demonstrate that osteogenic and adipogenic differentiation are reciprocally regulated by exogenous polyamines. PMID:23794266

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grain polyphenol oxidase (PPO) activity can cause discoloration of wheat (Triticum aestivum L.) food products. Five crosses (PI 117635/Antelope; Fielder/NW03681; Fielder/Antelope; NW07OR1070/Antelope; NW07OR1066/OR2050272H) were selected to study the genetic inheritance of PPO activity. STS marker...

  10. NADPH oxidase activity is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation

    PubMed Central

    MacFarlane, P M; Satriotomo, I; Windelborn, J A; Mitchell, G S

    2009-01-01

    Phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is a form of spinal, serotonin-dependent synaptic plasticity that requires reactive oxygen species (ROS) formation. We tested the hypothesis that spinal NADPH oxidase activity is a necessary source of ROS for pLTF. Sixty minutes post-AIH (three 5-min episodes of 11% O2, 5 min intervals), integrated phrenic and hypoglossal (XII) nerve burst amplitudes were increased from baseline, indicative of phrenic and XII LTF. Intrathecal injections (∼C4) of apocynin or diphenyleneiodonium chloride (DPI), two structurally and functionally distinct inhibitors of the NADPH oxidase complex, attenuated phrenic, but not XII, LTF. Immunoblots from soluble (cytosolic) and particulate (membrane) fractions of ventral C4 spinal segments revealed predominantly membrane localization of the NADPH oxidase catalytic subunit, gp91phox, whereas membrane and cytosolic expression were both observed for the regulatory subunits, p47phox and RAC1. Immunohistochemical analysis of fixed tissues revealed these same subunits in presumptive phrenic motoneurons of the C4 ventral horn, but not in neighbouring astrocytes or microglia. Collectively, these data demonstrate that NADPH oxidase subunits localized within presumptive phrenic motoneurons are a major source of ROS necessary for AIH-induced pLTF. Thus, NADPH oxidase activity is a key regulator of spinal synaptic plasticity, and may be a useful pharmaceutical target in developing therapeutic strategies for respiratory insufficiency in patients with, for example, cervical spinal injury. PMID:19237427

  11. Cytochrome C oxidase activity in germinating Phaseolus vulgaris l. seeds: Effects of carbon monoxide

    SciTech Connect

    Caughey, W.S. ); Sowa, S.; Roos, E.E.

    1989-04-01

    Cytochrome c oxidase is a key bioenergetic enzyme required for seed germination. The enzyme was isolated from 2-day germinating beans and biochemically compared to its bovine heart counterpart. Carbon monoxide, which binds to the heme a{sub 3} site of cytochrome c oxidase, we used to probe O{sub 2} utilization activity in isolated enzyme, mitochondrial particles, and whole seeds. Bean seeds under 80% CO/20% O{sub 2} exhibited 46% growth inhibition as determined by root length. Reversible, dose-dependent partial inhibition of bean seed mitochondrial respiration was observed in the presence of CO; heart mitochondria had a more sensitive, less reversible response. Effects of CO on bean and bovine heart enzyme were similar. The close correlation of CO effects observed on seedling growth, mitochondrial respiration and cytochrome oxidase activity indicate an important role for this enzyme during the early stages of seed germination.

  12. Regional brain effects of sodium azide treatment on cytochrome oxidase activity: a quantitative histochemical study.

    PubMed

    Cada, A; Gonzalez-Lima, F; Rose, G M; Bennett, M C

    1995-12-01

    The objective of the present study was to determine if regional variation in brain cytochrome oxidase activity was observed following systemic administration of sodium azide. An image analysis system calibrated with internal standards of known cytochrome oxidase activity was used to quantify cytochrome oxidase in histochemically stained brain sections. Rats receiving chronic infusion of sodium azide (400 micrograms/hr), which were sacrificed after two weeks, showed a substantial decrease in brain cytochrome oxidase activity over those infused with saline. All of the 22 regions sampled from telencephalic, diencephalic, and mesencephalic levels, showed a significant activity reduction which ranged between 26% and 37%. The regions that appeared significantly more vulnerable to the sodium azide effects were the mesencephalic reticular formation and the central amygdala, which displayed the largest decrease in activity. In addition, interregional correlations of activity showed a deeply modified pattern of correlative metabolic activity between hippocampal, amygdaloid and cortical areas after azide treatment. The regional effects found were consistent with azide-induced learning and memory dysfunctions. PMID:8847994

  13. Thioredoxin-1/peroxiredoxin-1 as sensors of oxidative stress mediated by NADPH oxidase activity in atherosclerosis.

    PubMed

    Madrigal-Matute, Julio; Fernandez-Garcia, Carlos-Ernesto; Blanco-Colio, Luis Miguel; Burillo, Elena; Fortuño, Ana; Martinez-Pinna, Roxana; Llamas-Granda, Patricia; Beloqui, Oscar; Egido, Jesus; Zalba, Guillermo; Martin-Ventura, José Luis

    2015-09-01

    To assess the potential association between TRX-1/PRX-1 and NADPH oxidase (Nox) activity in vivo and in vitro, TRX-1/PRX-1 levels were assessed by ELISA in 84 asymptomatic subjects with known phagocytic NADPH oxidase activity and carotid intima-media thickness (IMT). We found a positive correlation between TRX-1/PRX-1 and NADPH oxidase-dependent superoxide production (r=0.48 and 0.47; p<0.001 for both) and IMT (r=0.31 and 0.36; p<0.01 for both) adjusted by age and sex. Moreover, asymptomatic subjects with plaques have higher PRX-1 and TRX plasma levels (p<0.01 for both). These data were confirmed in a second study in which patients with carotid atherosclerosis showed higher PRX-1 and TRX plasma levels than healthy subjects (p<0.001 for both). In human atherosclerotic plaques, the NADPH oxidase subunit p22phox colocalized with TRX-1/PRX-1 in macrophages (immunohistochemistry). In monocytes and macrophages, phorbol 12-myristate 13-acetate (PMA) induced NADPH activation and TRX-1/PRX-1 release to the extracellular medium, with a concomitant decrease in their intracellular levels, which was reversed by the NADPH inhibitor apocynin (Western blot). In loss-of-function experiments, genetic silencing of the NADPH oxidase subunit Nox2 blocked PMA-induced intracellular TRX-1/PRX-1 downregulation in macrophages. Furthermore, the PMA-induced release of TRX-1/PRX-1 involves the modulation of their redox status and exosome-like vesicles. TRX-1/PRX-1 levels are associated with NADPH oxidase-activity in vivo and in vitro. These data could suggest a coordinated antioxidant response to oxidative stress in atherothrombosis. PMID:26117319

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

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  15. Virtual Screening Analysis and In-vitro Xanthine Oxidase Inhibitory Activity of Some Commercially Available Flavonoids.

    PubMed

    Umamaheswari, Muthuswamy; Madeswaran, Arumugam; Asokkumar, Kuppusamy

    2013-01-01

    Allopurinol, the xanthine oxidase inhibitor, is the only drug available for the treatment of gout. We examined the xanthine oxidase inhibitory activity of some commercially available flavonoids such asepigallocatechin, acacatechin, myricetin, naringenin, daidzein and glycitein by virtual screening and in-vitro studies. The interacting residues within the complex model and their contact types were identified. The virtual screening analysis were carried out using AutoDock 4.2 and in-vitro xanthine oxidase inhibitory activity was carried out using xanthine as the substrate. In addition, enzyme kinetics was performed using LineweaverBurkplot analysis. Allopurinol, a known xanthine oxidase inhibitor was used as the standard. The docking energy ofglycitein was found to be -8.49 kcal/mol which was less than that of the standard (-4.47 kcal/ mol). All the selected flavonoids were found to exhibit lower binding energy (-8.08 to -6.03 kcal/ mol) than allopurinol. The docking results confirm that flavonoids showed greater inhibition of xanthine oxidase due to their active binding sites and lesser binding energies compared to allopurinol. This may be attributed to the presence of benzopyran ring in the flavonoids. In the xanthine oxidase assay, IC50 value of glycitein was found to be 12±0.86 μg/mL, whereas that of allopurinol was 24±0.28 μg/mL. All the remaining compounds exhibited IC50 values ranging between 22±0.64 to 62±1.18 μg/mL. In the enzyme kinetic studies, flavonoids showed competitive type of enzyme inhibition. It can be concluded that flavonoids could be a promising remedy for the treatment of gout and related inflammatory disorders. Further in-vivo studies are required to develop potential compounds with lesser side effects. PMID:24250638

  16. Virtual Screening Analysis and In-vitro Xanthine Oxidase Inhibitory Activity of Some Commercially Available Flavonoids

    PubMed Central

    Umamaheswari, Muthuswamy; Madeswaran, Arumugam; Asokkumar, Kuppusamy

    2013-01-01

    Allopurinol, the xanthine oxidase inhibitor, is the only drug available for the treatment of gout. We examined the xanthine oxidase inhibitory activity of some commercially available flavonoids such asepigallocatechin, acacatechin, myricetin, naringenin, daidzein and glycitein by virtual screening and in-vitro studies. The interacting residues within the complex model and their contact types were identified. The virtual screening analysis were carried out using AutoDock 4.2 and in-vitro xanthine oxidase inhibitory activity was carried out using xanthine as the substrate. In addition, enzyme kinetics was performed using LineweaverBurkplot analysis. Allopurinol, a known xanthine oxidase inhibitor was used as the standard. The docking energy ofglycitein was found to be -8.49 kcal/mol which was less than that of the standard (-4.47 kcal/ mol). All the selected flavonoids were found to exhibit lower binding energy (-8.08 to -6.03 kcal/ mol) than allopurinol. The docking results confirm that flavonoids showed greater inhibition of xanthine oxidase due to their active binding sites and lesser binding energies compared to allopurinol. This may be attributed to the presence of benzopyran ring in the flavonoids. In the xanthine oxidase assay, IC50 value of glycitein was found to be 12±0.86 μg/mL, whereas that of allopurinol was 24±0.28 μg/mL. All the remaining compounds exhibited IC50 values ranging between 22±0.64 to 62±1.18 μg/mL. In the enzyme kinetic studies, flavonoids showed competitive type of enzyme inhibition. It can be concluded that flavonoids could be a promising remedy for the treatment of gout and related inflammatory disorders. Further in-vivo studies are required to develop potential compounds with lesser side effects. PMID:24250638

  17. The Glu²¹⁶/Ser²¹⁸ pocket is a major determinant of spermine oxidase substrate specificity.

    PubMed

    Cervelli, Manuela; Angelucci, Emanuela; Stano, Pasquale; Leboffe, Loris; Federico, Rodolfo; Antonini, Giovanni; Mariottini, Paolo; Polticelli, Fabio

    2014-08-01

    SMO (spermine oxidase) and APAO (acetylpolyamine oxidase) are flavoenzymes that play a critical role in the catabolism of polyamines. Polyamines are basic regulators of cell growth and proliferation and their homoeostasis is crucial for cell life since dysregulation of polyamine metabolism has been linked with cancer. In vertebrates SMO specifically catalyses the oxidation of spermine, whereas APAO displays a wider specificity, being able to oxidize both N¹-acetylspermine and N¹-acetylspermidine, but not spermine. The molecular bases of the different substrate specificity of these two enzymes have remained so far elusive. However, previous molecular modelling, site-directed mutagenesis and biochemical characterization studies of the SMO enzyme-substrate complex have identified Glu²¹⁶-Ser²¹⁸ as a putative active site hot spot responsible for SMO substrate specificity. On the basis of these analyses, the SMO double mutants E216L/S218A and E216T/S218A have been produced and characterized by CD spectroscopy and steady-state and rapid kinetics experiments. The results obtained demonstrate that mutation E216L/S218A endows SMO with N¹-acetylspermine oxidase activity, uncovering one of the structural determinants that confer the exquisite and exclusive substrate specificity of SMO for spermine. These results provide the theoretical bases for the design of specific inhibitors either for SMO or APAO. PMID:24854736

  18. Listeriolysin O suppresses Phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection

    PubMed Central

    Lam, Grace Y.; Fattouh, Ramzi; Muise, Aleixo M.; Grinstein, Sergio; Higgins, Darren E.; Brumell, John H.

    2012-01-01

    Summary The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L. monocytogenes infection, a response that would adversely affect pathogen survival. However, secretion of LLO inhibits the NADPH oxidase by preventing its localization to phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O, a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit the NADPH oxidase. Our studies demonstrate that while the PLCs induce antimicrobial NADPH oxidase activity, this effect is alleviated by the pore-forming activity of LLO. Therefore, the combined activities of PLCs and LLO on membrane lysis and the inhibitory effects of LLO on NADPH oxidase activity allows L. monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst. PMID:22177565

  19. INVESTIGATIONS OF AMITRAZ NEUROTOXICITY IN RATS. III. EFFECTS ON MOTOR ACTIVITY AND INHIBITION OF MONOAMINE OXIDASE

    EPA Science Inventory

    The formamidine pesticide amitraz (AMZ) produces many behavioral and physiological changes in rats. o explore possible neurochemical mechanisms for the behavioral effects of AMZ, we examined the dose effect and time course of AMZ on motor activity, monoamine oxidase (MAO) and ace...

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

    SciTech Connect

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

    2010-10-08

    Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

  1. The Dysregulation of Polyamine Metabolism in Colorectal Cancer Is Associated with Overexpression of c-Myc and C/EBPβ rather than Enterotoxigenic Bacteroides fragilis Infection

    PubMed Central

    Snezhkina, Anastasiya V.; Lipatova, Anastasiya V.; Sadritdinova, Asiya F.; Kardymon, Olga L.; Fedorova, Maria S.; Kaprin, Andrey D.

    2016-01-01

    Colorectal cancer is one of the most common cancers in the world. It is well known that the chronic inflammation can promote the progression of colorectal cancer (CRC). Recently, a number of studies revealed a potential association between colorectal inflammation, cancer progression, and infection caused by enterotoxigenic Bacteroides fragilis (ETBF). Bacterial enterotoxin activates spermine oxidase (SMO), which produces spermidine and H2O2 as byproducts of polyamine catabolism, which, in turn, enhances inflammation and tissue injury. Using qPCR analysis, we estimated the expression of SMOX gene and ETBF colonization in CRC patients. We found no statistically significant associations between them. Then we selected genes involved in polyamine metabolism, metabolic reprogramming, and inflammation regulation and estimated their expression in CRC. We observed overexpression of SMOX, ODC1, SRM, SMS, MTAP, c-Myc, C/EBPβ (CREBP), and other genes. We found that two mediators of metabolic reprogramming, inflammation, and cell proliferation c-Myc and C/EBPβ may serve as regulators of polyamine metabolism genes (SMOX, AZIN1, MTAP, SRM, ODC1, AMD1, and AGMAT) as they are overexpressed in tumors, have binding site according to ENCODE ChIP-Seq data, and demonstrate strong coexpression with their targets. Thus, increased polyamine metabolism in CRC could be driven by c-Myc and C/EBPβ rather than ETBF infection. PMID:27433286

  2. Evolution of histamine oxidase activity for biotechnological applications.

    PubMed

    Rosini, Elena; Tonin, Fabio; Vasylieva, Natalia; Marinesco, Stephane; Pollegioni, Loredano

    2014-01-01

    Histamine is present to various degrees in many foods, and concentrations in fish samples are considered a good indicator of freshness and hygienic food quality. Seeking for innovative methods to quantify histamine in foods, we used a synthetic gene designed on the sequence of histamine oxidase from Arthrobacter crystallopoietes (HOD) as the starting point in this study to develop a biosensor. HOD was expressed in Escherichia coli cells with a yield of ∼7 mg protein/L of fermentation broth. Recombinant wild-type HOD oxidized histamine and tyramine whereas it was inactive toward putrescine and cadaverine (two amines present in fish samples). The putative residues involved in substrate binding were identified by an in silico docking procedure based on a model of the structure of HOD: site-saturation mutagenesis was performed on 8 positions. The most significant changes in kinetic properties were observed for the P143M HOD: this variant showed higher histamine affinity and lower substrate inhibition by tyramine than wild-type enzyme. Biosensor prototypes were produced using both the wild-type and the P143M variant HOD. These biosensors showed a good sensitivity and selectivity with respect to biogenic amines present in food specimens. Accordingly, the HOD-based biosensor was successfully used to assess histamine in fish samples, yielding values in good agreement with those obtained by HPLC analyses but in a few seconds and at a significantly lower cost per analysis. PMID:23995223

  3. Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

    PubMed Central

    Berthier, Sylvie; Hograindleur, Marc-André; Paclet, Marie-Hélène; Polack, Benoît; Morel, Françoise

    2012-01-01

    S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b558; and (iii) to determine the S100A8 consensus site involved in cytochrome b558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase activation

  4. Semicarbazide-sensitive amine oxidase activation promotes adipose conversion of 3T3-L1 cells.

    PubMed Central

    Mercier, N; Moldes, M; El Hadri, K; Fève, B

    2001-01-01

    Semicarbazide-sensitive amine oxidase (SSAO) is an amine oxidase related to the copper-containing amine oxidase family. The tissular form of SSAO is located at the plasma membrane, and is mainly expressed in vascular smooth muscle cells and adipocytes. Recent studies have suggested that SSAO could activate glucose transport in fat cells. In the present work, we investigated the potential role of a chronic SSAO activation on adipocyte maturation of the 3T3-L1 pre-adipose cell line. Exposure of post-confluent 3T3-L1 pre-adipocytes to methylamine, a physiological substrate of SSAO, promoted adipocyte differentiation in a time- and dose-dependent manner. This effect could be related to SSAO activation, since it was antagonized in the presence of the SSAO inhibitor semicarbazide, but not in the presence of the monoamine oxidase inhibitor pargyline. In addition, methylamine-induced adipocyte maturation was mimicked by 3T3-L1 cell treatment with other SSAO substrates. Finally, the large reversion of methylamine action by catalase indicated that hydrogen peroxide generated by SSAO was involved, at least in part, in the modulation of adipocyte maturation. Taken together, our results suggest that SSAO may contribute to the control of adipose tissue development. PMID:11513731

  5. Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

    PubMed

    Gerdemann, C; Eicken, C; Magrini, A; Meyer, H E; Rompel, A; Spener, F; Krebs, B

    2001-07-01

    The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed. PMID:11451442

  6. FcγR-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

    PubMed Central

    Tian, Wei; Li, Xing Jun; Stull, Natalie D.; Ming, Wenyu; Suh, Chang-Il; Bissonnette, Sarah A.; Yaffe, Michael B.; Grinstein, Sergio; Atkinson, Simon J.

    2008-01-01

    The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after phagosome internalization. We conclude that p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization. PMID:18711001

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

    NASA Technical Reports Server (NTRS)

    Morre, D. James; Morre, Dorothy M.; Ternes, Philipp

    2003-01-01

    The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.

  8. Activity of DL-alpha-Difluoromethylarginine and Polyamine Analogues against Cryptosporidium parvum Infection in a T-Cell Receptor Alpha-Deficient Mouse Model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The in vivo effectiveness of a series of conformationally restricted polyamine analogs alone and in combination with DL-alpha-difluoromethylarginine (DFMA) towards a T-cell receptor-alpha deficient mouse model infection of Cryptosporidium parvum was tested. Polyamine analogues were constructed from ...

  9. Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice

    PubMed Central

    Nagasu, Hajime; Satoh, Minoru; Kiyokage, Emi; Kidokoro, Kengo; Toida, Kazunori; Channon, Keith M; Kanwar, Yashpal S; Sasaki, Tamaki; Kashihara, Naoki

    2016-01-01

    Increased generation of reactive oxygen species (ROS) is a common denominative pathogenic mechanism underlying vascular and renal complications in diabetes mellitus. Endothelial NAD(P)H oxidase is a major source of vascular ROS, and it has an important role in endothelial dysfunction. We hypothesized that activation of endothelial NAD(P)H oxidase initiates and worsens the progression of diabetic nephropathy, particularly in the development of albuminuria. We used transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NAD(P)H oxidase, Nox2 (NOX2TG). NOX2TG mice were crossed with Akita insulin-dependent diabetic (Akita) mice that develop progressive hyperglycemia. We compared the progression of diabetic nephropathy in Akita versus NOX2TG-Akita mice. NOX2TG-Akita mice and Akita mice developed significant albuminuria above the baseline at 6 and 10 weeks of age, respectively. Compared with Akita mice, NOX2TG-Akita mice exhibited higher levels of NAD(P)H oxidase activity in glomeruli, developed glomerular endothelial perturbations, and attenuated expression of glomerular glycocalyx. Moreover, in contrast to Akita mice, the NOX2TG-Akita mice had numerous endothelial microparticles (blebs), as detected by scanning electron microscopy, and increased glomerular permeability. Furthermore, NOX2TG-Akita mice exhibited distinct phenotypic changes in glomerular mesangial cells expressing α-smooth muscle actin, and in podocytes expressing increased levels of desmin, whereas the glomeruli generated increased levels of ROS. In conclusion, activation of endothelial NAD(P)H oxidase in the presence of hyperglycemia initiated and exacerbated diabetic nephropathy characterized by the development of albuminuria. Moreover, ROS generated in the endothelium compounded glomerular dysfunctions by altering the phenotypes of mesangial cells and compromising the integrity of the podocytes. PMID:26552047

  10. Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility

    PubMed Central

    Smith, Madison A.; Gonzalez, Jesica; Hussain, Anjum; Oldfield, Rachel N.; Johnston, Kathryn A.; Lopez, Karlo M.

    2016-01-01

    Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM−1 cm−1. No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture. PMID:26942005

  11. Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility.

    PubMed

    Smith, Madison A; Gonzalez, Jesica; Hussain, Anjum; Oldfield, Rachel N; Johnston, Kathryn A; Lopez, Karlo M

    2016-01-01

    Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM(-1 )cm(-1). No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture. PMID:26942005

  12. Semicarbazide-sensitive amine oxidase activity of guinea pig dorsal skin.

    PubMed

    Buffoni, F; Cambi, S; Banchelli, G; Ignesti, G; Pirisino, R; Raimondi, L

    1994-01-01

    A semicarbazide-sensitive amine oxidase activity with a high affinity for benzylamine (Bz.SSAO) (E.C. 1.4.3.6) is present in guinea pig dorsal skin. This enzymic activity oxidized benzylamine, histamine, 1,4-methylhistamine and acetylputrescine and was inhibited by semicarbazide and by B24 (3,5-diethoxy-4-aminomethylpyridine), a selective inhibitor of Bz.SSAO enzymes. It cross reacted with the antibodies raised against pure pig plasma benzylamine oxidase. Immunohistochemistry showed that it was localized in fibroblasts. Bz.SSAO activity of guinea pig dorsal skin increased during the process of skin healing. A treatment of the wounds with 3 micrograms of b-FGF significantly accelerated the process of skin healing and the increase of Bz.SSAO activity. PMID:7931260

  13. Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells

    PubMed Central

    Kim, Da Jung; Kim, Yong Sik

    2015-01-01

    Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells. PMID:26221064

  14. An artificial di-iron oxo-protein with phenol oxidase activity

    PubMed Central

    Faiella, Marina; Andreozzi, Concetta; de Rosales, Rafael Torres Martin; Pavone, Vincenzo; Maglio, Ornella; Nastri, Flavia; DeGrado, William F; Lombardi, Angela

    2013-01-01

    Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site. PMID:19915535

  15. Polyamine metabolism in flax in response to treatment with pathogenic and non–pathogenic Fusarium strains

    PubMed Central

    Wojtasik, Wioleta; Kulma, Anna; Namysł, Katarzyna; Preisner, Marta; Szopa, Jan

    2015-01-01

    Flax crop yield is limited by various environmental stress factors, but the largest crop losses worldwide are caused by Fusarium infection. Polyamines are one of the many plant metabolites possibly involved in the plant response to infection. However, in flax plants the polyamine composition, genes involved in polyamine synthesis, and in particular their regulation, were previously unknown. The aim of this study was to investigate the polyamine synthesis pathway in flax and its involvement in response to pathogen infection. It is well established that polyamines are essential for the growth and development of both plants and fungi, but their role in pathogen infection still remains unknown. In our study we correlated the expression of genes involved in polyamine metabolism with the polyamine levels in plant tissues and compared the results for flax seedlings treated with two pathogenic and one non-pathogenic strains of Fusarium. We observed an increase in the expression of genes participating in polyamine synthesis after fungal infection, and it was reflected in an increase of polyamine content in the plant tissues. The highest level of mRNA was characteristic for ornithine decarboxylase during infection with all tested, pathogenic and non-pathogenic, Fusarium strains and the arginine decarboxylase gene during infection with the pathogenic strain of Fusarium culmorum. The main polyamine identified in the flax seedlings was putrescine, and its level changed the most during infection. Moreover, the considerable increase in the contents of cell wall-bound polyamines compared to the levels of free and conjugated polyamines may indicate that their main role during pathogen infection lies in strengthening of the cell wall. In vitro experiments showed that the polyamines inhibit Fusarium growth, which suggests that they play an important role in plant defense mechanisms. Furthermore, changes in metabolism and content of polyamines indicate different defense mechanisms

  16. Modulation of NADPH oxidase activation in cerebral ischemia/reperfusion injury in rats.

    PubMed

    Genovese, Tiziana; Mazzon, Emanuela; Paterniti, Irene; Esposito, Emanuela; Bramanti, Placido; Cuzzocrea, Salvatore

    2011-02-01

    NADPH oxidase is a major complex that produces reactive oxygen species (ROSs) during the ischemic period and aggravates brain damage and cell death after ischemic injury. Although many approaches have been tested for preventing production of ROSs by NADPH oxidase in ischemic brain injury, the regulatory mechanisms of NADPH oxidase activity after cerebral ischemia are still unclear. The aim of this study is identifying apocynin as a critical modulator of NADPH oxidase and elucidating its role as a neuroprotectant in an experimental model of brain ischemia in rat. Treatment of apocynin 5min before of reperfusion attenuated cerebral ischemia in rats. Administration of apocynin showed marked reduction in infarct size compared with that of control rats. Medial carotid artery occlusion (MCAo)-induced cerebral ischemia was also associated with an increase in, nitrotyrosine formation, as well as IL-1β expression, IκB degradation and ICAM expression in ischemic regions. These expressions were markedly inhibited by the treatment of apocynin. We also demonstrated that apocynin reduces levels of apoptosis (TUNEL, Bax and Bcl-2 expression) resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. This new understanding of apocynin induced adaptation to ischemic stress and inflammation could suggest novel avenues for clinical intervention during ischemic and inflammatory diseases. PMID:21138737

  17. Characteristics of cellular polyamine transport in prokaryotes and eukaryotes.

    PubMed

    Igarashi, Kazuei; Kashiwagi, Keiko

    2010-07-01

    Polyamine content in cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, there are two polyamine uptake systems, namely spermidine-preferential (PotABCD) and putrescine-specific (PotFGHI), which belong to the family of ATP binding cassette transporters. Putrescine-ornithine and cadaverine-lysine antiporters, PotE and CadB, each consisting of 12 transmembrane segments, are important for cell growth at acidic pH. Spermidine excretion protein (MdtJI) was also recently identified. When putrescine was used as energy source, PuuP functioned as a putrescine transporter. In Saccharomyces cerevisiae, there are four kinds of polyamine uptake proteins (DUR3, SAM3, GAP1 and AGP2), consisting of either 12 or 16 transmembrane segments. Among them, DUR3 and SAM3 mostly contribute to polyamine uptake. There are also five kinds of polyamine excretion proteins (TPO1-5), consisting of 12 transmembrane segments. Among them, TPO1 and TPO5 are the most active proteins. Since a polyamine metabolizing enzyme, spermidine/spermine N(1)-acetyltransferase, is not present in yeast, five kinds of excretion proteins may exist. The current status of polyamine transport in mammalian and plant cells are reviewed. PMID:20159658

  18. Activity of carbohydrate oxidases as influenced by wheat flour dough components.

    PubMed

    Degrand, L; Rakotozafy, L; Nicolas, J

    2015-08-15

    The carbohydrate oxidase (COXMn) from Microdochium nivale may well have desired functionalities as a dough and bread improver, similarly to Aspergillus niger glucose oxidase (GOX). COXMn catalyses the oxidation of various monosaccharides as well as maltooligosaccharides for which the best activity is obtained towards the maltooligosaccharides of polymerisation degrees 3 and 4. For the same activity towards glucose under air saturation, we show that COXMn exhibits a similar efficiency towards maltose as GOX towards glucose whatever the oxygen supply. Assays with COXMn show that no competition exists between carbohydrates naturally present in the wheat flour. We show that reaction products (d-glucono-δ-lactone and hydrogen peroxide) and the wheat flour dough component, ferulic acid, have no noticeable specific effect on the COXMn activity. The demonstrated differences in kinetics between COXMn and GOX allow predicting of differences in the functional behaviours of those enzymes during wheat flour dough formation. PMID:25794758

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

    PubMed Central

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

    2013-01-01

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

  20. Chemical Evidence for Potent Xanthine Oxidase Inhibitory Activity of Ethyl Acetate Extract of Citrus aurantium L. Dried Immature Fruits.

    PubMed

    Liu, Kun; Wang, Wei; Guo, Bing-Hua; Gao, Hua; Liu, Yang; Liu, Xiao-Hong; Yao, Hui-Li; Cheng, Kun

    2016-01-01

    Xanthine oxidase is a key enzyme which can catalyze hypoxanthine and xanthine to uric acid causing hyperuricemia in humans. Xanthine oxidase inhibitory activities of 24 organic extracts of four species belonging to Citrus genus of the family Rutaceae were assayed in vitro. Since the ethyl acetate extract of C. aurantium dried immature fruits showed the highest xanthine oxidase inhibitory activity, chemical evidence for the potent inhibitory activity was clarified on the basis of structure identification of the active constituents. Five flavanones and two polymethoxyflavones were isolated and evaluated for inhibitory activity against xanthine oxidase in vitro. Of the compounds, hesperetin showed more potent inhibitory activity with an IC50 value of 16.48 μM. For the first time, this study provides a rational basis for the use of C. aurantium dried immature fruits against hyperuricemia. PMID:26950105

  1. Reduced cytochrome oxidase activity in the retrosplenial cortex after lesions to the anterior thalamic nuclei.

    PubMed

    Mendez-Lopez, Magdalena; Arias, Jorge L; Bontempi, Bruno; Wolff, Mathieu

    2013-08-01

    The anterior thalamic nuclei (ATN) make a critical contribution to hippocampal system functions. Growing experimental work shows that the effects of ATN lesions often resemble those of hippocampal lesions and both markedly reduce the expression of immediate-early gene markers in the retrosplenial cortex, which still appears normal by standard histological means. This study shows that moderate ATN damage was sufficient to produce severe spatial memory impairment as measured in a radial-arm maze. Furthermore, ATN rats exhibited reduced cytochrome oxidase activity in the most superficial cortical layers of the granular retrosplenial cortex, and, to a lesser extent, in the anterior cingulate cortex. By contrast, no change in cytochrome oxidase activity was observed in other limbic cortical regions or in the hippocampal formation. Altogether our results indicate that endogenous long-term brain metabolic capacity within the granular retrosplenial cortex is compromised by even limited ATN damage. PMID:23660649

  2. Coumarins with monoamine oxidase inhibitory activity and antioxidative coumarino-lignans from Hibiscus syriacus.

    PubMed

    Yun, B S; Lee, I K; Ryoo, I J; Yoo, I D

    2001-09-01

    A previously undescribed coumarin and a new coumarino-lignan, together with the known compounds scopoletin and cleomiscosins A, C, and D, have been isolated from the root bark of Hibiscus syriacus, and their structures were assigned on the basis of various spectral studies. The coumarin analogue and scopoletin inhibited monoamine oxidase with moderate IC(50) values. The new coumarino-lignan and cleomiscosin C showed lipid peroxidation inhibitory activity comparable to vitamin E. PMID:11575966

  3. Acetaldehyde-induced cytotoxicity involves induction of spermine oxidase at the transcriptional level.

    PubMed

    Uemura, Takeshi; Tanaka, Yuka; Higashi, Kyohei; Miyamori, Daisuke; Takasaka, Tomokazu; Nagano, Tatsuo; Toida, Toshihiko; Yoshimoto, Kanji; Igarashi, Kazuei; Ikegaya, Hiroshi

    2013-08-01

    Ethanol consumption causes serious liver injury including cirrhosis and hepatocellular carcinoma. Ethanol is metabolized mainly in the liver to acetic acid through acetaldehyde. We investigated the effect of ethanol and acetaldehyde on polyamine metabolism since polyamines are essential factors for normal cellular functions. We found that acetaldehyde induced spermine oxidase (SMO) at the transcriptional level in HepG2 cells. The levels and activities of ornithine decarboxylase (ODC) and spermidine/spermine acetyltransferase (SSAT) were not affected by acetaldehyde. Spermidine content was increased and spermine content was decreased by acetaldehyde treatment. Knockdown of SMO expression using siRNA reduced acetaldehyde toxicity. Acetaldehyde exposure increased free acrolein levels. An increase of acrolein by acetaldehyde was SMO dependent. Our results indicate that cytotoxicity of acetaldehyde involves, at least in part, oxidation of spermine to spermidine by SMO, which is induced by acetaldehyde. PMID:23707493

  4. Assessing gibberellins oxidase activity by anion exchange/hydrophobic polymer monolithic capillary liquid chromatography-mass spectrometry.

    PubMed

    Chen, Ming-Luan; Su, Xin; Xiong, Wei; Liu, Jiu-Feng; Wu, Yan; Feng, Yu-Qi; Yuan, Bi-Feng

    2013-01-01

    Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography--mass spectrometry (cLC-MS) method for the sensitive assay of in-vitro recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA1, GA4, GA9, GA20). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-co-divinylbenzene-co-ethylene glycol dimethacrylate)(META-co-DVB-co-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62-0.90 fmol. We determined the kinetic parameters (K m) of recombinant GA3-oxidase in Escherichia coli (E. coli) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA9 to GA4 in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology. PMID:23922762

  5. Assessing Gibberellins Oxidase Activity by Anion Exchange/Hydrophobic Polymer Monolithic Capillary Liquid Chromatography-Mass Spectrometry

    PubMed Central

    Liu, Jiu-Feng; Wu, Yan; Feng, Yu-Qi; Yuan, Bi-Feng

    2013-01-01

    Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography – mass spectrometry (cLC-MS) method for the sensitive assay of in-vitro recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA1, GA4, GA9, GA20). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-co-divinylbenzene-co-ethylene glycol dimethacrylate)(META-co-DVB-co-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62–0.90 fmol. We determined the kinetic parameters (Km) of recombinant GA3-oxidase in Escherichia coli (E. coli) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA9 to GA4 in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology. PMID:23922762

  6. Propofol Attenuates Small Intestinal Ischemia Reperfusion Injury through Inhibiting NADPH Oxidase Mediated Mast Cell Activation

    PubMed Central

    Gan, Xiaoliang; Xing, Dandan; Su, Guangjie; Li, Shun; Luo, Chenfang; Irwin, Michael G.; Xia, Zhengyuan; Li, Haobo; Hei, Ziqing

    2015-01-01

    Both oxidative stress and mast cell (MC) degranulation participate in the process of small intestinal ischemia reperfusion (IIR) injury, and oxidative stress induces MC degranulation. Propofol, an anesthetic with antioxidant property, can attenuate IIR injury. We postulated that propofol can protect against IIR injury by inhibiting oxidative stress subsequent from NADPH oxidase mediated MC activation. Cultured RBL-2H3 cells were pretreated with antioxidant N-acetylcysteine (NAC) or propofol and subjected to hydrogen peroxide (H2O2) stimulation without or with MC degranulator compound 48/80 (CP). H2O2 significantly increased cells degranulation, which was abolished by NAC or propofol. MC degranulation by CP further aggravated H2O2 induced cell degranulation of small intestinal epithelial cell, IEC-6 cells, stimulated by tryptase. Rats subjected to IIR showed significant increases in cellular injury and elevations of NADPH oxidase subunits p47phox and gp91phox protein expression, increases of the specific lipid peroxidation product 15-F2t-Isoprostane and interleukin-6, and reductions in superoxide dismutase activity with concomitant enhancements in tryptase and β-hexosaminidase. MC degranulation by CP further aggravated IIR injury. And all these changes were attenuated by NAC or propofol pretreatment, which also abrogated CP-mediated exacerbation of IIR injury. It is concluded that pretreatment of propofol confers protection against IIR injury by suppressing NADPH oxidase mediated MC activation. PMID:26246867

  7. Determination of diamine oxidase in lentil seedlings by enzymic activity and immunoreactivity.

    PubMed

    Federico, R; Angelini, R; Cesta, A; Pini, C

    1985-09-01

    A competitive radioimmunoassay for the quantitation of diamine oxidase (EC 1.4.3.6) from Lens culinaris is reported. Specific antibodies raised in rabbits immunized with a homogeneous preparation of the enzyme were incubated with purified (125)I-enzyme and with either unlabeled diamine oxidase or plant material. Antigen-antibody complexes were isolated from the mixture by incubation with Staphylococcus protein A. The sensitivity of the test was about 5 nanograms in terms of enzyme protein. This assay was applied to the determination of the enzyme in extracts from lentil shoots grown either in the dark or in the light. Diamine oxidase activity and enzyme protein (as determined by radioimmunoassay) were measured during 7 days after germination. Both enzymic activity and enzyme protein declined slowly in the dark and rapidly in the light. These results indicate that fluctuation of the enzymic activity in this organ, both in the light and in the dark, are mediated via changes in the amount of the enzyme protein and not via the action of an inhibitor. PMID:16664402

  8. Effects of dietary carbohydrate on iron metabolism and cytochrome oxidase activity in copper-deficient rats

    SciTech Connect

    Johnson, M.A.; Henderson, J.

    1986-03-01

    The effects of dietary carbohydrate on the metabolism of iron and the activity of cytochrome oxidase were examined in Cu-deficient and Cu-adequate rats. Male rats (n = 36) were fed one of six diets which varied in copper level (Cu-: < 0.6 ppm or Cu+: 8.2 ppm) and carbohydrate type (cornstarch, sucrose or fructose). After 31 days, Cu- rats had 50% more iron in the liver and 38, 30 and 18% less iron in the tibia, spleen and kidneys, respectively, than Cu+ rats. The activity of cytochrome oxidase in the bone marrow, heart, and liver were 59%, 51%, and 43%, respectively, of the levels in Cu/sup +/ rats. The type of dietary carbohydrate significantly affected the development of anemia during copper deficiency. Cu-rats fed cornstarch, sucrose or fructose had hematocrit levels which were 92, 83 or 73%, respectively, of Cu+ rats. Similarly, the levels of iron in the tibias of Cu- rats fed cornstarch, sucrose or fructose were 69, 66 or 54%, respectively, of Cu+ rats. The hematocrit levels of Cu- rats were positively correlated to both tibia iron levels (r = 0.64, p < 0.005) and liver cytochrome oxidase activities (r = 0.50, p < 0.05). Thus, it appears that changes in the metabolism of iron may be involved with the development of anemia in Cu- rats fed fructose or sucrose.

  9. Autophagy Protein Rubicon Mediates Phagocytic NADPH Oxidase Activation in Response to Microbial Infection or TLR Stimulation

    PubMed Central

    Yang, Chul-Su; Lee, Jong-Soo; Rodgers, Mary; Min, Chan-Ki; Lee, June-Yong; Kim, Hee Jin; Lee, Kwang-Hoon; Kim, Chul-Joong; Oh, Byungha; Zandi, Ebrahim; Yue, Zhenyu; Kramnik, Igor; Liang, Chengyu; Jung, Jae U.

    2013-01-01

    Summary Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicon's actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection. PMID:22423966

  10. Enhanced hydrolysis of soluble cellulosic substrates by a metallocellulase with veratryl alcohol-oxidase activity

    SciTech Connect

    Evans, B.R.; Margalt, R.; Woodward, J.

    1995-12-31

    A cellulose enzyme fraction was separated from Trichoderma reesei Pulpzyme HA{trademark}, and its characteristics suggested that it was mainly composed of cellobiohydrolase II (CBH II). The covalent attachment of pentaammineruthenium (III) to this enzyme resulted in threefold and fourfold enhancements of its hydrolytic activity on carboxymethyl cellulose (CMC) and barley {beta}-glucan, respectively, as well as endowing it with veratryl alcohol-oxidase activity. Enhancement of hydrolysis was not affected by addition of tartrate or hydrogen peroxide to the reaction mixture. Both native and pentaammineruthenium modified enzymes had negligible activity on cellobiose and p-nitrophenyl {beta}-cellobioside (PNPC).

  11. NADH Oxidase Activity of Indoleamine 2,3-Dioxygenase*

    PubMed Central

    Rosell, Federico I.; Kuo, Hsin H.; Mauk, A. Grant

    2011-01-01

    The heme enzyme indoleamine 2,3-dioxygenase (IDO) was found to oxidize NADH under aerobic conditions in the absence of other enzymes or reactants. This reaction led to the formation of the dioxygen adduct of IDO and supported the oxidation of Trp to N-formylkynurenine. Formation of the dioxygen adduct and oxidation of Trp were accelerated by the addition of small amounts of hydrogen peroxide, and both processes were inhibited in the presence of either superoxide dismutase or catalase. Anaerobic reaction of IDO with NADH proceeded only in the presence of a mediator (e.g. methylene blue) and resulted in formation of the ferrous form of the enzyme. We propose that trace amounts of peroxide previously proposed to occur in NADH solutions as well as solid NADH activate IDO and lead to aerobic formation of superoxide and the reactive dioxygen adduct of the enzyme. PMID:21690092

  12. Involvement of protein kinase D in Fc gamma-receptor activation of the NADPH oxidase in neutrophils.

    PubMed Central

    Davidson-Moncada, Jan K; Lopez-Lluch, Guillermo; Segal, Anthony W; Dekker, Lodewijk V

    2002-01-01

    Protein kinases involved in the activation of the NADPH oxidase by Fc gamma receptors in neutrophils were studied. Of three different protein kinase C (PKC) inhibitors, Gö 6976 inhibited the NADPH oxidase completely, whereas bisindolylmaleimide I and Ro 31-8220 caused a 70-80% inhibition. Thus a Gö 6976-sensitive, bisindolylmaleimide I/Ro 31-8220-insensitive component contributes to NADPH oxidase activation induced by Fc gamma receptors. Down-regulation of PKC isotypes resulted in inhibition of Fc gamma-receptor-activated NADPH oxidase, but a down-regulation-insensitive component was still present. This component was sensitive to Gö 6976, but insensitive to Ro 31-8220. It has been shown previously that protein kinase D/PKC-mu (PKD) shows this same pharmacology in vitro. We show that PKD is present in neutrophils and that, in contrast with PKC isotypes, PKD is not down-regulated. Therefore PKD may participate in NADPH oxidase activation. To obtain direct evidence for this we adopted an antisense approach. Antisense PKD inhibited NADPH oxidase induced by Fc gamma-receptor stimulation by 50% and the Ro 31-8220-insensitive component in the activation was inhibited by antisense PKD. In vitro kinase assays showed that PKD is activated by presenting IgG-opsonized particles to neutrophils. Furthermore, PKD localizes to the area of particle intake in the cell and phosphorylates two of the three cytosolic components of the NADPH oxidase, p40(phox) and p47(phox). Taken together, these data indicate that Fc gamma receptors engage PKD in the regulation of the NADPH oxidase. PMID:11903052

  13. Legionella pneumophila requires polyamines for optimal intracellular growth.

    PubMed

    Nasrallah, Gheyath K; Riveroll, Angela L; Chong, Audrey; Murray, Lois E; Lewis, P Jeffrey; Garduño, Rafael A

    2011-09-01

    The Gram-negative intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophila expressing a translocation reporter consisting of the Bordetella pertussisa denylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2 induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophila replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophila proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila. PMID:21742865

  14. Effect of architecture on the activity of glucose oxidase/horseradish peroxidase/carbon nanoparticle conjugates.

    PubMed

    Ciaurriz, Paula; Bravo, Ernesto; Hamad-Schifferli, Kimberly

    2014-01-15

    We investigate the activity of glucose oxidase (GOx) together with horseradish peroxidase (HRP) on carbon nanoparticles (CNPs). Because GOx activity relies on HRP, we probe how the arrangement of the enzymes on the CNPs affects enzymatic behavior. Colorimetric assays to probe activity found that the coupling strategy affects activity of the bienzyme-nanoparticle complex. GOx is more prone than HRP to denaturation on the CNP surface, where its activity is compromised, while HRP activity is enhanced when interfaced to the CNP. Thus, arrangements where HRP is directly on the surface of the CNP and GOx is not are more favorable for overall activity. Coverage also influenced activity of the bienzyme complex, but performing the conjugation in the presence of glucose did not improve GOx activity. These results show that the architecture of the assembly is an important factor in optimization of nanoparticle-protein interfaces. PMID:24231087

  15. Regulation of cytochrome c oxidase activity by c-Src in osteoclasts

    PubMed Central

    Miyazaki, Tsuyoshi; Neff, Lynn; Tanaka, Sakae; Horne, William C.; Baron, Roland

    2003-01-01

    The function of the nonreceptor tyrosine kinase c-Src as a plasma membrane–associated molecular effector of a variety of extracellular stimuli is well known. Here, we show that c-Src is also present within mitochondria, where it phosphorylates cytochrome c oxidase (Cox). Deleting the c-src gene reduces Cox activity, and this inhibitory effect is restored by expressing exogenous c-Src. Furthermore, reducing endogenous Src kinase activity down-regulates Cox activity, whereas activating Src has the opposite effect. Src-induced Cox activity is required for normal function of cells that require high levels of ATP, such as mitochondria-rich osteoclasts. The peptide hormone calcitonin, which inhibits osteoclast function, also down-regulates Cox activity. Increasing Src kinase activity prevented the inhibitory effect of calcitonin on Cox activity and osteoclast function. These results suggest that c-Src plays a previously unrecognized role in maintaining cellular energy stores by activating Cox in mitochondria. PMID:12615910

  16. Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization

    PubMed Central

    Wang, Haibo; Jiang, Yanchao; Shi, Dallas; Quilliam, Lawrence A.; Chrzanowska-Wodnicka, Magdalena; Wittchen, Erika S.; Li, Dean Y.; Hartnett, M. Elizabeth

    2014-01-01

    Activation of Rap1 GTPase can improve the integrity of the barrier of the retina pigment epithelium (RPE) and reduce choroidal neovascularization (CNV). Inhibition of NADPH oxidase activation also reduces CNV. We hypothesize that Rap1 inhibits NADPH oxidase-generated ROS and thereby reduces CNV formation. Using a murine model of laser-induced CNV, we determined that reduced Rap1 activity in RPE/choroid occurred with CNV formation and that activation of Rap1 by 2′-O-Me-cAMP (8CPT)-reduced laser-induced CNV via inhibiting NADPH oxidase-generated ROS. In RPE, inhibition of Rap1 by Rap1 GTPase-activating protein (Rap1GAP) increased ROS generation, whereas activation of Rap1 by 8CPT reduced ROS by interfering with the assembly of NADPH oxidase membrane subunit p22phox with NOX4 or cytoplasmic subunit p47phox. Activation of NADPH oxidase with Rap1GAP reduced RPE barrier integrity via cadherin phosphorylation and facilitated choroidal EC migration across the RPE monolayer. Rap1GAP-induced ROS generation was inhibited by active Rap1a, but not Rap1b, and activation of Rap1a by 8CPT in Rap1b−/− mice reduced laser-induced CNV, in correlation with decreased ROS generation in RPE/choroid. These findings provide evidence that active Rap1 reduces CNV by interfering with the assembly of NADPH oxidase subunits and increasing the integrity of the RPE barrier.—Wang, H., Jiang, Y., Shi, D., Quilliam, L. A., Chrzanowska-Wodnicka, M., Wittchen, E. S., Li, D. Y., Hartnett, M. E. Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization. PMID:24043260

  17. Ascorbyl palmitate-loaded chitosan nanoparticles: characteristic and polyphenol oxidase inhibitory activity.

    PubMed

    Kim, Mi Kyung; Lee, Ji-Soo; Kim, Kwang Yup; Lee, Hyeon Gyu

    2013-03-01

    The aim of this study was to produce ascorbyl palmitate (AP)-loaded nanoparticles in order to inhibit polyphenol oxidase (PPO) in bananas. AP-loaded chitosan nanoparticles were prepared using acetic acid and citric acid (denoted as CS/AA and CS/CA nanoparticles, respectively). As the initial AP concentration increases, the particle size significantly decreases, and the zeta potential, entrapment and loading efficiency significantly increases. The PPO inhibitory activity of AP was effectively improved when AP was nano-encapsulated by chitosan compared to no encapsulation. These results suggest that chitosan nano-encapsulation can be used to enhance the PPO inhibitory activity of AP. PMID:23247266

  18. Structural basis of antizyme-mediated regulation of polyamine homeostasis

    PubMed Central

    Wu, Hsiang-Yi; Chen, Shin-Fu; Hsieh, Ju-Yi; Chou, Fang; Wang, Yu-Hsuan; Lin, Wan-Ting; Lee, Pei-Ying; Yu, Yu-Jen; Lin, Li-Ying; Lin, Te-Sheng; Lin, Chieh-Liang; Liu, Guang-Yaw; Tzeng, Shiou-Ru; Hung, Hui-Chih; Chan, Nei-Li

    2015-01-01

    Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN–Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation. PMID:26305948

  19. The xanthine oxidase activity in different of secondary transformed peat-moorsh soils

    NASA Astrophysics Data System (ADS)

    Styła, Katarzyna; Wojciech Szajdak, Lech

    2010-05-01

    The investigations were carried out on the transect of peatland 4.5 km long, located in the Agroecological Landscape Park host D. Chlapowski in Turew (40 km South-West of Poznań, West Polish Lowland). The sites investigation were located along Wyskoć ditch. The following material was taken from four chosen sites marked as Zbęchy, Bridge, Shelterbelt and Hirudo in two layers: acrotelm (0-50 cm) and catotelm (50-100 cm). The object of this study was to characterize the biochemical properties by the determination of the xanthine oxidase activity in two layers (acrotelm and catotelm) of the four different peat-moorsh soils used as meadow. The xanthine oxidase activity was determined spectrophotometrically by measuring uric acid formation at λmax=290 nm with xanthine as substrate. In peat-moorsh soil the highest activities of xanthine oxidasewas observed in the Shelterbelt and whereas the lowest - in Zbęchy, Bridge and Hirudo. Activities of this enzyme in peat-moorsh soil ranged from 5.96 to 19.51 μmol h-1g d.m soil. Increased activities of xanthine oxidase have been recorded on the depth 50-100 cm - catotelm (from 11.71 to 19.51 μmol h-1g d.m soil) in comparison with the depth 0-50 cm - acrotelm (from 5.96 to 14.64 μmol h-1g d.m soil). This work was supported by a grant No. N N305 3204 36 founded by Polish Ministry of Education.

  20. Regulation of cytochrome c- and quinol oxidases, and piezotolerance of their activities in the deep-sea piezophile Shewanella violacea DSS12 in response to growth conditions.

    PubMed

    Ohke, Yoshie; Sakoda, Ayaka; Kato, Chiaki; Sambongi, Yoshihiro; Kawamoto, Jun; Kurihara, Tatsuo; Tamegai, Hideyuki

    2013-01-01

    The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O2 concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O2 concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O2 concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments. PMID:23832349

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

    SciTech Connect

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

    1990-05-01

    Rabbits fed a regular laboratory diet supplemented with a high-fat milk containing xanthine oxidase (XO) were studied to determine the presence of active XO in the blood. A pilot feeding study, where rabbits consumed a high-fat diet containing xanthine oxidase, showed a correlation between dairy food consumption and XO activity in the blood. Antibody to dietary XO was also found. In a second study, rabbits were fed ad libitum the high-fat milk and blood serum samples were tested weekly for XO activity. No elevation in serum XO activity was found. A third study showed that serum XO activity was increased when rabbits were force fed the high-fat milk. The final study consisted of force feeding {sup 125}I-labeled XO to one rabbit to ascertain whether the observed increase in serum XO was due to dietary or endogenous XO. Isoelectric focusing of sera collected from the test rabbit strongly suggested that at least a portion of the serum XO contained the radioactive label. This is the first direct evidence showing the uptake of dietary active XO from the gut.

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

    SciTech Connect

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

    2009-04-15

    Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O{sub 2}{sup {center_dot}}{sup -} generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67{sup phox} siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91{sup phox} knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47{sup phox}, p67{sup phox} and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67{sup phox} siRNA. Exposure of MPMVEC obtained from gp91{sup phox} knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly

  3. Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental diabetes.

    PubMed

    Di Noia, Maria Antonietta; Van Driesche, Sarah; Palmieri, Ferdinando; Yang, Li-Ming; Quan, Shuo; Goodman, Alvin I; Abraham, Nader G

    2006-06-01

    Up-regulation of heme oxygenase (HO-1) by either cobalt protoporphyrin (CoPP) or human gene transfer improves vascular and renal function by several mechanisms, including increases in antioxidant levels and decreases in reactive oxygen species (ROS) in vascular and renal tissue. The purpose of the present study was to determine the effect of HO-1 overexpression on mitochondrial transporters, cytochrome c oxidase, and anti-apoptotic proteins in diabetic rats (streptozotocin, (STZ)-induced type 1 diabetes). Renal mitochondrial carnitine, deoxynucleotide, and ADP/ATP carriers were significantly reduced in diabetic compared with nondiabetic rats (p < 0.05). The citrate carrier was not significantly decreased in diabetic tissue. CoPP administration produced a robust increase in carnitine, citrate, deoxynucleotide, dicarboxylate, and ADP/ATP carriers and no significant change in oxoglutarate and aspartate/glutamate carriers. The increase in mitochondrial carriers (MCs) was associated with a significant increase in cytochrome c oxidase activity. The administration of tin mesoporphyrin (SnMP), an inhibitor of HO-1 activity, prevented the restoration of MCs in diabetic rats. Human HO-1 cDNA transfer into diabetic rats increased both HO-1 protein and activity, and restored mitochondrial ADP/ATP and deoxynucleotide carriers. The increase in HO-1 by CoPP administration was associated with a significant increase in the phosphorylation of AKT and levels of BcL-XL proteins. These observations in experimental diabetes suggest that the cytoprotective mechanism of HO-1 against oxidative stress involves an increase in the levels of MCs and anti-apoptotic proteins as well as in cytochrome c oxidase activity. PMID:16595661

  4. A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit.

    PubMed

    Garchery, Cécile; Gest, Noé; Do, Phuc T; Alhagdow, Moftah; Baldet, Pierre; Menard, Guillaume; Rothan, Christophe; Massot, Capucine; Gautier, Hélène; Aarrouf, Jawad; Fernie, Alisdair R; Stevens, Rebecca

    2013-01-01

    The regulation of carbon allocation between photosynthetic source leaves and sink tissues in response to stress is an important factor controlling plant yield. Ascorbate oxidase is an apoplastic enzyme, which controls the redox state of the apoplastic ascorbate pool. RNA interference was used to decrease ascorbate oxidase activity in tomato (Solanum lycopersicum L.). Fruit yield was increased in these lines under three conditions where assimilate became limiting for wild-type plants: when fruit trusses were left unpruned, when leaves were removed or when water supply was limited. Several alterations in the transgenic lines could contribute to the improved yield and favour transport of assimilate from leaves to fruits in the ascorbate oxidase lines. Ascorbate oxidase plants showed increases in stomatal conductance and leaf and fruit sugar content, as well as an altered apoplastic hexose:sucrose ratio. Modifications in gene expression, enzyme activity and the fruit metabolome were coherent with the notion of the ascorbate oxidase RNAi lines showing altered sink strength. Ascorbate oxidase may therefore be a target for strategies aimed at improving water productivity in crop species. PMID:22725103

  5. Cocaine reduces cytochrome oxidase activity in the prefrontal cortex and modifies its functional connectivity with brainstem nuclei

    PubMed Central

    Vélez-Hernández, M.E.; Padilla, E.; Gonzalez-Lima, F.; Jiménez-Rivera, C.A.

    2014-01-01

    Cocaine-induced psychomotor stimulation may be mediated by metabolic hypofrontality and modification of brain functional connectivity. Functional connectivity refers to the pattern of relationships among brain regions, and one way to evaluate this pattern is using interactivity correlations of the metabolic marker cytochrome oxidase among different regions. This is the first study of how repeated cocaine modifies: (1) mean cytochrome oxidase activity in neural areas using quantitative enzyme histochemistry, and (2) functional connectivity among brain regions using inter-correlations of cytochrome oxidase activity. Rats were injected with 15 mg/kg i.p. cocaine or saline for 5 days, which lead to cocaine-enhanced total locomotion. Mean cytochrome oxidase activity was significantly decreased in cocaine-treated animals in the superficial dorsal and lateral frontal cortical association areas Fr2 and Fr3 when compared to saline-treated animals. Functional connectivity showed that the cytochrome oxidase activity of the noradrenergic locus coeruleus and the infralimbic cortex were positively inter-correlated in cocaine but not in control rats. Positive cytochrome oxidase activity inter-correlations were also observed between the dopaminergic substantia nigra compacta and Fr2 and Fr3 areas and the lateral orbital cortex in cocaine-treated animals. In contrast, cytochrome oxidase activity in the interpeduncular nucleus was negatively correlated with that of Fr2, anterior insular cortex, and lateral orbital cortex in saline but not in cocaine groups. After repeated cocaine specific prefrontal areas became hypometabolic and their functional connectivity changed in networks involving noradrenergic and dopaminergic brainstem nuclei. We suggest that this pattern of hypofrontality and altered functional connectivity may contribute to cocaine-induced psychomotor stimulation. PMID:24505625

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

    PubMed

    McCarty, Mark F; DiNicolantonio, James

    2016-01-01

    Lysophosphatidic acid (LPA), generated in the microenvironment of cancer cells, can drive the proliferation, invasion, and migration of cancer cells by activating G protein-coupled LPA receptors. Moreover, in cancer cells that have metastasized to bone, LPA signaling can promote osteolysis by inducing cancer cell production of cytokines, such as IL-6 and IL-8, which can stimulate osteoblasts to secrete RANKL, a key promoter of osteoclastogenesis. Indeed, in cancers prone to metastasize to bone, LPA appears to be a major driver of the expansion of osteolytic bone metastases. Activation of NADPH oxidase has been shown to play a mediating role in the signaling pathways by which LPA, as well as RANKL, promote osteolysis. In addition, there is reason to suspect that Nox4 activation is a mediator of the feed-forward mechanism whereby release of TGF-beta from bone matrix by osteolysis promotes expression of PTHrP in cancer cells, and thereby induces further osteolysis. Hence, measures which can down-regulate NADPH oxidase activity may have potential for slowing the expansion of osteolytic bone metastases in cancer patients. Phycocyanin and high-dose statins may have utility in this regard, and could be contemplated as complements to bisphosphonates or denosumab for the prevention and control of osteolytic lesions. Ingestion of omega-3-rich flaxseed or fish oil may also have potential for controlling osteolysis in cancer patients. PMID:27571113

  7. Diamine oxidase plasma activities after treatment with heparin and jejunal morphometry in untreated coeliac disease.

    PubMed Central

    Corazza, G R; Ginaldi, L; Falasca, A; Strocchi, A; Rossi, C A; Quaglino, D; Gasbarrini, G

    1989-01-01

    Diamine oxidase plasma concentrations after treatment with heparin were measured and compared with the surface to volume ratio of jejunal biopsy samples assessed by a morphometric technique in patients with untreated and treated coeliac disease and in biopsied controls. As expected, enzyme activity was significantly lower in patients with untreated coeliac disease than in patients on a gluten-free diet and in biopsied controls. No difference was found between treated patients and biopsied controls. There was a significant overall correlation between plasma enzyme activity and surface to volume ratio of jejunal mucosa, although two untreated patients without an overt malabsorption syndrome but with a very low surface to volume ratio had normal enzyme activity. This study shows that in coeliac disease plasma diamine oxidase activity after treatment with heparin does not always mirror the extent of the jejunal lesions, particularly in those patients with minimal or unrelated symptoms who would benefit most from a valid screening test to identify their condition. PMID:2511229

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

    PubMed

    Pessatti, M; Fontana, J D; Furtado, M F; Guimãraes, M F; Zanette, L R; Costa, W T; Baron, M

    1995-01-01

    Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use of biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom. PMID:7668847

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

    SciTech Connect

    Pessati, M.L.; Fontana, J.D.; Guimaraes, M.F.

    1995-12-31

    Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom.

  10. Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

    SciTech Connect

    Doonan, C.J.; Wilson, H.L.; Rajagopalan, K.V.; Garrett, R.M.; Bennett, B.; Prince, R.C.; George, G.N.

    2009-06-02

    The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme sulfite oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.

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

    PubMed

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

    1987-11-01

    Eugenia uniflora is widely used in Paraguayan folk medicine. A hydroalcoholic extract of the leaves showed some central nervous system activity in hippocratic screening when given intraperitoneally, but little to no acute or subacute toxicity in doses up to 4200 mg/kg orally in BALB c mice. The LD50 of the extract was 220 mg/kg i.p. in mice. A decoction or infusion of the leaves is recommended for treating gout by native herbalists. The known flavonoids quercitrin, quercetin, myricitrin and myricetin were found to be responsible for the xanthine oxidase inhibitory action of the plant extract. PMID:3437769

  12. 4-Coumaroyl coenzyme A 3-hydroxylase activity from cell cultures of Lithospermum erythrorhizon and its relationship to polyphenol oxidase.

    PubMed

    Wang, Z X; Li, S M; Löscher, R; Heide, L

    1997-11-15

    A 4-coumaroyl-CoA 3-hydroxylase activity was purified 4600-fold from cell cultures of Lithospermum erythrorhizon. The enzyme showed a molecular mass of 42,400 +/- 1700 Da in gel chromatography and required ascorbate, NADH, or NADPH as cofactors. 4-Coumaroyl-CoA, 4-coumarate, p-cresol, and several other phenolic substances, but not tyrosine, were accepted as substrates for the hydroxylation. Besides hydroxylase activity, the enzyme showed diphenol oxidase activity. Both activities were inhibited by diethyldithiocarbamate or beta-mercaptoethanol, although at different concentrations. The enzyme showed striking similarity to a 4-coumaroyl-glucose 3-hydroxylase from sweet potato (Ipomoe batatas) roots, which has reportedly been purified to homogeneity and identified as a specific enzyme of chlorogenic acid biosynthesis. Close examination and comparison to a commercially available polyphenol oxidase, however, suggest that the enzyme activities purified from both Lithospermum and sweet potato are polyphenol oxidases rather than specific enzymes of secondary metabolism. PMID:9367532

  13. Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity

    PubMed Central

    Liu, Jian; Zhou, Jun; Xing, Da

    2012-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination. PMID:22448275

  14. NADPH oxidase activation is required for pentylenetetrazole kindling-induced hippocampal autophagy.

    PubMed

    Zhu, Xinjian; Shen, Kai; Bai, Ying; Zhang, Aifeng; Xia, Zhengrong; Chao, Jie; Yao, Honghong

    2016-05-01

    Growing evidence indicates that alterations in autophagy are present in a variety of neurological disorders, ranging from neurodegenerative diseases to acute neurological insults. Only recently has the role of autophagy in epilepsy started to be recognized. In this study, we used pentylenetetrazole (PTZ) kindling, which provides a model of chronic epilepsy, to investigate the involvement of autophagy in the hippocampus and the possible mechanisms involved. Our western blot results showed that autophagy-related proteins were significantly increased after the mice were fully kindled. In addition, immunofluorescence studies revealed a significant increase in the punctate accumulation of LC3 in the hippocampal CA1 region of fully PTZ-kindled mice. Consistent with the upregulation of ATG proteins and punctate accumulation of LC3 in the hippocampal CA1 region, autophagosomal vacuole formation was observed by an ultrastructural analysis, verifying the presence of a hippocampal autophagic response in PTZ-kindled mice. Increased oxidative stress has been postulated to play an important role in the pathogenesis of a number of neurological diseases, including epilepsy. In this study, we demonstrate that PTZ kindling induced reactive oxygen species (ROS) production and lipid peroxidation, which were accompanied by mitochondrial ultrastructural damage due to the activation of NADPH oxidase. Pharmacological inhibition of NADPH oxidase by apocynin significantly suppressed the oxidative stress and ameliorated the hippocampal autophagy in PTZ-kindled mice. Interestingly, pharmacological induction of autophagy suppressed PTZ-kindling progress and reduced PTZ-kindling-induced oxidative stress while inhibition of autophagy accelerated PTZ kindling progress and increased PTZ-kindling-induced oxidative stress. These results suggest that the oxidative stress induced by NADPH oxidase activation may play a pivotal role in PTZ-kindling process as well as in PTZ kindling-induced hippocampal

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

    PubMed

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

    2016-02-01

    Fasciolosis is an important cattle and human disease caused by Fasciola hepatica and Fasciola gigantica. One of the possible methods to control this problem is to interrupt the life cycle of Fasciola by killing its larva (redia and cercaria) in host snail. Molecular identification of cercaria larva of F. gigantica was done by comparing the nucleotide sequencing with adult F. gigantica. It was noted that nucleotide sequencing of cercaria larva and adult F. gigantica were 99% same. Every month during the year 2011-2012, in vivo treatment with 60% of 4 h LC50 of phyto cercaricides citral, ferulic acid, umbelliferone, azadirachtin and allicin caused significant inhibition of acetylcholinesterase (AChE) and cytochrome oxidase activity in the treated cercaria larva of F. gigantica. Whereas, activity of both enzymes were not significantly altered in the nervous tissues of vector snail Lymnaea acuminata exposed to same treatments. Maximum reduction in AChE (1.35% of control in month of June) and cytochrome oxidase (3.71% of control in the month of July) activity were noted in the cercaria exposed to 60% of 4 h LC50 of azadirachtin and allicin, respectively. PMID:26536397

  16. Monoamine oxidase B and free radical scavenging activities of natural flavonoids in Melastoma candidum D. Don.

    PubMed

    Lee, M H; Lin, R D; Shen, L Y; Yang, L L; Yen, K Y; Hou, W C

    2001-11-01

    Monoamine oxidase type B (MAO-B) activity and free radicals are elevated in certain neurological diseases. Four natural flavonoids, quercitrin, isoquercitrin, rutin, and quercetin, were isolated for the first time from the leaves of Melastoma candidum D. Don. They exhibited an inhibitory effect on MAO-B. These potent flavonoids were purified using bioassay-guided fractionation and were separated by Diaion, Sephadex LH-20, and MCI CHP20P columns. The IC(50) values of the four potent flavonoids, quercitrin, isoquercitrin, rutin, and quercetin on monoamine oxidase were 19.06, 11.64, 3.89, and 10.89 microM and enzyme kinetics analysis revealed apparent inhibition constants (K(i)) of 21.01, 2.72, 1.83, and 7.95 microM, respectively, on the substrate, benzylamine. The four potent compounds also exhibited hydroxyl radical scavenging activity as determined using a spin trapping electron spin resonance method. This suggests that the four flavonoids from M. candidum possess both MAO-B inhibitory and free radical scavenging activities. These important properties may be used for preventing some neurodegenerative diseases in the future. PMID:11714358

  17. Activity-stability relationships revisited in blue oxidases catalyzing electron transfer at extreme temperatures.

    PubMed

    Roulling, Frédéric; Godin, Amandine; Cipolla, Alexandre; Collins, Tony; Miyazaki, Kentaro; Feller, Georges

    2016-09-01

    Cuproxidases are a subset of the blue multicopper oxidases that catalyze the oxidation of toxic Cu(I) ions into less harmful Cu(II) in the bacterial periplasm. Cuproxidases from psychrophilic, mesophilic, and thermophilic bacteria display the canonical features of temperature adaptation, such as increases in structural stability and apparent optimal temperature for activity with environmental temperature as well as increases in the binding affinity for catalytic and substrate copper ions. In contrast, the oxidative activities at 25 °C for both the psychrophilic and thermophilic enzymes are similar, suggesting that the nearly temperature-independent electron transfer rate does not require peculiar adjustments. Furthermore, the structural flexibilities of both the psychrophilic and thermophilic enzymes are also similar, indicating that the firm and precise bindings of the four catalytic copper ions are essential for the oxidase function. These results show that the requirements for enzymatic electron transfer, in the absence of the selective pressure of temperature on electron transfer rates, produce a specific adaptive pattern, which is distinct from that observed in enzymes possessing a well-defined active site and relying on conformational changes such as for the induced fit mechanism. PMID:27315165

  18. Estrogen Attenuates Ischemic Oxidative Damage via an ERα-Mediated Inhibition of NADPH Oxidase Activation

    PubMed Central

    Zhang, Quan-Guang; Raz, Limor; Wang, Ruimin; Han, Dong; De Sevilla, Liesl; Yang, Fang; Vadlamudi, Ratna K.; Brann, Darrell W.

    2009-01-01

    The goal of this study was to elucidate the mechanisms of 17β-estradiol (E2) antioxidant and neuroprotective actions in stroke. The results reveal a novel extranuclear receptor-mediated antioxidant mechanism for E2 during stroke, as well as a hypersensitivity of the CA3/CA4 region to ischemic injury after prolonged hypoestrogenicity. E2 neuroprotection was shown to involve a profound attenuation of NADPH oxidase activation and superoxide production in hippocampal CA1 pyramidal neurons after stroke, an effect mediated by extranuclear ERα-mediated nongenomic signaling, involving Akt activation and subsequent phosphorylation/inactivation of Rac1, a factor critical for activation of NOX2 NADPH oxidase. Intriguingly, E2 nongenomic signaling, antioxidant action and neuroprotection in the CA1 region were lost after long-term E2 deprivation; and this loss was tissue-specific, as the uterus remained responsive to E2. Correspondingly, a remarkable loss of ERα, but not ERβ, was observed in the CA1 following long-term E2 deprivation, with no change observed in the uterus. As a whole, the study reveals a novel, membrane-mediated antioxidant mechanism in neurons by E2, provides support and mechanistic insights for a “critical period” of E2 replacement in the hippocampus, and demonstrates a heretofore unknown hypersensitivity of the CA3/CA4 to ischemic injury after prolonged hypoestrogenicity. PMID:19889994

  19. Histamine content, diamine oxidase activity and histamine methyltransferase activity in human tissues: fact or fictions?

    PubMed

    Hesterberg, R; Sattler, J; Lorenz, W; Stahlknecht, C D; Barth, H; Crombach, M; Weber, D

    1984-04-01

    To understand the role of histamine in the aetiology and pathogenesis of human diseases reliable data are urgently needed for the histamine content and for the activities of histamine-forming and -inactivating enzymes in human tissues. In order to make a substantial progress toward this aim a tissue-sampling programme during surgical interventions was carefully conceived and conducted. From March 1982 until January 1983 106 tissue specimens were taken from 56 patients who underwent surgery. Only healthy tissues, not injured or oedematous, and without adherent structures were taken by only one surgeon who was interested in this research and experienced in tissue preparation procedures in biochemistry. The times of 'warm' ischaemia during the operative procedures were visually estimated, the times between resection of the organs or specimens and deep-freezing of the tissues were precisely recorded. Compared to previous work in the literature and especially to our own work using the same assays for determination higher histamine contents were found in this study in most of the tissues, in particular in the gastrointestinal tract. Also the diamine oxidase activities were considerably higher in many organs, e.g. 3-4 times higher in the gastrointestinal tract when compared with those in publications of our group who used always the same analytical test. However, the histamine methyltransferase activities in this study were not at variance to those determined in previous investigations. Many of them were reported in this communication for the first time. Since the methods for histamine determination and those for measuring enzymic activities were not different in this study and in previous communications of our group we are convinced that the optimized tissue-sampling and -preparation techniques were responsible for the higher values in this communication. But the problem of the 'warm' ischaemia period could not be solved by sample-taking procedures of this type during

  20. Listeriolysin O suppresses phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection.

    PubMed

    Lam, Grace Y; Fattouh, Ramzi; Muise, Aleixo M; Grinstein, Sergio; Higgins, Darren E; Brumell, John H

    2011-12-15

    The intracellular bacterial pathogen Listeria monocytogenes produces phospholipases C (PI-PLC and PC-PLC) and the pore-forming cytolysin listeriolysin O (LLO) to escape the phagosome and replicate within the host cytosol. We found that PLCs can also activate the phagocyte NADPH oxidase during L. monocytogenes infection, a response that would adversely affect pathogen survival. However, secretion of LLO inhibits the NADPH oxidase by preventing its localization to phagosomes. LLO-deficient bacteria can be complemented by perfringolysin O, a related cytolysin, suggesting that other pathogens may also use pore-forming cytolysins to inhibit the NADPH oxidase. Our studies demonstrate that while the PLCs induce antimicrobial NADPH oxidase activity, this effect is alleviated by the pore-forming activity of LLO. Therefore, the combined activities of PLCs and LLO on membrane lysis and the inhibitory effects of LLO on NADPH oxidase activity allow L. monocytogenes to efficiently escape the phagosome while avoiding the microbicidal respiratory burst. PMID:22177565

  1. THREE POLYPHENOL OXIDASES FROM RED CLOVER (TRIFOLIUM PRATENSE) DIFFER IN ENZYMATIC ACTIVITIES AND ACTIVATION PROPERTIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidases (PPOs) oxidize o-diphenols to o-quinones, which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense L.). Production of o-quinones in red clover inhibits postharvest proteolysis during the ensiling proces...

  2. A cerium method for the ultracytochemical localization of monoamine oxidase activity.

    PubMed

    Fujimoto, T; Inomata, K; Ogawa, K

    1982-01-01

    A cytochemical method based on the complex formation between cerous ions and hydrogen peroxide is described for the ultrastructural localization of monoamine oxidase (MAO). First, the residual MAO activity after fixation was measured by a radiochemical assay technique and was found to be sufficiently retained for cytochemical detection. Although the Tris buffer used in the present method was found to be inhibitory to MAO, considerable activity was still retained after fixation and incubation in Tris. MAO activity, detected as precipitates of cerium perhydroxide, was observed in the mitochondrial outer compartment, mitochondrial cristae and perinuclear space of myocardial cells and endothelial cells of rat heart. MAO activity was also found along the plasma membrane of capillary endothelia. Omission of substrate from the incubation medium or pre-incubation with pargyline, a specific MAO inhibitor, drastically reduced the amount of deposits. The present cerium method seems promising because of its reproducibility and the high electron density of the reaction products. PMID:6174485

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

    PubMed

    Liu, Biwu; Huang, Zhicheng; Liu, Juewen

    2016-07-14

    Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ∼15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F(-) capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F(-) in water and in toothpastes, while no other tested anions can achieve the activity enhancement. PMID:27378306

  4. Thiolactomycin inhibits D-aspartate oxidase: a novel approach to probing the active site environment.

    PubMed

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

    2010-10-01

    D-Aspartate oxidase (DDO) and D-amino acid oxidase (DAO) are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of D-amino acids. While several functionally and structurally important amino acid residues have been identified in the DAO protein, little is known about the structure-function relationships of DDO. In the search for a potent DDO inhibitor as a novel tool for investigating its structure-function relationships, a large number of biologically active compounds of microbial origin were screened for their ability to inhibit the enzymatic activity of mouse DDO. We discovered several compounds that inhibited the activity of mouse DDO, and one of the compounds identified, thiolactomycin (TLM), was then characterized and evaluated as a novel DDO inhibitor. TLM reversibly inhibited the activity of mouse DDO with a mixed type of inhibition more efficiently than meso-tartrate and malonate, known competitive inhibitors of mammalian DDOs. The selectivity of TLM was investigated using various DDOs and DAOs, and it was found that TLM inhibits not only DDO, but also DAO. Further experiments with apoenzymes of DDO and DAO revealed that TLM is most likely to inhibit the activities of DDO and DAO by competition with both the substrate and the coenzyme, FAD. Structural models of mouse DDO/TLM complexes supported this finding. The binding mode of TLM to DDO was validated further by site-directed mutagenesis of an active site residue, Arg-237. Collectively, our findings show that TLM is a novel, active site-directed DDO inhibitor that will be useful for elucidating the molecular details of the active site environment of DDO. PMID:20603179

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  6. [Sulfite oxidase activity deficiency caused by cofactor molybdenum deficiency: A case of early severe encephalopathy].

    PubMed

    Durousset, C; Gay, C; Magnin, S; Acquaviva, C; Patural, H

    2016-03-01

    Neonatal seizure incidence is approximately 3.5/1000 live births. Inborn metabolic diseases account for approximately 1-4% of neonatal seizure cases. Among them, the catabolism anomaly of sulfite to sulfate caused by sulfite oxidase or cofactor molybdenum deficiency (MoCD) is a rare metabolic disorder in which neurological damage is similar to that found in neonatal asphyxia. We report the case of a newborn child with a MoCD. Born of related parents, this child had intrauterine growth retardation predominating on size diagnosed in the third trimester of pregnancy. After an uneventful birth, he presented convulsions at the 12th hour of life, confirmed by an electroencephalogram. Anticonvulsants and adjuvant treatments were ineffective; the child then required intubation at day 5 of life. The initial biological assessment found an elevated blood lactate level and the chromatography of amino acids showed a significant decrease of cystine and the abnormal presence of sulfocysteine, suggestive of a lack of sulfite oxidase activity. The uric acid level measured secondarily was low, suggesting a MoCD. Brain MRI was performed at day 5 for diffuse ischemic injury of different ages. After limiting acute care, the child died at day 14 of life. The genetic study of the child found a homozygous mutation c.564+1G>A in the MOCS2 gene, confirming the diagnosis of MoCD, present in the heterozygous state in both parents. Investigations in a logical sequence quickly suggested the MoCD diagnosis in presence of a low plasma concentration of cysteine, the abnormal presence of sulfocysteine, and low uric acid levels. The diagnosis of sulfite oxidase deficiency was made. Until now, no treatment has proven effective but a new treatment appears to be effective in cases with a MOCS1 mutation. PMID:26775885

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

    NASA Astrophysics Data System (ADS)

    Liu, Biwu; Huang, Zhicheng; Liu, Juewen

    2016-07-01

    Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement.Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement. Electronic supplementary information (ESI) available: Methods, TMB oxidation kinetics and control experiments. See DOI: 10.1039/c6nr02730j

  8. Wheat Germ Agglutinin Induces NADPH-Oxidase Activity in Human Neutrophils by Interaction with Mobilizable Receptors

    PubMed Central

    Karlsson, Anna

    1999-01-01

    Wheat germ agglutinin (WGA), a lectin with specificity for N-acetylglucosamine and sialic acid, was investigated with respect to its ability to activate the NADPH-oxidase of in vivo-exudated neutrophils (obtained from a skin chamber), and the activity was compared to that of peripheral blood neutrophils. The exudate cells responded to WGA, by both releasing reactive oxygen species into the extracellular milieu and producing oxygen metabolites intracellularly. The peripheral blood cells were unresponsive. To mimic the in vivo-exuded neutrophils with regards to receptor exposure, peripheral blood neutrophils were induced to mobilize their granules and vesicles to varying degrees (in vitro priming), prior to challenge with WGA. The oxidative response to WGA increased with increasing levels of granule mobilization, and the receptor(s) could be shown to reside in the secretory vesicles and/or the gelatinase granules in resting neutrophils. Several WGA-binding glycoproteins were detected in subcellular fractions containing these organelles. The extra- and intracellular NADPH-oxidase responses showed differences in sialic acid dependency, indicating that these two responses are mediated by different receptor structures. PMID:10377127

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

    SciTech Connect

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

    2010-04-15

    Dithiocarbamates (DTCs) are a class of compounds that are extensively used in agriculture as pesticides. As such, humans and wildlife are undoubtedly exposed to these chemicals. Although DTCs are thought to be relatively safe due to their short half lives, it is well established that they are teratogenic to vertebrates, especially to fish. In zebrafish, these teratogenic effects are characterized by distorted notochord development and shortened anterior to posterior axis. DTCs are known copper (Cu) chelators but this does not fully explain the observed teratogenic effects. We show here that DTCs cause malformations in zebrafish that highly resemble teratogenic effects observed by direct inhibition of a group of cuproenzymes termed lysyl oxidases (LOX). Additionally, we demonstrate that partial knockdown of three LOX genes, lox, loxl1 and loxl5b, sensitizes the developing embryo to DTC exposure. Finally, we show that DTCs directly inhibit zebrafish LOX activity in an ex vivo amine oxidase assay. Taken together, these results provide the first evidence that DTC induced teratogenic effects are, at least in part, caused by direct inhibition of LOX activity.

  10. Exogenous methyl jasmonate regulates cytokinin content by modulating cytokinin oxidase activity in wheat seedlings under salinity.

    PubMed

    Avalbaev, Azamat; Yuldashev, Ruslan; Fedorova, Kristina; Somov, Kirill; Vysotskaya, Lidiya; Allagulova, Chulpan; Shakirova, Farida

    2016-02-01

    The treatment of 4-days-old wheat seedlings with methyl jasmonate (MeJA) in concentration optimal for their growth (0.1 μM) resulted in a rapid transient almost two-fold increase in the level of cytokinins (CKs). MeJA-induced accumulation of CKs was due to inhibition of both cytokinin oxidase (CKX) (cytokinin oxidase/dehydrogenase, EC 1.5.99.12) gene expression and activity of this enzyme. Pretreatment of wheat seedlings with MeJA decreased the growth-retarding effect of sodium chloride salinity and accelerated growth recovery after withdrawal of NaCl from the incubation medium. We speculate that this protective effect of the hormone might be due to MeJA's ability to prevent the salinity-induced decline in CK concentration that was caused by inhibition of gene expression and activity of CKX in wheat seedlings. The data might indicate an important role for endogenous cytokinins in the implementation of growth-promoting and protective effects of exogenous MeJA application on wheat plants. PMID:26748373

  11. Role of polyamines and phospholipase D in maize (Zea mays L.) response to drought stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hydroponic experiment was conducted to elucidate the role of polyamines and phospholipase D (PLD) in regulating response of maize plants to drought stress (DS). During the early stage of DS, an increase in PLD activity, independent of polyamines contents, was mainly responsible for stomatal closure...

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

    PubMed Central

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

    2010-01-01

    The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H2O2 generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle- and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc and VIII, indicated that the abundance these polypeptides decreased, ranging from 11 to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology. PMID:20833144

  13. A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential.

    PubMed

    Murata, M; Nishimura, M; Murai, N; Haruta, M; Homma, S; Itoh, Y

    2001-02-01

    Polyphenol oxidase (PPO) is responsible for enzymatic browning of apples. Apples lacking PPO activity might be useful not only for the food industry but also for studies of the metabolism of polyphenols and the function of PPO. Transgenic apple calli were prepared by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistant gene and antisense PPO gene. Four KM-resistant callus lines were obtained from 356 leaf explants. Among these transgenic calli, three calli grew on the medium containing KM at the same rate as non-transgenic callus on the medium without KM. One callus line had an antisense PPO gene, in which the amount and activity of PPO were reduced to half the amount and activity in non-transgenic callus. The browning potential of this line, which was estimated by adding chlorogenic acid, was also half the browning potential of non-transgenic callus. PMID:11302173

  14. Induction of hepatoma carcinoma cell apoptosis through activation of the JNK-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-ROS self-driven death signal circuit.

    PubMed

    Zeng, Ke-Wu; Song, Fang-Jiao; Wang, Ying-Hong; Li, Ning; Yu, Qian; Liao, Li-Xi; Jiang, Yong; Tu, Peng-Fei

    2014-10-28

    As an efficient method for inducing tumor cell apoptosis, ROS can be constantly formed and accumulated in NADPH oxidase overactivated-cells, resulting in further mitochondrial membrane damage and mitochondria-dependent apoptosis. In addition, JNK mitogen-activated protein kinase (JNK MAPK) signal also acts as a vital candidate pathway for inducing tumor cell apoptosis by targeting mitochondrial death pathway. However, the relationship between NADPH oxidase-ROS and JNK MAPK signal still remains unclear. Here, we discovered a novel self-driven signal circuit between NADPH oxidase-ROS and JNK MAPK, which was induced by a cytotoxic steroidal saponin (ASC) in hepatoma carcinoma cells. NADPH oxidase-dependent ROS production was markedly activated by ASC and directly led to JNK MAPK activation. Moreover, antioxidant, NADPH oxidase inhibitor and specific knock-out for p47 subunit of NADPH oxidase could effectively block NADPH oxidase-ROS-dependent JNK activation, suggesting that NADPH oxidase is an upstream regulator of JNK MAPK. Conversely, a specific JNK inhibitor could inhibit ASC-induced NADPH oxidase activation and down-regulate ROS levels as well, indicating that JNK might also regulate NADPH oxidase activity to some extent. These observations indicate that NADPH oxidase and JNK MAPK activate each other as a signal circuit. Furthermore, drug pretreatment experiments with ASC showed this signal circuit operated continuously via a self-driven mode and finally induced apoptosis in hepatoma carcinoma cells. Taken together, we provide a proof for inducing hepatoma carcinoma cell apoptosis by activating the JNK-NADPH oxidase-ROS-dependent self-driven signal circuit pathway. PMID:25064608

  15. (/sup 11/C)clorgyline and (/sup 11/C)-L-deprenyl and their use in measuring functional monoamine oxidase activity in the brain using positron emission tomography

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1986-04-17

    This invention involves a new strategy for imaging the activity of the enzyme monoamine oxidase in the living body by using /sup 11/C-labeled enzyme inhibitors which bind irreversibly to an enzyme as a result of catalysis. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

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

    PubMed Central

    2010-01-01

    Background Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton. Results Here, we in addition to toxins use conditional expression of the major actin regulatory protein LIM kinase-1 (LIMK1), and shRNA knock-down of cofilin to modulate the cellular F/G-actin ratio in the Ra2 microglia cell line, and we use Fluorescence Recovery after Photobleaching (FRAP) in β-actin-YFP-transduced cells to obtain a dynamic measure of actin recovery rates (actin turn-over rates) in different F/G-actin states of the actin cytoskeleton. Our data demonstrate that stimulated NADPH oxidase function was severely impaired only at extreme actin recovery rates and F/G-actin ratios, and surprisingly, that any moderate changes of these parameters of the actin cytoskeleton invariably resulted in an increased NADPH oxidase activity. Conclusion moderate actin polymerization and depolymerization both increase the FMLP and PMA-stimulated NADPH oxidase activity of microglia, which is directly correlated with neither actin recovery rate nor F/G- actin ratio. Our results indicate that NADPH oxidase functions in an enhanced state of activity in stimulated phagocytes despite widely different states of the actin cytoskeleton. PMID:20825680

  17. Oxidase, superoxide dismutase, and hydrogen peroxide reductase activities of methanobactin from types I and II methanotrophs.

    PubMed

    Choi, Dong W; Semrau, Jeremy D; Antholine, William E; Hartsel, Scott C; Anderson, Ryan C; Carey, Jeffrey N; Dreis, Ashley M; Kenseth, Erik M; Renstrom, Joel M; Scardino, Lori L; Van Gorden, Garrett S; Volkert, Anna A; Wingad, Aaron D; Yanzer, Paul J; McEllistrem, Marcus T; de la Mora, Arlene M; DiSpirito, Alan A

    2008-08-01

    Methanobactin (mb) is a copper-binding chromopeptide that appears to be involved in oxidation of methane by the membrane-associated or particulate methane monooxygenase (pMMO). To examine this potential physiological role, the redox and catalytic properties of mb from three different methanotrophs were examined in the absence and presence of O(2). Metal free mb from the type II methanotroph Methylosinus trichosporium OB3b, but not from the type I methanotrophs Methylococcus capsulatus Bath or Methylomicrobium album BG8, were reduced by a variety of reductants, including NADH and duroquinol, and catalyzed the reduction of O(2) to O(2)(-). Copper-containing mb (Cu-mb) from all three methanotrophs showed several interesting properties, including reductase dependent oxidase activity, dismutation of O(2)(-) to H(2)O(2), and the reductant dependent reduction of H(2)O(2) to H(2)O. The superoxide dismutase-like and hydrogen peroxide reductase activities of Cu-mb were 4 and 1 order(s) of magnitude higher, respectively, than the observed oxidase activity. The results demonstrate that Cu-mb from all three methanotrophs are redox-active molecules and oxygen radical scavengers, with the capacity to detoxify both superoxide and hydrogen peroxide without the formation of the hydroxyl radicals associated with Fenton reactions. As previously observed with Cu-mb from Ms. trichosporium OB3b, Cu-mb from both type I methanotrophs stimulated pMMO activity. However, in contrast to previous studies using mb from Ms. trichosporium OB3b, pMMO activity was not inhibited by mb from the two type I methanotrophs at low copper to mb ratios. PMID:18372044

  18. Correlation of active site metal content in human diamine oxidase with trihydroxyphenylalanine quinone cofactor biogenesis .

    PubMed

    McGrath, Aaron P; Caradoc-Davies, Tom; Collyer, Charles A; Guss, J Mitchell

    2010-09-28

    Copper-containing amine oxidases (CAOs) require a protein-derived topaquinone cofactor (TPQ) for activity. TPQ biogenesis is a self-processing reaction requiring the presence of copper and molecular oxygen. Recombinant human diamine oxidase (hDAO) was heterologously expressed in Drosophila S2 cells, and analysis indicates that the purified hDAO contains substoichiometric amounts of copper and TPQ. The crystal structure of a complex of an inhibitor, aminoguanidine, and hDAO at 2.05 Å resolution shows that the aminoguanidine forms a covalent adduct with the TPQ and that the site is ∼75% occupied. Aminoguanidine is a potent inhibitor of hDAO with an IC(50) of 153 ± 9 nM. The structure indicates that the catalytic metal site, normally occupied by copper, is fully occupied. X-ray diffraction data recorded below the copper edge, between the copper and zinc edges, and above the zinc edge have been used to show that the metal site is occupied approximately 75% by copper and 25% by zinc and the formation of the TPQ cofactor is correlated with copper occupancy. PMID:20722416

  19. Mouse aldehyde-oxidase-4 controls diurnal rhythms, fat deposition and locomotor activity

    PubMed Central

    Terao, Mineko; Barzago, Maria Monica; Kurosaki, Mami; Fratelli, Maddalena; Bolis, Marco; Borsotti, Andrea; Bigini, Paolo; Micotti, Edoardo; Carli, Mirjana; Invernizzi, Roberto William; Bagnati, Renzo; Passoni, Alice; Pastorelli, Roberta; Brunelli, Laura; Toschi, Ivan; Cesari, Valentina; Sanoh, Seigo; Garattini, Enrico

    2016-01-01

    Aldehyde-oxidase-4 (AOX4) is one of the mouse aldehyde oxidase isoenzymes and its physiological function is unknown. The major source of AOX4 is the Harderian-gland, where the enzyme is characterized by daily rhythmic fluctuations. Deletion of the Aox4 gene causes perturbations in the expression of the circadian-rhythms gene pathway, as indicated by transcriptomic analysis. AOX4 inactivation alters the diurnal oscillations in the expression of master clock-genes. Similar effects are observed in other organs devoid of AOX4, such as white adipose tissue, liver and hypothalamus indicating a systemic action. While perturbations of clock-genes is sex-independent in the Harderian-gland and hypothalamus, sex influences this trait in liver and white-adipose-tissue which are characterized by the presence of AOX isoforms other than AOX4. In knock-out animals, perturbations in clock-gene expression are accompanied by reduced locomotor activity, resistance to diet induced obesity and to hepatic steatosis. All these effects are observed in female and male animals. Resistance to obesity is due to diminished fat accumulation resulting from increased energy dissipation, as white-adipocytes undergo trans-differentiation towards brown-adipocytes. Metabolomics and enzymatic data indicate that 5-hydroxyindolacetic acid and tryptophan are novel endogenous AOX4 substrates, potentially involved in AOX4 systemic actions. PMID:27456060

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

    PubMed

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

    2004-08-01

    Nitroalkane oxidase (NAO), a flavoprotein cloned and purified from Fusarium oxysporum, catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones, with the production of H2O2 and nitrite. In this paper, the crystallization and preliminary X-ray data analysis of three crystal forms of active nitroalkane oxidase are described. The first crystal form belongs to a trigonal space group (either P3(1)21 or P3(2)21, with unit-cell parameters a = b = 103.8, c = 487.0 A) and diffracts to at least 1.6 A resolution. Several data sets were collected using 2theta and kappa geometry in order to obtain a complete data set to 2.07 A resolution. Solvent-content and Matthews coefficient analysis suggests that crystal form 1 contains two homotetramers per asymmetric unit. Crystal form 2 (P2(1)2(1)2(1); a = 147.3, b = 153.5, c = 169.5 A) and crystal form 3 (P3(1) or P3(2); a = b = 108.9, c = 342.5 A) are obtained from slightly different conditions and also contain two homotetramers per asymmetric unit, but have different solvent contents. A three-wavelength MAD data set was collected from selenomethionine-enriched NAO (SeMet-NAO) in crystal form 3 and will be used for phasing. PMID:15272176

  1. A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity

    PubMed Central

    Larrondo, Luis F.; Salas, Loreto; Melo, Francisco; Vicuña, Rafael; Cullen, Daniel

    2003-01-01

    Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe2+, with a Km close to 2 μM. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity. PMID:14532088

  2. Genomic organisation, activity and distribution analysis of the microbial putrescine oxidase degradation pathway.

    PubMed

    Foster, Alexander; Barnes, Nicole; Speight, Robert; Keane, Mark A

    2013-10-01

    The catalytic action of putrescine specific amine oxidases acting in tandem with 4-aminobutyraldehyde dehydrogenase is explored as a degradative pathway in Rhodococcus opacus. By limiting the nitrogen source, increased catalytic activity was induced leading to a coordinated response in the oxidative deamination of putrescine to 4-aminobutyraldehyde and subsequent dehydrogenation to 4-aminobutyrate. Isolating the dehydrogenase by ion exchange chromatography and gel filtration revealed that the enzyme acts principally on linear aliphatic aldehydes possessing an amino moiety. Michaelis-Menten kinetic analysis delivered a Michaelis constant (K(M)=0.014 mM) and maximum rate (Vmax=11.2 μmol/min/mg) for the conversion of 4-aminobutyraldehyde to 4-aminobutyrate. The dehydrogenase identified by MALDI-TOF mass spectrometric analysis (E value=0.031, 23% coverage) belongs to a functionally related genomic cluster that includes the amine oxidase, suggesting their association in a directed cell response. Key regulatory, stress and transport encoding genes have been identified, along with candidate dehydrogenases and transaminases for the further conversion of 4-aminobutyrate to succinate. Genomic analysis has revealed highly similar metabolic gene clustering among members of Actinobacteria, providing insight into putrescine degradation notably among Micrococcaceae, Rhodococci and Corynebacterium by a pathway that was previously uncharacterised in bacteria. PMID:23906496

  3. Mouse aldehyde-oxidase-4 controls diurnal rhythms, fat deposition and locomotor activity.

    PubMed

    Terao, Mineko; Barzago, Maria Monica; Kurosaki, Mami; Fratelli, Maddalena; Bolis, Marco; Borsotti, Andrea; Bigini, Paolo; Micotti, Edoardo; Carli, Mirjana; Invernizzi, Roberto William; Bagnati, Renzo; Passoni, Alice; Pastorelli, Roberta; Brunelli, Laura; Toschi, Ivan; Cesari, Valentina; Sanoh, Seigo; Garattini, Enrico

    2016-01-01

    Aldehyde-oxidase-4 (AOX4) is one of the mouse aldehyde oxidase isoenzymes and its physiological function is unknown. The major source of AOX4 is the Harderian-gland, where the enzyme is characterized by daily rhythmic fluctuations. Deletion of the Aox4 gene causes perturbations in the expression of the circadian-rhythms gene pathway, as indicated by transcriptomic analysis. AOX4 inactivation alters the diurnal oscillations in the expression of master clock-genes. Similar effects are observed in other organs devoid of AOX4, such as white adipose tissue, liver and hypothalamus indicating a systemic action. While perturbations of clock-genes is sex-independent in the Harderian-gland and hypothalamus, sex influences this trait in liver and white-adipose-tissue which are characterized by the presence of AOX isoforms other than AOX4. In knock-out animals, perturbations in clock-gene expression are accompanied by reduced locomotor activity, resistance to diet induced obesity and to hepatic steatosis. All these effects are observed in female and male animals. Resistance to obesity is due to diminished fat accumulation resulting from increased energy dissipation, as white-adipocytes undergo trans-differentiation towards brown-adipocytes. Metabolomics and enzymatic data indicate that 5-hydroxyindolacetic acid and tryptophan are novel endogenous AOX4 substrates, potentially involved in AOX4 systemic actions. PMID:27456060

  4. The Antimicrobial Activity of Marinocine, Synthesized by Marinomonas mediterranea, Is Due to Hydrogen Peroxide Generated by Its Lysine Oxidase Activity

    PubMed Central

    Lucas-Elío, Patricia; Gómez, Daniel; Solano, Francisco; Sanchez-Amat, Antonio

    2006-01-01

    Marinocine is a broad-spectrum antibacterial protein synthesized by the melanogenic marine bacterium Marinomonas mediterranea. This work describes the basis for the antibacterial activity of marinocine and the identification of the gene coding for this protein. The antibacterial activity is inhibited under anaerobic conditions and by the presence of catalase under aerobic conditions. Marinocine is active only in culture media containing l-lysine. In the presence of this amino acid, marinocine generates hydrogen peroxide, which causes cell death as confirmed by the increased sensitivity to marinocine of Escherichia coli strains mutated in catalase activity. The gene coding for this novel enzyme was cloned using degenerate PCR with primers designed based on conserved regions in the antimicrobial protein AlpP, synthesized by Pseudoalteromonas tunicata, and some hypothetical proteins. The gene coding for marinocine has been named lodA, standing for lysine oxidase, and it seems to form part of an operon with a second gene, lodB, that codes for a putative dehydrogenase flavoprotein. The identity of marinocine as LodA has been demonstrated by N-terminal sequencing of purified marinocine and generation of lodA mutants that lose their antimicrobial activity. This is the first report on a bacterial lysine oxidase activity and the first time that a gene encoding this activity has been cloned. PMID:16547036

  5. Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells.

    PubMed

    Pitari, G; D'Andrea, G; Salucci, M L; Rossi, A; Avigliano, L

    1998-08-01

    Ascorbate oxidase activity and immunoreactivity were evaluated in crude tissue extracts obtained from callus cell cultures induced by green zucchini sarcocarp and grown in the presence of tunicamycin, a powerful N-glycosylation inhibitor. Tunicamycin at 2 or 4 microg ml(-1) blocked cell growth within a couple of weeks, although a sustained cell viability was observed in the same period. A significant inhibition of total protein synthesis was observed at 10 and 15 days of culture time, with a decrease of 30% and 43% respectively when cells were grown in the presence of 2 microg ml(-1) tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 microg ml(-1). After the same culture times ascorbate oxidase specific activity assayed in crude tissue extracts showed increases of about 1.9-fold and 3.5-fold (10 days) and 1.7-fold and 3.1-fold (15 days) at 2 and 4 microg ml(-1) tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the same growing times. Lectin-blot, based on the use of concanavalin A, indicated a marked decrease of glycosylated proteins in tunicamycin-treated cultures. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarcely recognized the enzyme expressed in tunicamycin-treated cells; on the contrary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures. PMID:9870353

  6. Copper supplementation restores cytochrome c oxidase activity in cultured cells from patients with SCO2 mutations.

    PubMed Central

    Salviati, Leonardo; Hernandez-Rosa, Evelyn; Walker, Winsome F; Sacconi, Sabrina; DiMauro, Salvatore; Schon, Eric A; Davidson, Mercy M

    2002-01-01

    Human SCO2 is a nuclear-encoded Cu-binding protein, presumed to be responsible for the insertion of Cu into the mitochondrial cytochrome c oxidase (COX) holoenzyme. Mutations in SCO2 are associated with cardioencephalomyopathy and COX deficiency. Studies in yeast and bacteria have shown that Cu supplementation can restore COX activity in cells harbouring mutations in genes involving Cu transport. Therefore we investigated whether Cu supplementation could restore COX activity in cultured cells from patients with SCO2 mutations. Our data demonstrate that the COX deficiency observed in fibroblasts, myoblasts and myotubes from patients with SCO2 mutations can be restored to almost normal levels by the addition of CuCl(2) to the growth medium. PMID:11931660

  7. NADPH Oxidase Dependent NLRP3 Inflammasome Activation Plays an Important Role in Lung Fibrosis by Multi-Walled Carbon Nanotubes

    PubMed Central

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

    2015-01-01

    The purpose of this communication is to elucidate the key role of NADPH oxidase in NLRP3 inflammasome activation and generation of pulmonary fibrosis by multi-walled carbon nanotubes (MWCNTs). Although it is known that oxidative stress plays a role in pulmonary fibrosis by single-walled CNTs, the role of specific sources of reactive oxygen species (ROS), including NADPH oxidase, in inflammasome activation remains to be clarified. In this study, three long aspect ratio (LAR) materials (MWCNTs, SWCNTs, and silver nanowires) are used to compare with spherical carbon black and silver nanoparticles for their ability to trigger oxygen burst activity and NLRP3 assembly. All LAR materials but not spherical nanoparticles induce robust NADPH oxidase activation and respiratory burst activity in THP-1 cells, which are blunted in p22phox deficient cells. NADPH oxidase is directly involved in lysosome damage by LAR materials, as demonstrated by decreased cathepsin B release and IL-1β production in p22phox deficient cells. Reduced respiratory burst activity and inflammasome activation are also observed in bone marrow-derived macrophages from p47phox deficient mice. Moreover, p47phox deficient mice have reduced IL-1β production and lung collagen deposition in response to MWCNTs. Lung fibrosis is also suppressed by N-acetyl-cysteine (NAC) in wild type animals exposed to MWCNTs. PMID:25581126

  8. Probing Oxygen Activation Sites in Two Flavoprotein Oxidases Using Chloride as an Oxygen Surrogate

    SciTech Connect

    Kommoju, Phaneeswara-Rao; Chen, Zhi-wei; Bruckner, Robert C.; Mathews, F. Scott; Jorns, Marilyn Schuman

    2011-08-16

    A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX-chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX-chloride complex and a ternary MSOX-chloride-MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.

  9. Inhibition of guinea pig aldehyde oxidase activity by different flavonoid compounds: An in vitro study.

    PubMed

    Siah, Maryam; Farzaei, Mohammad Hosein; Ashrafi-Kooshk, Mohammad Reza; Adibi, Hadi; Arab, Seyed Shahriar; Rashidi, Mohammad Reza; Khodarahmi, Reza

    2016-02-01

    Aldehyde oxidase (AO), a cytosolic molybdenum-containing hydroxylase, is predominantly active in liver and other tissues of mammalian species and involved in the metabolism of extensive range of aldehydes and nitrogen-containing compounds. A wide range of natural components including polyphenols are able to interfere with AO-catalyzed reactions. Polyphenols and flavonoids are one of the extensive secondary plant metabolites ubiquitously present in plants considered an important part of the human diet. The aim of the present study was to investigate inhibitory effect of selected phenolic compounds from three subclasses of aurone, flavanone and phenolic lactone compounds on the activity of AO, spectrophotometrically. AO enzyme was partially purified from liver of guinea pig. Then, inhibitory effects of 10 flavonoid compounds including 8 derivatives of 2-benzylidenebenzofuran-3(2H)-ones, as well as naringenin and ellagic acid on the activity of aldehyde oxidase were assessed compared with the specific inhibitor of AO, menadione. Among the phenolic compounds with inhibitory effects on the enzyme, ellagic acid (IC50=14.47 μM) was the most potent agent with higher inhibitory action than menadione (IC50=31.84 μM). The mechanisms by which flavonoid compounds inhibit AO activity have been also determined. The inhibitory process of the assessed compounds occurs via either a non-competitive or mixed mode. Although flavonoid compounds extensively present in the nature, mainly in dietary regimen, aurones with promising biological properties are not widely distributed in nature, so synthesis of aurone derivatives is of great importance. Additionally, aurones seem to provide a promising scaffold in medicinal chemistry for the skeleton of new developing drugs, so the results of the current study can be valuable in order to better understanding drug-food as well as drug-drug interaction and also appears to be worthwhile in drug development strategies. PMID:26722818

  10. Evidence of a novel transduction pathway mediating detection of polyamines by the zebrafish olfactory system.

    PubMed

    Michel, W C; Sanderson, M J; Olson, J K; Lipschitz, D L

    2003-05-01

    To better understand the full extent of the odorant detection capabilities of fish, we investigated the olfactory sensitivity of zebrafish to a monoamine and several polyamines using electrophysiological and activity-dependent labeling techniques. Electro-olfactogram (EOG) recording methods established the relative stimulatory effectiveness of these odorants as: spermine > spermidine approximately agmatine > glutamine > putrescine >or= cadaverine >or= histamine > artificial freshwater. The detection threshold for the potent polyamines was approximately 1 micromol l(-1). Cross-adaptation experiments suggested that multiple receptors are involved in polyamine detection. Three observations indicated that polyamine signaling may involve a transduction cascade distinct from those used by either amino acids or bile salts. Like bile salts and the adenylate cyclase activator forskolin, but unlike amino acid odorants, polyamines failed to stimulate activity-dependent labeling of olfactory sensory neurons with the cation channel permeant probe agmatine, suggesting a signaling pathway different from that used by amino acid stimuli. Also supporting distinct amino acid and polyamine signaling pathways is the finding that altering phospholipase C activity with the inhibitor U-73122 significantly reduced amino acid-evoked responses, but had little effect on polyamine- (or bile salt-) evoked responses. Altering cyclic nucleotide-mediated signaling by adenylate cyclase activation with forskolin, which significantly reduced responses to bile salts, failed to attenuate polyamine responses, suggesting that polyamines and bile salts do not share a common transduction cascade. Collectively, these findings suggest that polyamines are a new class of olfactory stimuli transduced by a receptor-mediated, second messenger signaling pathway that is distinct from those used by amino acids or bile salts. PMID:12682101

  11. Conjugation of chitosan nanoparticles with biogenic and synthetic polyamines: A delivery tool for antitumor polyamine analogues.

    PubMed

    Chanphai, P; Tajmir-Riahi, H A

    2016-11-01

    We report the conjugation of chitosan nanoparticles with biogenic polyamines spermine (spm), spermidine (spmd) and synthetic polyamines 3,7,11,15-tetrazaheptadecane.4HCl (BE-333) in aqueous solution. Multiple spectroscopic methods, thermodynamic parameters and molecular modeling were used to analyse polyamine bindings to chitosan nanoparticles. Thermodynamic parameters ΔS, ΔH and ΔG showed that polyamines bind protein through H-bonding and hydrophobic contacts with biogenic polyamines form more stable conjugates than synthetic polyamines. As polymer size increases the stability of polyamine-chitosan conjugate increases. The loading efficacy was 40-50% for polyamine-chitosan conjugates. Modeling showed that polyamine-protein interaction is spontaneous and chitosan nanoparticles can be used for delivery of antitumor polyamine analogues. PMID:27516317

  12. Polyamine levels as related to growth, differentiation and senescence in protoplast-derived cultures of Vigna aconitifolia and Avena sativa

    NASA Technical Reports Server (NTRS)

    Kaur Sawhney, R.; Shekhawat, N. S.; Galston, A. W.

    1985-01-01

    We have previously reported that aseptically cultured mesophyll protoplasts of Vigna divide rapidly and regenerate into complete plants, while mesophyll protoplasts of Avena divide only sporadically and senesce rapidly after isolation. We measured polyamine titers in such cultures of Vigna and Avena, to study possible correlations between polyamines and cellular behavior. We also deliberately altered polyamine titer by the use of selective inhibitors of polyamine biosynthesis, noting the effects on internal polyamine titer, cell division activity and regenerative events. In Vigna cultures, levels of free and bound putrescine and spermidine increased dramatically as cell division and differentiation progressed. The increase in bound polyamines was largest in embryoid-forming callus tissue while free polyamine titer was highest in root-forming callus. In Avena cultures, the levels of total polyamines decreased as the protoplast senesced. The presence of the inhibitors alpha-difluoromethyl-arginine (specific inhibitor of arginine decarboxylase), alpha-difluoromethylornithine (specific inhibitor of ornithine decarboxylase) and dicyclohexylamine (inhibitor of spermidine synthase) reduced cell division and organogenesis in Vigna cultures. Addition of low concentration of polyamines to such cultures containing inhibitors or removal of inhibitors from the culture medium restored the progress of growth and differentiation with concomitant increase in polyamine levels.

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

    PubMed

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

    2014-08-01

    This study aimed to explore the potential of polished adlay, brown adlay, adlay bran, and adlay hull to prevent and treat hyperuricemia. Brown adlay extract effectively decreased the serum uric acid levels of oxonate-induced hyperuricemic rats. Free and bound phenolic extracts from these materials contained significant amounts of phenolics, with free phenolics dominated by chlorogenic acid and p-coumaric acid while bound phenolics dominated by p-coumaric acid and ferulic acid. Free and bound phenolics of adlay bran exhibited significant xanthine oxidase inhibition activities, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities, oxygen radical absorbance capacities, and superoxide radical scavenging activities. Adlay bran phenolics could be effective xanthine oxidase inhibitors and radical scavengers. p-Coumaric acid is a xanthine oxidase inhibitor with strong superoxide radical scavenging activity. However, ferulic acid is a xanthine oxidase inhibitor with weak superoxide radical scavenging activity. Chlorogenic acid is a superoxide radical scavenger with weak xanthine oxidase inhibitory activity. PMID:25029106

  14. NADPH Oxidase- and Mitochondria-derived Reactive Oxygen Species in Proinflammatory Microglial Activation: A Bipartisan Affair?

    PubMed Central

    Bordt, Evan A.; Polster, Brian M.

    2014-01-01

    Microglia are the resident immune cells of the brain and play major roles in central nervous system development, maintenance, and disease. Brain insults cause microglia to proliferate, migrate, and transform into one or more activated states. Classical M1 activation triggers the production of proinflammatory factors such as tumor necrosis factor- α (TNF-α), interleukin-1β (IL-1β), nitric oxide (NO), and reactive oxygen species which, in excess, can exacerbate brain injury. The mechanisms underlying microglial activation are not fully understood, yet reactive oxygen species are increasingly implicated as mediators of microglial activation. In this review, we highlight studies linking reactive oxygen species, in particular hydrogen peroxide derived from NADPH oxidase-generated superoxide, to the classical activation of microglia. In addition, we critically evaluate controversial evidence suggesting a specific role for mitochondrial reactive oxygen species in the activation of the NLRP3 inflammasome, a multiprotein complex that mediates the production of IL-1β and IL-18. Finally, the limitations of common techniques used to implicate mitochondrial ROS in microglial and inflammasome activation, such as the use of the mitochondrially-targeted ROS indicator MitoSOX and the mitochondrially-targeted antioxidant MitoTEMPO, are also discussed. PMID:25091898

  15. Recombinant human diamine oxidase activity is not inhibited by ethanol, acetaldehyde, disulfiram, diethyldithiocarbamate or cyanamide.

    PubMed

    Bartko, Johann; Gludovacz, Elisabeth; Petroczi, Karin; Borth, Nicole; Jilma, Bernd; Boehm, Thomas

    2016-08-01

    Human diamine oxidase (hDAO, EC 1.4.3.22) is the key enzyme in the degradation of extracellular histamine. Consumption of alcohol is a known trigger of mast cell degranulation in patients with mast cell activation syndrome. Ethanol may also interfere with enzymatic histamine degradation, but reports on the effects on DAO activity are controversial. There are also conflicting reports whether disulfiram, an FDA-approved agent in the treatment of alcohol dependence, inhibits DAO. We therefore investigated the inhibitory potential of ethanol and disulfiram and their metabolites on recombinant human DAO (rhDAO) in three different assay systems. Relevant concentrations of ethanol, acetaldehyde, and acetate did not inhibit rhDAO activity in an in vitro assay system using horseradish peroxidase (HRP) -mediated luminol oxidation. The aldehyde dehydrogenase (ALDH; EC 1.2.1.3) inhibitors cyanamide and its dimer dicyanamide also had no effect on DAO activity. In one assay system, the irreversible ALDH inhibitor disulfiram and its main metabolite diethyldithiocarbamate seemed to inhibit DAO activity. However, the decreased product formation was not due to a direct block of DAO activity but resulted from inhibition of peroxidase employed in the coupled system. Our in vitro data do not support a direct blocking effect of ethanol, disulfiram, and their metabolites on DAO activity in vivo. PMID:27401969

  16. Sphingomyelinase promotes oxidant production and skeletal muscle contractile dysfunction through activation of NADPH oxidase

    PubMed Central

    Loehr, James A.; Abo-Zahrah, Reem; Pal, Rituraj; Rodney, George G.

    2015-01-01

    Elevated concentrations of sphingomyelinase (SMase) have been detected in a variety of diseases. SMase has been shown to increase muscle derived oxidants and decrease skeletal muscle force; however, the sub-cellular site of oxidant production has not been elucidated. Using redox sensitive biosensors targeted to the mitochondria and NADPH oxidase (Nox2), we demonstrate that SMase increased Nox2-dependent ROS and had no effect on mitochondrial ROS in isolated FDB fibers. Pharmacological inhibition and genetic knockdown of Nox2 activity prevented SMase induced ROS production and provided protection against decreased force production in the diaphragm. In contrast, genetic overexpression of superoxide dismutase within the mitochondria did not prevent increased ROS production and offered no protection against decreased diaphragm function in response to SMase. Our study shows that SMase induced ROS production occurs in specific sub-cellular regions of skeletal muscle; however, the increased ROS does not completely account for the decrease in muscle function. PMID:25653619

  17. Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation.

    PubMed

    Zielonka, Jacek; Zielonka, Monika; VerPlank, Lynn; Cheng, Gang; Hardy, Micael; Ouari, Olivier; Ayhan, Mehmet Menaf; Podsiadły, Radosław; Sikora, Adam; Lambeth, J David; Kalyanaraman, Balaraman

    2016-03-25

    Using high throughput screening-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of the NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O2 (̇̄)), hydrogen peroxide (H2O2), and peroxynitrite (ONOO(-)), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO(-)formation in activated macrophages. A new diagnostic marker product for ONOO(-)is reported. We conclude that the newly developed high throughput screening/reactive oxygen species assays could also be used to identify potential inhibitors of ONOO(-)formed from Nox2-derived O2 (̇̄)and nitric oxide synthase-derived nitric oxide. PMID:26839313

  18. Dopamine-β-oxidase activity in man, using hydroxyamphetamine as substrate

    PubMed Central

    Sjoerdsma, A.; von Studnitz, W.

    1963-01-01

    Hydroxyamphetamine was administered orally to five human subjects in daily doses of 26 to 118 mg. Approximately half the dosage administered was recoverable in the urine as unchanged drug (free plus conjugated) and 3.7 to 9.1% was excreted as the β-hydroxylated metabolite, hydroxynorephedrine (free plus conjugated). Since conversion of hydroxyamphetamine to hydroxynorephedrine occurs in vitro by the action of dopamine-β-oxidase, a simple method is suggested for measuring the activity of this enzyme and the effect of its inhibitors in man. No impairment of β-hydroxylation was noted in an adrenalectomized subject. The β-hydroxylation of hydroxyamphetamine in vivo has not been described previously. PMID:13977820

  19. Following Glucose Oxidase Activity by Chemiluminescence and Chemiluminescence Resonance Energy Transfer (CRET) Processes Involving Enzyme-DNAzyme Conjugates

    PubMed Central

    Niazov, Angelica; Freeman, Ronit; Girsh, Julia; Willner, Itamar

    2011-01-01

    A hybrid consisting of glucose oxidase-functionalized with hemin/G-quadruplex units is used for the chemiluminescence detection of glucose. The glucose oxidase-mediated oxidation of glucose yields gluconic acid and H2O2. The latter in the presence of luminol acts as substrate for the hemin/G-quadruplex-catalyzed generation of chemiluminescence. The glucose oxidase/hemin G-quadruplex hybrid was immobilized on CdSe/ZnS quantum dots (QDs). The light generated by the hybrid, in the presence of glucose, activated a chemiluminescence resonance energy transfer process to the QDs, resulting in the luminescence of the QDs. The intensities of the luminescence of the QDs at different concentrations of glucose provided an optical means to detect glucose. PMID:22346648

  20. Following glucose oxidase activity by chemiluminescence and chemiluminescence resonance energy transfer (CRET) processes involving enzyme-DNAzyme conjugates.

    PubMed

    Niazov, Angelica; Freeman, Ronit; Girsh, Julia; Willner, Itamar

    2011-01-01

    A hybrid consisting of glucose oxidase-functionalized with hemin/G-quadruplex units is used for the chemiluminescence detection of glucose. The glucose oxidase-mediated oxidation of glucose yields gluconic acid and H(2)O(2). The latter in the presence of luminol acts as substrate for the hemin/G-quadruplex-catalyzed generation of chemiluminescence. The glucose oxidase/hemin G-quadruplex hybrid was immobilized on CdSe/ZnS quantum dots (QDs). The light generated by the hybrid, in the presence of glucose, activated a chemiluminescence resonance energy transfer process to the QDs, resulting in the luminescence of the QDs. The intensities of the luminescence of the QDs at different concentrations of glucose provided an optical means to detect glucose. PMID:22346648

  1. Activation of NADPH oxidase 1 increases intracellular calcium and migration of smooth muscle cells.

    PubMed

    Zimmerman, Matthew C; Takapoo, Maysam; Jagadeesha, Dammanahalli K; Stanic, Bojana; Banfi, Botond; Bhalla, Ramesh C; Miller, Francis J

    2011-09-01

    Redox-dependent migration and proliferation of vascular smooth muscle cells (SMCs) are central events in the development of vascular proliferative diseases; however, the underlying intracellular signaling mechanisms are not fully understood. We tested the hypothesis that activation of Nox1 NADPH oxidase modulates intracellular calcium ([Ca(2+)](i)) levels. Using cultured SMCs from wild-type and Nox1 null mice, we confirmed that thrombin-dependent generation of reactive oxygen species requires Nox1. Thrombin rapidly increased [Ca(2+)](i), as measured by fura-2 fluorescence ratio imaging, in wild-type but not Nox1 null SMCs. The increase in [Ca(2+)](i) in wild-type SMCs was inhibited by antisense to Nox1 and restored by expression of Nox1 in Nox1 null SMCs. Investigation into potential mechanisms by which Nox1 modulates [Ca(2+)](i) showed that thrombin-induced inositol triphosphate generation and thapsigargin-induced intracellular calcium mobilization were similar in wild-type and Nox1 null SMCs. To examine the effects of Nox1 on Ca(2+) entry, cells were either bathed in Ca(2+)-free medium or exposed to dihydropyridines to block L-type Ca(2+) channel activity. Treatment with nifedipine or removal of extracellular Ca(2+) reduced the thrombin-mediated increase of [Ca(2+)](i) in wild-type SMCs, whereas the response in Nox1 null SMCs was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases, restored the thrombin-induced increase of [Ca(2+)](i) in Nox1 null SMCs. Migration of SMCs was impaired with deficiency of Nox1 and restored with expression of Nox1 or the addition of sodium vanadate. In summary, we conclude that Nox1 NADPH oxidase modulates Ca(2+) mobilization in SMCs, in part through regulation of Ca(2+) influx, to thereby promote cell migration. PMID:21810651

  2. Polyamines and drug oxidations.

    PubMed

    Chapman, S K

    1976-01-01

    The addition of spermine or of spermidine to rat liver assay systems produced marked changes in a number of microsomal drug oxidations. The hydroxylation of aniline and the N-demethylation of ethylmorphine were both enhanced with concentrations of 1-10 mM spermine or of spermidine. The results with putrescine on ethylmorphine metabolism were less dramatic, and no effect could be observed with putrescine in studies with other drug substrates. In contrast to the enhancing effects, inhibition was observed when spermine or spermidine was added to p-nitroanisole O-demethylation assay mixtures, and no effect was observed in assays for acetanilide hydroxylation. The inhibiting and enhancing effects of the polyamines can be observed in assays containing liver preparations from both male and female rats, and those from rats pretreated with phenobarbital or 3-methylcholanthrene. In all studies, the alterations were kinetically noncompetitive. The effects were shown to be independent of the NADPH-generating system and the cation requirements, and were not mediated through an interaction with NADPH-cytochrome c reductase. The possibility is considered that the enhancing and inhibiting effects may be related to the ability of these polycations to bind to microsomal membranes and cause alterations at different sites of substrate interaction. PMID:10139

  3. Cdc42-Dependent Activation of NADPH Oxidase Is Involved in Ethanol-Induced Neuronal Oxidative Stress

    PubMed Central

    Wang, Xin; Ke, Zunji; Chen, Gang; Xu, Mei; Bower, Kimberly A.; Frank, Jacqueline A.; Zhang, Zhuo; Shi, Xianglin; Luo, Jia

    2012-01-01

    It has been suggested that excessive reactive oxygen species (ROS) and oxidative stress play an important role in ethanol-induced damage to both the developing and mature central nervous system (CNS). The mechanisms underlying ethanol-induced neuronal ROS, however, remain unclear. In this study, we investigated the role of NADPH oxidase (NOX) in ethanol-induced ROS generation. We demonstrated that ethanol activated NOX and inhibition of NOX reduced ethanol-promoted ROS generation. Ethanol significantly increased the expression of p47phox and p67phox, the essential subunits for NOX activation in cultured neuronal cells and the cerebral cortex of infant mice. Ethanol caused serine phosphorylation and membrane translocation of p47phox and p67phox, which were prerequisites for NOX assembly and activation. Knocking down p47phox with the small interfering RNA was sufficient to attenuate ethanol-induced ROS production and ameliorate ethanol-mediated oxidative damage, which is indicated by a decrease in protein oxidation and lipid peroxidation. Ethanol activated cell division cycle 42 (Cdc42) and overexpression of a dominant negative (DN) Cdc42 abrogate ethanol-induced NOX activation and ROS generation. These results suggest that Cdc42-dependent NOX activation mediates ethanol-induced oxidative damages to neurons. PMID:22662267

  4. Measurement of polyphenol oxidase activity using optical waveguide lightmode spectroscopy-based immunosensor.

    PubMed

    Kim, Namsoo; Kim, Woo-Yeon

    2015-02-15

    Polyphenol oxidase (PPO) is an important quality index during food processing involving heat-treatment and sensitive determination of PPO activity has been a critical concern in the food industry. In this study, a new measurement of PPO activity exploiting an optical waveguide lightmode spectroscopy-based immunosensor is presented using a polyclonal anti-PPO antibody that was immobilized in situ to the surface of a 3-aminopropyltriethoxysilane-treated optical grating coupler activated with glutaraldehyde. When analysed with a purified PPO fraction from potato tubers, a linear relationship was found between PPO activities of 0.0005607-560.7U/mL and the sensor responses obtained. The sensor was applicable to measurement of PPO activity in real samples that were prepared from potato tubers, grapes and Kimchi cabbage, and the analytical results were compared with those obtained by a conventional colorimetric assay measuring PPO activity. When tested for long-term stability, the sensor was reusable up to 10th day after preparation. PMID:25236218

  5. Blueberry polyphenol oxidase: Characterization and the kinetics of thermal and high pressure activation and inactivation.

    PubMed

    Terefe, Netsanet Shiferaw; Delon, Antoine; Buckow, Roman; Versteeg, Cornelis

    2015-12-01

    Partially purified blueberry polyphenol oxidase (PPO) in Mcllvaine buffer (pH=3.6, typical pH of blueberry juice) was subjected to processing at isothermal-isobaric conditions at temperatures from 30 to 80 °C and pressure from 0.1 to 700 MPa. High pressure processing at 30-50 °C at all pressures studied caused irreversible PPO activity increase with a maximum of 6.1 fold increase at 500 MPa and 30 °C. Treatments at mild pressure-mild temperature conditions (0.1-400 MPa, 60 °C) also caused up to 3 fold PPO activity increase. Initial activity increase followed by a decrease occurred at relatively high pressure-mild temperature (400-600 MPa, 60 °C) and mild pressure-high temperature (0.1-400 MPa, 70-80 °C) combinations. At temperatures higher than 76 °C, monotonic decrease in PPO activity occurred at 0.1 MPa and pressures higher than 500 MPa. The activation/inactivation kinetics of the enzyme was successfully modelled assuming consecutive reactions in series with activation followed by inactivation. PMID:26041182

  6. Activation of Polyphenol Oxidase in Dormant Wild Oat Caryopses by a Seed-Decay Isolate of Fusarium avenaceum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Incubation of dormant wild oat (Avena fatua L., isoline M73) caryopses for 1 to 3 days with Fusarium avenaceum seed-decay isolate F.a.1 induced activity of the plant defense enzyme polyphenol oxidase (PPO). Both extracts and leachates obtained from F.a.1-treated caryopses had decreased abundance of ...

  7. Defining the role of tyrosine and rational tuning of oxidase activity by genetic incorporation of unnatural tyrosine analogs.

    PubMed

    Yu, Yang; Lv, Xiaoxuan; Li, Jiasong; Zhou, Qing; Cui, Chang; Hosseinzadeh, Parisa; Mukherjee, Arnab; Nilges, Mark J; Wang, Jiangyun; Lu, Yi

    2015-04-15

    While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr. PMID:25672571

  8. Levels of Polyamines and Kinetic Characterization of Their Uptake in the Soybean Pathogen Phytophthora sojae

    PubMed Central

    Chibucos, M. Constantine; Morris, Paul F.

    2006-01-01

    Polyamines are ubiquitous biologically active aliphatic cations that are at least transiently available in the soil from decaying organic matter. Our objectives in this study were to characterize polyamine uptake kinetics in Phytophthora sojae zoospores and to quantify endogenous polyamines in hyphae, zoospores, and soybean roots. Zoospores contained 10 times more free putrescine than spermidine, while hyphae contained only 4 times as much free putrescine as spermidine. Zoospores contained no conjugated putrescine, but conjugated spermidine was present. Hyphae contained both conjugated putrescine and spermidine at levels comparable to the hyphal free putrescine and spermidine levels. In soybean roots, cadaverine was the most abundant polyamine, but only putrescine efflux was detected. The selective efflux of putrescine suggests that the regulation of polyamine availability is part of the overall plant strategy to influence microbial growth in the rhizosphere. In zoospores, uptake experiments with [1,4-14C]putrescine and [1,4-14C]spermidine confirmed the existence of high-affinity polyamine transport for both polyamines. Putrescine uptake was reduced by high levels of exogenous spermidine, but spermidine uptake was not reduced by exogenous putrescine. These observations suggest that P. sojae zoospores express at least two high-affinity polyamine transporters, one that is spermidine specific and a second that is putrescine specific or putrescine preferential. Disruption of polyamine uptake or metabolism has major effects on a wide range of cellular activities in other organisms and has been proposed as a potential control strategy for Phytophthora. Inhibition of polyamine uptake may be a means of reducing the fitness of the zoospore along with subsequent developmental stages that precede infection. PMID:16672477

  9. Evaluation of Xanthine Oxidase Inhibitory Potential and In vivo Hypouricemic Activity of Dimocarpus longan Lour. Extracts

    PubMed Central

    Sheu, Shi-Yuan; Fu, Yuan-Tsung; Huang, Wen-Dar; Chen, Yung-Ann; Lei, Yi-Chih; Yao, Chun-Hsu; Hsu, Feng-Lin; Kuo, Tzong-Fu

    2016-01-01

    Background: Longan is a fruit tree known to contain many phenolic components, which are capable of protecting people from oxidative damage through an anti-inflammatory mechanism. It may be also worthwhile to study the effect on lowering uric acid activity. Materials and Methods: This study investigates the lowering of uric acid using longan extracts, including flowers, pericarps, seeds, leaves, and twigs, on potassium-oxonate-induced hyperuricemia mice and its inhibitory actions against xanthine oxidase (XO) activities. Results: The findings revealed that ethyl acetate fraction of longan extracts exhibited strong XO-inhibitory activity, and the flower extracts (IC50 = 115.8 μg/mL) revealed more potent XO-inhibitory activity to those of pericarps (118.9 μg/mL), twigs (125.3 μg/mL), seeds (262.5 μg/mL), and leaves (331.1 μg/mL) in vitro. In addition, different dosages of longan extract (50–100 mg/kg) were administered to hyperuricemic mice. The lowering effect of longan extracts on uric acid at 75 mg/kg markedly reduced plasma uric acid levels in decreasing order: Flowers (80%) > seeds (72%) > pericarps (64%) > twigs (59%) > leaves (41%), compared with allopurinol (89%). Finally, 10 isolated phytochemicals from longan flowers were then examined in vitro. The results indicated that proanthocyanidin A2 and acetonylgeraniin A significantly inhibited XO activity in vitro. This is the first report providing new insights into the urate-reducing effect of phenolic dimer and hydrolyzable tannin, which can be developed to potential hypouricemic agents. SUMMARY Longan flower extracts possess more potent XO-inhibitory activity than pericarps, twigs, seeds, and leaves in vitroThe lowering effect of longan flowers and seeds extracts markedly reduced plasma uric acid levels as compared to allopurinol in vivoThe extract proanthocyanidin A2 and acetonylgeraniin A were demonstrated potent XO inhibitory activity in vitro Abbreviations used: PO: Potassium-oxonate, XO: xanthine

  10. Peroxygenase and Oxidase Activities of Dehaloperoxidase-Hemoglobin from Amphitrite ornata

    PubMed Central

    2015-01-01

    The marine globin dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of monohaloindoles, a previously unknown class of substrate for DHP. Using 5-Br-indole as a representative substrate, the major monooxygenated products were found to be 5-Br-2-oxindole and 5-Br-3-oxindolenine. Isotope labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H2O2, indicative of a previously unreported peroxygenase activity for DHP. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. It was found that 5-Br-3-oxindole, a precursor of the product 5-Br-3-oxindolenine, readily reduced the ferric enzyme to the oxyferrous state, demonstrating an unusual product-driven reduction of the enzyme. As such, DHP returns to the globin-active oxyferrous form after peroxygenase activity ceases. Reactivity with 5-Br-3-oxindole in the absence of H2O2 also yielded 5,5′-Br2-indigo above the expected reaction stoichiometry under aerobic conditions, and O2-concentration studies demonstrated dioxygen consumption. Nonenzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5′-Br2-indigo, and together suggest a newly identified oxidase activity for DHP. PMID:24791647

  11. Potato and mushroom polyphenol oxidase activities are differently modulated by natural plant extracts.

    PubMed

    Kuijpers, Tomas F M; van Herk, Teunie; Vincken, Jean-Paul; Janssen, Renske H; Narh, Deborah L; van Berkel, Willem J H; Gruppen, Harry

    2014-01-01

    Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the outcome of such screening, this study compared the effect of 60 plant extracts on the activity of PPO from mushroom ( Agaricus bisporus , AbPPO) and PPO from potato ( Solanum tuberosum , StPPO). Some plant extracts had different effects on the two PPOs: an extract that inhibited one PPO could be an activator for the other. As an example of this, the mate ( Ilex paraguariensis ) extract was investigated in more detail. In the presence of mate extract, oxygen consumption by AbPPO was found to be reduced >5-fold compared to a control reaction, whereas that of StPPO was increased >9-fold. RP-UHPLC-MS analysis showed that the mate extract contained a mixture of phenolic compounds and saponins. Upon incubation of mate extract with StPPO, phenolic compounds disappeared completely and saponins remained. Flash chromatography was used to separate saponins and phenolic compounds. It was found that the phenolic fraction was mainly responsible for inhibition of AbPPO and activation of StPPO. Activation of StPPO was probably caused by activation of latent StPPO by chlorogenic acid quinones. PMID:24344979

  12. Developmental onset of mixed-function oxidase activity in preimplantation mouse embryos

    SciTech Connect

    Filler, R.; Lew, K.J.

    1981-11-01

    Two-cell embryos, obtained from the C57BL/6N and DBA/2N strains, were cultured in media that supported in vitro differentiation and that contained (/sup 3/H)benzo(a)pyrene. High-pressure liquid chromatography of the activated intermediates formed during in vitro early embryonic development indicated that the onset of polynuclear aromatic hydrocarbon activation coincided with blastocyst formation. Comparison of individual oxygenated intermediates metabolically formed from embryos genetically ''responsive'' or ''nonresponsive'' to aromatic hydrocarbons revealed significant quantitative differences in the production of dihydrodiol, quinone, and phenolic derivatives. In addition to exhibiting basal mixed-function oxidase activity, blastocysts were also responsive to enzymatic induction when exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. The presence of operative metabolite-detoxifying pathways was also assayed. Enzymatic treatment of water-soluble metabolites with ..beta..-glucuronidase or arylsulfatase revealed that neither glucuronic acid conjugates nor sulfate ester derivatives were present. These data, therefore, provide direct evidence that late preimplanation mouse embryos (day 3 1/2 of gestation) are similar to later developmental stages in having the enzymatic capability for xenobiotic activation and enzyme induction but are dissimilar with respect to their detoxification mechanisms(s). Moreover, the ability of preimplantation embryos to activate directly polynuclear aromatic hydrocarbon to bioreactive intermediates may be of importance in assessing the ontological susceptibility of the developing embryo to carcinogenic or teratogenic chemicals.

  13. Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production.

    PubMed

    Bal-Price, Anna; Matthias, Anita; Brown, Guy C

    2002-01-01

    Cultured rat microglia produced extracellular superoxide at a rate of 814 +/- 52 pmol/min/million cells when stimulated with phorbol 12-myristate 13-acetate (PMA) as measured by extracellular cytochrome c reduction. This superoxide production resulted in a rapid rate of superoxide dismutase-sensitive nitric oxide (NO) breakdown (155 +/- 30 pmol of NO/min/million cells) when NO was added to PMA stimulated microglia. Lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-activated microglia produce NO at the rate of 145 +/- 42 pmol/min/million cells and activated astrocytes at the rate of 51 +/- 9 pmol/min/million cells as estimated by NO electrode. Both types of cells maintained a steady-state level of 0.5-0.7 microm NO, only in the presence of L-arginine. Addition of PMA to activated microglia (but not activated astrocytes) caused the rapid and complete disappearance of all extracellular NO (but was restored in the presence of superoxide dismutase) followed by the production of peroxynitrite (as measured by urate-sensitive oxidation of dihydrorhodamine). Co-incubation of activated microglia with cerebellar granule neurones resulted in NO inhibition of neuronal respiration, but this was rapidly removed by PMA-induced breakdown of the NO. Thus, microglial NADPH oxidase can regulate the bioavailability of NO and the production of peroxynitrite. PMID:11796745

  14. 2-Arylquinazolin-4(3H)-ones: Inhibitory Activities Against Xanthine Oxidase.

    PubMed

    Zafar, Humaira; Saad, Syed M; Perveen, Shahnaz; Arshia; Malik, Rizwana; Khan, Ajmal; Khan, Khalid M; Choudhary, Muhammad I

    2016-01-01

    2-Arylquinazolin-4(3H)-ones (1-25) were synthesized, and evaluated for their xanthine oxidase inhibitory activity. Significant to moderate activities were exhibited by the compounds 1-3, 7, 9, 13-15, 19-21, and 23 with IC50 between 2.80 - 28.13 µM as compared to the standard allopurinol (IC50 (IC50 = 2.01 ± 0.01 µM). Compounds 4-6, 8, 11-12, 16-18, 22, and 24 demonstrated a weak activity with IC50 values 44.60 - 112.60 µM. Nonetheless, compounds 10 and 25 did not show any activity. Amongst all derivatives, compound 2, containing a C-4´ dimethylamino group, was the most potent inhibitor of the enzyme with an IC50 value comparable to the standard. Kinetics studies on the most active compounds (2, 7, 9, 14, 15, 19, and 20) were conducted in order to determine their modes of inhibition and dissociation constants Ki. Some of the compounds of 2-arylquinazolin-4(3H)-one series were thus identified as potential leads for further studies towards the treatment of hyperuricemia and gout. PMID:26256588

  15. Alteration of serum semicarbazide-sensitive amine oxidase activity in chronic renal failure.

    PubMed

    Nemcsik, J; Szökö, E; Soltész, Zs; Fodor, E; Toth, L; Egresits, J; Tábi, T; Magyar, K; Kiss, I

    2007-01-01

    Despite recent intensive investigations, physiological and pathological role of semicarbazide-sensitive amine oxidase (SSAO) is far from clear. In this study, serum SSAO activity was determined, radiochemically, in various groups of uremic patients: haemodialysed (HD), peritoneally dialysed (PD) and those receiving conservative treatment but still not dialysed (ND), as well as in controls. Reduced enzyme activity was found in HD uremic patients before and after dialysis treatment, compared to controls (5260 +/- 862 and 6011 +/- 958 pmol/h/ml vs. 8601 +/- 283 pmol/h/ml, p < 0.01 and p < 0.05, respectively). The activity was slightly lower in PD, and normal in ND patients. In HD patients SSAO activity was also determined by an assay based on the formation of hydrogen peroxide, and was found to be elevated compared to controls (2384 +/- 323 pmol/h/ml vs. 1437 +/- 72 pmol/h/ml, p < 0.05). The elevated serum SSAO activity measured through the detection of the enzyme-generated hydrogen peroxide in HD patients might indicate its contribution to the accelerated atherosclerotic disease observed in uremia. PMID:17431736

  16. Mutation at a Strictly-Conserved, Active-Site Tyrosine in the Copper Amine Oxidase Leads to Uncontrolled Oxygenase Activity†

    PubMed Central

    Chen, Zhi-wei; Datta, Saumen; DuBois, Jennifer L.; Klinman, Judith P.; Mathews, F. Scott

    2010-01-01

    The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ), and the subsequent oxidative deamination of substrate amines. Because the same active-site pocket must facilitate both reactions, individual active-site residues may serve multiple roles. We have examined the roles of a strictly-conserved active-site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically, and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild-type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy-intermediates in cofactor biogenesis, but also indicates the critical control of oxidation chemistry that can be conferred by a single active-site residue. PMID:20684524

  17. Regulatory effects of polyamines on membrane-bound acetylcholinesterase

    PubMed Central

    Kossorotow, A.; Wolf, H. U.; Seiler, N.

    1974-01-01

    The effects of putrescene, spermidine and spermine on membrane-bound acetylcholinesterase from human erythrocyte `ghosts' and the solubilized enzyme of the electric organ of the electric eel were studied by kinetic methods. Measurements were made by using a photometric method which made it possible to record the enzyme reaction in the steady-state phase. Substrate-concentration-dependent activation and inhibition of acetylcholinesterase by polyamines is similar to that by Na+, K+, Ca2+, Mg2+ and certain quaternary and bisquaternary amines. The kinetics suggest an allosteric reaction mechanism. On the basis of the kinetic results a role for the polyamines as modulators of synaptic acetylcholinesterase is proposed. PMID:4462573

  18. Effect of the polyamine analogue N1,N11-diethylnorspermine on cell survival and susceptibility to apoptosis of human chondrocytes.

    PubMed

    Stanic, Ivana; Cetrullo, Silvia; Facchini, Annalisa; Stefanelli, Claudio; Borzì, Rosa Maria; Tantini, Benedetta; Guarnieri, Carlo; Caldarera, Claudio Marcello; Flamigni, Flavio

    2008-07-01

    Chondrocyte survival is closely linked to cartilage integrity, and forms of chondrocyte apoptotic death can contribute to cartilage degeneration in articular diseases. Since growing evidence also implicates polyamines in the control of cell death, we have been investigating the role of polyamine metabolism in chondrocyte survival and apoptosis. Treatment of human C-28/I2 chondrocytes with N(1),N(11)-diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, inhibited polyamine biosynthesis and induced polyamine catabolism, thus rapidly depleting all main polyamines. DENSPM did not increase significantly caspase activity, but provoked a late cell death associated to DNA fragmentation. A short treatment with DENSPM did not reduce cell viability when given alone, but enhanced caspase-3 and -9 activation in chondrocytes exposed to tumor necrosis factor-alpha (TNF) and cycloheximide (CHX). A longer treatment with DENSPM however reduced caspase response to TNF plus CHX. Depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not cause cell death and contrasted apoptosis by decreasing caspase activities. In conclusion, following DENSPM treatment, C-28/I2 chondrocytes are initially sensitized to caspase 9-dependent apoptosis in the presence of TNF and CHX and may eventually undergo a late and mainly caspase-independent cell death in the absence of other stimuli. Moreover, these results indicate that a reduction of polyamine levels not only leads to inhibition of cell proliferation, but also of caspase-mediated pathways of chondrocyte apoptosis. PMID:18231987

  19. Polyamine Uptake, Kinetics, and Competition among Polyamines and between Polyamines and Inorganic Cations.

    PubMed

    Pistocchi, R; Bagni, N; Creus, J A

    1986-02-01

    Polyamine uptake, the kinetics of this uptake, and the competition among polyamines and between polyamines and inorganic cations were studied in petals of Saintpaulia ionantha Wendl. Uptake experiments using (14)C-labeled polyamines were carried out on single petals, at room temperaure (20 degrees C) and in the light. The results show that putrescine, spermidine, and spermine uptake was dependent on the external pH and occurred up to high external polyamine concentrations with K(m) values of 8.6, 1.2, and 2.1 millimolar, respectively, with spermidine being the most absorbed at low concentration (17 micromolar). Putrescine and spermidine did not seem to compete for the same site of absorption. Furthermore, putrescine and spermidine uptake was not inhibited by Ca(2+), Mg(2+), and K(+) at the same concentrations (17 micromolar), whereas 1.7 millimolar Ca(2+) inhibited and K(+) enhanced spermidine uptake. The intracellular localization of the absorbed putrescine was determined using two different methods. Very little label was found in the apoplast, while most of it was localized in the 98,500g supernatant. According to our data the vacuole, which represents a substantial part of Saintpaulia parenchyma cells, could be a site of putrescine accumulation. 2,4-Dinitrophenol and diethylstilbestrol did not inhibit uptake; however, at 0 degrees C there was a 35% inhibition of spermidine uptake, compared with the controls kept at 20 degrees C as well as a 68% inhibition with 20 millimolar NaSCN. PMID:16664661

  20. Molecular Basis of Reduced Pyridoxine 5′-Phosphate Oxidase Catalytic Activity in Neonatal Epileptic Encephalopathy Disorder*

    PubMed Central

    Musayev, Faik N.; Di Salvo, Martino L.; Saavedra, Mario A.; Contestabile, Roberto; Ghatge, Mohini S.; Haynes, Alexina; Schirch, Verne; Safo, Martin K.

    2009-01-01

    Mutations in pyridoxine 5′-phosphate oxidase are known to cause neonatal epileptic encephalopathy. This disorder has no cure or effective treatment and is often fatal. Pyridoxine 5′-phosphate oxidase catalyzes the oxidation of pyridoxine 5′-phosphate to pyridoxal 5′-phosphate, the active cofactor form of vitamin B6 required by more than 140 different catalytic activities, including enzymes involved in amino acid metabolism and biosynthesis of neurotransmitters. Our aim is to elucidate the mechanism by which a homozygous missense mutation (R229W) in the oxidase, linked to neonatal epileptic encephalopathy, leads to reduced oxidase activity. The R229W variant is ∼850-fold less efficient than the wild-type enzyme due to an ∼192-fold decrease in pyridoxine 5′-phosphate affinity and an ∼4.5-fold decrease in catalytic activity. There is also an ∼50-fold reduction in the affinity of the R229W variant for the FMN cofactor. A 2.5 Å crystal structure of the R229W variant shows that the substitution of Arg-229 at the FMN binding site has led to a loss of hydrogen-bond and/or salt-bridge interactions between FMN and Arg-229 and Ser-175. Additionally, the mutation has led to an alteration of the configuration of a β-strand-loop-β-strand structure at the active site, resulting in loss of two critical hydrogen-bond interactions involving residues His-227 and Arg-225, which are important for substrate binding and orientation for catalysis. These results provide a molecular basis for the phenotype associated with the R229W mutation, as well as providing a foundation for understanding the pathophysiological consequences of pyridoxine 5′-phosphate oxidase mutations. PMID:19759001

  1. Serotonin 2A and 2B receptor-induced phrenic motor facilitation: differential requirement for spinal NADPH oxidase activity

    PubMed Central

    MacFarlane, P.M.; Vinit, S.; Mitchell, G.S.

    2011-01-01

    Acute intermittent hypoxia (AIH) facilitates phrenic motor output by a mechanism that requires spinal serotonin (type 2) receptor activation, NADPH oxidase activity and formation of reactive oxygen species (ROS). Episodic spinal serotonin (5-HT) receptor activation alone, without changes in oxygenation, is sufficient to elicit NADPH oxidase-dependent phrenic motor facilitation (pMF). Here we investigated: 1) whether serotonin 2A and/or 2B (5-HT2a/b) receptors are expressed in identified phrenic motor neurons, and 2) which receptor subtype is capable of eliciting NADPH-oxidase-dependent pMF. In anesthetized, artificially ventilated adult rats, episodic C4 intrathecal injections (3 × 6µl injections, 5 min intervals) of a 5-HT2a (DOI) or 5-HT2b (BW723C86) receptor agonist elicited progressive and sustained increases in integrated phrenic nerve burst amplitude (i.e. pMF), an effect lasting at least 90 minutes post-injection for both receptor subtypes. 5-HT2a and 5-HT2b receptor agonist-induced pMF were both blocked by selective antagonists (ketanserin and SB206553, respectively), but not by antagonists to the other receptor subtype. Single injections of either agonist failed to elicit pMF, demonstrating a need for episodic receptor activation. Phrenic motor neurons retrogradely labeled with cholera toxin B fragment expressed both 5-HT2a and 5-HT2b receptors. Pre-treatment with NADPH oxidase inhibitors (apocynin and DPI) blocked 5-HT2b, but not 5-HT2a-induced pMF. Thus, multiple spinal type 2 serotonin receptors elicit pMF, but they act via distinct mechanisms that differ in their requirement for NADPH oxidase activity. PMID:21223996

  2. Association between remote organ injury and tissue polyamine homeostasis in acute experimental pancreatitis - treatment with a polyamine analogue bismethylspermine.

    PubMed

    Jin, Hai-Tao; Lämsä, Teemu; Nordback, Panu H; Hyvönen, Mervi T; Grigorenko, Nikolay; Khomutov, Alex R; Nordback, Isto; Räty, Sari; Pörsti, Ilkka; Alhonen, Leena; Sand, Juhani

    2011-01-01

    Experimental pancreatitis is associated with activation of polyamine catabolism. The polyamine analog bismethylspermine (Me(2)Spm) can ameliorate pancreatic injury. We investigated the roles of polyamine catabolism in remote organs during pancreatitis and explored the mechanism of polyamine catabolism by administering Me(2)Spm. Acute pancreatitis was induced by an infusion of 2 or 6% taurodeoxycholate before Me(2)Spm administration. Blood, urine and tissues were sampled at 24 and 72 h to assess multi-organ injury and polyamine catabolism. The effect of Me(2)Spm on mortality in experimental pancreatitis was tested separately. Liver putrescine levels were elevated following liver injury. Me(2)Spm increased the activity of spermidine/spermine N(1)-acetyltransferase (SSAT) and depleted the spermidine, spermine or putrescine levels. Lung putrescine levels increased, and SSAT and spermine decreased following lung injury. Me(2)Spm enhanced the activity of SSAT and decreased the spermidine and spermine levels. Renal injury was manifested as an increase in creatinine or a decrease in urine output. Decreases in kidney SSAT, spermidine or spermine and an increase in putrescine were found during pancreatitis. In the 2% taurodeoxycholate model, Me(2)Spm decreased urine output and raised plasma creatinine levels. Me(2)Spm increased SSAT and decreased polyamines. Excessive Me(2)Spm accumulated in the kidney, and greater amounts were found in the 6% taurodeoxycholate model in which this mortality was not reduced by Me(2)Spm. In the 2% taurodeoxycholate model, Me(2)Spm dose-dependently induced mortality at 72 h. Like pancreatic injury, remote organ injury in pancreatitis is associated with increased putrescine levels. However, Me(2)Spm could not ameliorate multi-organ injury. Me(2)Spm administration was associated with significant renal toxicity and induced mortality, suggesting that the current dose is too high and needs to be modified. PMID:22001988

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

    PubMed Central

    Sasabe, Jumpei; Suzuki, Masataka; Imanishi, Nobuaki; Aiso, Sadakazu

    2014-01-01

    It has been proposed that D-amino acid oxidase (DAO) plays an essential role in degrading D-serine, an endogenous coagonist of N-methyl-D-aspartate (NMDA) glutamate receptors. DAO shows genetic association with amyotrophic lateral sclerosis (ALS) and schizophrenia, in whose pathophysiology aberrant metabolism of D-serine is implicated. Although the pathology of both essentially involves the forebrain, in rodents, enzymatic activity of DAO is hindbrain-shifted and absent in the region. Here, we show activity-based distribution of DAO in the central nervous system (CNS) of humans compared with that of mice. DAO activity in humans was generally higher than that in mice. In the human forebrain, DAO activity was distributed in the subcortical white matter and the posterior limb of internal capsule, while it was almost undetectable in those areas in mice. In the lower brain centers, DAO activity was detected in the gray and white matters in a coordinated fashion in both humans and mice. In humans, DAO activity was prominent along the corticospinal tract, rubrospinal tract, nigrostriatal system, ponto-/olivo-cerebellar fibers, and in the anterolateral system. In contrast, in mice, the reticulospinal tract and ponto-/olivo-cerebellar fibers were the major pathways showing strong DAO activity. In the human corticospinal tract, activity-based staining of DAO did not merge with a motoneuronal marker, but colocalized mostly with excitatory amino acid transporter 2 and in part with GFAP, suggesting that DAO activity-positive cells are astrocytes seen mainly in the motor pathway. These findings establish the distribution of DAO activity in cerebral white matter and the motor system in humans, providing evidence to support the involvement of DAO in schizophrenia and ALS. Our results raise further questions about the regulation of D-serine in DAO-rich regions as well as the physiological/pathological roles of DAO in white matter astrocytes. PMID:24959138

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

    SciTech Connect

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

    1983-09-01

    Narcotic withdrawal is often accompanied by an atypical depression which responds to resumption of narcotics. It was hypothesized that methadone might exert its antidepressant effects through monoamine oxidase (MAO) inhibition. The current study examined /sub 3/H-methadone distribution in rat brain and effects on regional MAO activity with acute doses (2.5 mg/kg) which approximate those found during chronic methadone maintenance in man. Limbic areas (amygdala, basomedial hypothalamus, caudate-putamen, hippocampus, preoptic nucleus), as well as pituitary and liver were assayed for MAO activity and methadone concentration. MAO activities did not differ significantly in acute methadone or saline-treated cage-mates at 1 or 24 hr. The concentrations of methadone at 1 hr ranged between 17 and 223 ng/100 mg wet wt tissue in the preoptic nucleus and pituitary, respectively. No significant correlation was found between change in MAO activity (MAO methadone/MAO saline) and methadone concentration in any region at 1 or 24 hr. This study does not support the hypothesis that methadone acts as an antidepressant through MAO inhibition, at least not following acute administration of this exogenous opioid.

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

    PubMed

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

    2015-11-01

    Chrysin, a bioactive flavonoid, was investigated for its potential to inhibit the activity of xanthine oxidase (XO), a key enzyme catalyzing xanthine to uric acid and finally causing gout. The kinetic analysis showed that chrysin possessed a strong inhibition on XO ability in a reversible competitive manner with IC50 value of (1.26±0.04)×10(-6)molL(-1). The results of fluorescence titrations indicated that chrysin bound to XO with high affinity, and the interaction was predominately driven by hydrogen bonds and van der Waals forces. Analysis of circular dichroism demonstrated that chrysin induced the conformational change of XO with increases in α-helix and β-sheet and reductions in β-turn and random coil structures. Molecular simulation revealed that chrysin interacted with the amino acid residues Leu648, Phe649, Glu802, Leu873, Ser876, Glu879, Arg880, Phe1009, Thr1010, Val1011 and Phe1013 located within the active cavity of XO. The mechanism of chrysin on XO activity may be the insertion of chrysin into the active site occupying the catalytic center of XO to avoid the entrance of xanthine and causing conformational changes in XO. Furthermore, the interaction assays indicated that chrysin and its structural analog apigenin exhibited an additive effect on inhibition of XO. PMID:26275460

  6. Xanthine oxidase inhibitory activities and crystal structures of methoxyflavones from Kaempferia parviflora rhizome.

    PubMed

    Nakao, Kikuyo; Murata, Kazuya; Deguchi, Takahiro; Itoh, Kimihisa; Fujita, Takanori; Higashino, Masayuki; Yoshioka, Yuri; Matsumura, Shin-Ichi; Tanaka, Rika; Shinada, Tetsuro; Ohfune, Yasufumi; Matsuda, Hideaki

    2011-01-01

    Kaempferia parviflora (KP), a Zingiberaceae plant, is used as a folk medicine in Thailand for the treatment of various symptoms, including general pains, colic gastrointestinal disorders, and male impotence. In this study, the inhibitory activities of KP against xanthine oxidase (XOD) were investigated. The extract of KP rhizomes showed more potent inhibitory activity (38% at 500 µg/ml) than those of the other Zingiberaceae plants tested. Ten methoxyflavones were isolated from the KP extract as the major chemical components and their chemical structures were elucidated by X-ray crystallography. The structurally confirmed methoxyflavones were subjected to the XOD inhibitory test. Among them, 3,5,7,4',5'-pentamethoxyflavone and 3',4',5,7-tetramethoxyflavone showed inhibitory activities (IC(50) of 0.9 and >4 mM, respectively) and their modes of inhibition are clarified as competitive/non-competitive mixed type. To the best of our knowledge, this is the first report to present the inhibitory activities of KP, 3,5,7,4',5'-pentamethoxyflavone and 3',4',5,7-tetramethoxyflavone against XOD. PMID:21720029

  7. A sensitive method to assay the xanthine oxidase activity in primary cultures of cerebellar granule cells.

    PubMed

    Atlante, A; Valenti, D; Gagliardi, S; Passarella, S

    2000-11-01

    Since xanthine oxidase (XO, Xanthine:oxidoreductase, E.C.1.2.3.22) is a key enzyme in reactive oxygen specie formation which plays a major role in cell oxidative stress, the availability of a sensitive and simple assay useful to detect its activity in monolayer cell cultures is worthwhile. In order to achieve this, we developed a method in which the conversion of pterine into isoxanthopterin is monitored fluorimetrically. Temperature assay was 50 degrees C. The activity of XO was detected in cerebellar granule cells exposed to glutamate. Since XO is formed from protease-dependent xanthine dehydrogenase processing, its activity appearance was found to be prevented by the protease inhibitor, leupeptin, as well as the glutamate NMDA-receptor inhibitor, MK-801, and the Ca(++) complexing agent, EGTA. The reported novel protocol, at variance with a conventional method, is shown to be a simple, fast, sensitive and relatively cheap method to assay XO activity. In addition, the reported assay can be applied to any cell type in culture. PMID:11086257

  8. Cardiolipin linoleic acid content and mitochondrial cytochrome c oxidase activity are associated in rat skeletal muscle.

    PubMed

    Fajardo, Val Andrew; McMeekin, Lauren; Saint, Caitlin; LeBlanc, Paul J

    2015-04-01

    Cardiolipin (CL) is an inner-mitochondrial membrane phospholipid that is important for optimal mitochondrial function. Specifically, CL and CL linoleic (18:2ω6) content are known to be positively associated with cytochrome c oxidase (COX) activity. However, this association has not been examined in skeletal muscle. In this study, rats were fed high-fat diets with a naturally occurring gradient in linoleic acid (coconut oil [CO], 5.8%; flaxseed oil [FO], 13.2%; safflower oil [SO], 75.1%) in an attempt to alter both mitochondrial CL fatty acyl composition and COX activity in rat mixed hind-limb muscle. In general, mitochondrial membrane lipid composition was fairly resistant to dietary treatments as only modest changes in fatty acyl composition were detected in CL and other major mitochondrial phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). As a result of this resistance, CL 18:2ω6 content was not different between the dietary groups. Consistent with the lack of changes in CL 18:2ω6 content, mitochondrial COX activity was also not different between the dietary groups. However, correlational analysis using data obtained from rats across the dietary groups showed a significant relationship (p = 0.009, R(2) = 0.21). Specifically, our results suggest that CL 18:2ω6 content may positively influence mitochondrial COX activity thereby making this lipid molecule a potential factor related to mitochondrial health and function in skeletal muscle. PMID:25727371

  9. Vascular smooth muscle cell proliferation depends on caveolin-1-regulated polyamine uptake

    PubMed Central

    Grossi, Mario; Rippe, Catarina; Sathanoori, Ramasri; Swärd, Karl; Forte, Amalia; Erlinge, David; Persson, Lo; Hellstrand, Per; Nilsson, Bengt-Olof

    2014-01-01

    Much evidence highlights the importance of polyamines for VSMC (vascular smooth muscle cell) proliferation and migration. Cav-1 (caveolin-1) was recently reported to regulate polyamine uptake in intestinal epithelial cells. The aim of the present study was to assess the importance of Cav-1 for VSMC polyamine uptake and its impact on cell proliferation and migration. Cav-1 KO (knockout) mouse aortic cells showed increased polyamine uptake and elevated proliferation and migration compared with WT (wild-type) cells. Both Cav-1 KO and WT cells expressed the smooth muscle differentiation markers SM22 and calponin. Cell-cycle phase distribution analysis revealed a higher proportion of Cav-1 KO than WT cells in the S phase. Cav-1 KO cells were hyper-proliferative in the presence but not in the absence of extracellular polyamines, and, moreover, supplementation with exogenous polyamines promoted proliferation in Cav-1 KO but not in WT cells. Expression of the solute carrier transporters Slc7a1 and Slc43a1 was higher in Cav-1 KO than in WT cells. ODC (ornithine decarboxylase) protein and mRNA expression as well as ODC activity were similar in Cav-1 KO and WT cells showing unaltered synthesis of polyamines in Cav-1 KO cells. Cav-1 was reduced in migrating cells in vitro and in carotid lesions in vivo. Our data show that Cav-1 negatively regulates VSMC polyamine uptake and that the proliferative advantage of Cav-1 KO cells is critically dependent on polyamine uptake. We provide proof-of-principle for targeting Cav-1-regulated polyamine uptake as a strategy to fight unwanted VSMC proliferation as observed in restenosis. PMID:25301005

  10. Surface modification of an epoxy resin with polyamines and polydopamine: The effect on the initial electroless copper deposition

    NASA Astrophysics Data System (ADS)

    Schaubroeck, David; Mader, Lothar; De Geyter, Nathalie; Morent, Rino; Dubruel, Peter; Vanfleteren, Jan

    2014-06-01

    This paper describes the influence of polydopamine and polyamine surface modifications of an etched epoxy cresol novolak (ECN) resin on the initial electroless copper deposition. Three different strategies to introduce polyamines on a surface in aqueous environment are applied: via polyethyleneimine adsorption (PEI), via polydopamine and via polyamines grafted to polydopamine. Next, the influence of these surface modifications on the catalytic palladium activation is investigated through X-ray photoelectron spectroscopy (XPS) analysis. Finally, the initial electroless copper deposition on modified epoxy surfaces is evaluated using SEM and Energy Dispersive Spectroscopy (EDS). Grafted polyamines on polydopamine surface modifications result in a large increase of the initial deposited copper.

  11. Aliphatic polyamines in physiology and diseases.

    PubMed

    Ramani, D; De Bandt, J P; Cynober, L

    2014-02-01

    Aliphatic polyamines are a family of polycationic molecules derived from decarboxylation of the amino acid ornithine that classically comprise three molecules: putrescine, spermidine and spermine. In-cell polyamine homeostasis is tightly controlled at key steps of cell metabolism. Polyamines are involved in an array of cellular functions from DNA stabilization, and regulation of gene expression to ion channel function and, particularly, cell proliferation. As such, aliphatic polyamines play an essential role in rapidly dividing cells such as in the immune system and digestive tract. Because of their role in cell proliferation, polyamines are also involved in carcinogenesis, prompting intensive research into polyamine metabolism as a target in cancer therapy. More recently, another aliphatic polyamine, agmatine, the decarboxylated derivative of arginine, has been identified as a neurotransmitter in mammals, and investigations have focused on its effects in the CNS, notably as a neuroprotector in brain injury. PMID:24144912

  12. NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A*S⃞

    PubMed Central

    Frasch, S. Courtney; Berry, Karin Zemski; Fernandez-Boyanapalli, Ruby; Jin, Hyun-Sun; Leslie, Christina; Henson, Peter M.; Murphy, Robert C.; Bratton, Donna L.

    2008-01-01

    Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation. PMID:18824544

  13. ASIC-like Currents in Freshly Isolated Cerebral Artery Smooth Muscle Cells are Inhibited by Endogenous Oxidase Activity

    PubMed Central

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

    2011-01-01

    Background/Aims: The aim of this study was to determine if VSMC ASIC-like currents are regulated by oxidative state. Methods: We used whole-cell patch clamp of isolated mouse cerebral VSMCs to determine if 1) reducing agents, such as DTT and GSH, and 2) inhibition of endogenous oxidase activity from NADPH and Xanthine oxidases potentiate active currents and activate electrically silent currents. Results: Pretreatment with 2 mM DTT or GSH, increased the mean peak amplitude of ASIC-like currents evoked by pH 6.0 from 0.4 ± 0.1 to 14.9 ± 3.6 pA/pF, and from 0.9 ± 0.3 to 11.3 ± 2.4 pA/pF, respectively. Pretreatment with apocynin, a NADPH oxidase inhibitor, mimics the effect of the reducing agents, with the mean peak current amplitude increased from 0.9 ± 0.5 to 7.0 ± 2.6 pA/pF and from 0.5 ± 0.2 to 26.4 ± 6.8 pA/pF by 50 and 200 μM apocynin, respectively. Pretreatment with allopurinol, a xanthine oxidase inhibitor, also potentiates the VSMC ASIC-like activity. Conclusion: These findings suggest that VSMC ASIC-like channels are regulated by oxidative state and may be inhibited by basal endogenous oxidative sources such as NADPH and xanthine oxidase. PMID:21325830

  14. Induction of mixed-function oxidase activity in mouse lymphoid tissues by polycyclic aromatic hydrocarbons

    SciTech Connect

    Griffin, G.D.; Egan, B.Z.; Lee, N.E.; Burtis, C.A.

    1986-01-01

    Polycyclic aromatic hydrocarbon (PAH) exposure can cause mixed-function oxidase (MFO) enzyme induction in certain tissues of various organisms. Measurements of such induction might serve as a useful bioindicator of human exposure to PAHs, provided readily obtainable human tissues can be utilized for such measurements. The authors have investigated the MFO activity in various lymphoid tissues of the C3H mouse as a model system and have studied the effect of systemic PAH treatment on such enzyme activity. An MFO enzyme assay was used to measure the activity of 7-ethoxyresorufin deethylase, an enzyme activity that may be specific for the cytochrome P-448 subset of MFO enzymes (those enzymes that are induced in cells or tissues following PAH administration). Intraperitoneal injection of mice with 180 mg/kg (4.6 mg) benzo(a)pyrene (BaP) or 160 mg/kg (4.0 mg) 3-methylcholanthrene (MC) produced a significant induction in MFO activity in mouse spleen S9 fractions 48 h after the injection. Induction ratios (induced activity/control activity) between 4 and 5 were seen with BaP; MC produced induction ratios of 2.5-3.0. Enzyme activity was not induced in the spleen within 16 h following BaP or MC administration. Other experiments indicated that MFO activity could be induced in thymus cells 48 h after either BaP or MC treatment. Treatment with BaP or MC did produce significant enzyme induction in the liver and lung tissues from the animals both 16 and 48 h after chemical treatment.

  15. Arginase 2 deficiency reduces hyperoxia-mediated retinal neurodegeneration through the regulation of polyamine metabolism.

    PubMed

    Narayanan, S P; Xu, Z; Putluri, N; Sreekumar, A; Lemtalsi, T; Caldwell, R W; Caldwell, R B

    2014-01-01

    Hyperoxia treatment has been known to induce neuronal and glial death in the developing central nervous system. Retinopathy of prematurity (ROP) is a devastating disease in premature infants and a major cause of childhood vision impairment. Studies indicate that, in addition to vascular injury, retinal neurons are also affected in ROP. Using an oxygen-induced retinopathy (OIR) mouse model for ROP, we have previously shown that deletion of the arginase 2 (A2) significantly reduced neuro-glial injury and improved retinal function. In the current study, we investigated the mechanism of A2 deficiency-mediated neuroprotection in the OIR retina. Hyperoxia treatment has been known to induce neuronal death in neonates. During the hyperoxia phase of OIR, a significant increase in the number of apoptotic cells was observed in the wild-type (WT) OIR retina compared with A2-deficient OIR. Mass spectrometric analysis showed alterations in polyamine metabolism in WT OIR retina. Further, increased expression level of spermine oxidase was observed in WT OIR retina, suggesting increased oxidation of polyamines in OIR retina. These changes were minimal in A2-deficient OIR retina. Treatment using the polyamine oxidase inhibitor, N, N'-bis (2, 3-butadienyl)-1, 4-butanediamine dihydrochloride, significantly improved neuronal survival during OIR treatment. Our data suggest that retinal arginase is involved in the hyperoxia-induced neuronal degeneration in the OIR model, through the regulation of polyamine metabolism. PMID:24556690

  16. Inhibition of monoamine oxidase-A activity in rat brain by synthetic hydrazines: structure-activity relationship (SAR).

    PubMed

    Dar, Ahsana; Khan, Khalid M; Ateeq, Humayun S; Khan, Shagufta; Rahat, Shagufta; Perveen, Shahnaz; Supuran, Claudiu T

    2005-06-01

    A series of hydrazine derivatives was synthesized in order to evaluate their monoamine oxidase A (MAO-A) inhibitory effects. MAO-A inhibitory activity of 4-tosyl benzoic acid carbohydrazide was quite potent, similarly to that of the corresponding 4-benzyloxy-benzoic acid carbohydrazide and its N-cyanoethylated derivative. Structural variations of these compounds, such as the replacement of the 4-substitutent, of the aromatic ring on which the carbohydrazide moiety is grafted, as well as cyclization of the hydrazide moiety in five- or six-membered rings caused either significant decline or complete loss of MAO inhibitory properties. The most active compound (4-tosyl benzoic acid carbohydrazide) was also subjected to the forced swim test, an animal model of depression, eliciting a marked reduction in immobility time in rats, without affecting the locomotor activity, implying that it possesses anti-depressant properties due to inhibition of MAO type-A. PMID:16119198

  17. Global gene expression profiling of the polyamine system in suicide completers.

    PubMed

    Fiori, Laura M; Bureau, Alexandre; Labbe, Aurélie; Croteau, Jordie; Noël, Simon; Mérette, Chantal; Turecki, Gustavo

    2011-06-01

    In recent years, gene expression, genetic association, and metabolic studies have implicated the polyamine system in psychiatric conditions, including suicide. Given the extensive regulation of genes involved in polyamine metabolism, as well as their interconnections with the metabolism of other amino acids, we were interested in further investigating the expression of polyamine-related genes across the brain in order to obtain a more comprehensive view of the dysregulation of this system in suicide. To this end, we examined the expression of genes related to polyamine metabolism across 22 brain regions in a sample of 29 mood-disordered suicide completers and 16 controls, and identified 14 genes displaying differential expression. Among these, altered expression of spermidine/spermine N1-acetyltransferase, spermine oxidase, and spermine synthase, has previously been observed in brains of suicide completers, while the remainder of the genes represent novel findings. In addition to genes with direct involvement in polyamine metabolism, including S-adenosylmethionine decarboxylase, ornithine decarboxylase antizymes 1 and 2, and arginase II, we identified altered expression of several more distally related genes, including aldehyde dehydrogenase 3 family, member A2, brain creatine kinase, mitochondrial creatine kinase 1, glycine amidinotransferase, glutamic-oxaloacetic transaminase 1, and arginyl-tRNA synthetase-like. Many of these genes displayed altered expression across several brain regions, strongly implying that dysregulated polyamine metabolism is a widespread phenomenon in the brains of suicide completers. This study provides a broader view of the nature and extent of the dysregulation of the polyamine system in suicide, and highlights the importance of this system in the neurobiology of suicide. PMID:21208503

  18. Flagellin-induced NADPH oxidase 4 activation is involved in atherosclerosis

    PubMed Central

    Kim, Jinoh; Seo, Misun; Kim, Su Kyung; Bae, Yun Soo

    2016-01-01

    It is widely accepted that bacterial infection-mediated inflammation facilitates development of atherosclerosis by activating toll-like receptor (TLR) signaling system. We reasoned that NADPH oxidases (Nox), required for TLR-mediated inflammatory response, are involved in atherogenesis. Here, we show that the activation of Nox4 through TLR5 regulates the inflammation of the endothelium and in atherogenesis. Flagellin-induced interaction between the COOH region of Nox4 and the TIR domain of TLR5 led to H2O2 generation, which in turn promoted the secretion of pro-inflammatory cytokines including IL-8, as well as the expression of ICAM-1 in human aortic endothelial cells (HAECs). Knockdown of the Nox4 in HAECs resulted in attenuated expressions of IL-8 and ICAM-1 leading to a reduction in the adhesion and trans-endothelial migration of monocytes. Challenge of recombinant FliC (rFliC) to the ApoE KO mice with high-fat diet (HFD) resulted in significantly increased atherosclerotic plaque sizes compared to the saline-injected mice. However, an injection of rFliC into the Nox4ApoE DKO mice with HFDs failed to generate atherosclerotic plaque, suggesting that Nox4 deficiency resulted in significant protections against rFliC-mediated atherogenesis. We conclude that TLR5-dependent Nox4 activation and subsequent H2O2 generation play critical roles for the development of atherosclerosis. PMID:27146088

  19. Characterization of germin-like protein with polyphenol oxidase activity from Satsuma mandarine.

    PubMed

    Cheng, Xi; Huang, Xingjian; Liu, Siyu; Tang, Mi; Hu, Wanfeng; Pan, Siyi

    2014-07-01

    Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris-HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65°C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity. PMID:24845377

  20. Flagellin-induced NADPH oxidase 4 activation is involved in atherosclerosis.

    PubMed

    Kim, Jinoh; Seo, Misun; Kim, Su Kyung; Bae, Yun Soo

    2016-01-01

    It is widely accepted that bacterial infection-mediated inflammation facilitates development of atherosclerosis by activating toll-like receptor (TLR) signaling system. We reasoned that NADPH oxidases (Nox), required for TLR-mediated inflammatory response, are involved in atherogenesis. Here, we show that the activation of Nox4 through TLR5 regulates the inflammation of the endothelium and in atherogenesis. Flagellin-induced interaction between the COOH region of Nox4 and the TIR domain of TLR5 led to H2O2 generation, which in turn promoted the secretion of pro-inflammatory cytokines including IL-8, as well as the expression of ICAM-1 in human aortic endothelial cells (HAECs). Knockdown of the Nox4 in HAECs resulted in attenuated expressions of IL-8 and ICAM-1 leading to a reduction in the adhesion and trans-endothelial migration of monocytes. Challenge of recombinant FliC (rFliC) to the ApoE KO mice with high-fat diet (HFD) resulted in significantly increased atherosclerotic plaque sizes compared to the saline-injected mice. However, an injection of rFliC into the Nox4ApoE DKO mice with HFDs failed to generate atherosclerotic plaque, suggesting that Nox4 deficiency resulted in significant protections against rFliC-mediated atherogenesis. We conclude that TLR5-dependent Nox4 activation and subsequent H2O2 generation play critical roles for the development of atherosclerosis. PMID:27146088

  1. Activation of Membrane NADPH Oxidase Associated with Lysosome-Targeted Acid Sphingomyelinase in Coronary Endothelial Cells

    PubMed Central

    Bao, Jun-Xiang; Jin, Si; Zhang, Fan; Wang, Zheng-Chao; Li, Ningjun

    2010-01-01

    Abstract This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O2·− production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs. Antioxid. Redox Signal. 12, 703–712. PMID:19761405

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

    NASA Technical Reports Server (NTRS)

    Morre, D. James

    2002-01-01

    The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

  3. Nitric Oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation

    PubMed Central

    Qian, Jin; Chen, Feng; Kovalenkov, Yevgeniy; Pandey, Deepesh; Moseleley, M. Arthur; Foster, Matthew W.; Black, Stephen M.; Venema, Richard C.; Stepp, David W.; Fulton, David J.R.

    2012-01-01

    The NADPH oxidases (Nox) are a family of transmembrane oxidoreductases that produce superoxide and other reactive oxygen species (ROS). Nox5 was the last of the conventional Nox isoforms to be identified and is a calcium-dependent enzyme that does not depend on accessory subunits for activation. Recently, Nox5 was shown to be expressed in human blood vessels and therefore the goal of current study was to determine whether nitric oxide (NO) can modulate Nox5 activity. Endogenously produced NO potently inhibited basal and stimulated Nox5 activity and inhibition was reversible with chronic, but not acute exposure to L-NAME. Nox5 activity was reduced by NO donors, iNOS, eNOS and in endothelial cells and LPS-stimulated smooth muscle cells in a manner dependent on NO concentration. ROS production was diminished by NO in an isolated enzyme activity assay replete with surplus calcium and NADPH. There was no evidence for NO-dependent changes in tyrosine nitration, glutathiolation or phosphorylation of Nox5. In contrast, there was evidence for the increased nitrosylation of Nox5 as determined by the biotin-switch assay and mass spectrometry. Four S-nitrosylation sites were identified and of these, mutation of C694 dramatically lowered Nox5 activity, NO-sensitivity and biotin-labeling. Furthermore, co-expression of the denitrosylation enzymes thioredoxin (Trx1) and GSNO reductase (GSNOR) prevented NO-dependent inhibition of Nox5. The potency of NO against other Nox enzymes was Nox1≥Nox3>Nox5>Nox2 whereas Nox4 was refractory. Collectively, these results suggest that endogenously produced NO can directly S-nitrosylate and inhibit the activity of Nox5. PMID:22387196

  4. The Role of NADPH Oxidases (NOXs) in Liver Fibrosis and the Activation of Myofibroblasts

    PubMed Central

    Liang, Shuang; Kisseleva, Tatiana; Brenner, David A.

    2016-01-01

    Chronic liver injury, resulted from different etiologies (e.g., virus infection, alcohol abuse, nonalcoholic steatohepatitis (NASH) and cholestasis) can lead to liver fibrosis characterized by the excess accumulation of extracellular matrix (ECM) proteins (e.g., type I collagen). Hepatic myofibroblasts that are activated upon liver injury are the key producers of ECM proteins, contributing to both the initiation and progression of liver fibrosis. Hepatic stellate cells (HSCs) and to a lesser extent, portal fibroblast, are believed to be the precursor cells that give rise to hepatic myofibroblasts in response to liver injury. Although, much progress has been made toward dissecting the lineage origin of myofibroblasts, how these cells are activated and become functional producers of ECM proteins remains incompletely understood. Activation of myofibroblasts is a complex process that involves the interactions between parenchymal and non-parenchymal cells, which drives the phenotypic change of HSCs from a quiescent stage to a myofibroblastic and active phenotype. Accumulating evidence has suggested a critical role of NADPH oxidase (NOX), a multi-component complex that catalyzes reactions from molecular oxygen to reactive oxygen species (ROS), in the activation process of hepatic myofibroblasts. NOX isoforms, including NOX1, NOX2 and NOX4, and NOX-derived ROS, have all been implicated to regulate HSC activation and hepatocyte apoptosis, both of which are essential steps for initiating liver fibrosis. This review highlights the importance of NOX isoforms in hepatic myofibroblast activation and the progression of liver fibrosis, and also discusses the therapeutic potential of targeting NOXs for liver fibrosis and associated hepatic diseases. PMID:26869935

  5. The Role of NADPH Oxidases (NOXs) in Liver Fibrosis and the Activation of Myofibroblasts.

    PubMed

    Liang, Shuang; Kisseleva, Tatiana; Brenner, David A

    2016-01-01

    Chronic liver injury, resulted from different etiologies (e.g., virus infection, alcohol abuse, nonalcoholic steatohepatitis (NASH) and cholestasis) can lead to liver fibrosis characterized by the excess accumulation of extracellular matrix (ECM) proteins (e.g., type I collagen). Hepatic myofibroblasts that are activated upon liver injury are the key producers of ECM proteins, contributing to both the initiation and progression of liver fibrosis. Hepatic stellate cells (HSCs) and to a lesser extent, portal fibroblast, are believed to be the precursor cells that give rise to hepatic myofibroblasts in response to liver injury. Although, much progress has been made toward dissecting the lineage origin of myofibroblasts, how these cells are activated and become functional producers of ECM proteins remains incompletely understood. Activation of myofibroblasts is a complex process that involves the interactions between parenchymal and non-parenchymal cells, which drives the phenotypic change of HSCs from a quiescent stage to a myofibroblastic and active phenotype. Accumulating evidence has suggested a critical role of NADPH oxidase (NOX), a multi-component complex that catalyzes reactions from molecular oxygen to reactive oxygen species (ROS), in the activation process of hepatic myofibroblasts. NOX isoforms, including NOX1, NOX2 and NOX4, and NOX-derived ROS, have all been implicated to regulate HSC activation and hepatocyte apoptosis, both of which are essential steps for initiating liver fibrosis. This review highlights the importance of NOX isoforms in hepatic myofibroblast activation and the progression of liver fibrosis, and also discusses the therapeutic potential of targeting NOXs for liver fibrosis and associated hepatic diseases. PMID:26869935

  6. Elevated NADPH oxidase activity contributes to oxidative stress and cell death in Huntington's disease

    PubMed Central

    Valencia, Antonio; Sapp, Ellen; Kimm, Jeffrey S.; McClory, Hollis; Reeves, Patrick B.; Alexander, Jonathan; Ansong, Kwadwo A.; Masso, Nicholas; Frosch, Matthew P.; Kegel, Kimberly B.; Li, Xueyi; DiFiglia, Marian

    2013-01-01

    A mutation in the huntingtin (Htt) gene produces mutant Htt and Huntington's disease (HD), a neurodegenerative disorder. HD patients have oxidative damage in the brain, but the causes are unclear. Compared with controls, we found brain levels of NADPH oxidase (NOX) activity, which produces reactive oxygen species (ROS), elevated in human HD postmortem cortex and striatum and highest in striatum of presymptomatic individuals. Synaptosome fractions from cortex and striatum of HD140Q/140Q mice had elevated NOX activity at 3 months of age and a further rise at 6 and 12 months compared with synaptosomes of age-matched wild-type (WT) mice. High NOX activity in primary cortical and striatal neurons of HD140Q/140Q mice correlated with more ROS and neurite swellings. These features and neuronal cell death were markedly reduced by treatment with NOX inhibitors such as diphenyleneiodonium (DPI), apocynin (APO) and VAS2870. The rise in ROS levels in mitochondria of HD140Q/140Q neurons followed the rise in NOX activity and inhibiting only mitochondrial ROS was not neuroprotective. Mutant Htt colocalized at plasma membrane lipid rafts with gp91-phox, a catalytic subunit for the NOX2 isoform. Assembly of NOX2 components at lipid rafts requires activation of Rac1 which was also elevated in HD140Q/140Q neurons. HD140Q/140Q mice bred to gp91-phox knock-out mice had lower NOX activity in the brain and in primary neurons, and neurons had normal ROS levels and significantly improved survival. These findings suggest that increased NOX2 activity at lipid rafts is an early and major source of oxidative stress and cell death in HD140Q/140Q neurons. PMID:23223017

  7. Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase

    PubMed Central

    Marín-Navarro, Julia; Roupain, Nicole; Talens-Perales, David; Polaina, Julio

    2015-01-01

    Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described. PMID:26642312

  8. Xanthine oxidase inhibitory activity of the methanolic extracts of selected Jordanian medicinal plants

    PubMed Central

    Hudaib, Mohammad M.; Tawaha, Khaled A.; Mohammad, Mohammad K.; Assaf, Areej M.; Issa, Ala Y.; Alali, Feras Q.; Aburjai, Talal A.; Bustanji, Yasser K.

    2011-01-01

    Background: The search for novel xanthine oxidase (XO) inhibitors with a higher therapeutic activity and fewer side effects are desired not only to treat gout but also to combat various other diseases associated with the XO activity. At present, the potential of developing successful natural products for the management of XO-related diseases is still largely unexplored. In the present study, we have screened the methanolic extracts of various Jordanian medicinal plants for their XO inhibitory activities using an optimized protocol. Materials and Methods: The methanolic extracts of 23 medicinal plants, belonging to 12 families, were tested in vitro, at 200 μg/ml concentrations, for their XO inhibitory potential. The dose-dependent inhibition profiles of the most active plants were further evaluated by estimating the IC50 values of their corresponding extracts. Results: Six plants were found most active (% inhibition more than 39%). These plants are Salvia spinosa L. (IC50 = 53.7 μg/ml), Anthemis palestina Boiss. (168.0 μg/ml), Chrysanthemum coronarium L. (199.5 μg/ml), Achillea biebersteinii Afansiev (360.0 μg/ml), Rosmarinus officinalis L. (650.0 μg/ml), and Ginkgo biloba L. (595.8 μg/ml). Moreover, four more plants, namely Lavandula angustifolia Mill. (28.7% inhibition), Helianthemum ledifolium (L.) Mill. (28.4%), Majorana syriaca (L.) Kostel. (25.1%), and Mentha spicata L. (22.5%) showed a XO inhibitory activity in the range of 22–30%. Conclusion: The study showed that many of the tested plant species are potential sources of natural XO inhibitors that can be developed, upon further investigation, into successful herbal drugs for treatment of gout and other XO-related disorders. PMID:22262935

  9. Coimmobilization of acetylcholinesterase and choline oxidase on gold nanoparticles: stoichiometry, activity, and reaction efficiency.

    PubMed

    Keighron, Jacqueline D; Åkesson, Sebastian; Cans, Ann-Sofie

    2014-09-30

    Hybrid structures constructed from biomolecules and nanomaterials have been used in catalysis and bioanalytical applications. In the design of many chemically selective biosensors, enzymes conjugated to nanoparticles or carbon nanotubes have been used in functionalization of the sensor surface for enhancement of the biosensor functionality and sensitivity. The conditions for the enzyme:nanomaterial conjugation should be optimized to retain maximal enzyme activity, and biosensor effectiveness. This is important as the tertiary structure of the enzyme is often altered when immobilized and can significantly alter the enzyme catalytic activity. Here we show that characterization of a two-enzyme:gold nanoparticle (AuNP) conjugate stoichiometry and activity can be used to gauge the effectiveness of acetylcholine detection by acetylcholine esterase (AChE) and choline oxidase (ChO). This was done by using an analytical approach to quantify the number of enzymes bound per AuNP and monitor the retained enzyme activity after the enzyme:AuNP synthesis. We found that the amount of immobilized enzymes differs from what would be expected from bulk solution chemistry. This analysis was further used to determine the optimal ratio of AChE:ChO added at synthesis to achieve optimum sequential enzyme activity for the enzyme:AuNP conjugates, and reaction efficiencies of greater than 70%. We here show that the knowledge of the conjugate stoichiometry and retained enzyme activity can lead to more efficient detection of acetylcholine by controlling the AChE:ChO ratio bound to the gold nanoparticle material. This approach of optimizing enzyme gold nanoparticle conjugates should be of great importance in the architecture of enzyme nanoparticle based biosensors to retain optimal sensor sensitivity. PMID:25167196

  10. Localization of hydrogen peroxide accumulation and diamine oxidase activity in pea root nodules under aluminum stress.

    PubMed

    Sujkowska-Rybkowska, Marzena; Borucki, Wojciech

    2014-02-01

    Aluminum (Al) is one of the environmental stressors that induces formation of reactive oxygen species (ROS) in plants. Hydrogen peroxide (H2O2) and H2O2-generated apoplast diamine oxidase (DAO) activity were detected cytochemically via transmission electron microscopy (TEM), in pea (Pisum sativum L.) root nodules exposed to high (50 μM AlCl3, for 2 and 24h) Al stress. The nodules were shown to respond to Al stress by disturbances in infection thread (IT) growth, bacteria endocytosis, premature degeneration of bacteroidal tissue and generation of H2O2 in nodule apoplast. Large amounts of peroxide were found at the same sites as high DAO activity under Al stress, suggesting that DAO is a major source of Al-induced peroxide accumulation in the nodules. Peroxide distribution and DAO activity in the nodules of both control plants and Al-treated ones were typically found in the plant cell walls, intercellular spaces and infection threads. However, 2 h Al treatment increased DAO activity and peroxide accumulation in the nodule apoplast and bacteria within threads. A prolonged Al treatment (24 h) increased the H2O2 content and DAO activity in the nodule apoplast, especially in the thread walls, matrix and bacteria within infection threads. In addition to ITs, prematurely degenerated bacteroids, which occurred in response to Al, were associated with intense staining for H2O2 and DAO activity. These results suggest the involvement of DAO in the production of a large amount of H2O2 in the nodule apoplast under Al stress. The role of reactive oxygen species in pea-Rhizobium symbiosis under Al stress is discussed. PMID:24246127

  11. Methionine sulfoxide reductase: chemistry, substrate binding, recycling process and oxidase activity.

    PubMed

    Boschi-Muller, Sandrine; Branlant, Guy

    2014-12-01

    Three classes of methionine sulfoxide reductases are known: MsrA and MsrB which are implicated stereo-selectively in the repair of protein oxidized on their methionine residues; and fRMsr, discovered more recently, which binds and reduces selectively free L-Met-R-O. It is now well established that the chemical mechanism of the reductase step passes through formation of a sulfenic acid intermediate. The oxidized catalytic cysteine can then be recycled by either Trx when a recycling cysteine is operative or a reductant like glutathione in the absence of recycling cysteine which is the case for 30% of the MsrBs. Recently, it was shown that a subclass of MsrAs with two recycling cysteines displays an oxidase activity. This reverse activity needs the accumulation of the sulfenic acid intermediate. The present review focuses on recent insights into the catalytic mechanism of action of the Msrs based on kinetic studies, theoretical chemistry investigations and new structural data. Major attention is placed on how the sulfenic acid intermediate can be formed and the oxidized catalytic cysteine returns back to its reduced form. PMID:25108804

  12. Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase.

    PubMed

    Li, Jian; Lawton, Thomas J; Kostecki, Jan S; Nisthal, Alex; Fang, Jia; Mayo, Stephen L; Rosenzweig, Amy C; Jewett, Michael C

    2016-02-01

    Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an alternative to traditional recombinant protein expression, we show the ability to use cell-free protein synthesis (CFPS) to produce complex MCO proteins with high soluble titers. Specifically, we report the production of MCOs in an Escherichia coli-based cell-free transcription-translation system. Total yields as high as 1.2 mg mL(-1) were observed after a 20-h batch reaction. More than 95% of the protein was soluble and activity was obtained by simple post-CFPS addition of copper ions in the form of CuSO4 . Scale-up reactions were achieved from 15 to 100 µL without a decrease in productivity and solubility. CFPS titers were higher than in vivo expression titers and more soluble, avoiding the formation of inclusion bodies. Our work extends the utility of the cell-free platform to the production of active proteins containing copper cofactors and demonstrates a simple method for producing MCOs. PMID:26356243

  13. Adsorption and Catalytic Activity of Glucose Oxidase Accumulated on OTCE upon the Application of External Potential

    PubMed Central

    Benavidez, Tomás E.; Torrente, Daniel; Marucho, Marcelo; Garcia, Carlos D.

    2014-01-01

    This article describes the adsorption of glucose oxidase (GOx) onto optically transparent carbon electrodes (OTCE) under the effect of applied potential and the analysis of the enzymatic activity of the resulting GOx/OTCE substrates. In order to avoid electrochemical interferences with the enzyme redox center, control electrochemical experiments were performed using flavin adenine dinucleotide (FAD) and GOx/OTCE substrates. Then, the enzyme adsorption experiments were carried out as a function of the potential applied (ranged from the open circuit potential to +950 mV), the pH solution, the concentration of enzyme, and the ionic strength on the environment. The experimental results demonstrated that an increase in the adsorbed amount of GOx on the OTCE can be achieved when the potential was applied. Although the increase in the adsorbed amount was examined as a function of the potential, a maximum enzymatic activity was observed in the GOx/OTCE substrate achieved at +800 mV. These experiments suggest that although an increase in the amount of enzyme adsorbed can be obtained by the application of an external potential to the electrode, the magnitude of such potential can produce detrimental effects in the conformation of the adsorbed protein and should be carefully considered. As such, the article describes a simple and rational approach to increase the amount of enzyme adsorbed on a surface and can be applied to improve the sensitivity of a variety of biosensors. PMID:25261840

  14. Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice.

    PubMed

    Hosseini, Mohsen; Mahfouf, Walid; Serrano-Sanchez, Martin; Raad, Houssam; Harfouche, Ghida; Bonneu, Marc; Claverol, Stephane; Mazurier, Frederic; Rossignol, Rodrigue; Taieb, Alain; Rezvani, Hamid Reza

    2015-04-01

    Xeroderma pigmentosum type C (XP-C) is characterized mostly by a predisposition to skin cancers and accelerated photoaging, but little is known about premature skin aging in this disease. By comparing young and old mice, we found that the level of progerin and p16(INK4a) expression, β-galactosidase activity, and reactive oxygen species, which increase with age, were higher in young Xpc(-/-) mice than in young Xpc(+/+) ones. The expression level of mitochondrial complexes and mitochondrial functions in the skin of young Xpc(-/-) was as low as in control aged Xpc(+/+)animals. Furthermore, the metabolic profile in young Xpc(-/-) mice resembled that found in aged Xpc(+/+) mice. Furthermore, premature skin aging features in young Xpc(-/-) mice were mostly rescued by inhibition of nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) activity by using a NOX1 peptide inhibitor, suggesting that the continuous oxidative stress due to overactivation of NOX1 has a causative role in the underlying pathophysiology. PMID:25437426

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

    PubMed

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

    2016-01-01

    Ferulic acid has structural similarity with curcumin which is being reported for its monoamine oxidase (MAO) inhibitory activity. Based on this similarity, we designed a series of ferulic acid amides 6a-m and tested for their inhibitory activity on human MAO (hMAO) isoforms. All the compounds were found to inhibit the hMAO isoforms either selectively or non-selectively. Nine compounds (6a, 6b, 6g-m) were found to inhibit hMAO-B selectively, whereas the other four (6c-f) were found to be non-selective. There is a gradual shift from hMAO-B selectivity (6a,b) to non-selectivity (6c-f) as there is an increase in chain length at the amino terminus. In case of compounds having an aromatic nucleus at the amino terminus, increasing the carbon number between N and the aromatic ring increases the potency as well as selectivity toward hMAO-B. Compounds 6f, 6j, and 6k were subjected to membrane permeability and metabolic stability studies by in vitro assay methods. They were found to have a better pharmacokinetic profile than curcumin, ferulic acid, and selegiline. In order to understand the structural features responsible for the potency and selectivity of 6k, we carried out a molecular docking simulation study. PMID:26592858

  16. Regional brain cytochrome oxidase activity in beta-amyloid precursor protein transgenic mice with the Swedish mutation.

    PubMed

    Strazielle, C; Sturchler-Pierrat, C; Staufenbiel, M; Lalonde, R

    2003-01-01

    Cytochrome oxidase activity was examined in a transgenic mouse model of Alzheimer's disease with overexpression of the 751 amino acid isoform of beta-amyloid precursor protein with the Swedish mutation under control of the murine thy-1 promoter. The neuritic plaques, abundantly localized in the hippocampus and anterior neocortical areas, showed a core devoid of enzymatic activity surrounded by higher cytochrome oxidase activity at the sites of the dystrophic neurites and activated glial cells. Quantitative measures, taken only in the healthy-appearing regional areas without neuritic plaques, were higher in numerous limbic and non-limbic regions of transgenic mice in comparison with controls. Enzymatic activity was higher in the dentate gyrus and CA2-CA3 region of the hippocampus, the anterior cingulate and primary visual cortex, two olfactory structures, the ventral part of the neostriatum, the parafascicularis nucleus of the thalamus, and the subthalamic nucleus. Brainstem regions anatomically related with altered forebrain regions were more heavily labeled as well, including the substantia nigra, the periaqueductal gray, the superior colliculus, the medial raphe, the locus coeruleus and the adjacent parabrachial nucleus, as well as the pontine nuclei, red nucleus, and trigeminal motor nucleus. Functional brain organization is discussed in the context of Alzheimer's disease. Although hypometabolism is generally observed in this pathology, the increased cytochrome oxidase activity obtained in these transgenic mice can be the result of a functional compensation on the surviving neurons, or of an early mitochondrial alteration related to increased oxidative damage. PMID:12732258

  17. Perturbation of Polyamine Catabolism Can Strongly Affect Root Development and Xylem Differentiation1[W

    PubMed Central

    Tisi, Alessandra; Federico, Rodolfo; Moreno, Sandra; Lucretti, Sergio; Moschou, Panagiotis N.; Roubelakis-Angelakis, Kalliopi A.; Angelini, Riccardo; Cona, Alessandra

    2011-01-01

    Spermidine (Spd) treatment inhibited root cell elongation, promoted deposition of phenolics in cell walls of rhizodermis, xylem elements, and vascular parenchyma, and resulted in a higher number of cells resting in G1 and G2 phases in the maize (Zea mays) primary root apex. Furthermore, Spd treatment induced nuclear condensation and DNA fragmentation as well as precocious differentiation and cell death in both early metaxylem and late metaxylem precursors. Treatment with either N-prenylagmatine, a selective inhibitor of polyamine oxidase (PAO) enzyme activity, or N,N1-dimethylthiourea, a hydrogen peroxide (H2O2) scavenger, reverted Spd-induced autofluorescence intensification, DNA fragmentation, inhibition of root cell elongation, as well as reduction of percentage of nuclei in S phase. Transmission electron microscopy showed that N-prenylagmatine inhibited the differentiation of the secondary wall of early and late metaxylem elements, and xylem parenchymal cells. Moreover, although root growth and xylem differentiation in antisense PAO tobacco (Nicotiana tabacum) plants were unaltered, overexpression of maize PAO (S-ZmPAO) as well as down-regulation of the gene encoding S-adenosyl-l-methionine decarboxylase via RNAi in tobacco plants promoted vascular cell differentiation and induced programmed cell death in root cap cells. Furthermore, following Spd treatment in maize and ZmPAO overexpression in tobacco, the in vivo H2O2 production was enhanced in xylem tissues. Overall, our results suggest that, after Spd supply or PAO overexpression, H2O2 derived from polyamine catabolism behaves as a signal for secondary wall deposition and for induction of developmental programmed cell death. PMID:21746808

  18. Modulation of the NMDA receptor by polyamines

    SciTech Connect

    Williams, K.; Romano, C.; Dichter, M.A.; Molinoff, P.B. )

    1991-01-01

    Results of recent biochemical and electrophysiological studies have suggested that a recognition site for polyamines exists as part of the NMDA receptor complex. The endogenous polyamines spermine and spermidine increase the binding of open-channel blockers and increase NMDA-elicited currents in cultured neutrons. These polyamines have been termed agonists at the polyamine recognition site. Studies of the effects of natural and synthetic polyamines on the binding of ({sup 3}H)MK-801 and on NMDA-elicited currents in cultured neurons have led to the identification of compounds classified as partial agonists, antagonists, and inverse agonists at the polyamine recognition site. Polyamines have also been found to affect the binding of ligands to the recognition sites for glutamate and glycine. However, these effects may be mediated at a site distinct from that at which polyamines act to modulate the binding of open-channel blockers. Endogenous polyamines may modulate excitatory synaptic transmission by acting at the polyamine recognition site of the NMDA receptor. This site could represent a novel therapeutic target for the treatment of ischemia-induced neurotoxicity, epilepsy, and neurodegenerative diseases.

  19. Polyamine Uptake in Carrot Cell Cultures 1

    PubMed Central

    Pistocchi, Rossella; Bagni, Nello; Creus, José A.

    1987-01-01

    Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca2+. The effects of Ca2+ were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca2+ and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca2+ concentration range between 10 micromolar and 1 millimolar. La3+ nullified the stimulatory effect of 10 micromolar Ca2+ on putrescine uptake and that of 1 millimolar Ca2+ on spermidine uptake. La3+ at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule. PMID:16665446

  20. Polyamine changes during senescence and tumorogenesis in plants.

    PubMed

    Srivastava, B I

    1987-09-14

    Putrescine, spermidine, spermine and two unknowns designated as A and B were detected in first seedling leaves of barley (Hordeum vulgare L. var. Wolfe). The levels of these polyamines in first seedling leaves from 4-day-old barley plants grown in darkness or in light were comparable and did not change significantly after exposure of dark grown plants to light for 24 h. No significant consistent changes in the amounts of above polyamines, except perhaps decline in spermidine, were noted during senescence of intact or excised first seedling leaves of barley and this spermidine decline was suppressed during retardation of senescence of excised leaves by 10 mg/l kinetin in the dark. In addition, putrescine, spermidine, spermine, cadaverine and diaminopropane (0.2 mM, 1 mM, 10 mM) had no effect on senescence of excised barley leaves in the dark and both spermine and spermidine induced bleaching of the leaves in the light. Both spermine and spermidine (approx. 10 mM) inhibited RNase and DNase activities but stimulated phosphodiesterase activity (assayed with bis-p-nitrophenyl phosphate as substrate) in crude soluble extracts from barley leaves. Purified snake venom phosphodiesterase activity assayed with RNA as substrate was, however, stimulated by 300-400% by 7-14 mM spermine or spermidine indicating similar possibilities for barley phosphodiesterase. These results together with the presence of multiple species of these enzymes and a decline in net soluble RNase and DNase activities during senescence in barley leaves reported previously, make it unlikely that inhibition of RNase activity in vitro by polyamines could be correlated with their effect on senescence. Putrescine, spermidine and spermine were detected in normal and crown gall tumor tissue cultures of tobacco (Nicotiana tabacum var Wisconsin 38) and in tobacco mosaic virus (TMV)-infected freshly excised pith tissue from tobacco which represented non-proliferating tissue. The level of all three polyamines was

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

    PubMed

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

    2016-08-01

    The aim of this study is to examine women with polycystic ovary syndrome (PCOS) to determine the relationship between xanthine oxidase (XO) and oxidative stress, inflammatory status, and various clinical and biochemical parameters. In this cross-sectional study a total of 83 women including 45 PCOS patients and 38 healthy women were enrolled. We collected blood samples for XO and superoxide dismutase (SOD) activity, hormone levels, cholesterol values, and inflammatory markers. Body mass index (BMI) , waist-to-hip ratio (WHR), and blood pressure were assessed. Blood samples were taken for hormonal levels, cholesterol levels, fasting plasma glucose (FPG), fasting plasma insulin (FPI), homeostatic model assessment-insulin resistance (HOMA-IR) index, quantitative insulin sensitivity check index (QUICKI), C-reactive protein (CRP), white blood cell and neutrophil counts, XO and SOD activities. The basal hormone levels, triglyceride (TG) levels, TG/HDL-C (high density lipoprotein-cholesterol) ratios FPG, FPI and HOMA-IR levels were higher in PCOS patients compared to controls (p<0.05). Platelet and plateletcrit (PCT) values, CRP, and XO activity were significantly increased, however SOD activity was decreased in PCOS patients (p<0.001). XO activity was positively correlated with LH/FSH and TG/HDL ratios, CRP, PCT, FPG, FPI, and HOMA-IR, and negatively correlated with QUICKI levels. In conclusion, XO is a useful marker to assess oxidative stress in PCOS patients. Positive correlations between XO and inflammatory markers and cardiovascular disease risk factors suggest that XO plays an important role in the pathogenesis of PCOS and its metabolic complications. PMID:27295433

  2. NMDA Receptor-Mediated Activation of NADPH Oxidase and Glomerulosclerosis in Hyperhomocysteinemic Rats

    PubMed Central

    Zhang, Chun; Yi, Fan; Xia, Min; Boini, Krishna M.; Zhu, Qing; Laperle, Laura A.; Abais, Justine M.; Brimson, Christopher A.

    2010-01-01

    Abstract This study investigated the role of NMDA receptor in hyperhomocyteinemia (hHcys)-induced NADPH oxidase (Nox) activation and glomerulosclerosis. Sprague–Dawley rats were fed a folate-free (FF) diet to produce hHcys, and a NMDA receptor antagonist, MK-801, was administrated. Rats fed the FF diet exhibited significantly increased plasma homocysteine levels, upregulated NMDA receptor expression, enhanced Nox activity and Nox-dependent O2.− production in the glomeruli, which were accompanied by remarkable glomerulosclerosis. MK-801 treatment significantly inhibited Nox-dependent O2.− production induced by hHcys and reduced glomerular damage index as compared with vehicle-treated hHcys rats. Correspondingly, glomerular deposition of extracellular matrix components in hHcys rats was ameliorated by the administration of MK-801. Additionally, hHcys induced an increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) expression and a decrease in matrix metalloproteinase (MMP)-1 and MMP-9 activities, all of which were abolished by MK-801 treatment. In vitro studies showed that homocysteine increased Nox-dependent O2.− generation in rat mesangial cells, which was blocked by MK-801. Pretreatment with MK-801 also reversed homocysteine-induced decrease in MMP-1 activity and increase in TIMP-1 expression. These results support the view that the NMDA receptor may mediate Nox activation in the kidney during hHcys and thereby play a critical role in the development of hHcys-induced glomerulosclerosis. Antioxid. Redox Signal. 13, 975–986. PMID:20406136

  3. Manganese(IV) Oxide Production by Acremonium sp. Strain KR21-2 and Extracellular Mn(II) Oxidase Activity

    PubMed Central

    Miyata, Naoyuki; Tani, Yukinori; Maruo, Kanako; Tsuno, Hiroshi; Sakata, Masahiro; Iwahori, Keisuke

    2006-01-01

    Ascomycetes that can deposit Mn(III, IV) oxides are widespread in aquatic and soil environments, yet the mechanism(s) involved in Mn oxide deposition remains unclear. A Mn(II)-oxidizing ascomycete, Acremonium sp. strain KR21-2, produced a Mn oxide phase with filamentous nanostructures. X-ray absorption near-edge structure (XANES) spectroscopy showed that the Mn phase was primarily Mn(IV). We purified to homogeneity a laccase-like enzyme with Mn(II) oxidase activity from cultures of strain KR21-2. The purified enzyme oxidized Mn(II) to yield suspended Mn particles; XANES spectra indicated that Mn(II) had been converted to Mn(IV). The pH optimum for Mn(II) oxidation was 7.0, and the apparent half-saturation constant was 0.20 mM. The enzyme oxidized ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] (pH optimum, 5.5; Km, 1.2 mM) and contained two copper atoms per molecule. Moreover, the N-terminal amino acid sequence (residues 3 to 25) was 61% identical with the corresponding sequence of an Acremonium polyphenol oxidase and 57% identical with that of a Myrothecium bilirubin oxidase. These results provide the first evidence that a fungal multicopper oxidase can convert Mn(II) to Mn(IV) oxide. The present study reinforces the notion of the contribution of multicopper oxidase to microbially mediated precipitation of Mn oxides and suggests that Acremonium sp. strain KR21-2 is a good model for understanding the oxidation of Mn in diverse ascomycetes. PMID:17021194

  4. The Arabidopsis COX11 Homolog is Essential for Cytochrome c Oxidase Activity

    PubMed Central

    Radin, Ivan; Mansilla, Natanael; Rödel, Gerhard; Steinebrunner, Iris

    2015-01-01

    Members of the ubiquitous COX11 (cytochrome c oxidase 11) protein family are involved in copper delivery to the COX complex. In this work, we characterize the Arabidopsis thaliana COX11 homolog (encoded by locus At1g02410). Western blot analyses and confocal microscopy identified Arabidopsis COX11 as an integral mitochondrial protein. Despite sharing high sequence and structural similarities, the Arabidopsis COX11 is not able to functionally replace the Saccharomyces cerevisiae COX11 homolog. Nevertheless, further analysis confirmed the hypothesis that Arabidopsis COX11 is essential for COX activity. Disturbance of COX11 expression through knockdown (KD) or overexpression (OE) affected COX activity. In KD lines, the activity was reduced by ~50%, resulting in root growth inhibition, smaller rosettes and leaf curling. In OE lines, the reduction was less pronounced (~80% of the wild type), still resulting in root growth inhibition. Additionally, pollen germination was impaired in COX11 KD and OE plants. This effect on pollen germination can only partially be attributed to COX deficiency and may indicate a possible auxiliary role of COX11 in ROS metabolism. In agreement with its role in energy production, the COX11 promoter is highly active in cells and tissues with high-energy demand for example shoot and root meristems, or vascular tissues of source and sink organs. In COX11 KD lines, the expression of the plasma-membrane copper transporter COPT2 and of several copper chaperones was altered, indicative of a retrograde signaling pathway pertinent to copper homeostasis. Based on our data, we postulate that COX11 is a mitochondrial chaperone, which plays an important role for plant growth and pollen germination as an essential COX complex assembly factor. PMID:26734017

  5. Rotenone Activates Phagocyte NADPH Oxidase through Binding to Its Membrane Subunit gp91phox

    PubMed Central

    Zhou, Hui; Zhang, Feng; Chen, Shih-heng; Zhang, Dan; Wilson, Belinda; Hong, Jau-shyong; Gao, Hui-Ming

    2011-01-01

    Rotenone, a widely used pesticide, reproduces Parkinsonism in rodents and associates with increased risk for Parkinson’s disease. We previously reported rotenone increased superoxide production through stimulating microglial phagocyte NADPH oxidase (PHOX). The present study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91phox, the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91phox. Functional studies showed both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91phox/p22phox) and cytosolic subunits (p67phox and p47phox). Rotenone-elicited extracellular superoxide release in p47phox-deficient macrophages suggested rotenone enabled to activate PHOX through a p47phox-independent mechanism. Increased membrane translocation of p67phox, elevated binding of p67phox to rotenone-treated membrane fractions, and co-immunoprecipitation of p67phox and gp91phox in rotenone-treated wild-type and p47phox-deficient macrophages indicated p67phox played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91phox. Rac1, a Rho-like small GTPase, enhanced p67phox-gp91phox interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91phox; such an interaction triggered membrane translocation of p67phox, leading to PHOX activation and superoxide production. PMID:22094225

  6. The Arabidopsis COX11 Homolog is Essential for Cytochrome c Oxidase Activity.

    PubMed

    Radin, Ivan; Mansilla, Natanael; Rödel, Gerhard; Steinebrunner, Iris

    2015-01-01

    Members of the ubiquitous COX11 (cytochrome c oxidase 11) protein family are involved in copper delivery to the COX complex. In this work, we characterize the Arabidopsis thaliana COX11 homolog (encoded by locus At1g02410). Western blot analyses and confocal microscopy identified Arabidopsis COX11 as an integral mitochondrial protein. Despite sharing high sequence and structural similarities, the Arabidopsis COX11 is not able to functionally replace the Saccharomyces cerevisiae COX11 homolog. Nevertheless, further analysis confirmed the hypothesis that Arabidopsis COX11 is essential for COX activity. Disturbance of COX11 expression through knockdown (KD) or overexpression (OE) affected COX activity. In KD lines, the activity was reduced by ~50%, resulting in root growth inhibition, smaller rosettes and leaf curling. In OE lines, the reduction was less pronounced (~80% of the wild type), still resulting in root growth inhibition. Additionally, pollen germination was impaired in COX11 KD and OE plants. This effect on pollen germination can only partially be attributed to COX deficiency and may indicate a possible auxiliary role of COX11 in ROS metabolism. In agreement with its role in energy production, the COX11 promoter is highly active in cells and tissues with high-energy demand for example shoot and root meristems, or vascular tissues of source and sink organs. In COX11 KD lines, the expression of the plasma-membrane copper transporter COPT2 and of several copper chaperones was altered, indicative of a retrograde signaling pathway pertinent to copper homeostasis. Based on our data, we postulate that COX11 is a mitochondrial chaperone, which plays an important role for plant growth and pollen germination as an essential COX complex assembly factor. PMID:26734017

  7. Polyamine metabolism and osmotic stress. I. Relation to protoplast viability

    NASA Technical Reports Server (NTRS)

    Tiburcio, A. F.; Masdeu, M. A.; Dumortier, F. M.; Galston, A. W.

    1986-01-01

    Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, and Vigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.

  8. Polyamine metabolism and osmotic stress. I. Relation to protoplast viability.

    PubMed

    Tiburcio, A F; Masdeu, M A; Dumortier, F M; Galston, A W

    1986-01-01

    Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, and Vigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro. PMID:11539086

  9. Sulfite Oxidase Activity Is Essential for Normal Sulfur, Nitrogen and Carbon Metabolism in Tomato Leaves

    PubMed Central

    Brychkova, Galina; Yarmolinsky, Dmitry; Batushansky, Albert; Grishkevich, Vladislav; Khozin-Goldberg, Inna; Fait, Aaron; Amir, Rachel; Fluhr, Robert; Sagi, Moshe

    2015-01-01

    Plant sulfite oxidase [SO; E.C.1.8.3.1] has been shown to be a key player in protecting plants against exogenous toxic sulfite. Recently we showed that SO activity is essential to cope with rising dark-induced endogenous sulfite levels in tomato plants (Lycopersicon esculentum/Solanum lycopersicum Mill. cv. Rheinlands Ruhm). Here we uncover the ramifications of SO impairment on carbon, nitrogen and sulfur (S) metabolites. Current analysis of the wild-type and SO-impaired plants revealed that under controlled conditions, the imbalanced sulfite level resulting from SO impairment conferred a metabolic shift towards elevated reduced S-compounds, namely sulfide, S-amino acids (S-AA), Co-A and acetyl-CoA, followed by non-S-AA, nitrogen and carbon metabolite enhancement, including polar lipids. Exposing plants to dark-induced carbon starvation resulted in a higher degradation of S-compounds, total AA, carbohydrates, polar lipids and total RNA in the mutant plants. Significantly, a failure to balance the carbon backbones was evident in the mutants, indicated by an increase in tricarboxylic acid cycle (TCA) cycle intermediates, whereas a decrease was shown in stressed wild-type plants. These results indicate that the role of SO is not limited to a rescue reaction under elevated sulfite, but SO is a key player in maintaining optimal carbon, nitrogen and sulfur metabolism in tomato plants. PMID:27135342

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

    SciTech Connect

    Silanikove, Nissim Shapiro, Fira; Leitner, Gabriel

    2007-11-23

    The aims of this study were to test the hypothesis that the substrates of xanthine oxidase (XO), xanthine and hypoxanthine, are consumed while the milk is stored in the gland between milkings, and to explore how XO activity responds to bacteria commonly associated with subclinical infections in the mammary gland. Freshly secreted milk was obtained following complete evacuation of the gland and induction of milk ejection with oxytocin. In bacteria-free fresh milk xanthine and hypoxanthine were converted to uric acid within 30 min (T{sub 1/2} {approx} 10 min), which in turn provides electrons for formation of hydrogen peroxide and endows the alveolar lumen with passive protection against invading bacteria. On the other hand, the longer residence time of milk in the cistern compartment was not associated with oxidative stress as a result of XO idleness caused by exhaustion of its physiological fuels. The specific response of XO to bacteria species and the resulting bacteria-dependent nitrosative stress further demonstrates that it is part of the gland immune system.

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

    PubMed Central

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  12. Substance P Exacerbates Dopaminergic Neurodegeneration through Neurokinin-1 Receptor-Independent Activation of Microglial NADPH Oxidase

    PubMed Central

    Chu, Chun-Hsien; Qian, Li; Chen, Shih-Heng; Wilson, Belinda; Oyarzabal, Esteban; Jiang, Lulu; Ali, Syed; Robinson, Bonnie; Kim, Hyoung-Chun

    2014-01-01

    Although dysregulated substance P (SP) has been implicated in the pathophysiology of Parkinson's disease (PD), how SP affects the survival of dopaminergic neurons remains unclear. Here, we found that mice lacking endogenous SP (TAC1−/−), but not those deficient in the SP receptor (neurokinin-1 receptor, NK1R), were more resistant to lipopolysaccharide (LPS)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic neurodegeneration than wild-type controls, suggesting a NK1R-independent toxic action of SP. In vitro dose–response studies revealed that exogenous SP enhanced LPS- and 1-methyl-4-phenylpyridinium (MPP+)-induced dopaminergic neurodegeneration in a bimodal manner, peaking at submicromolar and subpicomolar concentrations, but was substantially less effective at intermediate concentrations. Mechanistically, the actions of submicromolar levels of SP were NK1R-dependent, whereas subpicomolar SP-elicited actions required microglial NADPH oxidase (NOX2), the key superoxide-producing enzyme, but not NK1R. Subpicomolar concentrations of SP activated NOX2 by binding to the catalytic subunit gp91phox and inducing membrane translocation of the cytosolic subunits p47phox and p67phox. The importance of NOX2 was further corroborated by showing that inhibition or disruption of NOX2 blocked subpicomolar SP-exacerbated neurotoxicity. Together, our findings revealed a critical role of microglial NOX2 in mediating the neuroinflammatory and dopaminergic neurodegenerative effects of SP, which may provide new insights into the pathogenesis of PD. PMID:25209287

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

    NASA Astrophysics Data System (ADS)

    Abbasi, Mahboube; Amiri, Razieh; Bordbar, Abdol-Kalegh; Ranjbakhsh, Elnaz; Khosropour, Ahmad-Reza

    2016-02-01

    Immobilized proteins and enzymes are widely investigated in the medical field as well as the food and environmental fields. In this study, glucose oxidase (GOX) was covalently immobilized on the surface of modified iron oxide magnetic nanoparticles (MIMNs) to produce a bioconjugate complex. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to the size, shape and structure characterization of the MIMNs. Binding of GOX to these MIMNs was confirmed by using FT-IR spectroscopy. The stability of the immobilized and free enzyme at different temperature and pH values was investigated by measuring the enzymatic activity. These studies reveal that the enzyme's stability is enhanced by immobilization. Further experiments showed that the storage stability of the enzyme is improved upon binding to the MIMNs. The results of kinetic measurements suggest that the effect of the immobilization process on substrate and product diffusion is small. Such bioconjugates can be considered as a catalytic nanodevice for accelerating the glucose oxidation reaction for biotechnological purposes.

  14. Magnetic colorimetric immunoassay for human interleukin-6 based on the oxidase activity of ceria spheres.

    PubMed

    Peng, Juan; Guan, Jufang; Yao, Huiqin; Jin, Xiaoyong

    2016-01-01

    A novel magnetic colorimetric immunoassay strategy was designed for sensitive detection of human interleukin-6 (IL-6) using ceria spheres as labels. Ceria spheres showed excellent oxidase activity, which can directly catalyze the oxidation of substrate o-phenylenediamine (OPD) to a stable yellow product, 2,3-diaminophenazine (oxOPD). The absorbance of oxOPD was recorded to reflect the level of IL-6. The relatively mild conditions made the immunoassay strategy more robust, reliable, and easy. A linear relationship between absorbance intensity and the logarithm of IL-6 concentrations was obtained in the range of 0.0001-10 ng mL(-1) with a detection limit of 0.04 pg mL(-1) (S/N = 3). The colorimetric immunoassay exhibited high sensitivity and specificity for the detection of IL-6. This immunoassay has been successfully applied in the detection of IL-6 in serum samples and can be readily extended toward the on-site monitoring of cancer biomarkers in serum samples. PMID:26416691

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

    PubMed

    Martinkova, Pavla; Opatrilova, Radka; Kruzliak, Peter; Styriak, Igor; Pohanka, Miroslav

    2016-05-01

    Magnetic particles (MPs) are currently used as a suitable alternative for peroxidase in the construction of novel biosensors, analytic and diagnostic methods. Their better chemical and thermal stabilities predestine them as appropriate pseudo-enzymatic catalysts. In this point of view, our research was focused on preparation of simply and fast method for immobilization of glucose oxidase onto surface of MPs with peroxidase-like activity. Spectrophotometric method (wavelength 450 nm) optimized for glucose determination using modified MPs has been successfully developed. Concentration curve for optimization of method was assayed, and Michaelis-Menten constant (K m) calculated, maximum reaction rate (V max), limit of detection, and correlation coefficient were determined to be 0.13 mmol/l (2.34 mg/dl), 1.79 pkat, 3.74 µmol/l (0.067 mg/dl), and 0.996, respectively. Interferences of other sugars such as sucrose, sorbitol, deoxyribose, maltose, and fructose were determined as well as effect of substances presenting in plasma (ascorbic acid, reduced glutathione, trolox, and urea). Results in comparison with positive and negative controls showed no interferences of the other sugars and no influence of plasma substances to measuring of glucose. The constructed method showed corresponding results with linear dependence and a correlation coefficient of 0.997. Possibility of repeated use of modified MPs was successfully proved. PMID:27041274

  16. LRPPRC mutation suppresses cytochrome oxidase activity by altering mitochondrial RNA transcript stability in a mouse model.

    PubMed

    Xu, Fenghao; Addis, Jane B L; Cameron, Jessie M; Robinson, Brian H

    2012-01-01

    LRPPRC (leucine-rich pentatricopeptide repeat-containing) has been shown to be essential for the maturation of COX (cytochrome c oxidase), possibly by stabilizing RNA transcripts of COXI, COXII and COXIII genes encoded in mtDNA (mitochondrial DNA). We established a mouse 'gene-trap' model using ES cells (embryonic stem cells) in which the C-terminus of LRPPRC has been replaced with a β-geo construct. Mice homozygous for this modification were found to be subject to embryonic lethality, with death before 12.5 dpc (days post-coitum). Biochemical analysis of MEFs (mouse embryonic fibroblasts) isolated from homozygous mutants showed a major decrease in COX activity, with slight reductions in other respiratory chain complexes with mtDNA encoded components. Constructs of LRPPRC containing different numbers of PPRs (pentatricopeptide repeats) were expressed as recombinant proteins and tested for their ability to bind to the COXI mRNA transcript. Full binding required the first 19 PPR motifs. A specific segment of COXI mRNA was identified as the binding target for LRPPRC, encoded by mouse mtDNA nucleotides 5961-6020. These data strongly suggest that LRPPRC is involved in the maturation of COX, and is involved in stabilizing of mitochondrial mRNAs encoding COX transcripts. PMID:21880015

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

    PubMed

    Hatanaka, Elaine; Dermargos, Alexandre; Hirata, Aparecida Emiko; Vinolo, Marco Aurélio Ramirez; Carpinelli, Angelo Rafael; Newsholme, Philip; Armelin, Hugo Aguirre; Curi, Rui

    2013-01-01

    The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47 (phox) phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47 (phox) mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts. PMID:23579616

  18. Polyamine biosynthesis is critical for growth and differentiation of the pancreas

    PubMed Central

    Mastracci, Teresa L.; Robertson, Morgan A.; Mirmira, Raghavendra G.; Anderson, Ryan M.

    2015-01-01

    The pancreas, in most studied vertebrates, is a compound organ with both exocrine and endocrine functions. The exocrine compartment makes and secretes digestive enzymes, while the endocrine compartment, organized into islets of Langerhans, produces hormones that regulate blood glucose. High concentrations of polyamines, which are aliphatic amines, are reported in exocrine and endocrine cells, with insulin-producing β cells showing the highest concentrations. We utilized zebrafish as a model organism, together with pharmacological inhibition or genetic manipulation, to determine how polyamine biosynthesis functions in pancreatic organogenesis. We identified that inhibition of polyamine biosynthesis reduces exocrine pancreas and β cell mass, and that these reductions are at the level of differentiation. Moreover, we demonstrate that inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, phenocopies inhibition or knockdown of the enzyme deoxyhypusine synthase (DHS). These data identify that the pancreatic requirement for polyamine biosynthesis is largely mediated through a requirement for spermidine for the downstream posttranslational modification of eIF5A by its enzymatic activator DHS, which in turn impacts mRNA translation. Altogether, we have uncovered a role for polyamine biosynthesis in pancreatic organogenesis and identified that it may be possible to exploit polyamine biosynthesis to manipulate pancreatic cell differentiation. PMID:26299433

  19. Antioxidant, α-glucosidase and xanthine oxidase inhibitory activity of bioactive compounds from maize (Zea mays L.).

    PubMed

    Nile, Shivraj H; Park, Se W

    2014-01-01

    Chemical investigations into maize (Zea mays L.) kernels yielded phenolic compounds, which were structurally established using chromatographic and spectroscopic methods. The isolated phenolic compounds from maize kernel were examined in vitro for their antioxidant abilities by DPPH (2,2-diphenyl-1-picryl hydrazine) radical, OH radical scavenging activity, and reducing ability, along with α-glucosidase and xanthine oxidase (XO) inhibition. The isolated maize phenolics revealed significant xanthine oxidase and α-glucosidase inhibitory activity to that of allopurinol and acarbose in vitro and in vivo, respectively. The kinetics study with xanthine oxidase revealed competitive type of inhibition by isolated maize vanillic acid (M2), ferulic acid (M5), 3'-methoxyhirsutrin (M7), and peonidin-3-glucoside (M10) as compared to control allopurinol. Overall, with few exceptions, all the phenolic compounds from maize kernel revealed significant biological activities with all parameters examined. Also, the phenolic compounds from maize were found to be more reactive toward DPPH radical and had considerable reducing ability and OH radical scavenging activity. These findings suggest that maize kernel phenolic compounds can be considered as potential antioxidant, α-glucosidase, and XO inhibitory agents those might be further explored for the design of lead antioxidant, antidiabetic and antigout drug candidates using in vivo trials. PMID:23957301

  20. Inactivation of nitroalkane oxidase upon mutation of the active site base and rescue with a deprotonated substrate.

    PubMed

    Valley, Michael P; Fitzpatrick, Paul F

    2003-07-23

    Mutation of Asp402 in nitroalkane oxidase to Asn or Ala inactivates the enzyme with neutral nitroethane as substrate, but the activity can be rescued with the nitroethane anion. The V/K values of the D402N and D402A enzymes with the nitroethane anion are independent of pH, whereas the V/K values of the wild-type and D402E enzymes are pH dependent with both the protonated and the deprotonated forms of nitroethane. Moreover, although the V/K value of the D402E enzyme with neutral nitroethane is 20-fold less than that of the wild-type enzyme, there is only a 2-fold difference in the V/K values with the nitroethane anion. These results are fully consistent with a primary role for Asp402 as the active site base in nitroalkane oxidase which abstracts the substrate alpha-proton. PMID:12862464

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

    PubMed Central

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

    2013-01-01

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

  2. Arsenic, cadmium, mercury and nickel stimulate cell growth via NADPH oxidase activation.

    PubMed

    Mohammadi-Bardbori, Afshin; Rannug, Agneta

    2014-11-10

    Exposure to metals and metalloids including arsenic, cadmium, mercury, and nickel has been a worldwide health problem for several decades. The aim of this study was to learn how metal-induced oxidative stress triggers cell proliferation, a process of great significance for cancer. NADPH oxidase (NOX) activity and cell proliferation were measured as endpoints in both NOX-deficient and NOX-proficient cells. The X chromosome linked CGD (X-CGD) human promyelocytic leukemia PLB-985 cells lacking gp91phox and the X-CGD cells re-transfected with gp91phox (X-CGD-gp91(phox)) were used together with immortalized human keratinocyte cells (HaCaT). The cells were exposed to different concentrations of the metals alone or together with the NOX inhibitor, diphenyleneiodonium (DPI). We found that the studied metals increased NOX activity. They stimulated cell proliferation in HaCaT and X-CGD-gp91(phox) cells at concentrations below 1μM but not in the X-CGD cells that lack functional NOX. Addition of DPI attenuated the metal-induced cell proliferation. At concentrations above 1μM these metals inhibited cell proliferation. Based on these findings, we propose that many environmental pollutants, including metals and also endogenous NOX-activators such as oxidants and growth factors, interfere with cell growth kinetics by increasing the levels of the diffusible molecule H2O2. Here, we provide evidence that NOXs is central to the mechanism of metal-mediated reactive oxygen species production and stimulation of cell proliferation. PMID:25446860

  3. The monoamine oxidase inhibitory activity of essential oils obtained from Eryngium species and their chemical composition.

    PubMed

    Klein-Júnior, Luiz Carlos; Dos Santos Passos, Carolina; Tasso de Souza, Tiago Juliano; Gobbi de Bitencourt, Fernanda; Salton, Juliana; de Loreto Bordignon, Sérgio Augusto; Henriques, Amélia Teresinha

    2016-06-01

    Context Monoamine oxidase (MAO) inhibitors are used in the treatment of depression, anxiety disorders, and the symptomatic treatment of Parkinson's disease. Eryngium, the most representative of the Apiaceae family, is well known for the presence of essential oils (EOs), which have already demonstrated MAO inhibitory potential. Objective The objective of this study is to evaluate the MAO inhibitory capacity of the EOs obtained from Eryngium floribundum Cham. & Schlecht. (EF), E. eriophorum Cham. & Schlecht. (EE), E. nudicaule Lam. (EN), E. horridum Malme (EH), and E. pandanifolium Cham. & Schlecht. (EP). Materials and methods EOs were obtained from fresh whole plants by hydrodistillation (3 h). Chemical analyses were performed by GC/MS using apolar and polar columns, with oven temperature from 60 to 300 °C at 3 °C/min. The MAO-A and -B activities were evaluated in vitro by an end-point method using kynuramine as the substrate and mitochondrial suspension or human recombinant enzymes as the enzymatic source. DMSO 2%, clorgyline 10(-7) M, and pargyline 10(-6) M were used as controls. Results and discussion EFEO, EEEO, ENEO, EHEO, and EPEO GC/MS analysis showed (E)-caryophyllene (4.9-10.8%), germacrene D (0.6-35.1%), bicyclogermacrene (10.4-17.2), spathulenol (0.4-36.0%), and globulol (1.4-18.6%) as main constituents. None of the EOs inhibited MAO-A activity (4 and 40 μg/mL). However, EHEO inhibited MAO-B activity with an IC50 value of 5.65 μg/mL (1-200 μg/mL). Pentadecane (10 μM), its major constituent (53.5%), did not display significant MAO-B inhibition. Conclusion The study demonstrates the promising application of Eryngium species as a source of potential central nervous system bioactive secondary metabolites, specially related to neurodegenerative disorders. PMID:26810928

  4. Characterization and cDNA sequence of Bothriechis schlegeliil-amino acid oxidase with antibacterial activity.

    PubMed

    Vargas Muñoz, Leidy Johana; Estrada-Gomez, Sebastian; Núñez, Vitelbina; Sanz, Libia; Calvete, Juan J

    2014-08-01

    Snake venoms are complex mixtures of proteins including l-amino acid oxidase (lAAO). A lAAO (named BslAAO) with a mass of 56kDa and a theoretical Ip of 5.79, was purified from Bothriechis schlegelii venom through size-exclusion, ion exchange and affinity chromatography. The entire protein sequence of 498 amino acids, was determined from cDNA using reverse-transcribed mRNA isolated from venom gland. The enzyme showed dose-dependent inhibition of bacterial growth. BslAAO showed inhibitory effect against S. aureus with a MIC of 4μg/mL and a MBC of 8μg/mL. Against Acinetobacter baumannii, showed a MIC of 2μg/mL and MBC of 4μg/mL, No effect was observed in Escherichia coli. This antibacterial activity was inhibited by catalase, indicating that antimicrobial activity was due to H2O2 production. BslAAO did not show any cytotoxic activity toward mouse myoblast cell line C2C12 or peripheral blood mononuclear cells. The enzyme oxidated l-Leu, with a Km of 16.37μM and a Vmax of 0.39μM/min. Snake venoms lAAOs, are potential frames of different therapeutics molecules since these enzymes exhibit low MICs and MBCs and show to be harmless to human cells due to microorganisms being generally several fold more sensitive to reactive oxygen species than human tissues. PMID:24875315

  5. Epithelial-to-Mesenchymal Transition in Podocytes Mediated by Activation of NADPH Oxidase in Hyperhomocysteinemia

    PubMed Central

    Zhang, Chun; Xia, Min; Boini, Krishna M.; Li, Cai-Xia; Abais, Justine M.; Li, Xiao-Xue; Laperle, Laura A.; Li, Pin-Lan

    2012-01-01

    The present study tested the hypothesis that hyperhomocysteinemia (hHcys) induces podocytes to undergo epithelial-to-mesenchymal transition (EMT) through the activation of NADPH oxidase (Nox). It was found that increased homocysteine (Hcys) level suppressed the expression of slit diaphragm-associated proteins, P-cadherin and zonula occludens-1 (ZO-1) in conditionally immortalized mouse podocytes, indicating the loss of their epithelial features. Meanwhile, Hcys remarkably increased the abundance of mesenchymal markers, such as fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA). These phenotype changes in podocytes induced by Hcys were accompanied by enhanced superoxide (O2.−) production, which was substantially suppressed by inhibition of Nox activity. Functionally, Hcys significantly enhanced the permeability of the podocyte monolayer coupled with increased EMT, and this EMT-related increase in cell permeability could be restored by Nox inhibitors. In mice lacking gp91phox (gp91−/−), an essential Nox subunit gene, hHcys-enhanced podocyte EMT and consequent glomerular injury were examined. In wild-type (gp91+/+) mice, hHcys induced by a folate-free (FF) diet markedly enhanced expression of mesenchymal markers (FSP-1 and α-SMA) but decreased expression of epithelial markers of podocytes in glomeruli, which were not observed in gp91−/− mouse glomeruli. Podocyte injury, glomerular sclerotic pathology, and marked albuminuria observed in gp91+/+ mice with hHcys were all significantly attenuated in gp91−/− mice. These results suggest that hHcys induces EMT of podocytes through activation of Nox, which represents a novel mechanism of hHcys-associated podocyte injury. PMID:21647593

  6. Sustained activation of proton channels and NADPH oxidase in human eosinophils and murine granulocytes requires PKC but not cPLA2α activity

    PubMed Central

    Morgan, Deri; Cherny, Vladimir V; Finnegan, Alison; Bollinger, James; Gelb, Michael H; DeCoursey, Thomas E

    2007-01-01

    The prevailing hypothesis that a signalling pathway involving cPLA2α is required to enhance the gating of the voltage-gated proton channel associated with NADPH oxidase was tested in human eosinophils and murine granulocytes. This hypothesis invokes arachidonic acid (AA) liberated by cPLA2α as a final activator of proton channels. In human eosinophils studied in the perforated-patch configuration, phorbol myristate acetate (PMA) stimulation elicited NADPH oxidase-generated electron current (Ie) and enhanced proton channel gating identically in the presence or absence of three specific cPLA2α inhibitors, Wyeth-1, pyrrolidine-2 and AACOCF3 (arachidonyl trifluoromethyl ketone). In contrast, PKC inhibitors GFX (GF109203X) or staurosporine prevented the activation of either proton channels or NADPH oxidase. PKC inhibition during the respiratory burst reversed the activation of both molecules, suggesting that ongoing phosphorylation is required. This effect of GFX was inhibited by okadaic acid, implicating phosphatases in proton channel deactivation. Proton channel activation by AA was partially reversed by GFX or staurosporine, indicating that AA effects are due in part to activation of PKC. In granulocytes from mice with the cPLA2α gene disrupted (knockout mice), PMA or fMetLeuPhe activated NADPH oxidase and proton channels in a manner indistinguishable from the responses of control cells. Thus, cPLA2α is not essential to activate the proton conductance or for a normal respiratory burst. Instead, phosphorylation of the proton channel or an activating molecule converts the channel to its activated gating mode. The existing paradigm for regulation of the concerted activity of proton channels and NADPH oxidase must be revised. PMID:17185330

  7. AMPK activation by glucagon-like peptide-1 prevents NADPH oxidase activation induced by hyperglycemia in adult cardiomyocytes.

    PubMed

    Balteau, Magali; Van Steenbergen, Anne; Timmermans, Aurélie D; Dessy, Chantal; Behets-Wydemans, Gaetane; Tajeddine, Nicolas; Castanares-Zapatero, Diego; Gilon, Patrick; Vanoverschelde, Jean-Louis; Horman, Sandrine; Hue, Louis; Bertrand, Luc; Beauloye, Christophe

    2014-10-15

    Exposure of cardiomyocytes to high glucose concentrations (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase (NOX2). NOX2 activation is triggered by enhanced glucose transport through a sodium-glucose cotransporter (SGLT) but not by a stimulation of glucose metabolism. The aim of this work was to identify potential therapeutic approaches to counteract this glucotoxicity. In cultured adult rat cardiomyocytes incubated with 21 mM glucose (HG), AMP-activated protein kinase (AMPK) activation by A769662 or phenformin nearly suppressed ROS production. Interestingly, glucagon-like peptide 1 (GLP-1), a new antidiabetic drug, concomitantly induced AMPK activation and prevented the HG-mediated ROS production (maximal effect at 100 nM). α2-AMPK, the major isoform expressed in cardiomyocytes (but not α1-AMPK), was activated in response to GLP-1. Anti-ROS properties of AMPK activators were not related to changes in glucose uptake or glycolysis. Using in situ proximity ligation assay, we demonstrated that AMPK activation prevented the HG-induced p47phox translocation to caveolae, whatever the AMPK activators used. NOX2 activation by either α-methyl-d-glucopyranoside, a glucose analog transported through SGLT, or angiotensin II was also counteracted by GLP-1. The crucial role of AMPK in limiting HG-mediated NOX2 activation was demonstrated by overexpressing a constitutively active form of α2-AMPK using adenoviral infection. This overexpression prevented NOX2 activation in response to HG, whereas GLP-1 lost its protective action in α2-AMPK-deficient mouse cardiomyocytes. Under HG, the GLP-1/AMPK pathway inhibited PKC-β2 phosphorylation, a key element mediating p47phox translocation. In conclusion, GLP-1 induces α2-AMPK activation and blocks HG-induced p47phox translocation to the plasma membrane, thereby preventing glucotoxicity. PMID:25128166

  8. Identification of a mammalian vesicular polyamine transporter

    PubMed Central

    Hiasa, Miki; Miyaji, Takaaki; Haruna, Yuka; Takeuchi, Tomoya; Harada, Yuika; Moriyama, Sawako; Yamamoto, Akitsugu; Omote, Hiroshi; Moriyama, Yoshinori

    2014-01-01

    Spermine and spermidine act as neuromodulators upon binding to the extracellular site(s) of various ionotropic receptors, such as N-methyl-d-aspartate receptors. To gain access to the receptors, polyamines synthesized in neurons and astrocytes are stored in secretory vesicles and released upon depolarization. Although vesicular storage is mediated in an ATP-dependent, reserpine-sensitive fashion, the transporter responsible for this process remains unknown. SLC18B1 is the fourth member of the SLC18 transporter family, which includes vesicular monoamine transporters and vesicular acetylcholine transporter. Proteoliposomes containing purified human SLC18B1 protein actively transport spermine and spermidine by exchange of H+. SLC18B1 protein is predominantly expressed in the hippocampus and is associated with vesicles in astrocytes. SLC18B1 gene knockdown decreased both SLC18B1 protein and spermine/spermidine contents in astrocytes. These results indicated that SLC18B1 encodes a vesicular polyamine transporter (VPAT). PMID:25355561

  9. Exosomes from hypoxic endothelial cells have increased collagen crosslinking activity through up-regulation of lysyl oxidase-like 2.

    PubMed

    de Jong, Olivier G; van Balkom, Bas W M; Gremmels, Hendrik; Verhaar, Marianne C

    2016-02-01

    Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations, research on exosome-ECM interactions is limited. Here, we investigate whether the exosome-associated lysyl oxidase family member lysyl oxidase-like 2 (LOXL2) is involved in ECM remodelling. We found that LOXL2 is present on the exterior of endothelial cell (EC)-derived exosomes, placing it in direct vicinity of the ECM. It is up-regulated twofold in EC-derived exosomes cultured under hypoxic conditions. Intact exosomes from hypoxic EC and LOXL2 overexpressing EC show increased activity in a fluorometric lysyl oxidase enzymatic activity assay as well as in a collagen gel contraction assay. Concordantly, knockdown of LOXL2 in exosome-producing EC in both normal and hypoxic conditions reduces activity of exosomes in both assays. Our findings show for the first time that ECM crosslinking by EC-derived exosomes is mediated by LOXL2 under the regulation of hypoxia, and implicate a role for exosomes in hypoxia-regulated focal ECM remodelling, a key process in both fibrosis and wound healing. PMID:26612622

  10. Reductive half-reaction of nitroalkane oxidase: effect of mutation of the active site aspartate to glutamate.

    PubMed

    Valley, Michael P; Fitzpatrick, Paul F

    2003-05-20

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707]. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the DeltaG(++) for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase. PMID:12741843

  11. Synthesis and theoretical calculations of carbazole substituted chalcone urea derivatives and studies their polyphenol oxidase enzyme activity.

    PubMed

    Nixha, Arleta Rifati; Arslan, Mustafa; Atalay, Yusuf; Gençer, Nahit; Ergün, Adem; Arslan, Oktay

    2013-08-01

    Synthesis of carbazole substituted chalcone urea derivatives and their polyphenol oxidase enzyme activity effects on the diphenolase activity of banana tyrosinase were evaluated. Tyrosinase has been purified from banana on an affinity gel comprised of Sepharose 4B-L-tyrosine-p-aminobenzoic acid. The results showed that most of the compounds (3,4,5a,5d-h) inhibited and some of them (5c,5i-l) activated the tyrosinase enzyme activity. The molecular calculations were performed using Gaussian software for the synthesized compounds to explain the experimental results. PMID:22803668

  12. Polyamines in Eukaryotes, Bacteria, and Archaea.

    PubMed

    Michael, Anthony J

    2016-07-15

    Polyamines are primordial polycations found in most cells and perform different functions in different organisms. Although polyamines are mainly known for their essential roles in cell growth and proliferation, their functions range from a critical role in cellular translation in eukaryotes and archaea, to bacterial biofilm formation and specialized roles in natural product biosynthesis. At first glance, the diversity of polyamine structures in different organisms appears chaotic; however, biosynthetic flexibility and evolutionary and ecological processes largely explain this heterogeneity. In this review, I discuss the biosynthetic, evolutionary, and physiological processes that constrain or expand polyamine structural and functional diversity. PMID:27268252

  13. Spatial and temporal distribution of genes involved in polyamine metabolism during tomato fruit development.

    PubMed

    Tsaniklidis, Georgios; Kotsiras, Anastasios; Tsafouros, Athanasios; Roussos, Peter A; Aivalakis, Georgios; Katinakis, Panagiotis; Delis, Costas

    2016-03-01

    Polyamines are organic compounds involved in various biological roles in plants, including cell growth and organ development. In the present study, the expression profile, the accumulation of free polyamines and the transcript localisation of the genes involved in Put metabolism, such as Ornithine decarboxylase (ODC), Arginine decarboxylase (ADC) and copper containing Amine oxidase (CuAO), were examined during Solanum lycopersicum cv. Chiou fruit development and maturation. Moreover, the expression of genes coding for enzymes involved in higher polyamine metabolism, including Spermidine synthase (SPDS), Spermine synthase (SPMS), S-adenosylmethionine decarboxylase (SAMDC) and Polyamine oxidase (PAO), were studied. Most genes participating in PAs biosynthesis and metabolism exhibited an increased accumulation of transcripts at the early stages of fruit development. In contrast, CuAO and SPMS were mostly expressed later, during the development stages of the fruits where a massive increase in fruit volume occurs, while the SPDS1 gene exhibited a rather constant expression with a peak at the red ripe stage. Although Put, Spd and Spm were all exhibited decreasing levels in developing immature fruits, Put levels maxed late during fruit ripening. In contrast to Put both Spd and Spm levels continue to decrease gradually until full ripening. It is worth noticing that in situ RNA-RNA hybridisation is reported for the first time in tomato fruits. The localisation of ADC2, ODC1 and CuAO gene transcripts at tissues such as the locular parenchyma and the vascular bundles fruits, supports the theory that all genes involved in Put biosynthesis and catabolism are mostly expressed in fast growing tissues. The relatively high expression levels of CuAO at the ImG4 stage of fruit development (fruits with a diameter of 3 cm), mature green and breaker stages could possibly be attributed to the implication of polyamines in physiological processes taking place during fruit ripening. PMID

  14. High glycolate oxidase activity is required for survival of maize in normal air.

    PubMed

    Zelitch, Israel; Schultes, Neil P; Peterson, Richard B; Brown, Patrick; Brutnell, Thomas P

    2009-01-01

    A mutant in the maize (Zea mays) Glycolate Oxidase1 (GO1) gene was characterized to investigate the role of photorespiration in C4 photosynthesis. An Activator-induced allele of GO1 conditioned a seedling lethal phenotype when homozygous and had 5% to 10% of wild-type GO activity. Growth of seedlings in high CO2 (1%-5%) was sufficient to rescue the mutant phenotype. Upon transfer to normal air, the go1 mutant became necrotic within 7 d and plants died within 15 d. Providing [1-14C]glycolate to leaf tissue of go1 mutants in darkness confirmed that the substrate is inefficiently converted to 14CO2, but both wild-type and GO-deficient mutant seedlings metabolized [1-14C]glycine similarly to produce [14C]serine and 14CO2 in a 1:1 ratio, suggesting that the photorespiratory pathway is otherwise normal in the mutant. The net CO2 assimilation rate in wild-type leaves was only slightly inhibited in 50% O2 in high light but decreased rapidly and linearly with time in leaves with low GO. When go1 mutants were shifted from high CO2 to air in light, they accumulated glycolate linearly for 6 h to levels 7-fold higher than wild type and 11-fold higher after 25 h. These studies show that C4 photosynthesis in maize is dependent on photorespiration throughout seedling development and support the view that the carbon oxidation pathway evolved to prevent accumulation of toxic glycolate. PMID:18805949

  15. Structural Characterization of Mutations at the Oxygen Activation Site in Monomeric Sarcosine Oxidase

    SciTech Connect

    Schuman Jorns, Marilyn; Chen, Zhi-wei; Mathews, F. Scott

    2010-04-30

    Oxygen reduction and sarcosine oxidation in monomeric sarcosine oxidase (MSOX) occur at separate sites above the si- and re-faces, respectively, of the flavin ring. Mutagenesis studies implicate Lys265 as the oxygen activation site. Substitution of Lys265 with a neutral (Met, Gln, or Ala) or basic (Arg) residue results in an {approx}10{sup 4}- or 250-fold decrease, respectively, in the reaction rate. The overall structure of MSOX and residue conformation in the sarcosine binding cavity are unaffected by replacement of Lys265 with Met or Arg. The side chain of Met265 exhibits the same configuration in each molecule of Lys265Met crystals and is nearly congruent with Lys265 in wild-type MSOX. The side chain of Arg265 is, however, dramatically shifted (4-5 {angstrom}) compared with Lys265, points in the opposite direction, and exhibits significant conformational variability between molecules of the same crystal. The major species in solutions of Lys265Arg is likely to contain a 'flipped-out' Arg265 and exhibit negligible oxygen activation, similar to Lys265Met. The 400-fold higher oxygen reactivity observed with Lys265Arg is attributed to a minor (<1%) 'flipped-in' Arg265 conformer whose oxygen reactivity is similar to that of wild-type MSOX. A structural water (WAT1), found above the si-face of the flavin ring in all previously determined MSOX structures, is part of an apparent proton relay system that extends from FAD N(5) to bulk solvent. WAT1 is strikingly absent in Lys265Met and Lys265Arg, a feature that may account for the apparent kinetic stabilization of a reductive half-reaction intermediate that is detectable with the mutants but not wild-type MSOX.

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

    PubMed

    Li Lee, Mui; Chung, Ivy; Yee Fung, Shin; Kanthimathi, M S; Hong Tan, Nget

    2014-04-01

    King cobra (Ophiophagus hannah) venom L-amino acid oxidase (LAAO), a heat-stable enzyme, is an extremely potent antiproliferative agent against cancer cells when compared with LAAO isolated from other snake venoms. King cobra venom LAAO was shown to exhibit very strong antiproliferative activities against MCF-7 (human breast adenocarcinoma) and A549 (human lung adenocarcinoma) cells, with an IC50 value of 0.04±0.00 and 0.05±0.00 μg/mL, respectively, after 72-hr treatment. In comparison, its cytotoxicity was about 3-4 times lower when tested against human non-tumourigenic breast (184B5) and lung (NL 20) cells, suggesting selective antitumour activity. Furthermore, its potency in MCF-7 and A549 cell lines was greater than the effects of doxorubicin, a clinically established cancer chemotherapeutic agent, which showed an IC50 value of 0.18±0.03 and 0.63±0.21 μg/mL, respectively, against the two cell lines. The selective cytotoxic action of the LAAO was confirmed by phycoerythrin (PE) annexin V/7-amino-actinomycin (AAD) apoptotic assay, in which a significant increase in apoptotic cells was observed in LAAO-treated tumour cells than in their non-tumourigenic counterparts. The ability of LAAO to induce apoptosis in tumour cells was further demonstrated using caspase-3/7 and DNA fragmentation assays. We also determined that this enzyme may target oxidative stress in its killing of tumour cells, as its cytotoxicity was significantly reduced in the presence of catalase (a H2O2 scavenger). In view of its heat stability and selective and potent cytotoxic action on cancer cells, king cobra venom LAAO can be potentially developed for treating solid tumours. PMID:24118879

  17. NADPH Oxidase NOX4 Mediates Stellate Cell Activation and Hepatocyte Cell Death during Liver Fibrosis Development

    PubMed Central

    Sancho, Patricia; Mainez, Jèssica; Crosas-Molist, Eva; Roncero, César; Fernández-Rodriguez, Conrado M.; Pinedo, Fernando; Huber, Heidemarie; Eferl, Robert; Mikulits, Wolfgang; Fabregat, Isabel

    2012-01-01

    A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2−/−/p19ARF−/−, Stat3Δhc/Mdr2−/−) and a model of experimental induced fibrosis (CCl4) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes. PMID:23049784

  18. Microfluidic Devices Integrating Microcavity Surface-Plasmon-Resonance Sensors: Glucose Oxidase Binding-Activity Detection

    PubMed Central

    Amarie, Dragos; Alileche, Abdelkrim; Dragnea, Bogdan; Glazier, James A.

    2010-01-01

    We have developed miniature (≈1 μm diameter) microcavity surface-plasmon-resonance sensors (MSPRS), integrated them with microfluidics and tested their sensitivity to refractive-index changes. We tested their biosensing capability by distinguishing the interaction of glucose oxidase (Mr 160 kDa) with its natural substrate (β-D-glucose, Mr 180 Da) from its interactions with non-specific substrates (L-glucose, D-mannose and 2-deoxy-D-glucose). We ran the identical protocol we had used with the MSPRS on a Biacore 3000 instrument using their bare gold chip. Only the MSPRS was able to detect β-D-glucose binding to glucose oxidase. Each MSPRS can detect the binding to its surface of fewer than 35,000 glucose-oxidase molecules (representing 9.6 fg or 60 zmol of protein), about 106 times fewer than classical surface-plasmon-resonance biosensors. PMID:19968248

  19. Increasing the catalytic activity of Bilirubin oxidase from Bacillus pumilus: Importance of host strain and chaperones proteins.

    PubMed

    Gounel, Sébastien; Rouhana, Jad; Stines-Chaumeil, Claire; Cadet, Marine; Mano, Nicolas

    2016-07-20

    Aggregation of recombinant proteins into inclusion bodies (IBs) is the main problem of the expression of multicopper oxidase in Escherichia coli. It is usually attributed to inefficient folding of proteins due to the lack of copper and/or unavailability of chaperone proteins. The general strategies reported to overcome this issue have been focused on increasing the intracellular copper concentration. Here we report a complementary method to optimize the expression in E. coli of a promising Bilirubin oxidase (BOD) isolated from Bacillus pumilus. First, as this BOD has a disulfide bridge, we switched E.coli strain from BL21 (DE3) to Origami B (DE3), known to promote the formation of disulfide bridges in the bacterial cytoplasm. In a second step, we investigate the effect of co-expression of chaperone proteins on the protein production and specific activity. Our strategy allowed increasing the final amount of enzyme by 858% and its catalytic rate constant by 83%. PMID:27165502

  20. Cytotoxicity of, and DNA damage by, active oxygen species produced by xanthine oxidase.

    PubMed

    Chiricolo, M; Tazzari, P L; Abbondanza, A; Dinota, A; Battelli, M G

    1991-10-21

    Toxicity to Raji cells of the xanthine oxidase/hypoxanthine system is related to the formation of single-strand DNA breaks. DNA damage was proportional to the concentration of xanthine oxidase and to the time of exposure. It was prevented by the absence of hypoxanthine, or by the presence of allopurinol, or both superoxide dismutase and catalase. The release of 51Cr from damaged cells was detectable 12 h after the inhibition of cloning efficiency and the production of DNA breakage. These data suggest that DNA damage induced by the oxygen products precedes the severe lesion to the cellular membrane. PMID:1936259

  1. Type I Helicobacter pylori Lipopolysaccharide Stimulates Toll-Like Receptor 4 and Activates Mitogen Oxidase 1 in Gastric Pit Cells

    PubMed Central

    Kawahara, Tsukasa; Teshima, Shigetada; Oka, Ayuko; Sugiyama, Toshiro; Kishi, Kyoichi; Rokutan, Kazuhito

    2001-01-01

    Guinea pig gastric pit cells express an isozyme of gp91-phox, mitogen oxidase 1 (Mox1), and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phox). Helicobacter pylori lipopolysaccharide (LPS) and Escherichia coli LPS have been shown to function as potent activators for the Mox1 oxidase. These cells spontaneously secreted about 10 nmol of superoxide anion (O2−)/mg of protein/h under LPS-free conditions. They expressed the mRNA and protein of Toll-like receptor 4 (TLR4) but not those of TLR2. LPS from type I H. pylori at 2.1 endotoxin units/ml or higher stimulated TLR4-mediated phosphorylations of transforming growth factor β-activated kinase 1 and its binding protein 1 induced TLR4 and p67-phox and up-regulated O2− production 10-fold. In contrast, none of these events occurred with H. pylori LPS from complete or partial deletion mutants of the cag pathogenicity island. Lipid A was confirmed to be a bioactive component for the priming effects, while removal of bisphosphates from lipid A completely eliminated the effects, suggesting the importance of the phosphorylation pattern besides the acylation pattern for the bioactivity. H. pylori LPS is generally accepted as having low toxicity; however, our results suggest that type I H. pylori lipid A may be a potent stimulator for innate immune responses of gastric mucosa by stimulating the TLR4 cascade and Mox1 oxidase in pit cells. PMID:11401977

  2. Augmented EGF receptor tyrosine kinase activity impairs vascular function by NADPH oxidase-dependent mechanism in type 2 diabetic mouse.

    PubMed

    Kassan, Modar; Ait-Aissa, Karima; Ali, Maha; Trebak, Mohamed; Matrougui, Khalid

    2015-10-01

    We previously determined that augmented EGFR tyrosine kinase (EGFRtk) impairs vascular function in type 2 diabetic mouse (TD2). Here we determined that EGFRtk causes vascular dysfunction through NADPH oxidase activity in TD2. Mesenteric resistance arteries (MRA) from C57/BL6 and db-/db- mice were mounted in a wired myograph and pre-incubated for 1h with either EGFRtk inhibitor (AG1478) or exogenous EGF. The inhibition of EGFRtk did not affect the contractile response to phenylephrine-(PE) and thromboxane-(U46619) or endothelium-dependent relaxation (EDR) to acetylcholine in MRA from control group. However, in TD2 mice, AG1478 reduced the contractile response to U46619, improved vasodilatation and reduced p22phox-NADPH expression, but had no effect on the contractile response to PE. The incubation of MRA with exogenous EGF potentiated the contractile response to PE in MRA from control and diabetic mice. However, EGF impaired the EDR and potentiated the vasoconstriction to U46619 only in the control group. Interestingly, NADPH oxidase inhibition in the presence of EGF restored the normal contraction to PE and improved the EDR but had no effect on the potentiated contraction to U46619. Vascular function improvement was associated with the rescue of eNOS and Akt and reduction in phosphorylated Rho-kinase, NOX4 mRNA levels, and NADPH oxidase activity. MRA from p47phox-/- mice incubated with EGF potentiated the contraction to U46619 but had no effect to PE or ACh responses. The present study provides evidence that augmented EGFRtk impairs vascular function by NADPH oxidase-dependent mechanism. Therefore, EGFRtk and oxidative stress should be potential targets to treat vascular dysfunction in TD2. PMID:26036345

  3. Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

    PubMed Central

    Moomaw, Ellen W.; Hoffer, Eric; Moussatche, Patricia; Salerno, John C.; Grant, Morgan; Immelman, Bridget; Uberto, Richard; Ozarowski, Andrew; Angerhofer, Alexander

    2013-01-01

    Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site. PMID:23469254

  4. Changes in Cytokinin Content and Cytokinin Oxidase Activity in Response to Derepression of ipt Gene Transcription in Transgenic Tobacco Calli and Plants.

    PubMed Central

    Motyka, V.; Faiss, M.; Strand, M.; Kaminek, M.; Schmulling, T.

    1996-01-01

    Metabolic control of cytokinin oxidase by its substrate was investigated in planta using wild-type (WT) and conditionally ipt gene-expressing transgenic (IPT) tobacco (Nicotiana tabacum L.) callus cultures and plants. The derepression of the tetracycline (Tc)-dependent ipt gene transcription was followed by a progressive, more than 100-fold increase in total cytokinin content in IPT calli. The activity of cytokinin oxidase extracted from these calli began to increase 16 to 20 h after gene derepression, and after 13 d it was 10-fold higher than from Tc-treated WT calli. An increase in cytokinin oxidase activity, as a consequence of elevated cytokinin levels, was found in detached leaves (8-fold after 4 d) and in roots of intact plants (4-fold after 3 d). The partially purified cytokinin oxidase from WT, repressed IPT, and Tc-derepressed IPT tobacco calli exhibited similar characteristics. It had the same broad pH optimum (pH 6.5-8.5), its activity in vitro was enhanced 4-fold in the presence of copper-imidazole, and the apparent Km(N6-[[delta]2iso-pentenyl]adenine) values were in the range of 3.1 to 4.9 [mu]M. The increase in cytokinin oxidase activity in cytokinin-overproducing tissue was associated with the accumulation of a glycosylated form of the enzyme. The present data indicate the substrate induction of cytokinin oxidase activity in different tobacco tissues, which may contribute to hormone homeostasis. PMID:12226431

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in meristems isolated from field-g...

  6. Structural basis of Ornithine Decarboxylase inactivation and accelerated degradation by polyamine sensor Antizyme1

    PubMed Central

    Wu, Donghui; Kaan, Hung Yi Kristal; Zheng, Xiaoxia; Tang, Xuhua; He, Yang; Vanessa Tan, Qianmin; Zhang, Neng; Song, Haiwei

    2015-01-01

    Ornithine decarboxylase (ODC) catalyzes the first and rate-limiting step of polyamine biosynthesis in humans. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis. Excessive accumulation of polyamines has a cytotoxic effect on cells and elevated level of ODC activity is associated with cancer development. To maintain normal cellular proliferation, regulation of polyamine synthesis is imposed by Antizyme1 (AZ1). The expression of AZ1 is induced by a ribosomal frameshifting mechanism in response to increased intracellular polyamines. AZ1 regulates polyamine homeostasis by inactivating ODC activity and enhancing its degradation. Here, we report the structure of human ODC in complex with N-terminally truncated AZ1 (cAZ1). The structure shows cAZ1 binding to ODC, which occludes the binding of a second molecule of ODC to form the active homodimer. Consequently, the substrate binding site is disrupted and ODC is inactivated. Structural comparison shows that the binding of cAZ1 to ODC causes a global conformational change of ODC and renders its C-terminal region flexible, therefore exposing this region for degradation by the 26S proteasome. Our structure provides the molecular basis for the inactivation of ODC by AZ1 and sheds light on how AZ1 promotes its degradation. PMID:26443277

  7. Intestinal NADPH oxidase 2 activity increases in a neonatal rat model of necrotizing enterocolitis.

    PubMed

    Welak, Scott R; Rentea, Rebecca M; Teng, Ru-Jeng; Heinzerling, Nathan; Biesterveld, Ben; Liedel, Jennifer L; Pritchard, Kirkwood A; Fredrich, Katherine M; Gourlay, David M

    2014-01-01

    Necrotizing enterocolitis (NEC) is a complication of prematurity. The etiology is unknown, but is related to enteral feeding, ischemia, infection, and inflammation. Reactive oxygen species production, most notably superoxide, increases in NEC. NADPH oxidase (NOX) generates superoxide, but its activity in NEC remains unknown. We hypothesize that NOX-derived superoxide production increases in NEC. Newborn Sprague-Dawley rats were divided into control, formula-fed, formula/LPS, formula/hypoxia, and NEC (formula, hypoxia, and LPS). Intestinal homogenates were analyzed for NADPH-dependent superoxide production. Changes in superoxide levels on days 0-4 were measured. Inhibitors for nitric oxide synthase (L-NAME) and NOX2 (GP91-ds-tat) were utilized. RT-PCR for eNOS, NOX1, GP91phox expression was performed. Immunofluorescence studies estimated the co-localization of p47phox and GP91phox in control and NEC animals on D1, D2, and D4. NEC pups generated more superoxide than controls on D4, while all other groups were unchanged. NADPH-dependent superoxide production was greater in NEC on days 0, 3, and 4. GP91-ds-tat decreased superoxide production in both groups, with greater inhibition in NEC. L-NAME did not alter superoxide production. Temporally, superoxide production varied minimally in controls. In NEC, superoxide generation was decreased on day 1, but increased on days 3-4. GP91phox expression was higher in NEC on days 2 and 4. NOX1 and eNOS expression were unchanged from controls. GP91phox and p47phox had minimal co-localization in all control samples and NEC samples on D1 and D2, but had increased co-localization on D4. In conclusion, this study proves that experimentally-induced NEC increases small intestinal NOX activity. All components of NEC model are necessary for increased NOX activity. NOX2 is the major source, especially as the disease progresses. PMID:25517730

  8. Structure of Prokaryotic Polyamine Deacetylase Reveals Evolutionary Functional Relationships with Eukaryotic Histone Deacetylases

    SciTech Connect

    P Lombardi; H Angell; D Whittington; E Flynn; K Rajashankar; D Christianson

    2011-12-31

    Polyamines are a ubiquitous class of polycationic small molecules that can influence gene expression by binding to nucleic acids. Reversible polyamine acetylation regulates nucleic acid binding and is required for normal cell cycle progression and proliferation. Here, we report the structures of Mycoplana ramosa acetylpolyamine amidohydrolase (APAH) complexed with a transition state analogue and a hydroxamate inhibitor and an inactive mutant complexed with two acetylpolyamine substrates. The structure of APAH is the first of a histone deacetylase-like oligomer and reveals that an 18-residue insert in the L2 loop promotes dimerization and the formation of an 18 {angstrom} long 'L'-shaped active site tunnel at the dimer interface, accessible only to narrow and flexible substrates. The importance of dimerization for polyamine deacetylase function leads to the suggestion that a comparable dimeric or double-domain histone deacetylase could catalyze polyamine deacetylation reactions in eukaryotes.

  9. Xanthine oxidase, but not neutrophils, contributes to activation of cardiac sympathetic afferents during myocardial ischaemia in cats

    PubMed Central

    Tjen-A-Looi, Stephanie C; Fu, Liang-Wu; Longhurst, John C

    2002-01-01

    Activation of cardiac sympathetic afferents during myocardial ischaemia causes angina and induces important cardiovascular reflex responses. Reactive oxygen species (ROS) are important chemical stimuli of cardiac afferents during and after ischaemia. Iron-catalysed Fenton chemistry constitutes one mechanism of production of hydroxyl radicals. Another potential source of these species is xanthine oxidase-catalysed oxidation of purines. Polymorphonuclear leukocytes (PMNs) also contribute to the production of ROS in some conditions. The present study tested the hypothesis that both xanthine oxidase-catalysed oxidation of purines and neutrophils provide a source of ROS sufficient to activate cardiac afferents during ischaemia. We recorded single-unit activity of cardiac afferents innervating the ventricles recorded from the left thoracic sympathetic chain (T1-5) of anaesthetized cats to identify the afferents' responses to ischaemia. The role of xanthine oxidase in activation of these afferents was determined by infusion of oxypurinol (10 mg kg−1, i.v.), an inhibitor of xanthine oxidase. The importance of neutrophils as a potential source of ROS in the activation of cardiac afferents during ischaemia was assessed by the infusion of a polyclonal antibody (3 mg ml−1 kg−1, i.v.) raised in rabbits immunized with cat PMNs. This antibody decreased the number of circulating PMNs and, to a smaller extent, platelets. Since previous data suggest that platelets release serotonin (5-HT), which activates cardiac afferents through a serotonin receptor (subtype 3,5-HT3 receptor) mechanism, before treatment with the antibody in another group, we blocked 5-HT3 receptors on sensory nerve endings with tropisetron (300 μg kg−1, i.v.). We observed that oxypurinol significantly decreased the activity of cardiac afferents during myocardial ischaemia from 1.5 ± 0.4 to 0.8 ± 0.4 impulses s−1. Similarly, the polyclonal antibody significantly reduced the discharge frequency of

  10. Chcanges in Germinability and Activities of Polyphenol Oxidase and Peroxidase in Seeds of Pentaclethramacrophylla During Lowtemperature Treatment

    NASA Astrophysics Data System (ADS)

    Udosen, I. R.; Nkang, A. E.; Sam, S. M.

    2012-07-01

    Activities of peroxidase (POD) and polyphenol Oxidase (PPO) were investigated in seeds of Pentaclethramacrophylla during low temperature treatment. The seeds from the small-sized fruits (variety A) and those of the big-sized fruits (variety B) showed high germination, with maximum germination values ranging between 60 ñ 90%. Low temperature treatment did not significantly (P< 0.5) affect maximum germination values. Activities of POD and PPO increased initially (2-4 days) but declined with prolonged (6ñ8 days) low temperature treatment.

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

    PubMed Central

    McCarty, Mark F.

    2015-01-01

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

  12. Effects of IFN-γ on intracellular trafficking and activity of macrophage NADPH oxidase flavocytochrome b558

    PubMed Central

    Casbon, Amy-Jo; Long, Matthew E.; Dunn, Kenneth W.; Allen, Lee-Ann H.; Dinauer, Mary C.

    2012-01-01

    Flavocytochrome b558, the catalytic core of the phagocyte NADPH oxidase (NOX2), mediates electron transfer from NADPH to molecular oxygen to generate superoxide, the precursor of highly ROS for host defense. Flavocytochrome b558 is an integral membrane heterodimer consisting of a large glycosylated subunit, gp91phox, and a smaller subunit, p22phox. We recently showed in murine macrophages that flavocytochrome b558 localizes to the PM and Rab11-positive recycling endosomes, whereas in primary hMDMs, gp91phox and p22phox reside in the PM and the ER. The antimicrobial activity of macrophages, including ROS production, is greatly enhanced by IFN-γ, but how this is achieved is incompletely understood. To further define the mechanisms by which IFN-γ enhances macrophage NADPH oxidase activity, we evaluated changes in flavocytochrome b558 expression and localization, along with NADPH oxidase activity, in IFN-γ stimulated RAW 264.7 cells and primary murine BMDMs and hMDMs. We found that enhanced capacity for ROS production is, in part, a result of increased protein expression of gp91phox and p22phox but also demonstrate that IFN-γ induced a shift in the predominant localization of gp91phox and p22phox from intracellular membrane compartments to the PM. Our results are the first to show that a cytokine can change the distribution of macrophage flavocytochrome b558 and provide a potential, new mechanism by which IFN-γ modulates macrophage antimicrobial activity. Altogether, our data suggest that the mechanisms by which IFN-γ regulates antimicrobial activity of macrophages are more complex than previously appreciated. PMID:22822009

  13. Anticancer drugs and hyperthermia enhance cytotoxicity induced by polyamine enzymatic oxidation products.

    PubMed

    Marra, M; Agostinelli, E; Tempera, G; Lombardi, A; Meo, G; Budillon, A; Abbruzzese, A; Giuberti, G; Caraglia, M

    2007-08-01

    A correlation between regulation of cell proliferation and polyamine metabolism is described. The latter can enter protein synthesis through the modification of eukaryotic initiation factor 5A (eIF5A) and the formation of the peculiar amino acid hypusine. Specific inhibitors of hypusine formation induce apoptosis that can be potentiated by the combination with cytokines such as interferonalpha (IFNalpha) that itself decreases hypusine synthesis. We have also demonstrated that the concomitant treatment of cancer cells with IFNalpha and the protein synthesis inhibitor fusion protein TGFalpha/Pseudomonas Aeruginosa toxin synergize in inducing cancer cell growth inhibition. Another way used by polyamines to induce apoptosis is the generation of intracellular oxidative stress through the interaction with bovine serum amine oxidase (BSAO). This enzyme used simultaneously to spermine induces apoptosis, necrosis, inhibition of cell proliferation and inhibition of DNA and protein synthesis in several cell types. The enzymatic oxidation products of polyamine, H2O2 and aldehyde(s) cause these effects. We have recently found that the cytotoxicity of anti-cancer agents, either etoposide or docetaxel, in cancer cells is potentiated in the presence of BSAO/Spermine. In conclusion, polyamine metabolites could be useful in the design of new therapeutic strategies. PMID:17610128

  14. Polyamines are Inhibitors of Gastric Acid Secretion

    NASA Astrophysics Data System (ADS)

    Ray, Tushar K.; Nandi, Jyotirmoy; Pidhorodeckyj, Nykolai; Meng-Ai, Zhou

    1982-03-01

    The naturally occurring organic polycations such as spermine and spermidine inhibit histamine-stimulated gastric acid secretion by bullfrog gastric mucosa in vitro; spermine is much more potent than spermidine. Unlike the H2 receptor antagonists, the polyamines are completely ineffective from the nutrient side and are effective only from the secretory side of the chambered mucosa. The polyamine effects could be reversed by increasing K+ concentration in the secretory solution. Studies with isolated gastric microsomal vesicles demonstrate that the polyamines do not inhibit the gastric H+,K+-ATPase but greatly decrease the ATPase-mediated uptake of H+ under appropriate conditions. For the latter effects the presence of polyamine within the vesicle interior was found to be essential. Our data strongly suggest an uncoupling of the gastric H+,K+-ATPase system by the polyamines. The therapeutic potential of these and similar compounds in the treatment of hyperacidity and peptic ulcer is discussed.

  15. Ornithine Decarboxylase, Polyamines, and Pyrrolizidine Alkaloids in Senecio and Crotalaria

    PubMed Central

    Birecka, Helena; Birecki, Mieczyslaw; Cohen, Eric J.; Bitonti, Alan J.; McCann, Peter P.

    1988-01-01

    When tested for ornithine and arginine decarboxylases, pyrrolizidine alkaloid-bearing Senecio riddellii, S. longilobus (Compositae), and Crotalaria retusa (Leguminosae) plants exhibited only ornithine decarboxylase activity. This contrasts with previous studies of four species of pyrrolizidine alkaloid-bearing Heliotropium (Boraginaceae) in which arginine decarboxylase activity was very high relative to that of ornithine decarboxylase. Unlike Heliotropium angiospermum and Heliotropium indicum, in which endogenous arginine was the only detectable precursor of putrescine channeled into pyrrolizidines, in the species studied here—using difluoromethylornithine and difluoromethylarginine as the enzyme inhibitors—endogenous ornithine was the main if not the only precursor of putrescine converted into the alkaloid aminoalcohol moiety. In S. riddellii and C. retusa at flowering, ornithine decarboxylase activity was present mainly in leaves, especially the young ones. However, other very young organs such as inflorescence and growing roots exhibited much lower or very low activities; the enzyme activity in stems was negligible. There was no correlation between the enzyme activity and polyamine or alkaloid content in either species. In both species only free polyamines were detected except for C. retusa roots and inflorescence—with relatively very high levels of these compounds—in which conjugated putrescine, spermidine, and spermine were also found; agmatine was not identified by HPLC in any plant organ except for C. retusa roots with rhizobial nodules. Organ- or age-dependent differences in the polyamine levels were small or insignificant. The highest alkaloid contents were found in young leaves and inflorescence. PMID:16665870

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

    PubMed

    Nana, Fernand W; Hilou, Adama; Millogo, Jeanne F; Nacoulma, Odile G

    2012-01-01

    This paper describes a preliminary assessment of the nutraceutical value of Amaranthus cruentus (A. cruentus) and Amaranthus hybridus (A. hybridus), two food plant species found in Burkina Faso. Hydroacetonic (HAE), methanolic (ME), and aqueous extracts (AE) from the aerial parts were screened for in vitro antioxidant and xanthine oxidase inhibitory activities. Phytochemical analyses revealed the presence of polyphenols, tannins, flavonoids, steroids, terpenoids, saponins and betalains. Hydroacetonic extracts have shown the most diversity for secondary metabolites. The TLC analyses of flavonoids from HAE extracts showed the presence of rutin and other unidentified compounds. The phenolic compound contents of the HAE, ME and AE extracts were determined using the Folin-Ciocalteu method and ranged from 7.55 to 10.18 mg Gallic acid equivalent GAE/100 mg. Tannins, flavonoids, and flavonols ranged from 2.83 to 10.17 mg tannic acid equivalent (TAE)/100 mg, 0.37 to 7.06 mg quercetin equivalent (QE) /100 mg, and 0.09 to 1.31 mg QE/100 mg, respectively. The betacyanin contents were 40.42 and 6.35 mg Amaranthin Equivalent/100 g aerial parts (dry weight) in A. cruentus and A. hybridus, respectively. Free-radical scavenging activity expressed as IC50 (DPPH method) and iron reducing power (FRAP method) ranged from 56 to 423 µg/mL and from 2.26 to 2.56 mmol AAE/g, respectively. Xanthine oxidase inhibitory activities of extracts of A. cruentus and A. hybridus were 3.18% and 38.22%, respectively. The A. hybridus extract showed the best antioxidant and xanthine oxidase inhibition activities. The results indicated that the phytochemical contents of the two species justify their traditional uses as nutraceutical food plants. PMID:24281664

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

    PubMed Central

    Nana, Fernand W.; Hilou, Adama; Millogo, Jeanne F.; Nacoulma, Odile G.

    2012-01-01

    This paper describes a preliminary assessment of the nutraceutical value of Amaranthus cruentus (A. cruentus) and Amaranthus hybridus (A. hybridus), two food plant species found in Burkina Faso. Hydroacetonic (HAE), methanolic (ME), and aqueous extracts (AE) from the aerial parts were screened for in vitro antioxidant and xanthine oxidase inhibitory activities. Phytochemical analyses revealed the presence of polyphenols, tannins, flavonoids, steroids, terpenoids, saponins and betalains. Hydroacetonic extracts have shown the most diversity for secondary metabolites. The TLC analyses of flavonoids from HAE extracts showed the presence of rutin and other unidentified compounds. The phenolic compound contents of the HAE, ME and AE extracts were determined using the Folin–Ciocalteu method and ranged from 7.55 to 10.18 mg Gallic acid equivalent GAE/100 mg. Tannins, flavonoids, and flavonols ranged from 2.83 to 10.17 mg tannic acid equivalent (TAE)/100 mg, 0.37 to 7.06 mg quercetin equivalent (QE) /100 mg, and 0.09 to 1.31 mg QE/100 mg, respectively. The betacyanin contents were 40.42 and 6.35 mg Amaranthin Equivalent/100 g aerial parts (dry weight) in A. cruentus and A. hybridus, respectively. Free-radical scavenging activity expressed as IC50 (DPPH method) and iron reducing power (FRAP method) ranged from 56 to 423 µg/mL and from 2.26 to 2.56 mmol AAE/g, respectively. Xanthine oxidase inhibitory activities of extracts of A. cruentus and A. hybridus were 3.18% and 38.22%, respectively. The A. hybridus extract showed the best antioxidant and xanthine oxidase inhibition activities. The results indicated that the phytochemical contents of the two species justify their traditional uses as nutraceutical food plants. PMID:24281664

  18. Advanced glycation endproducts induce apoptosis of endothelial progenitor cells by activating receptor RAGE and NADPH oxidase/JNK signaling axis

    PubMed Central

    Chen, Jianfei; Jing, Jun; Yu, Shiyong; Song, Minbao; Tan, Hu; Cui, Bin; Huang, Lan

    2016-01-01

    Elevated levels of advanced glycation endproducts (AGEs) is an important risk factor for atherosclerosis. Dysfunction of endothelial progenitor cells (EPCs), which is essential for re-endothelialization and neovascularization, is a hallmark of atherosclerosis. However, it remains unclear whether and how AGEs acts on EPCs to promote pathogenesis of atherosclerosis. In this study, EPCs were exposed to different concentrations of AGEs. The expression of NADPH and Rac1 was measured to investigate the involvement of NADPH oxidase pathway. ROS was examined to indicate the level of oxidative stress in EPCs. Total JNK and p-JNK were determined by Western blotting. Cell apoptosis was evaluated by both TUNEL staining and flow cytometry. Cell proliferation was measured by 3H thymidine uptake. The results showed that treatment of EPCs with AGEs increased the levels of ROS in EPCs. Mechanistically, AGEs increased the activity of NADPH oxidase and the expression of Rac1, a major component of NADPH. Importantly, treatment of EPCs with AGEs activated the JNK signaling pathway, which was closely associated with cell apoptosis and inhibition of proliferation. Our results suggest that the RAGE activation by AGEs in EPCs upregulates intracellular ROS levels, which contributes to increased activity of NADPH oxidase and expression of Rac1, thus promoting cellular apoptosis and inhibiting proliferation. Mechanistically, AGEs binding to the receptor RAGE in EPCs is associated with hyperactivity of JNK signaling pathway, which is downstream of ROS. Our findings suggest that dysregulation of the AGEs/RAGE axis in EPCs may promote atherosclerosis and identify the NADPH/ROS/JNK signaling axis as a potential target for therapeutic intervention. PMID:27347324

  19. Cyclic AMP and Polyamines Overcome Inhibition by Myelin-Associated Glycoprotein through eIF5A-Mediated Increases in p35 Expression and Activation of Cdk5.

    PubMed

    He, Huifang; Deng, Kangwen; Siddiq, Mustafa M; Pyie, Aung; Mellado, Wilfredo; Hannila, Sari S; Filbin, Marie T

    2016-03-01

    Inhibitory molecules associated with CNS myelin, such as myelin-associated glycoprotein (MAG), represent major obstacles to axonal regeneration following CNS injury. Our laboratory has shown that elevating levels of intracellular cAMP, via application of the nonhydrolyzable analog dibutyryl cAMP (dbcAMP), can block the inhibitory effects of MAG and myelin. We have also shown that elevation of cAMP results in upregulation of arginase I and increased polyamine synthesis. Treatment with putrescine or spermidine blocks myelin-mediated inhibition of neurite outgrowth, but the mechanism underlying this effect has not yet been elucidated. Here we show that cyclin-dependent kinase 5 (Cdk5) is required for dbcAMP and putrescine to overcome MAG-mediated inhibition. The ability of dbcAMP and putrescine to overcome inhibition by MAG is abolished in the presence of roscovitine, a Cdk inhibitor that has greater selectivity for Cdk5, and expression of dominant negative Cdk5 abolishes the ability of dbcAMP or putrescine to enhance neurite outgrowth in the presence of MAG. Importantly, dbcAMP and putrescine increase expression of p35, the neuron-specific activator of Cdk5, and rat DRG neurons transduced with HSV overexpressing p35 can overcome inhibition by MAG. The upregulation of p35 by putrescine is also reflected in increased localization of p35 to neurites and growth cones. Last, we show that putrescine upregulates p35 expression by serving as a substrate for hypusine modification of eIF5A, and that this hypusination is necessary for putrescine's ability to overcome inhibition by MAG. Our findings reveal a previously unknown mechanism by which polyamines may encourage regeneration after CNS injury. PMID:26961960

  20. Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes.

    PubMed

    Kasi, Viswanath; Bodiga, Sreedhar; Kommuguri, Upendra Nadh; Sankuru, Suneetha; Bodiga, Vijaya Lakshmi

    2011-07-01

    Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47(phox) phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress. PMID:21651898

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

    SciTech Connect

    Park, Joonghoon; Park, Eok; Ahn, Bong-Hyun; Kim, Hyoung Jin; Park, Ji-hoon; Koo, Sun Young; Kwak, Hyo-Shin; Park, Heui Sul; Kim, Dong Wook; Song, Myoungsub; Yim, Hyeon Joo; Seo, Dong Ook; Kim, Soon Ha

    2012-08-15

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

  2. Involvement of Pyruvate Oxidase Activity and Acetate Production in the Survival of Lactobacillus plantarum during the Stationary Phase of Aerobic Growth▿ †

    PubMed Central

    Goffin, Philippe; Muscariello, Lidia; Lorquet, Frederique; Stukkens, Aline; Prozzi, Deborah; Sacco, Margherita; Kleerebezem, Michiel; Hols, Pascal

    2006-01-01

    In addition to the previously characterized pyruvate oxidase PoxB, the Lactobacillus plantarum genome encodes four predicted pyruvate oxidases (PoxC, PoxD, PoxE, and PoxF). Each pyruvate oxidase gene was individually inactivated, and only the knockout of poxF resulted in a decrease in pyruvate oxidase activity under the tested conditions. We show here that L. plantarum has two major pyruvate oxidases: PoxB and PoxF. Both are involved in lactate-to-acetate conversion in the early stationary phase of aerobic growth and are regulated by carbon catabolite repression. A strain devoid of pyruvate oxidase activity was constructed by knocking out the poxB and poxF genes. In this mutant, acetate production was strongly affected, with lactate remaining the major end product of either glucose or maltose fermentation. Notably, survival during the stationary phase appeared to be dramatically improved in the poxB poxF double mutant. PMID:17012588

  3. Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation

    PubMed Central

    Terry-Lorenzo, Ryan T.; Chun, Lawrence E.; Brown, Scott P.; Heffernan, Michele L. R.; Fang, Q. Kevin; Orsini, Michael A.; Pollegioni, Loredano; Hardy, Larry W.; Spear, Kerry L.; Large, Thomas H.

    2014-01-01

    The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, ‘compound 2’ [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki=7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors. PMID:25001371

  4. Effect of ethanol, carbon tetrachloride, and methyl ethyl ketone on butanol oxidase activity in rat lung and liver

    SciTech Connect

    Carlson, G.P. )

    1989-01-01

    Tha ability of the rat liver to oxidize 2-butanol via a cytochrome P-450-mediated mixed-function oxidase reaction is well known. The purpose of this study was to examine this microsomal alcohol oxidizing system in rat lung and determine if it could be altered by treatments that inhibit or induce this activity. 2-Butanol was incubated with microsomal preparations from male rats, and methyl ethyl ketone production was measured by gas chromatography. The rate was six to eight times lower in lung than in liver. Administration of low doses of ethanol (0.5 ml/kg and 1.0 ml/kg) ip for 7 d did not alter activity in the liver but was inhibitory in the lung, as was a high dose of 3.0 ml/kg in the liver. Carbon tetrachloride (1.0 ml/kg, ip) decreased activity in both tissues, especially the lung. The effects of the two inhibitors were not additive. Methyl ethyl ketone induced 2-butanol oxidation in both tissues. The lung possesses butanol oxidase activity that is alterable by both inhibitors and inducers.

  5. Polyamine metabolic canalization in response to drought stress in Arabidopsis and the resurrection plant Craterostigma plantagineum

    PubMed Central

    Bartels, Dorothea; Koncz, Csaba; Altabella, Teresa

    2011-01-01

    In this work, we have studied the transcriptional profiles of polyamine biosynthetic genes and analyzed polyamine metabolic fluxes during a gradual drought acclimation response in Arabidopsis thaliana and the resurrection plant Craterostigma plantagineum. The analysis of free putrescine, spermidine and spermine titers in Arabidopsis arginine decarboxylase (adc1–3, adc2–3), spermidine synthase (spds1–2, spds2–3) and spermine synthase (spms-2) mutants during drought stress, combined with the quantitative expression of the entire polyamine biosynthetic pathway in the wild-type, has revealed a strong metabolic canalization of putrescine to spermine induced by drought. Such canalization requires spermidine synthase 1 (SPDS1) and spermine synthase (SPMS) activities and, intriguingly, does not lead to spermine accumulation but to a progressive reduction in spermidine and spermine pools in the wild-type. Our results suggest the participation of the polyamine back-conversion pathway during the drought stress response rather than the terminal catabolism of spermine. The putrescine to spermine canalization coupled to the spermine to putrescine back-conversion confers an effective polyamine recycling-loop during drought acclimation. Putrescine to spermine canalization has also been revealed in the desiccation tolerant plant C. plantagineum, which conversely to Arabidopsis, accumulates high spermine levels which associate with drought tolerance. Our results provide a new insight to the polyamine homeostasis mechanisms during drought stress acclimation in Arabidopsis and resurrection plants. PMID:21330782

  6. Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps.

    PubMed

    Csomós, Krisztián; Kristóf, Endre; Jakob, Bernadett; Csomós, István; Kovács, György; Rotem, Omri; Hodrea, Judit; Bagoly, Zsuzsa; Muszbek, Laszlo; Balajthy, Zoltán; Csősz, Éva; Fésüs, László

    2016-01-01

    Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system. PMID:27512953

  7. NADPH oxidase activation contributes to native low-density lipoprotein-induced proliferation of human aortic smooth muscle cells

    PubMed Central

    Park, Il Hwan; Hwang, Hye Mi; Jeon, Byeong Hwa; Kwon, Hyung-Joo; Hoe, Kwang Lae; Kim, Young Myeong; Ryoo, Sungwoo

    2015-01-01

    Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-θ (PKCθ) and protein kinase C-β (PKCβ) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCθ and PKCβ stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox−/− mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated h

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

    PubMed

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

    2008-07-01

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

  9. Polyphenolic composition, antioxidant activity, and polyphenol oxidase (PPO) activity of quince (Cydonia oblonga Miller) varieties.

    PubMed

    Wojdyło, Aneta; Oszmiański, Jan; Bielicki, Paweł

    2013-03-20

    Phytochemical profiles (phenolic compounds, L-ascorbic acid, antioxidant and PPO activities) of 13 different quince varieties and 5 genotypes were studied. Polyphenols were identified by LC-PDA-QTof/MS and quantified by UPLC-PDA and UPLC-FL. A total of 26 polyphenolic compounds found in quince tissues were identified and presented: 9 flavan-3-ols ((-)-epicatechin, procyanidin B2, 3 procyanidin dimers and trimers, and 1 tetramer); 8 hydroxycinnamates, derivatives of caffeoylquinic and coumaroylquinic acid; and 9 kaempferol and quercetin derivatives. The content of total polyphenols was between 1709.43 (genotype 'S1') and 3436.56 mg/100 g dry weight ('Leskovač'). Flavan-3-ols, which are the major class of quince polyphenols, represented between 78 and 94% of the total polyphenolic compounds. The activity of PPO enzyme ranged from 709.85 to 1284.59 ΔU/min, and that of L-ascorbic acid ranged from 5.86 to 26.42 mg/100 g. Some quince varieties and their products characterized by a higher content of phenolic compounds may be selected to promote their positive effect on health. PMID:23461298

  10. Active Site and Loop 4 Movements with Human Glycolate Oxidase: Implications for Substrate Specificity and Drug Design

    SciTech Connect

    Murray,M.; Holmes, R.; Lowther, W.

    2008-01-01

    Human glycolate oxidase (GO) catalyzes the FMN-dependent oxidation of glycolate to glyoxylate and glyoxylate to oxalate, a key metabolite in kidney stone formation. We report herein the structures of recombinant GO complexed with sulfate, glyoxylate, and an inhibitor, 4-carboxy-5-dodecylsulfanyl-1, 2,3-triazole (CDST), determined by X-ray crystallography. In contrast to most {alpha}-hydroxy acid oxidases including spinach glycolate oxidase, a loop region, known as loop 4, is completely visible when the GO active site contains a small ligand. The lack of electron density for this loop in the GO-CDST complex, which mimics a large substrate, suggests that a disordered to ordered transition may occur with the binding of substrates. The conformational flexibility of Trp110 appears to be responsible for enabling GO to react with a-hydroxy acids of various chain lengths. Moreover, the movement of Trp110 disrupts a hydrogen-bonding network between Trp110, Leu191, Tyr134, and Tyr208. This loss of interactions is the first indication that active site movements are directly linked to changes in the conformation of loop 4. The kinetic parameters for the oxidation of glycolate, glyoxylate, and 2-hydroxy octanoate indicate that the oxidation of glycolate to glyoxylate is the primary reaction catalyzed by GO, while the oxidation of glyoxylate to oxalate is most likely not relevant under normal conditions. However, drugs that exploit the unique structural features of GO may ultimately prove to be useful for decreasing glycolate and glyoxylate levels in primary hyperoxaluria type 1 patients who have the inability to convert peroxisomal glyoxylate to glycine.

  11. NOX2β: A Novel Splice Variant of NOX2 That Regulates NADPH Oxidase Activity in Macrophages

    PubMed Central

    Guida, Elizabeth; King, Paul T.; Sobey, Christopher G.; Drummond, Grant R.

    2012-01-01

    Nox2 oxidase is one isoform in a family of seven NADPH oxidases that generate reactive oxygen species (ROS) and thereby contribute to physiological and pathological processes including host defense, redox signaling and oxidative tissue damage. While alternative mRNA splicing has been shown to influence the activity of several Nox-family proteins, functionally relevant splice variants of Nox2 have not previously been identified. We immunoscreened several mouse tissues and cells for the presence of truncated Nox2 proteins and identified a 30 kDa protein in lung, spleen and macrophages. RT-PCR analysis of mRNA from primary and immortalised (RAW264.7) mouse macrophages, and from human alveolar macrophages, identified a truncated Nox2 transcript which, upon sequence analysis, was found to be a product of the ‘exon skipping’ mode of alternative splicing, lacking exons 4–10 of the Nox2 gene. The predicted protein is comparable in size to that identified by immunoscreening and contains two transmembrane helices and an extended cytosolic C-terminus with binding sites for NADPH and the Nox organiser protein p47phox. Importantly, selective siRNA-mediated knockdown of the transcript reduced expression of the 30 kDa protein in macrophages, and suppressed phorbol ester-stimulated ROS production by 50%. We thus provide the first evidence that Nox2 undergoes alternative mRNA splicing to yield a 30 kDa protein – herein termed Nox2β – that regulates NADPH oxidase activity in macrophages from mice and humans. The discovery of Nox2β paves the way for future examination of its role in physiological and pathological processes. PMID:23118986

  12. Structures and Mechanism of the Monoamine Oxidase Family

    PubMed Central

    Gaweska, Helena; Fitzpatrick, Paul F.

    2011-01-01

    Members of the monoamine oxidase family of flavoproteins catalyze the oxidation of primary and secondary amines, polyamines, amino acids, and methylated lysine side chains in proteins. The enzymes have similar overall structures, with conserved FAD-binding domains and varied substrate-binding sites. Multiple mechanisms have been proposed for the catalytic reactions of these enzymes. The present review compares the structures of different members of the family and the various mechanistic proposals. PMID:22022344

  13. Polyamines and abiotic stress tolerance in plants

    PubMed Central

    Gill, Sarvajeet Singh

    2010-01-01

    Environmental stresses including climate change, especially global warming, are severely affecting plant growth and productivity worldwide. It has been estimated that two-thirds of the yield potential of major crops are routinely lost due to the unfavorable environmental factors. On the other hand, the world population is estimated to reach about 10 billion by 2050, which will witness serious food shortages. Therefore, crops with enhanced vigour and high tolerance to various environmental factors should be developed to feed the increasing world population. Maintaining crop yields under adverse environmental stresses is probably the major challenge facing modern agriculture where polyamines can play important role. Polyamines (PAs)(putrescine, spermidine and spermine) are group of phytohormone-like aliphatic amine natural compounds with aliphatic nitrogen structure and present in almost all living organisms including plants. Evidences showed that polyamines are involved in many physiological processes, such as cell growth and development and respond to stress tolerance to various environmental factors. In many cases the relationship of plant stress tolerance was noted with the production of conjugated and bound polyamines as well as stimulation of polyamine oxidation. Therefore, genetic manipulation of crop plants with genes encoding enzymes of polyamine biosynthetic pathways may provide better stress tolerance to crop plants. Furthermore, the exogenous application of PAs is also another option for increasing the stress tolerance potential in plants. Here, we have described the synthesis and role of various polyamines in abiotic stress tolerance in plants. PMID:20592804

  14. Hexose Oxidase-Mediated Hydrogen Peroxide as a Mechanism for the Antibacterial Activity in the Red Seaweed Ptilophora subcostata.

    PubMed

    Ogasawara, Kimi; Yamada, Kenji; Hatsugai, Noriyuki; Imada, Chiaki; Nishimura, Mikio

    2016-01-01

    Marine algae have unique defense strategies against microbial infection. However, their mechanisms of immunity remain to be elucidated and little is known about the similarity of the immune systems of marine algae and terrestrial higher plants. Here, we suggest a possible mechanism underlying algal immunity, which involves hexose oxidase (HOX)-dependent production of hydrogen peroxide (H2O2). We examined crude extracts from five different red algal species for their ability to prevent bacterial growth. The extract from one of these algae, Ptilophora subcostata, was particularly active and prevented the growth of gram-positive and -negative bacteria, which was completely inhibited by treatment with catalase. The extract did not affect the growth of either a yeast or a filamentous fungus. We partially purified from P. subcostata an enzyme involved in its antibacterial activity, which shared 50% homology with the HOX of red seaweed Chondrus crispus. In-gel carbohydrate oxidase assays revealed that P. subcostata extract had the ability to produce H2O2 in a hexose-dependent manner and this activity was highest in the presence of galactose. In addition, Bacillus subtilis growth was strongly suppressed near P. subcostata algal fronds on GYP agar plates. These results suggest that HOX plays a role in P. subcostata resistance to bacterial attack by mediating H2O2 production in the marine environment. PMID:26867214

  15. Hexose Oxidase-Mediated Hydrogen Peroxide as a Mechanism for the Antibacterial Activity in the Red Seaweed Ptilophora subcostata

    PubMed Central

    Ogasawara, Kimi; Yamada, Kenji; Hatsugai, Noriyuki; Imada, Chiaki; Nishimura, Mikio

    2016-01-01

    Marine algae have unique defense strategies against microbial infection. However, their mechanisms of immunity remain to be elucidated and little is known about the similarity of the immune systems of marine algae and terrestrial higher plants. Here, we suggest a possible mechanism underlying algal immunity, which involves hexose oxidase (HOX)-dependent production of hydrogen peroxide (H2O2). We examined crude extracts from five different red algal species for their ability to prevent bacterial growth. The extract from one of these algae, Ptilophora subcostata, was particularly active and prevented the growth of gram-positive and -negative bacteria, which was completely inhibited by treatment with catalase. The extract did not affect the growth of either a yeast or a filamentous fungus. We partially purified from P. subcostata an enzyme involved in its antibacterial activity, which shared 50% homology with the HOX of red seaweed Chondrus crispus. In-gel carbohydrate oxidase assays revealed that P. subcostata extract had the ability to produce H2O2 in a hexose-dependent manner and this activity was highest in the presence of galactose. In addition, Bacillus subtilis growth was strongly suppressed near P. subcostata algal fronds on GYP agar plates. These results suggest that HOX plays a role in P. subcostata resistance to bacterial attack by mediating H2O2 production in the marine environment. PMID:26867214

  16. Catalytic activities of fungal oxidases in hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate-based microemulsion.

    PubMed

    Zhou, Gui-Ping; Zhang, Yun; Huang, Xi-Rong; Shi, Chuan-Hong; Liu, Wei-Feng; Li, Yue-Zhong; Qu, Yin-Bo; Gao, Pei-Ji

    2008-10-01

    For hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF(6)]), an H(2)O-in-[BMIM][PF(6)] microemulsion could be formed in the presence of nonionic surfactant Triton X-100 (TX-100). In such a medium, both lignin peroxidase (LiP) and laccase could express their catalytic activity with the optimum molar ratio of H(2)O to TX-100 at 8.0 for LiP and >20 for laccase, and the optimum pH values at 3.2 for LiP and 4.2 for laccase, respectively. As compared with pure or water saturated [BMIM][PF(6)], in which the two oxidases had negligible catalytic activity due to the strong inactivating effect of [BMIM][PF(6)] on both enzymes, the use of the [BMIM][PF(6)]-based microemulsion had some advantages. Not only the catalytic activities of both fungal oxidases greatly enhanced, but also the apparent viscosity of the medium decreased. PMID:18602799

  17. Tryptamine serves as a proligand of the AhR transcriptional pathway whose activation is dependent of monoamine oxidases.

    PubMed

    Vikström Bergander, Linda; Cai, Wen; Klocke, Bernward; Seifert, Martin; Pongratz, Ingemar

    2012-09-01

    The function of the aryl hydrocarbon receptor (AhR) in mediating the biological effect to environmental pollutants is well established. However, accumulated evidence indicates a wide range of physiological and pathological functions mediated by the AhR, suggesting the existence of endogenous AhR ligand(s). The nature of an AhR ligand remain elusive; however, it is known that the AhR is activated by several compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin or the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole. In this study, we show that physiological concentrations of tryptamine (TA) lead to induction of cytochrome P4501A1 transcription through an AhR-dependent mechanism. In addition, we show that activation of the AhR by TA requires a functional monoamino oxidase system, suggesting that TA acts as an AhR proligand possibly by converting to a high-affinity AhR ligand. Taken together, we show a possible mechanism, through which AhR signaling is activated by endogenous conversion of TA involving monoamine oxidases. PMID:22865928

  18. Regulation of polyamine synthesis in plants. Annual progress report

    SciTech Connect

    Malmberg, R.L.

    1993-02-09

    Polyamines are small positively charged compounds that have been hypothesized to be involved in a wide variety of plant physiological and development functions. The regulation of the polyamine synthesis pathway is uniquely interesting because of the existence of two pathways to putrescine synthesis, and the consequent questions of how these two pathways are compartmentalized and how they interact with each other. The specific directions our research is taking are: (1) A characterization of arginine decarboxylase regulation; we have discovered two post-translational mechanisms for regulating arginine decarboxylase activity. One of these is a novel protease that clips the arginine decarboxylase pre-protein to activate it. We would like to understand this activating protease better, determine its mechanism of action, and determine its importance in the overall scheme of arginine decarboxylase regulation. (2) We have begun a similar characterization of ornithine decarboxylase by purifying it from plants. (3) We are characterizing the polyamine mutant collection we have developed. (4) Finally, we have begun to characterize the evolution of arginine decarboxylase, as an additional approach that could shed light on its functions in plants. Our intent is to understand arginine decarboxylase structure and regulation in detail, and then to further explore regulatory differences between ornithine and arginine decarboxylases.

  19. Divergent regulation of the key enzymes of polyamine metabolism by chiral alpha-methylated polyamine analogues.

    PubMed

    Hyvönen, Mervi T; Howard, Michael T; Anderson, Christine B; Grigorenko, Nikolay; Khomutov, Alex R; Vepsäläinen, Jouko; Alhonen, Leena; Jänne, Juhani; Keinänen, Tuomo A

    2009-09-01

    The natural polyamines are ubiquitous multifunctional organic cations which play important roles in regulating cellular proliferation and survival. Here we present a novel approach to investigating polyamine functions by using optical isomers of MeSpd (alpha-methylspermidine) and Me2Spm (alpha,omega-bismethylspermine), metabolically stable functional mimetics of natural polyamines. We studied the ability of MeSpd and Me2Spm to alter the normal polyamine regulation pathways at the level of polyamine uptake and the major control mechanisms known to affect the key polyamine metabolic enzymes. These include: (i) ODC (ornithine decarboxylase), which catalyses the rate-limiting step of polyamine synthesis; (ii) ODC antizyme, an inhibitor of ODC and polyamine uptake; (iii) SSAT (spermidine/spermine N1-acetyltransferase), the major polyamine catabolic enzyme; and (iv) AdoMetDC (S-adenosyl-L-methionine decarboxylase), which is required for the conversion of putrescine into spermidine, and spermidine into spermine. We show that the stereoisomers differ in their cellular uptake and ability to downregulate ODC and AdoMetDC, and to induce SSAT. These effects are mediated by the ability of the enantiomers to induce +1 ribosomal frameshifting on ODC antizyme mRNA, to suppress the translation of AdoMetDC uORF (upstream open reading frame) and to regulate the alternative splicing of SSAT pre-mRNA. The unique effects of chiral polyamine analogues on polyamine metabolism may offer novel possibilities for studying the physiological functions, control mechanisms, and targets of the natural polyamines, as well as advance therapeutic drug development in cancer and other human health-related issues. PMID:19522702

  20. An antifungal gamma-pyrone and xanthones with monoamine oxidase inhibitory activity from Hypericum brasiliense.

    PubMed

    Rocha, L; Marston, A; Kaplan, M A; Stoeckli-Evans, H; Thull, U; Testa, B; Hostettmann, K

    1994-08-01

    A new gamma-pyrone (hyperbrasilone), three known xanthones (1,5-dihydroxyxanthone, 5-hydroxy-1-methoxyxanthone and 6-deoxyjacareubin) and betulinic acid have been isolated from a dichloromethane extract of stems and roots of Hypericum brasiliense. Their structures were established by spectroscopic methods (UV, EI-MS, 1H and 13C NMR) and that of the gamma-pyrone was confirmed by X-ray crystallography. Hyperbrasilone and the xanthones were all antifungal against Cladosporium cucumerinum, while the three xanthones showed differing degrees of inhibition of monoamine oxidase A and B. PMID:7765428

  1. Studies on Polyphenol Content, Activities and Isozymes of Polyphenol Oxidase and Peroxidase During Air-Curing in Three Tobacco Types 1

    PubMed Central

    Sheen, S. J.; Calvert, J.

    1969-01-01

    The change in polyphenol content in the primed leaves of burley, flue-cured, and Turkish tobaccos during air-curing was related to the activities and isozymes of polyphenol oxidase and peroxidase. The quantity of chlorogenic acid was rapidly reduced during the first week of curing. The decrease in rutin content during curing was less significant, especially when the concentration of chlorogenic acid was high in leaf tissues. This result was further confirmed by in vitro assays with partially purified tobacco polyphenol oxidase. The polyphenol oxidase activity did not differ at any stage of curing in the 3 tobaccos. When the activity was measured by the oxidation of 3,4-dihydroxyphenylalanine it rose rapidly during the first day of curing and then decreased sharply so that in the fully cured leaf only 15% activity remained. The increase in activity was not observed when chlorogenic acid was used as the substrate. A similar level of peroxidase activity was found in the 3 tobaccos before curing. Peroxidase activities increased rapidly during the first 24 hr of curing, declined thereafter, and remained highest in the flue-cured tobacco, less in the Turkish line, and least in the burley at the end of curing process. By polyacrylamide gel block electrophoresis, 10 peroxidase isozyme bands, 2 cationic and 8 anionic, appeared identical in all 3 tobaccos. When catechol replaced benzidine-2 HCl as the electron donor, 1 cationic and 2 anionic peroxidase isozymes did not form. Of interest is that the same 10 peroxidase isozyme bands also exhibited polyphenol oxidase activities when treated with 3,4-dihydroxyphenylalanine or chlorogenic acid. Results suggest that in the crude tobacco leaf extract the peroxidase and polyphenol oxidase may associate as protein complexes, and peroxidase isozymes may differ in electron-donor requirements. Isozyme patterns for both oxidases at various curing intervals differed only quantitatively. Images PMID:16657046

  2. Kinetic evidence that methionine sulfoxide reductase A can reveal its oxidase activity in the presence of thioredoxin.

    PubMed

    Kriznik, Alexandre; Boschi-Muller, Sandrine; Branlant, Guy

    2014-04-15

    The mouse methionine sulfoxide reductase A (MsrA) belongs to the subclass of MsrAs with one catalytic and two recycling Cys corresponding to Cys51, Cys198 and Cys206 in Escherichia coli MsrA, respectively. It was previously shown that in the absence of thioredoxin, the mouse and the E. coli MsrAs, which reduce two mol of methionine-O substrate per mol of enzyme, displays an in vitro S-stereospecific methionine oxidase activity. In the present study carried out with E. coli MsrA, kinetic evidence are presented which show that formation of the second mol of Ac-L-Met-NHMe is rate-limiting in the absence of thioredoxin. In the presence of thioredoxin, the overall rate-limiting step is associated with the thioredoxin-recycling process. Kinetic arguments are presented which support the accumulation of the E. coli MsrA under Cys51 sulfenic acid state in the presence of Trx. Thus, the methionine oxidase activity could be operative in vivo without the action of a regulatory protein in order to block the action of Trx as previously proposed. PMID:24632144

  3. Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase from Escherichia coli

    SciTech Connect

    Weidner, Annett; Neumann, Piotr; Wille, Georg; Stubbs, Milton T.; Tittmann, Kai

    2008-03-01

    The peripheral membrane flavoprotein pyruvate oxidase from E. coli has been crystallized in the full-length form and as a proteolytically activated truncation variant lacking the last 23 amino acids at the C-terminus. The thiamine diphosphate- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from Escherichia coli (EcPOX) has been crystallized in the full-length form and as a proteolytically activated C-terminal truncation variant which lacks the last 23 amino acids (Δ23 EcPOX). Crystals were grown by the hanging-drop vapour-diffusion method using either protamine sulfate (full-length EcPOX) or 2-methyl-2,4-pentanediol (Δ23 EcPOX) as precipitants. Native data sets were collected at a X-ray home source to a resolution of 2.9 Å. The two forms of EcPOX crystallize in different space groups. Whereas full-length EcPOX crystallizes in the tetragonal space group P4{sub 3}2{sub 1}2 with two monomers per asymmetric unit, the crystals of Δ23 EcPOX belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and contain 12 monomers per asymmetric unit.

  4. Polyamines are essential in embryo implantation: expression and function of polyamine-related genes in mouse uterus during peri-implantation period.

    PubMed

    Zhao, Yue-Chao; Chi, Yu-Jing; Yu, Yong-Sheng; Liu, Ji-Long; Su, Ren-Wei; Ma, Xing-Hong; Shan, Chun-Hua; Yang, Zeng-Ming

    2008-05-01

    Polyamines are key regulators in cell growth and differentiation. It has been shown that ornithine decarboxylase (Odc) was essential for post-implantation embryo development, and overexpression of spermidine/spermine N1-acetyltransferase will lead to ovarian hypofunction and hypoplastic uteri. However, the expression and function of polyamine-related genes in mouse uterus during early pregnancy are still unknown. In this study we investigated the expression, regulation, and function of polyamine-related genes in mouse uterus during the peri-implantation period. Odc expression was strongly detected at implantation sites and stimulated by estrogen treatment. The expression of Odc antizyme 1 and spermidine/spermine N1-acetyltransferase was also highly shown at implantation sites and regulated by Odc or polyamine level in uterine cells. Embryo implantation was significantly inhibited by alpha-difluoromethylornithine, an Odc inhibitor. Moreover, the reduction of Odc activity caused by alpha-difluoromethylornithine treatment was compensated by the up-regulation of S-adenosylmethionine decarboxylase gene expression. Collectively, our results indicated that the coordinated expression of uterine polyamine-related genes may be important for embryo implantation. PMID:18202119

  5. Depletion of intracellular polyamines relieves inward rectification of potassium channels.

    PubMed Central

    Shyng, S L; Sha, Q; Ferrigni, T; Lopatin, A N; Nichols, C G

    1996-01-01

    Two different approaches were used to examine the in vivo role of polyamines in causing inward rectification of potassium channels. In two-microelectrode voltage-clamp experiments, 24-hr incubation of Xenopus oocytes injected with 50 nl of difluoromethylornithine (5 mM) and methylglyoxal bis(guanylhydrazone) (1 mM) caused an approximate doubling of expressed Kir2.1 currents and relieved rectification by causing an approximately +10-mV shift of the voltage at which currents are half-maximally inhibited. Second, a putrescine auxotrophic, ornithine decarboxylase-deficient Chinese hamster ovary (O-CHO) cell line was stably transfected with the cDNA encoding Kir2.3. Withdrawal of putrescine from the medium led to rapid (1-day) loss of the instantaneous phase of Kir2.3 channel activation, consistent with a decline of intracellular putrescine levels. Four days after putrescine withdrawal, macroscopic conductance, assessed using an 86Rb+ flux assay, was approximately doubled, and this corresponded to a +30-mV shift of V1/2 of rectification. With increasing time after putrescine withdrawal, there was an increase in the slowest phase of current activation, corresponding to an increase in the spermine-to-spermidine ratio over time. These results provide direct evidence for a role of each polyamine in induction of rectification, and they further demonstrate that in vivo modulation of rectification is possible by manipulation of polyamine levels using genetic and pharmacological approaches. PMID:8876254

  6. Expression and distribution of genes encoding for polyamine-metabolizing enzymes in the different zones of male and female mouse kidneys.

    PubMed

    Levillain, Olivier; Ramos-Molina, Bruno; Forcheron, Fabien; Peñafiel, Rafael

    2012-11-01

    The role of polyamines in renal physiology is only partially understood. Moreover, most of the data on the enzymes of polyamine metabolism come from studies using whole kidneys. The aim of the present study was to analyze the mRNA abundance of the genes implicated in both the polyamine biosynthetic and catabolic pathways in different renal zones of male and female mice, by means of the quantitative reverse transcription-polymerase chain reaction. Our results indicate that there is an uneven distribution of the different mRNAs studied in the five renal zones: superficial cortex, deep cortex, outer stripe of the outer medulla (OS), inner stripe of the outer medulla (IS), and the inner medulla + papilla (IM). The biosynthetic genes, ornithine decarboxylase (ODC) and spermine synthase, were more expressed in the cortex, whereas the mRNAs of the catabolic genes spermine oxidase (SMO) and diamine oxidase were more abundant in IS and IM. The genes involved in the regulation of polyamine synthesis (AZ1, AZ2 and AZIN1) were expressed in all the renal zones, predominantly in the cortex, while AZIN2 gene was more abundant in the OS. ODC, SMO, spermidine synthase and spermidine/spermine acetyl transferase expression was higher in males than in females. In conclusion, the genes encoding for the polyamine metabolism were specifically and quantitatively distributed along the corticopapillary axis of male and female mouse kidneys, suggesting that their physiological role is essential in defined renal zones and/or nephron segments. PMID:22562773

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

    PubMed Central

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

    2010-01-01

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

  8. Extraction of rice bran extract and some factors affecting its inhibition of polyphenol oxidase activity and browning in potato.

    PubMed

    Boonsiripiphat, Kunnikar; Theerakulkait, Chockchai

    2009-01-01

    The extraction conditions of rice bran extract (RBE), including extraction ratio, extraction time, and extraction temperature, were studied in relation to enzymatic browning inhibition in potato. The inhibitory effect of RBE on potato polyphenol oxidase (PPO) activity and its total phenolic compound content were highest at an extraction ratio of 1:3 (rice bran:water, w/v), extraction time of 30 min, and extraction temperature of 40 degrees C. RBE showed the most inhibitory effect on PPO activity at pH 6.5. However, the inhibitory effect of RBE on potato PPO activity and its total phenolic compound content were decreased at the higher temperature and longer time. PMID:19291577

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

    NASA Astrophysics Data System (ADS)

    Qi, De-Qiang; Yu, Chuan-Ming; You, Jin-Zong; Yang, Guang-Hui; Wang, Xue-Jie; Zhang, Yi-Ping

    2015-11-01

    A series of pyrazole-based 1,3,4-oxadiazole derivatives were rationally designed and synthesized in good yields by following a convenient route. All the newly synthesized molecules were fully characterized by IR, 1H NMR and elemental analysis. Eight compounds were structurally determined by single crystal X-ray diffraction analysis. The fluorescence properties of all the compounds were investigated in dimethyl sulfoxide media. In addition, these newly synthesized compounds were evaluated for in vitro inhibitory activity against commercial enzyme xanthine oxidase (XO) by measuring the formation of uric acid from xanthine. Among the compounds synthesized and tested, 3d and 3e were found to be moderate inhibitory activity against commercial XO with IC50 = 72.4 μM and 75.6 μM. The studies gave a new insight in further optimization of pyrazole-based 1,3,4-oxadiazole derivatives with excellent fluorescence properties and XO inhibitory activity.

  10. Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism.

    PubMed

    Yang, Ting; Peleli, Maria; Zollbrecht, Christa; Giulietti, Alessia; Terrando, Niccolo; Lundberg, Jon O; Weitzberg, Eddie; Carlström, Mattias

    2015-06-01

    Oxidative stress contributes to the pathogenesis of many disorders, including diabetes and cardiovascular disease. Immune cells are major sources of superoxide (O2(∙-)) as part of the innate host defense system, but exaggerated and sustained O2(∙-) generation may lead to progressive inflammation and organ injuries. Previous studies have proven organ-protective effects of inorganic nitrite, a precursor of nitric oxide (NO), in conditions manifested by oxidative stress and inflammation. However, the mechanisms are still not clear. This study aimed at investigating the potential role of nitrite in modulating NADPH oxidase (NOX) activity in immune cells. Mice peritoneal macrophages or human monocytes were activated by lipopolysaccharide (LPS), with or without coincubation with nitrite. O2(∙-) and peroxynitrite (ONOO(-)) formation were detected by lucigenin-based chemiluminescence and fluorescence techniques, respectively. The intracellular NO production was measured by DAF-FM DA fluorescence. NOX isoforms and inducible NO synthase (iNOS) expression were detected by qPCR. LPS increased both O2(∙-) and ONOO(-) production in macrophages, which was significantly reduced by nitrite (10µmol/L). Mechanistically, the effects of nitrite are (1) linked to increased NO generation, (2) similar to that observed with the NO donor DETA-NONOate, and (3) can be abolished by the NO scavenger carboxy-PTIO or by the xanthine oxidase (XO) inhibitor febuxostat. Nox2 expression was increased in activated macrophages, but was not influenced by nitrite. However, nitrite attenuated LPS-induced upregulation of iNOS expression. Similar to that observed in mice macrophages, nitrite also reduced O2(∙-) generation in LPS-activated human monocytes. In conclusion, XO-mediated reduction of nitrite attenuates NOX activity in activated macrophages, which may modulate the inflammatory response. PMID:25724690

  11. Polyamines and uremia.

    PubMed

    Campbell, R A

    1987-01-01

    PAs are intracellular regulators of growth and anabolic processes. Toxic properties of PAs are conferred according to the increasing number of cationic charges. In uremia, PA accumulation occurs both in and outside the cell. Decreased PA synthesis and blunted PA pathway responses suggest PAs may participate in cellular down regulation. This would explain the lack of tissue responses to elevated plasma hormones, a feature of uremia. Such homeostatic control could prevent life-threatening PA toxicity due to an imbalance between production, degradation and excretion. The concurrent rise in plasma PA oxidative activity, while adversely influencing behavior of lymphocytes and neutrophils reduces the likelihood of direct PA toxicity. In addition, preliminary observation of increased glomerular size and capillary area of intact kidneys in PA treated mice suggest there could be a PA dependent adaptive enhancement of renal excretion in circumstances of decreased renal mass due to disease, thus, compensatory renal hypertrophy. It is important to further inspect the non-uremic chronic hyperpolyaminemias as they occur in man. The continued search for parallel adverse systemic and local effects will be useful in strengthening the base for understanding homeostatic consequences of PA accumulation unobserved by the complex mixture of uremic metabolites. In conclusion, we have examined some known regulatory and toxic properties of PAs and related these to features of the uremic syndrome. Available information on the various ramifications of PA dysmetabolism in uremia, as with other suspect toxins, is in part circumscribed and indirect. Caution is to be exercised in evaluating the new and provisional PA related cause and effect relationships suggested here.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3447450

  12. Polyamines and regulation of ripening and senescence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyamines (PAs) are small polycationic, biogenic amines that influence many biochemical and physiological processes such as cell division, cell elongation, flowering, fruit set and development, fruit ripening and senescence. Significant information about PA action has emerged from indirect studies ...

  13. Stress and polyamine metabolism in fungi

    PubMed Central

    Valdés-Santiago, Laura; Ruiz-Herrera, José

    2013-01-01

    Fungi, as well as the rest of living organisms must deal with environmental challenges such as stressful stimuli. Fungi are excellent models to study the general mechanisms of the response to stress, because of their simple, but conserved, signal-transduction and metabolic pathways that are often equivalent to those present in other eukaryotic systems. A factor that has been demonstrated to be involved in these responses is polyamine metabolism, essentially of the three most common polyamines: putrescine, spermidine and spermine. The gathered evidences on this subject suggest that polyamines are able to control cellular signal transduction, as well as to modulate protein-protein interactions. In the present review, we will address the recent advances on the study of fungal metabolism of polyamines, ranging from mutant characterization to potential mechanism of action during different kinds of stress in selected fungal models. PMID:24790970

  14. Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

    PubMed

    López-Cruz, Roberto I; Pérez-Milicua, Myrna Barjau; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal-Vertiz, Jaime A; de la Rosa, Alejandro; Vázquez-Medina, José P; Zenteno-Savín, Tania

    2014-05-01

    Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas. PMID:24530799

  15. The urinary MHPG/creatinine ratio and its relationship to platelet monoamine oxidase activity in abstinent alcoholics.

    PubMed

    Farren, C K; Tipton, K F

    1999-01-01

    This study was designed to assess the baseline noradrenergic turnover of subgroups of postwithdrawal abstinent alcoholics and healthy controls. The method chosen was an overnight fasting urine sample of the breakdown product of norepinephrine, MHPG, related to urinary creatinine. A comparison was made with platelet monoamine oxidase activity and also within subgroups of the study population. This study found no difference between alcoholics and controls, nor between subgroups of postwithdrawal alcoholics in their level of urinary MHPG corrected for creatinine, and no significant correlation with major subject characteristics or with platelet monoamine oxidase. There was a trend, however, towards a significant correlation with duration of abstinence from alcohol, and there was a correlation with a history of fighting when drinking alcohol, but not with sociopathic traits overall. Within the type 2 alcoholics there was a significant correlation with a history of fighting when drinking and a negative correlation with behavioral tolerance to alcohol. It is possible that only the subset of type 2 alcoholics with certain antisocial characteristics have noradrenergic abnormalities. Although no statistical difference was found between the different groups under study, the information is helpful in increasing understanding of the noradrenergic system in abstinent alcoholics. PMID:20575773

  16. Body pool and synthesis of ascorbic acid in adult sea lamprey (Petromyzon marinus): An agnathan fish with gulonolactone oxidase activity

    PubMed Central

    Moreau, Régis; Dabrowski, Konrad

    1998-01-01

    Although many vertebrates can synthesize ascorbic acid (vitamin C), it is still unclear from the evolutionary perspective when the ability to synthesize the vitamin first appeared in the animal kingdom and how frequently the trait has been lost. We report here ascorbic acid biosynthesis ability in sea lamprey (Petromyzon marinus) which represent the most ancient vertebrate lineage examined thus far for presence of gulonolactone oxidase, the enzyme catalyzing the terminal step in biosynthesis of vitamin C. This finding supports the view that the ancestors of living vertebrates were not scurvy prone and that the loss of gulonolactone oxidase activity subsequently occurred several times in vertebrate phylogeny. Adult sea lamprey allocate significant amounts of ascorbic acid to the gonads to guaranty high-quality gametes. Tissue stores of ascorbate were maintained by de novo synthesis (1.2–1.3 mg of ascorbic acid/300-g sea lamprey per day at 15°C) while sea lamprey fast during spawning migration. We estimate that the in vivo daily renewal rate of ascorbate is 4–5% of the whole-body ascorbate pool based on measurement of its biosynthesis and concentration in the whole animal. PMID:9707638

  17. [Effect of abiotic elicitation on the sanguinarine production and polyphenol oxidase activity in the suspension culture of Eschscholtzia californica CHAM].

    PubMed

    Bilka, František; Balážová, Andrea; Bilková, Andrea; Holková, Ivana

    2013-08-01

    Elicitation of plant in vitro cultures represents a biotechnological tool to improve the production of secondary metabolites. In this study, the effect of AgNO3 and CdCl2 on the sanguinarine production by the suspension culture of Eschscholtzia californica CHAM. was investigated. Elicitors were added to the cultures at the 14th day of subcultivation and their effect on the sanguinarine production was evaluated after a 48 h exposure. AgNO3 at the concentration of 0.075 mmol.l-1 and CdCl2 at the concentration of 4 mmol.l-1 induced a ca. 5.2- and 5.6-multiple increase in sanguinarine synthesis, respectively. This amount represents probably the maximal production, because a further increase in the elicitors concentrations did not increase sanguinarine production. Both abiotic elicitors induced a polyphenol oxidase specific activity increase. Polyphenol oxidase is probably involved in the biosynthesis of sanguinarine at the level of dopamine formation. Dopamine is a precursor of (S)-norcoclaurine, the first intermediate with the benzylisoquinoline structure. PMID:24047145

  18. Pigment epithelium-derived factor stimulates skeletal muscle glycolytic activity through NADPH oxidase-dependent reactive oxygen species production.

    PubMed

    Carnagarin, Revathy; Carlessi, Rodrigo; Newsholme, Philip; Dharmarajan, Arun M; Dass, Crispin R

    2016-09-01

    Pigment epithelium-derived factor is a multifunctional serpin implicated in insulin resistance in metabolic disorders. Recent evidence suggests that exposure of peripheral tissues such as skeletal muscle to PEDF has profound metabolic consequences with predisposition towards chronic conditions such as obesity, type 2 diabetes, metabolic syndrome and polycystic ovarian syndrome. Chronic inflammation shifts muscle metabolism towards increased glycolysis and decreased oxidative metabolism. In the present study, we demonstrate a novel effect of PEDF on cellular metabolism in mouse cell line (C2C12) and human primary skeletal muscle cells. PEDF addition to skeletal muscle cells induced enhanced phospholipase A2 activity. This was accompanied with increased production of reactive oxygen species in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner that triggered a shift towards a more glycolytic phenotype. Extracellular flux analysis and glucose consumption assays demonstrated that PEDF treatment resulted in enhanced glycolysis but did not change mitochondrial respiration. Our results demonstrate that skeletal muscle cells express a PEDF-inducible oxidant generating system that enhances glycolysis but is sensitive to antioxidants and NADPH oxidase inhibition. PMID:27343430

  19. Molecular mechanism underlying promiscuous polyamine recognition by spermidine acetyltransferase.

    PubMed

    Sugiyama, Shigeru; Ishikawa, Sae; Tomitori, Hideyuki; Niiyama, Mayumi; Hirose, Mika; Miyazaki, Yuma; Higashi, Kyohei; Murata, Michio; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Kashiwagi, Keiko; Igarashi, Kazuei; Matsumura, Hiroyoshi

    2016-07-01

    Spermidine acetyltransferase (SAT) from Escherichia coli, which catalyses the transfer of acetyl groups from acetyl-CoA to spermidine, is a key enzyme in controlling polyamine levels in prokaryotic cells. In this study, we determined the crystal structure of SAT in complex with spermidine (SPD) and CoA at 2.5Å resolution. SAT is a dodecamer organized as a hexamer of dimers. The secondary structural element and folding topology of the SAT dimer resemble those of spermidine/spermine N(1)-acetyltransferase (SSAT), suggesting an evolutionary link between SAT and SSAT. However, the polyamine specificity of SAT is distinct from that of SSAT and is promiscuous. The SPD molecule is also located at the inter-dimer interface. The distance between SPD and CoA molecules is 13Å. A deep, highly acidic, water-filled cavity encompasses the SPD and CoA binding sites. Structure-based mutagenesis and in-vitro assays identified SPD-bound residues, and the acidic residues lining the walls of the cavity are mostly essential for enzymatic activities. Based on mutagenesis and structural data, we propose an acetylation mechanism underlying promiscuous polyamine recognition for SAT. PMID:27163532

  20. Type IX Ehlers-Danlos syndrome and Menkes syndrome: the decrease in lysyl oxidase activity is associated with a corresponding deficiency in the enzyme protein.

    PubMed Central

    Kuivaniemi, H; Peltonen, L; Kivirikko, K I

    1985-01-01

    Type IX of the Ehlers-Danlos syndrome (E-D IX) and the Menkes syndrome are X-linked recessively inherited disorders characterized by abnormalities in copper metabolism. These abnormalities are associated with a severe reduction in the activity of lysyl oxidase, the extracellular copper enzyme that initiates crosslinking of collagens and elastin. No increase in this deficient enzyme activity was obtained when culture media from fibroblasts of patients with E-D IX or the Menkes syndrome were incubated with copper under various conditions in vitro. A distinct, although small, increase in lysyl oxidase activity was obtained, however, when copper-supplemented media were used during culturing of the fibroblasts, although even under these conditions, the enzyme activity in the media from the affected cells remained markedly below that of the controls. Immunoprecipitation, dot-blotting, and immunoperoxidase staining experiments with antisera to human lysyl oxidase indicated that fibroblasts from patients with E-D IX or the Menkes syndrome do not secrete into their medium, or contain inside the cell, any significant amounts of a copper-deficient, catalytically inactive lysyl oxidase protein. These findings appear to be consistent with the hypothesis that synthesis of the lysyl oxidase protein itself is impaired. The possibility is not excluded, however, that a copper-deficient enzyme protein may be synthesized in normal amounts but become degraded very rapidly inside the cell. The failure to obtain any large increase in the deficient lysyl oxidase activity upon various forms of copper administration suggests that it may not be possible to obtain any significant improvement in the connective tissue manifestations of these disorders by copper therapy. Images Fig. 1 Fig. 2 PMID:9556668

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

    NASA Astrophysics Data System (ADS)

    Olea, David; Faure, Chrystel

    2003-09-01

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

  2. Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function

    PubMed Central

    Sokolovska, Anna; Becker, Christine E.; Eddie Ip, WK; Rathinam, Vijay A.K.; Brudner, Matthew; Paquette, Nicholas; Tanne, Antoine; Vanaja, Sivapriya K.; Moore, Kathryn J.; Fitzgerald, Katherine A.; Lacy-Hulbert, Adam; Stuart, Lynda M.

    2013-01-01

    Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates a number of functions of these organelles that allow them to participate in processes essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3-inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3-inflammasome and caspase-1 in host defense. PMID:23644505

  3. Effect of physical exercise on changes in activities of creatine kinase, cytochrome c oxidase and ATP levels caused by ovariectomy.

    PubMed

    Siebert, Cassiana; Kolling, Janaína; Scherer, Emilene B S; Schmitz, Felipe; da Cunha, Maira Jaqueline; Mackedanz, Vanize; de Andrade, Rodrigo B; Wannmacher, Clovis M D; Wyse, Angela T S

    2014-09-01

    The reduction in the secretion of ovarian hormones, principally estrogen, is a consequence of menopause. Estrogens act primarily as female sex hormones, but also exert effects on different physiological systems including the central nervous system. The treatment normally used to reduce the symptoms of menopause is the hormone therapy, which seems to be effective in treating symptoms, but it may be responsible for adverse effects. Based on this, there is an increasing demand for alternative therapies that minimize signs and symptoms of menopause. In the present study we investigated the effect of ovariectomy and/or physical exercise on the activities of energy metabolism enzymes, such as creatine kinase (cytosolic and mitochondrial fractions), pyruvate kinase, succinate dehydrogenase, complex II, cytochrome c oxidase, as well as on ATP levels in the hippocampus of adult rats. Adult female Wistar rats with 90 days of age were subjected to ovariectomy (an animal model widely used to mimic the postmenopausal changes). Thirty days after the procedure, the rats were submitted to the exercise protocol, which was performed three times a week for 30 days. Twelve hours after the last training session, the rats were decapitated for subsequent biochemical analyzes. Results showed that ovariectomy did not affect the activities of pyruvate kinase, succinate dehydrogenase and complex II, but decreased the activities of creatine kinase (cytosolic and mitochondrial fractions) and cytochrome c oxidase. ATP levels were also reduced. Exercise did not produce the expected results since it was only able to partially reverse the activity of creatine kinase cytosolic fraction. The results of this study suggest that estrogen deficiency, which occurs as a result of ovariectomy, affects generation systems and energy homeostasis, reducing ATP levels in hippocampus of adult female rats. PMID:24810635

  4. Factors influencing diamine oxidase activity and γ-aminobutyric acid content of fava bean (Vicia faba L.) during germination.

    PubMed

    Yang, Runqiang; Chen, Hui; Gu, Zhenxin

    2011-11-01

    Factors (germination time, spectra, temperature, pH, and chemical inhibitors) influencing diamine oxidase (DAO, EC 1.4.3.6) activity and γ-aminobutyric acid (GABA) content of fava bean (Vicia faba L.) during germination were investigated in this study. DAO activity significantly increased in germinating seeds but varied with different organs. The enzyme activity was higher in shoot than that in cotyledon, hypocotyl, and radicle. When seeds were germinated in the dark, DAO activity was 2.35-, 2.00-, 2.36-, 4.40-, and 1.67-fold of that under white, red, blue, green, and yellow spectra, respectively. The optimum germination temperature and pH value for increasing DAO activity were 30 °C and 3.0, respectively. The DAO activity was inhibited significantly by aminoguanidine and sodium ethylenediamine tetracetate, while it was activated by CuCl(2) and CaCl(2). Germinating at an appropriate temperature and pH, 30% of GABA formation was supplied by DAO. Calcium was related to the regulation of DAO activity and GABA accumulation. PMID:21942768

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

    PubMed

    Zsengellér, Zsuzsanna K; Rosen, Seymour

    2016-09-01

    The renal biopsy is a dynamic way of looking at renal disease, and tubular elements are an important part of this analysis. The mitochondria in 20 renal biopsies were examined by immunohistochemical (electron transport chain enzyme: cytochrome C oxidase IV [COX IV]) and enzyme histochemical methods (COX), both by light and electron microscopy. The distal convoluted tubules and thick ascending limbs showed the greatest intensity in the COX immunostains and enzyme activity in controls. The degree of mitochondrial COX protein and enzyme activity diminished as the tubules became atrophic. With proximal hypertrophic changes, there was great variation in both COX activity and protein expression. In contrast, in three cases of systemic lupus erythematosus, biopsied for high-grade proteinuria, the activity was consistently upregulated, whereas protein expression remained normal. These unexpected findings of heterogeneous upregulation in hypertrophy and the dyssynchrony of protein expression and activity may indicate mitochondrial dysregulation. Functional electron microscopy showed COX activity delineated by the intense mitochondrial staining in normal or hypertrophic proximal tubules. With atrophic changes, residual small mitochondria with diminished activity could be seen. With mitochondrial size abnormalities (enlargement and irregularity, adefovir toxicity), activity persisted. In the renal biopsy, mitochondrial analysis is feasible utilizing immunohistochemical and enzyme histochemical techniques. PMID:27578326

  6. Activation of mouse macrophages causes no change in expression and function of phorbol diesters' receptors, but is accompanied by alterations in the activity and kinetic parameters of NADPH oxidase.

    PubMed Central

    Berton, G; Cassatella, M; Cabrini, G; Rossi, F

    1985-01-01

    Mouse peritoneal macrophages activated in vivo by the injection of Corynebacterium parvum release larger amounts of superoxide anion (O2-) than macrophages from control mice when stimulated with phorbol myristate acetate (PMA). The biochemical bases for this enhanced response of activated macrophages have been investigated by studying the expression and function of receptors for the stimulant, and the activity of the enzyme NADPH oxidase which is responsible for the production of O2- in leucocytes. Studies of binding of phorbol dibutyrate, an agent closely related to PMA, showed that the affinity constants (Kds) and the number of binding sites were the same in resident and activated peritoneal macrophages. The activity of the NADPH oxidase was, however, different in the two macrophage populations which differ in their capacity to release O2-. NADPH oxidase activity was studied in macrophage monolayers after lysis with deoxycholate. The main features of this activity were as follows: stimulation of macrophages with PMA or zymosan caused an increase in NADPH-dependent O2- production; NADPH oxidase activity in the lysates followed the same dose-response curve for different concentrations of PMA as O2- release by intact macrophages; O2- release by intact macrophages could be fully accounted for by NADPH-dependent O2- production by macrophage lysates; activity was strictly substrate-specific, in that NADH could not substitute for NADPH; after stimulation with PMA or zymosan, NADPH oxidase activity was higher in lysates of C. parvum-activated macrophages than in lysates of resident macrophages; NADPH oxidase activities of activated and resident macrophages differed markedly in their kinetic parameters. The NADPH oxidase of macrophages activated by C. parvum or trehalose dimycolate of mycobacterial origin displayed a five to seven times lower Km compared to the enzyme in resident macrophages. PMID:2981767

  7. Interaction of Platelet Activating Factor, Reactive Oxygen Species Generated by Xanthine Oxidase, and Leukocytes in the Generation of Hepatic Injury After Shock/Resuscitation

    PubMed Central

    Yamakawa, Yasuhiko; Takano, Manabu; Patel, Mayur; Tien, Nevin; Takada, Tadahiro; Bulkley, Gregory B.

    2000-01-01

    Objective To evaluate the putative relation of platelet activating factor (PAF), xanthine oxidase, reactive oxidants, and leukocytes in the pathogenesis of hepatic injury after shock/resuscitation (S/R) in vivo. Background Reactive oxygen metabolites generated by xanthine oxidase at reperfusion have been found to trigger postischemic injury in many organs, including the liver. However, the precise linear sequence of the mechanism of consequent hepatic injury after S/R remains to be characterized. Methods Unheparinized male rats were bled to a mean blood pressure of 45 ± 3 mmHg. After 2 hours of shock, they were resuscitated by reinfusion of shed blood (anticoagulated with citrate-phosphate-dextrose) and crystalloid and observed for the next 6 or 24 hours. Results S/R caused the oxidation of hepatic glutathione and generated centrolobular leukocyte accumulation at 6 hours, followed by predominantly centrolobular hepatocellular injury at 24 hours. Each of these components was attenuated by PAF inhibition with WEB 2170, xanthine oxidase inhibition with allopurinol, antioxidant treatment with N-acetylcysteine, or severe leukopenia induced by vinblastine. In each case, the degree of leukocyte accumulation at 6 hours correlated with the hepatocellular injury seen at 24 hours. However, xanthine oxidase inhibition with allopurinol failed to attenuate further the small level of residual hepatocellular injury seen in leukopenic rats. Conclusion These findings suggest that reactive oxidants generated by xanthine oxidase at reperfusion, stimulated by PAF, mediate hepatocellular injury by triggering leukocyte accumulation, primarily within the centrolobular sinusoids. PMID:10714632

  8. P2X7 receptor-NADPH oxidase axis mediates protein radical formation and Kupffer cell activation in carbon tetrachloride-mediated steatohepatitis in obese mice.

    PubMed

    Chatterjee, Saurabh; Rana, Ritu; Corbett, Jean; Kadiiska, Maria B; Goldstein, Joyce; Mason, Ronald P

    2012-05-01

    While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl(4)-treated hepatocytes and generating redox-mediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and posttranslational nitration, primarily in Kupffer cells, at 24h post-CCl(4) administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase and P2X7 receptor-dependent, correlated well with the release of TNF-α and MCP-2 from Kupffer cells. The Kupffer cells in CCl(4)-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms. PMID:22343416

  9. Deficiency of Rac1 Blocks NADPH Oxidase Activation, Inhibits Endoplasmic Reticulum Stress, and Reduces Myocardial Remodeling in a Mouse Model of Type 1 Diabetes

    PubMed Central

    Li, Jianmin; Zhu, Huaqing; Shen, E; Wan, Li; Arnold, J. Malcolm O.; Peng, Tianqing

    2010-01-01

    OBJECTIVE Our recent study demonstrated that Rac1 and NADPH oxidase activation contributes to cardiomyocyte apoptosis in short-term diabetes. This study was undertaken to investigate if disruption of Rac1 and inhibition of NADPH oxidase would prevent myocardial remodeling in chronic diabetes. RESEARCH DESIGN AND METHODS Diabetes was induced by injection of streptozotocin in mice with cardiomyocyte-specific Rac1 knockout and their wild-type littermates. In a separate experiment, wild-type diabetic mice were treated with vehicle or apocynin in drinking water. Myocardial hypertrophy, fibrosis, endoplasmic reticulum (ER) stress, inflammatory response, and myocardial function were investigated after 2 months of diabetes. Isolated adult rat cardiomyocytes were cultured and stimulated with high glucose. RESULTS In diabetic hearts, NADPH oxidase activation, its subunits' expression, and reactive oxygen species production were inhibited by Rac1 knockout or apocynin treatment. Myocardial collagen deposition and cardiomyocyte cross-sectional areas were significantly increased in diabetic mice, which were accompanied by elevated expression of pro-fibrotic genes and hypertrophic genes. Deficiency of Rac1 or apocynin administration reduced myocardial fibrosis and hypertrophy, resulting in improved myocardial function. These effects were associated with a normalization of ER stress markers' expression and inflammatory response in diabetic hearts. In cultured cardiomyocytes, high glucose–induced ER stress was inhibited by blocking Rac1 or NADPH oxidase. CONCLUSIONS Rac1 via NADPH oxidase activation induces myocardial remodeling and dysfunction in diabetic mice. The role of Rac1 signaling may be associated with ER stress and inflammation. Thus, targeting inhibition of Rac1 and NADPH oxidase may be a therapeutic approach for diabetic cardiomyopathy. PMID:20522592

  10. Cigarette smoke-induced kinin B1 receptor promotes NADPH oxidase activity in cultured human alveolar epithelial cells.

    PubMed

    Talbot, Sébastien; Lin, James Chi-Jen; Lahjouji, Karim; Roy, Jean-Philippe; Sénécal, Jacques; Morin, André; Couture, Réjean

    2011-07-01

    Pulmonary inflammation is an important pathological feature of tobacco smoke-related lung diseases. Kinin B1 receptor (B1R) is up-regulated in the rat trachea chronically exposed to cigarette-smoke. This study aimed at determining (1) whether exposure to total particulate matter of the cigarette smoke (TPM) can induce B1R in human alveolar epithelial A549 cells, (2) the mechanism of B1R induction, (3) the functionality of de novo synthesized B1R, and (4) the role of B1R in TPM-induced increase of superoxide anion (O₂(●⁻)) level. Results show that A549 cells exposed to 10 μg/ml TPM increased O₂(●⁻) level along with B1R (protein and mRNA) and IL-1β mRNA. In contrast, B2R and TNF-α mRNA were not affected by TPM. The increasing effect of TPM on O₂(●⁻) level was not significantly affected by the B1R antagonist SSR240612. TPM-increased B1R mRNA was prevented by co-treatments with N-acetyl-l-cysteine (potent antioxidant), diphenyleneiodonium (NADPH oxidase inhibitor), IL-1Ra (interleukin-1R antagonist) and SN-50 (specific inhibitor of NF-kB activation) but not by pentoxifylline (TNF-α release inhibitor), indomethacin and niflumic acid (COX-1 and -2 inhibitors). Stimulation of B1R with a selective agonist (des-Arg⁹-BK, 10 μM; 30 min) increased O₂(●⁻)production which was prevented by apocynin and diphenyleneiodonium (NADPH oxidase inhibitors). Data suggest that the increased expression of B1R by TPM in A549 cells is mediated by oxidative stress, IL-1β and NF-kB but not by cyclooxygenases or TNF-α. The amplification of O₂(●⁻) levels via the activation of B1R-NADPH oxidase may exacerbate pulmonary inflammation and contribute to the chronicity of tobacco smoke-related lung diseases. PMID:21600945

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

    PubMed

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

    2009-12-01

    In vitro studies demonstrated that alternative oxidase (AOX) is biochemically regulated by a sulfhydryl-disulfide system, interaction with alpha-ketoacids, ubiquinone pool redox state and protein content among others. However, there is still scarce information about the in vivo regulation of the AOX. Cucumis sativus wild-type (WT) and MSC16 mutant plants were grown under two different light intensities and were used to analyze the relationship between the amount of leaf AOX protein and its in vivo capacity and activity at night and day periods. WT and MSC16 plants presented lower total respiration (V(t)), cytochrome oxidase pathway (COP) activity (v(cyt)) and alternative oxidase pathway (AOP) activity (v(alt)) when grown at low light (LL), although growth light intensity did not change the amount of cytochrome oxidase (COX) nor AOX protein. Changes of v(cyt) related to growing light conditions suggested a substrate availability and energy demand control. On the other hand, the total amount of AOX protein present in the tissue does not play a role in the regulation neither of the capacity nor of the activity of the AOP in vivo. Soluble carbohydrates were directly related to the activity of the AOP. However, although differences in soluble sugar contents mostly regulate the capacity of the AOP at different growth light intensities, additional regulatory mechanisms are necessary to fully explain the observed results. PMID:19493308

  12. [The Xanthine Oxidase Inhibitory Activity and Hypouricemic Effects of Crude Drugs Obtained from the Silkworm in Mice].

    PubMed

    Tanaka, Ryuichirou; Miyata, Yuuma; Minakuchi, Naoki; Murakami, Ayako; Sakazaki, Fumitoshi

    2015-01-01

    This study evaluated the effects of crude drugs obtained from the silkworm in mice with oxonic acid-induced hyperuricemia using xanthine oxidase inhibitory activity and plasma uric acid levels. The plasma uric acid level was analyzed using an improved HPLC with UV detection (HPLC-UV) method, which enabled high-sensitivity analysis of a microliter of plasma. Using this method, we evaluated natural products administered orally to the hypouricemic mice. The plasma uric acid level of mice administered a water-soluble extract from silkworm larvae with botrytis (used in traditional Chinese medicine to reduce wind, lower blood pressure, and change platelet coagulation) was significantly lower than in the control group 1, 2, and 3 h after treatment. In addition, water soluble extracts from a fungus (NBRC 31161) metabolite and silkworm pupae and larvae reduced the plasma uric acid levels in mice compared with the control group. PMID:26423873

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-11-01

    Creatine kinase (CK: adenosine-5-triphosphate-creatine phosphotransferase) is an important enzyme of muscle cells; the presence of a large amount of the enzyme in blood serum is a biomarker of muscular injuries, such as acute myocardial infarction. This work describes a bi-enzyme (glucose oxidase and hexokinase based) biosensor for rapid and convenient determination of CK activity by measuring the rate of ATP production by this enzyme. Simultaneously the biosensor determines glucose concentration in the sample. Platinum disk electrodes were used as amperometric transducers. Glucose oxidase and hexokinase were co-immobilized via cross-linking with BSA by glutaraldehyde and served as a biorecognition element of the biosensor. The biosensor work at different concentrations of CK substrates (ADP and creatine phosphate) was investigated; optimal concentration of ADP was 1mM, and creatine phosphate - 10 mM. The reproducibility of the biosensor responses to glucose, ATP and CK during a day was tested (relative standard deviation of 15 responses to glucose was 2%, to ATP - 6%, to CK - 7-18% depending on concentration of the CK). Total time of CK analysis was 10 min. The measurements of creatine kinase in blood serum samples were carried out (at 20-fold sample dilution). Twentyfold dilution of serum samples was chosen as optimal for CK determination. The biosensor could distinguish healthy and ill people and evaluate the level of CK increase. Thus, the biosensor can be used as a test-system for CK analysis in blood serum or serve as a component of multibiosensors for determination of important blood substances. Determination of activity of other kinases by the developed biosensor is also possible for research purposes. PMID:26452867

  15. Lymphatic diamine oxidase secretion stimulated by fat absorption is linked with histamine release.

    PubMed

    Ji, Yong; Sakata, Yasuhisa; Li, Xiaoming; Zhang, Chao; Yang, Qing; Xu, Min; Wollin, Armin; Langhans, Wolfgang; Tso, Patrick

    2013-04-15

    Diamine oxidase (DAO) is abundantly expressed in mammalian small intestine catalyzing the oxidative breakdown of polyamines and histamine. The aim of this study was to determine the relationship between stimulation of intestinal diamine oxidase secretion with intestinal fat absorption and histamine release. Conscious intestinal lymph fistula rats were used. The mesenteric lymph ducts were cannulated and intraduodenal tubes were installed for the infusion of Liposyn II 20% (an intralipid emulsion). Lymphatic DAO activity and protein secretion were analyzed by radiometric assay and Western blot, respectively. Lymphatic histamine concentration was measured by ELISA. Infusion of Liposyn II (4.43 kcal/3 ml) resulted in a ~3.5-fold increase in lymphatic DAO protein secretion and DAO activity, peaking at 1 h and lasting for 3 h. Liposyn II infusion also increased the lymphatic histamine release, a substrate for DAO. To determine the relationship of DAO release with histamine release, histamine was administered intraperitoneally (10 mg/kg) in fasting rats and resulted in a significant doubling in lymphatic DAO activity, supporting a link between histamine and DAO. In addition, ip administration of the histamine H4 receptor antagonist JNJ7777120 significantly reduced the Liposyn II-induced DAO output by 65.9%, whereas H(1) (pyrilamine maleate), H(2) (ranitidine), and H(3) (thioperamide maleate) receptor antagonists had little effect. We conclude that DAO secretion may contribute to the catabolism of histamine released during fat absorption and this is probably mediated through the histamine H(4) receptor. PMID:23413254

  16. Ligation of FcγR Alters Phagosomal Processing of Protein via Augmentation of NADPH Oxidase Activity.

    PubMed

    Balce, Dale R; Rybicka, Joanna M; Greene, Catherine J; Ewanchuk, Benjamin W; Yates, Robin M

    2016-07-01

    Proteolysis and the reduction of disulfides, both major components of protein degradation, are profoundly influenced by phagosomal redox conditions in macrophages. We evaluated the activation of phagocytic receptors that are known to influence activation of the phagocyte NADPH oxidase (NOX2), and its effect on phagosomal protein degradation. Population-based and single phagosome analyses of phagosomal chemistries in murine macrophages revealed that activation of NOX2 via the Fcγ receptor (FcγR) during phagocytosis decreased rates of proteolysis and disulfide reduction. Immunoglobulin G (IgG)-stimulated reactive oxygen species (ROS) production and the inhibition of phagosomal proteolysis and disulfide reduction were dependent on NOX2, FcγR and protein kinase C (PKC)/spleen tyrosine kinase (Syk) signaling. In contrast, low levels of ROS production were observed following the phagocytosis of unopsonized beads, which resulted in higher rates of phagosomal proteolysis and disulfide reduction. Phagosomes displayed autonomy with respect to FcγR-mediated differences in NOX2 activation and proteolysis, as phagosomes containing unopsonized cargo retained low NOX2 activation and high proteolysis even in the presence of phagosomes containing IgG-opsonized cargo in the same macrophage. These results show that opsonization of phagocytic cargo results in vastly different phagosomal processing of proteins through the FcγR-triggered, PKC/Syk-dependent local assembly and activation of NOX2. PMID:27020146

  17. Hemin/G-quadruplexes as DNAzymes for the fluorescent detection of DNA, aptamer-thrombin complexes, and probing the activity of glucose oxidase.

    PubMed

    Golub, Eyal; Freeman, Ronit; Niazov, Angelica; Willner, Itamar

    2011-11-01

    Hemin/G-quadruplex catalyzes the H(2)O(2)-mediated oxidation of Amplex Red to the fluorescent product resorufin. This process is implemented to develop hairpin nucleic acid structures for the detection of DNA, to probe the catalytic activity of glucose oxidase, to use the thrombin-aptamer complex as a catalytic readout structure, and to quantitatively analyze telomere chain composition. PMID:21881641

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. Selective labelling of cell-surface polyamine-binding proteins on leukaemic and solid-tumour cell types using a new polyamine photoprobe.

    PubMed Central

    Felschow, D M; Mi, Z; Stanek, J; Frei, J; Porter, C W

    1997-01-01

    Polyamine transport is an active process which contributes to the regulation and maintenance of intracellular polyamine pools. Although the biochemical properties of polyamine transport in mammalian cells have been extensively studied, attempts to isolate and characterize the actual protein(s) have met with limited success. As one approach, photoaffinity labelling of cell surface proteins using a polyamine-conjugated photoprobe may lead to the identification of polyamine-binding proteins (pbps) associated with the transport apparatus and/or other regulatory responses. In a previous study [Felschow, MacDiarmid, Bardos, Wu, Woster and Porter (1995) J. Biol. Chem. 270, 28705-28711], we demonstrated that the photoprobes N4-ASA-spermidine and N1-ASA-norspermine [where the ASA (azidosalicylamidoethyl) group represents the photoreactive moiety] competed effectively with polyamines for transport and selectively labelled two major pbps at 118 and 50 kDa on the surface of murine and human leukaemia cells. In the present study, a new and more potent polyamine-conjugated photoprobe, N1-ASA-spermine, has been synthesized and used to develop a method based on detergent lysis for identifying putative cell-surface pbps on solid-tumour cell types. Transport kinetic assays showed that the new photoprobe competed with spermidine uptake with an apparent Ki of 1 microM, a value 20-50-fold lower than those of earlier probes. In L1210 cells, the new probe identified pbp50 and pbp118 thus reaffirming their identity as pbps. Two new bands were also detected. In A549 human lung adenocarcinoma cells, N1-ASA-spermine identified pbps at 39, 62, 73 and 130 kDa, the latter believed to be a size variant of pbp118. The presence of pbp130/118 in two very different cell types suggests the generality of the protein among mammalian cell types as well as its importance for further study. The high affinity of the photoprobe for the polyamine-transport system strongly suggests that at least some of the

  20. NADPH oxidase activity is essential for Keap1/Nrf2-mediated induction of GCLC in response to 2-indol-3-yl-methylenequinuclidin-3-ols.

    PubMed

    Sekhar, Konjeti R; Crooks, Peter A; Sonar, Vijayakumar N; Friedman, David B; Chan, Jeff Y; Meredith, Michael J; Starnes, Joseph H; Kelton, Kathy R; Summar, Samantha R; Sasi, Soumya; Freeman, Michael L

    2003-09-01

    Glutamate cysteine ligase, the rate-limiting enzyme for the synthesis of glutathione, represents an important component of chemoprevention paradigms. GCLC and GCLM, the genes encoding glutamate cysteine ligase subunits, are induced by indoles, such as indomethacin. Novel functionalized indole analogues and other structurally related compounds were synthesized and used for a comparative structure analysis of GCLC induction. Use of mouse embryo fibroblasts null for Nrf2 (nuclear factor-erythroid 2p45-related transcription factor) and HepG2 cells overexpressing Keap1 demonstrated that indole analogue-mediated GCLC expression was regulated by Nrf2-Keap1 interactions. Indole analogues capable of inducing GCLC were found to increase NADPH oxidase activity. Indole analogues unable to induce GCLC did not increase oxidase activity. HepG2 cells transfected with FLAG/Keap1 were exposed to indomethacin, and the redox state of Keap1 cysteine residues was assessed. The data indicated that Keap1 exhibited several oxidation states that were sensitive to indomethacin treatment. These indomethacin-mediated changes in thiol oxidation states were suppressed by diphenyleneiodonium, a NADPH oxidase inhibitor. Diphenyleneiodonium also suppressed indole analogue-mediated increases in GCLC mRNA. In summary, the use of the indole analogues identified NADPH oxidase activity as a novel upstream activity regulating Nrf2/Keap1 signaling of GCLC, provided data supporting the hypothesis that Keap1 is a downstream effector for oxidase activity, and afforded in vivo data to support the hypothesis that Keap1 thiols can act as molecular sensors of reactive oxygen species. Finally, the comparative structure analysis suggests that 2-indol-3-yl-methylenequinuclidin-3-ols may represent a prototype for the development of novel chemopreventative agents able to activate Keap1/Nrf2 signaling. PMID:14500406

  1. Abortiporus biennis tolerance to insoluble metal oxides: oxalate secretion, oxalate oxidase activity, and mycelial morphology.

    PubMed

    Graz, Marcin; Jarosz-Wilkołazka, Anna; Pawlikowska-Pawlega, Bozena

    2009-06-01

    The ability of Abortiporus biennis to tolerate and solubilize toxic metal oxides (Cu(2)O, Al(2)O(3), ZnO, CuFe(2)O(4)Zn, CdO, and MnO(2)) incorporated into agar media was investigated and the growth rate, oxalic acid secretion, and mycelial morphology were monitored. Among the tested metal oxides, formation of clear zones underneath the mycelium growing on Cu(2)O- and ZnO-amended plates was observed. ZnO, CdO and Cu(2)O caused the highest rate of fungal growth inhibition. An increased level of oxalic acid concentration was detected as a response of A. biennis to the presence of Cu(2)O, MnO(2), ZnO and CuFe(2)O(4)Zn in growth medium. The oxalate oxidase (OXO) was found to be responsible for oxalic acid degradation in A. biennis cultivated in metal-amended media. An increased level of OXO was observed in media amended with Cu(2)O, ZnO and MnO(2). Confocal microscopy used in this study revealed changes in mycelial morphology which appeared as increased hyphal branching, increased septation and increased spore number. PMID:18985279

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-06-26

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

  4. Accumulation of free polyamines enhances the antioxidant response in fruits of grafted tomato plants under water stress.

    PubMed

    Sánchez-Rodríguez, E; Romero, L; Ruiz, J M

    2016-01-15

    Polyamines, small aliphatic polycations, have been suggested to play key roles in a number of biological processes. In this paper, attempts were made to investigate the possibility of improving antioxidant response of tomato fruits in relation with endogenous free polyamines content. We studied the reactive oxygen species and polyamines content, and antioxidant and polyamine-biosynthesis enzyme activities in fruits of ungrafted and grafted tomato plants under moderate water stress. We used a drought-tolerant cultivar (Zarina) and drought-sensitive cultivar (Josefina) to obtain reciprocal graft, selfgraft and ungraft plants. Fruits contained higher endogenous polyamine content during the course of the experiment relative to the control, coupled with higher arginine decarboxylase and spermine synthase activities in Zarina ungrafted and ZarxJos. In these cultivars, tomato fruits showed a lower reactive oxygen species generation and higher catalase and superoxide dismutase activities, suggesting that a higher content in polyamines (especially spermine) exerted a positive effect on antioxidant systems. All of these data suggest that spermine leads to more effective reactive oxygen species scavenging (less tissue damage) in tomato fruits, which may function collectively to enhance dehydration tolerance. PMID:26687637

  5. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity

    PubMed Central

    Deep, Gagan; Kumar, Rahul; Jain, Anil K.; Dhar, Deepanshi; Panigrahi, Gati K.; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P.; Oberlies, Nicholas H.; Agarwal, Rajesh

    2016-01-01

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1–5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47phox). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity. PMID:26979487

  6. Graviola inhibits hypoxia-induced NADPH oxidase activity in prostate cancer cells reducing their proliferation and clonogenicity.

    PubMed

    Deep, Gagan; Kumar, Rahul; Jain, Anil K; Dhar, Deepanshi; Panigrahi, Gati K; Hussain, Anowar; Agarwal, Chapla; El-Elimat, Tamam; Sica, Vincent P; Oberlies, Nicholas H; Agarwal, Rajesh

    2016-01-01

    Prostate cancer (PCa) is the leading malignancy among men. Importantly, this disease is mostly diagnosed at early stages offering a unique chemoprevention opportunity. Therefore, there is an urgent need to identify and target signaling molecules with higher expression/activity in prostate tumors and play critical role in PCa growth and progression. Here we report that NADPH oxidase (NOX) expression is directly associated with PCa progression in TRAMP mice, suggesting NOX as a potential chemoprevention target in controlling PCa. Accordingly, we assessed whether NOX activity in PCa cells could be inhibited by Graviola pulp extract (GPE) that contains unique acetogenins with strong anti-cancer effects. GPE (1-5 μg/ml) treatment strongly inhibited the hypoxia-induced NOX activity in PCa cells (LNCaP, 22Rv1 and PC3) associated with a decrease in the expression of NOX catalytic and regulatory sub-units (NOX1, NOX2 and p47(phox)). Furthermore, GPE-mediated NOX inhibition was associated with a strong decrease in nuclear HIF-1α levels as well as reduction in the proliferative and clonogenic potential of PCa cells. More importantly, GPE treatment neither inhibited NOX activity nor showed any cytotoxicity against non-neoplastic prostate epithelial PWR-1E cells. Overall, these results suggest that GPE could be useful in the prevention of PCa progression via inhibiting NOX activity. PMID:26979487

  7. Hydrogen Peroxide Linked to Lysine Oxidase Activity Facilitates Biofilm Differentiation and Dispersal in Several Gram-Negative Bacteria▿

    PubMed Central

    Mai-Prochnow, Anne; Lucas-Elio, Patricia; Egan, Suhelen; Thomas, Torsten; Webb, Jeremy S.; Sanchez-Amat, Antonio; Kjelleberg, Staffan

    2008-01-01

    The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria. PMID:18502869

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

    PubMed

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

    2016-10-01

    Leptin is a protein involved in the regulation of food intake and in the immune and inflammatory responses, among other functions. Evidences demonstrate that obesity is directly associated with high levels of leptin, suggesting that leptin may directly link obesity with the elevated cardiovascular and renal risk associated with increased body weight. Adverse effects of leptin include oxidative stress mediated by activation of NADPH oxidase. The aim of this study was to evaluate the effect of L-carnitine (LC) in rat renal epithelial cells (NRK-52E) exposed to leptin in order to generate a state of oxidative stress characteristic of obesity. Leptin increased superoxide anion (O2 (•) -) generation from NADPH oxidase (via PI3 K/Akt pathway), NOX2 expression and nitrotyrosine levels. On the other hand, NOX4 expression and hydrogen peroxide (H2 O2 ) levels diminished after leptin treatment. Furthermore, the expression of antioxidant enzymes, catalase, and superoxide dismutase, was altered by leptin, and an increase in the mRNA expression of pro-inflammatory factors was also found in leptin-treated cells. LC restored all changes induced by leptin to those levels found in untreated cells. In conclusion, stimulation of NRK-52E cells with leptin induced a state of oxidative stress and inflammation that could be reversed by preincubation with LC. Interestingly, LC induced an upregulation of NOX4 and restored the release of its product, hydrogen peroxide, which suggests a protective role of NOX4 against leptin-induced renal damage. J. Cell. Biochem. 117: 2281-2288, 2016. © 2016 Wiley Periodicals, Inc. PMID:26918530

  9. Polyamines in Pollen: From Microsporogenesis to Fertilization

    PubMed Central

    Aloisi, Iris; Cai, Giampiero; Serafini-Fracassini, Donatella; Del Duca, Stefano

    2016-01-01

    The entire pollen life span is driven by polyamine (PA) homeostasis, achieved through fine regulation of their biosynthesis, oxidation, conjugation, compartmentalization, uptake, and release. The critical role of PAs, from microsporogenesis to pollen–pistil interaction during fertilization, is suggested by high and dynamic transcript levels of PA biosynthetic genes, as well as by the activities of the corresponding enzymes. Moreover, exogenous supply of PAs strongly affects pollen maturation and pollen tube elongation. A reduction of endogenous free PAs impacts pollen viability both in the early stages of pollen development and during fertilization. A number of studies have demonstrated that PAs largely function by modulating transcription, by structuring pollen cell wall, by modulating protein (mainly cytoskeletal) assembly as well as by modulating the level of reactive oxygen species. Both free low-molecular weight aliphatic PAs, and PAs conjugated to proteins and hydroxyl-cinnamic acids take part in these complex processes. Here, we review both historical and recent evidence regarding molecular events underlying the role of PAs during pollen development. In the concluding remarks, the outstanding issues and directions for future research that will further clarify our understanding of PA involvement during pollen life are outlined. PMID:26925074

  10. Evaluation of antioxidant and xanthine oxidase inhibitory activity of different solvent extracts of leaves of Citrullus colocynthis

    PubMed Central

    Nessa, Fazilatun; Khan, Saeed A.

    2014-01-01

    Background: Citrullus colocynthis is a folk medicinal plan of United Arab Emirates. Several studies on this plant reported and focused on the biological and toxicological profile of fruits pulp. The present study focused on the antioxidant potency of leaf extract of this plant. Aim: To evaluate the antioxidant and xanthine oxidase (XO) inhibitory activities of C. colocynthis by chemical method. Materials and Methods: Four different solvent extracts (methanol-CCM, methanol: water (1:1)-CCMW, chloroform-CCC and hexane-CCH) of leaves of C. colocynthis were investigated for their free radical scavenging activity using DPPH radical as a substrate, lipid peroxidation (LPO) inhibitory activity using a model system consisting of β-carotene-linoleic acid, superoxide radical scavenging activity (enzymatically/nonenzymatically) and XO inhibitory activity. A dose response curve was plotted for determining SC50 and IC50 values for expressing the results of free radical scavenging activity and XO inhibitory activities respectively. Results: The high polyphenolic content of CCM and CCMW extract showed highest antioxidant activity irrespective the method used for this investigation. The overall results decreased in the order of: CCM > CCMW > CCC > CCH. CCH extract was inactive towards chemically generated superoxide radical and poor DPPH radical scavengers. The results of LPO inhibitory activities of leaves extract (0.1, 0.5 and 1.0 mg/mL) also decreased in the order of: CCM > CCMW > CCC > CCH. Overall 1.0 mg/mL leaves extract showed highest antioxidant potency amongst the studied concentration. Conclusion: CCMW and CCM extract of C. colocynthis exhibited promising antioxidants and XO inhibitory activities. PMID:25002802

  11. Exposure of cardiomyocytes to angiotensin II induces over-activation of monoamine oxidase type A: implications in heart failure.

    PubMed

    Manni, Maria Elena; Zazzeri, Marina; Musilli, Claudia; Bigagli, Elisabetta; Lodovici, Maura; Raimondi, Laura

    2013-10-15

    Several evidences indicate that increased cardiac mitochondrial monoamine oxidase type A (MAO-A) activity associates with a failing phenotype. Till now, the mechanism underlying such relation is largely unknown. We explored the hypothesis that exposure of cardiomyocytes to AT-II caused activation of MAO-A and also of catalase and aldehyde dehydrogenase activities, enzymes involved in degrading MAO's end products. Left ventricular cardiomyocytes were isolated from normoglycemic (N) and streptozotocin-injected (50 mg/kg) rats (D) treated or not treated with losartan (20 mg/kg/day in drinking water; DLos and NLos, respectively), a type 1 receptor (AT1) antagonist, for 3 weeks. In each group of cells, MAO, catalase and aldehyde dehydrogenase activities were measured radiochemically and spectrophotometrically. The same enzymes were also measured in HL-1 immortalized cardiomyocytes not exposed and exposed to AT-II (100 nM for 18 h) in the absence and in the presence of irbesartan (1 μM), an AT1 antagonist. MAO-A catalase and aldehyde dehydrogenase activities were found significantly higher in D, than in N cells. MAO-A positively correlated with catalase activity in D cells. MAO-A and aldehyde dehydrogenase but not catalase over-activation, were prevented in DLos cells. Similarly, MAO-A activity, but not catalase and aldehyde dehydrogenase increased significantly in HL-1 cells acutely exposed to AT-II and this increase was prevented when irbesartan, an AT1 antagonist was present. Over-activation of cardiomyocyte MAO-A activity is among acute (18 h) and short-term (2-weeks of diabetes) cardiac effects of AT-II and a novel target of AT1 antagonists, first line treatments of diabetic cardiomyopathy. PMID:24012905

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

    PubMed

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

    2015-11-01

    The effect of ultrasound treatment on particle size, color, viscosity, polyphenol oxidase (PPO) activity and microstructure in diluted avocado puree was investigated. The treatments were carried out at 20 kHz (375 W/cm(2)) for 0-10 min. The surface mean diameter (D[3,2]) was reduced to 13.44 μm from an original value of 52.31 μm by ultrasound after 1 min. A higher L(∗) value, ΔE value and lower a(∗) value was observed in ultrasound treated samples. The avocado puree dilution followed pseudoplastic flow behavior, and the viscosity of diluted avocado puree (at 100 s(-1)) after ultrasound treatment for 1 min was 6.0 and 74.4 times higher than the control samples for dilution levels of 1:2 and 1:9, respectively. PPO activity greatly increased under all treatment conditions. A maximum increase of 25.1%, 36.9% and 187.8% in PPO activity was found in samples with dilution ratios of 1:2, 1:5 and 1:9, respectively. The increase in viscosity and measured PPO activity might be related to the decrease in particle size. The microscopy images further confirmed that ultrasound treatment induced disruption of avocado puree structure. PMID:25899308

  13. Antimicrobial activity and hydrophobicity of edible whey protein isolate films formulated with nisin and/or glucose oxidase.

    PubMed

    Murillo-Martínez, María M; Tello-Solís, Salvador R; García-Sánchez, Miguel A; Ponce-Alquicira, Edith

    2013-04-01

    The use of edible antimicrobial films has been reported as a means to improve food shelf life through gradual releasing of antimicrobial compounds on the food surface. This work reports the study on the incorporation of 2 antimicrobial agents, nisin (N), and/or glucose oxidase (GO), into the matrix of Whey protein isolate (WPI) films at pH 5.5 and 8.5. The antimicrobial activity of the edible films was evaluated against Listeria innocua (ATCC 33090), Brochothrix thermosphacta (NCIB10018), Escherichia coli (JMP101), and Enterococcus faecalis (MXVK22). I